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InduePlant

PROCEEDINGS OFASTUDY GROUP MEETING,BUENOS AIRES 16-20 NOVEMBER 197 ORGANIZED BY THE JOINT FAO/IAEA DIVISION OF ATOMIC ENERGY IN FOODAND AGRICULTURE

INDUCED MUTATIONS AND PLA N T IMPROVEMENT

PA N E L PROCEEDINGS SERIES

INDUCED MUTATIONS AND PLANT IMPROVEMENT

PROCEEDINGS OF A L A T IN AM E R IC A N STUD Y GROUP M E E T IN G

ON INDUCED M U TATIO N S AN D P L A N T IM PR O V E M E N T O RG AN IZED B Y THE

JO INT F AO / IA E A DIVISION OF A TO M IC EN E R G Y IN FOOD AND AG R IC U LTU R E

AN D H ELD IN BUENOS AIRES, 16-20 N O VEM BER 1970

IN T E R N A T IO N A L A TO M IC EN E R G Y A G E N C Y V IE N N A , 1972

These proceedings are dedicated to the m em ory of the Swedish scien tist

N ILS NYBO M ( t 9 July 1970)

He was a p ioneer in developing induced mutation techniques

fo r vegeta tive ly propagated plants

INDUCED M U TATIO N S AND P L A N T IM PR O V E M E N T IA E A , V IEN N A , 1972

STI/PUB/297

P rin te d b y th e IA E A in A u s tr ia

J an u ary 1 9 7 2

FOREWORD

A Latin A m erican Study Group M eeting on Induced Mutations and Plant Im provem ent was held on 16-20 N ovem ber 1970 in Buenos A ire s . The Joint F A O / IA E A D iv is ion o f A tom ic E nergy in Food and A gricu ltu re organ ized the m eeting, which was generously supported by the O rgan ization o f A m e r ican States through th e ir In te r-A m erican N u clear E nergy Com m ission , by the host Governm ent, and by the governm ents o f 15 M em ber States o f FAO and IA E A who sent experts from th e ir countries to partic ipate.

The purpose o f the m eeting was to p rovide an opportunity fo r plant b reed ers and genetic ists o f La tin A m erican countries to m eet experts in mutation breed ing, to learn from th e ir experiences and to discuss the p ossib le ro le o f mutation techniques fo r plant im provem ent in Latin A m erica .

The m eeting consisted o f lectu res by acknowledged experts from d ifferen t parts o f the world , reports from Latin A m erican plant b reed ers and genetic ists , and discussions. The com plete proceed ings are published in the present book. P apers are printed in th e ir o r ig in a l language with abstracts in both E nglish and Spanish. The discussions a re in English . To enable those who do not readboth languages ea s ily to get a better and qu icker understanding o f the m ateria l, some o f the figu re captions and table tit les a re presented in both English and Spanish.

CONTENTS

B reed ing with natural and induced v a r ia b il it y ............................................. 3B . S i g u r b j ô r n s s o n

The genetic arch itectu re o f phenotype patterns in b a r le y ........................ 7Â . G u s t a f s s o nD iscussion ....................................................................................................... 12

Plant conform ation and y ie ld ........................................................................... 13R . S . L o o m i s a n d W . A . W i l l i a m sD iscussion ....................................................................................................... 24

P L A N T BREEDING IN L A T IN A M E R IC A

Plant breed ing in Latin A m e r ic a .................................................................... 29A . G r o b m a nD iscussion ....................................................................................................... 41

Considerations on breed ing m eth ods.............................................................. 43F . G . B r i e g e rD iscussion ....................................................................................................... 47

E l m ejoram ien to de las plantas por inducción de mutacionesen L a t in o a m é r ic a ............................................................................................ 49E . A . F a v r e tD iscussion ....................................................................................................... 60

P o ss ib ilit ie s and im plications o f mutation breed ing in J a m a ic a ........... 61C . A . P a n t o n a n d T . M e n e n d e zD iscussion ....................................................................................................... 66

Induced seed -coat co lour mutations in beans and th e ir s ign ificancefo r bean im provem ent ................................................................................... 67С . С . M o hD iscussion .......................................................................................... ,............ 72

IN D U CTIO N OF M U TATIO N S

Com parative genetic e ffec ts o f d ifferen t physica l mutagensin h igher p la n ts ................................................................................................ 75H . H . S m i t hD iscussion ....................................................................................................... 93

Advances in methods o f mutagen treatm ent ............................................... 95C . F . K o n z a k , I r e n e M . W i c k h a m a n d M. J. D e K o c k D iscussion ....................................................................................................... 118

INTRODUCTORY PAPERS

E ffic ien cy o f m utagenesis ............................................................................... 121H . G a u l , G. F r i m m e l , T . G i c h n e r andE . U l o n s k a

Mutagenic sp ec ific ity in flow erin g plants: Facts and prospects .......... 141R . A . N i la nD iscussion ....................................................................................................... 150

PR IN C IP LE S OF M U TA T IO N BREEDING

Mutational reconstruction o f crop ideotypes ............................................. 155M . S . S w a m i n a t h a nD iscussion ....................................................................................................... 170

U tiliza tion o f induced chrom osom al aberrations: Translocations,duplications and tr isom ies in barley ........................................................ 173A . H a g b e r g , G. P e r s s o n a nd G. H a g b e r gD iscussion ....................................................................................................... 182

M utagenesis applied to durum wheat: Resu lts and p e r s p e c t iv e s .......... 183G . T . S c a r a s c i a - M u g n o z z a , D. B a g n a r a and A . B o z z i n iD iscussion ....................................................................................................... 197

Combination o f mutated genes as an additional too l in plant breeding . 199W . G o t t s c h a l kD iscussion .................................................................................. ..... ............... 216

M U T A T IO N BREEDING IN VARIOUS CROPS

Mutation breed ing fo r y ie ld and kernel perform ance in spring barley . 221H . H a n s e l , W . S i m o n and K . E h r e n d o r f e rD iscussion ...................................................................................................... 233

Mutation breed ing in w h e a t ............................................................................... 23 7K a t a r i n a B o r o j e v i c a n d S. B o r o j e v i cD iscussion ....................................................................................................... 250

Mutations and phys io log ica l reaction to se ve ra l chem ical mutagensin peanuts, A rach is hypogaea L ................................................................... 253A . A s h r iD iscussion ............................................................................. ......................... 263

Studies o f combined treatm ent o f A c e r negundo L . seeds withgrowth regu lators and mutagens ................................................................ 265G . F . P r i v a l o vD iscussion ....................................................................................................... 274

Investigation o f the eco logy o f the mutant gene ......................................... 277K . K . S i d o r o v a a n d V . V . K h v o s t o v a

Mutation breed ing in soybeans .............................................................. .. 285F . К . S . K o o

Im provem ent o f v ege ta tive ly propagated plantsby ion izin g radiation ..................................................................................... 293C . B r o e r t j e sD iscussion .................................................................................................. .. . 298

S PE C IF IC O BJECTIVES OF M U TA T IO N BREEDING

B reed ing and screen ing techniques fo r seed protein im provem entin crop plants ................................................................................................... 303O . P . K a m r aD iscussion ....................................................................................................... 314

Induced va ria tion in quantitatively inherited characters ........................ 317R . D . B r o c k , H . F . S h a w and D . F . C a l l e nD iscussion ....................................................................................................... 321

Induced mutation research in wheat .............................................................. 323C . F . K o n z a kD iscussion ....................................................................................................... 329

V aria tion in protein quantity and quality induced in barleyby EMS treatm ent .......................................................................................... 331H . D o l lD iscussion ....................................................................................................... 341

M utagenesis o f a fluctuating character: g ra in dormancyin K ristin a b a r l e y ............................................................................................ 343Â . G u s t a f s s o n , U . L u n d q v i s t , J . K u c e r a a n d J . G h a t n e k a rD iscussion ....................................................................................................... 348

Induced mutations and w inter barley im provem ent .......... ......................... 349R . A . N i 1 a n

M U TA T IO N RESEARCH AND U T IL IZ A T IO N OF INDUCED M U TANTS IN L A T IN A M E R IC A

Reacción frente a Puccin ia recóndita t r it ic i de las líneas derivadas del cruzam iento entre la variedad de tr igoSinvalocho M A y su mutante inducida ..................................................... 355F . L . M u j i c a , E . F . A n t o n e l l i y H. P . C e n o zD iscussion ....................................................................................................... 363

A soc iac ión entre la condición harinosa y los componentesprote icos del endosperm a en la mutante opaque-2 .............................. 365J . C o r r e n t i y R u t M . S o l a r iD iscussion ....................................................................................................... 368

Rendim iento y estabilidad en m ezclas de mutantes de cebada ............. 369A . v o n d e r P a h l e nD iscussion ....................................................................................................... 384

Com paración entre los efectos del metanosulfonato de etiloy los rayos X en la inducción de mutaciones en Capsicum annuum L . 387H . M . Z u b r z y c k i y A . v o n d e r P a h l e nD iscussion ...................................................................................................... 396

Developm ent o f gossypol-g land less strains o f c o t t o n .............................. 397M . G u t i e r r e z , J . V r d o l j a k and A . R i c c i a r d i

Estudio sobre la variab ilidad inducida por etilm etansulfonato (EMS) en ca racteres cuantitativos de plantas hermanas de mutantesd rásticos en tr igo ( T . vu lga re ) con propósitos de s e le c c ió n ............. 405R . T r u j i l l o F i g u e r o a y M . J . R í o s B e t a n c o u r t D iscussion ....................................................................................................... 423

Mutaciones inducidas y program a de m ejoram iento del tr ig o en Chile 425I . R a m í r e z A r a y a , C . S a n z de C o r t á z a r y С . F . K o n z a kDiscussion ...................................................................................................... 432

E l m ejoram ien to genético del tr igo en e l B ra s ily las posib ilidades de u tilizac ión de mutaciones inducidas ................ 435E . A . O s o r i oD iscussion ....................................................... ............................................... 441

In form aciones sobre la inducción de mutaciones en tr igomediante la irrad iac ión de sem illas ....................................................... 443E . P e i x o t o G o m e sD iscussion ............... ....................................................................................... 445

F recu encia de mutaciones inducidas por radiación gamma y metanosulfonato de etilo en d iferentes estados degerm inación de las sem illas de fr i jo l ( Phaseolus vu lgaris L . ) . . . . 447F . D e l g a d o de l a F l o r B .D iscussion ...................................................................................................... 451

Irrad iac ión de sem illa de sorgo ( Sorghum vu lga re ) y de tr igo(T riticu m vu lgare) con rayos gamma ................................................... 453G . d e A l b a , N . R e y e s y A . H e r n a n d e sD iscussion ...................................................................................................... 455

Inducción a r t if ic ia l de mutantes en papa c r io lla(Solanum phureja Juz. et Buk. ) ................................................................ 457P . L . G ó m e z C u e r v o y N. E s t r a d a R a m o sD iscussion ...................................................................................................... 468

Obtención de g ira so les a n d ro e s té r ile s .......................................................... 471C . R e m u s s i , H. S a u m e l l y G. V i d a lD iscussion ...................................................................................................... 474

Mutaciones inducidas por irrad iac ión en e l p era lPackham 's Trium ph ..................................................................................... 475F . R o b yD iscussion ...................................................................................................... 484

E l uso de radiaciones en e l m ejoram iento del banano(Musa sapientum L . ) ................................................................................... .. 485J . V e l e z F o r t u ñ o y A . C e d e ñ o M a l d o n a d oD iscussion ....................................................................................................... 489

Some b io lo g ica l e ffec ts o f post-treatm ent with cysteineon gam m a-irrad ia ted r ic e seeds ............................................................ 491A . A n d oD iscussion ....................................................................................................... 500

E fecto del d im etil sulfóxido sobre la radiosensib ilidad del esperm a de D rosoph ila m elanogaster en dos estados de sumaduración ...................................................................................................... 501B e a t r i z M a z a r B a r n e t tD iscussion ...................................................................................................... 504

Investigation o f a possib le mutagenic e ffec t o f ju ice fromirrad ia ted banana ............................................................................................ 505A . B l u m e n s c h e i n , M . M . I g r e j a and M . A . V a l é r i oDiscussion ......................................................................................... 510

GENERAL DISCUSSION

The ro le o f induced mutations in plant im provem ent ............................. 513R . D . B r o c kD iscussion ....................................................................................................... 520

Summary o f G enera l D iscussion .............................................................. 523

APPE N D IX : L IS T OF M U TAN T V AR IE T IE S ............................................. 526

L IS T OF P A R T IC IP A N T S ................................................................................. 545

INTRODUCTORY PAPERS

BREEDING WITH N ATU R AL AND INDUCED VARIABILITY

B. SIGURBJÔRNSSONJoint FAO/IAEA Division of Atomic Energy

in Food and Agriculture,International Atomic Energy Agency,Vienna

' It has long been known that induced mutations could be useful fo r the solution o f sp ec ific p rob lem s where m ore conventional methods w ere insu ffic ien t. The few people who experim ented with this technique had some success wh ile they w ere laying the groundwork fo r its fu ll exploitation by other plant b reed ers . Some o f these w orkers have joined in com piling their experience to produce a Manual on Mutation B reeding, which has just been published as IA E A Techn ical Reports S eries No. 119.

Recent experience and indeed success in plant breed ing has c lea r ly shown that the m astering o f mutation breed ing techniques m ay becom e cru c ia l to fu rther success in the breed ing of many crop spec ies . There a re two m ain reasons fo r th is. One is that fo r som e crops, espec ia lly the ce rea ls , the breed ing intensity has been so great that fo r some eco lo g ic a l reg ions it w il l be in creas in g ly d ifficu lt to ach ieve fu rther p rogress drawn so le ly from germ plasm already existing and read ily ava ilab le.The h ighest-y ie ld ing genotypes in the w orld co llections a re indeed those v a r ie t ie s which have been b red recen tly and a re s t ill in use. O ther a g ro nom ic ch a racteris tics , such as d isease res is tance, a re a lso becom ing in c reas in g ly d ifficu lt to loca te naturally, and even i f they a re found, the prob lem s o f in corporating these tra its into h igh -perform ance genotypes m ay present form idab le d ifficu lties .

The m ore recen t e ffo rts to im prove the nutritional quality o f cerea ls , esp ec ia lly by adjusting the amino acid balance, have run into considerable d ifficu lties in that, fo r exam ple, h igh -lys in e genotypes a re found only ve ry ra re ly . These are some o f the reasons why plant b reed ers a re again paying m ore attention to the poss ib ilit ies o ffe red by induction o f mutations for the tra its they seek. The best-known contem porary plant b reeder,Dr. Norm an Borlaug, is aware o f these p rob lem s and is in terested in exp loring the p o ss ib ilit ies o ffe red by the mutation breed ing technique. He had planned to partic ipate in this Study Group M eeting and to g ive a paper h ere . I was so rry to learn that he had to cancel his participation , but I am sure we a ll understand his reasons caused by an event beyond his con tro l! I know I speak fo r a ll o f us here when I express our m ost s in cere congratulations to Dr. Borlaug on rece iv in g the Nobel Peace P r iz e . I know of no plant b reeder, o r fo r that m atter, no one concerned with the fight against hunger in the w orld , who m ore deserved this highest award fo r work to wards peace in the w orld than Norm an Borlaug. F o r us as plant b reeders this award is indeed encouraging and underlines the im portance o f plant b reed ing, not only fo r the im provem ent o f agricu ltu ra l crops to in crease the liv in g standard fo r the fa rm er, but also fo r the s tr ive to feed a grow ing w orld population and thus fo r the s tr iv e towards w orld peace and prosperity .

3

4 SIGURBJÓRN SSON

The other reason fo r the increasing attention being paid to induced mutations is the rap id and alarm ing eros ion o f our genetic resou rces .These resou rces a re v ita l i f sustained p rogress in plant breed ing is to be expected. Aeons o f evolution have sculptured fa m ilie s , species, va r ie tie s and races o f crops, m ost o f which i f lost would take aeons to produce again. We cannot p red ict our agricu ltu ra l and food production needs m ore than a few decades ahead. Y e t the present gene resou rces must se rve us fo r centuries to com e. Our present resou rces o f plant genes, even i f they have no value in breed ing to m eet our present needs, a re the basic r e sources fo r the future when one considers the com plex ity and duration o f natural evolution.

The rap id ly expanding human population is resu lting in the cultivation o f m ore v irg in land with the simultaneous destruction o f the native plarits which grew there. M oreover, the new fie ld s as w ell as the old agricu ltu ra l areas in many countries a re being sown with h igh ly-b red superior va r ie t ie s which unfortunately a re rooted in a v e ry narrow genetic base. The need fo r im m ediate and thorough exploration o f plant gene resou rces and their safe conservation is th ere fo re o f im m ediate and urgent concern.

Mutation w orkers , whose daily bread is the creation and manipulation o f genetic va r ia b ility , a re acutely aware o f the dangers resu lting from the genetic eros ion taking p lace. Because they a re working with the a rt if ic ia l crea tion o f va r ia b ility to enhance genetic resou rces , they know best our lim ited ab ility to sculpture genotypes and create new variants through the use o f a r t if ic ia l m utagenesis.

N everth e less , mutation induction is a re a l and proven way to create varia tion within a crop va r ie ty , and fo r agricu ltu re and food production as practised today, a r t if ic ia l m utagenesis o ffe rs a poss ib ility fo r induction o f d es ired attributes, perhaps some o f those that e ith er cannot be found naturally o r have been lost. When no gene fo r res istance to a particu lar ra ce o f a particu lar d isease organ ism can be found, plant b reeders have no obvious a lternative other than attempting mutation induction.

I have outlined two reasons why plant b reeders a re now paying m ore attention to mutation induction and mutation breed ing. Th ere is a third reason a lso . The method does not only seem to be p rom ising in theory but has in the las t few yea rs a lready g iven r is e to a number of agronom ica lly s ign ifican t crop v a r ie t ie s . A t the Symposium on Induced Mutations in P lan ts, held in Pullm an, Washington, in 1969, I reported 77 re g is te red mutant crop va r ie t ie s . A t the tim e o f the present m eeting, this total has in creased to 93. In addition, there are a number o f mutant va r ie t ie s on the m arket, e sp ec ia lly o f ornam entals, o f which I do not have detailed in form ation . A l is t o f mutant va r ie tie s appears in the Appendix. The rate o f re lea se o f 90 o f these va r ie tie s has been as fo llow s: B e fo re 1950,1 va r ie ty ; between 1950 and 1961, 15 va r ie t ie s , and between 1962 and 1970, 74 v a r ie t ie s . It thus appears that the use o f induced mutations is in creas in g rap id ly , m ostly as a resu lt o f recen t and rap id im provem ent in mutagen treatm ent techniques and a better understanding o f the nature and behaviour o f induced mutants. The mutant va r ie t ie s have been r e leased in the fo llow ing crops: C erea ls , 36 (barley 15, wheat 11, r ic e 5, oats 5); legu m es,15; ornam entals, 32; and 10 va r ie t ie s o f various other crops. X -ra y s have been used m ost frequently fo r mutation induction, e sp ec ia lly in the past, and have resu lted in the re lea se o f 63 va r ie t ie s . G am m a-rays have given r is e to 16 va r ie t ie s , neutrons to 13, and a che-

NATURAL AND INDUCED VARIABILITY 5

m ica l mutagen has so fa r resu lted in 1 com m erc ia l v a r ie ty . D irect m u ltip lication o f mutants has given r is e to 81 va r ie t ie s , and 12 have a risen as a resu lt o f c ro sses in vo lv in g induced mutants. The la tte r figu re is probably v e ry conserva tive since it is d ifficu lt to obtain in form ation on induced mutations in ped ig rees . Induced mutants a re , fo r exam ple, v e ry w idely used by p riva te and other plant b reeders in Europe fo r b a r le y as w e ll as ornam entals, but it is d ifficu lt to obtain in form ation on this point.

It is even m ore d ifficu lt to obtain in form ation about acreage under mutant v a r ie t ie s . The present conservative estim ates put this at 3-6 m illion hectares , at a value o f $750 - 1500 m illion .

Thus, it seem s that mutation breed ing has com e o f age as an e ffic ien t plant b reed ing method. L e t us hope that unchecked genetic eros ion w ill not lead to a situation w here the induction o f mutations becom es the only way to secure h igh -perform ance crop va r ie t ie s , but can rem ain , as now, a usefu l com plem entary method.

I hope this m eeting w ill help you decide i f and when it is pro fitab le to re s o r t to induced mutations in your plant breed ing p rogram s, and to som e extent, help you in making better use o f this technique. Means o f manipulating the mutation p rocess should be as eas ily ava ilab le to you as those fo r m anipu lating'hybrid ization and se lection . It is rea lly not a choice o f e ither one method o r the other, since they should a ll be part o f your arsena l. P lant breed ing has but one goal: im provem ent o f plants.The better-equ ipped one is to reach that goal, the qu icker the resu lts.

THE GENETIC ARCHITECTURE OF PHENOTYPE PATTERNS IN BARLEY*

Á. GUSTAFSSON

Institute of Genetics,Lund, Sweden

Abstract-Resumen

T H E G E N E T IC A R C H IT E C T U R E O F P H E N O T Y P E P A T T E R N S IN B A R L E Y .

A s e r ie s o f p h e n o ty p ic p a tte rn s in b a r le y h a v e b e e n stu d ie d w ith re g a rd to t h e ir g e n e t i c b a c k g ro u n d s in

th e la s t d e c a d e . S e v e n d i f f e r e n t p h e n o ty p e p a tte rn s are d iscu sse d fro m th is p o in t o f v ie w in th e a r t ic le

( c h lo r o p la s t and c h lo r o p h y ll m u ta tio n s , e c e r i f e r u m an d e r e c t o id e s t y p e s , p ra e m a tu ru m and in te r m e d iu m

m u ta tio n s , as w e l l as m u ta tio n s fo r m ild e w r e s is t a n c e ) . E a ch p a tte r n h a s a g e n e t i c b a c k g ro u n d c o n s is t in g

o f n u m e ro u s or s e v e r a l g e n e s an d a l l e l e s h a v in g d is c r e te e f f e c t s . E s p e c ia l ly in f o r m a t iv e in th is r e s p e c t are

th e c h lo r o p h y ll an d e c e r i f e r u m m u ta tio n s . S o m e , l ik e th e e c e r i f e r u m and e r e c t o id e s t y p e s , show a p ro

n o u n c e d m u ta g e n s p e c i f i c i t y . In a d d it io n to c h a n g e s in g e n e s and a l l e l e s le a d in g to d is c r e te e f f e c t s th e re

o c c u r in a l l p h e n o ty p e p a tte rn s n u m e ro u s m u ta tio n s w ith s m a ll ( m in o r , q u a n t i t a t iv e ) e f f e c t s . T h e term s

m a c r o - an d m ic r o m u t a t io n s , as w e l l as o l i g o - an d p o ly g e n e s are d is c u s s e d . A n ew d e n o ta tio n " c r i to m u ta -

t io n , c r i t o g e n e , c r i t o a l l e l e " — fro m th e G re e k w o rd " k r i t ik o s ” ( a b l e to d is c e r n , t o s e p a r a te ) — is in tr o d u c e d

t o g e th e r w ith its c o u n te r p a r t " a c r i t o m u t a t io n , a c r i t o g e n e , a c r i t o a l l e l e " , w h ic h im p lie s le ss e a s i l y d is c e r n ib le

p h e n o t y p ic a l l y .

L A E S T R U C T U R A G E N E T IC A D E M O D E L O S F E N O T IP IC O S EN L A C E B A D A .

En e l ú lt im o d e c e n io se h a n e s tu d ia d o u n a s e r ie d e m o d e lo s f e n o t fp ic o s e n la c e b a d a c o n r e s p e c to a

sus fo n d o s g e n é t i c o s . En la m e m o r ia se e x a m in a n s ie te m o d e lo s d i fe r e n te s d e fe n o tip o s d e sd e e s te p u n to d e

v is t a ( m u t a c io n e s c lo r o p lá s t ic a s y c l o r o f í l i c a s , t ip o s e c e r if e r u m y e r e c t o id e s , m u ta c io n e s p ra e m a tu r u m e

in t e r m e d iu m , a s f c o m o m u ta c io n e s p a ra lo g r a r la r e s is te n c ia a l o f d io ) . C a d a u n o d e e l l o s p o se e un fo n d o

g e n é t i c o fo rm a d o por n u m ero so s o v a r io s g e n e s y a le lo s d e e f e c to s d is c r e to s . E s p e c ia lm e n t e s i g n if i c a t iv a s

a e s te r e s p e c t o son la s m u ta c io n e s d e la c l o r o f i la y d e l e c e r i f e r u m - A lg u n a s , c o m o lo s tip o s e c e r if e r u m

y e r e c t o i d e s , e x h ib e n u n a e s p e c i f i c id a d m u t a g é n ic a p r o n u n c ia d a . A d e m á s d e lo s c a m b io s e n g e n e s y a le lo s

qu e o r ig in a n e f e c t o s d is c r e to s , en tod o s lo s m o d e lo s fe n o tfp ic o s se p ro d u c e n n u m e ro sa s m u ta c io n e s c o n

e f e c t o s s e c u n d a r io s ( m e n o r e s , c u a n t i t a t iv o s ) . El a u to r d is c a te lo s té rm in o s m a c r o y m ic r o m u t a c io n e s , a s f

c o m o lo s d e o l ig o y p o l ig e n e s . El a u to r u t i l i z a u n a s e r ie d e n u e v o s té rm in o s « c r i t o m u t a c ió n , c r i t o g e n e ,

c r i t o a l e l o » — d e l g r ie g o k r ít ik ó s ( c a p a z d e d is c e r n ir , d e se p a ra r) — y sus a n tó n im o s « a c r i t o m u t a c ió n ,

a c r i t o g e n e , a c r i t o a l e l o » , c o n l a s i g n i f i c a c ió n d e m e n o s f á c i l d e d is c e r n ir d e sd e e l p u n to d e v is t a f e n o t f p ic o .

In his textbook o f 1909 Johannsen coined the Germ an equivalents of the term s "genotype" and "phenotype". "G ene" and "phene", as w e ll as " is o g en ic " and "isoph en ic", a lso com e from this outstanding book. The term "phene" has not gained such common usage as the term "g en e ". The "phene1 concept, how ever, was extensive ly discussed, fo r instance, by Turesson (1922) and Stubbe (1949). Turesson talked o f genophenes, the various poss ib le reaction types o f a genotype. Stubbe, in his turn, analysed what he ca lled the "H eterogen ie g le ich er Phane", a phenomenon a lready treated by Johannsen (1926, p. 167, and previously), v iz . that isophenic individuals need not be isogen ic . Stubbe r e fe r re d to e a r lie r work in D rosophila and Hordeum and enum erated a se r ie s o f cases in Antirrh inum , where one and the same mutant phenotype is caused by d ifferen t genes, fo r instance,

* P a r t o f th e re s e a rc h re p o rte d in th is p a p e r h a s b e e n c a r r ie d o u t u n d er R e se a rc h A g r e e m e n t w it h the

I n t e r n a t io n a l A t o m ic E n e rg y A g e n c y N 0 .3 5 8 / C F .

7

8 GUSTAFSSON

eram osa (2 genes), choripeta la (3 genes), heroína (6 genes), narrow -leaved (17 genes). The expressions "phenocopy", "phenogenetics", "phenogenesis", "phenodeviant", etc. (R iege r et al. 1968) fu rther indicate the usefulness o f the words "phene" and "phenotype" in term inology.

In the fo llow ing I shall concentrate m y discussion on the analysis in ba rley , w here numerous data indicate a rem arkab ly heterogen ic basis o f s e v e ra l fundamental patterns o f phenotypic characters (Gustafsson et a l. 1969). A lis t o f these and other heterogen ic characters can be found in the survey by N ilan (1964) on the cytology and genetics o f barley .

The fo llow ing phene patterns are under intense genetic study:(1) Chloroplast and ch lorophyll mutations (von W ettste in et a l. 1971,

Gustafsson 1940, 1969a and unpub 1., W a lles 1968);(2) E cer ife ru m mutations (Lundqvist et al. 1962, 1968, von W ettstein-

Knowles 1969, F e s te r and S^gaard 1969, S^gaard unpubl. );(3) E recto ides mutations (P ersson 1969, P ersson and Hagberg 1969,

Gustafsson 1941, 1969a and unpubl. );(4) Praem aturum mutations (Gustafsson et al. 1960, D orm ling and

Gustafsson 1969, Gustafsson 1969b);(5) B rev iaris ta tu m mutations (Kugera unpubl., Gustafsson et a l. 1969);(6) Interm edium (contrasting hexastichon) mutations (Gustafsson 1969a,

Gustafsson and Lundqvist unpubl. );(7) Mutations leading to m ildew res is tan ce (N ilan 1964, F a v re t 1965

and unpubl., Jorgensen 1969, Gustafsson 1969b and unpublished data).

The occu rrence o f numerous genes and a lle le s with d isc re te e ffec ts is ch a rac te r is tic o f these phene patterns. The number o f genes responsib le fo r the ch lorophyll mutations ( sensu la to ) was calculated by Gustafsson (1954) to be approxim ately 250 - 300 in barley . In fact, von W ettstein et al. (19 71) have assigned 198 re c e s s iv e lethal ch lorop last mutants to m ore than 80 loc i, but conclude that fo r the albina phenotype alone s e ve ra l hundred genes m ay be responsib le . A conspicuous phene type is represen ted by "a lboxantha", where a ll induced and spontaneous a lle le s belong to a single locus. M ost o f the ch lorophyll mutations eas ily recorded a re o f a d iscrete character; in s e v e ra l cases their fu ll expression depends on ligh t intensity and tem perature. Certain ly, in addition, numerous mutations, a lle le s and genes may lead to sligh t changes in ch lorop last structure and function o r in pigm ent content and ra tio .

The profound investigations concerning the ecerife ru m character p e r fo rm ed by the authors lis ted w ill not be discussed in deta il h ere. The ch ie f resu lt is that the wax form ation o f stem s, leaves and spikes is in various ways broken down and changed. Phene patterns with distinct b iochem ical and subm icroscop ica l features a r is e . Gene lo c i have been iden tified up to a number o f 45-50, s e ve ra l with numerous a lle le s . Many m ore genes are certa in ly to be iden tified in the intense hybrid ization and linkage p rogram o f the authors. Th ere is no reason to doubt that there a re a lso numerous m utations o f the wax system involv ing slight phene changes, d ifficu lt to define and analyse.

In the case o f the erec to id es character around 30 gene lo c i are known, som e o f them with distinct phene ch a racteris tics , so that the corresponding lo c i can be distinguished s im p ly by a phenotypical inspection. Cases o f s ligh tly deviating erec to id es phenotypes ex is t. To a grea t extent they have been excluded from linkage and a lle lism tests . A contrast to this group o f

PHENOTYPE PATTERNS IN BARLEY 9

dense-eared mutations is form ed by the la x -ea red varian ts. One spec ific set o f mutants and a lle le s belong to a single locus. The la x -ea red phenotypes o f mutants in this locus possess five stamens instead o f three.Num erous dense- and la x -ea red mutants only s ligh tly deviate from the parent phenotype.

Praem aturum mutations are m ostly o f a m ore o r less quantitative type. D isc re te ly segrega tin g mutants have been assigned to at least fiv e o r s ix gene lo c i. O ften mutants in locus m at-a, to which the two well-know n mutants erec to id es 16 ( e r t - o 16) and the m arket va r ie ty M ari (e a r ly 8) belong, a re in addition ch aracterized by photoperiod insensitiv ity . The d iffe ren ce in heading tim e and m aturity o f M ari and its parent Bonus v a r ie s between a few and 20 o r 30 days (o r m ore ) depending on the photo- and th erm o-period applied (" r e la t iv e v ia b il ity " ). S evera l hundred mutants, eas ily screened by th e ir ea r ly heading, deviate from th e ir parents in heading tim e by one o r two days, o r m ore frequently part o f a day. No doubt, numerous genes are invo lved in such ea rlin ess .

The brev ia ris ta tum s a re now under spec ia l study. So fa r ten gene lo c i, each locus with a s e r ie s o f a lle le s , have been iden tified . The phenotypical express ion va r ie s considerably. One locus is noticeab le by the strik ing intraplant d ifferen tia tion in awn length o f term ina l and la te ra l spikes.Mutants ex ist with a s e r ie s of phenotype gradations from alm ost com plete awnlessness to a lm ost norm al awn developm ent (ch a rac te ris tic o f the parent v a r ie ty ). It has been suggested (N ilan 1964, p. 121) that the uncertainty of c lass ifica tion and the fa ilu re to determ ine the in teraction among n on -a lle les have resu lted in an overestim ate o f the number of genes invo lved in the in heritance o f awns. The induced mutations studied so fa r indicate a fa ir ly high number of genes responsib le fo r d isc re te phenotypic changes. A lso in this group o f mutations d ifferen t genes lead to d iffe ren ces in phene exp res sion. A la rg e number o f genes can be proposed fo r the sm a ll changes o f awn length, d ifficu lt to analyse except under s tr ic t ly con tro lled phytotron conditions and a fte r accurate b iom etric analysis.

In p rog ress is an analysis o f the in term edium mutants, which in genera l are r e c e s s iv e to the norm al nutans parent. Six lo c i have been identified .In contrast, the hexastichon type, eas ily induced by mutation and monogenic, has an evident e ffec t when heterozygous, g iv ing a m ore o r less in term edium lik e appearance, with the side flo re ts p a rtia lly developed and fe r t i le . The induced hexastichon mutations are a lle lic to the spontaneous gene v on chrom osom e 2.

A m ost in teres tin g phene com plex is re la ted to res is tance against powdery m ildew, w ith its numerous ra ces . N ilan (loc . c it. ) an dF avre t (lo c . c it . and unpubl.) have indicated a la rg e number o f a lle le s and genes on chrom osom e 5, which m ay poss ib ly have orig inated by repeated gene duplication. N ilan reports the p re vious studies o f res is tan ce . Spontaneous genes fo r res is tance appear to be m ostly dominant, while many induced mutations fo r res is tan ce a re r e c e s s ive (Jorgensen loc . cit. ) and deviate from the spontaneous sources in reaction type. In appropriate tests the induced mutations a re ea s ily d is covered , although ra re ly deviating in m orpho log ica l ch arac te ris tics .

In sum m ary it can be stated that numerous gene lo c i a re often in vo lved in the phenotypical expression of a character pattern. Th ere o c curs, as o r ig in a lly proposed fo r Antirrhinum and Hordeum , a sort o f "group m utab ility ". S im ilar phenes can be caused by d iffe ren t a lle le s in a gene locus but a lso by mutations in d ifferen t genes. These appear to be spread

10 GUSTAFSSON

ove r the genom e. An exception is the m ildew genes m ostly lo ca lized to chrom osom e 5. On the other hand, a lle le s w ithin one and the sam e locus m ay m arked ly deviate with rega rd to th e ir d egree o f phenotypical expression, and d ifferen t genes m ay lead to s trik ing ly d ifferen t reactions o f the phene pattern. In a lm ost e v e ry group o f characters analysed, s e ve ra l o r numerous mutations with d iscrete e ffects a re iso lated , in addition to the numerous mutations with insign ifican t o r slight changes in phenotype. The dense- o r la x -ea red , as w ell as the praematurum mutants a re excellen t exam ples o f such a contrast.

W ith regard to dominance behaviour m ost o f the mutations induced are re c e s s iv e , but cases of semidominant o r even dominant behaviour occur, fo r instance in the case of erecto ides , praem aturum and hexastichon mutations. An in terestin g case was described in e r t - d 14 (Gustafsson and von W ettstein 1957), where the phene "ea r density" has a re c ess iv e background, but the phene "long f ir s t in ternode" g ives dominance in crossings with the parent type.

In mutation analysis o f h igher plants there is now a distinct need fo r a te rm in o log ica l d ifferen tia tion o f the phene and gene e ffec ts . In my Pullman paper o f 1969, I r e fe r r e d to the ambiguous use and meaning of such denotations as "m orp h o log ica l" and "p h ys io lo g ica l", "m a c ro " and "m ic ro " mutations, "o l ig o " ("m a jo r " ) and "p o ly " ("m in o r " ) genes, etc.

The term s m acro - and m icrom utations have since long been used in a d ifferen t sense on the genotypic as w e ll as the phenotypic le v e l (loc . cit. ).In 193 7 I proposed these expressions to be used on the "gen otyp ica l", not the "phenotyp ica l" le v e l. Besides, the application of "m a c ro " and "m ic ro " fo r d ifferen t phenotypic p roperties is often inadequate and obscure (in spite o f the exp ress ive denomination of, fo r exam ple, Gaul 1965). The term s "p o ly - and o ligogen es " a re defined by M ather (1941, p. 160; 1943, p. 34) in the fo llow ing way: "Q ualita tive varia tion is usually m onogenic o r digenic in inheritance . . . In contrast to this, quantitative varia tion m ay be said to be po lygen ic" . "D iffe ren ces distinguishing species m ay be o ligogen ic ,.i. e. they m ay be con tro lled by a sm all number o f genes having e ffects la rg e when com pared with the non-heritable fluctuation, and hence leading to sharp segrega tion ". (H ere re fe ren ce m ay be made also to the definitions in the g lossary of R ie g e r et a l. 1968).

It is im m ed iate ly evident that the numerous mutated genes responsib le fo r the ece r ife ru m character, g iving sharp segrega tin g e ffec ts , cannot be ca lled o ligogen ic (from the Greek: oligo; sm all, a few ). N or can they, accord ing to M ather1 s defin ition (o r the common use o f the expression ) be ca lled polygen ic, s ince they a re sharp in appearance and segregation .The hexastichon character may, o f course, be r e fe r r e d to as "o lig ogen ic " (one gene; strong e ffec ts ), but possib ly not the "in term ed ium " character with its m inim um number o f s ix genes, and defin ite ly not the m ildew r e sistance, erecto ides o r chlorophyll mutants, which a re s im ila r in the high number o f genes to the ecer ife ru m patterns. The genes responsib le a re numerous ("p o ly " ) but have sharp, often la rg e e ffects ("o lig o " , according to M a th e r 's defin ition ). In this connection I w ill r e fe r the reader to Johannsen's c r it ic ism o f the term "po lygen " (1926, p. 489).

F o r the reason o f p rec isen ess I here propose the term "critom utation " (re la ted to G reek: kritikós; able to d iscern , to separate; c f. a lso the English w ords c r it ic , c r it ic a l, c r ite r ion ). A critom utation ( a critogene, a c r ito a lle le ) is a mutation (gene, a lle le ), which is eas ily d iscern ib le in

PHENOTYPE PATTERNS IN BARLEY 11

its e ffec ts and sharp in segregation and linkage tests. The phenotype e x p ression m ay va ry but a critom utation is always ea s ily recove red , when adequate methods of analysis a re applied.

The corresponding p re fix fo r mutations, genes and a lle le s , participating in quantitative phene patterns, having sligh t, unsharp, "m in o r " e ffects , w ill be "a c r ito " m eaning not eas ily d iscern ib le o r distinguishable in analysis 1.

It w ill be reca lled that with the eve r fin er methods o f analysis in c o n tro lle d environm ental and b iochem ica l studies acritom utations and acr ito - genes (a lle le s ) may be p ro g re ss iv e ly tra n s fe rred to the critogroup .

R E F E R E N C E S

D O R M L 1N G , 1 . , G U S T A F S S O N , A . , 1 9 6 9 , P h y to tro n c u l t iv a t io n o f e a r ly b a r le y m u ta n ts , T h e o r . a p p l . G e n e t .

39 : 5 1 .

F A V R E T , E . A . , 1 9 6 5 , " In d u c e d m u ta tio n s in b r e e d in g fo r d is e a s e r e s is t a n c e " , T h e U se o f In d u c e d M u ta tio n s

in P la n t B re e d in g ( R e p . F A O / IA E A T e c h . M e e t in g , R o m e , 1 9 6 4 ) , P e rg a m o n P ress, O x fo rd : 5 2 1 .

F E ST E R , R . , S 0 G A A R D , B . , 1 9 6 9 , T h e l o c a l i z a t i o n o f e c e r i f e r u m l o c i in b a r l e y , H e re d ita s 6 1 : 3 2 7 .

G A U L , H . , 1 9 6 5 , " T h e c o n c e p t o f m a c r o - and m ic r o - m u ta t io n s an d re s u lts o n in d u c e d m ic r o - m u ta t io n s

in b a r l e y " , T h e U se o f In d u ce d M u ta t io n s in P la n t B re e d in g ( R e p . F A O / IA E A T e c h . M e e t in g , R o m e , 1 9 6 4 ) ,

P e rg a m o n P re s s , O x fo r d : 4 0 7 .

G U S T A F S S O N , Á . , 1 9 3 7 , S tu d ie s o n th e g e n e t i c b a s is o f c h lo r o p h y ll f o r m a tio n an d th e m e c h a n is m o f in d u c e d

m u t a t in g , H e r e d ita s 24: 3 3 .

G U S T A F S S O N , Â . , 1 9 4 0 , T h e m u ta tio n sy s te m o f th e c h lo r o p h y ll a p p a r a tu s , Lu nds U n iv . Á rssk r. N . F .

A v d . 2 . 3 6 1 1 : 1 .

G U S T A F S S O N , Â . , 1 9 4 1 , M u t a t io n e x p e r im e n t s in b a r l e y , H e r e d ita s 2 7 : 2 2 5 .

G U S T A F S S O N , Â . , 1 9 5 4 , " S tu d ie s o n th e e x p e r im e n t a l c o n t r o l o f th e m u ta tio n p r o c e s s " , R a d io b io l .

S y m p . , L iè g e : 2 8 2 ,

G U S T A F S S O N , A . , 1 9 6 9 a , " A s tu d y o n in d u c e d m u ta tio n s in p lan ts': In tro d u c to ry a d d r e s s " , In d u c e d M u ta

tio n s in P la n ts ( P r o c . S y m p . P u llm a n , 1 9 6 9 ) , I A E A , V ie n n a : 9 .

G U S T A F S S O N , Á . , 1 9 6 9 b , " P o s i t iv e M u ta t io n e n und ih re V e r w e n d u n g in d e r Z t ic h tu n g h o c h le is t e n d e r

G e r s t e n * S o r t e n " , A r b e i t s t a g . 1 9 6 9 A r b e i ts g e m e in s c h a ft S a a t z u c h t l e i t e r , G u m p e n s te in : 6 3 .

G U S T A F S S O N , Á . , H A G B E R G , A . , L U N D Q V IS T , U . , 1 9 6 0 , T h e in d u c t io n o f e a r ly m u ta n ts in Bonus

b a r l e y , H e r e d ita s _46: 6 7 5 .

G U S T A F S S O N , A . , H A G B E R G , A . , L U N D Q V IS T , U . , P E R S SO N , G . , 1 9 6 9 , A p ro p o se d sy s te m fo r th e c o l

l e c t io n o f b a r le y m u ta n ts a t S v a l ô f , H e re d ita s _62: 4 0 9 .

G U S T A F S S O N , Â . , v o n W E T T S T E I N , D . , 1 9 5 7 , " M u ta t io n e n u n d M u ta t io n s z O c h tu n g " , H a n d b u ch d e r

P fL a n z e n z ü c h tu n g I , 2 . A u f l . , V e r l a g - P a u l - P a r e y , H a m b u r g - B e r lin : 6 1 2 .

J O H A N N S E N , W . , 1 9 0 9 , 1 9 2 6 , E le m e n te d e r e x a k t e n E r b l ic h k e it s le h r e , 1 . , 3 . A u f l . , G u s t a v - F is c h e r -

V e r l a g , J e n a .

JÔ R G E N SE N , J . H . , 1 9 6 9 , "A n a l l e l i c s e r ie s o f m u ta n t g e n e s f o r p o w d e r y - m ild e w r e s is ta n c e in b a r l e y " , 2nd In t .

S y m p . B a r le y G e n e t . P u llm a n , 1 9 6 9 ( in p re s s).

L U N D Q V IS T , U . , v o n W E T T S T E IN , D . , 1 9 6 2 , In d u c t io n o f e c e r i f e r u m m u ta n ts in b a r le y b y io n i z in g r a d ia

tio n s an d c h e m i c a l m u ta g e n s I , H e r e d ita s 4 8 : 3 4 2 .

L U N D Q V IS T , U . , v o n W E T T S T E IN -K N O W L E S , P e n n y , v o n W E T T S T E I N , D . , 1 9 6 8 , In d u c t io n o f e c e r i f e r u m

m u ta n ts in b a r le y b y io n iz in g r a d ia t io n s and c h e m i c a l m u ta g e n s I I , H e r e d ita s 5 9 : 4 7 3 .

1 I a m in d e b te d to P ro fe sso r G . B e n d z , U n iv e r s it y o f C o p e n h a g e n , fo r th e c o in in g o f th e se te r m s .

12 GUSTAFSSON

M A T H E R , К . , 1 9 4 1 , V a r ia t io n an d s e le c t i o n o f p o ly g e n ic c h a r a c t e r s , J . G e n e t . 4 1 : 1 5 9 .

M A T H E R , K . , 1 9 4 3 , P o ly g e n ic in h e r it a n c e an d n a tu ra l s e le c t i o n , B i o l . R e v ._ 1 8 : 3 2 .

N IL A N , R . A . , 1 9 6 4 , T h e c y t o l o g y an d g e n e t ic s o f b a r l e y , M o n o g r . S u p p l, N o .3 , R e s. S t u d . , W a sh in g to n

S ta te U n iv . 3 2 1 : 1 .

PE R S SO N , G . , 1 9 6 9 , A n a t t e m p t to f in d s u it a b le g e n e t i c m a rk e rs fo r d e n s e - e a r l o c i in b a r le y I , H e re d ita s 62: 2 5 .

PE R S SO N , G . , H A G B E R G , A . , 1 9 6 9 , In d u c e d v a r ia t io n in a q u a n t i ta t iv e c h a r a c t e r in b a r l e y . M o rp h o lo g y

an d c y t o g e n e t i c s o f e r e c t o id e s m u ta n ts , H e re d ita s 6 1 : 1 1 5 .

RIEGER, R . , M I C H A E U S , A . , G R EEN , M . M . , 1 9 6 8 , A G lo ssa ry o f G e n e t ic s and C y t o g e n e t ic s , 3rd Edn,

S p r in g e r * V e r la g , N ew Y o r k .

S T U B B E , H . # 1 9 4 9 , l ib e r H e te r o g e n ie g l e ic h e r P h â n e , B i o l . Z b l . j î8 : 4 9 2 .

T U R E S S O N , G . , 1 9 2 2 , T h e g e n o t y p ic a l resp o n se o f th e p la n t s p e c ie s to th e h a b i t a t , H e re d ita s 3: 2 1 1 .

V o n W E T T S T E ÍN , D . , H E N N IN G S E N , K . W . , B O Y N T O N , I . E . . K A N N A N G A R A . G . C . . N IE LSE N , O . F . ,

1 9 7 1 , " T h e g e n i c c o n t r o l o f c h lo r o p la s t d e v e lo p m e n t in b a r l e y " , A u to n o m y an d B io g e n e s is o f M ito c h o n d r ia

a n d C h lo r o p la s t s , A m s te r d a m ( in p re s s).

V o n W E T T S T E IN -K N O W L E S , P e n n y , 1 9 6 9 , " T h e m o le c u la r p h e n o ty p e s o f th e e c e r i f e r u m m u ta n ts ” , 2nd In t .

S y m p . B a r le y G e n e t . P u llm a n , 1 9 6 9 ( in p ress).

W A LLE S, B . , 1 9 6 8 , Biochemical mutants affected in chloroplast morphogenesis, H e ie d it a s 5 9 ; 3 4 6 .

D I S C U S S I O N

G. DE A L B A : What types and doses o f radiation have been used?A . GUSTAFSSON: A l l types between 100 and 20 000 rad.H. G AU L: As your Swedish mutation group has shown, a mutation

in an e rt locus may resu lt in a la rge phenotypic change as w e ll as in a sm a ll one. Would it not be m ore appropriate to speak o f "c r ito a lle le s "(o r a s im ila r te rm ) instead o f "c r ito g en es "? M oreover, I do not like the introduction o f you r term s "critom utations" and "acritom utations", because I cannot see a d ifferen ce to the established term s "m acrom utations" and "m icrom u tations".

A . GUSTAFSSON: I shall possib ly include tlje term "c r ito a l le le " in the fina l v e rs ion o f m y m anuscript.

A . GROBM AN: In the analysis which you presented on mutation spectra in barley , there was no re fe ren ce made to mutations resu lting from u ltrav io le t radiation . Is there any in form ation ava ilab le on the effects on mutation spectra and mutation frequencies re la t iv e to other mutation- induction treatm ents, from your work on barley?

A . GUSTAFSSON: UV has been used as a mutagenic agent in ba rley as w e ll as in other crop plants, irrad ia tin g pollen gra ins. Mutations do a r is e in this way. The mutation spectrum w ill be d ifferen t to that a fter X -irra d ia tion o f po llen gra ins, gross chrom osom al rearrangem ents (translocations, etc. ) lacking, w hereas factor mutations (fo r instance o f the ch loro phyll mutation type) frequently a r is e .

PLAN T CONFORMATION AND YIELD

R. S. LOOMIS, W .A . WILLIAMS

University of California, Davis,United States of America

Abstract-Resumen

P L A N T C O N F O R M A T IO N A N D Y Œ L D .

T h e p o t e n t ia l i t ie s a r e e x a m in e d fo r c r o p y i e ld e n h a n c e m e n t th ro u g h g e n e t i c m o d i f i c a t io n o f th e

p r o d u c t io n p ro c e sse s o f p la n t c o m m u n it ie s . P h o to sy n th e sis an d r e s p ir a t io n p ro c e sse s a r e le s s a m e n a b le t o c h a n g e

th a n a r e a s p e c ts o f p la n t c o n f o r m a t io n r e s u lt in g f r o m c h a n g e s in t h e d is tr ib u t io n o f p h o to s y n th a te s in g r o w th .

S im u la t io n m o d e ls a r e d e s c r ib e d w h ic h m a y a id in p r e d ic t in g th e in f lu e n c e s w h ic h s m a ll c h a n g e s in m o r p h o lo g y

a n d p h y s io lo g y w o u ld h a v e o n y i e l d .

M O R F O L O G IA D E L A S P L A N T A S Y R E N D IM IE N T O .

Los a u to re s e s tu d ia n la s p o s ib il id a d e s d e a u m e n t a r e l r e n d im ie n to d e lo s c u l t iv o s m e d ia n te l a m o d i f i c a c ió n

g e n é t i c a d e lo s p r o c e s o s d e p r o d u c c ió n en c o l e c t iv id a d e s d e p la n t a s . Los p ro c e s o s d e fo to s ín te s is y r e s p ir a c ió n

so n m e n o s s u s c e p tib le s d e a l t e r a c ió n q u e lo s a s p e c to s m o r fo ló g ic o s d e la s p la n t a s re s u lta n te s d e c a m b io s e n la

d is tr ib u c ió n d e lo s p ro d u c to s d e fo to s ín te s is e n e l c r e c im ie n t o . S e d e s c r ib e n m o d e lo s d e s im u la c ió n q u e p u e d e n

s e r v ir d e a y u d a p a ra p r e d e c ir lo s p o s ib le s e f e c t o s d e p e q u e ñ a s v a r ia c io n e s m o r fo ló g ic a s y f i s io l ó g ic a s so b re e l

r e n d im ie n t o .

1. INTRO D U C TIO N

Plant breed ing continues to se rve as the p rincipa l mechanism fo r ach ieving advances in crop production. In m ost instances, we have approached the prob lem of plant im provem ent with a c le a r ly defined set o f goals — fo r exam ple, res istance o r to lerance to a spec ific pest o r to an environm ental event such as daylength o r frost, o r a hybrid advantage is sought. The b reed ing e ffo r t can gen era lly p roceed rapid ly toward the se lected goal because appropriate techniques ex ist fo r em p ir ica l se lection and evaluation o f the new genetic combinations.

In contrast, e ffo rts at mutation breeding despite its great potential have been less successfu l. One reason seem s to be that we cannot eas ily d irect the mutations towards particu lar goa ls . As a resu lt, only a sm all number of the v iab le mutations which we might produce w ill be useful fo r a particu lar p rob lem . The mutation b reed er then is faced with the question o f whether there is any p rac tica l use fo r the in teresting sets o f genes which he accumula tes. He is like a person who invents a can opener but hasn't yet invented a can.

Th is dilem m a, while o f particu lar concern to the mutation b reeder, is an im portant and grow ing issue in a ll aspects o f applied b io logy. Th is can be seen by phrasing the question in another way: G iven an understanding o f ab io -m o lecu la r o r physio log ica l p rocess , o r g iven a new m orphologica l descrip tion , can we p red ict how this w ill influence the behaviour of the whole organism as an individual and in a community? Our sc iences have been orien ted m ore towards reductionist research which seeks to provide an understanding o f m echanism s. W hile the ultim ate aim o f reduction ist r e search is to perm it in tegration , i . e . rational explanations of the whole, we

13

14 LOOMIS and WILLIAMS

have not put equivalent e ffo rt into in tegrative research [1 ]. Idea lly the in teg ra tive techniques would perm it us to p red ict the u tility o f a particu lar tra it. At present, our best a lternative is tim e-consum ing and expensive.We must incorporate the tra it into a suitable com m erc ia l va r ie ty and then conduct a se r ie s o f fie ld tr ia ls to obtain an em p ir ica l answer as to whether the tra it is advantageous. The p rocess is made ea s ie r i f an established analogy ex ists — fo r exam ple, i f we know how earlin ess o r lodging resistance re la tes to fie ld perform ance. H ow ever, it is not so easy to detect sm all d iffe ren ces in behaviour that contribute to genera l y ie ld -a b ility . F o r exam ple, w ill a 5% change in lea f area have a g rea te r influence on y ie ld than a 5% change in photosynthesis rate?

In this paper we w ill consider various aspects o f crop production s y s tem s fo r th e ir.su scep tib ility to im provem ent through mutation breeding, and how techniques o f m odel building and sim ulation can be used in in tegrative research .

2. THE CRO P PRO D U CTIO N SYSTEM

Crop production system s bas ica lly are means fo r capturing so la r energy through photosynthesis and storing the energy in the chem ical bonds of products useful to ou rse lves o r to our anim als. Patterns o f energy and m ateria ls flow through such system s can be sum m arized in flux d iagram s

Energy losses ^

1,586,500' kcal

SUNLIGHT ENERGY 1,800,000 kcal per sq. m eter

PHOTOSYNTHESIS (GROSS)------ fc- 2,000 kcal Energy

F I G . l . M o d e ls o f tw o s im p le g r a z in g sy s te m s in C a l i f o r n ia : a n ir r ig a te d p a stu re w ith a l f a l f a ( le f t ) a n d a d r y

la n d c o m m u n it y o f ro se c l o v e r ( r i g h t ) . T h e a n n u a l f lo w o f e n e r g y th ro u g h p la n ts an d b e e f a n im a ls (b o x e s ) is

sh o w n b y a rro w s . M o st o f th e in c o m in g s o la r e n e r g y is d is s ip a te d th ro u g h t ra n s p ir a tio n an d o th e r e n e r g y e x c h a n g e s

( e n e r g y " lo s s e s " ) . P o rtio n s o f th e e n e r g y c o n v e r t e d in p h o to sy n th e s is a r e lo s t th ro u g h r e s p ir a t io n an d u n co n su m e d

p o rt io n s o f th e p la n t s . S e e M a c F a d y e n [2] fo r o th e r e x a m p le s o f p r o d u c t io n s y s te m s .

( F IG . 1 . M o d e lo s d e dos s is te m a s s e n c i l lo s d e a l im e n t a c ió n d e l g a n a d o e n C a l i fo r n ia : u n p a s to d e a l f a l f a

e n r e g a d í o ( a l a iz q u ie r d a ) y o tro d e t r é b o l ro sa e n s e c a n o ( T r i f o l iu m h ir tu m , a la d e r e c h a ) . S e in d ic a p o r

m e d io d e f le c h a s e l f lu jo a n u a l d e e n e r g ía a t ra v é s d e la s p la n ta s y d e l g a n a d o v a c u n o ( c a s i l l a s ) . L a m a y o r

p a r t e d e l a e n e r g ía s o la r in c id e n t e se d is ip a p o r t ra n s p ir a c ió n y o tro s fe n ó m e n o s d e in t e r c a m b io e n e r g é t ic o

( « p é rd id a s » d e e n e r g ía ) . P o r la r e s p ir a c ió n y la s p a r te s n o c o n s u m id a s d é l a s p la n t a s , se p ie r d e u n a f r a c c ió n

d e l a e n e r g ía tra n s fo rm a d a e n e l p r o c e s o d e fo t o s ín t e s is . V é a n s e o tro s e je m p lo s d e s is te m a s d e p r o d u c c ió n e n

M a c F a d y e n [ 2 ] . )

PLANT CONFORMATION AND YIELD 15

which deta il the inputs and losses o f energy and the s ize o f the standing crop (F ig . 1). One's attention is drawn to a s e r ie s o f im portant p rocesses :

1. The e ffic ien cy o f energy capture in photosynthesis.2. The loss o f energy through resp iration associated with ce llu la r

maintenance and growth.3. The partition ing o f the accumulated energy pool among various parts

o f the standing crop , some o f which form the econom ica lly useful y ie ld .

Our concern is to m ax im ize the amount o f energy which accumulates as y ie ld whether in protein , lip id , o r carbohydrate. In e ffic ien c ies in any o f these p rocesses represen t " le a k s " in the system which, i f plugged, should serve to in crease y ie ld . Thus, weeds serve as com petitors in light in te rception and thus serve as a leak in the firs t p rocess, photosynthesis. A fte r f ir s t considering m odelling techniques, we w ill exam ine b r ie fly how each of these three p rocesses a ffects y ie ld .

3. M ODEL BUILD ING IN IN TE G R A T IV E RESEARCH

3.1. P rop ertie s o f m odels

A m odel represen ts a sum m ary o f a coherent body o f experim ental data in a lo g ica l structure. The m odel may be stated as a concept, fo r exam ple, the concept o f a gene, o r m ay be sum m arized in a m athem atical relationship such as E = M c 2. The experim enta l data may be sum m arized in e ith er causal o r assoc ia tive statements depending upon our current le v e l o f understanding o f the p rocesses involved . The "u ltim ate" m odel would consist o f the sim plest set o f rigorous m athem atical equations needed fo r a com plete descrip tion of the system .

M odels of plant behaviour becom e too com plex fo r analytical m athem atics since in teractive elem ents, feedback, feed -fo rw ard , and threshold phenomena must be considered . The situation is made m ore com plex by the need to consider s e ve ra l leve ls o f organization . Y ie ld is a p roperty o f a plant community and the tra its which we w ish to consider fo r th e ir e ffec t on y ie ld are p roperties o f tissues, organs or whole plants. H igh-speed d ig ita l com puters and, m ore recen tly , dynam ic-system sim ulation languages, have made it possib le to attempt quantitative m odels o f plant behaviour, in dependent o f our ab ility to cope with the situation as a problem in m athem atics . M odels o f com plex p rocesses such as photosynthesis, transpiration , ion absorption, and growth have been developed fo r the organ, individual plant, and community le v e ls o f organization .

One o f the key features o f such m odels, like the rea l system s which they sim ulate, is that they have p roperties beyond those which can be d is covered by analysis o f th e ir iso la ted components. Th is resu lts from the way the parts (o r subm odels) o f the system are coupled together. Thus, the va lid ity o f a m odel and hence its u tility in in tegra tive research , e .g . in pred icting the influence o f a mutated gene, depend upon how w e ll we understand the system . N eed less to say, there are many gaps in our knowledge o f plant p rocesses . Attem pts at m odel building re v ea l these gaps and help estab lish p r io r it ie s fo r needed research [1 ,3 ] .


3.2. Models o f production p rocesses

S evera l v e ry useful static m odels o f photosynthesis have been developed using lea f area as the arch itectu ra l elem ent [4, 5 ]. These w ere stimulated by the ea r ly work o f Boysen-Jensen [6 ], Monsi and Saeki [7] and N ich iporovich [8 ] . D eW it 's [4] m odel considers d irect and diffuse sunlight d istribution in gen era lized fo liage canopies. Optical p roperties o f leaves and th e ir photosynthetic functions, C 0 2 p ro files , and other factors may be treated as va r iab les . The m odel by Duncan et al. [5] treats the canopy in m ore deta il by dividing it into a se r ie s o f horizonta l la yers , each with a spec ific le a f area and angle o f d isplay. In both m odels, each hour o f the day (s o la r positions) is considered as a d iscrete situation and is simulated separate ly . The hourly resu lts are tota lled to learn the daily production.

R ecently , DeW it and B rouw er [9, 10] have attempted a dynamic sim u lation m odel o f plant growth. The m odel is designed fo r m aize and starts with phys io log ica l le v e l in form ation, a descrip tion o f the seed ling plant, and w eather data as a basis fo r grow ing single plants o r whole com m unities. Calculations fo r a long s e r ie s o f "s ta te va r iab les " which describe the plant are done fo r each hour and returned as input data along with changing weather data as input fo r the next hour. Th is is like a "m oving p ictu re" in contrast to the " s t i l l p ic tu res" obtained with d iscrete sim ulations.

4. PHOTOSYNTHESIS IN P L A N T COM M UNITIES

4 .1 . Canopy arch itecture

Through experim ent and sim ulations, a good deal o f in form ation has accumulated about community photosynthesis. F irs t , we know that the th eore tica l upper lim it in e ffic ien cy o f energy conversion is near 5.2% of tota l so la r irrad iance or about 11.7% o f the photosynthetically active rad iation (P A R ) [11 ]. Such high e ffic ien c ies are achieved only in dim light since light response curves fo r PN (net photosynthesis o f individual leaves ) are cu rv i- lin ea r and e ffic ien cy declines as the lea f is irrad ia ted m ore intensely. Thus the manner o f lea f d isplay re la tive to the sun 's position becom es im portant. Th is has been considered in the d iscrete sim ulation m odels with the finding that w ith typ ica l lea f display, maximum e ffic ien cy in light conve rs ion during m idsum m er in the tem perate zone may be only about 70% as great as the th eore tica l lim it [4, 5] . Some rea l com m unities have been found to achieve this rate o f conversion although m ost of the best values are n ea rer 50%.

The sim ulations also have shown that production rate is particu larly sensitive to so la r radiation patterns, lea f area index, photosynthetic capab ility o f the leaves , and to a le s s e r extent, to le a f optical p roperties , COz concentration, and to canopy arch itecture [4, 5, 12 ]. F o r the lea f display ch a racteris tics o f m ost plants, the c r it ic a l fac to r seem s to be percent cover since there is lit t le change in production rate as le a f area index increases beyond that requ ired fo r near com plete in terception o f light (F ig . 2). This corresponds to B rou gham 's [13] concept o f a c r it ic a l lea f area index. At low le a f densities (below L=2.0 with random d ispersion ), there is some advantage in horizonta l lea f d isp lay. Only with v e ry dense canopies ( L =6 and above), do e rec t leaves have a m arked advantage.


F IG . 2 . S im u la t io n s o f d a i l y c r o p g r o w th r a te ( C ) fo r m a i z e an d c l o v e r c o m m u n it ie s ( h ig h an d lo w p h o t o

s y n th e t ic c a p a b i l i t y ) as a fu n c t io n o f l e a f a r e a in d e x ( L ) . T h r e e s im p le c a n o p ie s w ith l e a f a n g le ( r e la t i v e to

g ro u n d ) o f 0°, 45®, or 90®. M o st r e a l c o m m u n it ie s b e h a v e s im ila r to t h e 4 5 ° c a s e s . S o la r an d s k y l ig h t d a ta o f

3 8 ° N fo r 1 J u ly .

( F I G . 2 . S im u la c ió n d e l r it m o d ia r io d e c r e c im ie n t o d e p la n t a s ( С ) e n c o l e c t iv id a d e s d e m a í z y d e t ré b o l

( a l t a y b a j a c a p a c id a d d e fo to s ín te s is ) e n fu n c ió n d e l ín d ic e de s u p e r f ic ie d e la s h o ja s ( L ) . T r e s d o s e le s f o l ia r e s

s im p le s c o n h o ja s e n á n g u lo s ( r e s p e c to d e l te rre n o ) d e O®, 4 5 * o 9 0 °. L a m a y o r ía d e la s c o l e c t iv id a d e s r e a le s

se c o m p o r t a n d e fo r m a s im ila r a l c a s o d e lo s 4 5 e. Los d a to s r e la t iv o s a a c t i v i d a d s o la r y lu m in o s id a d so n lo s

c o r r e s p o n d ie n te s a 38®N e n d e j u l i o . )

In this regard , it is in teresting that r ic e breeding e ffo rts at the In te rnational R ice Research Institute w ere aimed sp ec ifica lly towards developing e re c t- le a fed types. Th is seem s to have been m otivated in part by a c o r r e lation between erec t leaves, s ilic a content, and s t if f straw im portant fo r n itrogen to lerance [14] and, inpart, by ea r ly predictions by Japanese w orkers o f an advantage in light distribution fo r the erec t character. However, it is on ly recen tly that the new va r ie tie s have been grown at the lea f densities shown in F ig . 2 to be requ ired to lend a m arked advantage to e rec t leaves (Yoshida, personal communication; F ig . 12-7 in Chandler [15] where L = 10 was e x ceeded ). Only a few experim ents have been done with other species to test these opinions regard ing e rec t leaves; while the resu lts tend to confirm the sim ulation pred iction , m ore experim ents are needed [12, 16] . Plant b reeders should also g ive attention to the v e r t ic a l d istribution o f leaves in re la tion to lea f width [17] pa rticu la rly fo r plants with opposite phyllotaxy. In dwarfed species, the v e r t ic a l separation o f leaves should be maintained at tw ice lea f'w id th o r m ore to ensure diffuse light penetration.

4 .2 . L ea f p roperties

The im portance o f photosynthetic capability is also apparent from F ig . 2. Our in form ation on the photosynthetic behaviour o f crop species is im proving rap id ly as better techniques are applied to the problem ; much o f the o ld er in form ation on lea f rates is o f litt le value. Th ere are two basic groups of plants o f im portance in agricu lture; 1) a high capacity group including m aize, sugar cane, sorghum, other trop ica l grasses , and some dicotyledons


such as Amaranthus and certa in A tr ip lex spp; and 2) a low er capacity group including wheat and other tem perate grasses , sugar beet, cotton, sunflower, bean and other dicotyledons. Relevant features o f the groups are contrasted in Tab le I [18 ].

R esearch at s e ve ra l laboratories is now d irected at considering in ter- and in traspec ific varia tion in P N in detail [19, 20 ]. Thus fa r, both C3 and C4 plants [21, 22] have been found together only among A tr ip lex and Panicum spp. H ow ever, considerab le in traspec ific va r ia b ility is found among both C3 and C4 plants [21,22]. These studies are stim ulated by the presumed corre la tion o f PN with y ie ld , but PN can be negatively co rre la ted with y ie ld under some circum stances. L .T . Evans {personal communication) pointed out that PN in the flag lea f o f wheat upon which grain y ie ld is c lo se ly dependent, declined over an evolu tionary genotypic se r ie s while gra in y ie ld increased m arkedly. The y ie ld in creases apparently have come from changes in the partitioning ratio.

The basic p roperties o f the photosynthetic apparatus seem rather s im ila r fo r a ll h igher plants and thus not eas ily a ltered through induced mutations. Chlorophyll defic ien t mutants are common but they tend to low er productivity ra ther than to enhance it (3 to 4 mg chi dm"2 lea f surface is requ ired fo r m axim al P^ in most sp ec ies ). There is m ore room fo r varia tion in secondary p roperties including such aspects as lea f res istances to gaseous diffusion, canopy arch itecture, and plant response to crowding in dense stands.

T A B L E I. PH O TO SYN TH E TIC SYSTEMS IN CRO P PLA N TS

C h a r a c te rL o w c a p a c i t y

s p e c ie s

H ig h c a p a c i t y

s p e c ie s

N e t p h o t o s y n t h e t ic r a te ( P ^ ) in

f u l l sun, m g C 0 2 d " 2 h - 1 2 5 - 4 5 4 5 - 6 0

C a r b o n p a th C a lv i n c y c l e , f ix H a t c h - S la c k p a th , f ix

C 0 2 w ith C 3 a c id C 0 2 w ith C 4 a c id

C 0 2 c o m p e n s a tio n p o in t ,

p p m V / V C 0 2 >40 < 10

In c re a s e P^j in lo w o x y g e n Y e s N o

D if fe r e n c e s a ls o e x is t in l e a f a n a to m y and o th e r c h a r a c t e r is t ic s .

5. P A R T IT IO N IN G OF PH O TO SYN TH ATES TO R E SPIR A T IO NAND GROW TH

Evans' observation with wheat ca lls attention to the e ffic ien cy with which the p rim ary production o f plants may be used in growth and resp iration . Whole plant resp ira tion losses over the diurnal period usually amount to 20 to 40% or m ore o f the daily whole plant P^ fo r herbaceous species. The la rg e r rates occur with old plants (la rg e biom ass and hence high m aintenance), high tem peratures and/or dim light. Com pared to photosynthesis we know re la t iv e ly litt le about the magnitude, b iochem ical controls and other features about resp ira tion needed fo r determ in ing whether genetic manipulations are possib le .


5.1. Resp iration

R esp ira tion is usually c lose ly linked o r "coup led" [23] with other b io chem ical a c tiv ities . Th ere may be circum stances when resp ira tion can becom e uncoupled, and hence "w aste fu l" but m ost resp ira tion seem s to represen t a n ecessary cost to the plant. Two main aspects o f resp ira tion may be distinguished: resp ira tion associated with growth (R G); and re sp ira tion associated with maintenance (E M ). B iochem ica l ac tiv ities such as n itrate assim ila tion and synthesis of nucleic acids, proteins and c e ll walls are associated with growth and a ll requ ire energy. RM probably re la tes to protein turnover, active gradients and other continuing functions. M cC ree [25] found in c lo v e r that they could distinguish a resp ira tion component which was p roportiona l to current photosynthesis and which then presum ably r e flected growth. The res idua l portion o f the resp ira tion was co rre la ted to plant weight and presum ably was R M.

The amount o f C 0 2 re leased in resp ira tion is re la ted to the amounts of sugar combusted and hence to energy used in RG and RM. How ever, highly reduced m ateria ls such as lip ids contain a h igher energy content than does sugar. Thus, while sugar is resp ired and weight is lost, energy tends to be conserved . The CO2/O2 exchange ratio re fle c ts the sum o f photosynthesis and resp ira tion and hence the energy balance. M yers [24] was able to p red ict the C 0 2/02 exchange ratio of algae from С, H, O, and N com position o f the algae and a knowledge o f whether they w ere assim ila ting N 03 o r NH| ions. One might calculate C 0 2/02 ratios fo r higher plant tissues and thus p red ict the energy requ irem ents from resp ira tion (de V r ies at W ageningen has tr ied this accord ing to DeW it, personal com m unication ). De V r ie s ' calculations suggest that in young grow ing tissues, the production from sugars o f substrates fo r protein , lip ids and c e ll w a lls generate m ore A T P (a high energy organ ic phosphorus compound produced in resp ira tion ) than is subsequently used up in synthetic p rocesses . H is tentative conclusion is that this surplus A T P would m eet a ll RM requ irem ents o f the new ly synthesized tissues and additional sugars would not be requ ired fo r R M.

R esp ira to ry activ ities of various plant tissues are va riab le but, i f coupling is c lose , these varia tions re fle c t varia tions in other aspects o f tissue m etabolism . S evera l varian ts o f the basic carbon pathway fo r the oxidation o f sugars do ex ist in higher plants: most im portant are the hexosemonophosphate shunt (an alternate to norm al g ly co ly s is ) and certa in steps re la tin g to the conversion o f lip ids to sugars. W hile it may be possib le to e lim inate through mutation breeding certa in "unnecessary" resp ira to ry pathways or reduce demand by elim inating unnecessary chem ica l products, we have litt le experience o r in form ation on which to make pred ictions. The la rge percentage o f resp ira tion appears to be the consequence o f growth and developm ent about which we can make independent decisions.

5.2' . D istribution o f photosynthate

Turning to the other aspect o f the partition ing problem , nam ely the distribution o f photosynthates to storage, new roots, leaves , stem s and other organs, one finds that the potentials fo r change through mutation breed ing is w e ll established [26 ]. The varia tions in number and re la tive s ize of plant organs is v e ry great in response to environm ent, and the pattern of this p lastic ity , as in the case o f branching, can be controlled


genetica lly . Two features deserve attention here: the dynamic charactero f growth and developm ent; and, the ro le o f sim ulation m odels in p re dicting the consequence o f sm all changes in the con tro l system s.

M onsi [2 7] and Monsi and Murata [28] discuss the consequences to the plant o f vary ing the partition ing ratio among leaves , stem s, roots and storage . F igu re 3 shows the structure o f a plant system a fter s e ve ra l production cyc les w ith various distribution ratios. As this is repeated o ver a long period o f tim e, v e ry la rge d ifferences in plant m orphology can be generated from sm all d ifferences in partition ing.

0 = 1-1=3 МФ2 1 = 2Ф1 2'M'I ЗНФ0

A L L O C A TI O N RATIO

F I G .3 . T h e w e ig h t o f v a r io u s p la n t p a rts ( le a v e s , L ; s te m s . S ; ro o ts , R; an d s to re d m a t e r ia l , M ) fo r a

h y p o t h e t ic a l s e e d l in g p la n t ( t 0) a n d a f te r s e v e r a l g ro w th c y c l e s (tg + 5) w ith f i v e d i f f e r e n t a l l o c a t io n r a t io s . In

e a c h c y c l e , n e w p h o to s y n th a te s p ro d u c e d in p ro p o rtio n t o l e a f w e ig h t w e r e a l l o c a t e d t o L : S : R : M a c c o r d in g t o

th e in d ic a t e d r a t io s . A n a d ju s tm e n t w a s m a d e fo r r e s p ira t io n lo s s e s . N o te th e a p p a r e n t stro n g a d v a n ta g e

fro m a la r g e a l l o c a t i o n t o l e a f g r o w th ( e x t r e m e r ig h t ) . In r e a l p la n ts th e a l l o c a t io n r a t io c h a n g e s c o n t in u o u s ly

d u e t o in t e r n a l f u n c t io n a l b a la n c e s ( a f t e r M o n si and M u ra ta [ 2 8 ] ) .

( F I G . 3 . P eso d e la s d is tin ta s p a r te s d e u n a p la n ta ( h o ja s . L ; t a l lo s , S ; r a f e e s , R ; y m a t e r i a l a c u m u la d o , M)

e n e l c a s o de u n a p lá n t u la h i p o t é t ic a ( t 0) y a l c a b o d e v a r io s c i c l o s d e c r e c im ie n t o ( t 0 + 5 ) c o n c i n c o r a z o n e s

d i fe r e n te s d e d is tr ib u c ió n . En c a d a c i c l o , lo s n u ev o s p ro d u c to s d e f o to s ín te s is o b te n id o s e n p r o p o r c ió n a l p e s o

d e la s h o ja s se h a n a t r ib u id o a L : S ; R : M s e g ú n l a s ra z o n e s in d ic a d a s . S e h a e f e c t u a d o u n r e a ju s te c o r r e s p o n d ie n te

a la s p é rd id a s p o r r e s p ir a c ió n . O b s é r v e s e l a m a n if ie s t a v e n t a ja d e a tr ib u ir u n a t a z ó n e l e v a d a a l c r e c im ie n t o

d e la s h o ja s ( e x t r e m o d e r e c h o ) . En la r e a l id a d , la r a z ó n d e d is tr ib u c ió n en la s p la n t a s v a r ía d e u n m o d o c o n t in u o

d e b id o a lo s e q u il ib r io s f u n c io n a le s in te rn o s ( s e g ú n M o n si y M u ra ta [ 2 8 ] ) . )

Of particu lar im portance here is the ratio o f lea f a rea to root surface. L eaves serve to capture sunlight and produce additional substrates and it would appear as a good stra tegy for the plant to m axim ize le a f growth in ea r ly growth periods. Roots, on the other hand, must also re c e iv e substra tes since nutrients and w ater are essen tia l to the rest o f the plant. Two con tro l system s are operating here. F irs t , there appears to be a p r io r ity fo r use o f available substrates with lea f growth f ir s t fo llow ed by stem growth and root growth [29 ]. Second, these p r io r it ie s may be overridden when w ater s tress o r nutrient defic ienc ies , resu lting from inadequate root surface, re s tr ic t lea f growth and thus allow a g rea te r proportion o f the availab le substra tes to be used in root growth [30]. Thus a "functional balance" is established in the partition ing o f substrates between roots and leaves [10 ].


Other functional balances, as between fru it growth and vegeta tive growth, and le a f m esophyll and vascu lar tissues [31 ], are evident in a ll aspects o f plant growth.

During the past 40 yea rs , plant physio logists have focussed strongly on the ro le o f hormones in con trolling growth and developm ent, and we have departed from the tendency of e a r l ie r w orkers who v iew ed most whole plant prob lem s in term s o f substrate distribution [32 ]. However, M itch e ll's [33] studies o f t il le r in g in ryeg rass , as an exam ple, show that hormones and substrates can both regu late the same system . What rem ains unclear in most cases is whether the hormones perm it (o r induce?) growth which se rves then to m ob ilize transport o f substrates towards the growth centre, o r i f the hormone induces transport which perm its growth. W hether hormones act as sw itches o r engines, our p rincipa l concern is with whether substrate supply is adequate fo r the capacity o f various tissues to grow , and what p r io r it ie s ex is t when substrate is lim iting .

5 .3 . M odels o f plant growth and development

There appears to be a lm ost unlim ited opportunity fo r genetic manipulation o f these control system s. However, as with photosynthesis and re sp ira tion, we are s t i l l faced with the issue o f how to pred ict the e ffec t on com munity y ie ld o f a sm all change in some physio log ica l o r m orphologica l tra it. The dynamic sim ulation m odel fo r plant growth now being developed by DeW it and B rouw er [3, 9, 10, 30] shows considerab le prom ise in this regard .

The vegeta tive growth o f corn (em ergence to tasse lin g ) was chosen by DeW it and B rouw er [9] fo r th e ir in itia l dynamic m odel o f plant growth, the E lem en tary Crop Growth Sim ulator (E LC R O S ). T h e ir ob jective was to m odel the p rocesses o f photosynthesis, resp iration , transp iration , and growth at the tissue and organ le v e ls o f organization . These and other subsystems w ere in tegrated to output at the community (c rop ) le v e l in term s o f dry m atter produced and consumed.

I f it takes about 50 days from em ergence to tasseling, a suitable in te r v a l o v e r which to advance tim e is about one hour. A one-hour in terva l also is short enough to take into account diurnal in fluences. The rates o f photosynthesis, resp ira tion , tránspiration , and growth are calculated hourly from the state o f the crop as characterized by the le v e l of re s e rv es , the weight, area and structure o f the lea f canopy, and the weight and activ ity of the roots as they, in turn, are influenced by the basic environm ental factors . F igu re 4 is a block diagram o f D eW it's m odel core and the submodel o f root growth. O ther submodels o f ELCROS are handled in a s im ila r fashion.H ourly values fo r weather are generated from daily weather data fo r ra d iation, tem perature, dew point, and wind speed. Soil and a ir tem peratures are g iven a diurnal pattern by im posing appropriate sine functions. Photosynthesis is calculated o ff- lin e in the d iscrete sim ulation described above [4] and introduced as a tabular function o f the e levation o f the sun, the degree of cloudiness, and the le a f a rea o f the crop canopy. R esp ira tion is computed as a function o f tem perature, re s e rv es , and the nitrogen content and age o f the plant parts. T ransp ira tion is m odelled as a subsystem involving the heat capacity o f the leaves , vapour and energy function, and the balance between w ater absorbing and evaporating surfaces. Growth is sim ulated taking into account the influence o f tem perature, re s e rv es , age o f tissues, and the degree o f w ater s tress .


4 T S )

F I G .4 . R e la t io n a l d ia g r a m fo r th e c e n t r a l p o r t io n o f E L C R O S . A rro w s r e p re s e n t f lo w o f m a t e r i a l ; b o x e s a re

a c c u m u la t e d w e ig h t o f r e s e rv e s (R E S), le a v e s ( W L V ) , s te m s ( W S T ) , a n d ro o ts (W R T ) ; c i r c l e s re p re se n t in fo r m a

t io n b it s . RGRR is r e l a t iv e g ro w th r a te o f ro o ts , re s e rv e s n o t l im it in g , as d e te r m in e d b y s o i l t e m p e r a tu r e ( T S ) ,

a n d PG R W is th e p o s s ib le g ro w th r a te o b ta in e d b y m u lt ip ly in g RGRR У С , th e p o rt io n s o f th e ro o ts c a p a b l e o f

g r o w th . T h e v a l v e sy m b o ls a r e a c t u a l g r o w th ra te s - fo r ro o ts th e PGRW in fo r m a t io n is c o m b in e d in fo r m a t io n

on RES an d its a v a i l a b i l i t y t o ro o ts t o o b ta in GRW, th e r a te a t w h ic h ro o ts in c r e a s e in w e ig h t . PHR is p h o t o

s y n th e s is r a t e an d RSP is r e s p ir a t io n . T h e a c t u a l m o d e l d e s c r ib e s r e a l p la n t s in te rm s o f m o re th a n 20 s ta te

v a r ia b le s (b o x e s ) an d th e l in e s o f m a t e r ia l an d in fo r m a t io n f lo w a r e q u ite c o m p le x ( fr o m B ro u w er a n d D e W it [ 3 0 ] ) .

( F IG . 4 , D ia g r a m a d e r e la c io n e s c o rre s p o n d ie n te s a l a p a r t e c e n t r a l d e E L C R O S ( E le m e n t a r y C r o p G ro w th

S im u la t o r ) . L as H e c h a s re p re s e n ta n e l f lu jo de m a t e r i a l ; la s c a s i l l a s re p r e s e n ta n e l p e s o a c u m u la d o d e

re s e rv a s (R E S), h o ja s ( W L V ) , t a l lo s ( W S T ) , y r a íc e s ( W R T) ; lo s c i r c u io s r e p re s e n ta n u n id a d e s (b its ) d e in f o r m a

c i ó n . RGRR e s la v e l o c id a d r e l a t iv a d e c r e c im ie n t o d e la s r a íc e s , s ie n d o la s r e s e rv a s n o l im it a t iv a s , d e te r m in a d a

p o r la t e m p e r a tu r a d e l s u e lo ( T S ) y PGR W es la v e lo c id a d d e c r e c im ie n t o p o s ib le o b te n id a m u lt ip l ic a n d o

RGRR x С , o s e a , la s p a r te s d e la s r a í c e s c a p a c e s d e c r e c im ie n t o . Los s ím b o lo s c o n v á lv u la s r e p re s e n ta n v e l o

c id a d e s r e a le s d e c r e c im ie n t o e n e l c a s o d e la s r a íc e s , la in f o r m a c ió n PGRW e s in f o r m a c ió n c o m b in a d a so b re

RES y su d is p o n ib i lid a d p a ra la s r a íc e s , a f in d e o b te n e r G RW , r it m o a l c u a l a u m e n t a n e n p e s o la s r a íc e s .

PHR e s la v e l o c id a d d e fo to s ín te s is y RSP es l a r e s p ir a c ió n . Este m o d e lo d e s c r ib e p la n ta s r e a le s e n fu n c ió n d e

m á s 20 v a r ia b le s d e e s ta d o ( c a s i l la s ) y la s l ín e a s de c i r c u la c ió n d e m a t e r ia le s e in f o r m a c ió n son m u y c o m p le ja s

( to m a d o d e B ro u w e r y D e W it [ 3 0 ] ) . )

A number o f resu lts obtained with the p re lim in ary m odel w ere presented recen tly [30] . F ie ld experim ents with corn w ere conducted in C aliforn ia , Iowa, and The Netherlands, and the appropriate weather data applied to the m odel. In a ll three cases the slope of the grand period o f growth is sim ulated quite w e ll. The sim ulated exponential growth period , however, was ea r ly in C a lifo rn ia and late in The Netherlands. DeW it and B rouw er have proposed, as a potential source im provem ent fo r the model, a study o f the so il tem perature s im u lator and o f the e ffec t o f so il tem perature on ea rly growth.

Our group is working on a s im ila r m odel fo r sugar beet to simulate the growth o f the crop from em ergence to harvest, including consideration o f the main econom ica lly important product, the sugar accumulated in the storage root [34 ] .

6 . CONCLUSION

Thus far, m odels and experim ents have perm itted only broad gen era lizations regard ing the behaviour of production system s. W hether o r not the sim ulation m odels can deal with subtle d ifferences in plant form and physiology

PL А К Т CONFORMATION AND YIELD 23

rem ains to be seen. It should be noted that highly com plex m odels such as ELCRO S begin to behave like rea l plants in term s o f the in teractions among the various subsystem s. In this sense they have bu ilt-in hom eostatic m echanism s. In fact, this is the only reason they work, and the reason why input va riab les can be presented as means without stochastic p roperties . The resu lt is that the p roperties o f these m odels, like a ll m odels, are dependent upon the quality o f our opinions about plant physiology. A l l too often, our in form ation is o f poor quality. In this regard , physio logists need to g ive much m ore attention to developing sim ple techniques fo r m onitoring physio lo g ica l p rocesses and fo r screen ing genetic m ateria ls . A good example of the kind of ingenuity needed is seen in the techniques em ployed in screen ing wheat co llections fo r photosynthetic types [20] .

A second feature o f the problem is that sim ulation m odels requ ire m assive amounts of input in form ation . Given a new mutation, it would be too tim e-consum ing to gather new in form ation on a ll o f its physio log ica l p rocesses . Fortunately, ELCROS is based w e ll enough on genera l princip les that in form ation from one plant can be used fo r pred icting the behaviour of another. As a consequence, such sim ulation models have th e ir g rea test u tility in com parative analyses — finding out what happens when a certa in param eter is changed in se ve ra l ways — and this is p rec ise ly the kind o f question posed by a gene mutation.

R E F E R E N C E S

[ 1 ] L O O M IS , R . S . , S im u la t io n : a n in t e g r a t iv e t o o l in cro p r e s e a r c h , H o r t . S c i . 4 ( 19 6 9 ) 1 4 .

[2 ] M a c F A D Y E N , A . A . , "E n e rg y H o w in e c o s y s t e m s a n d its e x p lo i t a t i o n b y g r a z in g " , G ra z in g in T e r r e s tr ia l

a n d M a r in e E n v iro n m e n ts (C R IS P , D . J . , E d .) , B la c k w e l l , L o n d o n ( 1 9 6 4 ) .

[3 ] D e W IT , C . T . , " D y n a m ic c o n c e p t s in b io l o g y " , P r o d u c t iv it y o f P h o to s y n th e t ic S y s te m s : M o d e ls and

M eth o d s ( P r o c .I B P T e c h . M e e t . T r e b o n , C z e c h o s lo v a k ia , 1 9 7 0 ) , P U D O C , T h e N e th e r la n d s ( in p ress , 1 9 7 0 ) .

[4 ] D e W I T , C . T . , P h o to sy n th e s is o f l e a f c a n o p ie s , V e r s l . la n d b a u w k . O n d e r z . ( W a g e n in g e n ) 6 6 3 ( 1 9 6 5 ) 5 7 .

[5 ] D U N C A N , W . G . , L O O M IS , R . S . , W IL L IA M S , W . A . , H A N A U , R . . A m o d e l fo r s i m u la t in g p h o t o

sy n th e s is in p la n t c o m m u n it ie s , H i lg a r d ia 38 ( 1 9 6 7 ) 1 8 1 .

[6 ] B O Y S E N -J E N S E N , P . , D ie S to ffp r o d u k t io n d e r P f la n z e n , F is c h e r , J en a ( 1 9 3 2 ) .

[7 ] M O N S I, М . , S A E K I, T . , Ü b er d e n L ic h t fa k t o r in d e n P f la n z e n g e s e l ls c h a f t e n u n d s e in e B e d e u tu n g fu r

d ie S to ffp r o d u k t io n , J a p .J .B o t . .1 4 ( 1 9 5 3 ) 2 2 .

[8 ] N IC H IP O R O V IC H , A . A . , P h o to sy n th e s is an d th e th e o ry o f o b ta in in g h ig h y i e ld s , 1 5 th T i m i r y a z e v L e c tu r e ,

M o s c o w , I z d .A N S S R ( 1 9 5 6 ) .

[9 ] D e W I T , C . T . , BR OU W ER , R . , Ü b er e in d y n a m is c h e s M o d e l l d e s v e g e t a t i v e n W a ch stu m v o n P f la n z e n -

b e s tà n d e n , A n g e w .B o t . 4 2 ( 19 6 8 ) 1 .

[ 1 0 ] D e W I T , C . T . , BR OU W ER, R ., " T h e s im u la t io n o f p h o to s y n th e t ic s y s te m s " . P r o d u c t iv it y o f P h o to s y n th e t ic

S y s te m s : M o d e ls an d M e th o d s ( P r o c . ШР T e c h .M e e t .T r e b o n , C z e c h o s lo v a k ia , 1 9 7 0 ) , P U D O C , T h e

N e th e r la n d s ( in p ress, 1 9 7 0 ) .

[ 1 ] ] L O O M IS , R . S . , W IL L IA M S , W . A . , M a x im u m c r o p p r o d u c t iv i t y : a n e s t im a t e . C ro p S c i . 3 ( 1 9 6 3 ) 6 7 .

[ 1 2 ] L O O M IS , R . S . , W IL L IA M S , W . A . , " P r o d u c t iv i t y an d th e m o r p h o lo g y o f c ro p stan d s : p a tte rn s w ith

l e a v e s " , C h . 3 , P h y s io lo g i c a l A s p e c ts o f C ro p Y i e l d ( E A S T IN , J . D . , e t a l . , Eds), A m e i . S o c . A g r o n . ,

M a d iso n , W is . ( 1 9 6 9 ) .

[ 1 3 ] B R O U G H A M , R .W . , T h e r e la t io n s h ip b e t w e e n th e c r i t i c a l l e a f a r e a , t o t a l c h lo r o p h y ll c o n t e n t , an d

m a x im u m g ro w th r a te o f s o m e p a stu re an d c ro p p la n ts , A n n .B o t . N . S . 24 ( I9 6 0 ) 4 6 3 .

[ 1 4 ] Y O S H ID A , S . , N A V A S E R O , S . A . , R A M IR E Z , E . A . , E ffe c ts o f s i l i c a a n d n itr o g e n su p p ly o n s o m e l e a f

c h a r a c te r s o f th e r ic e p la n t , P I . S o il 3 1 ( 1 9 6 9 ) 4 8 .

[ 1 5 ] C H A N D L E R , R . F . , " P la n t m o r p h o lo g y an d stan d g e o m e t r y in r e la t io n to n it r o g e n " , C h . 1 2 , P h y s io lo g ic a l

A s p e c t s o f C ro p Y i e l d ( E A S T I N , J . D . , e t a l . , E d s), A m e r . S o c . A g r o n . , M a d iso n , W is . ( 1 9 6 9 ) .

[ 16 ] P E N D L E T O N , J . W . , F ie ld in v e s t ig a t io n s o f th e re la t io n s h ip o f l e a f a n g le in c o m ( Z e a m a y s L . ) t o g r a i n

y i e ld a n d a p p a r e n t p h o to s y n th e s is , A g r o n . J . 60 ( 19 6 8 ) 4 2 2 .


[ 1 7 ] N IC H IP O R O V IC H , A . A . , P ro p e rt ie s o f p la n t c ro p s as a n o p t ic a l s y s te m , S o v ie t P I .P h y s io l . 8 ( 1 9 6 1 ) 4 2 8 .

( T r a n s i . f ro m F i z i o lo g i y a R a st. 8, 5 3 6 ).

[18 ] D O W N T O N , W . F . S . , T R E G U N N A , E . B . , C a r b o n d io x id e c o m p e n s a tio n — its r e la t io n to p h o to s y n th e t ic

c a r b o x y la t io n r e a c t io n s , s y s te m a t ic s o f th e G r a m in e a e , a n d l e a f a n a to m y , C a n .J .B o t . 4 6 ( 19 6 8 ) 2 0 7 .

[ 1 9 ] K R EN ZER , E . G . , M O S S , D . N . , C a r b o n d io x id e c o m p e n s a t io n in g ra ss e s , C ro p S c i . 9 ( 1 9 6 9 ) 6 1 9 .

[20 ] M E N Z , K . M . , S c re e n in g fo r p h o to s y n th e t ic e f f i c i e n c y , C r o p S c i . 9 ( 19 6 9 ) 6 9 2 .

[ 2 1 ] E L -S H A R K A W Y , M . A . , H E S K E T H , J . , M U R A M O T O , H . , L e a f p h o t o s y n t h e t ic r a te s an d o th e r g ro w th

c h a r a c t e r is t ic s a m o n g 26 s p e c ie s o f G o ssy p iu m , C r o p S c i . 5 ( 1 9 6 5 ) 1 7 3 .

[2 2 ] D U N C A N , W . G . , H E S K E T H , J . D . , N e t p h o to sy n th e s is ra te s , r e l a t iv e l e a f g ro w th r a te s , and l e a f n u m b e r

o f 22 r a c e s o f m a i z e g ro w n a t l ig h t te m p e r a tu r e s . C r o p S c i . 8 ( 1 9 6 8 ) 6 7 0 .

[2 3 ] BEEVERS, H . , R e sp ira to ry M e t a b o lis m o f H a n ts , R o w -P e te rs o n , E v an sto n , 1 1 1 . ( 1 9 6 1 ) .

[2 4 ] M Y E R S, J . , " T h e p a tte r n o f p h o to sy n th e s is in C h l o r e l la " , C h . 1 7 , P h o to sy n th e sis in P la n ts ( F R A N C K , J . ,

L O O M IS , W . E . , E d s), Io w a S ta t e Press, A m e s ( 1 9 4 9 ) .

[2 5 ] M cC R E E , K . J . , " A n e q u a t io n fo r r a te o f r e s p ira t io n o f w h ite c l o v e r p la n ts g ro w n u n d er c o n t r o lle d

c o n d it io n s " , P r o d u c t iv it y o f P h o to s y n th e t ic S y s te m s : M o d e ls an d M e th o d s ( P r o c .I B P T e c h . M e e t .T r e b o n ,

C z e c h o s lo v a k ia , 1 9 7 0 ) , P U D O C , T h e N e th e r la n d s ( in p ress, 1 9 7 0 ) .

[2 6 ] IA E A / F A O , M a n u a l o n M u ta t io n B r e e d in g , T e c h .R e p . S e r . N o . 1 1 9 , IA E A , V ie n n a ( 1 9 7 0 ) .

[2 7 ] M O N S I, М . , D ry m a t t e r r e p r o d u c tio n in p la n ts . I . S c h e m a ta o f d r y - m a t t e r r e p r o d u c tio n , B o t .M a g . ,

T o k y o 7 7 ( 1 9 6 0 ) 8 1 .

[2 8 ] M O N S I, М . , M U R A T A , Y . , " D e v e lo p m e n t o f p h o t o s y n t h e t ic s y s te m s as in f lu e n c e d b y m a t t e r d is tr ib u tio n " ,

P r o d u c t iv it y o f P h o to s y n th e t ic S y s te m s : M o d els an d M e th o d s , ( P r o c . IBP T e c h . M e e t .T r e b o n , C z e c h o

s lo v a k i a , 1 9 7 0 ) ,P U D O C , T h e N e th e r la n d s ( in press, 1 9 7 0 ) .

[2 9 ] L O O M IS , R . S . , U LR IC H , A . , R esp o n ses o f s u g a r b e e ts to n itr o g e n d e f i c ie n c y as in f lu e n c e d b y p la n t

c o m p e t i t i o n , C ro p S c i . 2 (19 6 2 ) 3 7 .

[3 0 ] BR OU W ER, R . , D e W I T , C . T . , " A s im u la t io n m o d e l o f p la n t g r o w th w ith s p e c ia l a t te n t io n t o ro o t g ro w th

a n d its c o n s e q u e n c e s ” . R oot G ro w th ( W H IT T IN G T O N , W . J . , E d .) , B u tte rw o rth , L o n d o n ( 1 9 6 9 ) .

[ 3 1 ] FISH ER, F . J . F . , A d is c u s s io n o f l e a f m o rp h o g e n e s is in R a n u n c u lu s h irtu s , N . Z . J 1 . S c i . 3 (19 6 0 ) 6 8 5 .

[3 2 ] H E SLO P H A R R ISO N , J . , " D e v e lo p m e n t , d i f f e r e n t ia t io n an d y i e l d " , P h y s io lo g ic a l A s p e c ts o f C ro p Y ie ld

( E A S T I N , J . E . , e t a l . , Eds), A m e r . S o c . A g r o n . , M a d iso n , W i s . ( 1 9 6 9 ) .

[ 3 3 ] M IT C H E L L , K . J . , I n f lu e n c e o f l ig h t a n d te m p e ra tu re o n th e g r o w th o f ry e g ra ss ( L o U u m s p p .) . I I . T h e

c o n t r o l o f la t e r a l b u d d e v e lo p m e n t , P h y s io lo g ia P I. 6 ( 1 9 5 3 ) 4 2 5 .

[3 4 ] L O O M IS , R . S . , F IC K , G . W . , W IL L IA M S , W . A . , " D y n a m ic s im u la t io n s o f h ig h e r p la n t g r o w th " , ( P r o c .

C o n f . A p p l .C o n t . S y s te m s , S im u l .L a n g . ) , A C M / IE E E / S H A R E / S C i, S an F r a n c is c o ( 1 9 6 9 ) .

D I S C U S S I O N

H. SMITH: In the course o f the natural evolution o f h igher plantsthere would seem to have been a high se lec tive advantage to maximum utilization o f light energy. Is there any evidence on this?

R. S. LOOMIS: Our m easurem ents o f lea f photosynthesis have not beengo. It is only recen tly that our m easurem ents o f le a f photosynthesis have been made under good conditions (e sp ec ia lly high turbulence o f the a ir ), so that we might make meaningful com parisons among o r within spec ies . In addition, few physio logists have taken an in terest in evolu tionary com parisons. H ow ever, I am fa m ilia r with two situations worth commenting on. The firs t is that C a lv in -cyc le plants seem to possess the basic p rocess to which the Hatch-Slack enzym es w ere added la ter. How they w ere added independently in a w ide range o f evolu tionary d ivergent species is an in teresting problem (Downton and Tregunna o f the U n ivers ity o f B ritish Columbia have severa l papers on th is ). The second is a report (unpublished) by Evans o f CSIHO C anberra that an evolu tionary series in wheat o ve r which there was a marked increase in gra in y ie ld , showed a marked decline in photosynthesis o f the flag lea f. The y ie ld increase must have come from im provem ents in partitioning.


A. ASHRI: In your m odels you assume optimum nutritional le ve ls andother environm ental factors , am I co rrec t? How do you include the e ffec t o f d iseases?

R .S . LOOMIS: Thus far, m ost simulation m odels have dealt with"optim um " conditions since they are the easiest and also m ost gen era lizab le . H ow ever, they can deal w ith d iseases, com petition by weeds and environ m ental s tress i f we have the p roper input data. In d isc re te m odels o f photosynthesis we have considered the e ffec t o f a v iru s on sugar beet p ro ductivity with a good co rre la tion with fie ld resu lts . In our dynamic sim u lation m odel o f the sugar beet, n itrogen nutrition has been introduced as a va r iab le because fa rm ers control sugar beet growth and quality by con trolling n itrogen fe rtiliza tion .

B. SIGURBJORNSSON: You re fe rred to h igh-capacity and low -capac ityspecies with re ga rd to photosynthetic activity. Do you believe that it would be worthwhile fo r plant b reede rs to attempt to change low -capacity species into h igh -capacity ones?

R. S. LOOMIS: Y es , and extensive screen ing program s are underway atseve ra l cen tres. These program s have as th e ir ob jective the detection o f Hatch-Slack plants in species norm ally fo llow ing the Calvin cyc le . Thus far, they have not found the two mechanisms within the same spec ies . Two genera, A tr ip lex and Panicum have both types o f plants among th e ir species o ffe r in g the poss ib ility o f in te rsp ec ific tran s fer. O. BjOrkman at the C arneg ie Institution o f Washington, Stanford, C a liforn ia , is studying the genetics o f the A tr ip lex system . My understanding is that re la t iv e ly few genes are involved, some re la ted to lea f anatomy and some concerned with enzym es in the carbon pathway. When BjOrkman is fin ished perhaps we can judge whether these genes may be subject to mutation.

In addition, a la rge amount o f va r iab ility ex ists within the Calvin group and within the Hatch-Slack group. This va r ia b ility is re la ted to a large number o f genes con tro lling d ifferen t features o f the photosynthesis p rocess . Th ere is an exce llen t chance o f im proving the photosynthetic e ffic ien cy of m ost o f our econom ica lly useful plants.

PLA N T BREEDING IN LATIN AMERICA

P LA N T BREEDING IN LATIN AMERICA

A . GROBMAN

Northrup, King and C o , ,Lima, Peru

Abstract-Resumen

P L A N T BR EED IN G IN L A T I N A M E R IC A .

P la n t b r e e d in g h a s b e e n in th e fo re fro n t o f a g r ic u lt u r a l r e s e a r c h in L a t in A m e r i c a a n d has m a d e c o n t r i

b u tio n s to t h e e c o n o m ic s o f t h e L a tin A m e r i c a n c o u n tr ie s o f h u n d re d s o f m il l io n s o f d o l la r s . G r e a t a t te n t io n

w a s d e d ic a t e d in th e p a st t o t r a d i t io n a l t r o p ic a l e x p o r t cro p s , b u t t h e e x p l o s iv e p o p u la t io n g ro w th d e m a n d e d

a s h ift o f e f fo r t s t o fo o d c r o p s . P la n t b r e e d in g a c h ie v e m e n t s an d r e m a in in g p r o b le m s a r e p r e s e n te d fo r

v a r io u s c ro p s s u c h as m a i z e , b e a n s , r ic e , w h e a t , c o t to n , p o t a to e s , c o f f e e , b a n a n a s , su g a r c a n e an d so rg h u m .

T h e im p r o v e m e n t o f p r o t e in q u a n t ity a n d q u a li ty in s t a p le fo o d c ro p s w i l l b e o f p a r t ic u la r im p o r t a n c e f o r t h e

fu t u r e . A lth o u g h g e n e t i c v a r ia b i l i t y in L a tin A m e r i c a is far fro m h a v in g b e e n s u f f ic i e n t ly t a p p e d fo r m o st

s p e c i e s , in d u c t io n o f m u ta tio n s c o u ld b e u s e fu l in a t t a c k in g s p e c i f i c p r o b le m s in in d iv id u a l s p e c ie s . S e v e r a l

o f s u c h p r o b le m s a r e d is c u s s e d .

LA F IT O T E C N IA EN A M E R IC A L A T I N A .

L a f i t o t e c n ia s e e n c u e n tr a e n l a v a n g u a r d ia d e la s in v e s t ig a c io n e s a g r íc o la s e n A m é r i c a L a tin a y h a

p r o p o r c io n a d o a l a e c o n o m ía d e lo s p a ís e s la t in o a m e r ic a n o s in g re s o s p o r v a lo r d e c e n te n a r e s d e m il lo n e s d e

d ó la r e s . En e l p a s a d o se h a p r e s ta d o g r a n a t e n c ió n a lo s c u lt iv o s t r a d ic io n a le s t r o p ic a le s d e stin a d o s a la

e x p o r t a c ió n , p e r o d e b id o a l r á p id o a u m e n to d e l a p o b la c ió n lo s e s tu d io s s e h a n c o n c e n tr a d o e n la s p la n ta s

a l i m e n t i c i a s . El a u to r e x p o n e la s r e a l i z a c io n e s f i t o t é c n ic a s y lo s p r o b le m a s p e n d ie n te s e n lo q u e r e s p e c t a a

u n a s e r ie d e c u l t iv o s t a l e s c o m o e l m a í z , la s a lu b ia s , e l a r r o z , e l t r ig o , e l a lg o d ó n , la s p a ta t a s , e l c a f é ,

lo s p lá t a n o s , l a c a ñ a d e a z ú c a r , y e l m i j o . En e l fu tu ro , se rá d e p a r t ic u la r im p o r t a n c ia m e jo r a r l a c a n tid a d

y c a l i d a d d e la s p r o te ín a s d e lo s c u l t iv o s a l im e n t i c io s b á s ic o s . A u n q u e e n A m é r i c a L a tin a d is ta m u c h o d e

se r a p r o v e c h a d a s u f i c ie n t e m e n t e l a v a r ia b i l id a d g e n é t i c a e n u n g ra n n ú m e ro d e e s p e c i e s , l a in d u c c ió n d e

m u ta c io n e s p o d r ía se r u n in s tru m e n to ú t i l p a ra a b o rd a r p r o b le m a s e s p e c í f i c o s e n e l c a s o d e d e te r m in a d a s

e s p e c i e s . E l a u to r e x a m in a a lg u n o s d e es to s p r o b le m a s .


Plant breeding in Latin A m er ica has trad itiona lly been one o f the, i f not the, strongest fie ld o f resea rch in m ost agricu ltu ra l research institutions o f the area . Since the m iddle part o f the decade o f 1930-40, A gr icu ltu ra l Experim ent Stations o f the area, pa rticu la rly in Argentina, B ra z il, Chile, M exico and Peru in itiated strong resea rch program s fo r the im provem ent o f cotton, wheat, coffee, m aize, r ic e and sugar cane, among other crops. The wheat w ork in A rgentina and Uruguay, led in itia lly by B oerge r , K le in and Backhouse started during the period 1912-1917 and again since 1923 [25] and was, perhaps, the p ioneering e ffo r t in plant breed ing in Latin A m er ica under e ffic ien t fie ld plot and labora tory techniques.

P ro g re s s iv e ly , since the ea r ly 1940*s, many national and international resea rch p rogram s in the area have expanded the ir a c tiv it ie s . The M exican P rog ram and the R o ck e fe lle r Foundation started the O ffice of Special Studies in the ea r ly 401 s, which engaged in itia lly a grea t part o f its talents and funds in breeding. The resu lt in tra in ing and resea rch o f this institu tion and its successors, including the present C IM M YT , a re w e ll known,

29

30 GROBMAN

and are in la rge m easure responsib le fo r the sign ificant in creases in p ro duction o f wheat in seve ra l countries, espec ia lly in southern and eastern A s ia .

These breed ing program s are bas ica lly oriented towards the attainment o f h igher y ie ld s and are quite stra igh tforw ard . Many of them, such as those fo r wheat, corn, r ic e , and beans, operate on v e r y la rge scales and in m ost cases with fie ld plantings made two or even three tim es per y ea r. Contributing to this are m ild c lim atic conditions that perm it split and success ive plantings and low cost o f labour.

F o r sheer s ize o f program s in term s o f number o f selections and crosses and en tries in fie ld tests the various wheat and corn program s based in Latin A m er ica would rate among the w orld 's la rges t.

What they are gaining in s ize , these program s have with few exceptions n ever made up in depth. M ost of them lack a c r it ic a l m ass o f trained personnel at the P h .D . or s im ila r le ve l, and few of them have been success fu l in reta in ing the ir trained personnel fo r a su ffic ien tly long tim e.This has created m ore often than not discontinuities in their program s and blank periods in ach ievem ents.

In spite o f this apparently sweeping genera lization , individual research institutions can be iden tified in Latin A m er ica where favourable conditions fo r resea rch in plant breeding have existed or are being created anew, through enhanced institutional support and tra in ing and h iring o f personnel with advanced degrees .

P lant breed ing resea rch is m ain ly an undertaking o f publicly supported institutions in Latin A m er ica . On a reg ion a l basis, m ore com m erc ia l resea rch is appearing now in the southern and centra l countries o f South A m er ica . W hile less than 10% o f the agricu ltu ra l resea rch stations in the Andean Zone countries a re p riva te ly supported [21], a v e ry considerab le part o f the m a ize , sorghum, and wheat breeding operations in Argentina, B ra z il, Chile, and P eru are p riva te ly financed.

The impending leg is la tion on va r ie ta l protection in the USA, and the poss ib ility o f s im ila r leg is la tion com ing into being in Latin A m erica — such as C h ile 's recen tly prom ulgated seed law, including va r ie ta l p ro tec tion - m ay d efin ite ly condition an expansion of p riva te seed im provem ent and production activ ities in the area [15].

Should this be so, while many plant b reeders would quite lik e ly be channeled into com m erc ia l a c tiv ities , many others would rem ain in basic resea rch backstopping the activ ities of the fo rm er, as g rea te r challenges would be posed to their c rea tiv ity [1 8].

It is the intention in this paper to present b r ie fly some o f the problem s o f resea rch in genetica l plant im provem ent in the Latin A m erican area, and to d escribe how conventional se lection and hybrid ization breeding techniques are being or w ill continue to be applied, together with the opportunities fo r a mutational breeding approach.

2. BREEDING PRO B LEM S IN SELECTED CROPS IN L A T IN AM E R IC A

The crops which are considered in this rev iew are only those with which the author has had some connection, e ither through d irec t activ ity o r as an associa te o r resea rch adm in istrator. Some crops not included may a lso invo lve s im ila r or m ore challenging problem s and prom ises in plant breeding research .

PLANT BREEDING IN LATIN AMERICA 31

2.1. M a ize (Z ea mays L . )

A g rea te r area is grown to m a ize than to any other crop in M exico, C en tra l and South A m er ica . F rom the hot dry trop ics (25 cm of ra in per y ea r on the Caribbean Coast o f Venezuela) to the humid trop ics (5 m of ra in per yea r in the Chocó area o f Colom bia) to the heights of the shores o f Lake T it ica ca (a lm ost 4000 m above sea le v e l), m a ize is grown in an am azing v a r ie ty o f plant, ear, and grain types. Th is va r ia b ility has been studied, analysed, and c la ss ified [4, 5, 10, 14, 16, 20, 34, 36, 42, 43, 45, 46]. O ver 270 races o f m a ize have been reported from Latin A m er ica (Tab le I).

T A B L E I. NUM BER OF RACES OF M A IZE IN L A T IN AM E R IC A N COUNTRIES

B o l iv ia 30

B r a z i l 33

C e n t r a l A m e r i c a 22

C h i l e 19

C o l o m b ia 23

C u b a 7

E c u a d o r 29

M e x ic o 32

P eru 50

V e n e z u e la 19

2 7 1

Th ree m ajor m a ize germ plasm banks in Latin A m erica , one in the USA and s eve ra l a ccesso ry banks hold seed o f over 10 000 fie ld co llections and ra c ia l com posites. In recen t yea rs , C IM M YT and seve ra l other program s have str ived to crea te new pools of va r iab ility by recom bin ing va r ie ta l se lections into in tra ra c ia l com posites. Some in tra- and in te rra c ia l com pos ites, subjected to m ild se lection p ressu re to a llow maximum genetic r e com bination under various environm ents, have also been developed and have been made ava ilab le to m a ize b reeders o f the area.

It is c lea r , th ere fo re , that there exists a v e ry la rg e and as yet m ostly untapped r e s e rv e o f genetic va r iab ility at the disposal o f m aize b reed ers . Some evaluation o f this m a teria l has been effected fo r com bining ab ility fo r gra in y ie ld , fo r res is tance against lea f- feed in g insects, e sp ec ia lly Spodoptera frug iperda , fo r res istance to stunting and striped v iru ses and fo r som e gra in quality tra its [1, 2, 39, 40].

M a ize im provem ent schem es have been based on line se lection and c ross in g in o rd er to obtain hybrids of e ither two, th ree o r four lines. H etero tic responses are v e ry la rg e in m aize fo r y ie ld and tra its related to y ie ld . Recent tr ia ls o f F j va r ie ta l hybrids at many stations have d is closed the g rea t p oss ib ilit ies fo r increasing y ie ld from enhanced heterotic responses o f w ide in te rra c ia l crosses in m a ize [6, 7, 31, 39, 40].

32 GROBMAN

It is w idely accepted that a considerable part o f the genetic variance associated with y ie ld in corn is o f either the additive or dominance sort. E p ista tic contributions a re originated m ostly in wide in te rra c ia l o r in te r va r ie ta l c ro sses . Sufficient additive variance fo r y ie ld has been d e te rm ined [39] in m a ize to suggest that m ass se lection schem es, when w ell planned and executed, could be conducive to enhance p rogress in y ie ld le v e ls at ra tes com parable to those obtained with conventional hybrid schem es [28]. Th is has been proved in s e ve ra l m ass se lection studies on m a ize in Latin A m erica , espec ia lly in M exico [22] where between 3 and 23% y ie ld im provem ent was obtained per cyc le o f selection . S im ila r w ork in m ass se lection with average ra tes of y ie ld im provem ent o f 3 - 9% per cyc le has been conducted in Colom bia, Honduras, N icaragua, and B ra z il.

Mass se lection in m a ize , fo llow ing irrad ia tion in ord er to increase v a r ia b ility in y ie ld and other quantitative tra its , has been studied by Gardner [12] and Lonnquist et al. [29]. Although im provem ent in y ie ld resu lted , it was not g rea te r in magnitude than that fo llow ing mass selection on com parable unirradiated populations in Latin A m erica .

Reduction in total genetic variance and correspond ingly in additive genetic variance may have occurred in a m ass se lection study reported by H allauer and Sears [17]. I f such proved to be the case, in la te r cyc les of m ass se lection procedures, b reeders in terested in m a ize population im provem ent could re s o r t to e ith er one o f two schem es in order to maintain a high genetic (and additive) variance: (a) irrad ia te seed o f their advanced cyc le of se lection populations p r io r to further selection ; (b ) introduce seed o f d ifferen t h igh -yie ld ing va r ie tie s in ord er to fo rm a com posite, w here g rea te r va r ia b ility could be induced [8 ].

G reat in terest in m od ified protein m a ize va r ie tie s has arisen recen tly . M a ize b reeders in m ost Latin Am erican countries are a c tive ly introducing opaque-2 and f lo u ry -2 genes in m a ize lines and populations. Severa l hybrids and populations have been re leased in a lim ited manner.

Genetic background e ffects o f the o rd er of a maximum o f 10% deviation above the m ean w ere found by Lam bert et al. [26] fo r percent lys ine per 100 g protein in a s e r ie s of 16 hybrids. Dumanovic and Denic [11] studying percent o f endosperm lys in e in one inbred line a fter gamma irrad iation , found in a succeeding generation a standard deviation of 0. 57, around a mean of 1. 98% o f lys in e o f total amino acids, while the amino acids constituted 11% o f endosperm sample weight. Th is would mean approxim ately 0.180% o f lys in e in the endosperm and 0. 05% fo r the standard deviation, o r in term s o f coe ffic ien t o f va riab ility , 2. 89%. Grobman et al. [16], w orking in Peru , determ ined fo r 22 fam ilies o f trop ica l m a ize lines hom ozygous fo r the opaque-2 gene, an average value o f 0.316% lys ine in endosperm with a standard deviation o f 0. 0898, which in term s o f coe ffic ien t o f v a r ia b ility equals 2. 84%. This in form ation suggests that the background e ffec t on re la t iv e varia tion o f endosperm lys ine obtained by Dumanovic and Denic from irrad ia tion o f one line equalled the background re la tiv e va ria tion found in 22 fam ilies involving 46 d ifferen t groups of trop ica l m a ize lin es . Each o f the la tte r had between one and three s is te r lines, so the induction e ffec t o f irrad ia tion appears to be o f a g rea te r re la t iv e magnitude in this case than the naturally present m odifying e ffec t. In com parison with the data o f Lam bert et a l . , the la rg es t deviate o f the group o f lines studied by Grobman et al. in term s o f percent lysine in the protein was at the le v e l o f 87% o f the mean (mean 3.04%, la rg es t high deviate


5.67% ). The la rge background o f m od ifie r factors suggests, on the other hand, the poss ib ility o f im m ediate la rge gains through some schem e o f recu rren t se lection fo r high percent lysine, without requ irem en t o f m utation induction. A s additive genetic variance should dim inish in la ter cyc les , mutagenic agents could be used to open up new va r ia b ility by a lterin g background m od ifie r genes.

Cytop lasm ic m ale s te r ility is now being used in Argen tina, B razil, Chile, Peru , and Central A m er ica fo r the production o f hybrid seed. The appearance o f a ra ce o f le a f blight (Helm inthosporium m ayd is ) highly v iru len t on "T e x a s " type cytoplasm s, w idely used by b reeders and seed producers in the USA and Latin A m erica , requ ires im m ediate action towards the solution o f this problem . Other than continuing with manual detasseling in seed fie ld s , one a lternative is to use new sources of cy to p lasm ic m ale s te r ility with res is tance to H. m ayd is . Th is solution would requ ire iden tification o f r e s to re rs and severa l seasons o f repeated back- c rosses fo r the tran s fe r o f both s te r ility and r e s to re r factors to selected lin es . Another a lternative could be the use of mutagens to e ffec t changes in m itochondria l o r p lastic DNA where presum ably the cytop lasm ic m ale s te r ility tra it is con trolled . Assum ing that the control s ite is located in the DNA strand o f one or few m itochondria and plastids, that this DNA is capable of mutating (as evidenced by structural protein changes in N eurospora [44 ]), that heterogeneity o r m itochondrial polym orphism (which has been shown even in the same c e lls ) could occur, and that a fter mutagenic agent treatm ent there could be a form ation o f c e ll lineages with heterogeneous populations o f m itochondria, d erived from the same egg c e ll, this second a lternative would deserve study. It could take the form o f mutagen treatm ent at a stage in developm ent o f the fem ale in florescences that could condition d ifferen t a ltered oosphere c e ll lin eages. Plants derived from seed pollinated on these treated plants with a common pollinator would be evaluated fo r H. m aydis res is tance and association with cytoplasm ic m ale s te r ility .

2. 2. Beans (P haseolus vu lgaris )

Beans together with corn constitute the basis o f the typ ica l Latin A m erican daily d iet. Beans supplement m a ize in the protein components which are short in the cerea l, such as lysine and tryptophane [3 ].

Serious problem s are encountered with bean genetic im provem ent. Production is lagging behind demand in seve ra l areas, notably B ra z il [23], the west coast o f South A m erica , and in Central A m er ica [33].

R esearch on bean im provem ent in Latin A m er ica has follow ed the pattern of co llec tion of native va r ie t ie s in germ plasm banks f ir s t . The Andean Zone germ plasm bank at M edellin , Colom bia, holds c lose to 1900 bean en tries . A s im ila r number is in storage at the reg iona l germ plasm bank in M exico .

Bean d iseases a re the m a jor lim itin g factor in y ie ld . B reeding work aim ed at obtaining res istan t selections against rust (U rom yces phaseoli), anthracnose (Colletotrichum lindemuthianum), angular le a f spot (Isariops is g r ie s e o la ), C ercospora le a f spot, bacteria l and v ir a l d iseases in Colom bia, M ex ico , B ra z il, P eru , and Centra l A m erica . R esistant o r to leran t bean selections to Xanthomonas phaseoli, m osaic, Isa r iop s is , and rust, have been obtained in Central A m er ica [30, 32].

34 GROBMAN

Breed ing methods invo lve selection o f lines through the ped igree method, crossing, backcrossing, and recu rren t selection . In Colom bia and B ra z il, irrad ia tion w ork has been ca rr ied out in ord er to increase va r ia b ility for quantitative and qualitative tra its in beans [23].

2.3. R ice

R ice breeding has had some important achievem ents in Latin A m erica .One o f the ea r ly successes in the m id -40 's was the introduction o f new indica type va r ie tie s to northern Peru . Subsequent hybrid ization and se lection schem es y ie lded im proved r ic e va r ie tie s which enabled Peru to alm ost double national y ie ld , and attain the fifth -h ighest r ic e y ie ld in the w orld .

Surinam now has one o f the la rgest and best executed r ic e breeding schem es in the w orld . Surinam va r ie tie s have achieved renown on account o f wide adaptation in the trop ics of Latin A m er ica . Argentina, B razil,M exico , Venezuela, Colom bia and Peru have independent r ic e breeding p ro je c ts .

A la rge p ro ject has been recen tly launched at Cali, Colom bia, by C IA T (jo in tly sponsored by R ock e fe lle r and F ord Foundations). International R ice R esearch Institute (IR R I) va r ie ties are being used at this and other cen tres to obtain h igh -y ie ld and n itrogen -responsive plants, with long grain and cooking quality characteris tics , these two la tter m ostly absent in IRR I va r ie t ie s .

E a rly r ic e va r ie tie s have been introduced by Japanese fa rm ers near Belem in B ra z il. Th ree and even four crops per yea r are being obtained there [23]. E arlin ess , res istance to P ir icu la r ia oryzae (b last), "Hoja B lanca" v irus, n itrogen -responsiveness, insensitiv ity to photoperiod, res is tance to lodging, long grain, good cooking quality, high m illin g p e r centage are ch aracteris tics sought by b reeders in new r ic e va r ie tie s .

Q uality consciousness in the Latin Am erican r ic e m arket lim its varia tion in gra in types. P r ic e s fo r gra in o f new va r ie tie s that deviate from accepted standards tend to drop. There are, th ere fo re , specific lim itations to outbreeding fr e e ly to low quality va r ie tie s , which would otherw ise have good agronom ic ch aracteris tics . The application of mutagens to induce va r iab ility in m a jor econom ic tra its could certa in ly be a m a jo r breed ing technique in this crop.

Although not yet under consideration in Latin A m erica , hybrid r ic e could eventually becom e an important, i f not the m a jor breeding schem e.A l l elem ents requ ired fo r success in a hybrid breeding schem e are a va ilable. Some com plex genetic engineering w ill be requ ired , however, before this idea becom es a rea lity .

2.4. Wheat

Wheat breeding in Latin A m erica started in 1912 in Uruguay. Th ree yea rs la te r it began in A rgentina. This country has had a distinguished r o l l o f honour o f wheat b reeders , who have accumulated a number o f b reed ing fir s ts , espec ia lly in m illin g and baking quality, making Argentin ian wheat accepted the w orld o ver . Since 1925 wheat y ie lds have alm ost doubled in A rgen tina from about 800 kg/ha to near 1500 kg/ha [25]. A s fe r t i l iz e r s are not used on wheat in Argentina this increase in y ie ld is alm ost com plete ly due to va r ie ta l im provem ent.


Argentina has a number o f p rivate companies (K le in , Buk, V ile la , among the o ldest) w e ll established in wheat breeding and seed production. IN TA , the public research institution o f Argentina, has fo r a number of yea rs a lso been creating im proved va r ie tie s and has re leased to private b reeders im proved genetic m ateria l. In addition, IN T A has supported a ll p rivate and public breeding p ro jects , with an excellen t screen ing system fo r se lection and incorporation into com m erc ia l v a r ie tie s of genes confe rr in g res is tance to seve ra l d iseases, espec ia lly rust.

B ra z il has centered its wheat breeding p rogram in the State o f R io Grande do Sul on resis tance to d iseases, particu larly Septoria and G ibbere lla . P rob lem s associated with aluminium tox ic ity in southern B raz ilian so ils requ ire attention to this ch a racteris tic when se lectin g wheat lin es . New private program s in wheat breeding have been recen tly launched in southern B ra z il in addition to State F ed era l and FAO p rogram s. Main ob jectives a re short-stem m ed, h igh-yield ing plants with res is tance to d iseases and with good m illin g and baking qualities.

Colom bian and Peruvian b reeders have also worked on stem and linea l rust res istance, with some success. New va r ie t ie s have been re leased in these countries.

In Chile, governm ent and private breeding program s have been su ccessful in launching new va r ie t ie s with good resistance to stem rust and ye llow rust (Puccin ia s t r i i fo rm is ).

C IM M YT , the international wheat program centered in M exico, has had a tremendous im pact on wheat production not only in that country, but also around the w orld . C IM M YT works c lo se ly with alm ost a ll public and p riva te wheat resea rch program s exchanging m ateria ls and in form ation.

The program operates jo in tly with IN IA in M exico in developing and testing new va r ie t ie s . Since the introduction of genes fo r sem i-dwarfness-, in sensitiv ity to photoperiod, high tille r in g , and n itrogen -responsiveness from N orin 10 and s im ila r Japanese wheats, through the United States Departm ent o f A gricu ltu re , to the M exican wheat breed ing program o f C IM M Y T , a la rge fam ily of such sem i-dw arf h igh -yie ld ing wheats was created . These are to a la rge extent responsib le fo r the breakthrough in wheat production which is occurring in se ve ra l countries around the world (35]. B esides the tra its mentioned, C IM M Y T 's extensive breeding program seeks to incorporate d isease res istance, and m illin g and baking qualities, using fo r these la tte r tra its some o f the proven Argen tin ian m ateria l, among other sou rces. R esearch on the nature o f s te r ility in wheat has d isc losed the presence o f chrom osom ic s te r il ity genes, which could be responsib le fo r previous e rra t ic behaviour o f fe r t i l i ty restora tion , a delaying fac to r in hybrid wheat fea s ib ility [9 ]. F e r t i l ity res tora tion lines have been created w ith vary ing degrees o f sp ec ific ity and have beenm ade availab le throughout the w orld . A breakthrough in T r it ic a le resea rch was made at C IM M YT in 1 968-6 9, when hexaploid T r it ic a le plants derived from a tr ip le cross o f T r it ic a le hexaploids had a high le v e l o f fe r t ility , coupled with good seed ch a racteris tics [9 ].

2 .5 . Cotton (Gossypium sp p .)

Advanced plant breeding on this species has taken p lace in the Latin A m erican area, pa rticu la rly in Argentina, B ra z il, Peru , Trin idad, and

3 6 GROBMAN

Venezuela. Gossypium hirsutum, which probably originated in southern M ex ico [19], is by fa r the m ost w idely grown species o f cotton on the A m erican continent. G. barbadense is the cultivated species o f cotton in the cen tra l and southern Peruvian coast areas, where it is known as the Tangiiis type.

A t the Im peria l C o llege o f T rop ica l A gricu ltu re , Trin idad, Harland [19] conducted an in terestin g and extensive p rogram on cotton genetics and breed ing during the 1920's and 30's. A t La M olina Station in Peru during the ea r ly 30's, a strong cotton breeding p rogram was initiated fo llow ing a s im ila r one started in 1928 at the p r iva te ly financed Cañete F a rm e r 's Assoc ia tion Experim ent Station. L a te r on, this Station was expanded and made valuable contributions, together with the public La M olina Station, in breeding h igh-yield ing long-stap le Tangüis v a r ie t ie s .

B ra z il in itiated its v e ry successfu l cotton breed ing program at the Agronom ic Institute near Campinas, Sâo Paulo. Other breeding program s are centered in northern B razil, which deal m ostly w ith Upland (G. hirsutum) cotton, except in sm a ll areas o f the north-east, w here im proved selections o f the o lder G. barbadense loca l va r ie ties have been developed, o f the type M ocó and Seridó.

In la tte r yea rs , y ie ld plateaux have been reached in breed ing program s invo lv ing line se lection within res tr ic ted species and quality groups. Conventional breeding can no longer be counted on to produce y ie ld b reak throughs in cotton. In terspec ific hybridization studies, and mutation in duction studies a re seriou sly lacking in the area. Opportunities fo r mutagen- induced va r iab ility , and in ter- and in tra -sp ec ific hybrid ization studies ex ist in this crop.

2.6. Potatoes (Solanum spp. )

A la rg e number o f va r ie tie s of potatoes a re grown to a g rea te r extent in the Andean Zone and to a le s s e r extent in M exico, B ra z il, Argentina, Venezuela, and Central A m erica .

A la rg e co llection o f potato cu ltivars from the Andean Zone has been assem bled by M in istry o f Agricu ltu re and Universidad A g ra r ia b reeders in Peru . Sm aller co llections a re under study in B oliv ia , Colom bia and M ex ico .

As the cu ltivated potatoes (S. andigenum and S. tuberosum ) a re p robably autotetraploids in o r ig in [27], breed ing prob lem s here a re m ore com plex than in diploid species. H ybrid ization and F i clonal selection have been the basic methods o f breeding. Recently, S. tuberosum XS. andigenum hybrids and the ir clonal selections have shown prom ise in the Andean Zone.

Chrom osom e reduction to the diploid leve l, cross ing and selection at that le v e l and subsequent chrom osom e doubling, has been advocated as a possib ly m ore potent breeding technique. I f this technique w ere to be fina lly adopted, opportunities would then fo llow fo r in crease in va riab ility at the diploid le v e l through the application o f mutation-inducing treatm ents. Mutation induction would m ost lik e ly be used not in connection with y ie ld in crease pro jects , but ra ther to effect changes fo r spec ific im provem ent in o therw ise high quality va r ie tie s .


2.7. C offee (C o ffea arab ica )

It is estim ated that o f the nearly seven b illion co ffee tre e s planted in the w orld , 85% a re in the A m erican continent [23]. C offee exports count s ign ifican tly in the econom ies o f Colombia, B ra z il, and other Latin A m erican countries.

C o ffee co llections have been assem bled and kept at the Campinas, SP, B ra z il resea rch station, at the IIC A Station in Tu rria lba , Costa R ica, and in F lo r id a , USA. A station at O eiras , Portugal, co-opera tes with other stations around the w orld in screen ing fo r d isease res is tance.

The la rg es t co ffee breeding p rogram in Latin A m er ica has been centered fo r many yea rs at the Instituto Agronóm ico, Campinas, SP,B ra z il. Genetic analysis o f co ffee has yielded knowledge on 35 genetic fa c to rs [24, 41]. Mutants have been obtained and studied, particu larly the angustifo lia type [23].

Another im portant breed ing program has been underway fo r some yea rs at the In te r-A m erican Institute o f A gricu ltu ra l Sciences at Turria lba , Costa R ica.

B reed ing ob jec tives a re y ie ld , grain quality, and res is tance to d is eases. New co ffee cu ltivars such as Mundo Novo and Caturra have p ro duced sign ificant y ie ld in creases o f the o rd er o f 100 - 300% in B ra z il and other countries [ 23].

A recen t rep ort in "F o re ign A gr icu ltu re ", a publication o f the United States Departm ent o f A gricu ltu re , indicates that on 27 January 1970, sym ptom s o f the p resence o f co ffee rust (H em ile ia vasta tr ix ) w ere detected fo r the f ir s t tim e in the W estern H em isphere. A zone o f o ver 1200 km in length by 350 km in width in the B razilian states o f Bahia, M inas G era is and E sp íritu Santo has been found to contain rust fungi on co ffee plants. C lea rly , this in itia l su rvey indicates serious danger to co ffee production in the W estern H em isphere, unless e ffic ien t con tro l m easures are adopted. Am ong such m easures o f specia l in terest is the iso lation and introduction o f genes conditioning res istance to H em ile ia in cu rren tly highly productive cu ltiva rs . Mutation induction in association with c o llections o f m a ter ia ls that m ight exhibit res is tance should be considered.

2.8. Banana (Musa pard isiaca )

Another im portant export crop fo r Latin A m erica is bananas. It is a lso a basic food component in the trop ica l areas o f the region .

In itia l resea rch on banana genetics and breeding was centered in Trin idad, at the Im peria l C ollege o f T rop ica l A gricu ltu re . This w ork has now been tran s fe rred to Jam aica (Banana Board R esearch Departm ent). Some breed ing w ork is a lso being done in Guadeloupe and M artin ique. The la rg es t p ro jec t is, how ever, the one conducted by the United F ru it Company with its base at L a L im a , Honduras. This company started breeding work in Panam a in 1932 b e fo re m oving to its present location . Two expeditions brought back 800 accessions from Malaya, Java, New Guinea, Solomon Islands and the Ph ilipp ines. B reed ing ob jectives are shorter plant type, la rge bunches, res is tan ce to Sigatoka and Panam a d isease . The c la ss ica l breed ing method started in Trin idad uses a G ross M ichel (3n) fem ale parthenocarpic clone c rossed with diploid clones, obtaining a tetrap lo id . O ther breeding methods in vo lve 4nX2n crosses , and 2nX 2n to obtain 3n.

3 8 GROBMAN

F o r this la tter method, fem ale clones are needed that restitu te to 22 chrom osome eggs. Some genetic engineering can be done to put genomes o f various species in m eio tic restitu tion types, to g ive b reeders the opportunity of w orking at the diploid le v e l. On account o f fa c ility o f vegetative propagation in bananas, mutation induction at the diploid le v e l, in addition to the use o f m eio tic restitu tion types, could o ffe r some in teresting poss ib ilit ies .

2 .9 . Sugar cane (Saccharum offic inarum )

In itia lly , im proved sugar cane va r ie tie s from Java with P .O . J. designations w ere w idely used in Latin A m erica . Severa l countries developed the ir own breed ing p rog ram s . A t Campos, Guanabara, B razil,CB clones w ere developed, which found grea t acceptance in that country.A t Casagrande, Peru , new clones with long vegeta tive period fo r irr iga ted conditions w ere bred and are planted today in Peru and Ecuador. P R clones have been produced at the Mayagüez and R io P ied ras stations in Puerto R ico . In Cuba, in itia l breeding e ffo rts begun in 1905 by private concerns [23] have been in tensified since 1949 at the Matanzas Station.

M exico, Colom bia, Venezuela and A rgentina also conduct breeding work . The Hawaiian Sugar P lan ters Experim ent Station co-opera tes in Ecuador and Peru in lo ca l breeding e ffo rts .

C lonal se lections from in ter- and in traspec ific hybrids are being used and have considerab ly im proved y ie ld , sugar content, and d isease res is tance.

2.10. Sorghum (Sorghum spp .)

Sorghum is fast becom ing one o f the m ost im portant gra in and forage species in Latin A m erica . On account o f its exceptional adaptability, sorghum F j hybrids developed in the USA by private companies com prise m ost o f the cultivated area in the W estern H em isphere. Co-ordinated breeding e ffo rts between United States and Latin Am érican locations, esp ec ia lly in Argentina, M exico and P eru are increasing the poss ib ilities o f further im provem ent in this crop.

High y ie ld , drought res istance, gra in quality ch a racteris tics , and res is tance to d iseases are factors that have been bred into sorghum hybrids. A w orld co llection o f over 6000 en tries is ava ilab le to breeders , and is being a ctive ly used.

Colch icine treatm ent o f sorghum has been shown to induce point mutations coupled with som atic reductions and subsequent chrom osom e doubling [38]. Th is is defin ite ly a v e ry in teresting breeding too l since it ensures the developm ent of homozygous mutants in one generation.

3. CONCLUSIONS

A s a whole, plant breeding in Latin A m erica has been in the fo re fron t o f agricu ltu ra l resea rch in the area. It is a lso beyond doubt that plant breeding resu lts have made sign ificant contributions to the econom ies of the Latin Am erican countries, worth hundreds o f m illions o f do lla rs .It is less w e ll known that there is a backlog of im proved genetic m ateria l, which fo r one reason or another is s t ill waiting to be fu lly u tilized by


fa rm ers , and which prom ises new heights in productivity in the forthcom ing yea rs .

We must recogn ize , however, that a number o f problem s ex ist o r have la te ly a risen in the im provem ent o f crops in the Latin A m erican area, which requ ire ingenuity o f approach, and the best possib le u tilization of human and m a ter ia l resou rces, which as a ru le a re not plentifu l.

W hile g rea te r attention was dedicated in the past to trad itiona l trop ica l export crops, the exp los ive growth of Latin A m erican populations is conditioning a shift of public support to the genetic im provem ent o f food crops. Since, with the exception o f the Southern Cone countries o f South A m erica , the m a jo r ity o f a ll other countries are defic ien t in animal proteins in the diet and th ere fo re r e ly on plant foods, s ign ificant new in teres t is deve lop ing in breed ing protein quality in addition to conventional agronom ic ch aracteris tics into im proved va r ie tie s in the area.

The va r ia b ility o f genetic pools and co llections o f cu ltivars availab le to b reeders in Latin A m erica is fa r from having been su ffic ien tly tapped fo r m ost spec ies . T h ere fo re , it m ay be sa fe ly pred icted that fu ll and in te lligen t u tiliza tion of these pools o f natural genetic va r ia b ility w ill take precedence o ver the induction o f new va r iab ility in the future s tra tegy of Latin A m erican plant b reeders . This is not to say, how ever, that there w ill not be spec ific prob lem s in individual species, where the induction o f mutations in genes with m a jor phenotypic e ffects o r in polygen ic series with m inor quantitative e ffec ts , could not be used. In fact, many such prob lem s, som e o f which have been lis ted in the present discussion, m erit attention at once.

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4 0 GROBMAN

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B o lsa C e r e a l e s 2 8 2 1 (19 6 9 ) 3 2 .

[2 6 ] L A M B E R T , R . J . , A L E X A N D E R , D . E . , D U D L E Y , J . W . , R e la t iv e p e r f o r m a n c e o f n o r m a l an d m o d if ie d

p r o t e in ( o p a q u e -2 ) m a i z e h y b r id s , C r o p S c i . 9 (19 6 9 ) 2 4 2 .

[2 7 ] L A M M , R . , C y t o g e n e t ic stu d ie s in S o la n u m s e c t . T u b e r a r iu m , H e r e d ita s 3 1 ( 1 9 4 5 ) 1 .

[2 8 ] L O N N Q U IS T , J . H . , A m o d i f i c a t io n o f th e e a r to ro w p r o c e d u r e fo r th e im p ro v e m e n t o f m a i z e p o p u la t io n ,

C ro p S c i . 4 ( 19 6 4 ) 2 2 7 .

[2 9 ] L O N N Q U IS T , J . H . , C O T A , O . , G A R D N E R , C . O . , E f fe c t o f m a ss s e le c t i o n an d t h e r m a l n e u tro n

ir r a d ia t io n o n g e n e t i c v a r ia n c e s in o p e n p o l lin a t e d s p e c ie s o f c o rn (Z e a m a y s L J , C r o p S c i . 6 (19 6 6 )

3 3 0 .

[3 0 ] M E N D O Z A , M . D . , "E n sa y o s d e r e n d im ie n to d e f r i jo l d e l P C C M C A r e a liz a d o s e n G u a t e m a la " ,

IIC A Z o n a N o r te P u b l. M is c . 6 7 (19 6 9 ) 9 1 .

[ 3 1 ] M O L IN A , G . J . , C o m p o r t a m ie n to d e r a z a s de m a í z y sus c r u z a s c o n T u x p e f io , V a n d e fio y S t i f f S ta lk

S y n t h e t ic e n C o t a x t l a , M . S . T h e s is , C h a p in g o , M e x ic o ( 1 9 6 4 ) .

[3 2 ] M O N T E N E G R O , J . , "R e s u lta d o s d e tres e x p e r im e n to s c o n v a r ie d a d e s d e f r i jo l e n H o n d u ra s, 1 9 6 6 " ,

П С А Z o n a N o r te P u b l. M is e . 6 7 (19 6 9 ) 9 7 .

[3 3 ] P IN C H IN A T , A . , " E l P C C M F y e l fo m e n to d e l c u l t iv o d e f r i jo l e n C e n t r o a m é r ic a " , I IC A Z o n a N o rte

P u b l. M is e . 6 7 (19 6 9 ) 6 3 .

[3 4 ] RAM IREZ-, E . , T I M O T H Y , D .H . , D I A Z , E . , G R A N T , U . J . , w ith N IC H O L S O N , G . E . , A N D E R S O N , E . ,

BR O W N , W . L . , R a c e s o f M a i z e in B o l iv ia , N A S -N R C P u b l. 7 4 7 ( 1 9 6 0 ) .

[3 5 ] R E IT Z , L . P . , S A L M O N , S . C . , O r ig in , h is to ry an d u se o f N o r in - 1 0 w h e a t , C r o p . S c i . £ ( 1 9 6 8 ) 6 8 6 .

[3 6 ] R O BER T S, L . M . , G R A N T , U . J . , R A M IR E Z , R . , H A T H E W A Y , W . H . , S M IT H , D . L . , w ith

M A N G E L S D O R F , P . C . , R a c e s o f M a iz e in C o lo m b ia , N A S -N R C P u b l. 5 10 ( 1 9 5 7 ) .

[ 3 7 ] R O B IN SO N , H . F . , C O M S T O C K , R . E . , H A R V E Y , P . H . , G e n e t ic v a r ia n c e s in o p e n p o l l i n a t e d v a r ie t ie s

o f c o r n , G e n e t ic s 40 ( 19 5 5 ) 4 5 .

[3 8 ] R O S S , J . G . , F R A N Z K E , C . J . , S C H U H , L . A . , S tu d ie s o n c o l c h i c i n e in d u c e d v a r ia n ts in so rg h u m ,

A g r o n . J . 4 6 ( 19 5 4 ) 1 0 .

[ 3 9 ] S A L H U A N A , W . , " E v a lu a c ió n d e p o b la c io n e s b á s ic a s p a ra e l d e s a r r o llo d e *fu tu ro s h íb rid o s e n l a c o s ta

d e l P e r u " , H a C o n f . M e j . M a í z , Z o n a A n d in a , Q u ito (19 6 6 ) 1 7 .

[4 0 ] S C H E U C H , F . , " S e l e c c i ó n d e p o b la c io n e s p a ra e l m e jo r a m ie n to d e l a v a r ie d a d A l a z a n " , H a C o n f .

M e j . M a í z , Z o n a A n d in a , Q u ito (19 6 6 ) 2 5 .

[ 4 1 ] S Y B E N G A , J . , G e n é t ic a y c i t o l o g ía d e l c a f é , T u r r ia lb a 10_ 3 (19 6 0 ) 8 2.

[4 2 ] T I M O T H Y , D . H . , P E Ñ A , B . , R A M IR E Z , R . , w ith BR O W N , W . L . , A N D E R S O N , E . , R a ces o f M a iz e

in C h i l e , N A S -N R C P u b l. 8 47 ( 1 9 6 1 ) .

[4 3 ] T I M O T H Y , D .H . , H A T H E W A Y , W . H . , G R A N T , U . J . , T O R R E G R O Z A , М., S A R R IA , C . D . ,

V A R E L A , A . , R a c e s o f M a iz e in E c u a d o r , N A S -N R C P u b l. 9 7 5 ( 1 9 6 3 ) .

[4 4 ] W A G N E R , R . P . , G e n e t ic s an d p h e n o g e n e t ic s o f m it o c h o n d r ia , S c ie n c e 16 3 (19 6 9 ) 1 0 2 6 .

[ 4 5 ] W E L L H A U S E N , E . J . , R O B E R T S, L . M . , H E R N A N D E Z , E . , w ith M A N G E L S D O R F , P . C . , R a ce s o f M a iz e

in M e x ic o , B u ssey In s t itu t io n s , H a rv a rd U n iv . ( 1 9 5 2 ) .

[ 4 6 ] W E L L H A U S E N , E . J . , F U E N T E S , A . , H E R N A N D E Z , A . , w ith M A N G E L S D O R F , P . C . , R a ce s o f M a iz e

in C e n t r a l A m e r i c a , N A S -N R C P u b l. 5 1 1 ( 1 9 5 7 ) .


D I S C U S S I O N

C. K R U LL : Perhaps fo r those not a c tive ly w orking in the corn breed ing industry, it m ight be worthwhile to c la r ify your point concerning the use o f cytop lasm ic m ale s te r ility in corn seed production in Argentina and other countries. As stated, it m ight leave the im press ion that m ost corn seed is produced with this system in Argentina while in p ractice less than 5% o f the hybrid corn seed is so produced.

A . GROBM AN: W hile it is true that in Argentina and m ost Latin A m erican countries hybrid seed corn production u tiliz in g cytop lasm ic m ale s te r ility (CMS) is v e ry lim ited yet, there are, how ever, cases where CMS use is w idespread. One such case is B razil, where tens o f thousands of tons o f hybrid seed corn a re being produced with CMS and fe r t i l ity res to ra tion system s. O thers are in Central A m er ica and M exico, where p riva te com panies a re a lready producing hybrid seed with the CMS system . Many other program s w ere ready to launch, or had started, hybrid seed production with CMS, when the recen t viru lent Helm inthosporum maydis ra ce hit hybrids produced with CMS o f the "T e x a s " type.

A . ASHRI: S evera l o f our m ost important crop plants have originated in Latin A m erica . I wonder i f you could describe som e o f the e ffo rts being made to p rese rve this valuable germ plasm?

A . GROBM AN: The f ir s t and m ost im portant organ ized e ffo rt to co llect, p reserve , and evaluate the va r iab ility availab le in crop plant spec ies that have orig inated in Latin A m erica was begun under sponsorship o f the "Indigenous M aize P rese rva tion C om m ittee" o f the U .S . National Academ y o f Sciences — National R esearch Council. Th is e ffo r t resu lted in the accumulation o f o ver 10 000 individual co llections which a re being p reserved as v iab le seed sam ples under co ld -d ry storage conditions at Chapingo, M exico , M edellin , Colom bia and P irac icaba , B ra z il. Duplicates a re kept at the National Seed Storage Labora tory at F o r t Collins, C o lo ., USA. Sm aller banks in s e ve ra l countries, such as the Peruvian area germ plasm bank are a lso maintained.

Bean (P . lunatus) co llections a re kept m ain ly in M exico , Colom bia, and north -east B ra z il, and a lso at Davis, C alif. Peanut co llections are m aintained at M anfred i E xperim enta l Station, A rgentina. An extensive co llection o f cacao clones and re la ted species is in existence at Itabuna, B ra z il, P ich ilingue, Ecuador, Tu rria lba , Costa R ica, and at Tingo M aria, P eru . O ver 2000 clones and va r ie tie s of cassava are being maintained at the C IA T Station in Colom bia. Potato species, va r ie t ie s and clones, are maintained liv e in Peru , M exico and B oliv ia . Recently, potato b reeders have started m aintaining potato va r iab ility as botanical seeds.

CONSIDERATIONS ON BREEDING METHODS

F. G. BRIEGER

Universidad de Campinas,Campinas, S. P ., Brazil

Abstract-Resumen

C O N S ID E R A T IO N S O N BREEDING M E T H O D S .

T h e c u r v e o f p o p u la t io n g ro w th and th a t o f a g r ic u lt u r a l p ro d u c t io n p e r u n it a r e a sho w a g e n e r a l and

w o r ld - w id e t e n d e n c y o f th e fo r m e r to o v e r ta k e th e l a t t e r . T h e r e is s t i l l in m o st a re a s a c o n s id e r a b le m a r g in

for im p r o v in g y i e ld b y p h y t o t e c h n ic a l p r o c e d u r e s , b u t th e g e n e t ic is t s m u st a ls o m a k e e ffo r ts to r e a c h a

m a x im u m im p r o v e m e n t o f c ro p s o n a w o r ld - w id e b a s is . In th is c o n n e c t io n , t h e r e h a s b e e n a d e f i n i t e s h ift

in r e c e n t y e a r s to s u b s titu te p e d ig r e e b r e e d in g w ith p o p u la t io n b r e e d in g , a c c e p t in g a c e r t a in g e n e t ic

p l a s t ic i t y , w h ic h w i l l a l lo w th e u se o f im p ro v e d c u l t iv a r p o p u la tio n s o v e r w id e r a re a s .

A n o th e r im p o r ta n t q u e s t io n is h o w to o b ta in th e g e n e t i c m a t e r i a l fo r fu tu re b r e e d in g w o rk . H e re , tw o

a lt e r n a t iv e s h a v e to b e c o n s id e r e d : e i th e r th e u se o f th e a c c u m u la t e d n a t u ra l g e n e re s e r v o ir , or th e p r o d u c t io n

o f n e w m u ta n ts th ro u g h th e a p p li c a t io n o f m u t a g e n ic a g e n ts . A s fa r as b r e e d in g t e c h n iq u e is c o n c e r n e d , th e re

d o e s n o t e x is t a n y e s s e n t ia l d i f f e r e n c e w h e th e r o n e starts w ith o n e or th e o th e r s o u rc e o f g e n e t i c m a t e r i a l .

S o - c a l le d sp o n ta n e o u s m u ta tio n s h a v e a c c u m u la t e d as g e n ic r e s e r v e d u rin g s o m e te n th o u sa n d y e a r s o f

e v o lu t io n an d a r e a v a i la b le in p r im i t i v e r a c e s an d o ld c u lt iv a r s . A lth o u g h n u m e ro u s a t te m p ts h a v e b e e n m a d e

to s a v e th e s e r a c e s and c u l t iv a r s , to o l i t t l e r e s e a r c h h a s b e e n c a r r ie d o u t o n th e m e th o d s o f p r e s e r v in g su ch

s a m p le s w it h o u t g e n i c lo ss . O n e a d v a n ta g e o f th e s e " n a tu r a l" so u rce s is th a t th e a c c u m u la t e d m u ta tio n s a re

a lr e a d y in e q u il ib r iu m w ith th e g e n o t y p e , w h e r e a s in d u c e d m u ta n ts a r e in th e b e g in n in g le ss b a la n c e d . It is

a ls o k n o w n t h a t s o m e ty p e s o f m u ta n ts a r e p ro d u c e d e a s i l y , w h i l e o th e r d e s ir e d c h a n g e s a r e гак-, e v e n t s . T h e

q u e s t io n is a ls o s t i l l u n d er d isc u ss io n as to w h e th e r o n e sh o u ld s e le c t fo r m u ta n ts w ith s m a ll q u a n t i ta t iv e

c h a n g e s , or w h e th e r m u ta n ts w it h s o m e e v id e n t m a jo r e f f e c t a r e m o r e p r o m is in g . T h e r e is n o w p o s i t iv e

p r o o f t h a t r a d ia t io n te c h n iq u e s h a v e re s u lte d in a n u m b e r o f n e w c u lt iv a r s , th o u g h s u c h a l is t d o es n o t a llo w

a c o m p a r a t iv e ju d g e m e n t o n th e r e l a t iv e v a lu e o f d i f f e r e n t b r e e d in g m e th o d s . It s e e m s th a t it w i l l s t i l l b e

n e c e s s a r y to im p r o v e m u t a g e n ic te c h n iq u e s . T h e r e c e n t b r e a k - th r o u g h s in im p r o v in g b a s ic c ro p s o f w o r ld

w id e im p o r t a n c e su ch as w h e a t , r i c e or m a i z e , h a v e n o t b e e n o b ta in e d b y a p p ly in g m u t a g e n ic te c h n iq u e s ,

bu t b y u s in g a c c u m u la t e d n a tu ra l m u ta n t g e n e s . T h u s, th e t r a d i t io n a l m e th o d o f s ta r t in g fro m th e a c c u m u la t e d

g e n e r e s e r v e s e e m s fo r th e t im e b e in g th e m o r e e f f i c i e n t o n e .

C O N S ID E R A C IO N E S A C E R C A D E LO S M E T O D O S D E M EJO RA D E ESPE CIES V E G E T A L E S .

L a c u r v a d e c r e c im ie n t o d e m o g r á f ic o y l a d e p r o d u c c ió n a g r í c o l a p o r u n id a d d e s u p e r f ic ie p re s e n ta n

l a m is m a e v o l u c i ó n g e n e r a l e n to d o e l m u n d o : l a p r im e r a t ie n d e a s itu a rse p o r e n c im a d e l a se g u n d a . En

c a s i to d a s la s r e g io n e s e x is t e aú n c o n s id e r a b le m a r g e n p a ra m e jo ra r e l r e n d im ie n to p o r p r o c e d im ie n t o s

f i t o t é c n ic o s , p e r o t a m b ié n lo s g e n e t is ta s d e b e n e s fo r z a r s e p o r m e jo ra r a l m á x im o la s p la n ta s d e c u l t i v o a

e s c a la m u n d ia l. En e s te a s p e c to , se h a o p e ra d o e n lo s ú lt im o s a ñ o s un c a m b io b ie n d e f in id o , e n e l se n tid o

d e su stitu ir l a s e le c c i ó n g e n e a l ó g ic a por l a s e le c c i ó n d e p o b la c io n e s , a c e p t a n d o u n a c i e r t a p la s t ic id a d g e n é t i c a ,

q u e p e r m íta e l e m p le o d e p o b la c io n e s m e jo ra d a s d e p la n ta s d e c u l t iv o e n z o n a s m á s e x te n s a s .

O tra c u e s t ió n im p o r t a n t e e s c ó m o o b te n e r e l m a t e r i a l g e n é t i c o p a ra lo s fu tu ro s tra b a jo s d e m e jo r a d e

e s p e c ie s . S e o fr e c e n do s a lt e r n a t iv a s : re c u rr ir a l a d o t a c ió n g é n i c a n a tu ra l a c u m u la d a , o b ie n a la

p r o d u c c ió n d e n u e v o s m u ta n te s m e d ia n te la a p l ic a c ió n d e m u tá g e n o s . Por lo q u e a la s t é c n i c a s d e s e le c c i ó n

se r e f ie r e , n o e x is t e d i fe r e n c ia e s e n c i a l a lg u n a , se a u n o u o tro e l m a t e r i a l g e n é t i c o d e p a r t id a . L as l la m a d a s

.m u ta c io n e s e s p o n tá n e a s se h a n a c u m u la d o e in c o rp o ra d o a l a d o t a c ió n g é n i c a a lo la r g o d e u nos d i e z m i l añ o s

d e e v o l u c i ó n y se e n c u e n tr a n e n la s e s p e c ie s p r im it iv a s y e n la s v ie ja s v a r ie d a d e s d e c u l t iv o . A u n q u e se h a n

r e a l i z a d o n u m e ro so s in te n to s p o r c o n s e r v a r e s ta s e s p e c ie s y v a r ie d a d e s , no se h a n in v e s t ig a d o lo s u f ic ie n t e lo s

m é th o d o s p a ra p r e s e r v a r la s sin p é r d id a s g é n ic a s . U n a d e la s v e n t a ja s d e e s ta s f u e n te s « n a t u r a l e s » es q u e la s

m u ta c io n e s a c u m u la d a s se e n c u e n tr a n y a e n e q u i l ib r io c o n e l g e n o t ip o , e n ta n to q u e lo s m u ta n te s in d u c id o s se

e n c u e n tr a n a l p r in c ip io m e n o s e q u il ib r a d o s . T a m b i é n e s sa b id o q u e a lg u n o s t ip o s d e m u ta n te s se p ro d u c e n

c o n f a c i l i d a d , m ie n tr a s q u e o tro s c a m b io s a p e te c id o s se c o n s ig u e n r a r a m e n t e . O tra c u e s t ió n q u e t o d a v ía se

d is c u te e s si se d e b e p ro c u ra r l a o b te n c ió n d e m u ta n te s c o n a lg ú n e f e c t o a c u s a d o d e im p o r t a n c ia . En l a

a c t u a l id a d , e x is t e n p ru e b a s c o n c lu y e n te s d e q u e la s t é c n ic a s d e i r r a d ia c ió n h a n d a d o o r ig e n a u n a s e r ie d e

n u e v a s v a r ie d a d e s d e c u l t iv o , a u n q u e a ú n n o es p o s ib le e m it i r un j u i c i o c o m p a r a t iv o so b re e l v a lo r r e la t iv o

43

44 BRIEGER

d e lo s d is tin to s m é to d o s d e m e jo r a g e n é t i c a d e la s p la n ta s . A l p a r e c e r , t o d a v ía e s n e c e s a r io p e r f e c c io n a r la s

t é c n ic a s m u tá g e n a s . Los r e c ie n t e s é x it o s o b te n id o s e n l a m e jo r a d e c u l t iv o s b á s ic o s d e im p o r t a n c ia m u n d ia l,

c o m o e l t r ig o , e l a r r o z o e l m a í z , n o se h a n lo g r a d o a p lic a n d o t é c n i c a s m u tá g e n a s , s in o r e c u r r ie n d o a lo s

g e n e s m u ta n te s n a tu ra le s a c u m u la d o s . Por ta n to , p a r e c e q u e , d e m o m e n to , e s m á s e f i c a z e l m é to d o

t r a d i c io n a l b a s a d o e n l a r e s e r v a g é n i c a a c u m u la d a .

The phytogeneticist and breeder has to date directed his work mainly in order to satisfy the special needs of a given region or of limited market requirements; the situation is now, however, rapidly changing. The curve of population growth and that of increased productivity per unit area of food crops show a clear general and world-wide tendency of the former to pass beyond the maximum limit of the latter, and it does not matter essentially if certain areas of the world have already reached this point, depending on food imports, while others still have a favourable balance. There still exists in most areas a considerable margin for improving yield by phyto- technical procedures, such as application of fertilizers, control of erosion and mechanization. The geneticist must make all possible efforts to improve crop varieties, both in quality such as the increase of adequate proteins in food crops, and in overall increase of yield per unit area, as has been done quite recently by Borlaug in wheat. This must also be done on a co-operative world-wide basis. To illustrate the actual situation,I will cite some data referring to maize production in Brazil, a cereal which must be considered as one of the basic food crops, either as a direct source of human nutrition or as an indirect source after transformation into animal or industrial products. Using improved seed sources and adequate techniques for cultivation, a yield of 6t/ha should be reached, and experimental data indicate at present a maximum of the order of 10t/ha. Actually the average for Brazil is still below 2t/ha!

Recently, there has been a definite shift in breeding methods to substitute pedigree breeding, which includes the hybrid corn technique, with population breeding, abandoning the requirement of maximum homogeneity and accepting a reasonable degree of homogeneity combined with a certain genetic plasticity, which would allow the use of improved cultivar populations over wider areas. Another question is the acquisition of the most promising genetic material as a starting point for breeding work, and here two alternatives are under discussion: the use of the accumulated gene reserve existing in old or indigenous races of crop plants or their wild relatives, or the production of new mutants through the application of mutagenic agents,i.e. radiation or chemical mutagens or both in combination. This is a point which should be discussed during these meetings. In this respect, I expect that my position here is rather difficult. Although in general the geneticists using traditional methods form a considerable majority, I am personally in these meetings part of an accentuated minority, especially when confronted by such an efficient and enthusiastic exponent of the radiation technique as Professor Gustafsson. It must be discussed whether one or the other technique offers special advantages or whether both should be applied in a parallel fashion. As far as breeding technique is concerned, there does not exist any essential difference whether one starts with one or the other source of genetic material.

There cannot be any doubt that innumerable mutations have accumulated as a genic reserve, first in the wild ancestors of crop plants and then

BREEDING METHODS 4 5

during thousands of years of domestication and cultivation, during which selective elimination was maintained at a very low level in consequence of the more primitive methods used. Intensified modern breeding work accentuating the need for a high degree of homogeneity, and the mass production of seeds by large seed producers supplying the necessary material over ever-growing areas, lead to a marked and rapid reduction of this gene reserve, and thus of the source for new breeding work. The increase of human populations will limit the extension of areas where wild relatives of crop plants have existed without human interference. Indigenous races or "Landsorten" will rapidly disappear owing to the spread of modern technology in agriculture. Thus, there is an increasing danger that the main sources for plant improvement by traditional methods will become rapidly depleted. It is possible to foresee that in the not very remote future, the lack of natural sources of genetic variability will make indispensable the artificial production of such genetic variability by mutation methods. That this situation can really occur, is shown by the following examples.

Some of the most successful pioneering work in radiation breeding has been that of Gregory on peanuts [ 1, 2, 3]. He was in fact in a situation where he had not at his disposal sufficient basic material for his program, and was far-removed geographically from the areas where wild peanuts occur in Brazil or where there are old indigenous races of this crop in South America. Such sources were therefore non-existent from a practial point of view, and thus radiation breeding represented for him the best practical method.

In maize a different situation arose. Owing to the high productivity and uniformity of modern Corn Belt Dent Corn mainly in the form of hybrids, many breeders considered it advantageous to substitute old varieties in other areas, but frequently the results did not correspond to this expectation. Corn Belt Dent is a type of rather recent origin, obtained in the first half of the last century by farmers in the USA from accidental crosses of rather limited sources of North Eastern Little Flint and Caribbean Dent, and then improved by concentrated scientific breeding, reducing genetic variability.Far better results are now obtained by using local or indigenous races of Mexican Dent, directly in Mexico and Central America, and after crossing with local adapted material in other regions.

We thus have to conclude that the applicability of the traditional breeding technique depends essentially upon the conservation of basic sources of genetic variability. Several attempts have been made to collect indigenous and local material and in this respect the initiatives taken by the great Russian geneticist Vavilov should be remembered. For maize, the National Academy of Sciences of the USA has taken the initiative of collecting on a large scale indigenous races of maize, establishing for this purpose three Centres, in Mexico for the Meso-American region, in Colombia and Peru for the Andean Region and in Brazil for the vast area of the South American lowlands, and has furnished funds for the collecting work. However, these collections have been made on a purely empirical basis and no research has been initiated to establish the methodology, based on principles of population genetics, both for obtaining samples of the right minimum size and for their maintenance during subsequent plantings without causing reductions of genetic variability. The program

4 6 BRIEGER

was considered concluded after the collecting and storing of samples and after the publication of descriptive data.

Although I am convinced that at present most crop plants still have sufficient genetic variability, and thus recommend the intensified use of the traditional techniques, the moment may come when mutation breeding becomes the only applicable technique, due to the loss of basic sources for the traditional technique. In this connection, the shift in breeding methods which is occurring on a large scale, i. e. the substitution of pedigree breeding by population breeding, may acquire special importance. Since population breeding automatically maintains a higher degree of genetic variability, the use of this technique might delay the destructive loss of existing genetic resources.

When comparing the efficiency of the traditional technique and of mutation breeding, one advantage of the former seems to me quite evident. It results from the fact that the mutations accumulated under natural conditions are on the whole already in a reasonable equilibrium with the genotype in general. In most mutants induced with mutagenic agents, the imbalance is considerable, and one faces the difficulty of selecting for balanced types.It is also known that the frequency of some types of mutations is considerably increased by mutagens, while others retain their normal low natural frequency. It seems to me still an open question as to what type of mutants should be selected after treatment, whether one should select for small quantitative mutants which after accumulation through selection increase productivity or widen adaptation, or whether mutants with some evident major effect are more promising. There is certainly positive proof, for instance in a list published in 1969 [4], that radiation technique has resulted in establishing a number of new cultivare, though such a list does not allow a comparative judgement on the relative value of the methods, since there is no compilation available as to how many new cultivare have been obtained during the same years by the traditional techniques and how much effort was involved in both instances. It must also be recognized that most recent break-throughs in improving basic crops of world-wide importance such as wheat, rice, or maize, have all been obtained not by applying mutagenic techniques, but by the traditional breeding technique, using accumulated natural mutant genes.

For the time being, therefore, the traditional method of starting from the accumulated gene reserve appears to me as the more efficient method, except in special cases, as long as a sufficient range of natural genetic variability remains at the disposal of the breeder. On the other hand, a further improvement of mutagenic techniques by intensified basic research seems not only justified, but necessary.

REFERENCES

[ 1 ] G R E G O R Y , W . C . , X - r a y b r e e d in g o f p e a n u ts (A r a c h is h y p o g a e a L . ) , A g ro n . J. 4 7 (19 5 5 ) 3 9 6 .

[ 2 ] G R E G O R Y , W . C . , " I n d u c tio n o f u s e fu l m u ta tio n s in th e p e a n u t " , G e n e t ic s in P la n t B re e d in g ,

B ro o k h a v e n S y m p . B io l. 9 , ( 1 9 5 6 ) 1 7 7 .

[ 3 ] G R E G O R Y , W . C . , " M u t a t io n f r e q u e n c y , m a g n itu d e o f c h a n g e an d th e p r o b a b i l i t y o f im p r o v e m e n t in

a d a p t a t io n ” , T h e U se o f In d u c e d M u ta tio n s in P la n t B re e d in g (R ep . F A O / I A E A T e c h . M e e t in g , R o m e,

1 9 6 4 ), P e rg a m o n Press, O x fo rd (19 6 5 ) 4 2 9 .

[ 4 ] SIG U R B JÔ R N SSO N , B ., M IC K E , A . , "P ro g ress in m u ta tio n b r e e d in g " , In d u ce d M u ta tio n s in P lan ts

( P ro c . S y m p . P u llm a n , 1 9 6 9 ) , IA E A , V ie n n a (19 6 9 ) 6 7 3 .

BREEDING METHODS 47

DISCUSSION

H. GAUL: You mentioned that it is not proven that Gregory would not have had the same success if he had used primitive or wild forms of peanuts. For a priori reasons, it is easier to use a mutation induced in an adapted and up-to-date variety, than to use the same or a similar character from a wild form. In a long-term project we studied in our laboratory the possibilities to dissolve the pleiotropic effect of macromutants in a changed genetic background obtained by crossing. We found it relatively easy to remove undesired characters but difficult to maintain the desired ones, such as disease resistance or straw-stiffness.

F.G. BRIEGER: Since Gregory did not carry out comparative experiments, it is hardly possible to state what results he might have obtained if choosing some race, cultivar, etc., of the main area where Arachis occurs and has been cultivated since prehistoric times. Considering this range in latitude and altitude, he might have found some type with a reasonable degree of adaptability and desirable characters.

H. GAUL: I agree that techniques for using mutations are not yet readily available inallogamous species. However, they are available in autogamous plants and in some vegetatively propagated species. Every plant breeder trained in the use of mutations canusethem with reasonable prospects of having success at least for certain breeding goals. Mutations should be used as a supplement to conventional methods. But research should of course be continued to increase the efficiency of the new method.

F. G. BRIEGER: It seems to me that there is agreement in so far as stated byGaul that mutagenic techniques are "supplementary to conventional methods", but I am also convinced that regarding the situation in Latin America traditional methods are more appropriate and promising, both for allogamous or autogamous or species with mixed reproduction.

A. GUSTAFSSON: You have not been able to find any natural source for rust resistance in Coffea. I think this would be a good opportunity for a large scale experiment using induced mutation. But you definitely need (1) a sufficiently effective screening method for resistance, (2) large amounts of M2, or preferably M3, progenies, and (3) patience. Personally I am convinced, relying on parallel results of resistance mutations in barley, oats and wheat, that you would discover similar resistance mutations also in Coffea.

F.G. BRIEGER: Evidently we agree in the present case. Since as far as we know, no indications are available of the occurrence of some degree of resistance to the coffea rust, Hemelia vastatrix, it would be useful to try to obtain some such mutations through mutagenic action.

E L MEJORAMIENTO DE LAS PLANTAS POR INDUCCION DE MUTACIONES EN LATINOAMERICA*

E.A. FAVRETCentro de Investigaciones en Ciencias Agronómicas,Castelar, Argentina

Abstract-Resumen

P L A N T IM P R O V E M E N T T H R O U G H IN D U C T IO N OF M U T A T IO N S IN L A T IN A M E R IC A .

T h e p la n t b r e e d e r w h o se a im s a r e t o in c r e a s e a g r o n o m ic y ie ld s , to im p ro v e p r o d u c t q u a l i t y an d to assu re

m o re s a fe t y o f c r o p , im i t a t e s in h is te c h n iq u e s th e m e c h a n is m s o f p la n t e v o lu t io n . A s s p o n ta n e o u s m u ta tio n s

h a v e p la y e d a b a s ic r o le in p la n t e v o lu t io n it se e m s l o g i c a l to c o n s id e r th e in d u c tio n o f m u ta tio n s a s a t o o l in

p la n t b r e e d in g a ls o in L a tin A m e r i c a . H o w e v e r , m a n y p la n t b re e d e rs s t i l l h a v e o b je c t io n s w h ic h fo c u s o n the

fo l lo w in g q u e s t io n s : Is in d u c e d g e n e t i c v a r ia b i l i t y id e n t ic a l w ith n a tu ra l v a r ia t io n ? I f th e a n s w e r is y e s , th e

n e x t q u e s t io n w o u ld p r o b a b ly b e , w h e th e r in d u c tio n o f m u ta tio n s is m o r e e f f i c i e n t th a n th e a t te m p t to s e le c t

fo r sp o n ta n e o u s o n e s . H o w e v e r , i f th e a n s w e r is n o , i t im p lie s t h a t w e a r e a b le to c r e a t e g e n e t i c v a r ia t io n

w h ic h d o e s n o t e x is t in n a t u r e . In th e l a t t e r c a s e , o n e w i l l b e f a c e d w ith t h e n e x t q u e s t io n , w h e th e r su ch

in d u c e d m u ta tio n s a r e n o t a lw a y s u n d e s ire d a n d d e t r im e n t a l . T o d a y , w e c a n c l e a r ly d e n y th a t q u e s t io n , b u t

t h e q u e s t io n w o u ld s t i l l r e m a in w h e th e r m u ta t io n b r e e d in g is n o t to o s o p h is t ic a te d an d to o e x p e n s iv e a m e th o d

fo r m o st o f th e L a tin A m e r i c a n c o u n tr ie s . T h is p a p e r a t te m p ts t o a n s w e r th e s e q u e st io n s b y g iv in g so m e

e x a m p le s o f p r a c t i c a l a c h ie v e m e n t s o f m u ta tio n b r e e d in g . For e x a m p le , 8 379 sp rin g b a r le y v a r ie t ie s o f th e

U S D A c o l l e c t i o n h a v e b e e n s c r e e n e d fo r e a r lin e s s . S o m e e x t r e m e ly e a r ly ty p e s w e r e h e a d in g w ith in 80 - 88 d a y s .

S im i la r ty p e s h a v e b e e n p r o d u c e d b y m u ta t io n in d u c tio n , b u t a ls o a t y p e w it h h e a d in g a f te r 40 - 4 5 d a y s h a s b e e n

fo u n d , w h ic h d id n o t o c c u r in th e c o l l e c t i o n . T h is e x a m p le sh o w s th a t in d u c tio n o f m u ta tio n s c a n le a d t o p la n t

ty p e s q u ite s im ila r t o th o se a v a i la b le in n a tu re , b u t th e e x a m p le a ls o d e m o n s tr a te s th a t th e t e c h n iq u e is c a p a b le

o f b r o a d e n in g th e e x is t in g g e n e t i c v a r i a b i l i t y .

M o re e x a m p le s a r e g iv e n in t h e p a p e r fo r o th e r e c o n o m ic a l l y im p o r ta n t c h a r a c te r s s u c h as lo d g in g r e

s is ta n c e , a d a p t a b i l i t y a n d d is e a s e r e s is t a n c e . It is a ls o p o in te d o u t t h a t e v e n m u ta n t a l l e l e s w h ic h a r e id e n t ic a l

w it h th o se e x is t in g in c o l l e c t io n s a r e n o t u s e le s s , b e c a u s e t h e y m a y h a v e b e e n in d u c e d in v a r ie t ie s w ith a

c o m p le t e l y d i f f e r e n t g e n e t i c b a c k g ro u n d , an d t h e n e w g e n o t y p e m ig h t b e m o re e a s i l y u sed in c r o s s -b r e e d in g or

d i r e c t l y d e v e lo p e d in t o a n e w v a r i e t y . It is c o n c lu d e d t h a t a r t i f i c i a l in d u c tio n o f m u ta tio n s íí; n o t o n ly

r e c o m m e n d a b le , b u t in d is p e n s a b le fo r th e fu tu re p ro g ress o f p la n t b r e e d in g in L a tin A m e r i c a . C e r t a i n ly , m o re

e f fo r ts a r e n e e d e d to a d a p t th e m e th o d s , d e v e lo p e d o r ig in a l ly fo r p l a n t s p e c ie s o f t e m p e r a te c l i m a t i c r e g io n s , to

th e s p e c ie s a n d e c o s y s t e m s o f t r o p ic a l r e g io n s .

EL M E J O R A M IE N T O D E L A S P L A N T A S PO R IN D U C C IO N D E M U T A C IO N E S EN L A T IN O A M E R IC A .

E l f i t o t é c n ic o q u e t ra ta d e a u m e n t a r e l r e n d im ie n to a g r o n ó m ic o , m e jo r a r l a c a l i d a d d e l p ro d u c to e i n c r e

m e n ta r la s e g u r id a d d e la c o s e c h a , im i t a c o n sus t é c n i c a s lo s m e c a n is m o s d e e v o lu c i ó n d e la s p la n t a s . C o m o

la s m u ta c io n e s e s p o n tá n e a s h a n d e s e m p e ñ a d o u n p a p e l fu n d a m e n ta l e n d ic h a e v o lu c ió n , p a r e c e l ó g i c o c o n s id e r a r

la in d u c c ió n d e m u ta c io n e s c o m o in s tr u m e n to d e s e le c c i ó n d e p la n t a s t a m b ié n en A m é r i c a L a t in a . S in e m b a r g o ,

m u c h o s f i t o t é c n ic o s a ú n p o n e n re p a ro s q u e se c e n tr a n e n la s s ig u ie n te s p r e g u n ta s : ¿Es l a v a r ia c ió n g e n é t i c a

in d u c id a id é n t ic a a la v a r ia c ió n n a tu ra l? S i la c o n t e s t a c ió n es a f i r m a t iv a , se tra ta a c o n t in u a c ió n d e sa b e r si

la in d u c c ió n d e m u ta c io n e s e s m á s e f i c a z q u e s e le c c i o n a r la s e s p o n tá n e a s . En c a m b io , s i l a c o n t e s t a c ió n es

n e g a t i v a , e l l o i m p l ic a q u e p o d e m o s c r e a r u n a v a r ia c ió n g e n é t i c a q u e n o e x is t e e n l a n a t u r a le z a . En e s te ú lt im o

c a s o , se p la n t e a o tra c u e s t ió n , a sa b e r , s i d ic h a s m u ta c io n e s in d u c id a s n o son s ie m p r e in d e s e a b le s y p e r ju d i

c i a l e s . H o y d ía , p o d e m o s a f ir m a r d e c id id a m e n te lo c o n tr a r io , p e r o su b siste e l in te r r o g a n te d e ¡si la s e le c c i ó n

p o r m u ta c ió n n o e s un m é t o d o e x c e s iv a m e n t e c o m p lic a d o y c o s to s o p a ra la m a y o r ía d e lo s p a ís e s d e A m é r i c a

L a t i n a . L a m e m o r ia t ra ta d e re s p o n d e r a e s ta s p re g u n ta s c i ta n d o a lg u n o s e je m p lo s d e lo s p ro g re so s p r á c t ic o s

* P u b lic a c ió n t é c n i c a G e n .4 5 2 d e l C e n t r o d e I n v e s t ig a c io n e s e n C i e n c i a s A g r o n ó m ic a s , I N T A , C a s t e la r .

P a r te d e la s in v e s t ig a c io n e s c o n s ig n a d a s e n e s ta m e m o r ia se l l e v ó a c a b o c o n a r r e g lo a l C o n t r a t o d e

in v e s t ig a c ió n № -435/RB d e l O Œ A .

49

50 FAVRET

c o n s e g u id o s m e d ia n te e l m e jo r a m ie n to p o r m u ta c ió n . Por e j e m p lo , se h a n e x a m in a d o c o n r e s p e c t o a l a p r e

c o c id a d 8 379 v a r ie d a d e s d e c e b a d a s p r im a v e r a le s d e l a c o l e c c i ó n U S D A . A lg u n o s tip o s m u y p r e c o c e s e s p ig a ro n

en 80 - 88 d ía s . S e h a n o b te n id o t ip o s s im ila r e s p o r m u ta c ió n in d u c id a , a s í c o m o un t ip o Que e s p ig a a los

40 - 4 5 d ía s , q u e n o se e n c o n tr a b a e n la c o l e c c i ó n . Este e j e m p l o d e m u e s tra q u e la in d u c c ió n d e m u ta c io n e s

p e r m ite o b te n e r t ip o s d e p la n t a a n á lo g o s a lo s n a tu ra le s , a s í c o m o q u e d ic h a t é c n i c a es c a p a z d e a m p lia r la

v a r ia c ió n g e n é t i c a n a t u r a l .

L a m e m o r ia c o n t ie n e o tro s e je m p lo s r e la t iv o s a d iv e rso s c a r a c t e r e s im p o r ta n te s e c o n ó m ic a m e n t e , t a le s

c o m o la r e s is t e n c ia a l e n c a m a d o , l a a d a p t a b i lid a d y l a r e s is t e n c ia a la s e n fe r m e d a d e s . En la m is m a se in d ic a

t a m b ié n q u e in c lu s o lo s a l e l o s m u ta n te s id é n t ic o s a lo s y a e x is t e n t e s e n la s c o l e c c io n e s no son in ú t i le s , p o rq u e

es p o s ib le q u e h a y a n s id o in d u c id o s e n v a r ie d a d e s c o n u n fo n d o g e n é t i c o t o t a lm e n t e d is tin to y e l n u e v o g e n o t ip o

p u e d a u t i l iz a r s e m á s f á c i lm e n t e e n c r u z a m ie n t o s o p a ra o b te n e r d ir e c t a m e n t e u n a n u e v a v a r ie d a d . E n c o n c lu c íó n ,

se d i c e q u e l a in d u c c ió n a r t i f i c i a l d e m u ta c io n e s e s n o s ó lo r e c o m e n d a b le s in o e s in d is p e n s a b le p a ra e l fu tu ro

p ro g re so d e la f i t o t e c n ia e n A m é r i c a L a t in a . D esd e lu e g o , se n e c e s i t a n m á s t ra b a jo s p a ra a d a p ta r lo s m é to d o s ,

e la b o r a d o s en p r in c ip io p a ra e s p e c ie s v e g e t a l e s de r e g io n e s d e c l i m a t e m p la d o , a la s e s p e c ie s y e c o s is t e m a s d e

la s r e g io n e s t r o p ic a le s .

GENERALIDADES

El mejoramiento genético de las plantas es un proceso dirigido a reunir en un individuo o población de individuos, caracteres hereditarios que tienden a:

1) obtener un mayor rendimiento agronómico1,2) mejorar la calidad del producto, y3) aumentar la seguridad de cosecha.

El proceso es dinámico porque los objetivos son cambiables a través del tiempo en los tres aspectos mencionados. En el primero porque el cultivo de una planta es el resultado de una interacción entre el genotipo y el ambiente y si este último se modifica, sea por un manejo más apropiado, empleo de fertilizantes, herbicidas, mecanización, etc. , el rendimiento máximo actual cambia en procura del rendimiento máximo potencial. En otras palabras, el cultivo de una planta es un ecosistema sujeto a las alteraciones provocadas por factores exógenos.

Dos caminos generales existen para aumentar el rendimiento. El primero orientado a obtener el máximo potencial sin tener mucho en cuenta el rendimiento económico, y en este caso se modifican simultáneamente el patrimonio genético, el ambiente y su interacción; mientras en el segundo, por el contrario, los costos de producción son de tanta importancia que pueden reducir las posibilidades técnicas y retardar el progreso del mejoramiento. Esto último sucede principalmente en aquellos países que deben necesariamente exportar su exceso de producción. Como la modificación del ambiente provoca generalmente un aumento en los costos de producción y un mayor riesgo a nivel del rendimiento económico, deja la opción únicamente al mejoramiento genético que conduce normalmente a un menor costo y

1 D e f in ic io n e s :

1 . R e n d im ie n to g e n é t i c o : c a p a c id a d p ro d u c t iv a d e u n g e n o t ip o o p r o m e d io d e u n c o n ju n t o d e

g e n o t ip o s en u n a m b ie n t e e s p e c i f i c a d o .

2 . R e n d im ie n to a g r o n ó m i c o : p r o d u c c ió n d e un e c o s is t e m a p o r u n id a d d e s u p e r f ic ie o u n i d a d d e t i e m p o

o a m b a s .

3 . R e n d im ie n to e c o n ó m ic o : r e n d im ie n to a g r o n ó m ic o e x p r e s a d o e n d i f e r e n c i a e n tre v a lo r e s d e c o s to s

y b e n e f i c io s , p a ra un m o m e n to y c i r c u n s ta n c ia d a d a .

INDUCCION DE MUTACIONES EN LATINOAMERICA 51

a un menor riesgo. Los países exportadores se encuentran muchas veces restringidos por tener que producir en condiciones económicas competitivas y, por consiguiente, deben optar por el segundo proceso que es más lento.

Con respecto a la «calidad» y desde que este término no puede tener más que validez circunstancial, los objetivos en este terreno son los más variables posibles. También, en este caso, los países exportadores son los más expuestos a su influencia por las exigencias de sus clientes, lo que está ejemplificado en los requerimientos cualitativos del trigo que están pasando de un concepto tecnológico a uno nutritivo.

La seguridad de cosecha, finalmente, tiene importancia sólo local y no sufre cambios, mientras los factores de incidencia no se alteren. En el caso de las enfermedades, sin embargo, los objetivos pueden variar continuamente porque los agentes etiológicos son organismos también sujetos a procesos de evolución biológica. La solución temporaria de un problema fitopatológico hace surgir muchas veces nuevos problemas. En un ecosistema pueden representar factores de mucho peso en la modificación de los objetivos.

La metodología para obtener los logros del mejoramiento genético es también cambiable. El aporte de nuevos conocimientos o la reinterpretación de las hipótesis previas, van tratando de hacer cada vez más eficiente el mejoramiento. En breve, significa aproximar al mínimo la diferencia entre rendimiento genético y rendimiento agronómico.

PROBLEMAS LATINOAMERICANOS CONCERNIENTES

Las plantas cultivadas, como los países, aunque por otras razones, pueden ser clasificadas como desarrolladas, no desarrolladas o con el eufemismo de en proceso de desarrollo, dependiendo del mejoramiento genético que se ha logrado. Su nivel de desarrollo puede estimarse por el aumento del rendimiento genético y/o del rendimiento agronómico ocurrido durante un lapso de tiempo, por ejemplo desde principios de siglo a la fecha.

No existe necesariamente una asociación entre la clasificación análoga de los países, basadas generalmente en la renta per cápita medida en valores monetarios y la de las plantas que se cultivan en el citado país. Afortunadamente existe perfecta comprensión en los países en desarrollo de trasfondo agropecuario, de que sus cultivos más importantes deben estar en la faz máxima posible de desarrollo genético (caso del café en Brasil, cereales en la Argentina).

La situación, en cambio, es diferente si se considera la metodología utilizada. En este caso, los países en desarrollo aunque tengan una tecnología agrícola adelantada, viven generalmente esperando el aporte del conocimiento de otros países más desarrollados. No poseen, en otras palabras, una metodología de avanzada o «pionera» que les permita progresar con un ritmo más acelerado que el promedio del mejoramiento genético.

Para los países de regiones templadas, el aprovechamiento del conocimiento introducido es más rápido que para los países tropicales o subtropicales. En estos últimos, la escasez de conocimientos particulares empeora en sumo grado el progreso genético esperado. En Latinoamérica esta situación es evidente. En consecuencia, es aconsejable y necesario que los países en desarrollo tomen conciencia de la necesidad de lograr una metodología de avanzada y conocimientos propios independientes que le permitan un mayor progreso genético que lo normal de sus cultivos.

52 FAVRET

HISTORIA DEL MEJORAMIENTO GENETICO

Se dice que el conocimiento de la historia permite la predicción del futuro. El mejoramiento genético de las plantas (y de los animales) comienza con su domesticación. Este es un proceso evolutivo donde el hombre, total o parcialmente consciente, imprime sus objetivos y la intensidad de selección. Por ambas razones difiere de la evolución natural.

En cambio, en cuanto se refiere a la metodología para la obtención de los resultados, el hombre ha imitado los mecanismos que ha estudiado e interpretado en condiciones naturales. Según el eminente investigador sueco Nilsson-Ehle, la naturaleza es el mejor criador.

La evolución biológica se explica por una adaptación paulatina de las plantas al ambiente mediante una acumulación progresiva de mutaciones en un mismo genotipo, siendo la selección el proceso por el cual unas mutaciones se fijan en la población y otras desaparecen. Las primeras son consideradas, por consiguiente, de utilidad para la especie y las segundas inútiles. Algunos mecanismos como la deriva genética («random drift») pueden fijar ocasionalmente genes que no son necesariamente útiles.

Cuatro aspectos genéticos han participado en la domesticación de las plantas o en la crianza de las plantas, a saber:

a) La ocurrencia de mutaciones que tienen un efecto aparentemente considerable en términos del desvío que presentan en relación con la forma original. Algunos investigadores aconsejan denominar a tales mutaciones, macromutaciones o aún mutaciones sistémicas (Swaminathan, 1965). Son de herencia simple y aveces aparecen como formas negativas o no progresivas. Sin embargo, el valor de una mutación depende primariamente del genotipo donde se ha producido.Ejemplos de esta naturaleza tenemos en los resultados logrados por el genetista aleman von Sengbush, en la domesticación de especies de lupinos, artificialmente acelerada por una hábil selección de formas libres de principios tóxicos o de frutos indéhiscentes. Asimismo, la particularidad de poseer raquis no frágil en la espiga permitió el primer paso de domesticación en los cereales. En ambos casos los caracteres están regulados por pocos factores hereditarios.

b) Pequeños reajustes del genotipo, incluyendo la incorporación de las macromutaciones en el fondo genético más aprovechable. Esta faz corresponde al «cross-breeding» desarrollado durante este siglo y que ha transformado el mejoramiento genético empírico en una disciplina científica. Se basa teóricamente en la segregación independiente o condicionada de los factores hereditarios.

c) Acumulación lenta de mutaciones que ofrecen pequeñas ventajas, sobre todo aquellas relacionadas con la seguridad de cosecha. Ello es realizado por distintos métodos como son la retrocruza, selección recurrente,etc.

d) Homogeneización de la población para que se produzca la máxima expresión del rendimiento agronómico. Esto está involucrado en la producción de cultivares homocigotos o de híbridos Fj y tiende a la obtención de cultivos donde todos sus integrantes son iguales. Las ideas modernas sobre el empleo de las mezclas de genotipos (multilíneas) no contradice la proposición anterior porque las proporciones de las mismas son constantes y óptimas para reducir los efectos competitivos entre las plantasy el aprovechamiento total del ambiente.


Las mutaciones, por consiguiente, son la base primaria necesaria para el mejoramiento genético y participan en los aspectos mencionados en los párrafos a) y c) anteriormente citados. En el primero, mediante la contribución de macromutaciones, en el segundo con las mutaciones de efecto acumulativo, incluyendo aquí las micromutaciones.

Aunque la diferenciación entre ambos tipos de mutaciones es imprecisa y sólo medible por la diferencia entre la forma mutante y la forma original, es aplicable cuando se quiere evaluar cual es la utilidad agronómica de las mismas.

VARIACION NATURAL E INDUCIDA

Existen tres fuentes de variación genética a disposición de la crianza de las plantas, a saber:

a) la variación espontánea, acumulada en el germoplasma actual y queha constituido, en la primera mitad de este siglo, la única o casi única aprovechable,

b) la presente en especies silvestres afines a las cultivadas y de las cuales los genes útiles pueden ser transferidos si las barreras de aislamiento génico lo permiten,

c) la inducción de mutaciones por medios artificiales, sea físicos o químicos, sobre el genotipo deseado y en el momento dado. Esta última contribución es el «leitmotiv» de esta reunión y muchas de sus facetas serán presentadas y discutidas más adelante por relevantes expertos.

La oportunidad de substituir la primera por alguna de las otras es difícil de establecer, pero antes de decidir su aplicación es necesario tratar de aclarar algunas cuestiones, entre otras;

1. ¿Es la variación inducida idéntica o equivalente a la variación natural?Si la contestación es afirmativa se requiere preguntar entonces cuál es el método más eficiente para obtener esa variante transportada a un cultivar. Si, por el contrario, hubiera la posibilidad de crear variación que no existe actualmente o, si bien ha existido, ya no se tiene más a disposición, la inducción artificial tiene ventajas, dependiendo de la contestación a la siguiente pregunta;

2. Las mutaciones inducidas, ¿presentan características secundarias indeseables que las hacen finalmente poco aprovechables?

3. Por último, una vez contestada negativamente la segunda pregunta, cabe averiguar si la metodología necesaria a utilizar en la mutagénesis articicial no es excesivamente compleja o sofisticada y tal vez fuera del alcance de fitotécnicos con pocos recursos. En otras palabras, si su empleo no debe quedar relegado sea a los países que pueden hacer metodología avanzada, sea para aquellos casos donde los métodos más convencionales han agotado sus posibilidades prácticas.

No es intención discutir en detalle las tres etapas mencionadas. Es nuestro propósito presentar solamente algunos ejemplos donde se pueda comparar la variación espontánea y la inducida en aquellos casos donde las macromutaciones pueden ser aprovechables, representando un marcado progreso en el mejoramiento genético.

54 FAVRET

Durante el año 1970 hemos revisado la reacción de las 8379 entradas de cebadas primaverales de la colección de la USDA y 1580 de cebadas invernales. Este material fue sembrado en Castelar el 11 / VI/ 70 y la espigazón medida en días desde la fecha de siembra. Nuestro interés reside en la evaluación de formas que puedan espigar en períodos muy cortos con temperatura adecuada, para lugares con corto período vegetativo.

Como se observa en el cuadro I, aproximadamente un 5% son formas drásticas. Es posible que este valor sea distinto al real, desde que en la colección existen duplicaciones de origen. La lista de formas muy precoces aparece en el cuadro II.

En los trabajos de mutagenesis artificial la aparición de mutantes precoces es un fenómeno relativamente común. El número de loci envueltos en el carácter precocidad es de 4 a 6, según los estudios de Gustafsson y colaboradores (1960), lo que permite prever un rendimiento de una mutante precoz cada 10 000 progenies M2 si el tratamiento es de 10 krads de radiaciones ionizantes, lo que lo hace accesible en términos de fitotecnia aplicada.

Dos mutantes han sido analizadas con mayor cuidado, una es Mari (ea-a8) caracterizada por ser un caso sujeto a influencia por el fotoperíodo; en la otra se ha afectado un locus distinto, M.C. 20 superprecoz y la mutante es, en este caso, insensible al fotoperíodo. En el caso de máximo desvío posible y si la temperatura es la adecuada, esta última mutante espiga en 40-45 días (cuadro III).

PRECOCIDAD

CUADRO I. PERIODO DESDE LA SIEMBRA A ESPIGAZON (EN DIAS) DE LAS ENTRADAS DE LA COLECCION DE CEBADAS PRIMAVERALES DE LA COLECCION DE USDA CULTIVADAS EN CASTELAR (fecha de siembra: junio de 1970)

P e r ío d o d e s ie m b ra

a e s p ig a z ó n

( d ía s )

F r e c u e n c ia

a b s o lu ta

F r e c u e n c ia

r e la t iv a

X 1 0 " 3

8 0 - 88 29 3 ,4

8 9 - 9 1 13 1» 5

9 2 - 9 5 34 4 ,0

9 6 - 98 53 6 ,3

9 9 - 1 0 2 330 3 9 ,0

1 0 3 - 1 0 5 269

1 0 6 - 1 0 7 2 2 5 1 3 5 ,9

1 0 8 - 1 1 1 18 9

1 1 2 - 1 1 3 4 5 6 J> 1 1 4 6 7 8 1 -

T o t a l 8379

INDUCCION DE MUTACIONES EN LATINOAMERICA 5 5

CUADRO II. LISTA DE VARIEDADES PRECOCES EN LA COLECCION INTERNACIONAL (USDA)

N o m b re P r o c e d e n c ia C . l .

A n d ie In d ia 728

C o r b e l C h in a 1 1 1 3

S v a lo fs S ix R o w ed S u e c ia 1 5 4 9

M u ly a n C h in a 2 4 5 3 ( 1 1 567)

P e g a n C h in a 2 4 5 4

- Japó n 2 5 5 1

- USRR 2 6 3 3

E a r ly F in la n d ia 4 2 1 3

A t s e l E E .U U . 6450

L a p in P o lo n ia 6 4 2 7

O l l i P o lo n ia 6 4 4 9 ( 1 1 2 9 6 y otros)

P o la rb y g S u e c ia 6 556

A la s k a E xpress A la s k a 7 50 5

N a r u m 8 2 5 - 4 2 A la s k a 8 9 2 2

R edsk o rn S u e c ia 9 873

F lo y a - 10 0 7 4

- - 9 944

B a n k u ti K o ra i H u n g ría 10 4 5 5 ( 1 2 0 2 7 , 1 2 972)

A sa C a n a d á 1 1 3 0 1 ( 1 1 3 0 7 )

S u m m e r b a r le y H u n g ría 1 1 9 5 9

- H u n g ría 1 1 9 6 5

J a p a n is c h e 4 5 6 H u n g ría 1 2 9 7 5

En comparación entre los loci afectados, Mari (ea-a8) es una mutación en un locus que ya se conoce en la naturaleza, como en el caso de Kinai № 5 (Favret y Frecha, 1967). En cambio, MC 20 SP no se encuentra repetido en el germoplasma mundial, según los estudios realizados hasta el momento. Aunque los mismos no han progresado lo suficiente como para concluir al respecto, es evidente que la mutagenesis artificial simula y aún amplía la variación natural.

56 FAVRET

CUADRO III. PERIODO DESDE LA SIEMBRA A ESPIGAZON (EN DIAS) DE DIVERSAS VARIEDADES Y MUTACIONES DE CEBADA,EN CASTELAR (año 1970)

V a r ie d a d e s y m u ta n te s

F e c h a d e s ie m b ra

2 6 - 6 - 7 0 1 6 - 7 - 7 0

Bonus 108 100

M a ri ( e a - a 8) 80 73

E a r ly - 7 ( e a - b 7 ) 100 9 4

M . H e d a 98 86

М . C . 20 SP 72 62

K ín a i № 5 8 1 7 3

A t s e l 80 7 3

M u ly a n 86 73

RESISTENCIA AL VUELCO

Aunque el número de loci envueltos en este caso es mayor que en el precedente, como lo demuestran los estudios realizados en cebada por distintos investigadores (cf. Hagberg y Persson, 1969), es decir, es un caso de herencia poligénica, el carácter no puede ser considerado de variación continua, desde que algunas mutaciones aún en condición simple presentan un marcado desvío en relación a las formas normales.

El citado carácter aparece asimismo bien representado en el germoplasma natural. Sin embargo, la ventaja de obtener nuevas formas sobre cultivares que poseen buenas condiciones agronómicas, apoyaría la aplicación de la mutagenesis artificial. Los resultados aplicados son ya apreciados para condiciones extremas de alta fertilización en el caso de la cebada y posiblemente lo mismo puede ocurrir también en trigo.

Cuando el estudio de las mutantes erectoides progrese aún más, es posible que se pueda discriminar cuales son los genes de mejor utilidad en los planes de mejoramiento. Recuérdese que una tendencia a la obtención de formas enanas, resistentes al vuelco y con capacidad productiva en altos niveles de fertilidad, es un objetivo actual común para numerosos cultivos incluidos formas perennes como árboles frutales.

ADAPTABILIDAD

Una variación interesante en los planes de mejoramiento ha sido la utilización de la combinación feliz obtenida en los planes de mejoramiento de trigo llevados a cabo por el Dr. Borlaug en México y otros países y por el Dr. Vogel y asociados en Washington State University. El conocimiento


actual permite sugerir que la reunión de algunas macromutaciones (resistencia al vuelco, insensibilidad al fotoperíodo) agregada a factores de productividad y resistencia a enfermedades, ha culminado en cultivares de muy alto rendimiento.

La base genética proviene del trigo Norin 10, que posee las dos primeras condiciones mencionadas. La adaptabilidad a distintos nichos ecológicos estaría asociada, según los estudios de Swaminathan (1968) y los nuestros (Favret et al. 1969) a la condición de insensibilidad al ácido gibe- rélico de Norin 10, condición que está regulada por un solo par génico. Swaminathan (1968) ha demostrado que la misma condición es válida para las nuevas variedades de arroz que poseen el factor «Dee-gee-Woo-gen» para enanismo, lo que es una llamativa coincidencia.

Con la misma hipótesis de trabajo, tanto Swaminathan como nosotros (op. cit. ) hemos determinado independientemente en cebada genes para insensibilidad al GA inducidos en tratamientos mutagénicos. En nuestro caso (Favret, E.A., Favret, G. С. y Malvárez, E., datos no publicados) tal condición es también debida a un solo par génico.

En éste, como en otros casos, es necesario conocer la acción de los genes incluidos en el término de macromutaciones si realmente se quieren aprovechar al máximo cuando se encuentran involucrados en los planes de mejoramiento (cf. Gustafsson, 1963).

RESISTENCIA A LAS ENFERMEDADES

El estado actual de los conocimientos nos dice que la resistencia específica a determinados organismos patógenos está regulado por un número elevado de genes. El sistema es poligénico pero análogamente al caso de los «erectoides» de cebada, .mencionados previamente, cada par génico es capaz por sí solo de otorgar una reacción de resistencia de utilidad agronómica.

En cebada, existen por lo menos 14 factores involucrados en la reacción al hongo Erysiphe graminis, agente causal del oídio. El hecho de que son factores específicos implica que en determinadas circunstancias el patógeno puede reunir en un solo genotipo los factores correspondientes de virulencia y, en consecuencia, parasitar a la variedad más resistente que el hombre pueda criar. Los genes del huésped mencionados han sido determinados en la colección de germoplasma natural.

Con la inducción artificial de mutaciones se logra repetir algunas de esas mutantes o formas alélicas que poseen distinta correspondencia con los genes de virulencia del patógeno. Aunque facilite al criador de plantas nuevas posibilidades de confrontarlo, el destino final parecería ser el mismo que con los alelos de origen espontáneo.

Sin embargo, ha sido posible detectar un gene en cebada capaz de controlar la reacción general al hongo, es decir, que en uno de sus estados alélicos otorga a su variedad portadora resistencia al mismo cualquiera sea la combinación genotípica. Se trata del gene designado ml-о, que aparece con una frecuencia de 1 X 10 "8 mut/progenie/R, esperada para el caso de un carácter monogénico. Diversos investigadores han inducido esta mutación en diferentes cultivares y en todos los casos el comportamiento ha sido similar (Favret, 1970).

58 FAVRET

CUADRO IV. SISTEMA PROPUESTO PARA CONTROLAR EL EFECTO DE UN PATOGENO CUANDO SE POSEEN GENES PARA REACCION ESPECIFICA Y NO ESPECIFICA, COMO EN EL MENCIONADO DE ml-о EN CEBADA PARA REACCION A Erysiphe graminis.Como este gene presenta secuelas indeseables desde el punto de vista agronómico, su combinación con un gene de reacción específica (por ej. Ml-m) obviaría el problema anterior y contribuiría a una resistencia permanente.

G e n e p a ra r e a c c ió n

e s p e c í f i c a

G e n e p a ra

r e a c c ió n

n o e s p e c í f i c a

R e a c c ió n a

la e n fe r m e d a d

P o s ib i lid a d e s de

a p a r ic ió n d e u na

r a z a p a tó g e n a

m l - 0 + M 1-+

A l e l o s u s c e p tib le A l e l o s u s c e p tib le S u s c e p tib le -

m l - o M 1- +

A l e l o r e s is te n te A l e l o s u s c e p tib le R e sisten te S í

m l - 0 + M l- m R e sisten te

A l e l o s u s c e p t ib le A l e l o re s is te n te p e r o d e fe c t u o s o N o

m l - o M l- m

A l e l o r e s is te n te A l e l o re s is te n te R e siste n te N o

Si la condición de no especificidad de la reacción al oídio se confirma, este gene pasará a ser un útil aliado para el fitotécnico en el desarrollo de nuevos cultivares. Si, por otra parte, esta variación puede encontrarse en otras especies homologas, las posibilidades de disminuir el efecto de las enfermedades aumentaría considerablemente.

A pesar de un estudio intenso del germoplasma natural durante estos últimos 25 años, tal clase de mutación no ha sido aún determinada. Los beneficios del empleo de la mutagenesis artificial, en este caso, son obvios.

El gene ml-о tiene «per se» alguna expresión detrimental en el organismo portador, pues induce a la formación de manchas necróticas y a una disminución en el crecimiento de algunos de sus órganos. En condiciones de ausencia del parásito, la línea original supera ligeramente el rendimiento de la línea mutante.

Este problema puede ser resuelto, por lo menos en las condiciones ensayadas en la Argentina, por la combinación de un gene para reacción específica al oídio con el mencionado gene ml-o . Por ser aquél epistático sobre éste, el efecto detrimental se elimina y la resistencia permanece en condición no específica (cuadro IV).

CONCLUSIONES

El empleo de la mutagénesis artificial para la obtención de macromutaciones es de evidente utilidad en el mejoramiento de las plantas. La dificultad de su aprovechamiento reside principalmente en la falta de conocimiento para discernir cuando estamos en presencia de una de ellas. La


única solución es acompañar a los estudios aplicados o mejor aún, anticiparse a los mismos tras el análisis de la acción génica.

La inducción artificial de mutaciones crea las mejores condiciones para obtener líneas isogénicas que permitan realizar esos estudios. El progreso en este campo rendirá excelentes dividendos en el mejoramiento genético de las plantas; o expresado en otros términos, el conocimiento de la planta que provee la inducción artificial de mutaciones es un requisito indispensable para el progreso de la fitotecnia.

Para países que requieren urgentemente realizar progresos fitotécnicos, los esfuerzos básicos que se realicen en ese campo parecen plenamente justificados en el futuro mediato.

En cambio, la repetición de tentativas de lograr pequeñas ventajas a través de la inducción de micromutaciones puede no competir con un eficiente progreso por el «cross-breeding» condicional. En cambio, esta situación puede ser distinta en países de alto desarrollo científico.

En las especies tropicales, la utilidad de tales estudios será posiblemente de mayor utilidad que para las especies genéticamente desarrolladas de las regiones templadas. Estamos, sin embargo, conscientes de que el manejo de plantas de distintas exigencias ambientales, que crean ecosistemas diferentes, que presentan estructuras y una dinámica poblacional distinta, son problemas que requieren otros estudios prioritarios, los cuales por otra parte, son necesarios cualesquiera sea el método empleado.

El uso de mutaciones inducidas en Latinoamérica ha tenido poca o muy poca trascendencia hasta el momento. Ello se ha debido, generalmente,I o) a que muy pocos centros de investigación se dedican a su inducción y estudio y 2o) al hecho de ser considerado un método «ciego» que lleva a resultados imprevisibles. Una nueva reinterpretación de las mutaciones, medida más como un factor para analizar la actividad fisiológica de la planta que a una mera selección de formas que difieren de la normal, permitirá crear las condiciones necesarias para considerar a la mutagenesis artificial como un método no sólo auxiliar sino también necesario para el progreso genético de las plantas.

BIBLIOGRAFIA

S W A M IN A T H A N , M . S . , « A c o m p a r is o n o f m u ta tio n in d u c tio n in d ip lo id s a n d p o l y p l o id s » , T h e U se o f

In d u ce d M u ta tio n s in P la n t B re e d in g ( R e p .F A O / I A E A T e c h .M e e t i n g , R o m e , 1 9 6 4 ) , P e rg a m o n Press, O x fo rd

( 1 9 6 5 ) 6 1 9 .

G U S T A F S S O N , A . , H A G B E R G , A . , L U N D Q V IS T , U . , T h e in d u c tio n o f e a r ly m u ta n ts in B onu s b a r le y , H e re d ita s

4 6 ( 19 6 0 ) 6 7 5 .

F A V R E T , E . A . , F R E C H A , J . H . , A l le l i s m te s t o f g e n e s fo r e a r lin e s s , B a r le y N e w s l . 10 ( 19 6 6 ) 1 2 1 .

PER SSO N , G . , H A G B E R G , A . , In d u c e d v a r ia t i o n in a q u a n t i ta t iv e c h a r a c t e r in b a r l e y . M o r p h o lo g y and

c y t o g e n e t i c s o f e r e c t o id e s m u ta n ts . H e r e d ita s ^ 1 ( 1 9 6 9 ) 1 1 5 .

S W A M IN A T H A N , M . S . , M u ta t io n b r e e d in g , P r o c .X I I I n t .C o n g r . G e n e t ., J ap a n 3 ( 19 6 8 ) 3 2 .

F A V R E T , E . A . , R Y A N , G . S . , M A L V A R E Z , E . M . , « M u t a c io n e s in d u c id a s q u e a f e c t a n a l c r e c im ie n t o i n i c ia l

de la c e b a d a » . In d u c e d M u ta tio n s in P la n ts ( A c t a s S im p . P u llm a n , 1 9 6 9 ), O IE A , V ie n a ( 19 6 9 ) 10 9 .

G U S T A F S S O N , A . , « M u t a t io n s an d th e c o n c e p t o f v i a b i l i t y » , R e c e n t P la n t B r e e d in g R e se a rc h , S v a lo f 1 9 4 6 -

1 9 6 1 , A lm q v is t an d W ik s e l l , S t o c k h o lm ( 1 9 6 3 ) .

F A V R E T , E . A . , « D i f f e r e n t c a t e g o r ie s o f m u ta tio n s fo r d is e a s e r e a c t io n in th e h o st o r g a n is m » , M u ta tio n

B re e d in g fo r D is e a s e R e s is ta n c e ( P r o c .P a n e l , V ie n n a , 1 9 7 0 ) , IA E A , V ie n n a ( 1 9 7 1 ) 1 0 7 .

60 FAVRET

В. SIGURBJORNSSON: I would like to refer not only to the excellentpaper just presented but also to the previous paper on breeding in Latin America by Dr. Brieger, in which he considered the relative merits of mutation breeding versus conventional breeding. In order to facilitate such comparison, it would be useful to know the following: a) How many plantbreeders in Latin America are using mutation breeding exclusively in their programs, and how many institutes base their plant breeding programs on induced mutations? b) How many plant breeders in Latin America are using conventional breeding methods exclusively in their programs, and how many institutes base their plant breeding programs on conventional methods?

E. A. FAVRET: That is an important and interesting question. Thenumber of plant breeders in the second group is much higher, as has been shown by Dr. Grobman. On the contrary much fewer plant breeders are using exclusively artificial mutagenesis. I guess less than ten. InCastelarwe are using both methods simultaneously.

A. GROBMAN: Complementing Mr. Favret's estimation of the number of workers in mutation breeding in Latin America, I agree that perhaps there are no more than half a dozen researchers dedicated full-time to mutation breeding. There are others, however, who at one time or another have used mutation induction techniques in their breeding programs with varying degrees of success, probably mostly inconclusive. It might be argued that the lack of comparable results from mutation induction techniques, to the standard breeding technique results, is due to the limited number of workers employing the former technique. I must restate, however, that the tremendous array of variability which exists in plants originating in Latin America, has and continues to be a strong motivating factor in the selection by Latin American plant breeders of methods that make immediate use of the naturally-occurring variability.

E.A. FAVRET: It is difficult to assess the success or failure of thetrials carried out thus far in the field of artificial mutagenesis. It is my impression that some oí this work has not been performed correctly.

It would also be necessary to evaluate these same parameters in terms of conventional improvement. This is obviously very difficult. Success or failure are terms which are very difficult to define.

G. DE ALBA: The Mexican institute responsible for research onplant improvement through induced mutations is the Plant Genetics Department of the Comisión Nacional de Energía Nuclear. It ha's three full-time experts. Three professors in the Department of Agronomy at the Monterrey Technological Institute are working part-time on a study of sorghum, wheat and sesame populations irradiated with S0Co gamma-rays.

D I SC US S I ON

POSSIBILITIES AND IMPLICATIONS OF M UTATION BREEDING IN JAMAICA

C.A. PANTONThe Department of Botany, University of the West Indies,Mona, Kingston

T. MENENDEZBanana Breeding Research Scheme, Banana Board,Kingston, Jamaica, W. I.

Abstract-Resumen

P O S S IB IL IT IE S A N D IM P L IC A T IO N S O F M U T A T I O N BREEDING IN J A M A I C A .

T h e n a tu re an d o r g a n iz a t io n o f a g r ic u lt u r a l r e s e a r c h in J a m a ic a is o u t l in e d an d t h e r e l e v a n c e o f

m u ta g e n e s is is d isc u sse d w ith r e f e r e n c e t o b a n a n a s , c o c o n u ts , s u g a r c a n e , a n d s o y b e a n s . A s u p p le m e n ta r y

r o le fo r m u ta t io n b r e e d in g is p ro p o se d . It is s u g g e s te d th a t r e c e n t tre n d s in p la n t c u ltu r e te c h n iq u e s c o u ld

o p e n u p n e w p o s s ib il it ie s fo r in c r e a s in g t h e s c o p e an d u se fu ln e ss o f in d u c e d m u ta tio n s . M e n t io n is m a d e

o f e x is t in g f a c i l i t i e s fo r m u ta t io n w o rk in J a m a ic a .

P O S IB IL ID A D E S Y C O N S E C U E N C IA S D E L M E J O R A M IE N T O D E E SP E C IE S POR M U T A C IO N EN J A M A I C A .

S e d e s c r ib e n a g ra n d e s rasgo s l a n a t u r a le z a y l a o r g a n iz a c ió n d e la s in v e s t ig a c io n e s a g r o n ó m ic a s

e n J a m a ic a , y se e x a m i n a l a im p o r t a n c ia d e l a m u ta g e n e s is e n r e l a c ió n c o n lo s p lá ta n o s , s o ja , c a ñ a d e

a z ú c a r y c o c o s . S e p ro p o n e u n a fu n c ió n s u p le m e n t a r ia p a ta e l m e jo r a m ie n t o d e e s p e c i e s p o r m u t a c ió n .

S e s u g ie r e q u e la s r e c ie n t e s t e n d e n c ia s e n la s t é c n i c a s d e c u l t iv o p o d r ía n b r in d a r n u e v a s p o s ib ilid a d e s d e

a u m e n t a r e l a l c a n c e y l a u t i l id a d d e la s m u ta c io n e s in d u c id a s . S e m e n c io n a n la s in s ta la c io n e s e x is t e n t e s

en J a m a ic a p a ra t r a b a jo s d e m u ta c ió n .

1. INTRODUCTION

The responsibility of increasing agricultural output to support growing populations with adequate food and proper levels of nutrition is one to which most developing nations are committed. The efforts being made to improve the quantity and quality of food attest to the dedication with which these nations are tackling this tremendous task. The way in which science and technology has been influencing this area of development provides ample evidence that developing nations are taking advantage of the scientific age.

Our discussion here is confined to Jamaica where on the whole we share the common experience of more than proportional demand for food increases due to rising incomes and changes in consumption habits as our population becomes increasingly urbanized.

In giving its wholehearted support to agricultural research, our Government underlines the importance to the nation and its development of increasing agricultural output. The agencies in Jamaica engaged in agricultural research include the Ministry of Agriculture and Fisheries, the University of the West Indies, certain commodity associations such as the Banana Board, the Coconut Industry Board and the Sugar Manufacturers Association, and private organizations like the Pioneer Hybrid Seed Corn

61

62 PANTON and MENENDEZ

Company with headquarters in the United States. While the Ministry of Agriculture and Fisheries undertakes a broad program covering chiefly vegetable, tree, forage, and tuberous root crops, the commodity associations are strictly concerned with the staple or export crops. The area of activity of the University is limited at present to special programs of a more regional nature exemplified by our current grain legume program which includes work on soybeans.

The arrangements for the financing of these programs range from direct government subventions in the case of the Ministry of Agriculture and Fisheries, to funds derived from the marketing of staple crops as in the case of the commodity associations. Assistance is also given by the Food and Agriculture Organization of the United Nations and the British Government. The banana breeding research scheme is supported by Jamaica, the Windward Islands and Trinidad. The work of the University is supported by the Regional Research Council, a Commonwealth Caribbean Organization, the Rockefeller Foundation and contributions from private sources.

Though the fragmentation of agricultural research may raise many questions regarding efficient utilization of our limited resources, there is a high degree of co-operation between the various organizations concerned that shows we are accepting the challenge of new and exciting discoveries in this field.

While crop improvement through plant breeding has been carried out for a number of years largely in the staple crops, such as bananas, sugar cane, coconuts and more recently certain grain legumes, its application to vegetables, our wide assortment of tuberous food crops, forages and other tree crops has been neglected.

It is, of course, understandable that the ability to earn foreign exchange, as well as to give employment to a large number of people, are factors which have greatly influenced the emphasis on the staples in which only conventional breeding has been practised up to now. In any program of plant breeding, however, one is always faced with certain basic problems depending on the nature of the crop. Slow release of variability, disintegration of gene blocks which would otherwise confer advantage if allowed to remain intact, and chromosome irregularities are but a few of these problems.

The alternative is the use of induced mutation. In the sense that this method has been shown to speed up variability and to facilitate point mutations in the genetic material without necessarily destroying otherwise desirable gene clusters, there is good reason to expect it to be a useful tool in our agriculture. In considering its possible application to agricultural research, we will illustrate by reference to only four crops but we infer that it could be extended to a number of others. The four crops are bananas, coconuts, sugar cane and soybeans.

2. APPLICATIONS OF MUTATION BREEDING

2. 1. Bananas

In banana breeding, the main problem is to derive suitable seedless dessert bananas from essentially seedless cultivars and seeded wild types (Simmonds (1966), Shepherd (1968) ). The taxonomy and origins of cultivated bananas have been described by Simmonds and Shepherd (1955).

M U TATIO N BREEDING IN JAMAICA 63

Success in the synthesis of either triploid or tetraploid commercial bananas depends on breeding suitable diploid parents which will contribute about a third or a quarter of the final genotypes. The variation available in diploid cultivars and wild types in Musa acuminata has only partially been exploited. The gene pool is represented in collections in Jamaica but much remains inaccessible in natural centres in the Far East.

Spontaneous mutations in bananas of M.acuminata origin have arisen several times to effect in particular a useful and seemingly dominant change to shorter stature. For example, the triploid Cavendish group now ranges from the Dwarf (Chinese) to Jamaican Lacatan, with apparently reversible mutation between Lacatan and the shorter Robusta.Gros Michel has mutated to the short clones Highgate and Cocos. Ratoons of the new tetraploid bananas bred from Highgate are intermediate in height but further reduction in stature may be desirable.

There are prospects for introducing dwarfing genes from the occasional pollen grains found in Cavendish bananas. However, if it is inferred that the tendency to dwarf mutation is also likely in diploids, then there seems a good case for the experimental induction of mutation in chosen diploids of complex origin or for their use as specific recurrent parents in existing breeding programs.

Two considerations may mitigate against the value of inducing mutation in bananas. To be useful, mutation in a diploid must be dominant if it is to be expressed in a foreseeable triploid or tetraploid commercial derivative. Irradiation of pieces of com from existing cultivars may lead to chimerical confusion likely to reduce the practical value of this method.

It can be seen, however, that there are several features that suggest the induction of point mutations would be a suitable technique. What is assumed to be a dominant and simply inherited mutation to a dwarf habit occurs naturally. Fertility barriers may hinder the incorporation of characters in existing genotypes. Some diploid breeding lines are highly developed and one might be reluctant to disturb their superior combination of genes by the lengthy process of hybridization. Finally, the commercial banana must be, and the diploid banana can be propagated vegetatively. Eventually, free cell culture and the production of embryos and plants by aggregation and culture of mutated cells may open up new possibilities in this area.

Other requirements in breeding bananas relate to resistance to specific diseases to which conventional breeding techniques would seem to be providing an answer, and problems of fruit quality, some of which have only recently been realized and are not yet fully understood.

The chemical mutagenesis of diploid bananas is already being investigated in Jamaica in the Banana Breeding Research Scheme. This relatively small program could well be expanded if and when new techniques become available, or if results are encouraging. Nevertheless, in a program of mutagenesis with the definite objective of inducing short stature, other useful mutants affecting bunch characteristics or resistance to disease might well emerge and would not be overlooked.

2. 2. Coconuts

Problems in coconut breeding relate directly to the large size of the plant and its seeds, and to the period of growth necessary between

64 PANTON and MENENDEZ

generations. The natural variation available from recent collections is likely to satisfy breeding in Jamaica for many years. Breeding at the Research Department of the Coconut Industry Board aims to provide increased yield of copra and oil from plants resistant to Lethal Yellowing Disease (Whitehead (1968)). It would seem unnecessary to pretend a need for induced mutation breeding with existing techniques.

This question of techniques, however, raises a very important consideration that in the future may well have far reaching impact on the value of mutagenesis in plant breeding. The production of free cell cultures for the rapid multiplication of good genotypes is becoming a practicality and is likely to revolutionize the results of breeding methods involving intercrossing and recombination in such large plantation crops as coconuts and oil palms, which are planted at low density and in which wide variations in production are found as a result of heterozygosity. The Coconut Industry Board is already engaged in this area of research with British technical assistance.

2. 3. Sugar cane

At the regional centre for sugar cane breeding in Barbados, mutation induction has been directed at the suppression of flowering in varieties of high yielding potential. The inverse relationship between flowering and the yield of cane have prompted the use of irradiation in sugar research. Rao (1969) irradiated buds of two varieties of sugar cane from which non-flowering and shy-flowering sub-clone mutants were selected. He found significant increases in weight/cane in one variety with one non-flowering mutant showing good yield, and seemingly promising. Earlier, Rao et al. (1966) reported the induction of red rot resistant mutants by gamma irradiation in a susceptible variety. The effect of mutation on genetic and physiological characters as shown here emphasize the importance of this technique to crop improvement in this species. There will probably be room for work of this nature, if or when sugar cane breeding is extended to Jamaica.

2.4. Soybeans

Investigations in soybeans have been in progress since 1966. The program which originated in Trinidad was extended to Jamaica where emphasis has been placed on selection for day neutrality yield, high oil and protein content (Radley (1968, 1969)). Combining-ability studies were made on material with potential for these characteristics following selection. Certain hybrids have exceeded their parents in the characteristics mentioned above.

Having regard to the possibility of significantly increasing the overall protein content or elevating the levels of limiting amino acids in seed protein as has been shown in maize, we have now chosen to supplement our work by mutation breeding. This has been made possible through the kind co-operation of Dr. K. S. Koo at the Puerto Rico Nuclear Centre.Material consisting of seeds of four parents and four of their F3 hybrids was subjected to 15, 20 and 25 kR gamma irradiation. This irradiated material has recently been planted out and results are therefore not yet available.

M U TATIO N BREEDING IN JAM AICA 65

The use of chemical mutagens is also being considered, but this will depend on obtaining certain equipment and facilities for which some assistance is now being sought from the International Atomic Energy Agency.

3. DISCUSSION

It follows from these considerations that there is a place for mutation breeding in Jamaica. Where the obvious need lies, work has already begun. Reference has been made to the use of free cell culture for the multiplication of certain heterozygous genotypes which would open up new vistas in mutagenesis.

Furthermore, exciting possibilities arise from current research into the production from pollen tube nuclei of haploid free cell cultures (Sunderland and Wicks (1970)), which can be induced to form haploid plants. Because it is possible to fuse isolated protoplasts in culture (Power et al. (1970)), one can envisage the formation of homozygous diploids resulting from fusion or doubling of these haploid cells. Recessive mutants could be uncovered without recourse to an orthodox Mj generation leading to an M2, and these could be multiplied quickly.

At present, facilities available in Jamaica for mutation studies are limited to one cobalt source used for therapy and located in one of our large hospitals; a number of X-ray machines located in hospitals and used chiefly for diagnostic treatment and a number of caesium sources restricted to industrial use. A limited amount of work could be done with the cobalt source but its location is inconvenient for prolonged treatment and frequent use.

Although equipment and other facilities are for the most part lacking, a reasonable amount of personnel trained in the handling of mutagenic agents and in plant breeding are available in Jamaica. Based on the examples given, it is suggested that mutation breeding has an important supplementary role in agricultural research in this country.

REFERENCES

POW ER, J . B . , C U M M IN S , S . E . , C O C K I N G , E. C . , 1 9 7 0 , F u s io n o f is o la t e d p la n t p ro to p la sts , N a tu re 2 2 5 : 1 0 1 6 .

R A D L E Y , R . W . , 19 6 8 , T h e P ro sp e cts fo r S o y b e a n P ro d u c t io n in T r in id a d an d T o b a g o , A g r ie . S o c . T r in id a d

an d T o b a g o , P a p e r N o . 9 0 3.

R A D L E Y , R. W . , 19 6 9 , S o y b e a n — A n e w c r o p fo r th e C a r ib b e a n , C a r ib b . F a r m in g 1 : 7 .

R A O , P. S . , 1 9 6 9 , P r e l im in a r y D a t a o f R a d ia t io n - in d u c e d M u ta n ts in S u g a r C a n e , R ep . W . I . S u g a r A ss. T e c h .

M e e t in g , T r in id a d , 1 9 6 9 .

R A O , T . J . , S R IN IV A S A N , K . , A L E X A N D E R , K . C . , 1 9 6 6 , A red ro t r e s is ta n t m u ta n t o f su g a r c a n e in d u c e d

b y g a m m a ir r a d ia t io n , P ro c . In d ia n A c a d . S c i . 1 6 4 : 2 2 4 .

S IM M O N D S , N . W . , SH EPH ERD , K . , 1 9 5 5 , T h e t a x o n o m y an d o r ig in s o f th e c u l t iv a t e d b a n a n a s , J. L in n . S o c .

B o ta n y , 5 5 3 5 9 : 3 0 2 .

SH EPH ERD , K . , 19 6 8 , B a n a n a b r e e d in g in th e W est In d ie s , P . A . N . S . , S e c t . B . 1 4 : 3 7 0 .

S IM M O N D S , N . W . t 1 9 6 6 , B a n a n a s, 2nd E d n , L o n g m a n s G r e e n , L o n d o n .

S U N D E R L A N D , N . . W I C K S , F . M . , 19 7 0 , C u l t iv a t io n o f h a p lo id p la n ts fro m t o b a c c o p o l le n , N a tu re 2 2 4 : 1 2 2 7 .

W H IT E H E A D , R. A . , 19 6 8 . S e l e c t i n g an d b r e e d in g c o c o n u t p a lm s r e s is ta n t t o l e t h a l y e l l o w i n g d is e a s e ,E u p h y t ic a 1 7 : 8 1 .

6 6 PANTON and MENENDEZ

DISCUSSION

M. S. SWAMINATHAN: I was interested in your suggestion that good genotypes of coconuts can be multiplied by cell cultures. Has this already been done successfully and if so, can you give some details concerning the tissue and technique used?

C. A. PANTON: The technique is currently being developed by a group of British scientists at Wye College, London.

INDUCED SEED-COAT COLOUR MUTATIONS IN BEANS AND THEIR SIGNIFICANCE FOR BEAN IMPROVEMENT*

C.C. MOHInter-American Institute of Agricultural Sciences,Turrialba, Costa Rica

Abstract-Resumen

IN D U C E D S E E D -C O A T C O L O U R M U T A T IO N S IN B E A N S A N D T H E IR S IG N IF IC A N C E F O R BEAN IM P R O V E M E N T .

T h e c o m m o n d ry b e a n ( P h a s e o lu s . v u lg a r is L .) is a b a s ic fo o d c r o p in m a n y L a tin A m e r i c a n c o u n tr ie s and

se rv e s as a m a jo r p r o te in s o u rc e in th e d a i ly d ie t o f th e p e o p le . In h a b ita n ts o f p a r t ic u la r a r e a s , h o w e v e r ,

p r e fe r o n ly b e a n s o f c e r t a in c o lo u r s , and in m o st a r e a s , r e d , y e l l o w an d w h it e b e a n s a r e m o r e c o n s u m e d . It

is k n o w n th a t m a n y b l a c k b e a n v a r ie t ie s a r e su p erio r in d is e a s e r e s is t a n c e and y i e l d p r o d u c t io n , b u t b e c a u s e

o f t h e ir s e e d - c o a t c o lo u r , t h e ir c u l t iv a t io n is l im it e d in m a n y a r e a s w h e r e th e y h a v e l i t t l e m a r k e t v a l u e .

By t r e a t in g t h e se e d s o f b l a c k b e a n v a r ie t ie s w ith e t h y l m e th a n e s u lp h o n a te or g a m m a - r a y s , i t is p o s s ib le to

in d u c e m u ta tio n s o f b ro w n , y e l l o w o r w h it e s e e d - c o a t c o lo u r . T h e s c r e e n in g t e c h n iq u e in w h ic h t h e s e e d - c o a t

c o lo u r m u ta n ts a r e c o r r e la t e d w ith g r e e n h y p o c o t y l c o lo u r p e r m its th e is o la t io n o f th e p o t e n t ia l m u ta n ts a t a

v e r y e a r ly s t a g e o f s e e d lin g d e v e lo p m e n t . T h u s , m u ta tio n b r e e d in g a p p e a rs to b e an e f f i c ie n t m e th o d fo r

im p ro v in g t h e s e e d - c o a t c o lo u r s in b e a n s . G e n e t ic stu d ie s o f s o m e m u ta n ts sho w th a t t h e in d u c e d s e e d - c o a t

c o lo u r c h a r a c te r s a r e m o n o g e n ic r e c e s s iv e s .

M U T A C IO N E S IN D U C ID A S EN EL C O L O R D E L A PIEL D E SE M IL L A S D E A L U B IA S Y SU IM P O R T A N C IA P A R A

EL M E J O R A M IE N T O D E L A S M IS M A S .

L a a lu b ia c o m iin ( P h a se o lu s v u lg a r is L .) e s un a l im e n t o b á s ic o q u e se c u l t i v a e n m u ch o s p a ís e s d e la

A m e r i c a L a t in a , d o n d e c o n s t it u y e u n a d e la s f u e n te s p r in c ip a le s d e p r o te ín a s e n l a a l im e n t a c ió n c o t id ia n a d e

l a p o b la c i ó n . N o o b s ta n te , e n c a d a r e g ió n lo s c o n s u m id o r e s p r e f ie r e n la s a lu b ia s d e d e te r m in a d o s c o lo r e s .

En l a m a y o r ía d e la s r e g io n e s , la s q u e m a s se c o n s u m e n son la s a lu b ia s p in ta s , la s a m a r i l la s y la s b la n c a s .

S e s a b e q u e m u c h a s v a r ie d a d e s d e a lu b ia s n e g ra s so n s u p e r io re s p o r su r e s is t e n c ia a la s e n fe r m e d a d e s y su

r e n d im ie n t o . D e b id o a l c o lo r d e l a c u b ie r t a s e m in a l , su c u l t iv o e s l im it a d o e n m u c h a s z o n a s e n q u e la s

a lu b ia s n e g r a s t ie n e n e s c a s a a c e p t a c ió n . T r a ta n d o la s s e m il la s d e la s v a r ie d a d e s d e a lu b ia s n e g ra s c o n m e t a n o -

s u lfo n a to d e e t i l o o c o n ra y o s g a m m a , e s p o s ib le in d u c ir m u ta c io n e s p a ra o b te n e r s e m il la s c o n c u b ie r t a m a rró n ,

a m a r i l la o b l a n c a . U t i l iz a n d o l a t é c n i c a d e s e le c c i ó n se g ú n l a c u a l es to s m u ta n te s se c o r r e la c io n a n c o n e l

v e r d e d e l h i p o c ó t i lo , s e p u e d e n a is la r lo s m u ta n te s p o t e n c ia le s e n u n a f a s e m u y t e m p r a n a d e l d e s a r r o llo d e la s

p lá n t u la s . Por t a n t o , l a s e le c c i ó n p o r m u ta c ió n p a r e c e ser u n m é to d o e f i c a z p a ra m e jo ra r e l c o lo r d e l a

c u b ie r ta s e m in a l e n la s a lu b ia s . Los e s tu d io s g e n é t i c o s d e a lg u n o s m u ta n te s h a n p u e sto d e m a n if ie s t o q u e lo s

c a r a c t e r e s in d u c id o s ( c o lo r d e l a c u b ie r t a s e m in a l) so n r e c e s iv o s m o n o g é n ic o s .

1. INTRODUCTION

The dry bean (Phaseolus vulgaris L. ) is a basic food crop in many Latin American countries and provides a major protein source in the daily diet of the people. Inhabitants of particular areas, however, have a specific preference for certain colours of bean, and in general, red, yellow, or white beans are more consumed than are the black beans. In some areas, white beans have a much higher market price than other colours. In Costa Bica, for example, white beans are almost twice the price of the black or red beans. Therefore, seed-coat colour is an important agronomic character that determines the marketability of a dry bean variety.

* R e s e a rc h su p p o rte d b y th e U . S . A t o m ic E n e rg y C o m m is s io n u n d er c o n t r a c t A T ( 3 0 - l ) - 2 0 4 3 .

P u b lic a t io n N o . N Y O - 2 0 4 3 - 2 2 6 .

67

6 8 МОН

It is known that many black bean varieties are superior in disease resistance and yielding capacity to other coloured bean varieties [1], but because of their seed-coat colour, their cultivation is very restricted in the areas where they have little mark et value. Previous experimental results indicated that the genetic factors governing the seed-coat colours in beans are mutable [2, 3]. This finding provides great promise for the mutation breeding of the seed-coat colour of beans. Coupled with the screening technique in which the potential seed-coat mutants can be isolated at the seedling stage, the induced mutation method appears to be practicable for seed-coat colour breeding.

2. MATERIALS AND METHODS

Three black bean varieties, namely San Fernando, Turrialba-1, and Porrillo, were used as the experimental materials. The seeds used for the present studies were from single plant selections and selfed for four or more generations to ensure genetic purity. The newly harvested seeds were dried in an oven at about 34°С for two or more weeks before the experiments were performed. The moisture content of seeds varied from 8 to 10% depending upon the variety.

The mutagens used for' the treatments were ethyl methane sulphonate (EMS) and gammaTays. For the EMS treatments, seeds were soaked in unbuffered EMS aqueous solution of 0. 04 to 0. 08M concentration for 6 to 12 h at 20°C. For gamma irradiation, the seeds were irradiated with an acute dose of gamma-rays from a 60Co source at a dose rate of 900 R/min. After the treatments, the seeds were grown in individual pots in the greenhouse until maturity.

The Mi plants were individually harvested and the seeds were sown in the soil box in the greenhouse for screening the induced seed-coat colour mutants.

3. SCREENING TECHNIQUE

The success of the induced mutation method for plant breeding is largely conditioned by the following two factors: 1) whether a plant species has the genetic potential to mutate to the desirable traits, and 2) ease of detecting the desirable mutants. For the first, knowledge of the genetic constitution of the species and information on the mutability of the desirable traits are essential. For the second, an efficient screening technique for isolating the induced desirable mutants is helpful. It is known that the seed-coat of beans has a wide range of colour variations in the natural population, and the mutability of the colour factors is known in some cas es. During the past year, a simple screening technique has been developed which makes possible the isolation of the induced mutants with relative ease.

The development of the screening technique for the seed colours was based on the observation of the plant characters of several hundred bean varieties collected in Latin America. Based on the observed results, there is a correlation between the seed-coat colour and the hypocotyl colour of the seedling [4]. Essentially, the seed colours can be divided into four principal groups: black, bayo (from deep brown to light yellow), red, and white. Of 93 black seed varieties studied, all produced red hypocotyls;

COLOUR M UTATIONS IN BEANS 69

TABLE I. CORRELATION BETWEEN SEED-COAT AND HYPOCOTYL COLOURS OF 271 BEAN VARIETIES

S e e d - c o a t c o lo u rH y p o c o t y l c o lo u r

R ed G re e nT o t a l v a r ie t ie s

B la c k 93 0 93

B a y o 38 2 1 59

Red 54 2 2 76

W h ite 0 4 3 4 3

T o t a l 18 5 87 2 7 1

of 43 white seed varieties observed, all produced green hypoeotyls.The bayo or red varieties could produce either red or green hypocotyls (Table I). Although these results did not show a perfect correlation, they provided a criterion to detect the possible seed-coat colour mutants induced from a black variety.

Under normal greenhouse conditions the bean seedlings emerge within a week after sowing. A green hypocotyl mutant can readily be identified from its red parental type. In this way thousands of unwanted seedlings can be discarded within a week, and the isolated mutants transplanted for further growth observation. Those considered as deleterious types can be quickly discarded and only those with normal appearance maintained to maturity for further seed colour identification.

4. RESULTS AND DISCUSSION

4.1. Induced seed-coat colour mutants

The results on the seed-coat colour mutants induced by EMS and gamma- rays in three black bean varieties are presented in Table II. From a total of 1524 Mi plants from various treatments tested, 20 independently induced mutants were found. The seed-coat colour mutants so far obtained varied from white, yellow, to various degrees of brown. All these mutants were associated with a change of hypocotyl colour from red to green. Most of the induced mutants had a morphology and growth habit similar to their parents, except that a few produced smaller but more abundant seeds. However, all the mutants produced white flowers instead of the red flowers of their parents.

Since most bean varieties have a relatively short life-cycle of three months, the time required from the start of the treatment to the achievement of the seed-coat colour mutants is six to nine months (two to three life-cycles). For this particular breeding objective, it is of advantage to use the induced mutation method. A backcrossing method probably would take many more generations of laborious bean crossing in order to achieve the same goal.

It is known that in a world bean collection, black seed varieties of high yield and disease resistance are abundant. The possibility of converting the strains unacceptable because of their seed colour into cultivars is worth exploring.

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C O L O U R M U T A T IO N S IN BEAN S

4.2. Genetics of seed-coat colour mutants

71

Genetic factors affecting the seed-coat colours of beans can be divided into three categories: 1) pigmentation factor, P; 2) complementary colour factors; and 3) modifying factors. The P factor by itself produces no colour in the seed-coat, but other complementary colour factors must depend upon the presence of the dominant P factor in order to express their colours. Once the dominant P is present, the complementary factors either produce a definite colour by themselves or interact to produce a wide range of colours. Modifying factors do not produce colours, but only influence the colour produced by the other factors.

The white seed mutants induced in the present experiments can be due to 1) absence of the dominant P factor, or 2) absence of all complementary colour factors. It is likely that the white seed mutants were due to the change from the dominant P to its recessive, or merely due to the result of a P deletion. Genetic results on the production of the Fx black seed hybrids from crosses between the white seed mutants and the P white seed testers lent support to the above explanation (Table III).

TABLE III. F2 SEED-COAT COLOUR OF HYBRIDS BETWEEN INDUCED WHITE SEED MUTANTS AND WHITE SEED GENETIC TESTERSa

9

C ross

X a *N o . o f Fi h y b rid s F i s e e d - c o a t c o lo u r

W h it e - 1 x L a m p r e c h t 2 1 4 14 a l l b l a c k

W h it e - 1 x S m it h 2 1 5 1 2 0 a l l b l a c k

S m it h 2 1 5 1 x W h it e -2 4 a l l b l a c k

a B oth L a m p r e c h t 2 1 4 an d S m it h 2 1 5 1 a r e w h it e s e e d g e n e t i c te s te rs w h ic h c a r r y th e d o m in a n t

p ig m e n ta t io n f a c t o r , P , b u t h a v e n o o th e r c o m p le m e n t a r y c o lo u r f a c t o r s .

The bayo seed-coat mutants were probably due to the change in complementary colour factors or modifying factors. Unfortunately, there are no genetic markers available at present to test precisely which genetic loci had mutated.

The inheritance of the induced seed-coat colours of some mutants (two white and two bayo) has been studied by crossing with their original black seed parents. Genetic results showed that the induced characters are recessive and inherited in a simple Mendelian manner.

REFERENCES

[ 1 ] Y G L E S IA S , G . E . , E stu d io so b re e l e f e c t o d e l a d e n s id a d d e s ie m b r a , h a b ito d e c r e c im ie n t o , c o lo r y

t a m a ñ o d e f r i jo l e n lo s e n s a y o s d e v a r ie d a d e s , P u b lic a c ió n M is c e lá n e a N o . 2 2 , I I C A , T u r r ia lb a ,

C o s t a R ic a (19 6 4 ) 3 9 .

[ 2 ] S W A M P , V . , G IL L , H . S . , X - r a y in d u c e d m u ta tio n s in F r e n c h b e a n , I n d ia n J .G e n e t . 28 (19 6 8 ) 4 4 .

[ 3 ] M O H , C . C . , S e e d - c o a t c o lo r c h a n g e s in d u c e d b y e t h y l m e t h a n e s u lfo n a t e in th e c o m m o n b e a n

( P h a se o lu s v u lg a r is L . ) , M u ta t io n R es. 7 (19 6 9 ) 4 6 9 .

[ 4 ] М О Н , C . C . , A L A N , J . J . , U n p u b lis h e d d a ta .

72 МОН

O.P. KAMRA: What is the frequency of mutation induction of this type? Is there any change in amino acid profile in these mutants?

С. С. MOH: When you treat 200 to 300 seeds of the black bean variety (San Fernando) that we used with EMS of 0. 04 to 0. 06 M, you would expect to have a seed coat colour mutant obtained from the M2 or M2 selfed progenies. We have no analysis of the amino acids in these induced mutants.

A. GROBMAN: Black beans are known to have an enzymatic inhibitor. When fed raw to experimental mice they may produce death within a very short time. I would like to comment that it would be of extreme importance to study the enzymatic inhibition pattern of some of your seed coat colour mutants in regard to this character. Since black beans are preferred in some areas, black seed-coat colour mutants with low enzymatic inhibition would be of great interest.

C.C. MOH: Thank you for your comment. I agree that it is of importance and interest to study whether this enzymatic inhibitor exists in the seed-coat colour mutants. I ’shall ask INCAP of Guatemala to see whether such a comparative study could be made for us.

H. HANSEL: I would like to ask you whether the black bean variety you used in your experiment had a special fungus resistance, and whether the induced white or yellow mutants combine this resistance with the light colour?

C.C. MOH: The black bean varieties used in our experiments are high-yielding strains in Central America and have a good resistance to general diseases in this area. Dr. R. Gamez of the University of Costa Rica tested for us the resistance to two virus diseases of a white mutant (mutated from the black variety, San Fernando), and found that the resistance is as good as the original black parent.

D I SC US S I ON

INDUCTION OF MUTATIONS

COMPARATIVE GENETIC EFFECTS OF DIFFERENT PHYSICAL MUTAGENS IN HIGHER PLANTS*

H.H. SMITHDepartment of Biology, Brookhaven National Laboratory,Upton, N.Y., United States of America

Abstract-Resumen

C O M P A R A T I V E G E N E T IC E F F E C T S O F D IFFER E N T P H Y S IC A L M U T A G E N S IN H IG H ER P L A N T S .

T h e r e l a t i v e g e n e t i c e f f e c t s in h ig h e r p la n ts o f p h y s ic a l m u ta g e n s th a t p r o d u c e d i f fe r e n t s p a t ia l d is t r i

b u tio n s o f e n e r g y in ir r a d ia te d tissu es a r e r e v ie w e d . T h e s e a r e m a i n ly fa s t ( " f is s io n " ) neutrons,, m o n o -

e n e r g e t ic a n d m ix e d s p e c tr u m e n e r g y n e u tro n s , an d r a d ia t io n s p r o d u c e d b y a c c e le r a t e d h e a v y io n s .

C o m p a r is o n s a r e m a d e w ith s p a rs e ly io n i z in g X - an d y - r a y s . T h e a v e r a g e g e n e t i c e f fe c t iv e n e s s fro m a l l

re s u lts c o m b in e d w as g r e a te s t (RBE= 7 1 . 5) fo r ra d ia t io n s o f d o s e a v e r a g e l in e a r e n e r g y t ra n s fe r (L E T ) o f 53

to 1 7 5 k e V / p m . T h e RBE w a s r e d u c e d to 13 fo r r a d ia t io n s o f lo w e r ( 7 - 2 3 k eV //jm ) o r h ig h e r (> 1 7 5 k e V / / jm ) L E T .

T r a c k s e g m e n t a n a ly s e s o f c r o s s - s e c t io n v a r ia t io n s w ith L E T in d ic a t e t h a t t h e t a r g e t s ite s fo r in d u c e d g e n e t i c

c h a n g e o r d a m a g e h a v e e f f e c t i v e th ic k n e s s e s o f th e o rd e r o f s i z e o f D N A m o l e c u le s . T h e r e is l i t t l e e v id e n c e

in h ig h e r p la n ts fo r m u t a g e n ic s p e c i f i c i t y a m o n g p h y s ic a l m u ta g e n s . C o n t r o l o v e r d i f f e r e n t ia l r e c o v e r y o f

m u ta tio n s fro m h ig h L E T r a d ia t io n s r e m a in s to b e e x p lo r e d .

E F E C T O S G E N E T IC O S C O M P A R A D O S D E D IV E R SO S M U T A G E N O S F IS IC O S EN L A S P L A N T A S SU PERIO RES.

S e p a sa r e v is t a a lo s e f e c t o s g e n é t ic o s r e la t iv o s e n la s p la n t a s su p e r io re s d e m u tá g e n o s f ís ic o s q u e

p r o d u c e n d is tr ib u c io n e s e s p a c i a le s d i fe r e n te s d e e n e r g ía e n lo s t e j id o s ir r a d ia d o s . S e t ra ta p r in c ip a lm e n t e

d e n e u tro n e s r á p id o s ( " d e f is ió n ” ) , n e u tro n e s m o n o e n e r g é t ic o s y d e e s p e c tr o e n e r g é t ic o m ix t o , y r a d ia c io n e s

p r o d u c id a s p o r io n e s p e s a d o s a c e l e r a d o s . S e e f e c t ú a n c o m p a r a c io n e s c o n ra y o s X y y d é b i lm e n t e io n iz a n t e s .

L a e f e c t i v i d a d g e n é t i c a m e d ia d e to d o s lo s r e s u lta d o s c o m b in a d o s r e s u lta m á x im a (EBR= 7 1 , 5) p a ra la s

r a d ia c io n e s c o n u n a t ra n s m is ió n l in e a l d e e n e r g ía (T L E ) m e d ia p o r do sis d e 5 3 a 1 7 5 k e V / ^ m . L a EBR se

r e d u c e a 13 e n e l c a s o d e r a d ia c io n e s d e T L E in fe r io r ( 7 - 2 3 k eV //im ) o su p e r io r (> 1 7 5 k e V / / jm ). El a n á lis is

(b a s a d o e n lo s s e g m e n to s d e t ra z a s ) d e la s v a r ia c io n e s d e la s e c c i ó n e f i c a z c o n la T L E r e v e l a q u e lo s

p u n to s o b la n c o s d o n d e o c u r r e n la s m o d i f i c a c io n e s o lo s d a ñ o s g e n é t ic o s in d u c id o s t ie n e n e s p e so re s e f e c t iv o s

d e l o rd e n d e m a g n itu d d e la s m o lé c u la s d e l A D N . En la s p la n ta s su p e r io re s e x is t e n p o c a s p ru e b a s d e

e s p e c i f i c id a d m u t a g é n ic a e n t r e lo s m u tá g e n o s f ís ic o s . Q u e d a n p o r in v e s t ig a r lo s fa c t o r e s d e te r m in a n te s

d e l a r e c u p e r a c ió n d i f e r e n c i a l d e la s m u ta c io n e s p r o d u c id a s p o r r a d ia c io n e s d e e le v a d a T L E .

1. INTRODUCTION

Differences in the spatial distribution of energy in irradiated tissues,i.e. in radiation quality, influence the effectiveness of a unit of absorbed dose. The different genetic effects of physical mutagens are due to, and can be specified in terms of, rates of energy loss of charged particles.The reason for the dependence of relative biological effectiveness (EBE) on radiation quality is incompletely understood largely because of our inadequate knowledge of the basic mechanisms of action and cellular response to radiation-induced damage [63]. The concept of linear energy transfer (LET), commonly expressed in units of kiloelectron volts per micron (keV/цт), is the most generally used index of radiation quality and gives a

5|C R e se a rc h c a r r ie d o u t a t B ro o k h a v e n N a t io n a l L a b o r a to r y u n d e r th e a u s p ic e s o f th e U . S . A t o m ic

E n e rg y C o m m is s io n . P a rt o f th e r e s e a r c h re p o r te d h a s b e e n c a r r ie d o u t u n d e r R e s e a rc h A g r e e m e n t s w ith

t h e I n t e r n a t io n a l A t o m ic E n e rg y A g e n c y N os 6 1 9 / C F an d 3 3 5 / C F .

75

7 6 SMITH

TABLE I. RBE VALUES FOR FAST ("FISSION") NEUTRONS VS. LOW LET RADIATIONS ON PLANT SPECIES IRRADIATED AS DRY SEEDS OR YOUNG SEEDLINGS

M a t e r ia l a n d s p e c ie s

ir r a d ia te d

C r it e r ia

(re sp o n se l e v e l , g e n e ra t io n )

L o w L E T

r a d ia t io nRBE

R e fe r e n c e

N 0 .

S e e d s — w h e a t C h r o m o s o m e a b e rra t io n s (R ) X 5 - 9 1

T r i t ic u m . 2 x . 4 x . 6 x

sp p .S e e d l in g h e ig h t (R: ) У 1 0 - 1 5 24

S e e d f e r t i l i t y (R x ) У 10 - 50 24

C h lo r o p h y l l m u ta tio n s (Rg) У 2 5 24

S e e d s — m a i z e M u t a t io n ( 1 / l e a f , R t ) X 76 4 1 , 4 4

Z e a m a y sM u ta t io n ( 2 / l e a f , R x) X 37 4 1 , 4 4

S e e d s e t ( D 50, R x) X 33 4 1 , 4 4

P o lle n f e r t i l i t y ( D 50, R x) X 2 2 4 1 , 4 4

P la n t h e ig h t ( D 50, R x) X 1 2 4 1 , 4 4

S u r v i v a l (D s o , Rx ) X 1 0 4 1 , 4 4

E m e r g e n c e ( D 50, R 2 ) X 1 0 4 1 , 4 4

E m e r g e n c e r e v e r s a l ( R x ) X 5 4 1 , 4 4

P la n t h e ig h t r e v e r s a l ( R L) X 3 4 1 , 4 4

P la n t s u r v iv a l ( R A) У 9 - 1 9 5 1

S t e r i l i t y (R x ) У 6 - 9 5 1

P la n t h e ig h t (R A) У 9 - 1 1 5 1

A lb in o m u ta tio n s ( 1 °}o, Rg) У 2 5 1

C h lo r o p h y l l m u ta tio n s ( 1 °}o, R 2) У 3 - 7 5 1

T o t a l m u ta tio n s (17o, R 2 ) У 9 - 1 5 5 1

S e e d s — t o b a c c o C o t y le d o n e m e r g e n c e ( D 3 7, R x ) X 4 - 9 5

N ic o t ia n a s p e c ie sL e a f d e v e lo p m e n t ( D 3 7, R¿) X 1 0 - 1 1 5

T u m o u r fo r m a tio n ( D 3 7, R t) X 14 5

S e e d s — V ig n a S e e d l in g g r o w th (D 50, R : ) У 1 2 8

s in e n s is

S e e d s — A r a b id o p s is S in g l e lo c u s m u ta tio n ( l f y , R ) У 16 1 3 , 14

t h a l ia n aS e e d se t ( D so, R x) X 1 2 46

P la n t h e ig h t ( D 50, R x) X 1 1 46

S u r v i v a l ( D 50, R j) X 9 46

S e e d s — N ig e l la C h r o m o s o m e a b e rra t io n s (R A) У 78 32

d a m a s c e n a S e e d se t ( D 50, R ¡) X 26 46

P o l le n f e r t i l i t y ( D s0, R j) X 2 1 46

P la n t h e ig h t (D s o , R x) X 16 46

S u r v iv a l ( D 50, R j) X 1 1 46

E m e r g e n c e (D 5 0 , R i) X 1 1 46

TABLE I (cont. )

MUTAGENS IN HIGHER,PLANTS 77

M a t e r ia l an d s p e c ie s C r it e r i a L o w L E T R e fe r e n c e

ir r a d ia te d (re sp o n se l e v e l , g e n e r a t io n ) r a d ia t io n N o .

S e e d s — p e a

P isu m s a tiv u m

S e e d se t ( D so, R x ) X 32 46

P o lle n f e r t i l i t y ( D 50, R i) X 30 46

P la n t h e ig h t ( D 50, R x) X 23 46

S u r v i v a l ( D so, R j) X 14 46

E m e r g e n c e ( D 50> R j) X 1 1 46

S e e d s — ra d ish S e e d se t ( D 50, R j) X 1 5 46

R a p h an u s s a t iv u mP o l le n f e r t i l i t y (D 5 0 , R i) X 18 46

D ry w e ig h t ( D 50, R i) X 1 1 46

S u r v iv a l (D 50, Rt ) X 1 2 ■46

S e e d s — to m a to S e e d s e t ( D 50, R x) X 14 46

L y c o p e r s ic o n e s c u le n tu mP o lle n f e r t i l i t y (D so, R ]) X 9 46

P la n t h e ig h t (D s0 , R j) X 8 46

S u r v iv a l ( D 50, R: ) X 7 46

E m e r g e n c e ( D s o , R ^ X 7 46

S e e d l in g g ro w th (D so, R j) У 56 6

S e e d s — o n io n

A l l i u m с е р а

S e e d l in g g ro w th (D 50, R j) У 48 6

S e e d s — ra p e

B ra s s ic a nap us

S e e d l in g g ro w th (D so, R i) У 65 6

S e e d s - c u c u m b e r

C u c u m is s a tiv u s

S e e d l in g g r o w th ( D 50, Rj ) У 65 6

S e e d s — f e s c u e grass

F e s tu c a a r u n d in a c e a

S e e d l in g g ro w th (D 50 , R j) У 2 0 6

S e e d s — c o t to n

G o s s y p iu m a rb o re u m

S e e d l in g g ro w th ( D 50, R t ) У 44 6

S e e d s - b a r le y

H o rd e u m v u lg a r e

S e e d l in g g ro w th (D 5 0 , R i) У 65 6

S e e d s — l e t t u c e

L a c t u c a s a t iv a

S e e d l in g g ro w th (D 5 0 , R j) У 44 6

S e e d s — f la x

L in u m u s ita t is s im u m

S e e d l in g g r o w th (D 50, R j) У 17 6

S e e d s — c r im s o n c l o v e r

T r i f o l iu m in c a r n a tu m

S e e d l in g g ro w th (D 50, R ^ У 77 6

S e e d s — r ic e

O r y z a s a t iv a

D r y w e ig h t (D 50, Щ У

У

2 , 5 ( o x i c ) 7

21.9 (anoxic) 7

78 SMITH

TABLE I (cont. )

M a t e r ia l an d s p e c ie s

ir r a d ia te d

C r it e r i a

(re sp o n se l e v e l , g e n e r a t io n )

L o w L E T

r a d ia t io nRBE

R e fe r e n c e

N 0 .

S e e d lin g s — b ro a d b e a n S u r v iv a l ( D 50, R i) У 4 . 6 1 1

V i c i a fa b aD r y w e ig h t ( D 50, R i) у 6 .9 1 1

P la n t h e ig h t ( D So , R i) У 6 . 6 1 1

P o lle n f e r t i l i t y ( D 5 0 , R i) У 6 . 6 1 1

S e e d lin g s — p e a S u r v iv a l (D 50 , R j) У 5 . 7 1 1

P isu m s a tiv u mD ry w e ig h t ( D 50, R j) У 4 . 6 1 1

P la n t h e ig h t (D 50, Ri ) У 4 . 7 1 1

S e e d lin g s — s u n flo w e r S u r v iv a l ( D 50, R :) У 4 . 9 1 1

H e lia n th u s an n u u sD ry w e ig h t ( D 5 0 , R i) У 8 .4 1 1

P la n t h e ig h t ( D 50, R: ) У 5 . 4 1 1

S e e d lin g s - ra d ish S u r v i v a l ( D 50, R j) У 5 . 4 1 1

RaDhanus sa tiv u mD ry w e ig h t ( D so, R j) У 8 . 1 1 1

S e e d lin g s — A ra b id o p s is S u r v iv a l (D 50, R j ) У 4 . 6 1 1

t h a l ia n aD ry w e ig h t ( D 50, R i) У 5 . 7 1 1

P o lle n f e r t i l i t y (D 50, R! ) У 3 . 9 1 1

rough measure of energy distribution in irradiated tissues. Though recognized as an unsatisfactory parameter for comparing physical mutagens, dose average LET is currently widely employed to characterize the paths of charged particles of specified energies in irradiated tissues.

The aim in this paper is to review the genetic effects on higher plants of radiations (physical mutagens) of different LET as reported in papers published during the last ten years (1961-1970). Results prior to this period have been reviewed extensively by Bora [2], by Gopal-Ayengar and Swaminathan [17] and by Smith [39].

A wide variety of experimental plants have been used as experimental materials. Some of the more favoured species have been Zea mays (maize), Triticum spp. (wheat), Hordeum vulgare (barley), Tradescantia spp. (spiderwort), Oryza sativa (rice), Arabidopsis thaliana (a short life-cycle crucifer), and Nigella damascena (devil-in-the-bush). The plants have been irradiated at different stages of development, thus involving different tissues: seed, seedlings, inflorescences and pollen. The stage or plant part irradiated seems not to influence consistently the magnitude of the RBE. The experimental materials, criteria of response, and computed RBE values are summarized for fast ("fission") neutrons in Table I, for monoenergetic and mixed spectrum neutron energies in Table II, and for high LET radiations produced by accelerated heavy ions in Table III.The RBE of a high LET radiation is expressed as the reciprocal of the ratio of dose to that of ordinary X- or y-rays to produce the "same" effect.

MUTAGENS IN HIGHER PLANTS 79

Seeds have been preferred experimental materials since they can be subjected, without impairment of function, to a wide range of radiation doses and of environmental conditions (temperature, moisture, atmospheric content and storage time). It has long been recognized that the environmental conditions during and after irradiation greatly influence the degree of response. This is particularly true for low LET radiations and particularly for seed irradiations where large differences in moisture or oxygen content may exist. The influence of post-irradiation availability of oxygen on RBE was demonstrated in two recent papers [7, 18] in which barley seeds of very low moisture content (1% and 2.7%) were exposed in vacuo to 14 MeV neutrons or to fission neutrons and the response was compared to that of 7 -irradiated seed under oxic versus anoxic conditions of post-irradiation hydration. The RBE's under oxic conditions were 2.5 and 3, while under anoxic conditions they were 22 and 29. Similarly, the effect of soaking seeds prior to irradiation affects the RBE. For example, dry (13% moisture) barley seeds compared to those soaked for 36 hours before irradiating with fission neutrons versus 250 kVp X-rays gave RBE's of 18 and 4. 7 respectively [46]. Also, for a single locus somatic mutation in maize [43], seeds of 6. 7% moisture gave an RBE for fission neutrons versus 250 kVp X-rays of 67, whereas after 36 hours of soaking prior to irradiation the RBE was reduced to 5. These changes in RBE were due largely to changes in sensitivity to low LET radiations.A "single locus somatic mutation", as used here, usually refers to a scorable cell lineage of mutant plant tissue arising mainly from loss of a dominant marker gene in somatic tissues of a heterozygous Ri plant.

These examples serve to show that a listing of RBE values is of little meaning unless the state of the plant materials and the environmental conditions under which the irradiations were carried out are controlled and specified. Although this information is not completely available for most of the experiments and their RBE's listed in the tables, nevertheless the irradiations were usually carried out in air, i.e. under oxic conditions, the seeds were usually listed as dry and dormant which means around 10 to 20% moisture, and since the seeds were not specified as stored they were in all probability sown soon after irradiation and under oxic conditions in moist soil.

All irradiations listed in Tables I, II and III were administered as acute single exposures. Response of dry seeds to low LET radiation was found to be independent of dose rate over a range of 1758 to 10.3 rad/min [47]. However, a still lower dose rate of 1500 R/20 h day (1.25 R/min) was more effective with dry seeds than equivalent acute doses [33] and this was attributed to accumulative effects of long-lived free radicals.In a "wet" system, e.g. plantlets of African violet (Saintpaulia) propagated from leaf cuttings [56], mutation frequency per absorbed dose was affected by dose rate of both fast neutrons and X-rays, and for the latter reached a peak at about 200 rad/min, then declined again.

The low LET radiations used for comparison with high LET were either 250 kVp X-rays or -y-rays, the latter most frequently from 60Co but in some cases 137Cs. The high LET radiations listed in Table I are fission neutrons, unmodified or slightly degraded, with a spectrum of energies peaking at 1 to 1. 5 MeV. Most of the work on thermal neutrons

2. CONDITIONS OF IRRADIATION

TABLE

II.

RBE

VALU

ES

FOR

MON

OEN

ERGETIC

AN

D MEA

N

OF

MIX

ED

SPECTRUM

NEUTRON

EN

ERG

IES

GIV

ING

HIGH

LE

T RADIA

TIO

NS

IN HIG

HER

PLAN

TS

80 SMITH

TABLE

II

(con

t. )


Spec

tru

m

of

ener

gie

s fro

m

nea

r ze

ro

to ab

out

120

keV

, bu

t m

ostl

y fro

m

100

dow

n to

1 k

eV

.

TABLE

II

(cont

.

82 SMITH


[20, 22, 37, 55] was not included in the tables because the absorbed dose based on the elemental composition of the plant material was not computed [30-32]. The genetic effect of the nuclear reaction 14N (n, p) 14C was evaluated recently by exposing dry tomato pollen to thermal neutrons [57] and the RBE, compared to y-rays, was estimated to be 1.5 to 3.0. In Table III various high LET radiations are listed, mainly accelerated heavy ions (4He to 40Ar); and also high energy it- mesons and protons which gave RBE's above unity probably due to high LET secondary particles from nuclear disintegrations.

3. CRITERIA OF GENETIC RESPONSE

The criteria used to measure the effects of high versus low LET radiations that are listed in the tables cover a wide range of responses. They have been scored in either the treated generation (R^ or its progeny (R2 generation). Some of these are obvious and direct measures of mutagenesis, such as the frequency of chlorophyll or other viable mutations in the R2 generation (see Refs [23-25, 27, 38, 51]) and single locus somatic mutation cell lineages in the Ri generation (see Refs [9, 12, 14-16, 19, 28,29, 38, 40-42, 44, 47, 49, 53, 54]).

Many of the criteria listed in the tables are not usually considered as genetic effects (e.g. reduced growth, fertility or survival), however, they have been included with the more conventional genetic effects for the following reasons: 1) From an extensive review of the literature, Davies and Evans [10] concluded that, "the overwhelming bulk of evidence would support the thesis that lethality is primarily a consequence of genetic damage".2) Sparrow [50] finds that for a particular amount of plant damage, the absorbed energy per average interphase chromosome volume does not vary greatly among higher plant species. 3) A plot of log fast neutron dose versus log X-ray dose to produce in maize equal effects in somatic mutations, as well as various measures of growth inhibition and reduced fertility, all fall along (Fig. 1) a single straight line (over four orders of magnitude) indicating a common genetic cause for the various responses [41, 44]. 4) The RBE for heavy ions with a wide range in dose averageLET was greatest at 74 to 172 keV/ijm [ 19, 45] for somatic mutation as well as tumour induction and growth retardation (Fig. 2). 5) Track segmentanalysis of cross-section variations with LET [19] indicates that target sites for the above three effects all have effective thicknesses of the order of size of DNA molecules. 6) The rise and subsequent fall with increasing dose of a specific somatic mutation in maize [48] can be fitted by a mathematical model in which the decline of the curve is due to cell killing which, like the "mutation", may also be accounted for by chromosome breakage and deletion. In view of these six considerations it was felt that the various criteria listed in the tables are probably all legitimate measures of genetic damage, if not mutagenesis per se.

4. GENERALIZED COMPARATIVE EFFECTIVENESS OF PHYSICAL MUTAGENS

Since response to high LET radiations maintains for the most part an exponential or straight line relation to dose, whereas low LET radiations

TABLE

III.

RBE

VALU

ES

FOR

HIGH

LE

T RADIA

TIO

NS

PRODUCED

BY

ACCELERATED

HEAVY

IONS

IN

HIG

HER

PLAN

TS

84 SMITH

TABLE

III

(cont

.MUTAGENS IN HIGHER PLANTS 85

86 SMITH

F IG . 1 . L o g a r ith m ic p lo t o f n in e c r i t e r ia o f e q u a l e f f e c t ( s e e T a b l e I , R efs [ 4 1 , 4 4 ] ) in m a i z e p la n ts g ro w n

fro m se e d s w h ic h h a d r e c e iv e d a w id e r a n g e o f d oses o f f is s io n n eu tro n s o r o f 250 k V p X - r a y s .

(F IG . 1 . R e p r e s e n ta c ió n l o g a r í t m ic a d e n u e v e c r i te r io s d e i g u a l e f e c t o ( v é a s e e l c u a d ro I , r e f . [ 4 1 , 4 4 ] ) e n

p la n ta s d e m a í z d e s a r r o lla d a s a p a r t ir d e s e m il la s s o m e tid a s a d o sis m u y v a r ia d a s d e n e u tro n e s d e f is ió n o d e

ra y o s X d e 250 k V p .)

show a power function or curvilinear relation, the magnitude of the RBE will vary with dose. In turn, the dose required to produce an effect will depend on the radiosensitivity of the genotype, the criterion of response scored, and the level of damage reached. Most of the somatic effects listed in the tables were scored at 50% reduction (D50) from unirradiated control values, or sometimes D30 or D37. Most mutations were scored at a level of 1 to 3%, based on numbers of mutant plants in the Ri generation or those giving mutants in the R2. Consequently, though the criteria in the tables are varied, they nevertheless to a considerable extent represent comparable levels of response. The variation among species in dose required to produce equal damage is inversely related to interphase chromosome volume for both low and high LET radiations [5, 11, 46, 50].


LET (keV/^ IN MERISTEM l-LOG SCALE

F I G .2 . P lo ts o f RBE fo r c a u s in g tu m o u rs , g ro w th in h ib it io n , a n d s o m a t ic (Rj) m u ta tio n s in A ra b id o p s is

( T a b l e III , R e f . [ 1 9 ] ) w ith 2 50 k V p X - r a y s v s . a c c e le r a t e d h e a v y io n s o f L E T as sh o w n o n t h e a b s c is s a .

W ith e a c h o f t h e io n s , e x c e p t H e ( fu ll e n e r g y ) , th e B ra g g p e a k w a s a d ju s te d to f a l l in t h e sh o o t m e r is te m

o f th e s e e d .

( F I G .2 , R e p r e s e n ta c ió n d e la E B R ( e f i c a c ia b io l ó g ic a r e la t iv a ) n e c e s a r ia p a ra c a u s a r tu m o r e s , la in h ib ic ió n

d e l c r e c im ie n t o y m u ta c io n e s s o m á tic a s ( R i) en e l g é n e r o A r a b id o p s is ( c u a d ro III , r e f . [ 1 9 J ) c o n ra y o s X

d e 2 5 0 k V p , e x p r e s a d a e n fu n c ió n d e lo s io n e s p e s a d o s a c e le r a d o s d e l a T L E in d ic a d a e n a b c is a s . En e l c a so

d e c a d a u no d e es to s io n e s , a e x c e p c ió n d e l H e ( e n e r g ía t o t a l ) , se h a a ju s ta d o e l m á x im o d e B ra g g d e fo rm a

q u e co rre s p o n d a a l m e r is te m o d e l b ro te d e la s e m i l la . )

As a result of seed irradiations with fast neutrons versus X-rays of six species compared on the basis of five criteria [46] it was shown that:1) Within a single species, the RBE varied inversely with the dose required to produce the different criteria of response. 2) For a single criterion of response, the RBE among species varied directly with the radiation sensitivity of the species. 3) As a consequence of the inverse relation of RBE to dose, with the more radiosensitive species and criteria the RBE's ranged from 21 to 32, whereas with the least sensitive species . and criteria the RBE's ranged from 7 to 12 (Table I, Ref. [46]).

In spite of what appears to be a rather haphazard series of RBE values depending on genotype, criterion, dose, and environmental variables, there is nevertheless, under constant conditions, a constant relation of neutron dose to X-ray dose to produce equal effects. This constant relation is expressed not only by the regular change in RBE values, but more succinctly, by a simple power equation. For maize, irradiated as dry seeds [41,44], this equation is:

log N = - 2.6+1.95 log X

where N and X are the fission neutron and 250 kVp X-ray doses required to produce an equal effect (Fig. 1, Table I, Refs [41, 44]). In the same

88 SMITH

100

50

20

10

оceI-з

0.5

0.2

0.1

0.05

0.02

0.01

Т Т Л r i — г

¿ Zeo ■ Níaello a Pi sum* Lvcooersicon• Arobidopsis о Roohonus

J _ l ___ 1 I I_____L J ___ L10 20 50 100 200

X - R A Y S (k ra d )

F I G . 3 . L o g a r ith m ic p lo t o f v a r io u s c r i t e r ia o f e q u a l r a d ia t io n e f f e c t in s ix s p e c ie s o f p la n ts e x p o s e d as se ed s

to f iss io n n eu tro n s v s . 2 50 k V p X - r a y s ( T a b le I, R e f . [ 4 6 ] ) .

( F I G .3 . R e p r e s e n ta c ió n lo g a r í t m ic a d e d iv e rso s c r i te r io s d e ig u a l e f e c t o r a d io ló g ic o en se is e s p e c ie s d e

p la n t a s e x p u e s ta s e n fo rm a d e s e m il la s a n eu tro n e s d e f is ió n , e n fu n c ió n d e ra y o s X d e 250 k V p ( c u a d r o I,

r e f . [ 4 6 ] ) . )

way, considering a number of criteria of response in a number of higher plant species (Fig. 3), the relation of neutron to X-ray dose at changing dose levels is shown more simply by a general regression rather than the changing RBE values as such. The actual RBE's for the points plotted in Fig.3 are shown with Ref. [46] in Table I. In summary, the wide range in tolerance to radiation shown by seeds of higher plants, and the widely different radiosensitivities of higher plant species, have made it possible to explore the relative biological effectiveness of high versus low LET radiations over an extremely wide range of doses. The changing relationships with changing dose levels can be expressed by a simple power function equation, perhaps more significantly than by a series of changing RBE values. The slope of the line can be taken to represent the relative importance of dose to dose-squared effects of the two radiations being compared.

MUTAGENS IN HIGHER PLANTS 8 9

TABLE IV. MEAN RBE VALUES FOR MUTAGENESIS IN HIGHER PLANTS

LE T

( k e V / д т )

M e a n RBE

s in g le

lo c u s d e le t io n

R a c h r o m o .

a b e rra t io n s

r 2

m u ta tio n sM e a n

7 - 2 3 20 5 , 7 1 5 1 3 .6

2 3 -5 3 48 5 1 18 3 9 .0

5 3 - 1 7 5 5 1 92 7 1 . 5

> 1 7 5 6 . 5 20 1 3 . 2

In order to obtain some general RBE values for plants that can be compared with those of other organisms, averages were calculated from the RBE's listed in the tables. The average value for all RBE's listed in Tables I, II and III is 22. The average for fission neutrons is 18 (Table I) and for all the other high LET radiations 25 (Tables II and III). Since a main concern in this report is with the effect of LET on the RBE for mutagenesis, the information in Tables I, II and III is summarized in Table IV, grouped according to different ranges in keV/jum as used by the International Commission on Radiological Protection. These RBE values are higher in general (and involve higher doses) than for other organisms tested, such as insects and mammals.

5. MUTAGEN SPECIFICITY AND CONTROL OF RECOVERY

There is little if any evidence from higher plants for differential specificity of mutagenesis by high versus low LET radiations, with the possible exception of eceriferum mutants in barley [21], which are characterized by absence or reduction of the wax coating on the epidermis.The mutants have been tested for allelism by diallelic crosses and two mutants are considered allelic when the Fj has an eceriferum phenotype. The 84 observed mutants induced by sparsely ionizing radiations (X- and 7 -rays) were distributed rather evenly among 31 differently located eceriferum loci. In contrast, the mutants produced by densely ionizing radiations (protons, neutrons and a-rays) showed a prominently skewed locus distribution in that no less than 36% of the 39 mutants produced at 16 loci were found to be located at a particular single locus. There is no evidence to explain the preferential effectiveness of high ionization density for this locus.

The possibility of exerting control over the products recovered from high LET radiations has not been adequately explored. As pointed out by Neary [63], "there are three stages of evolution of radiation damage,(1) various primary molecular lesions, (2) lesions remaining after completion of fast physico-chemical metionic reactions, (3) residual lesions after the intervention of post-irradiation cellular repair and bypass mechanisms. The three classes of lesions probably depend in different ways on the quality of the radiation and the presence of modifying agents". Low LET

90 SMITH

radiations show a preponderantly oxygen-dependent component to damage caused during irradiation which may persist under appropriate conditions for some time. These effects are attributed mainly to the production of free radicals, which interact with biologically important molecules in the presence of oxygen, and in time decay [59]. The degree of response can be modified during this time by controlling environmental conditions.The effects of high LET radiations consist predominantly of primary, irrepairable, oxygen-independent events. This has led to a general attitude that the effects of high LET radiations are not modifiable by post-exposure conditions.

The following three experimental results suggest that the products recovered after irradiation can be markedly influenced by environmental conditions occurring later in the recovery process, i.e. subsequent to physico-chemical metionic or free radical reactions: 1) Seedlings grown from barley seeds irradiated with fast neutrons showed less evidence of damage when recovered for 10 days under 24 h (i.e. continuous) light than under a 20-h daylength [64]. 2) Lyman and Haynes [62] observed a markedenhancement of viability in diploid yeast which had been irradiated with heavy ions, then stored up to 48 h at 30° С rather than planted immediately. Several lines of evidence indicated that the recovery was based on enzymatic post-irradiation processes, unrelated to the initial physicochemical reactions associated with absorption of the radiation, and that these involved a "bypass" rather than a direct repair mechanism.3) Arabidopsis plants grown from seeds irradiated with 7 -rays, then quenched of free radicals, showed greater elimination of damaged cells when cultures were maintained continuously at 27°С than when they were grown at 20° С for as little as 4 days during germination,then subsequently raised at 27°С [60]. The difference could also be demonstrated in second generation progeny which exhibited less genetic variance in flowering time and lower sterility in those populations derived from plants recovered in the continuous higher temperature.

These results are in line with concepts of mutagenic specificity considered by Auerbach [56, 57]. In view of our present knowledge that genes are comprised of different linear sequences of only four nucleotides, the expectation seems remote for finding mutagenic specificity (and hence directed mutation) at the gene level through direct differential reaction of mutagen with DNA. However, mutational change in the information carried by DNA, particularly in higher forms, is both preceded and followed by secondary steps that may act as multiple "sieves" which determine whether a change in DNA will take place and whether it will give rise to an observable population of mutated cells. It is at the level of these "sieves" that it may be possible to modify the environment (intra- or extracellular) to give control of the spectrum of mutants ultimately obtained.

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p a r t ic le s fro m B1 0 ( n , a ) L i 7 r e a c t io n fo r c y t o g e n e t i c e f f e c t s in e in k o r n w h e a t , R a d ia t . B o t. 3 ( 19 6 3 ) 29»

[2 6 ] M A T S U M U R A , S . , M A B U C H I, T . , D if fe r e n c e s in e f fe c t s o f y - r a y s an d fast n e u tro n s fro m P o -B e s o u r c e

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[ 2 7 ] M A T S U M U R A , S . , N E Z U , М . , " R e la t io n b e t w e e n p o ly p lo id y a n d e f fe c t s o f n e u tro n r a d ia t io n o n

w h e a t ” , E f fe c ts o f I o n iz in g R a d ia tio n s o n S e e d s ( P ro c . C o n f . K a r ls r u h e , 1 9 6 0 ), IA E A , V ie n n a ( 1 9 6 1 ) 5 4 3 .

[2 8 ] M IC K E , A . , S M IT H , H . H . , W O O D L E Y , R . G . , M A S C H K E , A . , R e la t iv e c y t o g e n e t i c e f f i c i e n c y o f

m u o n s a n d n " m eso n s in Z e a m a y s L . , P r o c . n a tn . A c a d . S c i . U S A 52 (19 6 4 ) 2 1 9 ,

[2 9 ] M IC K E , A . , S M I T H , H . H . , W O O D L E Y , R . G . , M A S C H K E , A . , R e la t iv e c y t o g e n e t i c e f f i c i e n c y o f

m u o n s a n d тг- m e s o n s in Z e a m a y s L . a n d its m o d i f ic a t io n b y p o s t - ir r a d ia t io n s t o r a g e , R a d ia t . R e s. 23

(19 6 4 ) 5 3 7 .

92 SMITH

[3 0 ] M O U T S C H E N , J . , M O U T S C H E N -D A H M E N , М . , " E f fe c t s o f t h e r m a l, m o n o e n e r g e t ic an d f is s io n n eu tro n s

o n N ig e l l a c h r o m o s o m e s " ( P r o c . S y m p . N e u tro n s in R a d io b io lo g y , O a k R id g e , T e n n . , N o v .1 9 6 9 ) ,

C O N F - 6 9 1 1 0 6 ( 19 7 0 ) 3 9 1 .

[ 3 1 ] M O U T S C H E N , J . , M O U T S C H E N -D A H M E N , М . , W O O D L E Y , R . G . , A R C H A M B E A U , J . D . , T h e RBE o f

h e a v y p a r t ic le s fro m t h e r e a c t io n B ^ t n . c O L i 7 fo r c h r o m o s o m e a b e r r a t io n s in N ig e l la d a m a s c e n a L . ,

R a d ia t . R e s . 3 4 (19 6 8 ) 4 8 8 .

[ 3 2 ] M O U T S C H E N , J . , M O U T S C H E N -D A H M E N , М . , W O O D L E Y , R . , G IL O T , J . , T h e r e l a t iv e b io l o g ic a l

e f f e c t iv e n e s s o f d i f f e r e n t k in d s o f r a d ia t io n s o n c h r o m o s o m e a b e r r a t io n s in N ig e l l a d a m a s c e n a s e e d ,

In t . J . R a d ia t . B io l. 1 5 (19 6 9 ) 5 2 5 .

[3 3 ] N A T A R A J A N , A . T . , M A R IC , M . M . , T h e t im e - in t e n s i t y f a c t o r in d ry s e e d ir r a d ia t io n , R a d ia t . B o t. 1

( 1 9 6 1 ) 1 .

[3 4 ] N E A R Y , G . J . , S A V A G E , C h r o m o s o m e a b e r r a t io n s a n d th e t h e o r y o f RBE. П . E v id e n c e fro m

t r a c k - s e g m e n t e x p e r im e n t s w ith p ro ton s a n d a lp h a p a r t ic le s , I n t . J . R a d ia t . B io l. 1 1 (19 6 6 ) 2 0 9 .

[ 3 5 ] N E A R Y , G . J . , P R E S T O N , R .J . , S A V A G E , J . R . K . , C h r o m o s o m e a b e r r a t io n s a n d t h e th e o r y o f RBE.

Ш . E v id e n c e fro m e x p e r im e n t s w ith so ft X - r a y s an d a c o n s id e r a t io n o f t h e e f f e c t s o f h a rd X - r a y s ,

I n t . J . R a d ia t . B io l. 12 ( 1 9 6 7 ) 3 1 7 .

[3 6 ] N E A R Y , G . J . , S A V A G E , J . R . K . , E V A N S , H . J . , W H IT T L E , J . , U l t im a t e m a x im u m v a lu e s o f th e

RBE o f fa s t n eu tro n s a n d g a m m a - r a y s fo r c h r o m o s o m e a b e r r a t io n s , I n t . J . R a d ia t . B io l. 6 (19 6 3 ) 1 2 7 .

[ 3 7 ] P E R E A U -L E R O Y , P . , T h e c o m p a r a t iv e g e n e t i c e f f e c t s o f g a m m a ra y s an d n eu tro n s o n b a r le y s e e d ,

R a d ia t . B o t . 8 ( 19 6 8 ) 2 3 9 .

[3 8 ] R A N A , R . S . , S W A M I N A T H A N , M . S . , R e la tio n s h ip b e t w e e n c h im e r a s an d m u ta tio n s in d u c e d b y

60C o y -r a y s a n d 2 M e V fa s t n eu tro n s a t s p e c i f i c l o c i in b re a d w h e a ts , R a d ia t . B o t . 7 (19 6 7 ) 5 4 3 .

[3 9 ] S M IT H , H . H . , " T h e r e a c t o r a s a t o o l fo r r e s e a r c h in p la n t s c ie n c e s a n d a g r i c u lt u r e " , P ro g r a m m in g

a n d U t i l i z a t io n o f R e s e a rc h R e a c to rs ( P r o c . IA E A S y m p . V ie n n a , 1 9 6 1 ) 1 , A c a d e m i c P ress, N ew Y o r k

( 19 6 2 ) 4 2 5 .

[4 0 ] S M IT H , H . H . , R e la t iv e b i o l o g i c a l e f fe c t iv e n e s s o f d i f fe r e n t ty p e s o f io n i z in g ra d ia t io n s : c y t o g e n e t i c

e f f e c t s in m a i z e , R a d ia t . R es. S u p p l. 7 ( 1 9 6 7 ) 1 9 0 .

[ 4 1 ] S M IT H , H . H . , N e u tro n ir r a d ia t io n o f se ed s as a t o o l in p la n t g e n e t ic s an d b r e e d in g , J a p . J. G e n e t .

S u p p l. 1 , 4 4 (19 6 9 ) 4 4 3 .

[4 2 ] S M IT H , H . H . , B A T E M A N , J . L . , Q U A S T L E R , H . , R O SSI, H . H . , "RBE o f m o n o e n e r g e t ic fast n eu trons:

c y t o g e n e t i c e f f e c t s in m a i z e ” , B i o lo g ic a l E ffe c ts o f N e u tro n a n d P ro to n I rra d ia t io n s ( P r o c . S y m p .

U p to n , 19 6 3 ) 2 , IA E A , V ie n n a (19 6 4 ) 2 3 3 .

[4 3 ] S M IT H , H . H . , C O M B A T T I , N . C . , " F a c to r s in f lu e n c in g v a r ia t io n in RBE in ir r a d ia t io n o f m a i z e s e e d s " ,

N e u tro n I r r a d ia t io n o f S e e d s , T e c h . R e p . S e r . N o . 7 6 ( A b s t r . ) , IA E A , V ie n n a ( 1 9 6 7 ) 2 6 .

[4 4 ] S M IT H , H . H . , C O M B A T T I , N . C . , R O SSI, H . H . , "R e sp o n se o f se e d s to ir r a d ia t io n w it h X - r a y s an d

n eu tro n s o v e r a w id e r a n g e o f d o s e s " , N e u tro n I r r a d ia t io n o f S e e d s I I , T e c h . R e p . S e r , N o . 9 2 , IA E A ,

V ie n n a ( 19 6 8 ) 3 .

[4 5 ] S M IT H , H . H . , H IR O N O , Y . , C O N K L IN , M . E . , L Y M A N , J . T . , "R e sp o n se s in m u ta t io n , g ro w th

in h ib it io n , t u m o r iz a t io n an d i s o z y m e m u lt i p l i c i t y fro m e x p o s in g se e d s to ir r a d ia t io n s o f d i f fe r e n t

l in e a r e n e r g y t r a n s f e r " , In d u c e d M u ta t io n s in P lan ts (P r o c . S y m p . P u llm a n , 19 6 9 ), I A E A , V ie n n a

(19 6 9 ) 2 3 1 .

[4 6 ] S M IT H , H . H . , J O S H U A , D . C . , C O M B A T T I , N . C . , T H O M P S O N . K . H . , " R e la t iv e e f fe c t s o f fiss io n

n e u tro n v s . X - i r r a d ia t io n o f se e d s o v e r a w id e r a n g e o f g e n o t y p e s , d o ses a n d m o is tu r e l e v e l s " , N e u tro n

I r r a d ia t io n o f S e e d s III , T e c h . R e p . S e r . , IA E A , V ie n n a ( in p re s s).

[ 4 7 ] S M IT H , H . H . , R O SSI, H . H . , E n e rg y r e q u ir e m e n ts an d r e l a t i v e b i o l o g i c a l e f fe c t iv e n e s s fo r p r o d u c in g

a c y t o g e n e t i c p h e n o m e n o n in m a i z e b y ir r a d ia t io n o f se ed s w ith X - r a y s a n d m o n o e n e r g e t ic n eu tro n s ,

R a d ia t . R es. 28 ( 19 6 6 ) 3 0 2 .

[4 8 ] S M IT H , H . H . , R O S S I, H . H . , KELLERER, A . ( in p r e p a r a t io n ) .

[4 9 ] S M IT H , H . H . , W O O D L E Y , R . G . , M A S C H K E , A . , C O M B A T T I , N . C . , R e la t iv e c y t o g e n e t i c

e f f i c i e n c y o f X - r a y s a n d 28 G e V p ro to n s in Z e a m a y s , R a d ia t . R e s. 2 5 ( 19 6 5 ) 2 4 1 .

[50 ] S P A R R O W , A . H . , " R e la t io n s h ip b e t w e e n c h r o m o s o m e v o lu m e a n d r a d ia t io n s e n s it iv i t y in p la n t c e l l s " ,

C e l l u l a r R a d ia tio n B io lo g y , W i l l ia m s a n d W ilk in s , B a lt im o r e ( 19 6 5 ) 1 9 9 .

[ 5 1 ] S T O I L O V , M . , E f fe c t o f g a m m a ra y s a n d fa st n eu tro n s o n d i f f e r e n t m a i z e v a r ie t ie s , C . r . A c a d . S c i .

A g r i e . B u lg . 1 (19 6 8 ) 2 2 1 .

[5 2 ] T R O I T S K Y , N . A . , B Y L IN S K Y , N . F . , P H IL IP P O V IC H , A . S . , " T h e m u t a g e n ic e f f e c t o f in t e r m e d ia t e

n e u tr o n s " , M e c h a n is m s o f M u ta t io n an d In d u c in g F a c to rs ( L A N D A , Z . , E d .) , A c a d e m ia , P ra g u e

(19 6 6 ) 1 7 5 .

[5 3 ] U N D E R B R IN K , A . G . , S P A R R O W , R . C . , SP A R R O W , A . H . , R O SSI, H . H . , "RBE o f X - r a y s an d

0 .4 3 M e V m o n o e n e r g e t ic n e u tro n s o n s o m a t ic m u ta tio n s a n d lo ss o f r e p r o d u c t iv e in t e g r i t y in T r a d e s c a n t ia


s ta m e n h a ir s " , (P r o c . S y m p . N eu tro n s in R a d io b io lo g y , O a k R id g e , T e n n . , N o v . 1 9 6 9 ) , C O N F - 6 9 1 1 0 6

( 19 7 0 ) 3 7 3 . A ls o R a d ia t . R e s. 4 4 ( 19 7 0 ) 1 8 7 .

[ 5 4 ] U N D E R B R IN K , A . G . , S P A R R O W , R . C . , SP A R R O W , A . H . , R O S S I, H . H . , R e la t iv e b i o l o g i c a l e f f e c t i v e

n ess o f 0 .4 3 M e V an d lo w e r e n e r g y n eu tro n s o n s o m a t ic a b e r r a t io n s a n d h a ir le n g th in T r a d e s c a n t ia

s ta m e n h a ir s , In t . J . R a d ia t . B io l, ( in p ress).

[ 5 5 ] Y A M A G U C H I , H . , " G e n e t i c e f f e c t s o f p i l e r a d ia t io n s in r i c e " , B i o lo g ic a l E f fe c t s o f N e u tro n a n d P ro to n

I r r a d ia t io n s ( P r o c . S y m p . U p to n , 19 6 3 ) 1 I A E A , V ie n n a (19 6 4 ) 3 7 1 .

[ 5 6 ] A U E R B A C H , C . , T h e r o le o f m u ta g e n s p e c i f i c i t y in m u ta tio n b r e e d in g , S o v ie t G e n e t . 2 (19 6 6 ) 1 .

[ 5 7 ] A U E R B A C H , C . , " A n a ly s is o f a c a s e o f m u ta g e n s p e c i f i c i t y in N e u io s p o ra c r a s s a " , M u t a t io n as a

C e l l u l a r P ro c e ss (W O L S T E N H O L M E , G . E . W . , O 'C O N N O R , M .~ E d s), C h u r c h i l l , L o n d o n (19 6 9 ) 6 6 .

[5 8 ] BROERTJES, C . , " D o s e - r a t e e f f e c t s in S a i n t p a u l ia " , M u ta tio n s in P la n t B re e d in g II ( P r o c . P a n e l

V ie n n a , 1 9 6 7 ) , I A E A , V ie n n a (19 6 8 ) 6 3 .

[5 9 ] C O N G E R , B . V . , N IL A N , R . A . , K O N Z A K , C . F . , T h e r o le o f w a t e r c o n t e n t in th e d e c a y o f o x y g e n -

s e n s it iv e s ite s in b a r le y se ed s d u rin g p o s t - ir r a d ia t io n h y d r a t io n , R a d ia t . R e s. 39 ( 19 6 9 ) 4 5 .

[6 0 ] D A L Y , K . , E f fe c t o f t e m p e r a tu r e o n s u r v iv a l o f g a m m a - i r r a d i a t e d A ra b id o p s is s e e d , E x p e r ie n tia

( in p re s s).

[ 6 1 ] E C O C H A R D , R . , A n a p p r o a c h to th e s tu d y o f g e n e t ic e f f e c t s fro m t h e 14N ( n , p ) 14C r e a c t io n fo r t h e r m a l

n e u tro n s , I n t . J . R a d ia t . B io l. 1 7 ( 19 7 0 ) 4 3 9 .

[6 2 ] L Y M A N , J . T . , H A Y N E S , R . H . , R e c o v e r y o f y e a s t a f te r e x p o s u r e to d e n s e ly io n iz in g r a d ia t io n , R a d ia t .

R e s. S u p p l. 7 ( 1 9 6 7 ) 2 2 2 .

[ 6 3 ] N E A R Y , G . J . , " S o m e g e n e r a l a s p e c ts o f r e l a t i v e b io l o g ic a l e f fe c t iv e n e s s o f r a d ia t io n o f d i f fe r e n t

q u a li t ie s o n c e l l s " , ( P r o c . S y m p . N e u tro n s in R a d io b io lo g y , O a k R id g e , T e n n . , N o v . 1 9 6 9 ) , C O N F - 6 9 1 1 0 6

(19 7 0 ) 1 5 3 .

[6 4 ] S M IT H , H . H . , M IK A E L S E N , K . , " C o m p a r a t iv e b io d o s im e tr y s tu d ie s o f f iss io n n e u tro n f a c i l i t i e s in

t h e B ro o k h a v e n M e d i c a l R e a c to r (U S A ) an d t h e A s tra R e a c to r ( S e ib e r s d o r f ) " , N e u tro n I r r a d ia t io n o f

S e e d s H I, T e c h . R e p . S e r . , IA E A , V ie n n a ( in p ress).

DISCUSSION

R. NILAN: I wish to support and emphasize Dr. Smithfs statement relative to controlling the seed environment when using sparsely ionizing radiations (X- and 7 -rays). We know that effects of a given dose of X- or 7 -rays can be altered by as much as 60% by the amount of oxygen and water in the seed, the temperature during and after treatment and by other factors. "Recipes" for controlling these factors leading to maximum effectiveness of radiation treatments and to reproducibility of experiments have been published in numerous papers and in the IAEA/FAO Manual on Mutation Breeding. It is recommended that these "recipes" be utilized in all seed irradiation experiments.

ADVANCES IN METHODS OF M UTAGEN TREATM ENT *

C.F. KONZAK, Irene M. WICKHAM, M.J. DeKOCK Department of Agronomy and Program in Genetics,Washington State University,Pullman, Wash., United States of America

Abstract-Resumen

A D V A N C E S IN M E T H O D S O F M U T A G E N T R E A T M E N T .

S o fa r m e th o d s o f m u ta g e n t r e a t m e n t a r e m o s t ly d e s ig n e d fo r r e s e a r c h o n m u ta g e n s an d th e m u ta tio n

p ro ce ss r a t h e r th a n fo r p r a c t i c a l a p p li c a t io n . T h e p a p e r p resen ts a r e v ie w and a n a ly s is o f s o m e r e c e n t w o rk

r e la t in g to p a r t ic u la r a s p e c ts o f t re a t m e n t s o f se ed s w ith v a r io u s m u t a g e n ic c h e m ic a l s o r g a m m a - r a y s . T h is

w o rk a im e d a t g r e a t e r p r e c is io n in m u ta t io n e x p e r im e n t s , in c r e a s e d m u t a g e n ic e f f i c i e n c y , g r e a te r m u t a g e n ic

s p e c i f i c i t y , in c r e a s e d in p u t e f f i c i e n c y an d m o r e c o n v e n ie n c e . T h e n e w d is c o v e r ie s an d d e v e lo p m e n ts a r e

c o n n e c t e d w it h th e f o l lo w in g fa c t o r s : (a ) pH and b u ffe r s , (b) t e m p e r a tu r e , ( c ) r e d r y in g , (d ) p o s t - t r e a t m e n t

w a s h in g , ( e ) t issu e h y d r a t io n , ( f) m e t a b o l ic s t a t e an d c e l l s t a g e , (g ) p o s t - t r e a t m e n t m o d i f i c a t io n b y

c h e m i c a l s , an d (h ) s o m e p h y s ic a l an d c h e m i c a l p ro p e rt ie s o f m u ta g e n s r e la t e d to t h e ir in v itr o and in v iv o

r e a c t i v i t y . S o m e p r e c a u tio n s fo r s t o r a g e an d u se o f p o te n t c h e m ic a l m u ta g e n s a r e m e n t io n e d .

PRO G RE SO S EN LO S M E T O D O S DE T R A T A M IE N T O M U T A G E N IC O .

H a sta a h o ra lo s m é to d o s d e t r a ta m ie n to m u t a g é n ic o es tá n c o n c e b id o s e n su m a y o r ía p a ra l a in v e s t ig a c ió n

d e lo s m u tá g e n o s y d e lo s p ro c e so s d e m u t a c ió n , y n o p a ra la a p l ic a c ió n p r á c t i c a . En la m e m o r ia se p a sa n r e v is t a

y a n a l i z a n a lg u n o s tra b a jo s r e c ie n t e s r e la t iv o s a a s p e c to s p a r t ic u la r e s d e l t r a ta m ie n to d e s e m il la s c o n d is tin to s

m u tá g e n o s q u fm ic o s o c o n ra y o s g a m m a . E sta la b o r se r e a l i z a c o n lo s s ig u ie n te s o b je t iv o s : m a y o r p r e c is ió n

en lo s e x p e r im e n t o s d e m u t a c ió n , m a y o r e f i c a c i a m u t a g é n ic a , m a y o r e s p e c i f i c id a d m u t a g é n ic a , m a y o r

e f e c t iv id a d d e lo s re c u rso s d e to d o g é n e r o u t i l iz a d o s y m a y o r s e n c i l l e z d e la s o p e r a c io n e s . Los n u e v o s d e s

c u b r im ie n t o s y p e r f e c c io n a m ie n t o s e s tá n r e la c io n a d o s c o n lo s s ig u ie n te s f a c t o r e s : a ) pH y t a m p o n e s , b ) t e m

p e r a tu r a , c ) l e d e s e c a c i ó n , d) l a v a d o p o ste r io r a l t r a t a m ie n t o , e ) h i d r a t a c ió n t is u la r , f ) e s ta d o m e t a b ó l ic o

y fa s e c e l u l a r , g ) m o d i f i c a c ió n d e sp u é s d e l t r a ta m ie n to co n a g e n te s q u ím ic o s , h ) a lg u n a s p ro p ie d a d e s

f ís ic a s y q u ím ic a s d e lo s m u tá g e n o s r e la c io n a d a s c o n su r e a c t iv id a d in v itr o e in v i v o . S e in d ic a n ta m b ié n

a lg u n a s p r e c a u c io n e s n e c e s a r ia s p a ra e l a lm a c e n a m ie n t o y e m p le o d e m u tá g e n o s q u ím ic o s m u y a c t i v o s .

1. INTRODUCTION

Considerable progress towards the attainment of application-oriented goals in mutation research has been achieved in the past five years. The interaction and co-ordination among members of the FAO/IAEA Study Group on Induced Mutations in Plants and their role in guiding others has contributed greatly to this progress. However, those interested in the practical applications should recognize that the methods of mutagen treatment employed in most induced mutation research are as yet designed to distinguish and evaluate parameters affecting the biological activity of the mutagen or to better understand the mutation process rather than meet more practical goals.

* S c i e n t i f i c P a p e r N o . 3 5 9 6 , C o l l e g e o f A g r ic u l t u r e , W ash in g to n S t a t e U n iv e r s i t y , P u llm a n , P r o je c ts

1 7 4 6 , 4 7 4 6 , 8 783 an d 1 0 6 8 . R e s e a rc h su p p o rte d in p a rt b y P u b lic H e a lth S e r v ic e g r a n t G M 1 0 8 3 8 - 1 0 , and

fu n d s p r o v id e d b y th e W a sh in g to n S t a t e U n iv e r s ity R e se a rc h and I n s t itu t io n a l G ra n ts C o m m itte e s ;, th e W a sh in g to n

A g r ic u l t u r a l R e s e a rc h C e n t e r , an d th e U . S . A t o m ic E n e rg y C o m m is s io n C o n t r a c t A T ( 4 5 - l ) - 2 2 2 1 . A E C P ap er

R L O - 2 2 2 1 - T 3 - 8 . P art o f th e r e s e a r c h r e p o rte d in th is p a p e r h a s b e e n e a r n e d o u t u n d er R e s e a rc h A g r e e m e n t s

w ith th e In te r n a t io n a l A t o m ic E n e rg y A g e n c y N o s 3 2 1 / C F and 6 1 5 / C F .

95

96 KONZAK et al.

It is in fact evident that despite the attention on the importance of basing treatment regimes on knowledge of the factors affecting mutagenic activity, still too much mutation work published today deals with empirical methods, including mutagen comparisons which have little meaning. Also, while there has been a marked and general improvement in the quantitative basis of mutation experiments reported in the literature, still too many studies involve only small size samples, and must be considered only exploratory.

The recently published Manual on Mutation Breeding [1] may be a timely and helpful guide to improved experiments. However, many important problems basic to the future of mutation research and breeding remain unsolved although recent discoveries have revealed new avenues of investigation that could yield fantastic dividends.

In this paper we shall discuss research bearing on basic problems and on new avenues of investigation. We shall consider the subject primarily from the standpoint of mutagen treatment methods including the philosophy and goals of certain pre-treatments and post-treatments. In order to focus attention on this aspect of mutation research, the scope of this paper will be restricted largely to treatments of seeds with some of the more potent alkylating mutagens. Some pertinent new results with gamma radiation of seeds also will be considered. We shall be concerned with methods'of mutagen treatment which pertain to these goals of mutation research and mutation breeding: 1) greater precision in mutation experiments, 2) increased mutagenic efficiency, 3) greater mutagenic specificity and 4) increased input efficiency and convenience.

In earlier papers from this laboratory [2,3,4] the systems concept has been used to envision the interrelation between the in vitro mutagen treatment medium, the biological material, and the interaction of the two. This concept with all its implied complexities may be an over-simplification although it has proved useful. However, the dimênsions of this concept may be greatly increased by the inclusion of systems associated with pre- and post-treatments and the post-mutagen treatment recovery and development phases of the biological material. The possible impact of an action by a component of one system on the effects of interactions of that component in another system also must be considered.

Some important new developments in mutagen treatment methods serve to illustrate ways in which certain important factors affect the observed biological actions of mutagens. These new developments relate to the roles of: 1) pH and buffers, 2) temperature, 3) redrying, 4) post-treatment washing, 5) tissue hydration, 6) metabolic state and cell stage, 7) posttreatment modification by chemicals, and 8) some physical and chemical properties by mutagens to their in vitro and in vivo reactivity.

2. FACTORS AFFECTING ACTIONS BETWEEN AND WITHIN SYSTEMS INVOLVED IN MUTAGEN TREATMENTS

2. 1. pH and buffers

A number of effects of pH and of different buffers in chemical mutagen treatment media can now be described. The effect obtained may be specific for each agent and the interactions may differ with different biological ma-

METHODS OF MUTAGEN TREATMENT 97

terials. Nawar [5], Wagner et al. [6] and more recently Nawar et al. [7] have demonstrated for some nitrogen mustards and the related ethyleneimine (El) that the hydrogen ion concentration determines the proportion of neutral (imine) form of mutagen molecules present in the medium. This aspect is of critical importance to the interaction of the mutagen with the biological system, since the external membranes of the biological system (in this case barley seeds) offer a barrier to the passage of the immonium (+ charged) form. Yet, the immonium form of the mutagen molecule is the most reactive in the in vitro system. There may be analogous systems interactions which involve other mutagens.

Experiments suggest that the biological effects of N-methyl-N-nitroso- urea (MNH) also may be influenced by various treatment conditions including pH. The rate of decomposition increases with 1) an increase in pH, 2) an increase in the concentration of phosphate buffer, and 3) an increase in light intensity (photolysis) [8]. The latter aspect will not be considered here.

Loveless and Hampton [9] and McCalla et al. [8] supposed that the decomposition of MNH yields nitrous acid (HNO2) at low pH while diazomethane is produced under alkaline conditions. Both of these are known mutagenic agents in some in vivo systems, but neither of these degradation products cause chloroplast mutations in Euglena [8 ]. Thus, the entry of MNH per se into cells is required for production of biological effects [8]. More recently Loveless and Hampton [9] arrived at a similar conclusion.

Formation of labile degradation products (HNO2 and diazomethane) in the mutagen solution or in the cell, may result in strikingly different biological effects when treatment conditions are changed, as a consequence of consecutive and/or competing reactions in the mutagen treatment system. Our results as well as those of Velemînskÿ and Gichner [10] indicate that pH of the mutagen solution has a very pronounced effect on mutagenic effectiveness. Phosphate buffer alone influences the growth of Himalaya barley seeds, var. Cl 620 (Table I). Immersion in pH3 buffer reduced the seedling height 11. 92% when compared with the pH7 buffer treatment. Likewise,45 582 M2 seedlings were produced by the pH7 buffer control but only 31 177 seedlings were produced by the pH3 buffer control. The MNH treatments caused less Mx seedling injury when applied in pH7 buffer than when applied in either pH6 or pH3 buffers. The number of Mj spikes and M2 seedlings produced was greatest for the pH7 treatment. Although the percent Mi spikes with mutations and percent M2 mutant seedlings were not different, the actual number of mutants produced was greater for the pH7 treatments because the survival was better. Mutagen concentration also appears to have a decisive role in determining mutagenic efficiency (mutations/% damage) under alkaline conditions (Table II). Mutagenic efficiency was highest at an MNH concentration of 1. 0 mM and pH7 and lowest under acid conditions. However, concentration of MNH appears to be less important in determining efficiency at low pH's.

The MNH-pH-seed interactions thus seem very complex indicating that the mutagenic and/or lethal effects of MNH may not be ascribable to either of its reactive degradation products, at least until they are produced inside the cell.

The interactions of pH and buffer type involving the biological system (pre-soaked Himalaya barley seeds, var. Cl 620) show that the induction of mutations by isopropyl methane sulphonate (iPMS) is largely unaffected by either pH or buffer type, whereas both have a significant effect on injury

TABLE

I. INFLUENCE

OF pH

AND

MUTAGEN

CONCENTRATION

ON THE

EFFECTS

OF N-

METH

YL-N

-NIT

ROSO

UREA

TREATMENTS

TO

BARLEY

SEEDS, VARIETY

HIMALAYA

CI 620

98 KONZAK et al.

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о t-ч ф О <t смо СОоосмо соо о3тз /■*> аS'S ТЗ ф •нсо о <j-чО о о Г—1Г-»о LOсмt-Чф а Си CMI—1со смсмсо т-ЧРйС0| л

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TABLE

III.

INFLUENCE

OF pH

AND

BUFFER

ON THE

EFFECTS

OF MUTAGEN

TREATMENTS

TO BARLEY

SEEDS, VARIETY

HIMALAYA Cl

620

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1 0 2 KONZAK et al.

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METHODS OF MUTAGEN TREATMENT 1 03

to the biological system — here measured in terms of plant survival (Table III). In contrast, with diethyl sulphate (dES) the pH and buffer type influence both mutation and Mi plant survival. The different in vitro reaction mechanisms of iPMS and dES may explain this. Recent studies [ 11 ] showed that the degradation rate of iPMS, reacting by an SN1 reaction mechanism was unaffected by pH, buffer type, or buffer concentration, whereas the degradation rate of EMS reacting by an SN2 reaction mechanism was little affected at pH3, increased moderately at pH7, and increased significantly at pHll, The pH7 phosphate-buffered diethyl sulphate unexplainably had the most severe effect on the biological system. However, consistent results are that pH8 Tris [2-amino-2(hydroxymethyl)-l, 3-propanediol] buffer or pHl 1 phosphate buffer favour relatively better survival at relatively high rates of mutation. This is illustrated by their higher mutagenic efficiencies (Table IV). These results demonstrate independent actions of pH and buffer on the biological system from those on the degradation of the mutagen. Belli and Cervigni [12] and our unpublished results show that barley seeds take up acid from the treatment medium. Thus, pH8 Tris buffer or pHll phosphate buffer appear appropriate for alkyl alka.ne sulphonate or diethyl sulphate treatments. The buffers may affect membranes of the seed testa without altering pH within the tissue. Thus, in certain systems treatments involving low pH buffers should be avoided to prevent adverse effects on biological systems. Recently, however, a different chemical, sodium azide, was found to be a most effective and efficient mutagen on Himalaya barley in the pH range below 7 (Table V) [13]. A yield of 17% mutant spikes from 4 h treatment with 10' 3 M azide at pH3 certainly classifies this chemical among the highly effective mutagens.Azide appears to deserve a place among the more efficient mutagens.The ratio, mutation yield/aberrant cells, shows that 10"3M azide treatments at both pH3 and pH7 are highly efficient. Since azide is known to act as a metabolic inhibitor and since the effective concentration range (10‘3M) appears to suggest such an action, the interactions of azide in the various systems will be considered later. However, the ionic state of the effective moiety also may be the critical factor as was noted earlier for El. The neutral form of the azide molecule is in fact more abundant in vitro at low pH than high pH (pK = 1. 9 X 10 5). Therefore, the effect of pH on the in vitro chemical system must be considered independent from, but in relation to, the molecules which pass through the biological membrane. Only these molecules may then interact with cell components. When damage to the membranes of the biological system can be avoided or minimized by taking advantage of their protective mechanisms, increased mutagenic effectiveness and efficiency may be achievable.

As a component of the post-treatment medium, phosphate buffers at higher pH levels have variously but inconsistently given beneficial effects [3, 14, 15, 16]. However, further study of the effects of buffers, "scavenger" chemicals, or nutrient media as post-treatments may be worthwhile.

2.2. Temperature

Increases in temperature are known to appreciably increase the rate of most biochemical reactions, whereas physical processes, such as diffusion, are comparatively little affected. Rates of biological reactions are commonly changed by a factor of two for each 10°C change in temperature.

104 KONZAK et al.

However, in vitro degradation reactions of the mutagen may be increased by as much as a factor of four per 10°C difference [3,4, 17]. In contrast, diffusion rates when they apply are affected in proportion to absolute temperature [18,19].

These temperature effects can be used to advantage in the following way: seeds can be immersed in mutagen solutions at low temperature, i. e. 0-10°C, for sufficient time to allow adequate infusion of the mutagen throughout the cells of the embryo. Then, the seeds so treated might be transferred to a fresh mutagen solution (or perhaps to a plastic bag immersed in water) at a temperature as high as 40°C to increase the in vivo reaction rate of the mutagen. The change from 0 to 40°C will increase the in vitro degradation rates of dES or EMS by about 200 times [3, 19]. The period of treatment at high temperature should be very short relative to that at low temperature. After these treatments, appropriate post-treatments such as washing can be applied.

The bases for use of the procedure are 1) the observations of Heiner [20] that mutation yield was increased by increasing the temperature after a long period of exposure to mutagen at low temperature, 2) the more recent demonstration by Walles [21] that the kinetics of the uptake of EMS are those typical of diffusion which change only very little per 10°C change in temperature, and 3) the degradation and probably in vivo reaction rates of such agents as ethyl methane sulphonate (EMS) and dES are increased by as much as a factor of three or even more per 10°C difference in temperature [19].

Our earlier experiments [3, 20] showed that treatment of seeds at low temperature (0-l0°C) with chemicals like dES were more efficient than treatment at high temperature (30°C). Further work on this aspect in view of new knowledge is probably merited. However, recently we found that temperature had no effect on the rate at which biological effects are induced by iPMS as long as adjustments in treatment time are made for the rates of mutagen degradation at different temperatures and the treatment times were long enough to achieve concentration equilibrium between the treatment medium and the seeds (Konzak et al., unpublished).

2.3. Redrying of seeds

The primary purpose of redrying seeds after mutagen treatment is to gain convenience in handling treated seeds. Dry seeds normally can be prepared ahead of time at the convenience of the invéstigator and stored or sent through mail. In addition, redrying of seeds facilitates machine planting, often at a considerable saving in labour and time with increased uniformity of placement. Some of the consequences of redrying are concerned with the control of delayed effects, to be discussed more fully with posttreatment washing. Suitable redrying procedures have not been fully tested for seeds treated with all of the more potent chemical mutagens, although the procedures for irradiated seeds seem satisfactory [22]. Delayed effects have been minimal upon redrying seeds treated with the faster reacting iPMS and dES, but have been severe if slower hydrolyzing agents such as EMS are used [3, 4, 15,16].

Gaul and associates [23, 24, 25]. found that seeds treated with relatively high concentrations of EMS and washed for 24 h at 24°C could be redried and stored at -20°C without any increase in damage. More recently Gichner

METHODS OF M UTAGEN TREATMENT 105

and Gaul [26] have determined that if EMS-treated barley seeds are postwashed in flowing water at 25°C for 24 h then redried at 40°C to about 5% moisture, they will suffer no increase in damage during storage. If similarly treated seeds were stored at 20 or 13% moisture, they suffered increased damage. In contrast, if the seeds were held at 30% moisture during storage, there was a decrease in the level of damage observed after a six-week storage period.

The general cause of delayed effects is active mutagen retained in the tissue after treatment. Redrying has the effect of rapidly increasing the in vivo mutagen concentration, and the period over which delayed effects can be expected will often be related to hydrolysis rate of the ■ mutagen [15]. Mutagen degradation products may cause appreciable damage in vivo. Redrying has proved an excellent technique for detection of small amounts of mutagen that prove damaging in vivo when the water content of seeds is reduced. Small amounts of mutagen may not be detected in wet systems because the greater water content can more effectively compete for and degrade any remaining mutagen.

2.4. Post-treatment washing

Delayed effects of mutagen treatments have long been known, and have been described from experiments with widely different types of chemicals and radiation [2, 3, 4, 27, 28, 29, 30]. Rates at which the delayed effects develop differ depending on the mutagenic agent and treatment conditions. However, the delayed effects have appeared to represent an uncontrollable effect.

The main delayed effects of radiation were traced to the induction of potentially damaging reactive chemical intermediates by sparsely ionizing radiation in relatively dry tissue. The stability and lifetime of the reactive intermediates interpreted as free radicals, was shown to be dependent on tissue water content [31]. Prevention of delayed effects can be achieved by using seeds of more than 12% water content for gamma-radiation treatment. Control of dose can be achieved by preventing exposure to oxygen or exposing to a high oxygen concentration by post-soaking in water to raise tissue water content sufficiently to quench or react any free radicals present [1, 22].

Delayed effects of chemical mutagens were first noted by Auerbach [29, 30] in treatments of Drosophila with nitrogen mustard. Studies on the delayed effects of chemical mutagens on seeds have been made extensively following reports from our laboratory [3,19]. As mentioned earlier, the delayed effects following treatment of seeds with EMS have perhaps received .the most attention since they have been the most difficult to bring under complete control [32, 33].

Data of Narayanan [15] demonstrated that the rates at which damage increased during storage following treatment and redrying was approximately proportional to the different in vitro degradation rates of EMS and dES. He found also that post-washing in flowing water was far more effective in reducing the post-treatment effects than was soaking in standing water. These results were confirmed and extended recently in experiments in our laboratory by N. Van Mung (1970, unpublished). Further, the relative lack of effectiveness of post-treatment soaking in a standing water system was demonstrated rather impressively by Satpathy and Arnason [34]. In

1 0 6 KONZAK et al.

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their study EMS treated seeds were rinsed four times, placed in water and then stored at four different temperatures for 12 and 24 h. The water in the storage vessels was changed after one hour. Results indicated a distinct influence of temperature on mutation frequency in barley (Qm between 0°C and 40°C ranging from 1.4 to 2. 5)1. These results indicated that there was active mutagen still present in the seeds, and that the mutagen concentration was not reduced appreciably by leaching into the water surrounding the seeds. In fact the surface wash and one hour storage water change probably accounts for the difference in Qm values observed in their experiment from that of Froese-Gertzen et al. [4, 19,35].

Our recent results also have a bearing on this problem. We found from preliminary experiments that post-washing at elevated temperature was in itself damaging to the growth of seedlings2 . The conclusion from these tests was that the increased damage during redrying was due largely to the sensitivity induced by washing at higher temperatures (15°C and 25°C).

Our washing involved immersing plastic net bags of treated seeds in a water-filled plastic sink with an overflow drain. Slowly flowing tap water was passed through a 60 ft coil of 5 mm i. d. tin-lined copper tubing, cooled by immersing it in a refrigerated water bath kept at 0 ± 0. 05°C. EMS- treated seeds post-washed in the flowing water at 3 ± 2°C appeared to be less sensitive to the washing and redrying post-treatments than those post-washed at 15 + 1°C (Table VI). Those seeds post-washed at 25 ± 1°C and then washed at either 15 ± 1°C or 3 + 2°C were injured most. However, these experiments showed post-washing for more than 48 h in flowing water at 3 ± 2°C seemed necessary if delayed effects were to be avoided when seeds were redried to about 10% moisture and stored at 23 ± 2°C.

With as much as 96 h post-washing at 3 ± 2°C, however, damage sustained by the washed and redried seeds still appeared to be greater than that shown by the non-dried seeds. But similarly post-treated controls (receiving no mutagen) showed increased levels of injury as storage progressed. Thus, when the data for the treated, washed and redried seeds were compared with similarly post-treated controls (Fig. 1), the results indicated that post-washing between 72 and 96 h in flowing water at 3 ± 2°C prior to redrying would be adequate to prevent the delayed effects.

These results need to be confirmed by analyses of induced mutation frequencies, Mi plant survival and Mi sterility estimates. Nevertheless, the evidence obtained so far suggests that as a means of stopping the mutagen treatment it is extremely difficult to achieve complete removal of the active mutagen retained in seeds by washing. The recent data of Gichner and

1 Q io = m u ta tio n f r e q u e n c y a t t e m p e r a tu r e t / m u t a t io n f r e q u e n c y a t t - 1 0 ° C .

2 C o n d it io n s o f t r e a t m e n t w e r e as fo l lo w s :

a ) P re s o a k — T h e H i m a la y a b a r le y s e e d s w e r e s o a k e d in f lo w in g ta p w a t e r fo r 24 h a t 3 + 2 ° C .

b) T r e a t m e n t - T h e se e d s w e r e t r e a t e d in 0 . 15 M E M S s o lu t io n p re p a re d in 0 . 1M pH8 T r is b u ffe r

fo r 2 h a t 2 0 ° C .

c ) P o s t - t r e a t m e n t W a sh in g — T h e se ed s w e r e w a sh e d in f lo w in g w a t e r a t th e in d ic a t e d te m p e r a tu r e

fo r th e v a r io u s t im e s o f w a s h in g .

d) D r y in g — A s a m p le o f se e d s o f e a c h t r e a t m e n t w a s p la n t e d i m m e d i a t e l y , an d th e r e m a in in g

se e d s w e r e d r ie d a t c a . 2 2 ° C u n d er a n e x h a u s t h o o d . T h e s u c c e s s iv e p la n t in g s w e r e m a d e a f te r

1 d a y , 1 , 2 , 3 an d 4 w e e k s o f d r y in g . S e e d lin g s w e r e c u ltu r e d a t 2 2 ± 2 ° С u n d er c o n s ta n t

i l lu m in a t io n w ith c a . 80 f t - c a n d le s c o o l w h it e f lu o r e s c e n t l ig h t a c c o r d in g to sta n d a rd p ro c e d u re s

d e s c r ib e d b y K o n z a k e t a l . [ 2 2 ] .

1 0 8 KONZAK et al.

F IG . 1 . I n f lu e n c e o f p o s t - t r e a t m e n t w a s h in g , r e d r y in g a n d s to r a g e o f H i m a la y a b a r le y se ed s o n t h e ir su b seq u en t

s e e d lin g g r o w th . E M S tr e a t e d s e e d s , c o m p a r e d w ith c o n tr o ls th a t r e c e iv e d s im ila r p o s t - w a s h in g , r e d r y in g an d

s to r a g e p o s t - t r e a t m e n t s .

Gaul [26] indicate that the mutagen dosage period can be effectively terminated by first washing then drying to a low moisture level. We found that post-washing at the temperature used by these authors was more injurious to hulless Himalaya barley than to the hulled barley. The higher temperature would also stimulate some increase in reaction of mutagen retained by the seeds, particularly during the early part of the post-washing period, allowing somewhat less control of dose than was achieved by our method.

Some basic questions can be posed from the results of these experiments. Gichner and Gaul [26] found that redrying at 40°C to low (ca. 5%) moisture effectively arrests the activity of EMS but does not if seeds are at 12% moisture and stored at room temperature. However, when similarly treated, post-treated, and redried seeds were stored at higher moisture (30%) there was a significant recovery. They interpreted this result as indicating action of cellular repair processes. If this is so, then whatever effect can be reversed by storage at 30% moisture probably also can be removed by extensive washing in our experiments. Since our seeds were treated with comparable but somewhat lower doses of mutagen and since


these authors still observed increased damage in treated, post-treated and redried seeds stored at 12 to 20% moisture, alternative explanations seem appropriate.

Our recent data suggest that most of the delayed effects may be due to active mutagen so tenaciously retained in the seed tissues, probably in lipids, etc., that only by persistent, extensive washing can the final amounts be removed. At 30% moisture there may be sufficient water available for retained active mutagen to be hydrolyzed. Another interpretation is also plausible but less likely. Some of the delayed effects may be due to re-alkylations which involve shifting of alkyl groups from phosphates in DNA to other acceptor molecules. Results from storage of post-treated seeds at 30% moisture by Gichner and Gaul [26] would suggest this interpretation may be valid, and because the alkyl groups should be susceptible to hydrolysis from the phosphates, these results would not be contradictory to those from our laboratory. In these experiments we may not only be dealing with the matter of dosage control, but rather we may also be concerned with the stages in the fixation of mutations.

For the researcher the validity of these hypotheses is testable. If, in fact, the delayed effects are caused only by retained mutagen, and the mutagen can be removed by extensive washing, increasing levels of biological effects should be obtained by increasing the EMS concentration in a step-wise fashion without any delayed effects. Tests also using the shorter-lived dES and iPMS3 would also be appropriate. In contrast, the extensive washing should nullify the expected dose response if the post-treatment interrupts the fixation of mutations via the action of biological repair mechanisms or hydrolysis. In each case, however, the mutagen treatment period should be relatively short to permit a distinction of the immediate from the after effects of the agent. The post-washing results reported by Bender and Gaul [23, 40] as well as those from our experiments appear to support the data of Walles [21]. As mentioned earlier, Walles showed that the rates of mutagen uptake and out-diffusion for barley seeds followed simple diffusion kinetics, i. e. the rates are proportional to the difference in absolute temperature, degrees K, rather than degrees C. Thus, the proportionate effect of a 10°C difference in temperature is small for diffusion, but can be large for chemical or biochemical reactions. However, the actual rates of mutagen uptake and out-diffusion are not the same, because of different diffusion constants of the various interfaces involved from the relatively simple treatment medium to the more colloidal complex in the seed tissues.

The low mutagen concentration in the seed is of consequence only when the seed is to be redried. In the highly hydrated system, the hydrolysis process and reactions with other solutes compete effectively to reduce the number of mutagen molecules able to cause biological effects. The drying process usually involves rather rapid loss of water from the tissue,

3 T h e d iffe ren t reac tion m echan ism o f iPM S com pared w ith dES and EMS has been described [3 6 ,3 7 ].

M o re re cen tly T u rtóczky and Ehrenberg [3 8 ] and Osterman e t a l. [3 9 ] suggest that c e l l k il l in g by these two

types o f agents m ay h a ve d iffe ren t bases. H ow eve r, the d e layed e ffe c ts they no te for dES and EMS as com pared

w ith iPM S m ay b e re la ted to the d iffe ren t possib ilities associated w ith their reac tion m echanism s and ch em ica l

properties (rea c tio n ra tes). N o in teractions w ith iPM S m ay have been observed because the ra te o f reac tion

o f iPM S w ith the b io lo g ic a l system is the sam e as its hydrolysis ra te . Th is is n o t necessarily so fo r dES and EMS.

1 1 0 KONZAK et al.

increasing the relative concentration of dissolved mutagen and the concentration of other materials in the tissue as well, perhaps forcing some reactions that might be less favoured in the "wet" system.

Gichner et al. [41] report somewhat different effects of post-treatment washing and redrying with the N-methyl-N-nitrosourea (MNH) and N-ethyl- N-nitrosourea (ENH) treatments although their studies did not include storage after redrying. Post-washing at 24°C for 6 h almost eliminated the effects of a3 h treatment with non-buffered 10 mM MNH or of a 12 h treatment with1. 6 mM MNH, whether or not the seeds were redried after the treatments. Somewhat different results were obtained with ENH. No post-washing after a3 h treatment at 24°C with 14 mM ENH resulted in death of all seeds,' whereas survival was appreciably increased with 6 h of washing. Washing as longas 24 h caused only slight and non-significant decreases in damage and mutation.

2. 5. Tissue hydration

The entry of chemical mutagens into seeds and other tissues is facilitated when they are fully hydrated [l] ; thus, it appears advisable to hydrate seeds before they are immersed in mutagen solutions.

2.6. Metabolic state and cell stage

2. 6. 1. Cell stage

Several reports now have demonstrated that the population of cells in the shoot apex of seeds may be mixed withregardto the proportion with 2C and 4C levels of DNA [42,43,44,45]. The results of experiments described above could be somewhat different depending upon whether the apical meristem cells of the seeds used for pre-soaking and mutagen treatment were 2C or 4C at the start of the presoak. Avanzi et al. [42,44,45] have reported results which suggest that year-to-year variation in the development of meristems of seeds may be a source of differences in sensitivity. They observed differences in sensitivity of two durum varieties in one experiment, but when the experiment was repeated with an attempt to correlate sensitivity with cellular DNA, no differences were observed. Another question about this research relates to whether the cell§ measured for DNA content in cereal seeds could actually represent the specific cells participating in the development of the tillers.

Ratios of mutations in mutant barley spikes generally suggest that the number of mutant seedlings represent one-half of a simple gene segregation. Gaul [46] has suggested from similar data that the primary tillers of barley arise from four meristem cells. However, the same data also suggest that the mutations occurred on the chromatid level or in a single Gi cell having the 2C content of DNA. Smaller mutant sectors in spikes also are occasionally observed. These might result from 4C cells.

2.6.2. Metabolic state

Pre-soaking of barley seeds under favourable conditions for initiating mitosis in primordial cells appears to bring about not only a general in-

METHODS OF MUTAGEN TREATMENT 1 1 1

crease in sensitivity to EMS and dES but there is also a marked increase in sensitivity after certain periods of pre-soaking, and then a decrease [16,47, 48, 49, 50, 51, 52]. Certain studies have suggested that the enhanced sensitivity is associated with the time at which the cells have initiated DNA synthesis. Studies indicate "pulse" or brief treatment with a high concentration of mutagen induce marked differences in the mutation spectrum depending upon the mitotic stage of the cell at the time of treatment [50]. Refinement of methods developed from these indications could represent a remarkable advance in the control of the response of cells to mutagens. Although these results seem to defy an alternative interpretation, greater precision in such experiments is obtainable, and confirmation using improved techniques would be worthwhile. Grant et al. [51] have described a procedure using 5-aminouracil to increase cell synchrony in seeds being prepared for mutagen treatment. This technique appears promising, and we have tried it with interesting results. We redried seeds after treatment and post-washing, and upon redrying found considerable injury when the seeds were grown in the laboratory for seedling measurements. However, the seeds sown in the field grew comparably well. Analyses of the progeny have not been completed so we do not know if the technique was successful. Even so, pre-soaking in aerated water appears to be effective in advancing the cell stage. It remains to be determined whether cells in the S-stage can be redried after the mutagen treatment and post-treatments have been applied.

The selection of the mutagen for such a study is important. The evidence suggests that EMS quite evidently is not a very good mutagen to use if precise data are to be obtained. The hydrolysis rate is far too slow, and complete out-diffusion of chemical retained in seeds after treatment is accomplished only by prolonged periods of washing. A far better agent with similar chemical properties but having a much faster rate of hydrolysis is dES. Since dES has proved to be one of the more powerful mutagens, its reaction rates in biological systems must also be rather fast relative to those of EMS. With dES, the concentrations and treatment times needed for "pulse" treatments will be far less than those required with EMS and much less post-washing would be required to remove the residual mutagen. Recently Gopal-Ayengar et al. [53] observed only an increasing sensitivity to dES treatment with increased pre-soaking. The increased sensitivity began with the time of 3H thymidine incorporation and continued to increase with increased 3H thymidine incorporation. However, their treatment times may have been too long for the detection of any peaks in sensitivity caused by the time of pre-soaking.

2. 7. Post-treatment modification by chemicals

Some particularly interesting work aimed at modifying the biological effects of mutagen treatments has been conducted in our laboratory by Sideris et al. [13] and Nilan [54]. In these studies, the chemical sodium azide was found to act most effectively as a respiratory inhibitor in barley seeds when used in pH3 phosphate buffer. There was a small effect at pH7 but very little at pHll. At pH3, an azide post-treatment of gamma-irradiated seeds appreciably increased the frequency of aberrant cells (cells with at least one chromosome or chromatid bridge or fragment). However, the

1 1 2 KONZAK et al.

effect of azide post-treatment on irradiated seeds was virtually negated if the azide solutions were saturated with oxygen rather than nitrogen gas.

When azide is administered as a post-irradiation treatment, the result is a marked increase in aberrant cells and a significant decrease from additivity in the yield of mutations, considerably lowering the mutagenic efficiency. The effect increases with radiation dose, supporting the hypothesis that the azide increases the ability of radiation-induced biological lesions (potential aberrations or mutations) to become realized in terms of aberrations.

Available data suggest that azide inhibits oxidoreductases (including catalase and peroxidases). In the absence of radiation, this inhibition may permit cellular peroxides to become effective as mutagens, whereas normally their destructive potential would be kept in check by the peroxidase. Similarly when azide is administered after irradiation the inhibition of oxidoreductases prevents these enzymes from inactivating peroxides produced following the radiation treatment.

The concentration of ionic state of azide which may pass more freely through the seed membrane or the rate at which azide is degraded in solution is influenced by pH. Whether azide might be metabolized by cells to yield mutagenic degradation products can only be speculated as a possible alternative to the suggested peroxide mechanism for the induction of mutations.

Modification of repair processes following mutagen treatment also appears to offer unusual potential for increasing both mutagenic effectiveness and more importantly mutagenic efficiency. Misrepair might also be increased by infusing such compounds as base analogues into the pool of substances available to the cell for its repair processes, or misrepair might be enforced by use of growth stimulants. Research in this direction begs for increased attention.

Mutagen combination treatments designed to take advantage of the different properties of mutagen types also seem to have potential for increasing mutagenic efficiency [55].

With radiation, there is evidence that greater specificity in the effects of gamma rays can be achieved by the use of pre-treatments with a base analogue such as 5-bromouracil deoxyriboside [56]. The mechanism of action has been postulated and the idea needs further study. Brock [57] also indicates that it may be possible to direct the induction of mutations by use of an analogue to activate the regulatory site on the cistron. Evidence suggests that the activated locus will then be more specifically susceptible to reaction with a mutagen. Changes induced in the "activated" locus would presumably then be subjected to the possibility of misrepair or mistakes during replication.

2.8. Some physical and chemical properties of mutagens to their in vitro and in vivo reactivity

Results of preliminary studies on the relation of basic chemical and physical properties of mutagens to their type of biological action were reported earlier from our laboratory [3,4,19,35], and although we have completed further extensive investigations with the sulphonates, the data have not yet been prepared for publication. Recent studies reported by Osterman et al. [39], and by Turtôczky and Ehrenberg [38] are aimed


at similar objectives. These reports are extensive, very detailed and complete. They merit intensive study especially by those interested in understanding some of the basics of mutagenesis, and by those planning further research, particularly with any of the chemicals they compared.Of particular importance is their comparison of mutagens in terms of the Swain-Scott substrate constant (s) which describes the dependence of bimolecular rate constant on nucleophilicity of the receptor molecule.

Their findings suggest some reasons why the sulphonates and related sulphates, EMS, dES and iPMS are among the most effective mutagens.Further analyses would be desirable comparing their results with those of new experiments in which the delayed effects of the mutagens are prevented. In particular, our results with iPMS, while agreeing with their data, suggest that iPMS may not be a good mutagen. Our data (unpublished) with both yeast and barley clearly show that the rate at which mutations are induced in these organisms is proportional to the degradation rate of iPMS in solution.

This affirms other data suggesting that with agents such as iPMS which react by an SN1 reaction mechanism, the carbonium ion intermediate is probably non-selective and is competed for by cell sites and water. Whether the lifetime of the carbonium ion of iPMS is sufficiently long to provide for some selectivity would be well to know; however, the evidence suggests it is not. On the other hand, EMS and dES react by an Sn2 mechanism for which rates depend upon the available concentration both of the mutagen and the nucleophile (OH- of water or nucleophilic receptive sites in the cell). No carbonium ion intermediate is formed.Thus, these mutagens have the potential to be selective although they may be relatively more selective for SH groups of proteins rather than mutation- yielding sites on DNA. The data of Osterman et al. [39] suggest that this may be the case for EMS and other alkyl sulphonates. However, the relative availability of SH in cells differs markedly with the stage of mitosis and metabolic state. Thus, the potential particularly of the faster-reacting dES may be better than may appear. Moreover, the factors controlling the reactive properties of other mutagens, particularly the alkyl-nitrosoureas and urethanes should be determined. Some of these compounds appear to have potential for greater mutagenic efficiency than the sulphonates [43, 58].

3. CONCLUSIONS

In this review and analysis of some recent work relating to particular aspects of mutagen treatments, we have attempted to focus our concern on features of particular kinds of treatments which may pertain to the achievement of such goals as 1) greater precision in mutation experiments,2) increased mutagenic efficiency, 3) greater mutagenic specificity, and 4) increased input efficiency and convenience.

Contributing to 1) greater precision in experiments, is the considerable new knowledge on factors affecting in vitro and in vivo reaction rates of mutagens, which has become available since 1964. Pre-soaking of seeds to facilitate the diffusion of mutagenic and other substances through membranes and to achieve better infusion of mutagens into the tissues has already become commonplace. Most studies using chemical mutagens on seeds in-

114 KONZAK et al.

elude pre-soaking as a routine method. Post-treatment washing in flowing water, preferably at low temperature is a relatively new procedure which can appreciably increase precision in mutation experiments because it permits greater control of the mutagen dose. Likewise, knowledge of the influence of pH and of particular buffers on the in vitro degradation rate of the mutagen helps the investigator plan treatments to maximize the mutagenic effectiveness and thus reduce waste of usually relatively expensive chemicals. Knowledge of the influence of pH on the composition of the mutagen solution with regard to the1 ability of potentially reactive molecules to pass through membranes is an important discovery. Knowing that a mutagen is more or less reactive at one pH or another is of little value if the in vivo system effectively prevents entry of mutagen molecules because of their electrical charge.

The use of increasingly larger populations in experiments with mutagens and better biometric analyses has increased the probability that the results obtained are representative and therefore more applicable.

Much progress also has been made toward 2) increasing mutagenic efficiency. The question whether treatment at one temperature or another will affect the efficiency of certain mutagen treatments is yet unclear. With agents such as iPMS there is no difference as long as there is sufficient time available for the mutagen to infuse the tissues, and times of treatments are adjusted to account for the effect of temperature on the in vitro reaction rate. With dES, EMS and nitroso compounds, the influence on mutagenic efficiency may be possible by adjustments in the treatment temperature.A number of recent experiments suggest that particular pre-treatment, treatment and post-treatment regimes can markedly affect mutagenic efficiency. Selection of mutagens for use based on knowledge of their described properties and reported potential is a first step. The second is to apply the mutagen under conditions conducive to increased efficiency.The fact that a mutagen may prove more effective (higher mutation yield per unit dose) under certain circumstances than another may be insignificant if it is also highly effective in causing unwanted biological effects. A good case in point is the relative mutagenic effectiveness of gamma radiation which usually induces the highest yield of mutations per rad in wet and active tissues. Yet, under the same conditions, the yields of unwanted biological effects may be even greater per unit dose. Thus in all cases consideration must be given to the ratio of induced desirable to undesirable changes, whether or not this involves some waste of mutagen, since one of the lowest costs of most experiments is for the mutagen used.

The influence of pH and buffers on the mutagen and on the interaction between the mutagen or degradation products and the biological system is independent of the influence of pH and buffers on the biological system. Knowledge of these interrelationships allows the selection of treatment regimes which will avoid unnecessary biological damage. The new discoveries on methods for influencing repair and misrepair processes following mutagen treatments offer unusual promise for the future. Recent studies on the DNA content of cells in seeds suggest that all seeds cannot be considered alike. Preliminary tests on cytochemical analyses may be useful to determine which lot of seeds may respond to treatment best in terms of mutation yields and to determine which pre-treatment regime is necessary to obtain greatest mutagenic efficiency.


Considerable progress also has been made toward objective 3) greater mutagenic specificity. Specificity depends first on the selection of mutagens, which because of their physical and chemical properties offer appropriate potential for specificity. The mutagens must then be applied under conditions conducive to their selective action. Among the more potent mutagens, MNH, ENH, and relatively fast reacting dES may offer potential for high mutagenic efficiency and for studies of mutagenic specificity due to biological factors. Modification of the mitotic state and administering "pulse" treatments with relatively high concentrations of these mutagens appear to offer special promise. However, the cell state in which the mutagenic agent is most effective may not be the one in which the mutagen is most efficient or specific in action. Post-treatments with chemicals to influence those misrepair processes leading to mutation offer exciting new possibilities. Post-treatments with base analogues, amino acids and metabolic stimulators may increase specificity. Certainly post-irradiation treatment with azide increases chromosome aberration yields relative to mutations, but the effects could be gene specific. The use of selective chemicals to block or stimulate certain biochemical pathways during treatment appears to be another new approach worthy of further investigations.

Our final concern in this analysis is about 4) input efficiency and convenience. Both of these are important if mutagen treatment methods areto find much use and favour in practical work. What adjustments in a mutagen treatment regime can be made, for example, to take most advantage of a small supply of mutagen which for some workers may be both rather expensive and difficult to obtain? In this case, possibly the sequence 1) pre-soak, 2) low temperature infusion, 3) higher temperature treatment,4) lower temperature post-treatment washing, should be considered for seeds. This procedure may also be applicable to some other tissues.For the sake of convenience, possibly the whole sequence of pre-treatments, post-treatments, etc. , can be completed in a single seven to eight hour day.If not, might certain treatments be made overnight, some the next day, etc. , at convenient times? Redrying of seeds after the sequence of treatments greatly increases the convenience with which they can be managed in a practical schedule, considering aspects of weather, other responsibilities of personnel, etc. With EMS, the long times of post-washing are not the most convenient, but can be managed readily without too much difficulty. If a refrigerated water bath is not available, perhaps an ice bath will be suitable. The method of redrying seeds to low moisture content also appears useful. With these post-treatment procedures, seeds can be treated long ahead of time and stored until ready for planting or may be mailed from place to place as needed. Many laboratories now place seeds in plastic net bags for convenience in handling throughout the treatment sequence. Colour-coded clips and tags as well as attaching sets of bags together on a string as we commonly do also may improve convenience. Various similar schemes using colour coding and identifying numbers corresponding to a fully detailed work plan and time schedule might be applied.

Special precautions for the storage and use of potent mutagens are advisable. Among the safest of the potent chemical mutagens discussed here are the alkylating sulphonates and sulphates. These are not known to be effective carcinogens but their potency as mutagens should in itself command caution. Virtually nothing is known about the possible carcinogenic activity of sodium azide, although it is a common biochemist's tool.

1X6 KONZAK et al.

The most dangerous among the potent mutagens are the nitroso compounds and the family of imines. Virtually all of these are potent carcinogens and should be handled with extreme caution. The nitrosoureas and similar nitroso compounds must be stored in a cool place and not allowed to warm appreciably above room temperature as they have a tendency to explode or decompose by spontaneous combustion. A further precaution is to keep the powders in plastic bags in relatively small metal rather than glass containers preferably stored in an explosion-proof refrigerator. Solutions prepared from either the nitroso compounds or the imines should be made up in or near a fume hood. It is particularly important to work in a well-ventilated area since diazomethane gas is liberated rather rapidly when the nitroso compounds are prepared at high pH. When handling liquid mutagens like the sulphonates and some imines, a propipette must be used to avoid accidents. For those who wish to use the potent mutagens but feel they may not have suitable laboratory conditions, or skill in handling dangerous substances, we advise getting the co-operation from another person who is skilled, or obtaining the service from one of the major mutation laboratories in treating samples. In any case the potential hazard of powerful mutagenic agents should not be underestimated.

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[ 1 ] F A O / IA E A , Manual on M u tation Breeding, T e ch . Rep. Ser. N o. 119, IA E A , V ienna (1970 ).

[ 2 ] K O N Z A K , C . F . , N IL A N , R. A . , HARLE, J .R . , HEINER, R .E . , Con tro l o f factors a ffe c t in g the response

o f plants to m utagens, Brookhaven Sym p. B io l. 14 (1961 ) 128.

[ 3 ] K O N Z A K , C .F . , N IL A N , R. A . , W AGNER, J. , FOSTER, R .J . , "E ffic ien t ch em ica l m u tagenesis".

T h e Use o f Induced M utations in Plant Breeding (F A O / IA E A T e c h .M e e t in g , R om e, 1964), Pergam on

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METHODS OF M UTAGEN TREATMENT 1 17

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1 1 8 KONZAK et al.

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Occurrence o f 2C (G j ) and 4C (G 2 ) n u c le i in the ra d ic le m eristem s o f dry seeds in T r iticu m durum D es f.,

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agents under d iffe ren t conditions o f trea tm en t, Indian J. G enet. PI. Breed. £5 (1965 ) 24.

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seeds to ethy l m ethane su lfonate, Z . V ererbLehre 96 (1965) 13.

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treatm ents at d iffe ren t periods in the S phase o f D N A synthesis, Curr. S c i. _37 24 (1968 ) 685.

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the chrom osom e c y c le " (P ro c . Second Oxford C hrom osom e C o n f . , 1967), Chrom osom es Today 2

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M utations in Plants (P ro c . Sym p. Pu llm an, 1969), IA E A , V ienna (1969 ) 245.

[5 3 ] G O P A L -A Y E N G A R , A . R . , RAO , N . S . , JOSHUA, D .C , "M o d ific a t io n o f the e f f ic ie n c y o f

d ie th y l sulphate in r ic e seeds presoaked in w a te r ", Induced Mutations in Plants (P ro c . Sym p. Pu llm an,

1969), IA E A , V ienna (1969 ) 271.

[5 4 ] N IL A N , R. A . , "M u tagen ic s p e c if ic ity in flow erin g plants: Facts and prospects", these Proceedings.

[5 5 ] DOLL, H . , SANDFAER, J . , "M u tagen ic e f fe c t o f gam m a rays, d ie th y l sulphate, e thy l m ethane

sulphonate and various com binations o f gam m a rays and the ch e m ic a ls " , Induced Mutations in Plants

(P ro c . Sym p. Pu llm an , 1969), IA E A , V ienna (1969 ) 195.

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M utations in Plants (P ro c .S ym p . Pu llm an , 1969), IA E A , V ienna (1969 ) 305.

[5 7 ] BROCK, R .D . , "In creas ing the s p e c if ic ity o f m u ta tion " Induced M utations in Plants (P ro c . Sym p.

Pullm an, 1969), IA E A , V ienna (1969 ) 93.

[5 8 ] H ESLOT, H . , FERRARY, R . , TEM PE, J . , T h e r e la t iv e m u tagen ic e ffe c ts o f som e nitrosam ines on

barley seeds, M u ta tion Res. 3 (1966 ) 354.

DISCUSSION

A. ASHRI: When prolonged pre-soaking and post-treatment washing is practised should the material be aerated?

C. F. KONZAK: At the moment, it is not possible to give a firm answer to such a question. When material is washed in flowing water it is somewhat aerated — adding O2 to the wash-water could even have negative effects — since in some materials at least a phenomenon called ’'water sensitivity" occurs which could have a negative influence.

A. HAGBERG: Dr. Konzak, do you have information on genetic variation in the response to treatment with different mutagenic chemicals? There is a physiological variation in the ripe seed (demonstrated by Lexander


among others) depending on the genotype. This influences water uptake, etc. , and must have an influence on the "real dose" received. In Kristina barley, for example, there seems to be quite another stage of metabolism in the ripe seed. This should be looked into and can certainly explain differences like the one between Volla and Himalaya barley,

C. F. KONZAK: I am sure some differences in biological responses must be due to differences in "real dose" resulting from influences of membranes. However, this can be checked by testing a range of doses. Regarding other aspects, I think there could be differences in 2 С and 4 С conditions in apical cells in seeds depending on genotype and environment (season), which could affect the observed yield of mutants. This has been discussed in the paper. In this case the only solution to the problem seems to be tests of seed produced under different conditions, finding those that have 2 C, since the sector size from these primordia will be larger, resulting in greater efficiency for the effort. These certainly are problems that merit further experimentation. I think we now have methods with sufficient control of modifying factors that decisive results can be expected.

A. ASHRI: We found in peanuts differences between varieties in physiological sensitivity to dES, EMS and other mutagens; it was expressed both in percent germination and seedling size.

C. F. KONZAK: The phenomenon described seems not uncommon and needs further analysis, particularly to determine what part if not all are due to "environmental" differences and what part might be truly genetic rather than physiological.

H. HANSEL: Y o u presented a very clear effect of the washing after EMS-treatment on the seedling height. I would like to ask you, whether the washing had any effect on the number of mutations per Mi spike or on the M2 mutant frequency?

C. F. KONZAK: We do not yet have adequate data, and our new experiments have not yet been fully analysed. However, Dr. Gaul has reported results which are referred to in my text. You must realize of course that washing does remove a contribution to dose, and it: should be expected that the yield of mutants following any given treatment may be reduced. What needs to be looked at is the mutagenic efficiency of a given dose or series of doses. There is a suggestion that washing treatments may offer inproved efficiency due to a lowering of unwanted damage. We should have more data on this later.

H. JIMENEZ: Did you utilize chemicals such as Atrazine as a practical means to detect differences in the photosynthesis rate for the selection in segregant populations? If so, would you consider its use advisable?

C. F. KONZAK: We have not used that chemical. However, if Atrazine could be used to distinguish such differences, obviously this could prove a valuable method to obtain more photosynthetically efficient mutants. Certainly the idea of a chemical sieve to select cereal mutants with greater PEP carboxylase activity seems an excellent idea, and should certainly be investigated, since the practical impact of a mutation for greater carboxylase efficiency could be considerable.

EFFICIENCY OF MUTAGENESIS

H. GAUL, G . FRIMMELAbteilung fur Pflanzengenetik der Gesellschaft fur Strahlenforschung, Koln-Vogelsang, Federal Republic o f Germany

T . GICHNERDepartment of Plant Physiology and Genetics,Institute o f Experimental Botany,Prague, Czechoslovakia

E. ULONSKABayer. Landessaatzuchtanstalt,Freising-Weihenstephan, Federal Republic of Germany

Abstract-Resumen

EFFIC IENCY OF M U TAG EN ESIS .

Our present know ledge about the e f f ic ie n c y and e f f ic a c y o f various mutagens and se lec tion m ethods

is sum m arized ; exam p les are presented from the litera tu re and from published and unpublished results o f

our labora tories . "E ff ic ie n c y " is defin ed as the ratio o f ch lo roph y ll m utations to le th a lity , in ju ry , s te r ility

or ch rom osom e m utations. "E f f ic a c y ” refers to the pow er o f a m utagen to produce useful m utations; the

num ber o f se lec ted m acro- or m icro-m utan ts is re la ted to the to ta l number o f plants w h ich w ere a va ila b le

fo r s e le c tio n .

Neutrons have a h igher e f f ic ie n c y com pared to X - and gam m a-rays . W hether or not the e f f ic a c y is

increased is s t i l l an open question. Th e e f f ic ie n c y o f EMS was found in barley to be h igher than that o f

M N H and o f ENH . C om pared to X -ra ys , the e f f ic ie n c y o f EMS fo r M x le th a lity in barley appeared to be

about f iv e tim es h igh er, and fo r M j s te r ility about four tim es h igh er. Pre-soak ing o f barley seeds resulted

in an e ven h igher e f f ic ie n c y o f EMS com pared w ith the treatm ent o f dry seeds.

Storage or sh ipping as w e l l as m a ch in e -d r illin g o f seeds treated w ith ch em ica l mutagens requires

their red ry ing . EM S-treated barley seeds can be a r t if ic ia lly redried a fte r 18 h or m ore post-washing at 25°C .

Redried seeds w ith the ordinary moisture con ten t (10 to 14°}o) m ay be stored at 0°C or a low er tem perature.

I f the m oisture le v e l is 5<7o or less, these "superdry seeds” can be stored a t room tem pera tu re. In both

instances the o r ig in a lly induced m utation rate and b io lo g ic a l dam age is m a in ta in ed w ithout any s ign ifican t

ch an ge.

In breed ing program s, m acro-m utan ts m ay be u tiliz ed in the first p la ce as cross parents, whereas m ic ro -

mutants m ay be used d ir e c t ly . Th e e f f ic a c y o f EMS to produce m ild ew resistant mutants in barley was found to be

tw ic e as h igh as that o f X -rays . In contrast to these m acro-m utan ts, m icro-m utan ts occur much m ore fr e

qu en tly . F o llow in g X - irrad ia tion o f barley seeds, M 2 plants o f norm al appearance w ere taken at random

and se lec tion was conducted in subsequent generations betw een their p rogen ies, n am e ly betw een M 3 and

M 8 strains. A m on g 1000 M 2 progen ies derived from fe r t ile M j spikes, there was on the a verage one w ith

a y ie ld c ap ac ity 10°jo h igher than the m other v a r ie ty . Because o f this r e la t iv e ly h igh frequ ency, i t is

recom m ended that the m icro -m u ta tion techn ique be used for a continuous im p rovem en t o f n ew ly-in trodu ced

va r ie ties and in this w ay to extend their l i f e - t im e .

EFIC IENC IA DE LA M U TAG E N E SIS .

Se hace una recap itu la c ión de los con oc im ien tos que actua lm en te se poseen acerca de la e f ic ie n c ia

y e f ic a c ia de los distintos m utágenos y m étodos de s e lecc ión ; se c itan e jem p los tom ados de la literatura

en la m ateria y de los resultados, tanto publicados c o m o inéd itos, de los laboratorios de los propios autores.

La « e f i c i e n c i a » se d e fin e c o m o la re la c ión entre m utaciones c lo ro fí l ic a s y le ta lid a d , les ión , esterilid ad o

m utaciones crom osóm icas . La « e f i c a c i a » se re fie re a la capac idad de un m utágeno para producir m utaciones

provechosas; e l número de los m acro o m icrom utan tes se leccionados se re la c ion a con e l núm ero to ta l de plantas

disponibles para la s e le cc ió n .

En com parac ión con los rayos X y gam m a , los neutrones presentan una e f ic ie n c ia superior. Si aum enta

o no la e f ic a c ia , es una cuestión que está aún por reso lver . Se ha com probado que, en la c ebada , e l MSE

121

1 2 2 GAUL et al.

(m etanosu lfonato de e t i lo ) tiene una e f ic ie n c ia m ayor que la M N H (N -m e t il-N -n itro s o -u re a ) y que la ENH

(N -e t i l-N -n it r o s o -u r e a ). En com parac ión con los rayos X , la e f ic ie n c ia d e l MSE para la le ta lid ad M t en

la cebada parece ser unas 5 veces superiory para la e sterilidad M x unas 4 v eces superior. Rem ojando p rev ia

m ente las sem illas de cebada , la e f ic ie n c ia d e l MSE resulta aún m ayor, en com paración con e l tratam iento

de sem illas secas.

El a lm acen am ien to o transporte, as f com o la siem bra m ecán ica de las sem illas tratadas con mutágenos

qu ím icos , requ ieren e l secado de las m ism as. Las sem illas de cebada tratadas con MSE se pueden secar a rti

f ic ia lm en te a l cabo de 18 horas o más de le vad o posterior a 25°C . Las sem illas secadas, cuyo con ten ido

de humedad s e a e lc o r r ie n te (d e l 10 a l 147o), se pueden a lm acenar a 0eC o a tem peratura in fe r io r . Sí e l

con ten ido de humedad es d e l Ъ°/о om en os , estas sem illas «s u p e r s e ç a s » se pueden a lm acenar a la tem pera

tura am b ien te . En ambos casos, se m antienen sin cam bios de im portancia la proporción de m utaciones y

3os daños b io ló g ico s o r ig in a lm en te inducidos.

En los program as fito té cn ico s , los m acrom utantes se pueden u t iliz a r en prim er lugar com o progenitores

de c ru zam ien to , en tanto que los m icrom utantes se pueden u t il iz a r d irec tam en te . Se ha com probado que

la e f ic a c ia del MSE para producir mutantes resistentes a l o id io en la cebada es e l dob le que la de los rayos X .

En contraste con estos m acrom utantes, los m icrom utantes se dan con m ucha m ayor frecu en c ia . Después

de irrad iar sem illa s de cebada con rayos X , se tom aron a l azar plantas M 2 de aspecto norm al y se l le v ó a

cabo una se le cc ión en las generaciones siguientes entre sus descend ien tes, a saber, entre las cepas M 3 y M 8 .

Entre 1000 descendientes M ¿ , proven ientes de espigas fé rtiles M L , hubo por térm ino m ed io uno con una

capac idad de rend im ien to un 10% m ayor que la variedad p rogen itora . Dada esta frecuencia re la tivam en te

e le v a d a , se recom ienda u t il iz a r la técn ica de la m icrom utac ión para m ejora r constantem ente las variedades

rec ien tem en te introducidas y prolongar as f su duración.

1. INTRODUCTION

For extensive use of mutants in breeding, the high efficiency of their production is essential. The concept of increasing the efficiency has been stressed by one of the authors since 1958 [1-4] . The term "efficiency" was used in a broad sense and included both induction and selection of mutants. Efficiency is now often used with a specific meaning and, in addition, may be distinguished from two other terms, namely effectiveness and efficacy.

"Effectiveness" has been defined as the ratio "factor mutations/dose" by Ehrenberg [5] and Konzak et al. [6]. The effectiveness of a mutagen is of theoretical importance, but does not have any immediate practical implication. Since radiations and mutagenic chemicals are relatively inexpensive, a less effective mutagen may still be used with a higher dose to obtain practical success. The effectiveness will not be discussed any further in this paper, except in one example.

"Efficiency", as used in the present paper, was recently defined as the ratio "factor mutations/biological damage", where the criteria for measuring the damage often are: seedling height, survival and mitotic and meiotic chromosome mutations [6, 7]. The efficiency of mutagens is of both theoretical and practical importance. Low biological damage of a mutagen permits the use of a high dose. For practical purposes, the aim is to obtain with a high dose a high efficiency. Of particular interest is also the ratio "factor mutations/sterility".

For defining the efficiency, four criteria are used in the present paper: lethality, i.e. % survival reduction; injury, i.e. % seedling-height reduction; sterility, i.e. % reduction in fertility as measured by seed set; and abnormal anaphases, expressed as a percentage of the first mitotic cycle of root tips after seed germination, where fragments and/or bridges are recorded.

EFFICIENCY OF MUTAGENESIS 1 2 3

Less clearly defined is the term "efficacy". We use it in a similar way to Gregory [8], and understand this term to mean the power of a mutagen to produce useful mutations. The effectiveness and efficiency of mutagens have been investigated almost without exception in experiments with chlorophyll mutations and not with viable mutations, although the former are usually considered as factor mutations. The chlorophyll-mutation rate is used as a test for the frequency of viable mutations. To determine the efficacy of mutant production is obviously much more difficult and time- consuming. Any estimate of the efficacy is not only subject to the mutagenic treatment, but also to the selection technique applied.

In the present paper, the efficiency of mutagenesis will be discussed first and followed by the efficacy. Examples will be presented from both the literature and from experiments of our laboratories; for any important details the reader is asked to consult the original publications.

2. NEUTRONS VERSUS X- OR GAMMA-IRRADIATION

Of primary importance to the biological action of neutrons is the higher ion density as compared with X- or gamma-irradiation. Many experiments were conducted to compare the efficiency of both types of ionizing radiation. Unfortunately, it is still difficult to draw reliable quantitative conclusions, because the results obtained vary considerably [9]. This variation has several reasons, the most important of which are; (1) the dosimetry of fast and particularly of thermal neutrons is contradictory and involves problems even today; (2) neutrons are contaminated with gamma.-rays;(3) the dose-effect curves obtained are usually different to those of X- and gamma-rays; and (4) in contrast to X- and gamma-irradiation of seeds, the effect of neutrons is relatively independent of secondary factors such as oxygen content, moisture content over a wide range, etc.; there is, consequently, only a little or no after-effect.

Qualitative conclusions which can be drawn from the use of thermal and fast neutrons are as follows [10, 11]: (1) more uniform biological effects on seeds as measured in seedling height and plant survival; and (2) higher frequencies of chromosome mutations and factor mutations as well as more sterility.

The latter (2) has been interpreted as a consequence of less extra- chromosomal damage. In any case, it is obvious that the efficiency of neutrons is higher than that of X- and gamma-irradiation, because it is possible to obtain more factor mutations, chromosome mutations and sterility per surviving Mj plant. However, reliable information is only available for chlorophyll mutations. Whether or not neutrons are also superior in the induction of viable and particularly of useful mutations is still an open question.

As regards the induction of micro-mutations (mutations of quantitative characters), Brock [12] recently concluded that the action of neutrons is similar to that of X- and gamma-rays; up to now, it has not yet been shown convincingly that neutrons are superior. On the other hand, differences in the mutagenic specificity of neutrons versus X- and gamma-rays have been found, as indicated with erectoides and eceriferum mutants of barley [13, 14].

1 2 4 GAUL et al.

Another possible way of increasing the efficiency of the production of factor mutations is the use of chemical or physical treatments in addition to irradiation. These secondary factors can be used before, during, or after the irradiation. In barley, some secondary factors have reduced the killing rate, but the frequency of chlorophyll mutations has remained the same [15] or has even increased [16]. For example, a sub-lethal heat treatment immediately after the irradiation increased the survival rate from 54% to 64% and also caused an increase in the chlorophyll-mutation rate from 0.6% to 1. 0%. An extra treatment of C02 combined with heat treatment increased the survival rate by almost half, without having a noticeable effect on the chlorophyll-mutation rate [16].

There are relatively few experiments on the effect of secondary factors (see [17]). What results are available do indeed point to the future possibilities of this line of research, but so far no decisive success has been achieved, i.e. an increase in the mutation rate several times that found with irradiation alone.

3 . S E C O N D A R Y F A C T O R S

4. CHEMICALS VERSUS RADIATIONS

Ethyl methane sulphonate (EMS) may still be considered to be the most powerful and most recommendable chemical mutagen for seed treatment, at least if one considers the ease of application and the control of aftereffects. As indicated in the barley example of Table I, the efficiency of the production of factor mutations, related to lethality, increases about five to eleven times when compared with X-rays, depending on the dose; the lower the dose the higher the efficiency [18]. When the efficiency is related to sterility, EMS is four times superior to X-rays and no dose effect is to be observed.

Following EMS-treatment of barley seeds, the ratio of fragments to bridges in the first mitotic cycle is considerably higher than after X- irradiation. The high number of fragments results also in a higher total frequency of abnormal anaphases, when the lethality is used as a basis for comparison [19]. The low frequency of chromosomal recombinations may be explained by the theory that breaks produced by EMS have the tendency of remaining latent for a longer time and of opening up later [4]. There are also reasons for the assumption that more small and minute structural changes are induced by EMS than by X-rays [19].

Mesken and Van der Veen [20] found in Arabidopsis thaliana a higher correlation of the sterility between the Mj and the M2 generations, when EMS was used instead of X-rays for the treatment. The authors explain this observation by the diplontic sterility induced by EMS, but not by X-rays. This interpretation is in agreement with that of Sato and Gaul [19], who concluded that in contrast to the effects of X-rays a large part of EMS-induced sterility has a diplontic and not a haplontic nature. Because the frequency of chlorophyll mutations is independent of the degree of sterility in individual Mt spikes, it has been suggested that for practical purposes, only fertile Mx spikes (or inflorescences) should be selected to be grown in M2 and subsequent generations [1, 20]. It has also been suggested by Mesken and Van der Veen [20] that as a basis for estimating the efficiency

EFFICIENCY OF MUTAGENESIS 125

TABLE I. LETHALITY, STERILITY IN Mj GENERATION AND M2-CHLOROPHYLL MUTATION FREQUENCY, RESULTING FROM X-IRRADIATION (150 kV, 133 R/min) AND EMS-TREATMENT (6 h, 24°C) OF BARLEY SEEDS

M utagen ic

treatm ent

C h lo roph y ll

mutants

(°!o)

Leth a lity E ffic ien cy S terility E ffic ien cy

10000 R 0.77 8 .5 0 .09 14 .3 0 .05

48 m M EMS 5.1 4 .9 1 .0 4 23 .3 0 .22

30 000 R 3 .1 63 .5 0 .05 55.1 0 .06

145 m M EMS 21.1 53 .4 0 .40 95 .2 0 .22

35 000 R 4 .9 86 .7 0.06 61.7 0 .08

169 m M EMS 24.1 82.1 0.29 97 .4 0 .25

40000 R 6 .6 98 .5 0.07 77.7 0 .08

194 m M EMS 3 0 .6 9 6 .2 0 .32 98 .2 0.31

the M2 sterility should be used instead of the Mi sterility. Such a procedure resulted in Arabidopsis in a decrease of the efficiency of EMS-treatment as compared with that of X-rays. However, the authors still found EMS three times more efficient than X-rays.

The different effects of the various radiations and chemical mutagens suggest investigations on the mutagen specificity. Alikhanian [21] found in Actinomyces antibioticus that repeated selection for the same character resulted in a saturation effect, if the same mutagen was used in each cycle of mutagenic treatment and selection. Thus it was not possible to intensify the respective character beyond a certain level. However, a change of the mutagen applied resulted in a further response to selection, i. e. in a further intensification of the character. Joshi and Frey [22] obtained in oats results on mutagen specificity which support those of Alikhanian.They measured the mutagen-induced genetic variance of several characters. This variance was found to be 16% higher following an alternating treatment of thermal neutrons and EMS than after treatment with only one of these mutagens.

5. CHEMICAL MUTAGENS

For chemical mutagens, as for radiations, seed treatment is still the most appropriate method, where practical aspects are concerned. Some of the alkylating substances are very powerful, and have sometimes been referred to as "supermutagens".

The following discussion will be limited to the efficiency and effectiveness of EMS, which has a direct alkylating effect, and of N-methyl-N- nitroso-urea (MNH) as well as N-ethyl-N-nitroso-urea (ENH), which supposedly both react via intermediate products. Because the treatment

126 GAUL et al.


is done by soaking the seeds in an aqueous solution, two consequences should be noted: (1) The reaction of alkylating substances with water (hydrolysis) depends on the treatment time, temperature and the speed of hydrolysis. Correspondingly, the concentration of the solution is reduced and the hydrolysis products, which may be toxic, are increased; (2) Redrying of the seeds, which is essential for the use of a drilling machine, leads to an increase of the concentration of the solution in the seeds. These two opposite effects give rise to the various treatment variations which make it difficult to compare the action of chemical mutagens with each other.

In Table II exactly comparable results are reported of the treatment of barley seeds with 3. 5 mM MNH, 14 mM ENH and 145 mM EMS, under the influence of different post-washing times. The treatment was conducted for three hours at a temperature of 24°C, and the post-washing was done at the same temperature. More details may be found elsewhere [23].A comparison of the data in Table II shows that the effectiveness of MNH and ENH is very much higher than that of EMS, because EMS requires a considerably higher concentration to obtain the same or a similar mutation rate. However, as mentioned in the Introduction, this has no practical importance, since all of these chemicals are relatively inexpensive, and their concentration can, therefore, easily be raised.

The efficiency of EMS is in contrast to the effectiveness higher than that of the nitroso-ureas; the mutation rate achieved is higher with comparable biological damage. The stronger toxicity of the nitroso-ureas limits their mutagenic action, so that under the present experimental conditions the maximal frequency of chlorophyll mutations is about 14%.By post-washing, the indirect mutagenic action of the nitroso-urea compounds is reduced. In contrast to this, washing of EMS-treated seeds results primarily in a decrease of physiological damage, whereas the mutation rate remains unchanged.

6. POST-WASHING AND REDRYING OF EMS-TREATEDBARLEY SEEDS

Post-washing has two main advantages: (1) Non-reacted EMS andhydrolysis products are removed from the seeds. Consequently the treatment time is more clearly defined, which then increases the reproduce- ability; and (2) It permits redrying of the seeds without subsequent lethality. Planting with a drilling machine, and storage, require redried seeds [24].

Alkylation and hydrolysis as well as the diffusion of the EMS-solution into and out of the seeds depends on the temperature. Consequently not only the duration of the post-washing must be long enough, but it is also important to select the optimal temperature. In Table III results are recorded for two post-washing times, 6 and 24 h, and also for a subsequent redrying of the seeds which were washed for 24 h. The treatment was done with 290 mM EMS at 25°C, and the post-washing was conducted at 10°, 20°, and 30°C. Details are to be found elsewhere [25].

The extension of the post-washing from 6 to 24 h leads, at each washing temperature, to a decrease of injury, lethality and sterility. Regarding the temperature, a maximum for the efficiency values may be noticed at 20°C for injury, lethality and sterility. At 20°C, maximal efficiency values are

1 2 8 GAUL et a l.

TABLE III. INJURY, LETHALITY, STERILITY IN M¡ GENERATION, AND M2- CHLOROPHYLL MUTATION FREQUENCY RESULTING FROM EMS-TREATMENT OF BARLEY SEEDS (290 mM, 3 h, 25°C) FOLLOWED BY 6 AND 24 h POST-WASHING (P) AT 10- 30°C WITHOUT REDRYING AND WITH REDRYING (R)

Post-

trea tm en t

C h loroph y ll

mutants

m

Injury E ffic ien cy Le th a lity E ffic ien cy S terility E ffic ien cy

6 P 10° 22 .5 2 2 .2 1.01 73 .5 0.31 77 .7 0 .29

6 P 20° 2 5 .4 35 .1 0.72 85.1 0.30 84 .3 0 .30

6 P 30° - 50 .3 - 100 - - -

24 P 10° 1 2 .4 1 1 .4 1.09 4 6 .5 0.27 50 .7 0 .24

24 P 20° 23.1 21.1 1 .09 64 .4 0 .36 81.1 0 .28

24 P 30° 20 .9 38 .9 0.54 95 .7 0 .22 95 .9 0 .22

24 P 10°R 1 7 .2 33 .0 0 .52 78.1 0 .22 74 .6 0 .23

24 P 20°R 2 1 .4 14.1 1 .52 53.7 0 .40 70 .2 0 .30

24 P 30°R 24 .3 4 4 .9 0.54 88 .5 0 .27 94 .9 0 .26

also obtained after redrying of the 24 h post-washed seeds. For the redrying procedure, the residual EMS in the seeds is without any detectable influence, if the washing is extended to 24 h [24, 25].

7. INFLUENCE OF PRE-SOAKING SEEDSON THE EMS-TREATMENT

By soaking the seeds the hydration of their cells is increased, which results in a faster diffusion of EMS in the embryo tissue [26]. The effect of treatment with EMS is therefore raised. The response of the seedling height of EMS-treated barley seeds to different pre-soaking times at 24°C is shown in Fig. 1 [27]. The upper curve refers to EMS-treatment which was followed by post-washing, whereas the lower curve was obtained from experiments without post-washing. Both curves show a decreasing seedling height as a consequence of pre-soaking. With post-washing the reduction of seedling height is most pronounced in the first four hours and without post-washing in the first eight hours. The two curves are significantly different after a pre-soaking time of four hours. The reduced injury shown by the curve with post-washing is obviously caused by an extraction of the mutagen and by an increased water-uptake of the seeds. Both effects combine and decrease the concentration of EMS and its hydrolysis products in the seeds.

In Table IV [27] , the pre-soaking time varies between 0 and 24 h; the treatment was carried out for 2 h with 242 mMEMS, and the post-washing time was 12 h. In addition to this EMS-treatment, a stronger dose, namely


•— • Without post-washing о--- о With post-washing (12 h)

O)

•o 20 0) о if)

0 5 10 15 20 25

Pre-soaking time (h)

F I G . l . E ffec t o f p re-soak ing (O ' 24 h , 24eC ) , w ith and w ithout post-wash ing (12 h , 24eC ) , on barley

seeds, which were treated in 242 m M EMS solution for 2 h at 24°C . Results o f one exper im en t w ith post

washing and poo led data o f two experim en ts w ithou t post-w ash ing. 95°jo con fid en ce in terva ls w ere cons

tructed fo llo w in g analysis o f va r ian ce .

( F I G . l . E fecto d e l rem o jo p rev io (0 - 24 h , 24eC ) , con y sin la vado posterior (1 2 h , 24 °C ), en sem illas

de cebada tratadas con solución 242 m M de MSE durante 2 h a 24eC . Resultados de un experim en to con

la vado posterior y datos com binados de dos experim entos sin d ich o la vad o . Los in terva les de con fian za

d e l 95^0 se han determ inado por análisis de v a r ia n za .)

290 mM EMS for 3 h, was applied to dry seeds followed by 12 h of postwashing. As can be seen, the latter treatment leads to the same chlorophyll- mutation frequency as those with pre-soaking. The results in Table IV demonstrate that the maximal efficiency for injury, lethality, sterility and abnormal anaphases is reached at a pre-soaking time of 8 h. An extension of the pre-soaking time does not result in a further increase of the efficiency. Two conclusions may therefore be drawn: (1) To achieve the same mutation rate, dry seeds require a higher dose of EMS than presoaked ones; and (2) Injury, lethality, and the frequency of abnormal anaphases are drastically reduced through pre-soaking, if an equal frequency of chlorophyll mutations in a treatment of dry seeds is used for a comparison. By pre-soaking, the efficiency of the production of chlorophyll mutations can thus be considerably raised for all the biological criteria indicated in the Table, except for the sterility.

The experiments summarized in Table IV did not include the effect of redrying. However, results of similar experiments clearly indicate that the efficiency caused by pre-soaking is also increased when the seeds were redried [28].

8. STORAGE OF MUTAGEN-TREATED SEEDS

X- and gamma-irradiation of seeds result in after-effects during the storage period; mutation rate and biological damage increase with increasing storage time. Since these after-effects depend on the moisture content of

130 GAUL et al.

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132 GAUL et al.

the seeds, it can be recommended for practical purposes to conduct irradiation and storage of the seeds at a moisture content of 12 to 14%. At this moisture level, after-effects are without any practical importance. The equilibration of the moisture content can be carried out in desiccators and the storage in plastic bags [4].

Storage of EMS-treated seeds is a much greater problem. If the postwashing is long enough (18 h or more), most of the EMS is removed from the seeds, so that it is possible to redry them without damage [24, 25]. However, if the seeds are stored at room temperature and at the ordinary moisture content (10 to 14%), then after-effects may still lead to lethality. The after-effects increase with the EMS-dose applied, storage time and storage temperature [29]; the moisture content of the seeds during the storage also plays an important role [30].

It has been shown by Bender and Gaul [24] that after-effects can be avoided by cold storage of EMS-treated, post-washed and redried seeds.The seeds were stored for up to 12 months at a temperature of - 20°C; subsequently it was found that a temperature of 0°C is sufficient [29].Under cold storage, the biological state reached after the redrying of the seeds can be maintained without change.

The significance of the moisture content during the storage period of barley seeds was recently demonstrated by Gichner and Gaul [30]. Some of the results obtained are summarized in Table V. As can be seen in the upper part of the Table, the moisture content of the seeds immediately after redrying (no storage) has no influence on the biological damage and the frequency of chlorophyll mutations. After storage at 25°C with moisture contents of 30, 20, 13, and 5%, drastic differences are to be observed during a storage period of four to six weeks. The four moisture contents were maintained by the use of desiccators with different concentrations of H2 S04 .

The moisture content of 30% resulted in a strong reduction of injury, lethality, sterility, abnormal anaphases and chlorophyll-mutation rate; the efficiency is considerably reduced. Storage at 20% moisture content increases the damage up to complete lethality after two weeks, while a moisture content of 13% results in lethality after three weeks storage.In contrast to this, storage at a 5% moisture level maintains the originally induced effects without any significant change. Thus "superdry" seeds can be stored at 25°C for at least six weeks and probably for as long as required.

It has been suggested that the effect of the residual EMS and the hydrolysis products as well as the repair processes, depend on the metabolic activity of the seeds [30]. The differential reaction of seeds with different moisture contentsmaybe explained by these two factors.

In seeds with a high moisture content, which results in a strongly increased metabolic activity, the repair processes prevail. With lower moisture content, the damaging effects of the residual EMS may be most important. Obviously in superdry seeds the physiological conditions are essentially the same as under cold storage. The metabolism appears to be completely blocked and consequently all after-effects are stopped.This means that the efficiency as obtained by EMS-treatment, post-washing and redrying without storage, is maintained.

A new method is therefore now available for practical EMS-treatment, which permits storage and shipping even of seeds treated with extremely high EMS-doses.


TABLE VI. SUMMARY OF MILDEW RESISTANT MUTANTS SELECTED BETWEEN 1962 AND 1968 AFTER TREATMENT OF SEEDS OF EIGHT SPRING BARLEYS WITH X-RAYS OR EMS

T rea tm en tN o . o f

M 2 plants a

C h loroph y ll

mutants

(°!°)

T o ta l no.

M ild ew resistant plants

C o m p le te ly resistant P a rtia lly resistant

per 100 000 per 100 000

X -rays 1034 710 1 .9 9 59 2 .9 0 2 .8 0

EMS 258 307 8 .45 32 6.97 5 .42

T o ta l 1293 017 91 3 .71 3 .33

aIn one case M 3 plants.

9. INDUCTION OF USEFUL MACRO-MU TATIONS

All mutants exert a manifold effect which is also called a pleiotropic effect. The magnitude of expression of this pleiotropic effect is very variable. It may be assumed that with increasing size of the mutative alteration, the pleiotropic effect increases. Increasing pleiotropy results in general in decreasing vitality, i.e. in a lower yield per unit area, because the pleiotropic "side effects" are usually deleterious [31].

This is the reason why macro-mutations can only rarely be used directly in plant breeding. However, macro-mutants may be very useful in cross-breeding, since they may carry desired characters which otherwise are to be found only in non-adapted varieties, primitive types or even in wild forms. In contrast to mutations in well-adapted varieties, such primitive forms require a number of back-crosses to transfer the desired "wild-character"; the expenditure in terms of work, space, etc. (i. e. money), is large. In addition, characters not known in the natural variability of the species concerned may be created by induced mutations in an up-to- date variety. The traits of these trans-specific mutations may be either completely new, or may have been lost in the species during the course of evolution [4].

The response to selection for mildew resistance in eight mutagenically treated spring barleys is taken as an example of the production of useful macro-mutants [32]. Selection was started in 1962 and repeated annually.Both X-rays and EMS were used so that the efficacy of alternative seed treatments can be compared.

X-irradiation was usually performed several times, i. e. repeated annually before selection was started, whereas EMS-treatment was commonly done only once. Selection was conducted in the greenhouse after artificial infection with mildew spores, which was done in the M2 generation, except for one case in M3 . The resistance of plants selected was confirmed in several subsequent generations. During the first phase of the program, race group С was used for the infection. In later years a

134 GAUL et al.

F IG .2. Th e behaviour o f M2 progenies o f V o lla barley from M 3 to M 8 generation during repeated positive

or n ega tive se lec tion for y ie ld , and non -se lec tion .

(F IG .2. C om portam ien to de los descendientes M 2 de cebada V o lla entre las generaciones M 3 y M 8 ,

durante una s e le cc ión repetida positiva o n ega tiva respecto d e l rend im ien to y sin s e le c c ió n .)

field population of mildew from Cologne was used for inoculation. The population from this location is extremely aggressive compared to other ones in Germany, and it infected a number of so-called resistant varieties of Central Europe.

A summary of the response to selection is shown in Table VI. "Completely resistant" means that no infection takes place after inoculation with the field population of Cologne. As can be seen, about 1. 3 million single plants were tested, and 91 mildew resistant barleys were selected.Of these, 43 are "completely resistant". If one compares the efficacy of repeated X-irradiation with EMS-treatment, it can be seen that the latter produced 2. 2 times as many resistant plants as the former. Regarding chlorophyll mutations, this factor of EMS to X-rays is 4. 2. The higher efficiency of EMS in the production of chlorophyll mutations agrees with other results (see Table I), but should not detract from the fact that the efficacy of EMS for the induction of mildew resistance is also higher than that of X-rays.

10. INDUCTION OF USEFUL MICRO-MUTATIONS

According to our concept, the pleiotropy of micro-mutations is less pronounced, and consequently the vitality is less reduced compared with macro-mutations. In some cases, micro-mutants may even be "super- vital", i. e. they may have a higher yield than their mother line. Micromutations occur much more frequently than macro-mutations. It is therefore concluded that useful micro-mutants can be found in a limited volume of selection material, which can thus be easily handled at most plant- breeding stations.


To test these ideas, we initiated a series of experiments with barley and wheat about ten years ago. These long-term experiments, which were split up into a system of sub-experiments, are still being continued, and the latest results obtained were recently reported [33] . In the present paper, the presentation of results will be restricted to an evaluation of the efficacy of selecting for micro-mutant strains affecting the kernel yield.

For a better understanding of the experiments, it is necessary to know that the controls, i.e. the non-mutagenically treated material, are handled , in exactly the same way as that which was treated with X-rays or EMS.In addition to the "Individual Control" (CI), in each experiment a "Bulk Control" (CB) is also used, which is a mixture of individual plants "rom the CI control. Thus the kernel yield of selection strains from mutagenically treated material may be either related to CI or to CB. Response to yield selection obtained in CI may be measured on the basis of the CB yield (no selection). The selection starts in the M3 generation and is done between strains. It is continued in subsequent generations, so that up to five selection steps were conducted. The selection intensity varied, but was usually about 30%. In most of these experiments, the first selection steps for yield were made in a positive and negative direction. In addition, a group of sub - experiments is transferred from generation to generation without selection.

To demonstrate typical results, a series of experiments with the spring barley Volla is taken as an example. After X-irradiation of seeds, these experiments were started with the selection of 2000 normal appearing M2 plants which were taken at random; these plants could not be distinguished from their mother variety Volla. The same number of plants was taken from the untreated Volla. In Fig. 2 [32, 33], relative yield values are used, which are related to the corresponding Individual Control, i.e. for "positive selection" in X-irradiated material, the values are related to positive selection in CI, for "without selection" to CB, and for "negative selection" in X-irradiated material to negative selection in CI.

The results obtained "without selection" demonstrate that the original reduction of the mean yield of the strains derived from irradiation decreases from generation to generation. The continuous increase of yield in the M2 progenies has a "self-operating" mechanism, because no artificial selection was applied. The obvious explanation for this rapid "self-improvement" appears to be the effect of natural selection. The reason may be both the lower competition ability and the lower seed production of segregating homo- zygot mutants.

However, the response to repeated artificial selection in a positive direction clearly surpasses the effect of natural selection. The M8 selection strains have a higher yield level than the strains of the Individual Control which were subjected to the same selection procedure.

Parallel with these theoretically exact studies, we conducted experiments where the selection procedure involved only practical aspects. The mutagenic treatment was done with X-rays and EMS. Only positive selection was used and no selection was carried out in the untreated control (CB). Some examples of micro-mutants are recorded in Table VII, which shows results of five selection strains each of the varieties Haisa П, Volla and Wisa. As can be seen, the selection strains have a yield increase of 5 to 10% compared with their mother variety [33].

136 GAUL et al.

TABLE VII. EXAMPLES OF HIGH YIELD MICRO-MUTANTS. RELATIVE VALUES RELATED TO THE CORRESPONDING MOTHER VARIETY

M utant T rea tm en tN o . o f

trials

N o . o f

locations

K ern e l y ie ld

(<7o o f the m other va r ie ty )

Range M ean

fr .1 2 X -rays 13

Haisa II

1 97 .5 - 113 .9 106 .6

fr .1 2 X -rays 4 1 99.1 - 118 .4 105 .8

X3/115 X -rays 5 1 100 .5 - 113 .6 105 .5

X2/107 X -rays 6 1 100 .6 - 108 .0 104 .9

X2/106 X -rays 6 1 101 .8 - 108 .0 104 .6

Summary 34 1 9 7 .5 - 118 .4 105 .5

68/178 X -rays 12

V o lla

4 9 4 .9 - 132 .2 105 .3

68/172 X -rays 6 4 1 0 2 .0 - 108 .0 105 .2

68/175 X -rays 6 4 9 9 .8 - 111 .7 105.2

68/183 X -rays 8 4 9 8 .7 - 114 .2 105.1

68/191 X -rays 9 2 9 7 .4 - 113 .4 104.9

Summary 41 4 9 4 .9 - 132 .2 105.1

68/110 EMS 4

W isa

3 103 .6 - 113 .6 109 .8

68/149 X -rays 2 2 9 9 .0 - 119 .2 _ 109.1

68/111 X -rays 8 2 9 1 .4 - 133 .7 108 .7

68/148 X -rays 2 2 9 9 .4 - 115 .9 107 .7

68/107 EMS 4 3 98 .3 - 111 .6 106 .0

Summary 20 3 9 1 .4 - 133.7 108 .4

T o ta l summary

Haisa I I , V o lla

o f

W isa 95 4 9 1 .4 - 133.7 106 .3


TABLE VIII. SUMMARY OF YIELD-SELECTION EXPERIMENTS WITH MICRO-MUTANTS IN THE THREE BARLEYS HAISA II,VOLLA AND WISA

The total number of strains of the three varieties before selection is recorded on the left. On the right, relative (weighed) yield means of selection strains established over three to five generations are shown and related to the corresponding Bulk Control (CB). Each yield mean results from three individual values, namely from that of the highest or lowest yielding selection strain of each of the three varieties.

(T A B L A V I I I . RESUMEN DE LOS EXPERIMENTOS DE SELECCION EN C U A N T O AL RENDIM IENTO

REALIZAD O S C O N M IC R O M U TA N TE S EN LAS VARIEDADES DE CEBADA H A IS A I I . V O LLA Y V IS A

A la izq u ie rd a , figu ra e l núm ero to ta l de cepas de las tres variedades antes de la s e le c c ió n . A la

derech a , se ind ican los rendim ientos m ed ios (ponderados) de las cepas se lecc ionadas, estab lec idos

a lo la rgo de 3 a 5 g en erac ion es , en re la c ión con e l con tro l g lo b a l (C B ) correspond ien te. Cada

ren d im ien to m ed io es e l resultado de tres va lo res ind iv idu a les , a saber, e l de la cep a se lecc ionada

de ren d im ien to m á x im o o m fn im o de cada una de las tres va r ied ad es .)

Strain

type

Num ber o f starting

M 3 strains

P os itive N ega tiv e

se lec tion se lection

Num ber o f

years tested

K e rn e l-y ie ld m ean o f the

highest and lowest strain

per va r ie ty

(°]o o f bulk con tro l, CB)

Pos itive N ega tiv e

s e lec tion se lec tion

C I a 2972 1339 3 - 5 (3 - 4 )d 105 .1 93 .5

M F b 2972 1339 3 - 5 ( 3 - 4 ) 110 .2 77 .9

M S c 2972 1339 4 - 5 (3 - 4) 106 .1 77 .1

aIn d iv idu a l con tro l.

b D erived from m utagen ic trea tm en t (X - ir ra d ia t io n ) and from fe r t i le M j spikes.

c D er ived from m utagen ic trea tm en t (Х - ir ra d ia tion ) and from p a rtia lly s terile M x spikes,

d Values in parenthesis re fe r to the n ega tive se lec tion .

To our surprise, selection in the Individual Control of the theoretically exact experiments was effective, as can be seen in Table VIII. Among 1000 control strains (CI), there was on the average one with a yield capacity 5% higher; and among 400, one with a yield capacity 6% lower than the mother variety, i. e. the non-selected control (CB). The starting M3 strains, 2972 and 1339 respectively, are divided by three to obtain these average values (i.e. the exact number of strains is 991 and 446 respectively, instead of 1000 and 400). This result shows that the seed samples of the three varieties were not genetically "pure lines". When the studies were started, we had according to the breeding history good reason to believe that we were dealing with pure lines. It was concluded from these studies that pure lines are an abstract model and do not exist in reality.

As can be seen in Table VIII, the efficacy of selection after mutagenic treatment was considerably higher than in the CI control, particularly in

138 GAUL et al.

the progenies of fertile Mj spikes. Among 1000 of these strains, there was on the average one with a yield capacity 10% higher, and among 400, one with a yield capacity 22% lower than the corresponding Bulk Control (CB).

According to these results, we believe that the efficacy of the selection for high-yielding micro-mutants is relatively high, so that it is comparable with conventional practical breeding work. It has been suggested, therefore, that the micro-mutation technique be used for a continuous improvement of newly-released varieties and particularly of top varieties. It may be expected that the yield potential can be further raised in a second, third or later cycle of mutagenic treatment and selection. If selection for micromutants is combined with "maintenance breeding", these varieties may continue to keep their superiority over freshly introduced varieties for a longer period of time [33]. For maintenance breeding with micro-mutations, a special breeding program has been designed, which is now being tested at several practical breeding stations.

ACKNOWLEDGEMENT

The financial support of the Association EURATOM-ITAL is appreciated.

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[ 8 ] GREGORY, W .C . , "T h e e f f ic a c y o f m utation b re ed in g " , M utation and Plant Breeding, Publ. N o . 891,

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neutron irrad ia tion on seeds o f b a r ley , GeneticsJ39 (1954) 240.

[1 1 ] M acK E Y , J . , Neutron and X -ra y experim ents in ba rley , H ereditas _37 (1951) 421.

[1 2 ] BROCK, R .D . , M utations in qu an tita tive ly inherited traits induced by neutron irrad ia tion , Radiat.

Bot. 10 (1970) 209.

[1 3 ] PERSSON, G . , HAGBERG, A . , Induced varia tion in a quantita tive character in b a r le y . M orphology

and cy to g en e tic s o f erec to id es mutants, Hereditas_61 (1969) 115.

[1 4 ] LU N D O U IS T , U . . V O N W E TTS TE IN -K N O W LE S , P . , V O N W E T T S T E IN , D . , Induction o f ecer ife ru m

mutants in barley by ion iz in g rad iation and ch em ica l m utagens. I I . Hereditas_59 (1968 ) 473.

[1 5 ] N IL A N , R .A . , R elation o f carbon d io x id e , oxygen and low tem perature to the injury and cy to g en e tic

e ffe c ts o f X -rays in b a r le y , G enetics J39 (1954) 943.

[1 6 ] G A U L , H . , D ie W irkung von RQntgenstrahlen in Verb indung m it C 0 2 , C o lch ic in und H itze au f Gerste,

Z . P flZ iich t. 38 (1957) 397.

[1 7 ] N IL A N , R .A . , K O N Z A K , C . F . , "In creasing the e f f ic ie n c y o f m utation induction ” , M utation and Plant

B reeding, Pub l. N o . 891, N A S -N R C , W ashington, D .C . (1961 ) 437.


[1 8 ] G A U L , H . , U ngew ôhn lich hohe M utationsraten b e i Gerste nach Anwendung von Âthylm ethansu lfonat

und Rôntgenstrahlen, Naturwissenschaften 49 (1962) 431.

[1 9 ] S A T O , М . , G A U L , H . , E ffec t o f e th y l m ethane sulfonate on the fe r t i l ity o f b a r le y , Rad iat. Bot. 2

(1967) 7.

[2 0 ] M ESKEN, М . , V A N DER VEEN, J .H . , T h e p rob lem o f induced s te r ility : A com parison betw een EMS

and X -rays in Arabidopsis tha liana , Euphytica 17 (1968) 363.

[2 1 ] A L IK H A N IA N , S .J ., "Som e pecu lia rities o f induced v a r ia b ility o f m ic ro -o rgan ism s ", induced Mutations

and T h e ir U t il iz a t io n , Erw in-Baur-Gedâchtnisvorlesungen IV , G atersleben , 1966, A k a d e m ie -V e r la g ,

Berlin (1967 ) 387.

[2 2 ] JOSH1, S .N . , FREY, K .J . , G en etic v a r ia b ility in oats from recurrent and alternate treatm ent with

physica l and ch e m ic a l m utagens, Rad iat. Bot. _7 (1967) 513.

[2 3 ] G ICHNER, T . , G A U L , H . , O M U RA, T . , Th e in flu ence o f p ost-trea tm en t washing and redrying o f

barley seeds on the m u tagen ic a c t iv ity o f N -m e th y l-N -n itro sou rea and N -e th y l-N -n itro sou rea ,

Rad iat. Bot. 8 (1968) 499.

[2 4 ] BENDER, K . f G A U L , H . , N achw asche, Rucktrocknung und Lagerung b e i AM S -beh an delten Gersten-

sam en, Rad iat. Bot. 6 (1966) 505.

[2 5 ] BENDER, K . , G A U L , H . , V ariieru ng der AM S-W irkung b e i Gerste durch Anwendung versch iedener

Behandlungs- und Nachwaschtem peraturen , Rad ia t. B ot, J (1967) 289.

[2 6 ] W ALLES, S . , Studies on the uptake o f e th y l m ethane su lfonate in to em bryos o f b a r le y , H ered itas 58

(1967 ) 95.

[2 7 ] FRIMMEL, G . , G A U L , H . , D ie Wirkung des Vorquellens von Gerstensam en au f den Behandlungseffekt

von Athylm ethansu lfonat (in p repara tion ).

[2 8 ] FRIM M EL, G . , G A U L , H . (unpublished).

[2 9 ] G ICHNER, T . , G A U L , H . (unpublished).

[3 0 ] G ICHNER, T . , G A U L , H . , Storage e f fe c t fo llo w in g trea tm en t o f barley seeds w ith e th y l m ethane

su lfonate. I . In flu ence o f seed m oisture con ten t, Radiat. Bot. (in press).

[3 1 ] G A U L , H . , "Studies on populations o f m icro-m utan ts in barley and wheat w ithout and w ith s e le c t io n ” ,

Induced M utations and T h e ir U t il iz a t io n , E rw in-Baur-Gedâchtnisvorlesungen IV , G aters leben , 1966,

. A k a d e m ie -V e r la g , Berlin (1967 ) 269.

[3 2 ] G A U L , H . , M utationen in der Züchtung von selbstbestâubenden G etre id ea rten , V ortrage fur P flan zen -

züch ter, DLG V e r la g , Frankfurt/M ain 12 (1969 ) 234.

[3 3 ] G A U L , H . P . K . , U LO N SK A , E . , ZU M ~W IN KEL, С . , BRAKER, G . , "M icro -m u ta tion s in flu enc ing

y ie ld in barley-Studies o ve r nine genera tions” , Induced M utations in Plants (P ro c . Sym p. Pu llm an ,

1969), IA E A , V ienna (1969) 375.

MUTAGENIC SPECIFICITY IN FLOWERING PLANTS: FACTS AND PROSPECTS*

R.A. NILANDepartment of Agronomy and Program in Genetics,Washington State University,Pullman, Wash., United States of America

Abstract-Resumen

M U TA G E N IC SPE C IF IC ITY IN FLOWERING PLA N T S : FA C T S A N D PROSPECTS.

T h e d irec ted con tro l o f the m utation process has been lon g desired , in order to b e a b le to induce

m utations at sp e c if ic lo c i and to produce at w i l l mutants w ith b e n e fic ia l va lu e fo r crop im p rovem en t. H ow ever,

s in ce a l l genes con ta in a lin ea r sequence o f the sam e four nucleotides, there are no apparent d iffe ren t sites for

m utagen s p e c if ic ity on the in tragen ic l e v e l . In sp ite o f this, som e con tro l o f the m utation process can be

a ch ieved . T h e paper provides and discusses som e exam ples as (a ) d iffe ren t ia l response o f certa in lo c i to

c h e m ic a l and physica l mutagens, (b ) a lte ra tion o f the m utation spectrum , ( c ) p re fe ren tia l induction o f

breaks a t sp ec ific ch rom osom e aberrations. T h e mechanisms in vo lv ed are v e ry l i t t l e understood. Future

research m igh t b e pa rticu la rly concerned w ith in tra ce llu la r processes that p recede or fo llo w the reaction

b e tw een m utagen and D N A ( " s e le c t io n s ie v e s " ), w ith induction o f m utations in a c t iv e versus in a c t iv e genes,

w ith s p e c if ic ity o f mutagens on cy top la sm ic D N A , and w ith further d eve lop m en t o f the techniques o f gen e tic

transform ation by in corporation o f exogenous D N A .

ESPECIFIC IDAD M U T A G E N IC A EN LAS FANERO G AM AS: REALIDADES Y PERSPECTIVAS.

Desde h a c e m ucho t iem p o se trata d e con tro la r y d ir ig ir e l proceso de m utación para poder inducir

m utaciones en lo c i e sp ec ífico s y producir a voluntad mutantes ú tiles para m ejorar las especies d e c u lt iv o .

Ahora b ien , c o m o todos los genes con tienen en sucesión lin ea l los m ism os cuatro nucleótidos, no hay lugares

ostens ib lem en te d iferen tes en cuanto a la e sp ec ific id ad m utagén ica a l n iv e l in tragén ico . A pesar d e e l lo ,

puede lograrse c ie r to con tro l d e l proceso d e m u tac ión . En la m em oria se dan y exam inan algunos e jem p los ,

ta les com o : a ) respuesta d ife ren c ia l d e c iertos lo c i a los mutágenos qu ím icos y fís icos ; b ) a lte ra c ión d e l

espectro d e m u tación ; c ) inducción p re feren te d e rupturas en aberraciones crom osóm icas e sp ec ífica s . Se

con ocen m uy m a l los m ecan ism os que in te rv ien en . Las in vestigaciones futuras podrían versar, en particu lar,

sobre los procesos in tracelu lares preceden tes o consigu ientes a la rea cc ión entre e l m utágeno y e l A D N ( « tam ices

de s e l e c c i ó n » ) , la inducción d e m utaciones en genes activos considerada en re la c ió n con la inducción en

genes in activos , la espec ifid ad d e los m utágenos en e l A D N c itop la sm á tico , y e l p e r fecc ion am ien to d e las

técn icas de transform ación gen é t ic a por in corporac ión d e A D N exógen o .

1. INTRODUCTION

A long desired goal of plant mutagenesis has been to control and direct the induced mutation process, i. e. induce mutations at specific loci and alter the mutation spectrum. For mutation breeding this goal is more restrictive since the desire here is to produce at will mutations that have a beneficial value for crop improvement. Most investigations to this end have involved the search for mutagens and mutagen treatments that react selectively with certain genes. However, exceptions to the randomly produced mutations in flowering plants have been rare even when certain chemical mutagens that may react specifically with DNA have been used.

* S c ien t if ic Paper N o .3599. C o l le g e o f A gricu ltu re , W ashington State U n ivers ity , Pu llm an , P ro jec t 1068.

C erta in data ob ta in ed on funds from the U . S. A to m ic Energy C om m ission C on tract A T (4 5 -1 )-2 2 2 1 . AEC

Paper R LO -2 2 2 1 -T 2 -5 . Part o f the research reported in this paper has been carried out under Research

A g reem en ts w ith the In ternational A to m ic Energy A g en cy Nos 321/CF and 615/CF.

141

142 NILAN

Of course we now know why the hoped for discovery of locus-specific mutagens has not been generally realized. It is because all genes contain a linear sequence of the same four nucleotides and thus provide no apparent differential sites for mutagen specificity.

In spite of these rather pessimistic observations concerning true mutagen specificity, there are a number of experimental results which suggest that some control of the mutation process can be achieved and perhaps even greater control is possible in flowering plants. For the most part the mechanisms behind the types of mutagen specificity that constitute this control are not known but it may be concluded they are probably not due to direct interactions between specific genes and specific mutagens. However, for the utilization of mutations in plant breeding a knowledge of the basic causes of mutagen specificity is not an immediate concern.

Our ignorance about mechanisms of mutagen specificity is not surprising since the complete story on how mutagens act to produce mutations and chromosome aberrations in most of our crop plants has not been written.This arises from the fact that details of chromosome structure and the placement of DNA in the chromosome are far too scanty for intelligent hypotheses concerning induction of mutations and chromosome breaks.These problems and some possible solutions have been reviewed in more detail elsewhere [ 1, 2].

Certain types of mutagen specificity in flowering plants that will be described briefly have similar end results to those from increasing the efficiency of mutagens for cytogenetic changes. This area also has been reviewed extensively in previous publications [3,4, 5] and will be covered by others at this meeting.

However, one important point should be made before going on. As plant breeders it is important to recognize that increasing the efficiency and specificity of mutagens for inducing point mutations are not the only important goals for plant mutagenesis and mutation breeding. There must also be concern for increasing the efficiency and specificity of the mutagens for inducing chromosomal breaks and aberrations. This is because new karyotypes and hence phenotypes, some of which may not be obtainable through induced point mutations and other plant breeding techniques, may be produced through various kinds of gross chromosome aberrations.Ways in which these different chromosome aberrations can contribute to "chromosome engineering" as a method of plant breeding have been previously discussed [4].

2. FACTS FOR MUTAGEN SPECIFICITY

The following briefly summarizes the experimental facts for several types of mutagen specificity and for some control of the mutation process in flowering plants. Certain of these facts have been reviewed previously [6, 7]. Most come from experiments where seeds are the main organs of mutagen treatment. Very little research on mutagenic specificity has been conducted with pollen [ 1] or any other tissue.

MUTAGENIC SPECIFICITY IN PLANTS 143

2.1. Intralocus or intragenic specificity

There is increasing evidence now that mutations within a locus (intralocus mutations) can be induced in flowering plants [ 1, 2, for reviews]. The best data come from investigations with maize pollen where a high degree of resolving power for detecting intralocus changes may be achieved with genes controlling pollen characteristics, e. g. waxy [ 8, 9].

Of course, intralocus or intragenic mutations are readily detected in certain prokaryotes (bacteria, viruses) by use of mutagens that react specifically with DNA. Furthermore, there are good indications that such mutations can be induced quite readily in eukaryotic plants such as Neurospora [ 10, 11].

What about specificity or directed mutation at the level of intragenic mutation? The best evidence for mutagen specificity within a gene has come from the well-known work of Benzer with the rll mutants of phage T4.He has found "hot spots" or preferential sites whose positions within a given gene depend upon the mutagen. However, the nature and the origin of these "hot spots" is obscure and they have been attributed to the effect of neighbouring nucleotides on the chemical reactivity of a given base.

Intralocus specificity among eukaryotic plants has not been reported for several well-studied genes in Neurospora or for the waxy gene of maize. Furthermore, until our genetic resolving power in flowering plants is tremendously increased it will be a considerable time before we can even detect mutations within a gene or cistron. Because of the basic nature of DNA with four repeating bases we may wonder if true mutagen selectivity at this level will ever be a reality.

2. 2. Interlocus specificity

With more detailed genetic analyses of induced mutants and the selection of more suitable loci for study, examples of interlocus specificity in flowering plants are increasing. Most of the well-known examples, which incidentally have occurred chiefly in barley, have been reviewed extensively [ 1, 7] and need not be discussed here in detail.

With increasing datait becomes ever more evident that some type of interlocus specificity occurs among some of the 26 known erectoides loci [12] and of the 44 known eceriferum loci [ 13,14].

At certain loci the number of mutants is now large enough for an analysis of interlocus specificity. As seen in Table I (after [ 12]), there are considerable differences among locus ert a (32 mutants), locus ert с (34 mutants) and locus ert d (26 mutants) in responses to physical and chemical mutagens. Ert a mutates more frequently after treatment with sparsely ionizing radiations (X- and 7 -rays) than after densely ionizing radiation treatments (neutrons). On the other hand, locus ert с responds more frequently to neutrons. A differential response of these loci to certain chemical mutagens is also becoming evident. In addition chromosome breaks occur relatively frequently at or near locus ert £ but not at locus ert a. Tests of heterogeneity indicate the differences in mutagen response of the different loci to be significant.

Certain loci of the eceriferum (cer) group also show differential responses to the chemical and physical mutagens [ 14].

144 NILAN

TABLE I. INDUCTION OF erectoides (ert) MUTATIONS BY DIFFERENT MUTAGENS IN BARLEY (After Persson and Hagberg [ 12])

e r t - lo c i

M utagen ert-a e r t-c ert-d

X - and y-rays 14 11 9

Neutrons, Protons 1 16 6

C h em ica ls 17 7 11

T o ta l 32 34 26

The genetic basis for this type of mutagen specificity is not known.It may relate to mutagen-specific chromosome breaking properties at or near the ert or cer loci in question. These properties are discussed in section 2. 4. and under "Prospects". Again it is difficult to believe that these cases of mutagen specificity are due to direct differential interactions of mutagen and DNA.

A high degree of specificity or direction of mutation exists with the so-called controlling elements and mutable loci [ 15, 16] and with the paramutation phenomenon [ 17] in maize and other species. Furthermore, there is now some indication that these types of mutations may be induced by mutagens [18] and in this way specific structural genes would be mutated.As has been indicated by McClintock and others, once these genes have mutated, it is possible by removing the controlling element to stabilize the mutations at any desired state or variant.

2. 3. Group mutability (alteration of mutation spectrum)

By far the most reported type of mutagen specificity in higher plants has been the alteration of the relative proportion or of the mutation spectrum of chlorophyll-deficient mutations. Undoubtedly these mutations have been used so extensively because of their ease of detection and frequent appearance following mutagen treatments. Many experiments, using a variety of plants, have claimed changes in the proportion of different classes of chlorophyll phenotypes, e.g. albina, xantha, viridis, etc. (the spectrum) or in the ratios of viable to lethal types produced by different mutagens or treatment conditions. The problems and pitfalls of experimenting with these types of mutations and the reservations about the validity of many reports of alteration of mutation spectrum have been described previously [ 1 ].

Nevertheless, substantial data on induced chlorophyll-deficient mutations have now demonstrated a significant difference between the spectra induced by alkylating chemicals and by sparsely ionizing radiations. Alkylating agents such as diethyl sulphate, ethyl methane sulphonate, and ethylenimine compared to X-rays or gamma-rays induce lower proportions of albina and higher proportions of viridis and xantha mutations. For example, results from several large experiments in barley have demonstrated that gamma-rays and diethyl sulphate induced 48. 6 and 30. 3% albina.


37. 7 and 44. 8% viridis and 4. 5 and 10. 2% xantha mutations, respectively [19]. These figures are based on large numbers of mutations and are significant at the 99% confidence interval. Similar data for other alkylating compounds have been reported in barley [20, 21] as well as in rice [22].

Whether these differences can be attributed to different relative frequencies of induced chromosome aberrations and/or sterility induced by the chemical mutagen compared to X-rays is not known. Alkylating agents induce few chromosome aberrations compared to X-rays blit do induce appreciable amounts of sterility. From a very careful analysis of the relation of sterility to mutation spectrum, Gustafsson [21] reported that the differences in mutation spectra between the chemical mutagens and radiation in barley occurred at several levels of fertility. Westergaard [ 23 ] suggested that the alkylating chemicals, because of their apparent slight effect on chromosomes, may induce a higher proportion of less-drastic mutations, such as viridis, to extreme mutations, such as albina, than produced by radiation.

Evidence for alterations of the spectrum of chlorophyll-deficient mutations by conditions of mutagen treatment have been reviewed [ 1].

2.4. Specificity of chromosome breakage

There is considerable data from numerous investigations that some of the chemical mutagens, such as maleic hydrazide, induce breaks in specific regions of the chromosome [24]. Often these specific regions are heterochromatic and it is possible that induced breaks in these regions might cause or be accompanied by mutations in neighbouring genes. The causes of the differences in chromosome breakage in heterochromatic versus euchromatic regions are unknown. Nevertheless, there are differences in chromosome coiling, condensation and undoubtedly chemistry between the two regions, and these or other differences may be distinguished by certain of the chemical mutagens. Whatever the cause, it is an area that needs further investigation in relation to mutagen specificity of chromosome breakage and possibly certain types of interlocus specificity.

2. 5. Ratio of mutations to chromosome aberrations

A type of mutagen specificity is the alteration of the ratio of mutations to chromosome aberrations induced by different mutagens. It is now well known that there are mutagens that induce relatively high frequencies of chromosome aberrations and very few mutations. On the other hand, the alkylating agents such as diethyl sulphate, ethyl methane sulphonate and others appear to induce high frequencies of mutations accompanied by relatively few chromosome aberrations. It has also been shown that for X-rays and gamma-rays and for some of the chemical mutagens the conditions under which the mutagen treatment is administered can be influential in altering the mutation frequency in relation to the chromosome aberration frequency. Thus, we now have mutagens and/or mutagen treatments which can produce high proportions of mutations or of chromosome aberrations, depending upon the need in a mutation breeding program.

A more recent development in this area has been the very marked alteration of the ratios of mutation to chromosome aberration frequencies we have obtained under the influence of the respiration inhibitor sodium

146 NILAN

azide. This chemical has been one of several respiration inhibitors employed in our laboratory in investigations to understand the pathways or mechanisms involved in the induction of chromosome aberrations and mutations in irradiated plant cells.

During the past several years we have accumulated a considerable amount of information about the effect of sodium azide in non-irradiated and irradiated barley cells [25]. It is most effective in inhibiting respiration when administered in a solution buffered at pH3 and much less effective or even increases respiration if the solution is at pHll. More specifically it strongly inhibits catalase activity in solutions buffered at pH3 and pH7 and mildly inhibits the activity of this enzyme at pHll. Furthermore, peroxidase activity is considerably inhibited by sodium azide in solutions buffered at pH3, and mildly inhibited at pH7 and at pHll.

Of more relevance to this paper is the effect of sodium azide on mutation (chlorophyll-deficient M2 seedlings) and chromosome aberration frequencies in non-irradiated and irradiated cells (Sideris, unpublished results).

Seeds (caryopses) of barley were irradiated with gamma-rays in the Washington State University 60Co source at 10 and 20 kR. Non-irradiated and irradiated seeds were soaked in 10"3 M sodium azide solutions buffered at pH3, 7 and 11 by 0.1M phosphate buffer. As can be seen in Table II sodium azide in solutions buffered at pH3 induces very high frequencies of chlorophyll-deficient mutations and relatively low frequencies of chromosome aberrations, e.g. 17.3 mutations per 100 Mi spikes to 4% aberrated cells, or about 4:1. In solutions at pH7 the ratio is much reduced (2 :1) while at pHll it is roughly 1:1. It should be noted that the pH3 solution alone induces no higher frequencies of mutations and chromosome aberrations than the pH7 solution.

In irradiated cells the ratio of mutations to chromosome aberrations is reversed with most treatments, yielding ratios of mutations to aberrations of about 1 :6 to 1:7. However, sodium azide in pH3 solutions produced higher frequencies of mutations and a ratio of 1 :3. This higher ratio appears to be solely due to the high mutation rate induced by sodium azide alone. In addition there appears to be an effect of sodium azide in pH3 solutions on induced chromosome aberration frequencies. Thus there is good indication that in the irradiated cells there is a synergistic action of sodium azide buffered at pH3 for the radiation-induced chromosome aberration but not for mutation frequencies.

Aberration frequencies scored at the second anaphase of mitosis and at late prophase of meiosis I indicate that this lack of a synergistic effect for mutations in the irradiated cells is not due to elimination of chromosome aberrations and hence mutations in the ontogeny of the plant. Thus, it may be concluded that some or part of the chlorophyll-deficient and presumably other mutations in flowering plants are not directly related or due to gross chromosomal aberrations.

Explanations for these data have been devised but none are supported by sufficient data. They involve possible differences in energy requirements or repair of chromosome breaks versus DNA lesions. Current studies on the effect of the sodium azide and/or irradiation treatments ' on the induction and repair of DNA lesions and the relation of these lesions to chromosome aberrations and/or mutations should help understand the cause of the different ratios of mutations to aberrations described above.


TABLE П. FREQUENCIES OF MUTATIONS (PER 100 Mx SPIKES) AND CHROMOSOME ABERRATIONS (% ABERRANT CELLS) INDUCED IN NON-IRRADIATED AND IRRADIATED CELLS TREATED WITH SODIUM AZIDE (10'3 M) SOLUTIONS BUFFERED AT pH3, 7 AND 11

0 kR

M utations (M )

or pH3 pH7 p H l l

Aberrations (A )

M - 0 .6 0 .6 1 .0

A - 0 .5 1 .0 0 .0

M + 17.3 7 .0 1 .4

A + 4 .0 3 .0 1.0

10 kR

M - 5 .3 3 .8 4 .6

A - 32 .0 26 .0 25 .0

M + 18 .4 6 .4 5 .1

A + 55.0 36 .0 28 .0

20 kR

M - 7 .9 8 .5 8 .5

A - 56.0 54.0 62.0

M + 24 .0 12.7 10.0

A + 83 .0 68.0 65 .0

3. PROSPECTS FOR MUTAGEN SPECIFICITY

Within recent years there have been several new ideas and experimental approaches presented in the literature that may help to explain some of the above facts and to increase mutagen specificity in plants.

3.1. Selection sieves

The concept of "selection sieves" as proposed by Auerbach [26] maintains that numbers and types of mutations may be controlled by several secondary steps or "sieves" which determine whether a change in DNA will take place and whether once it has taken place it will give rise to an observable mutation. She maintains that it is at the level of these "sieves" that specificities may be expected. "Sieves" preceding the reaction between mutagen and DNA are concerned with chemical changes that a mutagen may undergo before reaching the gene and the accessibility of the gene to the mutagen. They are influenced by strain, cell type,

148 NILAN

metabolic state and probably depend upon the degree of coiling of chromosomes and chromosome regions and on the amount and type of chromosomal components other than DNA. The "sieves", following the reaction between mutagen and DNA, determine which of the changes in DNA will eventually appear as an observable mutation. They include processes such as repair, transcription, translation and competitive cell growth. All of these "sieves" occur in flowering plants and it is probably here where explanations of already established mutagen specificity reside and where new approaches to control of the mutations appear most fruitful. Indeed analyses and tests of some of these possibilities have already been published [27].

3.2. Active versus non-active genes

There are some indications now that active genes are more accessible to certain mutagens than repressed or inactive ones. Brock [27] using E. coli has attempted to determine the validity of this idea with the ultimate goal of doing similar experiments in flowering plants. Certain mutagens such as diethyl sulphate and ethyl methane sulphonate mutated active genes with a higher frequency than inactive genes. Other mutagens, for example gamma-rays, produced the same mutagenic efficiency for both active and inactive genes. It would appear that if similar specificities occur in higher plants some control of the mutation process should be possible by applying appropriate mutagens when particular genes are active.

In the same vein, it has been suggested again from work with microorganisms that the maximum frequency of a given type of mutation occurs when the treatment is given at the time of DNA replication. It is also known that in certain test organisms DNA replication along the chromosome is asynchronous in time sequence. In some chromosomes replication commences from the centromere and proceeds towards the telomere, in others the reverse happens. There is a possibility, therefore, that groups of loci could be treated with short periods or pulses of mutagen during the S phase of DNA synthesis.

Swaminathan and Sarma [28] have pursued this idea with barley. They found that using pulse treatments of EMS during eight different stages of the S phase resulted in differential induction of chlorophyll-deficient mutations. Although the data are still few and preliminary they do suggest a fairly high degree of control of mutagen specificity by this technique.

3.3. Differential effects on genes in cytoplasm versus genes in nuclei

With our increasing understanding of the genetic control that occurs in plants through cytoplasmic DNA, particularly that residing in the mitochondria and the chloroplasts, the possibility of differential induction of mutations in the cytoplasm versus the nucleus becomes intriguing and important.

In a pilot experiment Shwaier et al. [ 29] examined the effect of mutagens on genes affecting respiration in the mitochondria and in the nucleus of yeast. In this system respiratory-deficient mutants are caused by damage in the nuclear DNA or in the mitochondrial DNA. A distinct difference in cytoplasmic versus nuclear mutagen specificity was observed between nitrous acid (NA) and N-nitroso-N-methylurethane (NMU). Nitrous acid was unable to induce cytoplasmic respiratory-deficient mutants

MUTAGENIC SPECIFICITY IN PLANTS 1 4 9

but proved to be highly efficient in the induction of nuclear mutants. In contrast NMU induced more cytoplasmic than nuclear respiratory-deficient mutants. The basis for the differential action of these mutagens on nuclear versus cytoplasmic DNA is not yet understood.

3.4. Transformation

There have now been several attempts to effect genetic transformation in flowering plants [ 30]. The most elegant and detailed work on this problem has been that of Ledoux and his colleagues in Belgium. They have conclusively demonstrated the uptake and incorporation of bacterial DNA into cells of barley and Arabidopsis [31]. Furthermore, such exogenous bacterial DNA appeared to bring about a genetical change in Arabidopsis at a specific locus where a defect that resulted in a chlorophyll-deficient phenotype occurred.This may be considered an example of a directed genetic change by using proper selection of bacterial DNA.

Once desired molecules of DNA are synthesized in the laboratory (as now appears possible after the synthesis of a yeast gene by Kh.orana and colleagues at the University of Wisconsin [32]) transformation experiments will become even more useful and probably provide one of the best ways for producing at will desired genetic changes in flowering plants.

REFERENCES

[ 1 ] N IL A H , R . A . , "N a tu re o f induced m utations in h igher p lan ts", Induced M utations and T h e ir U t iliz a t io n ,

P roc . Sym p. Erw in-Baur-Gedâchtnisvorlesungen IV ,. G atersleben , 1966, A k a d e m ie -V e r la g , B er lin (1967 ) 5.

[ 2 ] N IL A N , R .A . , KLEINHOFS, A . , SIDERIS, E .G . , "Structural and b io ch em ica l concepts o f m utations in

flow er in g p lan ts". Induced M utations in Plants (P ro c . Sym p. Pu llm an , 1969), IA E A , V ienna (1969 ) 35.

[3 3 K 0 N 2 A K , C .F . , N IL A N , R .A . , W AGNER, J ., FOSTER, R .J .. "E ffic ie n t ch e m ic a l m utagenesis'’ , Th e

U se o f Induced M utations in P lan t B reed ing (R ep . FAO /IAEA T e c h . M ee tin g , Rom e, 1964), Pergam on

Press, Oxford (1965 ) 49.

[ 4 ] N IL A N , R . A . , K O N Z A K , C .F . , W AGNER, J ., LEG AU LT, R .R ., "E ffec tiven ess and e f f ic ie n c y o f radiations

fo r inducing gen e tic and c y to g en e t ic ch a n ges ", T h e Use o f Induced M utations in P lan t B reed ing (R ep .

FAO /IAEA T e ch . M ee tin g , Rom e, 1964), Pergam on Press, Oxford (1965 ) 71.

[ 5 ] FA O / IA E A , M anual on M utation B reed ing, T e c h . Rep. Ser. N o .119, IA E A , V ien n a (1970 ).

[ 6 ] S M ITH , H .H . , "M u tagen ic s p e c if ic ity and d irec ted m u ta tion ", M utation and P lan t B reed ing, Publ.

N o .891, N A S -N R C , W ashington, D .C . (1 9 6 1 )4 1 3 .

[ 7 ] G USTAFSSO N , Â . , "A study on induced m utations in plants: In troductory address", Induced Mutations

in Plants (P ro c . Sym p. Pu llm an , 1969), IA E A , V ienna (1969 ) 9 .

[ 8 ] NELSON, O . E . , T h e w a xy locus in m a iz e . I . Intralocus recom b ination frequ ency estim ates b y po llen

and b y con ven tion a l analyses, G enetics 47 (1962 ) 737.

[ 9 ] NELSON, O .E . , T h e w a xy locus in m a iz e . I I . T h e lo ca tion o f the con tro llin g e lem en t a lle le s ,

G enetics 60 (1968 ) 507.

C IO ] M A LL IN G , H .V . , DE SERRES, F .J ., R e la tion b etw een com p lem en ta tion patterns and gen e tic a lterations

in nitrous a c id -in du ced ad-3B mutants o f Neurospora crassa, M utation Res. 4 (1967 ) 425.

[1 1 ] M A LL IN G , H . V . , DE SERRES, F .J . , Id en t if ic a t io n o f g en e tic a lterations b y e th y l m ethanesu lfonate in

Neurospora crassa, M utation Res. 6 (1968 ) 181.

[1 2 ] PERSSON, G . , HAGBERG, A . , Induced va r ia tion in a quan tita tive character in b a r le y . M orphology

and cy to g en e tic s o f erec to id es mutants, H ered itas 61 (1969 ) 178.

[1 3 ] LU N D Q V IS T , U . , W E T T S T E IN , D , von , Induction o f ecer ife ru m mutants in b a r le y b y io n iz in g radiations

and c h em ica l mutagens, H ered itas 48 (1962 ) 342.

[1 4 ] LU N D Q V IS T , U . . W E TTS TE IN -K N O W LE S , Penny von , W E TTS TE IN , D . von , Induction o f e cer ife ru m

mutants in b a r le y by io n iz in g rad iations and ch em ica l m utagens. I I . H ered itas 59 (1968 ) 473.

150 NILAN

[1 5 ] M cC L lN T O C K , Barbara, G enetic systems regu lating gen e expression during d eve lop m en t, D ev i

B io l. Suppl. 1 (1967 ) 84.

[1 6 ] PETERSON, P . A . , C on tro llin g e lem en ts and m utab le lo c i in m a ize : T h e ir rela tionsh ip to b a c te r ia l

ep isom es, G en etica 41 (1970 ) 33.

[1 7 ] BRINK, R .A . , STYLES, E .D . , A X TE LL , J .D . , Param utation: D irected gen e tic change, S c ien ce

159 3811 (1968 ) 161.

[1 8 ] NEUFFER. M .G . , S tab ility o f the suppressor e lem en t in two m utator systems at the A i locus in m a ize ,

G enetics 53 (1966 ) 541.

[1 9 ] N ILA N , R .A . , K O N Z A K , C . F . , "In creasing the e f f ic ie n c y o f m utation in d u c tio n ", M utation and Plant

B reeding, Publ. N o .891, N A S -N R C , W ashington, D .C . (1961 ) 437.

[2 0 ] EHRENBERG, L . , GU STAFSSO N , Â . , LU N D Q V IS T , U , , V ia b le mutants induced in b a r le y b y ion iz in g

radiations and ch em ica l m utagens, H ereditas 47 (1961 ) 243.

[2 1 ] GU STAFSSO N , Â . , P rodu ctive m utations induced in b a r le y by io n iz in g radiations and ch em ica l

mutagens, H ered itas 50 (1963 ) 211.

[2 2 ] RAO, N .S . , G O PA L-A YE N G A R , A .R . , "C om b in ed e ffe c ts o f therm al neutrons and d ie th y l sulphate

on m utation frequ ency and spectrum in r ic e ” , B io lo g ic a l E ffects o f Neutron and Proton Irradiations

(P ro c . Sym p. Upton, 1963) _1, IA E A , V ien n a (1964 ) 383.

[2 3 ] WESTERGAARD, М . , "A discussion o f m u tagen ic sp e c if ic ity . 1. S p e c ific ity on t h e ’ geograph ica l*

l e v e l " , C hem ische M utagenese, E rw in-Baur-Gedâchtnisvorlesungen I, A k a d em ie -V e r la g , Berlin

(1960 ) 116.

[2 4 ] K IH L M A N , B .A . , A ctions o f C hem ica ls on D iv id in g C e lls , P ren t ic e -H a ll, N ew Jersey (1 9 6 6 ).

[2 5 ] S1DERIS, E .G . , N IL A N , R .A . , K O N Z A K , C .F . , "R ela tionsh ip o f rad ia tion -induced d am age in ba r ley

seeds to the inh ib ition o f certa in oxidoreductases by sodium a z id e " , Induced Mutations in Plants

(P ro c . Sym p. Pu llm an , 1969), IA E A , V ienna (1969 ) 313.

[2 6 ] AUERBACH, C . , T h e ch em ica l production o f m utations, S c ien ce 158 3805 (1967 ) 1141.

[2 7 ] BROCK, R .D . , " In creas in g the sp e c if ic ity o f m u ta t io n " , Induced Mutations in Plants (P ro c . Sym p.

Pu llm an , 1969), IA E A , V ienna (1969 ) 93.

[2 8 ] S W A M IN A T H A N , M .S . , SARM A, N . P . , A lte ra tion o f the m utation spectrum in b a r le y through

treatm ents at d iffe ren t periods in the S phase o f D N A synthesis, Curr. S c i. 37 (1968 ) 685,

[2 9 ] SCHW AIER, R . , NASHED, N . . Z1M M ERM ANN, F . K . , M utagen sp e c ific ity in the induction o f ca ryo tic

versus cy top la sm ic re sp ira to ry -d e fic ien t mutants in yeast by nitrous acid and a lk y la tin g nitrosam ides,

M o le c . Gen . G enet. 102 (1968 ) 290.

[3 0 ] СОЕ, E .H . , J r., SARKAR, K . R . , Preparation o f nu c le ic acids and a g en e tic transform ation attem pt

in m a iz e , C rop S c i. £ (1 9 6 6 ) 432.

[3 1 ] LEDOUX, L . , H U AR T , R . , "F a te and possible ro le o f exogenous b ac te r ia D NA in b a r le y " (P roc .

2nd In t. B arley G enet. Sym p. Pu llm an , 1969), ( in press).

[3 2 ] TH O RN , N ancy, Th e test-tu be synthesis o f a gen e, B io S c ien ce 20 (1970 ) 917.

DISCUSSION

H. SMITH: There are three examples mentioned in my paper that bear on the possibility of utilizing post-irradiation environmental conditions to achieve some measure of differential control over the products of irradiation,i. e. of mutagenic specificity in its widest sense. These involve experiments with Arabidopsis (Daly), yeast (Lyman and Haynes), and barley (Smith and Mikaelsen).

H. GAUL: I wonder if you have information at hand about the reproducibility of the results of Ledoux1.

R. A. NILAN: The most recent information I have is that Ledoux is repeating the transformation experiment with Arabidopsis. Data from the second experiment has not yet been published.

1 Ref. 131 ] o f the paper.


M.S. SWAMINATHAN: Our research on the alteration of the mutation spectrum in barley through the administration of pulse treatments with alkylating agents during different stages in the S phase of DNA synthesis was not prompted by any experiment with micro-organisms. It was a logical extension of our finding that by synchronizing the time of treatment with EMS with the S phase of DNA replication the total frequency of mutations is greatly enhanced and that DNA replication along a chromosome is asynchronous. After having ascertained the duration of the S phase with the tritium labelling technique, we proceeded in 1967 with giving short duration treatments during different stages of the S phase. The results revealed some degree of specificity for characters controlled by single genes such as alboxantha among chlorophyll defects. In addition to EMS, we have used in such studies iPMS, dES and NMU. The available data indicate that the technique offers possibilities for altering the proportion of different types of mutations.

PRINCIPLES OF MUTATION BREEDING

MUTATIONAL RECONSTRUCTION OF CROP IDEOTYPES*

M .S . S W A M IN A T H A N

Indian Agricultural Research Institute, New Delhi, India

Abstract-Resumen

M U T A T IO N A L RECO NSTRUCTIO N OF CROP ID EOTYPES.

Understanding the factors w h ich govern p lan t p rodu ctiv ity m akes i t possible to design ideo types o f crop

v a r ie ties , which, i f r e a liz ed through p lant b reed ing , wou ld h elp to further increase food production . D esirab le

attributes o f such ideo types fo r the trop ics and subtropics are. e . g . h igh p rod u ctiv ity per day, photo- and

th erm o-in sen s itiv ity , h igh p rodu ctiv ity per unit o f water, h igh photosynthetic a b il ity and low photo -resp ira tion .

In India, m utation b reed ing techniques h ave b een used ve ry e f fe c t iv e ly in the d eve lop m en t o f crop va r ie t ie s

corresponding to such id eo types . T h e paper presents som e exam p les such as b reed ing o f new ideotypes o f wheat

fo r un irrigated areas, a lte r in g o f le a f characteristics in r ic e to p rom ote b etter l ig h t in tercep tion , the induction

o f ph o to -in sen s itiv ity in cotton , the im provem en t o f g ra in setting in T r i t ic a le , and the im provem en t o f seed

p rote in .

RECONSTRUCCION M U T A C IO N A L DE ID IO T IPO S VEGETALES.

El c on oc im ien to de los factores que r igen la productiv idad de las plantas p erm ite d e fin ir id io tipos de

variedades v e g e ta le s , que de conseguirse por f ito te cn ia , contribu irían a aum entar la producción de a lim en tos .

Las caracterís ticas ap e tec ib le s de estos id io tipos para las regiones trop ica les y subtropicales son, entre otras,

e le va d a productiv idad d iaria , fo to insensib ilidad y term oinsensib ilidad , e le va d a productiv idad por unidad de

vo lu m en de agua, e le vad a capac idad de fotosíntesis y ba ja fo to resp irac ión . En la India, las técn icas de m u ta

c ión se u tiliza n m uy e f ic a zm en te para la ob tenc ión de variedades de plantas correspondientes a dichos id io tipos

En la m em oria se presentan algunos e jem p los , ta les com o la ob tenc ión de nuevos id io tipos de tr igo de secano,

la a lte ra c ión de las ca racterísticas de las hojas d e la rro zp a ra aum entar la in te rcep c ión de la lu z , la inducción

de la fo to insensib ilidad en e l a lgodón , e l m e jo ram ien to de la f i ja c ió n de los granos en T r it ic a le y e l m e jo ra

m ien to de las proteínas de las sem illa s .

1. INTRODUCTION

The exploitation of heterosis in the form of commercial hybrids, the enhancement of the effects of additive gene action through synthetics and composites and the development of the concept of a dwarf plant type suited to good agronomy have all helped to raise the ceiling of yield in many crop plants to a level not considered possible before. Based on an understanding of the factors governing productivity, conceptual models or "ideotypes" [1] of crop varieties can be constructed, which, if realized through breeding, would help to achieve still higher yields. The following are some of the desirable attributes of crop ideotypes, particularly in the tropics and subtropics where crop growth is possible throughout the year; a) superior population performance; b) high productivity per day; c) high photosynthetic ability; d) low photo-respiration; e) photo- and therm о-insensitivity; f) high response to nutrients; g) high productivity per unit of water; h) multiple resistance to pests; i) better protein quantity and quality; and j) crop canopies that can retain and fix a maximum of C02 .

* Part o f the research reported in this paper has been carried out under Research A g reem en t No.338/CF

and Research Contracts w ith the In ternational A to m ic Energy A g en cy Nos 378/RB and 863/RB.

155

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CROP IDEOTYPES 157

It is such a shift in selection criteria that has provided special scope for deploying mutation breeding techniques very effectively in the development of crop varieties suited for present-day needs [2, 3]. As a result, several new crop varieties developed by mutation breeding have been released in recent years for commercial cultivation in India (Table I). The results of some recent research at the Indian Agricultural Research Institute on the practical realization of desirable ideotypes through mutation breeding are summarized in this paper.

2. BREEDING NEW IDEOTYPES OF WHEAT FOR UNIRRIGATED AREAS

Nearly 66% of the wheat area of about 14 million hectares in India depends for crop growth only on rainfall. Asana [4] has developed an ideotype for such areas, which is designed to achieve a maximization of the yield of the main tiller, thereby compensating for poor tillering, and the dissociation of undesirable associations such as earliness and poor root growth, and earliness and a low number of spikelets per ear. This ideotype (Fig.l) has the following attributes: a) large number of spikelets andgrains on the main ear through adventitious branching in the ear; b) long peduncle which forms a substantial proportion of the length of the stem, thereby increasing photosynthesis in the peduncle and flag leaf; c) semidwarf plant habit which helps to avoid lodging in fields supplied with fertilizer; d) about seven leaves on the main axis with a large flag leaf, the leaves preferably being arranged horizontally so as to permit better retention of dew and also better light interception; e) good growth of roots at a soil depth of 3-4 ft; f) emergence of ears at such a time that will permit grains to develop for at least five weeks at a mean maximum temperature of about 25°C.

A study was initiated by Mehta and Swaminathan (unpublished) in 1967 to ascertain the possibility of isolating genotypes with the above attributes.The material for the study consisted of an induced branched ear mutant of Triticum aestivum var. N.P. 797 [5] and tetraploid branched ear material derived from crosses between T. durum and turgidum var. mi rabile. The material was treated with both gamma-rays (20 and 30 kR), ethyl methane sulphonate (0.2%) and N-nitroso-N-methyl urea (0. 02%). Recurrent selection was practised for the desirable characters in M2 and later generations and the extent of progress made so far in achieving the desirable ideotype is summarized in Table II. The data indicate that the chances for realizing Asana1 s ideotype for unirrigated wheat are very high. The most promising selections are being subjected to a further cycle of mutagen treatment, selection and recombination.

3. ALTERING LEAF CHARACTERS IN RICE TO PROMOTE BETTERLIGHT INTERCEPTION

The introduction of the dwarfing gene in Or.yza sativa subspecies indica has helped to increase the yield of indica rices substantially through fertilizer application. In many high-yielding dwarf indicas, the total biological yield does not appear to be higher than in the tall indicas. The higher grain yield really arises from a higher harvest index (i.e. grain/straw ratio).

1 5 8 SWAMINATHAN

WHEAT IDEOTYPES

F IG . 1. C onceptua l m odels o f the wheat p lan t d eve lop ed by Donald [1 ] fo r m ax im u m production and by

Asana [4 ] for un irrigated cond itions. Donald has suggested a un icu lm habit w ith leaves which w i l l not shade

each other, w h ile Asana has proposed h o r izon ta l le a ves fo r in tercep tin g and reta in ing dew , a branched ear and a

deep root system . Such ideotypes are based on considerab le research in production p h ys io logy .

( F I G . l . M odelos teóricos de sendas plantas de tr igo , obtenidas por D onald [1 ] para un ren d im ien to m á x im o y

por Asana [4 ] para c u lt iv o de secano. D onald postula plantas un icanales, con hojas que no se dan sombra

m utuam ente, m ientras que Asana propone hojas h orizon ta les para in tercep tar y retener e l ro c ío , una espiga

ram ificad a y un sistem a rad icu lar profundo. Estos id io tipos se basan en m inuciosas in vestigac ion es de la

f is io lo g ía de la rep rod u cc ión .)

The data from a study conducted by Asana and Salunke (unpublished) in Taichung Native 1 ( a dwarf indica from Taiwan), Basmati 370 (a tall indica from India with long slender grains) and Jamuna (a dwarf, fine-grain variety developed at the IARI, by crossing Taichung Native 1 with Basmati 370 followed by four backcrosses to the Basmati parent) are given in Table III.A detailed analysis of the production physiological processes in these three varieties by Asana and Salunke indicated that the net assimilation rate decreases with a drop in light transmission about 30 days after transplanting (Figs 2a and 2b). The rapid fall in light intensity after 30 days appeared to adversely affect tillering and the realization of the full sink capacity. A smaller leaf size and a lower number of leaves hence appeared to be desirable attributes for promoting a higher net assimilation rate in the population as a whole.

CROP IDEOTYPES 159

TABLE II. PROGRESS MADE IN THE DEVELOPMENT OF A BRANCHED-EAR WHEAT STRAIN

Plant partC haracteristics o f the in it ia l

m a ter ia l in 1967

Characteristics o f se lections in

m u tagen -trea ted popu lations in 1970

Ear Erratic branching, low sp ike le t

fe r t i l ity and low 1000-kernel

w eigh t

Good branching w ith up to 19 ad

ven titious branches per spike, good

fe r t i l i ty w ith up to 207 grains per spike,

good 1000-kernel w eigh t

Peduncle Poor peduncle exertion Peduncle index h igh, p rom oting g rea ter

photosynthesis in the peduncle

Plant heigh t S em i-d w a r f to dw arf (less

than 100 cm )

S em i-d w a r f to dw arf (less than 100 cm )

Leaves L ea fy types with 14 to 15

lea ves on the m ain stem

T w o s in g le - le a f and severa l reduced

lea f-n u m b er mutants were iso lated

Roots V a riab le depths D eep root system , reach ing a so il depth

o f 3 -4 ft

Ear V ery la te (1 Î7 to 120 days from

sow ing to earin g )

Early mutants tak ing about 105 days

from sowing to earing w ere iso lated

TABLE III. YIELD CHARACTERISTICS IN DWARF AND TALL indica RICE VARIETIES

V a r ie ty

Ta ich u n g Native; - l Basm ati-370 Jamuna

F ie ld

Grain y ie ld (q/h a ) 74 .4

Straw y ie ld ( q/ha) 73 .3

T o ta l b io lo g ic a l y ie ld (q/ha ) 147.7

31 .1 52 .8

121 .0 , 63.7

152 .1 116 .5

Pert

Grain y ie ld (g/frlant) 91.0

Straw y ie ld (g / p la n t) 73 .4

Harvest index 55.4

7 2 .5 84 .7

116 .4 91 .2

3 8 .4 4 8 .2

In order to examine the influence of leaf characters such as number, size, orientation and senescence pattern on percentage light transmission and net assimilation rate, the rice varieties Taichung Native 1 and Tainan 3 were treated with a wide range of mutagens including gamma-rays, ethyl methane sulphonate, and nitrosomethyl urea. Mutants with altered leaf characteristics were isolated in M2, M3, and later generations (Siddiq, Singh and Swaminathan, unpublished). There was considerable variation in the size, number, alignment and ageing pattern of leaves in the mutants.

160 SWAMINATHAN

R E L A T IV E GR OW TH R A T E NE T A S S I M I L A T I O N R A T E

F IG .2 a . R e la tiv e grow th and net assim ila tion rates in three ind ica r ic e v a r ie t ie s .

Some of these mutants had associated changes in panicle characters, such as density and fertility. Mutants with normal panicles but altered leaf characters are currently being used in studies of light transmission and dry matter production.

Qualset et al. [6] have recently described a mutant in bread wheat which influences the structure of the crop canopy and thereby affords an opportunity to evaluate light interception and the physiological aspects of crop productivity. Evans and Dunstone [7] have concluded from a study of physiological changes in the evolution of yield in Triticum species that so far evolution has progressed through changes in grain and leaf size and in the proportion of dry weight mobilized to the grain. They, however, feel that while photosynthetic rate has not limited the evolution of yield in wheat up to the present, it may well limit further evolution. The proceedings of a recent Symposium on "Physiological Aspects of Crop Yield" [8] also make it clear that the next major break-through in increasing productivity may come from the development of plant types which possess an inherently greater potential for biological yield, since the advantage conferred by alterations in the harvest index has already been exploited to a great extent.

4. INDUCING PHOTO-INSENSITIVITY IN Gossypium hirsutum

Several varieties of G_. hirsutum have been developed in South India which possess a good yield potential and excellent fibre properties. These strains when grown at Delhi and other parts of North India start flowering only when the day length goes below 12 hours. With the onset of short days,

CROP IDEOTYPES 161

BETWEEN ROWS

NO. OF DAYS A F T E R TR AN SPL AN TI N G

F IG . 2b. L igh t transm ission in three in d ica r ic e va r ie t ie s .

the temperature also goes down and the yield of the varieties is poor. In order to isolate photo-insensitive mutants, Jain and Raut (unpublished) treated the long staple variety MCU-5 with different doses of gamrna-rays. The M2 populations were screened for flowering habit and several mutants combining good yield and staple length with an early flowering habit were isolated (Table IV). These data suggest that in addition to selection in segregating generations being carried out under diverse environments as suggested by Borlaug [9] and Finlay [10], selection in mutagen-treated populations may also prove valuable for breaking the barriers to wide adaptation based on photo- and thermal-sensitivity.

162 SWAMINATHAN

TABLE IV. CHARACTERISTICS OF SOME INDUCED MUTANTS IN Gossypium hirsutum var. MCU-5

M ater ia lG am m a-ray dose

(k R )Days to flow er

Y ie ld

Cg/plot)

Staple length

(m m )

M C U -5 - 120 25 29.0

Mutant N o . 1 5 66 165 26.0

Mutant N o . 2 5 62 300 2 7 .5

Mutant N o . 4 20 64 250 28 .7

Mutant N o . 6 20 69 200 3 1 .6

Mutant N o . 8 30 69 150 29.0

Mutant N o . 9 40 66 120 26 .0

M utant N o .10 50 63 240 26.0

TABLE V. VARIATION FOR GRAIN FERTILITY IN THE M3 GENERATION OF MUTAGEN-TREATED Triticale

N o . o f seeds

Strain T rea tm en t N o . o f plants studied per spike

Range Mean

6456-3-1 C ontrol 1000 0-70 40 .08

EMS (0 .2 % ) 1000 0-90 45 .52

6450-2-1 C ontrol 200 25-73 50.54

20 kR gam m a-rays 1000 0-58 36 .76

E M S (0 .2% ) 1000 0-86 37 .47

5027 C ontrol 200 8-78 4 7 .8 7

20 kR gam m a-rays ; 1000 0-101 48 .36

5. IMPROVEMENT OF GRAIN SETTING IN Triticales

Recent research has revealed that in hexaploid (2n = 42) Triticales, seed fertility can be improved greatly by selection, thereby indicating that sterility is under genetic control. There is evidence from the work of Dr. R. Riley and his co-workers at the Cambridge Plant Breeding Institute that in addition to disturbances in the breeding system, timing imbalance in meiotic stages leading to pairing and disjunctional abnormalities may be responsible for sterility in the amphidiploids between Triticum durum and and Secale cereale. Timing imbalance is known to be under genetic control and hence can be remedied by selecting appropriate gene combinations.

CROP IDEOTYPES 163

DBC VALUE

F IG .3 . D istribution o f DBC values in the M 2 genera tion o f the w heat va r .S h a rb a ti Sonora.

TABLE VI. ALL INDIA CO-ORDINATED NATIONAL TRIAL, 1969-70. PERCENTAGE OF PROTEIN (ON OVEN DRY BASIS) (Data of A. Austin, H.D. Singh, V.K. Hansias)

V a r ie ty

4 a

T rea tm en t

M jb Мгс

K a lyan sona 11.30 12.11 12.38

HD 1941 12.23 12.61 14.13

HD 1944 12.32 12.61 12.70

HD 1949 12.02 13.00 13.20

HD 1539 11.94 13.33 15.06

HD 1674 11.52 13.16 13 .25

Pusa Lerm a 11.77 14 .85 16 .45

EA 222-1 11.73 12.36 12.78

D 2117 12.91 13 .08 13 .75

WL 202 13.63 13 .79 14.09

NX 5645 12.32 13.46 14.26

UP 301 12 .87 13.92 14.55

a Ц , : N o fe r t i l iz e r ,

b М д : N 135 + P 67 + К 34 kg/ha.

c Мг : N 200 + P 100 + К 50 kg/ha.

1 6 4 SWAMINATHAN

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CROP IDEOTYPES 165

Some promising Triticales derived from crosses between T. durum and S. ce re ale were treated in 1967 by Sharma and Swaminathan (unpublished) with different doses of gamma-rays and EMS. Observations were recorded on the number of grains per spike, number, of spikelets per spike and the yield of the main spike in the M2 and M3 populations. The variability in the number of grains per spike was much greater in the mutagen-treated populations as compared to the control (Table V). The population has responded positively to selection.

Apart from variability for seed fertility, the Mgand M3 populations contained plants with plump grains, early maturity and dwarf stature. The seeds of these mutants are being multiplied for trials.

6. INDUCED VARIATION FOR PROTEIN PROPERTIES

6.1. Bread wheat

The development of rapid screening techniques such as the dye-binding- capacity (DBC) method for estimating the content of protein and basic amino acids has made the examination of large populations of mutagen-treated material possible. Studies by Anand Kumar (unpublished) in the bread wheat variety Sharbati Sonora have shown that the variation for DBC values is considerably enlarged by both gamma-ray and EMS treatments (Fig. 3). It is also of interest that some induced mutants, such as Pusa Lerma, a gamma-ray induced amber-grain mutant of the Mexican semi-dwarf and red-grain variety, Lerma Rojo, show a striking enhancement in protein content with fertilizer application (Table VI). The data in Table VI indicate that varieties differ in their ability to produce more protein with increased fertilizer application.

Pusa Lerma has given high yields in national trials conducted under the All-India Co-ordinated Wheat Improvement Programme in Central and Peninsular India. Its performance as compared to the best check, Kalyan Sona, is given in Table VII. Monosomie analysis has revealed that chromosome ЗА of Lerma Rojo carries genes both for grain colour and protein content [11].

6.2. Barley

Munck et al. [12] have studied the genetics of the spontaneously occurring high protein and high protein-cum-high lysine (Hiproly) character in barley.It is clear from their study that these traits are under simple genetic control. During a study of the M2 and M3 populations of a six-rowed spring barley variety N.P. 113 treated with gamma-rays, fast neutrons, EMS, and NMU, several dwarf and early mutants were observed (Bansal, unpublished). Some of these mutants had a good yield potential and one early mutant, В. M. 20, has a protein content of over 17% (Table VIII). This high protein mutant had normal fertility and grain development and was isolated in the treatment with 0. 3% EMS [13].

In the M2 progeny of EMS (0. 3%) treated N. P. 113, a mutant characterized by a depression on the dorsal side of the grain was isolated. This "notched grain" mutant was late by eight to ten days, had normal fertility and a 30% lower 1000-kernel weight. This mutant had over 17% protein content

1 6 6 SWAMINATHAN

T A B L E V I I I . C H A R A C T E R I S T I C S O F S O M E B A R L E Y M U T A N T S

Grain y ie ld Days to 1000-kernel w eigh t Protein content

(q/h a ) m aturity (g m )

N .P . 113 (c o n tro l) 54.1 120 4 2 .0 11.2

B .M . 10 52.6 100 3 7 .5 11 .2

B .M . 20 4 3 .9 95 36 .3 17.66

B .M . 21 55 .5 96 38 .4 14 .4

TABLE IX. PROTEIN CONTENT OF NOTCHED GRAIN MUTANT IN BARLEY VAR. N. P. 113

. , . . riU k C l l l _ _M ater ia l DBC va lu e

N .P . 113 (c o n tro l) (a ) 10.23 0 .21

(b ) 11 .24 0 .22

N otched m utant (a ) 18.29 0 .305

(b ) 17 .45 0 .30

TABLE X. PROTEIN PROPERTIES OF SOME MINOR MILLETS

Species and va r ie ty

Paspalum

scrob icu latum

Prote in Lysine Tryptophan

(% ) (g/100 g prote in ) (g/100 g protein )

E leucine corocana

I .E . 903 6 .23 2 .13 1 .44

I .E . 901 7 .97 2 .63 1 .36

Setaria ita lic a

1 .5 . 711 10 .02 2 .2 9 0 .81

1 .5 . 263 11 .56 2 .5 8 1 .05

Pan icum m ilia ceu m

I. PM 1640 11.60 4 .3 5 1.10

I. PM 1639 12.19 4 .1 5 1 .1 8

I. Ps 261 11.56 3 .5 5 0 .92

I.P s 19 * 12 .62 3 .7 8 1 .14

CROP IDEOTYPES 167

and it has been crossed with the parent strain to ascertain whether the notched grain character canbe separated from the high proteintrait (Table IX). The mutant had a lysine content of 3. 49 g per 100 g protein and this was only slightly lower than that of the parent.

6.3. Panicum miliaceum

This millet has excellent protein properties (Table X), but its yield potential is low. Hence, seeds of a few cultivars of this millet have been treated with mutagens, in order to isolate dwarf and fertilizer-responsive strains.

7. INDUCTION OF MUTATIONS AT THE HAPLOID LEVEL

With the standardization of a technique for the raising of haploids from anther cultures by Guha and Maheshwari [14], a way is now open for the large scale production of haploids and their use in mutation breeding. While the original finding was made in Datura innoxia, subsequent research has shown that haploids of Nicotiana tabacum [15, 16, 17, 18] and of Oryza sativa [19, 20] can be produced by in vitro anther culture. Nitsch and Pereau-Leroy [21] have used mutagens in culture media for inducing mutations at the haploid level in Nicotiana tabacum. Gamma-rays were more effective than EMS in yielding mutants [22].

For using this technique successfully, it is necessary to standardize techniques for getting consistently a large number of haploids. Since genotypic differences in the ability of pollen to form embryoids seem to exist,Guha and Svvaminathan [23] carried out a study with 20 different rice varieties. Since the techniques used may be of interest they are described in detail here.

Florets of 20 different varieties of rice were collected while still enclosed within the sheath. After sterilization in dilute chlorox solution, they were transferred to an inoculation chamber where the buds were dissected and five anthers were planted on a sterile nutrient medium. Blaydes' medium [24] for soybean callus cultures with 3% sucrose was used as the basal medium. Various growth substances such as indoleacetic acid (IAA),2, 4-dichlorophenoxyacetic acid (2,4-D), kinetin, yeast extract and coconut milk were also added to the basal medium when required. The media were sterilized by autoclaving at 15 lb/in2 for 15 min and solidified, with 0.8% agar. The pH was adjusted to 5.8. Cultures were kept at 25°C, for the initial three to four weeks in dark and then in light (warm white fluorescent,1000 ft-candles),

Anthers were planted at three stages of development — late meiotic and tetrad formation stage, uninucleate stage and on the day of anthesis. Young anthers implanted at the time of tetrad formation or earlier failed to grow on any media. When planted on the day of anthesis, when pollen grains are mature, most of the anthers opened liberating the pollen grains which germinated giving rise to pollen tubes. Only anthers containing uninucleate pollen grains showed differentiation of pollen embryoids. Therefore, further experiments were carried out only with anthers containing uninucleate pollen grains. These results are similar to those reported for tobacco pollen.

In the basal medium without any growth adjuvants, anthers degenerated two to three weeks after inoculation. The addition of coconut milk (15%)

168 SWAMINATHAN

TABLE XI. VARIATION IN EMBRYOID FORMATION IN RICE VARIETIES

N o . StrainN o . o f anthers

inocu latedResponse

Ind ica strains

1. Assam 271 160 Som e ca llu s ing o f p o llen grain , but no

em bryo id fo rm ation

2. Assam 10456 130 N o response

3. Assam 10303 80 Som e ca llu s ing o f the p o llen but no

em bryo id form ation

4 . Assam Loca l 100 N o response

5. Assam 5955 1735 11% anthers produced p o llen em bryo id

6. Assam 5 172 26*70 anthers produced p o llen em bryo id

7. T ripura 2-5744 186 N o response

8. Assam cu lture 100 N o response

9. Soft gra in typ e o f

W est Bengal 245 C a llu sing

10. Sabarm ati 230 N o response

11. IR -8 1576 0 .8 % anthers showed p o llen em bryoids

12. IR-532 100 N o response

13. Ta ich u n g N a t iv e -1 360 Po llen grains en larged but no d iv is ion

14. N .P , 130 600 N o response

15. B asm ati-370 1735 0 .3 % anthers produced p o llen em bryoids

16. K a la-B asm ati 20 N o response

17. S igadis 100 N o response

18. S igadis IR -8 hybrid

Japónica strains

148 N o response

19. Ta ichung 65 720 Sligh t callusing but no em bryoid

fo rm ation

20. Ta in an 3 480 N o response

and yeast extract (1000 ppm) stimulated many pollen grains to divide. The incorporation of 2 ppm IAA and 2 ppm 2, 4-D and 1 ppm kinetin further stimulated division; this medium was therefore generally used for the induction of mitosis in pollen grains.

Acetocarmine squashes of the anthers were prepared at regular intervals throughout the culture period to assess the state of development of pollen. After one week in culture, some of the pollen grains started enlarging and attained two to three times their original volume. In these grains, the cytoplasm stained very densely. In another week, they underwent mitosis and became binucleate, where both the cells were morphologically alike. Soon,

CROP IDEOTYPES 169

mitosis commenced in both the cells, which continued giving rise to multicellular pollen grains three to four weeks after planting. In older cultures, these multicellular masses (embryoids) burst out of their original wall and assumed a globular stage. If embryoids were dissected out at this stage and planted on a fresh medium containing coconut milk (15%) without auxin, they exhibited organized growth and developed into normal plantlets. Chromosome counts in root tips in several such seedlings revealed that they are haploids with 2n = 12. However, if embryoids were allowed to grow in the original medium where they arose, divisions in the pollen grains continued in an irregular manner resulting in a mass of callus. It appeared that during the initial stages of development, 'pollen embryoids' get attached to the wall of the anthers by a suspensor-like outgrowth. It is very easy to miss this attachment if anthers are squashed or subjected to even slight pressure. Careful dissections revealed this attachment, the anther wall apparently acting like aplacentum. However, after some time, the embryoids get detached from the anther wall and grow independently.

There was a wide variation in the 20 varieties studied with regard to their potential for giving rise to androgenic haploids through this technique. Some primitive cultivare from Assam, e.g. Assam-5, gave an excellent response (Table XI). Tropical japónica varieties from Taiwan such as Taichung 65 and Tainan-3 did not behave any differently from many of the indica varieties. The dwarf, high-yielding indica strain, IR-8, developed at the International Rice Research Institute, the Philippines, was found suitable for use in such studies. The promising varieties are now being used in studies on the induction of mutations at the haploid level.

REFERENCES

[11 D O N A LD , C .M . , P io c .3rd In t.W h ea t G enetics S ym p .. Canberra (1 9 6 8 ) 377.

[2 ] S W A M IN A T H A N , M .S . . P r o c .X I I In t.C on gr.G en e tic s , T o k y o 3 (1968 ) 327.

[3 ] S W A M IN A T H A N , M .S . . Induced Mutations in Plants (P ro c . S ym p. Pu llm an, 1969), IAE A , V ien n a (1 9 6 9 )

719.[4 ] A S A N A , R .D . , A N ew T ech n o lo g y for D ryland Farm ing, Ind ian A gr icu ltu ra l Research Institute (1970 ) 55.

[5 ] S W A M IN A T H A N , M .S . , CHOPRA, V . L . , SASTR Y , G .R .K . , C u rr .S c i. 3 5 (1 9 6 6 ) 91.

[6 ] Q U ALSET, O .O .. F IC K , G .N . , C O N S T A N T IN , M .J ., OSBORNE, T .S ., S c ience 169 (1970) 1090.

[7 ] EVANS , L . T . , DUNSTO NE, R .L . , Aust. J .b io l. S c i. 23 ( 1970) 725.

[81 E A S T IN , J .D . , H ASK IN S . F .A . , S U LL IV A N , C . Y . , V A N BAVEL, C . P .C .H .M . , P h ys io lo g ica l Aspects

o f Crop Y ie ld , A m erican S oc ie ty o f A gron om y (1 9 6 9 ).

[9 ] BORLAUG, N .E . , P ro c .3rd In t.W h ea t Genetics S ym p ., Canberra (1968 ) 1.

[1 0 ] F IN L A Y . K .W . , P ro c .3rd In t. W heat G enetics S ym p ., Canberra (1968 ) 403.

[1 1 ] JHA, M .P . , K AU L, A . K . , R A G H A V IA H , P . , S W A M IN A T H A N . M .S . , W heat In form ation S erv ice

(1971 ), ( in press).

[1 2 ] M U N C K , L . , KARLSSON, K .E . , HAGBERG, A . , EGGUM, B .O . , S c ien ce 168 (1970 ) 985.

[ 1 3 ] BANSAL, H .C . , C u rr .S c i. 3 9 (1 9 7 0 ) 424.

[1 4 ] G U H A, S .. M AH ESH W ARI, S .C . , N atu re, Lond . 204 (1964 ) 497.

[1 5 ] N A K A T A . K . , T A N A K A . М . , J ap .J .G en et. 43 (1968 ) 64.

[16 ] T A N A K A , М ., N A K A T A , K . , Jap .J .G en et. 44 (1969 ) 88.

[17 ] N IT S C H , J .P . , N IT S C H , C . , S c ien ce 163 (1969 ) 85.

[1 8 ] SUNDERLAND, N . , W IC K S . F .M . , N atu re. Lond. 224 (1969 ) 1227.

[1 9 ] N IIZ E K I, H . , OONO, K . , P roc.Japan A ca d . 44 (1968 ) 554.

[2 0 ] GUH A, S ., IYER. R .D . , G U PTA , N . . S W A M IN A T H A N , M .S . , C u rr .S c i. 39 ( 1970) 171.

[2 1 ] N IT S C H , J .P . , PEREAU-LEROY, P . , C .r .h e b d . Séanc. A c a d .S c i . , Paris 269 D (1 9 6 9 ) 1650.

[2 2 ] N IT S C H , J .P . , Phytom orphology 1 9 (1 9 6 9 ) 389.

170 SWAMINATHAN

[2 3 ] GUH A, S ., S W A M IN A T H A N , M .S . , S c ience (1971 ), ( in press).

[2 4 ] BLAYDES, D .F . , Ph ys io log ie P l. 19 (1966 ) 748.

[2 5 ] JAG ATH E SAN , D . , B H A T IA , C . , S W A M IN A T H A N , M .S . , Nature, Lond. 190 (1961) 468.

[26 ] VARUGHESE, G ., S W A M IN A T H A N , M .S . , Indian Fm g 17 ( 1967) 8.

[27 ] BALA N A R A S A IA H , D . , KU LKARN I, L .G .„ Indian Frog 18 (1969 ) U .

[28 ] V IS H N U SWARUP, GILL, H .S . , Indian J .G en et. 28 (1968 ) 44 .

[2 9 ] JA IN , H .K . , SUR, S .С . , R A U T , R .N . , Indian I .G e n e t . 22 (1962 ) 81.

DISCUSSION

R.S. LOOMIS: Many plant breeders are now seeking to incorporate intocrop varieties tolerance and responsiveness to high nutrition. They also need to be concerned with maintaining and improving the plants' efficiency in foraging, uptake and assimilation of nutrient elements since selections made in highly fertile environments might easily lead to lower efficiency. Would an increase in population density be a reasonable alternative to the branched- head ideotype for semi-arid regions?

M.S. SWAMINATHAN: I entirely agree that we should breed varietieswith ability to utilize nutrients efficiently. Such studies are in progress at the Indian Agricultural Research Institute and recent research by Mr. Singh, a graduate student working with Dr. R.G. Anderson, has shown that the bread wheat variety, Sharbati Sonora, is very efficient in the utilization of nutrients and has also the ability to respond to high nutrition.

Increasing the density of plant population is certainly possible through better tillage, moisture conservation and sowing methods. However, since there are negative correlations between early maturity and spikelet number and early maturity and deep root development, the development of a branched ear provides an opportunity to increase the sink capacity under a short growing season. The two approaches — agronomic and genetic — are not mutually exclusive but will have to be integrated to achieve a jump of a higher quantum in the yield of dryland wheat.

B. SIGURBJOr NSSON: With reference to the list of released mutantvarieties you presented, could you give us an indication of the total acreage in India under mutant varieties?

M.S. SWAMINATHAN: The wheat variety N. P. 836 is a tall strain andmay be occupying only a small area in the States of Bihar and Bengal since semi-dwarfs have practically replaced the tall varieties. Sharbati Sonora is very popular for late sowing and has given high yields under proper agronomic management. Its grain quality is excellent for making both leavened and unleavened bread. Its area is likely to be about 0. 5 million hectares. The rice strain, Jagannath, is a photo-sensitive variety and is very suitable for monsoon conditions. It was released only last year and it may occupy about 100 000 hectares by 1971. The castor strain Arura is fast replacing the older varieties in the Telergana region of Andhra Pradesh and it is now grown in over 100 000 hectares. It is likely to become the most widely cultivated castor strain soon.

A. GROBMAN: The ideotype you presented for a wheat plant as developed by Asana show leaves extending at right angles to the stem. How do you reconcile this model with the one presented by Dr. Loomis where leaves appear inserted at acute angles in the stem for optimum light utilization?

CROP IDEOTYPES 171

M.S. SWAMINATHAN: Dr. Asana1 s ideotype is for dryland wheat andthe leaf characteristics are intended for enabling the capture and retention of dew as well as for intercepting sunlight efficiently. Under dryland farming, it is difficult to achieve a high density of plant population, particularly in an agricultural system which does not involve mechanized operations. Dr. Loomis' model, like that of Dr. Donald to which I referred, would help to maximize yield under optimal conditions of water and nutrient supply.

C.F. KONZAK: In relation to the concept of plant type you presented,what is the evidence for absorption of moisture by leaves of cereals. I know that such a phenomenon can be demonstrated for Solanacae, but it has been my impression that a plan to absorb dew as a source of water is unrealistic. Certainly such a structure would have negative value in terms of available surface for pathogens. Please obtain evidence if available before this is published, if the matter is not only a hypothetical concept.

M.S. SWAMINATHAN: There is ample evidence that moisture isabsorbed through leaves; this is the very basis of foliar feeding with nutrients. Even if the cuticle does not permit direct absorption of moisture in the very early stages, the energy balance will be favourable, if there is dew on the leaves during the process of evapo-transpiration. In the later stages of growth, the water can get into the cells also. In dry areas, the problem of rusts and other diseases is not important. The ideotype I presented is based upon a very detailed understanding of the physiology of the plant and of the ecology of the regions for which the ideotype has been developed. If you have any evidence which disproves the currently held concepts of the production physiologists, I shall be happy to have it.

K. BOROJEVIC:. How many plants can you grow of your ideotype with horizontal leaf position?

M. S. SWAMINATHAN: It is the number of ears per unit area that isimportant for yield and not merely plants per given area. In our earlier varieties, many tillers were non-productive due to the asynchronous nature of the tillering habit. This is why in the new ideotype emphasis has been placed on the productivity of the main tiller. We may be able to get a density of about 500 spikes per square metre.

UTILIZATION OF INDUCED CHROMOSOMAL ABERRATIONS Translocations, duplications and trisomies in barley*

A. HAGBERG, G. PERSSON, G. HAGBERG Swedish Seed Association,Svalôf, Sweden

Abstract-Resumen

U T IL IZ A T IO N OF INDUCED CH RO M O SO M AL ABERRATIONS: TRANS L O C A T IO N S , D U PL IC A T IO N S AND

TR ISO M IC S IN BARLEY.

C y to g en e t ic a l analysis o f induced b a r ley mutants led to a la rg e c o lle c t io n o f translocation lines w ith

d iffe ren t break points in each chrom osom e. Th is m a te r ia l is not on ly useful fo r lo c a liz a t io n o f genes

and lin k age groups, but can b e used also as a to o l or source for the estab lishm ent o f new karyotypes and

new gen e orders in the ba r ley chrom osom e. M uch o f the present work on hybrid ba r ley is based on tertia ry

trisom ies, derived from an induced segm en ta l in terchange in the Swedish ba r ley v a r ie ty "Bonus".

U T IL IZ A C IO N DE LAS ABERRACIONES CRO M O SO M ICAS IN D U C ID AS : TR AN SLO C AC IO N E S, D U PLICAC IO NES

Y TR ISO M ICO S EN LA CEBADA.

C om o resultado d e l análisis c ito g en é t ic o d e mutantes inducidos de cebada se ha obten ido un gran

núm ero de lin eas de translocación con distintos puntos de ruptura en cada crom osom a. Esto no só lo es

ú til para la lo c a l iz a c ió n de los genes y de los grupos d e en la ce , sino que puede serv ir d e m ed io o base para

e s tab lecer nuevos cario tipos y nuevos órdenes de genes en e l crom osom a de la cebada. Gran parte de los

actuales trabajos sobre cebada h íbrida se basan en e jem p lares trisóm icos terc ia rios , derivados de un

in te rcam b io s egm en ta l inducido en la variedad de cebada sueca «B o n u s » .

In a presentation of this kind there is always a choice between a general survey and a narrow topic illustrating general principles by the presentation of one's own recent results. For many reasons barley is used as a model giant in Swedish mutation research, and in his papers at this meeting Ake Gustafsson covers some types of mutants induced and cytogenetically analysed. We have been concentrating mainly on a cytogenetic analysis of induced mutations which might have a practical utilization in barley breeding, since our group is engaged in barley breeding work. To us it is important to stress the great value of continuity in building up a cytogenetic material with a vast fund of information which can be us eful as a tool in future genetic and breeding projects of our crops.

Our main interest in induced chromosome aberrations started with the localization in barley chromosomes of the loci for some stiff straw erectoides mutants linked to translocation break points [1]. The induced reciprocal translocations — segmental interchanges — were used to establish the bridge between the linkage groups and the barley chromosomes [2]. A series of papers [3, 4, 5] have shown the usefulness and the limitations of reciprocal translocations in direct localization of genes in linkage groups. The incorporation of markers and linked induced

* Part o f the research reported in this paper has been carried out under Research A g reem en t w ith

the In ternational A to m ic Energy A gen cy No. 3 18/CF.

173

174 HAGBERG et al.

mutations in homozygous translocation stocks with suitably located break points gives us the possibility to reveal the relative position of the genes to break points and to each other [6].

Thus, it is useful to have a number of break points available in each chromosome to be used as cytogenetic markers and as a tool or source for the establishment of new karyotypes and new gene orders in the barley chromosomes. For this reason we have been collecting induced segmental interchanges which are isolated and identified [7]. The material of translocations available is listed in Table I, giving the distribution on the different chromosomes taking part in the exchange.

In each chromosome we thus have a considerable number of break points to choose from which is important for the above mentioned reason in linkage studies and also if you want to produce certain duplications or trisomies. If you cross the different translocations involving the same two chromosomes diallelicly, e. g. the 19 translocations between the chromosomes 4 and 6 (Table I), the configuration in the MI of meiosis in the Fj generation will give information on relative position of break points.

TABLE I. INDUCED TRANSLOCATIONS BETWEEN DIFFERENT PAIRS OF BARLEY CHROMOSOMES

Chrom osom e no. 7 6 5 4 3 2 1Sum o f

breaks

1 26 27 32 27 25 19 - 156

2 22 22 20 25 27 - 135

3 25 16 22 24 - ' 139

4 14 19 23 - 132

5 22 20 - 139

6 22 - 126

7 - 131

T o ta l sum o f breaks 958

Table II clearly demonstrates the grouping of the T-lines in two groups: crosses within each group giving 7 II (marked II in the Table) and crosses between groups giving 1 IV + 5 II (marked IV in the Table). There is only one exception in this diallele, namely T 377, which very often gives F} plants with low chiasma frequency in chromosomes 4 and 6 resulting in open chains or rod-bivalents. Initial pairing seems to be 1 IV + 5 II in all combinations and the break point is probably in the neighbourhood of the satellite constriction of chromosome 6.

The two actual groups differ in break position as previously demonstrated [8]. This is verified by root tip mitosis studies (Table III). If the break points are on the same side of the centromere in both chromosomes in the two translocations crossed, there is formed 7 II (T358 XT 551) as is the case when the breaks are on different sides of the centromere in both chromosomes (T 358 X T 161, T 551 X T 161).

INDUCED CHROMOSOMAL ABERRATIONS 175

1 IV + 5 II are obtained when in one chromosome the breaks are in the same arm and in the other chromosome the breaks are in different arms (T 555 X T 358, T 555 XT 551 and T 555 X T 161). Special cases are found when the breaks are in the centromere region and the centromere itself is broken, and when one break is close to the nucleol organizer and the pairing sometimes seems to be influenced by this location (e. g.T4-6; 377).

If the crosses between two different reciprocal translocations are studied in F2 there are possibilities of obtaining plants homozygous for duplications, for duplication-deletions and for deletions if they are viable.In no case so far in barley have viable deletions or even duplication - deletions been obtained. Detailed studies prove them in some cases to be already lethal in the gametes. Other cross combinations give a few heterozygous deletions. As predicted [9, 10], there are two types of relative break positions in the translocations crossed which result in duplications. These duplications are viable, but they occur less frequently than expected. The two types can be illustrated by the two crosses in Figs la and lb.

Out of the F2 family, six F2 plants were obtained which proved to be duplication plants with regard to the segment "x" between the a and d break points in chromosome 6 (Fig. 2).

Typical for this type of break position is that one of the break points in each translocation is exactly located in the same spot, in this case in the centromere of chromosome 7. This corresponds to type 2 с [8].

The other possible break position for production of duplication is type 2 b [8], where the breaks in both chromosomes are on the same side of the centromere, and the T-line with the distal break in one chromosome has the proximal break in the other (Fig. lb).

As illustrated in Fig. 3, the Fj plants form three types of viable gametes, and one which is not viable, since it is completely lacking nucleol organizer.

In the F2 family we checked randomly 59 plants for karyotype; the distribution is shown in Fig. 4.

Thus there is a deficit of homozygous duplication plants and also the heterozygous duplications are slightly less than expected. These plants are especially interesting as they give us a chance to study the dose effect of the nucleol organizer.

In addition to studies on dose effects, the duplicatiops can be useful among other things in the mapping of the chromosomes, where the duplications could be used as are the trisomies for the whole chromosomes. The duplications can give additional information on gene sequence as could translocations described earlier in this paper.

In a paper presented at the First International Barley Genetics Symposium in 1963, Ramage [ill suggested the production oí hybrid barley based on the use of tertiary trisomies balanced for a male sterility factor. Seven years later Ramage was able to release the first hybrid barley variety Hembar using the tertiary trisomies derived from the segmental interchange T2-7d induced by X-rays in Bonus barley. Much of the present work on hybrid barley is based on this tertiary trisomie type.

The system that Ramage suggested is illustrated in Fig. 5. Many problems are involved in the production of the female parent and of course in finding the best parents with extremely high combining ability and in

176 HAGBERG et al.

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178 HAGBERG et al.

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FIG. 1. T w o types o fr e la t iv e break positions in crossed translocations resulting in duplications (a ) M eiosis

o f F, = 1 IV + 5 II, ( b ) M eiosis o f Fi: 7 II.

I X i

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VIABLE

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obtaining a seed set high enough to secure an economic seed production. Another problem is to find a trisomie with a suitable productivity.

Wiebe and Ramage [12] presented a system (Fig. 6) allowing multiplication of trisomie stock not requiring diploids. In this system the break point in the tertiary trisomie should be closely linked to one or more male sterility genes and a lethal marker to control the genotype of the progeny. The T-lines are now being investigated from this point of view in the hope of finding still better combinations of suitable ms-genes and break points to give a new chromosome of suitable size resulting in an optimum vitality for the purpose. In this work the collection of induced T-lines and also all information made available are very useful. This is a very beautiful example of the efficient use of this kind of material in actual breeding projects. Such examples have up to now been rather scarce. I am convinced that they will become more common as we learn more about the genetics and cytogenetics of our crops.

1 8 0 HAGBERG et al.

H yb rid

FIG . 5. Use o f ba lanced tertia ry tr isom ie in m u ltip lica tion o f fem a le parent for co m m erc ia l production

o f hybrid barley.

(F IG . 5. E m pleo d e un tr isôm ico te rc ia r io equ ilib rado en la m u lt ip lic a c ió n de progenitores fem en inos para

la producción c o m e rc ia l d e cebada h íbrida. )


m s o ms a Ms A

4 A

m s о ms о Ms A

*

H yb rid

FIG. 6. Use o f ba lan ced tertia ry tr isom ie w ith lin k age betw een m a le s te r ility , the break point and a le th a l

gen e in m u ltip lic a tio n o f fe m a le parent for c o m m erc ia l production o f hybrid barley .

(F IG , 6. E m p leo de un tr isôm ico te rc ia r io equ ilib rado con en la ce entre la esterilid ad m ascu lina, e l punto

de ruptura y un gene le ta l en la m u lt ip lic a c ió n de progenitores fem en inos para la producción c o m e rc ia l

de cebada h íbrida. )

182 HAGBERG et al.

REFERENCES

[ 1 ] HAGBERG, A . , TJIO, J .H , , C y to lo g ic a l lo c a liz a t io n o f the translocation point for the barley

m utant erecto id es 7, Hereditas 36 (1950) 487.

[2 ] BURNHAM , C .R . , HAGBERG, A , , C y to g en e tic notes on chrom osom al interchanges in barley ,

H ereditas 42 ( 1956) 467.

[3 ] HAGBERG, A . , C y to g en e tic analysis o f induced m utations, G enet, agr. 12 (1960) 319.

[4 ] PERSSON, G . , A n a ttem pt to find su itab le gen e tic markers for d ense-ear lo c i in ba r ley I, Hereditas

62 (1969) 25.

[ 5 ] FESTER, T . , SOGÂRD, В ., T h e lo c a liz a t io n o f eceriferu m lo c i in b a rley , H ered itas 61 (1969 ) 327.

[ 6 ] PERSSON, G . , An a ttem pt to find su itab le g en e tic markers for d ense-ear lo c i in barley II, Hereditas

63 (1969) 1.

[ 7 ] HAGBERG, A . , "Induced structural mutations in ba rley , esp ec ia lly translocation , and their use in

further d irected production o f v a r ia t io n ", Mutations in P lant Breeding (P roc . Panel V ienna, 1966),

IA E A . V ienna (1966) 109.

[83 HAGBERG, A . , "U se o f induced translocations in d irec ted production o f dup lica tion s", T h e Use o f

Induced Mutations in Plant Breeding (Rep. F A O / IA E A T ech . M ee tin g , Rom e, 1964), Pergam on

Press, Oxford (1965 ), 741.

[ 9 ] G O P IN A TH , D. M . , BURNHAM, C . R . , A cy to g en e tic study in m a iz e o f d e fic ien cy -d u p lic a tio n

produced by crossing interchanges in vo lv in g the sam e chrom osom es, G enetics 41 (1956) 382.

[ 10] HAGBERG, A . , HAGBERG, G . , "U se o f induced structural rearrangem ents in plant b reed in g ",

Induced Mutations in Plants (P roc . Symp. Pu llm an, 1969), IA E A , V ienna (1969 ) 647.

[1 1 ] RAM AG E, R. T . , "C hrom osom e aberrations and th e ir use in genetics and b reed ing - translocations",

Proc. 1st Int. B arley G enet. S ym p ., W agen ingen ( 1964) 99.

[1 2 ] WIEBE, G. A . , RAM AG E, R. T . , "H ybrid b a r le y " , Proc. 2nd Int. Barley G enet. Sym p.

Pu llm an (1969 ), ( in press).

DISCUSSION

W. GOTTSCHA.LK: Could you give us some details concerning the distribution of the breaks over the chromosomes or the whole genome, respectively? Is it a random distribution or is there a marked increase of the number of breaks in different regions of the chromosomes, for instance in the centromere region?

A. HAGBERG: There seem to be more breaks around the constrictions especially the centromeres than in other regions. However, we have concentrated on translocations induced by radiation and, thus, we have found a rather good distribution all over the chromosomes.

O. P. KAMRA.: Prof. Hagberg, could you kindly tell us about the application of duplication techniques on segments with o-amylase activity or other similar characters in barley.

A. HAGBERG: One of the goals of producing a duplication of most of the short arm of chromosome 6 was to study the dose effect of an a-amylase-controlling gene (orange lemma). We started out with a base material of orange lemma which later proved not to be linked to an a-amylase-activity gene. The correct orange lemma is now transferred to T6-7a and T6-7d but the cross between them has not yet been studied.A group at the Scottish Plant Breeding Station is working on this project now.

MUTAGENESIS APPLIED TO durum WHEAT Results and Perspectives*

G.T. SCARASCIA-MUGNOZZAIstituto di Agronomía dell'Université di Bari

D. BAGNARA, A. BOZZINILaboratorio per le Applicazioni in Agricultura del CNEN,Centro Studi Nucleari della Casaccia, S. Maria di Galería,Roma, Italy

Abstract-Resumen

M UTAG ENESIS APPLIED T O durum W H E A T : RESULTS AND PERSPECTIVES.

T h e work started in 1955-56 w ith com parisons o f various mutagens such as X -rays , fast neutrons,

therm al neutrons, e th y len e im in e , d ie th y l sulphate and ethy l m ethane sulphonate. T h e program la te r

included research on id en t if ic a t io n and se le c tion o f mutants, studies regard ing the m od e o f inheritance

o f induced m utations and testing o f the agron om ica l va lu e .o f s e lec ted m utant lines. These agron om ica l

tests have been carried out for a number o f years at d ifferen t loca tions in Ita ly and w ere then expanded

w ith support from FA O and IA E A to in ternational tria ls, carried out in co -op e ra tion w ith numerous

institutes and the countries o f the M ed iterranean and Near East regions. T h ree mutant va r ie ties w ere o f f ic ia l ly

re leased , and a fourth one has reached the f in a l stage o f o f f ic ia l testing. T h e u t iliz a t io n o f induced mutants

in cross-breed ing programs appears to be ex trem e ly prom ising. A la rge number o f lin es show pos itive

transgressions and ou ty ie ld both parents by up to 4 (P]o,

It is concluded that m utagenesis has proved to be a va lu ab le to o l for g en e t ic im provem en t o f

T r it icu m durum. It can w iden the v a r ia b ility o f a g ro -econ om ic characteristics , produce factors w ith a

g en e t ic a l behaviou r d iffe ren t from the usual one, and p rovide genes w h ich are not or on ly ex cep tion a lly

found in germ plasm co llec tion s . U t il iz a t io n o f m utant lines in cross-breed ing programs has been found

to b e o ften o f grea t advan tage com pared w ith the use o f w ild species, p r im it iv e m a te r ia l or e ven old cu ltivars.

A P L I C A C I O N D E L A M U T A G E N E S IS A L T R IG O D U R O : R E S U L T A D O S Y P E R S P E C T IV A S .

Los trabajos d ieron com ien zo en 1955-1956, con com paraciones de diversos m utágenos com o rayos X ,

neutrones rápidos, neutrones térm icos, e t ilen im in a , su lfato de d ie t i lo y m etanosu lfonato de e t i lo . Posteriorm ente,

se ex ten d ieron a las in vestigac iones sobre id en t if ic a c ió n y s e le cc ión de mutantes, a l estudio d e la m oda lidad

de transm isión h ered ita ria d e las m utaciones inducidas, y a la com probac ión d e l va lo r agron óm ico de las

lín eas mutantes se lecc ionadas, Estas com probaciones se han lle v a d o a cabo a l o la rg o de una ser ie de años

en d iferen tes lugares de I ta lia y , posteriorm ente, con e l apoyo de la FAO y e l O IEA, se han am p liado a

esca la in tern ac ion a l, e jecu tándose en co lab o ra c ión con numerosas instituciones y con los países d e l M ed iterráneo

y d e l C ercan o O rien te. Se han au torizado o fic ia lm en te tres variedades mutantes y una cuarta ha a lcanzado

la fase f in a l de ensayos o f ic ia le s . Parece o frecer exce len tes perspectivas la u til iz a c ió n d e mutantes inducidos

en los programas de m e jo ram ien to por cruce. Un gran numero d e líneas m an ifiestan transgresiones positivas

y superan en ren d im ien to a ambos progenitores hasta en un 40°Jo.

Se l le g a a la conclusión de que la m utagénesis ha dem ostrado ser un va lio so instrum ento para e l

m e jo ram ien to g e n é t ic o d e l T r it icu m durum. Perm ite am p lia r la va riab ilid ad de las características

agroecon óm icas , producir factores con un com partam ien to g e n é t ic o d ife ren te d e l hab itua l, y obtener genes

que no se encuentran -o só lo ex c ep c io n a lm en te - en las co le cc ion es de p lasm a g e rm in a l. Se ha com probado

que la u til iz a c ió n de líneas mutantes en los programas de m e jo ram ien to por cruce es, en muchos casos, muy

venta josa com parada con e l em p le o de especies silvestres, m a te r ia l p r im it iv o o incluso plantas de c u lt iv o ya

trad ic iona les.

* C on tribu tion No. 284 from the Laboratorio per le A p p lic a z io n i in A g r ico ltu ra d e l CNEN,

C en tro Studi N u c lea r i C asacc ia , S. M aria d i G a le r ia , Rom a, Ita ly . Part o f the research reported in

this paper has been carried out under Research A g reem en t w ith the In ternational A to m ic Energy

A g en cy N o , 319/CF.

183

1 8 4 SCARASCIA-MUGNOZZA et al.

The Italian program on mutation breeding derives from projects planned by D'Amato and Scarascia in 1955-1956 which included the establishment of a plant radiogenetics laboratory and of a "gamma field". Such projects were brought into existence by the Italian Nuclear Research Committee at Casaccia Nuclear Research Centre.

The mutation breeding program has been gradually developed on various species of seed-propagated plants. These species were chosen as representatives of different genera, of different ploidy levels, and of different ontogenetic development; their economic importance in Italian agriculture was also taken into consideration. More recently, research on vegetatively propagated plants has also been started.

Triticum durum was the first species on which mutation breeding work was begun. The program on durum wheat started about 15 years ago and it was developed through investigations on radiobiology, mutagenesis, genetics, cytogenetics and breeding. It is reasonable to state that from these studies and experience on Triticum durum, information on some technical problems of general value for the application of mutagenesis to crop plants, as well as positive practical results for the improvement of durum wheat, have been gathered.

1 . I N T R O D U C T I O N

2. RADIOGENETICAL STUDIES

Seeds of several varieties have been treated with chemical mutagens such as ethylene imine (El), diethyl sulphate (dES), and ethyl methane sulphonate (EMS), and with mutagenic radiation such as X-rays and fast (Nf) and thermal (Nth) neutrons.

In a series of experiments and studies (from those published in 1962 by D'Amato et al. to the most recent published by Bozzini and Scarascia- Mugnozza in 1970), a comparison between chemical and physical mutagens was made. The relative efficiency of the mutagens was established by combining the results obtained from several varieties and analysing, on a spike-progeny basis, the M2 chlorophyll mutation frequency. In general, the relative efficiency of the mutagens was as follows: EI<dES<X <Nf<EMS<Nth (the range of mutation frequency being from 1 to 24%). For morphological mutations, the relative efficiency was slightly different: El <X < dES < EMS < N(h< Nf (the range of mutation frequency being from 1 to 41%).

Of interest here is the determination of a correlation between the relative frequency of chlorophyll mutation from greenhouse analysis, with the frequency of morphological mutation which can be detected later in field analyses. The correlation coefficient (r) was calculated to be 0.48, significant at 5% level. Chlorophyll mutation frequency compared to morphological mutation frequency, both obtained in field analysis, gave a correlation coefficient r = 0.65, significant at 1% level.

The knowledge acquired regarding the mode of inheritance of induced mutations is very important. From the studies carried out so far on this subject, it is possible to state that except in very few cases, all mutations investigated showed a recessive monogenic behaviour.

Dominant and monofactorial behaviour was ascertained for the culm- shortening mutation present in the mutant line Cp В 132 from "Cappelli"

MUTAGENESIS APPLIED TO WHEAT 185

(Bozzini and Scarascia-Mugnozza, 1967), and semidominant, but not definitely monofactorial behaviour for a mutation for kernel size in another mutant line (Cp CB 2) from "Cappelli" (Bagnara, Poukhalski and. Rossi, in press).

It is interesting to note that under monofactorial control appeared to be mutations affecting characters which are generally considered to have a polygenic basis, such as broadness of leaves (Bozzini, unpublished data), plant height, length of spike (Bozzini, 1964, 1965; Bagnara, 1967; Scarascia- Mugnozza and Bozzini, 1968), adventitious root number (Bozzini, unpublished data), etc. Such a finding is particularly valuable in plant breeding, since several of these characters are difficult to transfer from one variety to another because of the large number of factors involved.

Valid, also, are the demonstrations of the different localization of mutations controlling the same phenotypic expression. Bagnara (1967) ascertained that four short straw mutations entering a diallel cross program concern different loci; furthermore it was possible to isolate recombinants possessing two short straw mutations in homozygous conditions, with a further culm length reduction and an extremely high lodging resistance.The need and significance of such studies and findings is undeniable for practical breeding work. Some evidence from our work on durum wheat is presented below, together with the results of the combination breeding work, carried out with crosses between mutants and among mutants and varieties.

3. METHODS OF SELECTION OF MUTATIONS

The selection of mutated plants has been carried out by applying screening methods based on visual inspection or using appropriate techniques for the identification of the expected changes on a single plant basis. In general, the off-spring from treated seeds have been bred according to the spike-progeny method; less frequently, the bulk method was applied.After the selection of mutations in the M2 and M3 generations, great care was taken in the following two or three generations in order to make a preliminary selection of the mutants of possible value and interest for breeding purposes.

Simultaneously, careful observations were made on these mutants taking into consideration in addition to the main modified character the possible presence of more or less deleterious changes in other characters of agronomic importance, for example, excessive lateness, reduced fertility, pigment deficiency, increased disease susceptibility, and inferior seed quality.

Space-planted M3 and M4 progenies and also occasionally M5 progenies allowed the selection of individual plants carrying the potentially interesting mutation but free of unfavourable modifications in other important agro- economic characters. Using this technique, among about 2000 viable mutations selected so far, numerous useful mutations have been identified. They affect characteristics related to the improvement of durum wheat, such as culm-length, number of internodes, solid stem, size, number and disposition of leaves, lodging resistance, earliness, decreased yellow berry percentage, male sterility, and resistance to diseases. In this respect, it is interesting to note that Bozzini (1971) recently found that

TABLE

I.

NUM

BER

OF

MUTANT

LINES

TES

TED

IN TH

E PERIO

D

1961

TO

1967

186 SCARASCIA-MUGNOZZA et al.

<D Он5 и

 CO

sH T

ria

l ca

rrie

d ou

t in

an

exp

erim

enta

l fie

ld

near

F

ogg

ia;

tria

l "B

" w

as

carr

ied

out

un

der

irri

gate

d

con

dit

ion

s.

Tri

al

carr

ied

out

in an

ex

per

imen

tal

field

ne

ar

Pol

icor

o (M

ate

ra).

The

yiel

ds

(100

k

g/h

a)

mar

ked

with

th

e sa

me

cap

ita

l le

tter

, fo

r th

e sa

me

tria

l an

d fo

r th

e a

vera

ge,

are

not

sign

ific

an

tly

dif

fere

nt

at

0.

01

pro

bab

ilit

y le

ve

l,

acco

rdin

g to

Du

nca

n's

m

ult

iple

ra

nge

te

st.

Val

ues

fro

m

0 (n

o lo

dgi

ng

pre

sen

t)

to 5

(all

pl

ots

lod

ged

);

the

cap

ita

l le

tter

s h

ave

the

sam

e m

ean

ing

as

thos

e us

ed

for

yiel

d

figu

res.


two mutant lines selected for their good agronomic performance also show a higher level of resistance to Tilletia triticoides in comparison with the mother variety.

4. EXPERIMENTS IN ITALY FOR TESTING THE AGRONOMICALVALUE OF MUTANT LINES

Having at our disposal a large stock of different mutant lines, apparently endowed with good agronomic characteristics, it was possible to undertake a program aimed at ascertaining the concrete possibilities of the direct use as new varieties of the best mutants identified in the preliminary trials.

Starting from the M6 generation, large-scale field trials were first carried out in the regions around Rome in co-operation with local agricultural institutions, and around Bari (southern Italy) in co-operation with the Institute of Agronomy, University of Bari. Subsequently, the best mutant lines identified in these trials were tested in an increasing number of locations under the auspices of the Research Group on durum Wheat Breeding, which is sponsored by the Italian Research Council, and includes several university and non-university institutions in central and southern Italy, Sicily and Sardinia. Thus, a large network of agronomic trials was established and carried out for several years in these regions.The mutant lines were tested in comparison with the mother varieties (Cappelli, Garigliano, Grifoni, Russello, Aziziah), with good varieties of durum wheat cultivated in Italy (Capeiti, Patrizio, Camar 7, Sincape 9), and with new cross-bred lines obtained in Italy (Maristella, Ichnusa,Fuba 3, etc.) or elsewhere (for example, the North Dakota varieties,Ld 357, Lakota, Wells).

Hundreds of mutant lines have been analysed within the framework of this program, and the results are summarized in Table I. Although many well-established commercial durums were included as checks in the trials, performance of tested lines has been constantly referred to the variety "Cappelli", still widely cultivated in Italy and some other Mediterranean countries, and referred to as the standard varietj' for its good pasta making qualities. Furthermore, Cappelli was the only control kept constant through all our experiments. Table I shows that for each period of selection the mutants tested were divided into two groups: the "old" mutants, already evaluated in the previous period(s), and the "new" ones, entering the trials for the first time. The number of mutant lines tested is very high in the first period of trials, i. e. soon after their selection and testing in the first generation after treatment. However, selection was very drastic, since only one third of the first period mutants were considered worthy of further closer evaluation.

In successive periods the percentage of mutant lines superior to Cappelli increased, due apparently to the effectiveness of the previous selection. The decrement in the number of "new" mutants to be tested is mainly due to the fact that the mutagenic treatments of seeds of durum wheat varieties were reduced. The first results of another large experiment started in 1966 are now available in which physical and chemical mutagens were applied to seeds of the varieties Capeiti and Ld 357; numerous lines with enhanced earliness or with better standing ability are now under test.


It should be mentioned that most of the selection work for obtaining improved mutants was carried out for reduced plant height and lodging resistance (Scarascia-Mugnozza, 1965). In addition the ability of some induced mutants to increase their productivity in response to nitrogen fertilizers was thoroughly investigated.

The principal results of this evaluation of the agronomic potential of the best induced mutants, carried out at many locations and over many years, have been already published (see, for example, Scarascia-Mugnozza etal., 1964, 1965; Scarascia-Mugnozza, 1965; Scarascia-Mugnozza et al., 1966; Pacucci et al. , 1966; Scarascia-Mugnozza, 1966).

More recently, data on the behaviour of induced mutants in irrigated soils have also been gathered (Table II). From Table II it is evident that the variety Casteldelmonte shows an increase in productivity when irrigation is applied, and that the same variety reaches an average yield significantly higher than all other varieties, including the mother variety Grifoni and the cv. Capeiti, the cultivation area of which is at present rapidly increasing in Italy.

5. INTERNATIONAL CO-OPERATIVE TRIALS

Positive results have also been obtained from an international program, sponsored by the FAO and IAEA, for the assessment of the practical value of some of the above mentioned mutants in the Mediterranean and Near East regions. Multilocation nurseries have been operational since 1965-66 and the data of the first four years, during which reliable results have been obtained from 57 experimental fields spread over 16 countries, have already been processed. Each field trial included eight mutant lines; controls used were two Italian varieties, Cappelli and Capeiti, and two local varieties, freely chosen by each co-operator.

Although a complete analysis of the large amount of data collected has already been published (nine characteristics of each variety were measured in each trial, providing some useful information on the genotype- environment interaction), it seems useful to report here at least the yield performances of these lines tested in a wide range of environments.

Table III gives the average grain yields registered during the cooperative trials of the first three-year period. The highest yields shown are from two mutant lines (GA B125 and GR A145) and from Capeiti; these lines are significantly more productive than, for example, the overall mean of local varieties and the common control variety Cappelli.

The data in Table IV, which give the mean grain yields observed in 1968-69 (fourth year of trials), are in agreement with the previous ones. The highest yielding lines are again the variety Capeiti and the mutants GR A145, GA B125 and GA A7 (the latter two are sister lines derived from the variety Garigliano); the high value of the mean of the local varieties can be explained since in several trials bread wheat varieties were also included among them.

The good performance of the best mutant lines is also due to their earliness, which suited them to the rather dry climatic conditions of the spring prevalent in the region. However, the good yield potential of other less-early mutants, derived from Cappelli (CP B132 and CP C48), was evident in some years and in some locations when a mean grain yield of 6000 kg/ha was reached.


TABLE III. GRAIN YIELD OF durum WHEAT MUTANTS AND VARIETIES, 1965-1968

EntryY ie ld

(kg/ha)

Duncan's m u ltip le

range tesl:

IT 0 6 - G A В125 3348.24

C a p e it i 3325. 74

IT 0 9 - GR A 145 3263.38

IT 0 4 - C P A26 3221.89

IT 0 7 - G A A 7 3213. 33

IT 0 8 - C P В 132 3050.45

M ean o f a l l lo c a l va rie ties 3008,01

IT 0 2 - RS A 1 2995. 79

IT 0 5 - C P C48 2972, 90

IT O l - A Z В155 2878. 94

1ТОЗ - C P В 144 2816,54

C a p p e lli 2723. 02

TABLE IV. GRAIN YIELD OF durum WHEAT MUTANTS AND VARIETIES, 1968-1969

EntryY ie ld

(kg/ha )

Duncan's m u ltip le

range test

U N 02 C ap e it i

I T 09 GR A 145

IT 06 G A В125

M ean o f a l l lo c a l

va r ie ties

I T 07 G A A 7

IT 02 RS A 1

IT 01 A Z В155

IT 08 C P В132

IT 05 C P C48

IT 03 C P В 144

UN 03 Cappelli

3167

3122

2965

2930

2920

2829

2688

2656

2613

2435

2343



The statistically significant difference in the average grain yield (Table III) between the mutant lines CP В132 and CP C48 and their mother variety Cappelli, amounting in the following year (Table IV) to around 300 kg/ha, is a clear example of the potential of induced mutations, particularly if we remember the large adaptability of the variety Cappelli which is grown not only in Italy but also in other Mediterranean countries.

6. DIRECT USE OF MUTATIONS: VARIETIES ALREADY RELEASED

All these data clearly demonstrate that through mutations it is possible to obtain in durum wheat, lines of agronomic value giving a consistent improvement in the performance of this crop. From this extensive evaluation of mutant lines, sufficient evidence was gathered to warrant the registration and release to farmers of four mutant lines as new varieties.

Two lines from Cappelli (CP B132 and CP C48) were released in 1968 with the names "Castelporziano" and "Castelfusano", respectively.In 1969 the best mutant line derived from the variety Grifoni (GR A145) was released with the name "Casteldelmonte", while in 1970 registration has been requested for a line (GA. B125) isolated from the variety Garigliano, which will be released under the name "Castelnuovo".

Table V summarizes the main characteristics of these new varieties in comparison with their respective mother varieties. The data represent averages from experiments carried out in several years and locations.

The varieties Castelfusano and Castelporziano attain their best performances in those environmental conditions in which both soil fertility and water resources are not limiting factors, particularly during kernel ripening. These features make these varieties suitable for regions near the limits of the existing area of cultivation of durum wheat. Considering the increasing demand and economic importance of this crop, such a potential extension of areas of cultivation is a clear advantage.

The varieties Casteldelmonte and Castelnuovo are characterized by pronounced adaptability and yielding ability, reduced height (especially evident in the cv. Casteldelmonte which is about 80 cm high), satisfactory lodging resistance, and heading time earlier than the above mentioned mutant varieties from Cappelli. Earliness, together with a broad adaptability (particularly of cv. Castelnuovo, as shown in the F AO/IAEA, trials in the Mediterranean and Near East regions), suit these two varieties for potential extension in the areas of traditional cultivation of dtirum wheat.

7. USE OF MUTATIONS IN HYBRIDIZATION PROGRAMS

A. study of the use of mutations in hybridization programs was started for durum wheat in 1963. A series of crosses were made according to a diallel scheme between the varieties Cappelli, Lakota and four radiation-induced mutants for short culm from Cappelli (Table VI). The aims were 1) to study the genetic basis of the four mutations obtained from the same mother variety and affecting the same character, whether they were allelic or not, the mutual relationships of dominance and recessivity, the number of loci involved, and the extent of

192 SCARASCIA-M UGNOZZA et al.

feО

MUTAGENESIS APPLIED TO WHEAT 1 9 3

epistatic effects (Bagnara, 1967); 2) to transfer into Cappelli and Lakota genotypes some factors inducing a reduction of culm length; and 3) to obtain simultaneously in the same genotype, factors having a culm shortening effect.

F2, F3 and F4 generations were grown as spaced plant progenies. Particular emphasis was given to selection for short culm, early heading time and high seed setting. Bulking of homogeneous plants within a number of more promising F4 plant progenies gave rise to 257 lines, grown in a comparative trial in 1968-69. Results concerning three of the .most representative cross combinations will be briefly described.They are samples of as many types of combination as possible, i. e. mutant line X Lakota; mutant line X mother variety Cappelli; and mutant line X mutant line.

From the cross CP B132 X Lakota, 96 lines were tested. As far as productivity is concerned,95 out of 96 tested lines outyielded both parents; referred to both the best parent and the standard variety Cappelli, the highest and the average recorded yields were as follows:

% of best parent % of CappelliHighest yield 6201 kg/ha 134. 8 156. 8Average yield 5546 kg/ha 120. 5 140. 3

For height and lodging, it was observed that length of culm was widely variable; sometimes it was intermediate, but more often it was comparable to the CP B132 type. S.everal cases were recorded,however, in which the lines showed transgressive segregation, being shorter than CP B132.In general there was no lodging at all, or less than in CP B132, only 4 lines out of 96 (marked with the numbers 101, 102, 146 and 151) being more susceptible to lodging than CP B132. Regarding heading time, no new line headed earlier, or as early as, Lakota. Earliest new lines headed at the same time as Cappelli. In general, however, lateness of CP B132 was reduced. For kernel characters, test weight was generally lower than that of CP В132, which was reached only by lines 109, 160 and 319. Yellow berry percentages were usually high, with only a few lines as little affected as Cappelli.

From the backcross CP B132 X Cappelli, 18 new lines were tested. Regarding productivity, the highest and average yields, referred to the best parent and to the standard variety Cappelli, were as follows:

% of best parent % of CappelliHighest yield 5342 kg/ha 116. 1 135. 1Average yield 4806 kg/ha 104.5 121.5

For height and lodging, it was observed that all the lines from this backcross to mother variety recover almost completely the short culm of CP B132, with only two exceptions (lines 373 and 506) which are as tall as Cappelli. Lodging resistance was generally as high, or higher than CP B132. Heading times of new lines were intermediate between parental values; all new lines, however, were earlier than CP B132, in spite of their short straw. For kernel characters, parental test weights were generally recovered, with some positive transgression (lines 378 and 512, both coming from the same F2 ancestor plant). Values of yellow


berry percentage were variable, but some lines were less affected than Cappelli.

Twenty lines obtained from the cross between two mutant lines,CP B132 and CP C48, were tested. With regard to productivity, the highest and average yields, again referred to the productivity of the best parent and of the standard variety Cappelli, were as follows:

For height and lodging, it was observed that the lines fell into three different classes of culm length: culm length intermediate between parental values; culm as tall as Cappelli (lines 335 and 336); and transgress ive segregants, shorter than either parent, carrying in homozygous conditions both mutations for short straw. In lines belonging to the latter group, lodging is totally absent. All other lines generally lodge as much as parental lines, with some positive and negative transgression. Heading times were intermediate with reference to parental values. For kernel characters, test weight was in most cases much higher than in parental lines and in Cappelli. All the lines showed a lower percentage of yellow berry compared to CP Б132, and were very often better than Cappelli.

A. comprehensive evaluation of the above results for the three cross combinations, together with those concerning all other combinations, leads to the conclusion that the indirect use of mutations is a real source to be exploited for the improvement of durum wheat. This is confirmed by the promising lines which have already been isolated, for example, the lines in which it has been possible to combine the short culm and lodging resistance of CP B132 with the heading time, low yellow berry percentage and good seed weight of Cappelli, or those which, characterized by the presence of two homozygous mutations (from CP B132 and CP C48), are particularly productive and completely resistant to lodging and with a very low yellow berry percentage.

At present, several crossing programs are being carried out in which mutations from several varieties are employed. Many lines selected in the segregating generations are being tested in numerous locations, mainly in central and southern Italy, in order to assess their yielding ability, optimal agronomic environment and stability of performance.

8. PERSPECTIVES

On the basis of the experience gathered up to now and summarized in this paper, we think that it is reasonable to state that mutagenesis is another valuable tool for genetic improvement of Triticum durum.It is clearly possible to get a widening of the variability of agro-economic characteristics (culmlength, kernel quality and disease resistance), to acquire factors with a genetic behaviour different from the usual one, to induce genes, not yet or only exceptionally found in the germplasm collection, which control useful features for building up new types of durum wheat plants.

Highest yield 5638 kg/ha Average yield 5235 kg/ha

% of best parent 122. 5 113. 8

% of Cappelli 142. 6 132. 4

MUTAGENESIS APPLIED TO WHEAT

Another important point is that such changes can be induced in outstanding varieties. This means that in some cases these mutant lines can directly give origin to new varieties, which combine the good performance of the mother variety with the improvement induced by mutation in single agronomic traits.

Furthermore, the efficacy of using mutations in cross-breeding programs should be emphasized. Here again, the induction of mutations in high yielding varieties is a great advantage, if the associated mutations for other characters are not deleterious, compared with the use of related wild species, primitive material, or old cultivars.

TABLE VII. PRODUCTIVITY OF THE BEST F5 CROSSES AMONG cv. "CA.PPELLI" AND MUTANTS FROM "CAPPELLI"

Cross com b ination

H ighest F5 p roductiv ity

Abso lu te va lu e

(kg/ha)°jo o f best parent % o f cv . ’’C a p p e ll i"

C a p p e lli x C P В132 5342 116. 1 135. 1

C a p p e lli X CP C48 4873 106. 2 121.3

C a p p e lli x C P В144 4795 116.4 121. 3

CP В132 x C P C48 5638 122. 5 142. 6

C P В132 X C P B144 5654 122. 9 143, 0

CP В 132 x CP CB2 5327 115.8 134.7

C P С 48 X C P B244 4905 108.7 124, 1

C P С 48 x C P CB2 5092 112. 8 128. 8

C P В144 X C P CB2 4592 111. 5 116. 1

The results obtained by backcrossing to mother varieties, or crossing mutants with mutants or with other varieties, would therefore seem to merit close attention and great expectations for the future.

Our experience in direct and indirect use of mutations in Triticum durum supports the view (Scarascia-Mugnozza, 1966) that mutants can be carriers not only of the mutation for which they have been selected, but also of other mutations, phenotypically expressed with lower intensity, induced elsewhere in the genotype. If not detrimental, they contribute in differentiating both mutant lines from each other and each mutant line from the mother variety. When mutants are used in hybridization programs, such differences are likely to lead in segregating generations to new recombinant genotypes, whose phenotype, including yield (Table VII), can be appreciably different from the parental ones.

In this way, mutation breeding, as one of the breeding methods at our disposal, can help to provide the large selection of Triticum durum varieties required by the farmer.

196 SCARASCIA-M UGNOZZA et al.

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lines o f T r iticu m durum, X I I Conv. Soc. Ita l. Genet. A g r . , G enet, agr. 2 1 :3 1 3 .

B AG N ARA, D . , ROSSI, L, f PORRECA, G . , 1970, Im p íe g o di m utanti rad io indotti nel m ig lio ra m en to

g e n e t ic o d e l frum ento duro: m etod i e risu ltati, X IV Conv. Soc. Ita l. G enet. A g r . , G enet, agr. ( in press).

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BOGYO , T . P . , S C A R A S C IA -M U G N O Z Z A , G . T . , SIGURBJÔRNSSON, B ., B AG N ARA, D . , 1969, "A dap ta tion

studies w ith rad ia tion -indu ced durum wheat mutants” , Induced Mutations in Plants (P roc. Sym p. Pu llm an, 1969),

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Oxford : 375.

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and s te r ility m utations induced in durum w heat by radiations and ch em ica ls . M utation Res. 9: 589.

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ch lo roph y ll m utations in durum wheat induced by radiations and ch em ica ls , Radiat. Bot. 2: 217.

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DISCUSSION

A. GUSTAFSSON: Did I understand you correctly when you stated that you had dominant mutations for short straw and that these were highly fertile and productive?

G.T. SCARASCIA-MUGNOZZA: Yes, the mutant CP B132, which has given origin to a variety released in 1968 named "Castelporziano" which significantly out-yields the mother variety "Cappelli", carries a mutation for culm shortening which has a dominant and monofactorial behaviour.

R. TRUJILLO FIGUEROA: Which yield components were increased in your high yield mutants? Do you use a selection method to obtain high yield production mutants?

G. T. SCARASCIA-MUGNOZZA: In the first part of our mutation breeding work, the results of which I have shown today, the principal task was to select for culm shortening and lodging resistance. Among the several mutants so characterized, we found lines with good potential value. According to a large set of field trials, some of these mutants appearedto out-yield the mother variety, and this higher yielding ability was increased when nitrogen fertilizers were applied. Under these circumstances, we were interested in ascertaining the changes in yield components of the outstanding mutants. The conclusion is that the increase in yield is due to some positive (but rather small) changes in the number of seeds per spikelet and in kernel weight, but essentially it should be attributed to an increased average fertility of the spikes per plant (main and secondary spikes), whereas in the mother variety ("Cappelli") the fertility of the secondary spikes is clearly lower than in the main spike. We may presume that in the semi-dwarf mutant lines the competition among main and secondary spikes during the plant development is reduced.

H. GAUL: Your mutants certainly have a pleiotropic effect (in the broad sense of this term). I wonder if in your cross-breeding program you found variation of the character complex and if so, could you comment on this.

G.T. SCARASCIA-MUGNOZZA: I cannot deny, of course, the presence of pleiotropic effects of some induced mutations especially in reference to the work performed by Prof. Gottschalk. As a matter of fact, in the short straw mutants I was referring to, no pleiotropic effect has been found in the sense that no reduction or very small changes have been observed, for example, in spike length, kernel size, root system development. I am inclined to think that it is more frequent than we have so far supposed that a mutant, together with the main mutation for which it was selected might carry a constellation of minor mutations which could also affect characters of agronomical interest. The important point for mutation breeding purposes (either for direct or indirect use of mutations) is to select for individuals which carry the main mutation but which are free of detrimental effects of the other induced mutations which affect, less


intensely, other agronomic traits. If not detrimental, they contribute in differentiating the mutant lines among themselves. When mutants are used in cross-combination programs, such differences can give origin in segregating generations to new recombinations with phenotypes evidently different from the parental types.

H. HÀNSEL: First I wish to thank you for sending us your short straw mutants of Cappelli and for the permission to use them in cross breeding. Then I wish to ask you whether it is correct that when crossing one or the other of your mutants with the mother variety, you have obtained segregants which surpassed both parents in yield, and if this is correct, whether you think that the reason for this could be multiple mutations carried by the mutant, or whether you would prefer a different explanation.

G. T. SCARASCIA -MUGNOZZA: Several F5 lines, coming from crosses between mutants or backcrosses to the mother variety, out-yielded both parents. These results, as well as other positive transgressive, recombinants (e. g. in kernel characters), support the view that the four mutant lines used in this hybridization program are by no means carriers only of mutations for short straw, but also of other mutations, phenotypically expressed with lower intensity and induced elsewhere in the genotype.

A. A.SHRI: What type of segregation for yield did you obtain in the crosses of "Parent" X "Mutant" which gave high yielding derivatives? Was it discontinuous or continuous, indicative of polygenic segregation?

G. T. SCARASCIA-MUGNOZZA: The results I have described are referred to a group of Fg lines selected among the more promising F4 progenies. Dr. Bagnara, who is largely involved in the programs related to the indirect use of mutations, is now analysing all the genetical information obtained from this series of crosses made according to a diallel scheme.

B. SIGURBJÔRNSSON: You frequently cited the incidence of "yellow berry" as an indication of pasta or durum quality. Is this character, in your opinion, a good indicator of quality and are there other indicators which could be used to measure durum quality?

G. T. SCARASCIA -MUGNOZZA: I am completely aware that the percentage of "yellow berries" is a rough indication of the quality or better of the technological properties of a durum wheat. However, yellow berry is a very easy kernel character to measure and there is a relationship between this characteristic and the technological properties.According to my knowledge, the field of research on qualities and technological properties of a good durum wheat, which has to furnish a good semolina for pasta production, is practically uncovered.

Probably, themosturgent problems to be solved are 1) a complete knowledge of the parameters more strictly correlated to the technological properties of the semolina and more easy to .determine, and 2) the development of micromethods for screening even single plants in early generations of selection.

COMBINATION OF MUTATED GENES AS AN ADDITIONAL TOOL IN PLAN T BREEDING *

W. GOTTSCHALK Institute of Genetics,University of Bonn,Bonn, Federal Republic of Germany

Abstract-Resumen

C O M B IN A T IO N OF M U T A T E D GENES AS A N A D D IT IO N A L TO O L IN P L A N T BREEDING.

Som e X -ra y induced useful mutants o f Pisum w ere crossed w ith one another in order to un ite their useful

characters and to study the in teractions o f the m utated genes in question. Plants hom ozygous for two or m ore

m utated genes w ere se lec ted and d eve lop ed in to pure lines. T h e breed ing va lu e , p re ferab ly the seed production

o f these strains, was studied in the F4 -genera tion .

T h e com b in a tion o f the characters "h igh number o f ovu les per o va ry " (m utan t 68C ) and "s tem b ifu rca tion "

(m utan t 1201A) is possib le w ithou t any n ega tive in teractions be tw een the genes in vo lved . Other com binations,

how ever, result in a m arked decrease o f seed production , dem onstrating that there is a certa in disharmony

betw een the genes in question. Crosses betw een the h ig h -y ie ld in g fasc ia ted m utant 489C w ith other useful

mutants o f our c o lle c t io n resulted in a h igh number o f d ifferen t recom binations due to the c o m p lica ted gen o typ ic

constitu tion o f mutant 489C. Som e o f these recom binants represent a ve ry prom ising m a te r ia l fo r pea breed ing.

Furthermore, a c le a r heterosis e f fe c t was observed in a ll Fx -hybrids when the fasc ia ted m utant was used as one

o f the parents.

C O M B IN A C IO N DE GENES M U TA D O S CO M O H ERRAM IENTA A D IC IO N A L EN F IT O T E C N IA .

Se han cruzado entre sí determ inados mutantes favorab les de Pisum, inducidos con rayos X con e l fin

de com binar sus caracteres ventajosos y estudiar las in teracciones de los genes'm utados en cuestión. Se han

se lecc ion ado y desarrollado en líneas puras plantas h om oz igó ticas respecto de dos o más genes mutados. El

va lo r f ito té c n ic o , sobre todo la producción de sem illas de estas cepas, se ha estudiado en la generac ión F4 .

Resulta pos ib le com binar los caracteres « e l e v a d o número de óvu los por o v a r i o » (m utante 68C) y

«b i fu r c a c ió n d e l t a l l o » (m utan te 1201A), sin que se produzca ninguna in te racc ión n egativa entre los genes

de que se trata. Sin em bargo, otras com b inac iones dan lugar a un m arcado descenso de la producción de

sem illas, lo que demuestra que ex iste una c ie rta desarm onía entre los genes en cuestión. M ed ian te cruces

entre e l m utante fasc icu lado de a lto ren d im ien to 489C con otros mutantes favorab les de la c o le c c ió n del

autor, se ha obten ido un gran número de d iferen tes recom b inaciones, deb ido a la c o m p licad a constitución

gen o típ ica d e l m utante 489C . Algunas de estas recom b inac iones o frecen muy alentadoras perspectivas para

e l m e jo ram ien to d e l guisante. Adem ás, se ha observado un c la ro e fe c to de heterosis en todos los híbridos

F j , cuando e l m utante fa sc icu lado se u t il iz a com o uno de los progen itores.

1. INTRODUCTION

Comprehensive collections of mutants of different crops are available in many institutions and intensive efforts are being made in order to advance our knowledge on the composition of the respective genomes. A small group of these genotypes is already in use for breeding purposes, and about 70 commercial varieties developed by means of mutated genes [1, 2] have been released during the past few years. In some cases, the mutants in question gave rise directly to the varieties without using additional breeding methods,

* Th is work is supported by the M in istry o f Education and Sciences o f the Federa l R epublic o f

G erm any and by EU RATO M . T h e paper is ded ica ted to Professor Dr. W a lter Schum acher on his 70th

birthday.

199

200 GOTTSCHALK

while in other cases, the mutated genes were incorporated into the genomes of varieties or strains already existing. One of the next steps in the field of mutation breeding will be the combination of different mutant genes in order to unite the respective useful characters. Experience in this field is still very limited. It is generally known, however, that most mutant genes show a negative selection value. It appears that such a gene represents a foreign element within the genome causing a disturbance in its genic harmony. Therefore, it should be expected that the accumulation of several mutant genes in the same genome causes even more negative effects resulting in a general decrease of the physiological efficiency and fertility.

Many mutated genes are used as markers for gene localization. For this purpose, strains homozygous for five or even more mutated genes have been developed in order to cross them with newly arising mutants.These hybridizations are often not possible or can be carried out only with great difficulty because of the extreme reduction of the fertility of the marker stocks, and it is often necessary to use plants heterozygous for the mutant genes in question.

Fortunately, most of the mutants utilized for breeding purposes belong to the group of micromutations which often influence the selection value only insignificantly or not at all. Negative effects of the combined action of mutant genes in plant breeding are known from Triticum monococcum [3,4], Lupinus lut eus [5] and Oryza sativa [6], positive results were obtained in Antirrhinum [ 9 ], Hordeum [ 8, 9, 10, 11 ] and Oryza [ 6 ]. In general, however, more information concerning this problem is urgently needed.

In the present paper, the combined action of several useful mutant genes of the Pisum genome, in the homozygous as well as the heterozygous condition, is discussed.

2. THE MUTANTS

More than 800 X-ray and neutron induced mutants of Pisum sativum are available in our collection, but only a quarter of them is fertile. A very small group of genotypes shows genetically conditioned alterations in comparison with the initial line which could be of interest for pea breeding. We tried to increase the efficiency of some of these mutants by crossing them with one another in order to combine the respective mutated genes. The genotypes used for these hybridizations are as follows:

Mutant 46

This is a recessive early flowering and ripening genotype. Earliness is not due to a more rapid development in the early stages of ontogenesis in this case, but it is a matter of the position of the inflorescences on the stem. Plants of the initial line form their lowest inflorescences at the axil of the 11th - 13th foliage leaf while in mutant 46 they are formed at the 4th - 6th leaf. Therefore, it enters the flowering period 10-14 days earlier.

M UTATED GENES IN PLANT BREEDING 201

FIG . 1. Upper part o f the shoot o f the pea m utant 1201A showing stem b ifu rca tion . T h e leaves w ere rem oved

in order to show the ac tion o f the m utated gene.

(F IG . 1. Parte superior de la m ata d e l m utante d e l guisante 1201A, que muestra la b ifu rcac ión d e l ta llo .

Se han qu itado las hojas a f in de poner de m an ifies to la acc ión d e l gen m utado. )

Mutant 68C

This mutant represents a typical micromutation in the sense of Gaul [12]. The action of the recessive gene consists of a 30% increase in the number of ovules per carpel (Fig. 4). It is not possible to discern this gene action without having analysed the ovule number.

Mutant 1201A

The recessive gene causes a stem bifurcation in the late stages of ontogenetic development. Two corresponding stems develop in the upper part of the shoot resulting in an increase in the number of pods per plant (Fig. 1). The gene shows a reduction of its penetrance which influences the yielding capacity of the mutant negatively [13, 14, 15]; nevertheless, the seed production was somewhat better compared with the productivity of the initial line considering a many-year average.

202 GOTTSCHALK

FIG . 2 . Upper part o f the fa sc ia ted pea m utant 489C showing the considerab le increase in number o f pods

and their a ccu m u la tio n in the a p ica l reg ion o f the plant.

(F IG .2 . Parte superior d e l m utante fa sc icu lado d e l guisante 489C, que muestra e l considerab le aum ento

d e l número de vainas y su acu m u lac ión en la reg ión ap ica l de la p la n ta .)

Mutant 176A

Many plant organs such as leaflets, stipules, flowers, pods, and seeds are reduced in size by the action of this mutated gene. Seed production is somewhat superior to that of the control material. The mutant could be of some interest for the canning industry because of its small kernels. Kernel weight is only about 60% of the seed weight of the initial line. The behaviour of this gene is very interesting as far as some problems of basic genetic research are concerned. It behaves recessively when crossed with the initial line, but acts as a dominant gene when crossed with other specific mutated genes of our collection.

Mutant 489C

This genotype belongs to the group of fasciated pea mutants, one of which was already used by Mendel 120 years ago designated as Pisum umbellatum. Stem fasciation results in a considerable increase in thenumber of flowers and pods which are accumulated in the apical region of the plants (Fig. 2). It is the best mutant available in our collection as far as the seed production is concerned. The mean values for the character "number of seeds per plant" ranged between 132 and 199% of the corresponding control values during the past few years. Moreover, it is one of the most interesting genotypes we have because of its very complicated genetic situation.


F IG .3 . Y ie ld in g cap a c ity o f mutants 46, 68C, 176A, 489C and 1201A o f Pisum sativum in the past

years. Each po in t represents the m ean va lu e for the character "num ber o f seeds per p lan t" as a p ercen tage

o f the corresponding va lues o f the in it ia l lin e . T h e m eans o f the sam e m utant ob ta ined in d iffe ren t years

are connected by v e r t ic a l lines.

(F IG .3 . C apacidad de ren d im ien to de los mutantes 46, 68C, 176A, 489C y 1201A de Pisum sativum

en los ú ltim os anos. Cada punto in d ica e l va lo r m ed io correspondiente a l ca rácter «n ú m e ro de sem illas

por p la n ta » , en form a de po rcen ta je de los va lo res correspondientes de la lín ea in ic ia l. Los va lo res m edios

de l m ism o m utante obten idos en años d iferen tes se han unido por m ed io de lineas ve r t ica le s . )

The yielding properties of the mutants mentioned above are illustrated graphically in Fig. 3. Each point represents the mean value for the character "number of seeds per plant" related to the corresponding value of the initial line grown at the same location. The figure gives a good impression of the capacity of the mutants in question during the past 11 years. In general, it is obvious that the early ripening mutant 46 does not reach the level of the control material. Mutant 68C is more or less equivalent to the initial line; 1201A and 176A are somewhat better, while the fasciated mutant 489C exceeds the control values considerably.

3. COMBINED ACTION OF MUTATED GENES IN THE HOMOZYGOUS CONDITION

There are two possibilities for the utilization of combined action of mutated genes in plant breeding. Pure lines can be developed which contain the respective genes in the homozygous condition. This is primarily valid for self-fertilizing species. In cross-fertilizing species the heterozygous

204 GOTTSCHALK

F IG . 4. C om parison o f the number o f o vu les per o va ry ( l e f t ) and seeds per pod (r ig h t ) in mutants 46, 68C,

176A and 1201A, and in the double recess ive recom binants 68C/46, 68C/176, and 68C/1201A (F4 -

or F5 -genera tion , re sp ec tive ly , 1970).

(F IG .4. C om parac ión d e l número de óvu los por ova r io (parte izqu ie rd a ) y de sem illa s por va in a (parte derecha)

en los mutantes 46, 68C, 176A y 1201A, así com o en los recom binan tes recesivos dobles 68C/46, 68C/176

y 68C/1201A (g en era c ión F4 o F5> respec tivam en te ; 1970 ).)


condition of mutated genes could be of interest because of the utilization of the heterosis effect. Pisum sativum is not only a reliable self-pollinator, it is even a cleistogamous species. Nevertheless, a very clear heterosis was observed in our experiments after having crossed specific mutants of our collection with one another. Therefore, it will be useful to discuss the problem of the combined action of mutant genes separately with regard to their homozygous and heterozygous condition.

3.1. Combination of two mutant genes

As already mentioned, gene 68C causes an increase in the number of ovules per carpel. In 1970, a mean of 9. 64 ± 0. 35 ovules was obtained whereas the corresponding value of the initial line was only 7. 63 ± 0. 30.This gene action should lead to a general increase in seed production, which does not take place in mutant 68C, however, because a reduction of the number of pods per plant regularly occurs. The common occurrence of a useful and a negative feature caused by the same mutant pleiotropic gene can often be observed in mutation breeding. We incorporated such a gene into the genomes of mutants 46, 176A and 1201A in order to study the interaction of the respective genes. Plants homozygous for genes 68C/46, 68C/176A and 68C/1201A were selected in Рд-generation and propagated.The yielding capacity of these double recessive homozygous strains was evaluated in the F4 -generation. The results are given diagrammatically in Fig. 4.

Let us first consider the character "number of ovules per carpel".There are clear differences in this character between the initial line, mutant 46 and mutant 68C (Fig. 4, above left). Curves for plants homozygous for 68C as well as for 46 coincide nearly completely with that for 68C as far as the distribution of the ovule number is concerned. This is also valid for the recombinants 68C/176A and 68C/1201A as can be seen clearly from Fig. 4. This suggests that the action of gene 68C becomes effective without any restrictions under the double recessive conditions studied. It prevails completely over the corresponding action of genes 46, 176A and 1201A. But the situation changes when the character "number of seeds per pod" is considered (right-hand part of Fig. 4). Seed setting of mutant 68C is not as good as expected from the high number of ovules.The mean value was 4. 4 in 1970, only slightly different to the mean of the initial line (3.9). Of all ovules present in the ovaries of the initial line,51. 5% developed into seeds, whereas the proportion in mutant 68C was45. 7% only. Similar results were obtained in earlier generations. The mutant is obviously unable to utilize its favourable potential in an optimal way.

It is very interesting to compare this behaviour of mutant 68C with the double recessive strain 68C/46. There are no differences between these two genotypes with regard to the ovule number, but there are marked differences in the number of seeds per pod. The double recessive plants are not able to produce as many seeds as parent 68C in spite of the same number of ovules per ovary. The same situation was found in double recessive plants having the constitution 68C/176A (Fig. 4, middle). It seems that there are certain interactions between the mutated genes which negatively influence the yielding capacity of the double recessives. These interrelationships cannot be observed in double recessives of the constitution

2 0 6 GOTTSCHALK

TABLE I. THE MEANS FOR THE CHARACTERS "NUMBER OF OVULES PER OVARY" AND "NUMBER OF SEEDS PER POD" IN Pisum MUTANTS46, 68C, 176A, 1201 A, AND SOME RECOMBINATIONS IN COMPARISON TO THE VALUES OF THE INITIAL LINE

G enotype

Num ber o f ovu les

per ovary

M ean Percen tage o f

in it ia l lin e

Num ber o f seeds

per pod

M ean Percen tage o f

in it ia l lin e

Percen tage o f ovules

d eve lop ed in to seeds

In it ia l lin e 7 .63 100. 0 3 .93 100. 0 51.5

Mutants

46 7 .21 94. 5 2. 56 65 .1 35 .5

176A 7 .5 4 98.8 3 .65 92.9 4 8 .4

1201A 7.79 102.1 3, 80 96 .7 4 8 .8

68C 9 .64 126.3 4 .41 112 .2 4 5 .7

Recom binants

68С/ 46 9 .42 123. 5 3 .15 8 0 .2 33 .4

68С/ 176A 9 .4 4 123.7 3. 18 8 0 .9 33 .7

68C/1201A 9 .81 128.6 4. 19 106 .6 42.7

FIG . 5. F o lia g e le a f o f a p ea p lan t hom ozygous for the m utated genes Ü, a f and coch showing not an

a d d it iv e but a cu m u la tive action . R ight: le a f o f a norm al pea.

(F IG . 5. F o lla je de una planta de guisante h o m o z ig ó t ic a respecto de los genes mutados ti, a f y coch .

que muestra una a cc ión no ad itiva sino acu m u la tiva . A la derecha, ho ja de una p lanta norm al de gu isan te .)


68C/1201A. The curves for this material and for parent 68C coincide nearly completely (Fig. 4, below). The situation is elucidated in Table I which gives the mean values for the different genotypes studied.

Our results show that the union of genes 68C +46 as well as of 68C + 176A does not result in the combination of the useful characters of the respective mutants. Genes 46 and 176A display their complete action when united with gene 68C while the latter gene is not able to display its full action in this combination. The union of the characters caused by genes 68C and 1201A, however, is possible without any restrictions.This behaviour can be interpreted in the sense that there is a certain disharmony between genes 68C and 46 as well as between 68C and 176A, whereas full harmony exists between genes 68C and 1201A.

3.2. Combination of more than two mutant genes

The simplest and most common possibility of the co-operation of mutant genes is their additive action, resulting in the combination of the characters controlled by them. In exceptional cases, a cumulative action occurs, resulting in completely unexpected effects which cannot be predicted. An impressive example of such behaviour is the combined action, of the genes tl (acacia mutant: leaflets instead of tendrils), ai (afila mutant: branched tendrils instead of leaflets), and coch (cochleata mutant: tendrils or whole leaves instead of stipules) of the Pisum genome. Plants homozygous for these three genes develop highly divergent foliage leaves.The upper part of their rachis is sub-divided into five corresponding organs each of them repeatedly sub-divided. A tiny leaflet is formed at the end' of each lateral branch. The same structure is also formed in the stipules.In this way, very strange leaves arise containing more than 1000 leaflets but no tendrils at all (Fig. 5). Even a taxonomist would not be able to discern tltl afaf cochcoch individuals as representing a pea plant. More examples concerning the additive and cumulative action of mutant genes on the structure of plant organs can be found elsewhere [ 16, 17].

This problem can likewise be of interest with regard to the utilization of mutated genes in plant breeding. The combination of the useful characters of the fasciated pea mutant 489C with some other favourable genotypes of our collection may be taken as an example for demonstrating the difficulties as well as the prospects of this method. As mentioned above, mutant 489C shows an excellent seed production (Fig. 1) due to a large increase of the number of pods per plant as a consequence of stem fasciation. Such a high improvement of the grain yield (30-100%!) could never be reached by conventional breeding methods. In spite of this efficiency, the mutant cannot be directly used for pea breeding because the stems are too high and flowering and harvesting time is about ten days too late. Therefore, it was crossed with the early ripening mutant 46 in order to combine stem fasciation with earliness. Furthermore, hybridizations between 489C X 176A (small grains, narrow leaves) as well as 489C X 1201A (stem bifurcation) were carried out. All these mutants behave as recessive ones; consequently, dihybrid segregations should be expected in the F2- generations and there should be no difficulties in selecting plants homozygous for the respective two mutated genes and to develop them to strains.

Unexpectedly, a very complicated segregation was obtained which cannot be interpreted in the sense of a digenic mode of inheritance. In

2 0 8 GOTTSCHALK

the voluminous F2-generations of each of these three hybridizations, 20 to30 different phenotypes were selected; many of them could be developed to pure lines in the subsequent generations. This complicated situation was not observed after having crossed other mutants of our collection but it regularly occurred in all cases in which the fasciated mutant 489C was used as one of the parents. Consequently, it is assumed that this behaviour is due to this mutant. Our results can only be understood if we assume that it contains at least five or six mutated genes. An interpretation will be given in the discussion.

Plant breeding means hybridization, recombination and selection, and these methods were also used in our mutated material. The unexpected richness of phenotypes and genotypes in the F2-generations of the respective crossings represents not a disadvantage but an ideal situation for the selection of genotypes superior to the initial line as far as their breeding value is concerned.

An extraordinarily high genetic diversity was found after having crossed the fasciated mutant 489C with the narrow-leaved and small grained mutant 176A. The following characters were found in the F4-generation in pure lines of different genotypic constitutions: normal stem without any fasciation; extreme stem faciation as in mutant 489C, i. e. an accumulation of all flowers and pods in the most apical nodes of the plants; a relaxed degree of fasciation characterized by a distribution of flowers and pods over a longer part of the apical stem region; size of leaves, flowers, pods, and seeds as in the initial line; narrow leaves and pods, small flowers and seeds; flowering and ripening time as in the initial line; normal flowering, late ripening; late flowering, late ripening; strongly shortened internodes; shortened internodes; weakly shortened internodes; internode length as in the initial line; long inter nodes; very long internodes.

All these characters can be combined in specific ways resulting in a high genetical and morphological diversity. Not all recombinations could be selected which are theoretically conceivable; some other ones were only found in segregating families and are not yet available in the form of homozygous strains. Figures 6, 7 and8 illustrate only lines homozygous for specific combinations of the characters just mentioned. The true genotypic diversity of our F4 -material was considerably higher.

A survey on the diversity of the strains selected following hybridization of 489C X 176A is diagrammatically represented in Fig. 6, which shows the mean values for the characters "plant height" and "number of seeds per plant". Both these criteria are related to the corresponding means of the initial line = 100%. The material is subdivided into four groups as follows: group 1, the parental mutants 489C and 176A; group 2, non-fasciated strains; group 3, fasciated strains; and group 4, strains showing a relaxed degree of stem fasciation. Within each group the strains are arranged in an order of increasing internode length and plant height. Additional details concerning the morphology of the plants are given by the capital letters above each pair of columns as explained in the figure caption. These capitals do not represent gene designations, they are used only for the morphological characterization of the respective pure lines.

It is not necessary to explain all recombinations in detail, but I would like to point out a series of recombinants which show a marked reduction in their internode length while their seed production is not reduced as compared with the capacity of the initial line. This is for instance valid


FIG . 6. Survey on the p h ys io lo g ica l e f f ic ie n c y o f 19 d iffe ren t recom binants se lected in F2 fo llo w in g hybrid iza tion

o f mutants 489C (s tem -fa sc ia tion ) x 176A (narrow leaves ) and d eve lop ed to pure lines. T h e means o f the

characters "s tem len g th " ( l e f t co lu m n ) and "num ber o f seeds per p lan t" (r igh t co lum n ) o f these strains eva lu ated

in the F4 -gen era tion are used as c r ite r ia re la ted to the corresponding m eans o f the in it ia l lin e = lOOô. T h e

ca p ita l le tters above the colum ns are as fo llow s : В = norm al broad lea ves ; D = dim in ished s ize o f leaves ,

flow ers, pods, and seeds; I = in te rm ed ia te s ize o f lea ves ; N = norm al f low erin g and ripen ing t im e ;

L = la te ripen ing tim e.

(F IG .6. Exam en de la e f ic ie n c ia f is io ló g ic a de 19 recom binantes d iferen tes se leccionados en la gen erac ión

F2 a ra íz de la h ib ridac ión de mutantes 489C (fa sc icu la c ión d e l ta llo ) x 176A (hojas estrechas) y desarrollados

en líneas puras. Los va lo res m ed ios de los caracteres « lo n g itu d d e l t a l l o » (co lu m n a de la izqu ierda ) y

«n ú m e ro de sem illa s por p la n t a » (co lu m na de la derecha) de estas cepas, eva luados en la g en erac ión F4 ,

se u t iliz a n com o cr ite r io s re lacionados con los va lo res m ed ios correspondientes de la lín ea in ic ia l = ЮО'Уо.

Las mayúsculas que figu ran sobre las colum nas s ign ifican : В = hojas de anchura norm al; D = m enor tam año

de las hojas, flores, va inas y sem illas ; I = hojas de tam año in te rm ed io ; N = ép oca norm al d e flo rescen c ia

y m aduración ; L = época de m aduración ta rd ía .)

for strain С: short internodes (plant height = 75% of the corresponding mean of the initial line only); non-fasciated stem; normal leaves; late ripening. The seed production of this strain reached a mean of 162% obtained by evaluation of 361 plants grown in 8 replications. The high productivity was found in each plot; therefore, the mean is very reliable. Undoubtedly, this recombinant is promising for utilization in pea breeding in spite of its lateness.

A similar situation prevails in strains E, F, K, and S; but the high seed production is not necessarily correlated with late ripening. Strain G for instance shows the following characters: short internodes (plant height = 76%); stem relaxed fasciated; normal leaves; normal flowering and ripening time. Its yield was 133% compared with the initial line, considering 197 plants grown in 4 plots. The markedly reduced plant height of the strains just mentioned means an increased lodging resistance of this material when grown under field conditions.

Besides the short-stemmed genotypes, many different long-stemmed recombinants were selected and tested with regard to their yielding capacity. Most of them showed a seed production essentially superior to

210 GOTTSCHALK

FIG . 7. Survey on the e f f ic ie n c y o f 14 d iffe ren t recom binants se lec ted a fter having crossed mutants 489C

(s tem -fa sc ia tion ) x 1201A (s tem -b ifu rca tion ). T h e deta ils o f this figu re are arranged as in F ig . 6; the values

w ere obtained in 1970 from the F4 -genera tion . Th e cap ita l letters above the colum ns are as fo llow s:

N = norm al flow erin g and ripen ing t im e ; L = la te ripen ing t im e .

(F IG . 7. Exam en de la e f ic ie n c ia de 14 recom binantes d iferen tes se leccionados tras e l cruce de mutantes

489C (fa sc icu la c ión d e l ta llo ) x 1201A (b ifu rcac ión d e l ta llo ). Los e lem en tos de esta figura están dispuestos

com o en la figu ra 6 ; los va lores se obtuvieron en 1970 a partir de la generación F4 . Las mayúsculas que

figuran sobre las colum nas s ign ifican : N = ép oca norm al de flo rescen c ia y m aduración ; L = ép oca de

m aduración ta rd ía .)

that of the initial line, but the plants were too high. They cannot be used for cultivation in the field, but they could represent a useful material as garden peas. The best genotypes of this group are the strains D, Y and W, which have the following characteristics.

D: very long internodes (plant height about 135 cm = 190% of the initial line); stem non-fasciated; broad leaves; late ripening.

Y: very long internodes (plant height = 178%); stem non-fasciated; narrow leaves; late ripening.

W: long internodes (plant height = 163%); stem relaxed fasciated; broad leaves; late ripening.

The means for the character "number of seeds per plant" of these pure lines ranged from 158 - 179% of the corresponding control values. Strain V, comparable to W, but with an earlier ripening time, shows likewise a very good capacity for seed production (128%).

A similar situation was found in the other two crossings. Details concerning the relations between stem length, stem structure, ripening time and seed production of the whole material selected and tested can be seen in Figs 6, 7 and 8. Some strains of interest for pea breeding are the following.

From Б4 -generation 489C (stem fasciation) X 1201A (stem bifurcation) (Fig.7); the short-stemmed strains I, B, and С showing a very good seed production; and strain H (plant height, flowering and ripening time

M UTATED GENES IN PLANT BREEDING 2 11

FIG . 8. Survey on the e f f ic ie n c y o f 13 d iffe ren t recom binants se lec ted a fter hav in g crossed mutants 489C

(s tem -fa sc ia tion ) x 46 (earlin ess ). T h e deta ils o f the figu re are arranged as in F ig . 6; the values w ere

obtained in 1970 from the F4 -genera tion . T h e cap ita l letters above the colum ns are as fo llow s: E = ea rly

flo w e r in g and ripen ing ; N = norm al r ipen ing t im e ; L = la te ripen ing.

(F IG . 8. Exam en de la e f ic ie n c ia de 13 recom binantes d iferentes se leccionados tras e l cruce de. mutantes

489C (fa sc icu la c ión d e l ta llo ) X 46 (p reco c id ad ). Los e lem en tos de la figura están dispuestos com o en la

figura 6; los va lo res se ob tuvieron en 1970 a partir de la generac ión F4 . Las mayúsculas que figuran sobre

las colum nas s ign ifican : E = flo rescen c ia y m aduración tempranas; N = época de m aduración norm al;

L = m aduración ta rd ía .)

comparable with the initial line, stem relaxed fasciated, high seed production). Unfortunately, only a small plot containing 40 plants of this line was available in 19 70. It would be a very promising material if its good yielding capacity could be confirmed in later generations. It nearly reaches the productivity of the high-yielding parental mutant 489C, but it is superior to this genotype as far as plant height and ripening time are concerned.

From F4-generation 489C (stem fasciation) X 46 (earliness) (Fig. 8) : the short-stemmed strains D, M, N, C, and.К reach a seed production comparable with the initial line. The other short-stemmed recombinants selected are not of interest for breeding purposes because of their reduced grain yield. This is also valid with regard to strain H, the plants of which showed the greatest reduction in size obtained in our crossing experiments.The degree of stem fasciation is somewhat relaxed in these plants; furthermore, they contain the gene for earliness and form their lowest pods immediately above the ground. Consequently, many seeds are lost to birds or are damaged due to the high humidity of this region. The mean value for the seed production of this strain given in Fig. 8 is a potential value only. The exact amount of seeds harvested was considerably lower because of the losses just mentioned.

The recombination originally expected from this crossing has not yet been found. We are interested in getting a strain in which the characters stem fasciation, i. e. a high number of pods per plant, low position of flowers

212 GOTTSCHALK

FIG . 9. Com parison o f the v ita l i t y and cap ac ity o f som e Pisum mutants w ith their hybrids. T h e columns

represent the m eans for the characters "num ber o f seeds per p lan t" ( le f t ) and "s tem len g th " (r igh t),

re la ted to the corresponding m eans o f the in it ia l lin e = 100%.

a: m utant 489C ( le f t ) c : m utant 489C ( le f t )

mutant 176A (m id d le ) m utant 189 (m id d le )

hybrid 489C/176A (r igh t) hybrid 489C/189 (righ t)

b: m utant 48ЭС ( le f t ) d: m utant 489C ( le f t )

in it ia l lin e (m id d le ) m utant 26 (m id d le )

hybrid 48 9 C / in itia l lin e (r igh t) hybrid 489C/26 (righ t)

(F IG . 9. C om parac ión de la v ita lid a d y capacidad d e algunos mutantes de Pisum con sus híbridos. Las

colum nas representan los va lo res m ed ios de los caracteres «n ú m e ro d e sem illa s por p la n t a » (parte izqu ierda

de la figu ra ) y « lo n g itu d d e l t a l l o » (parte derecha), en re la c ión con los correspondientes va lo res m ed ios

de la lín ea in ic ia l = 100%. )

a: m utante 489C

m utante 176A

híbrido 489C/176A

(izqu ie rda )

(cen tro )

(derecha )

c : m utante 489C

m utante 189

h íbrido 489C/189

(izqu ie rd a )

(cen tro )

(derecha )

b: m utante 489C

lín ea in ic ia l

h íbrido 489C / lín ea in ic ia l

(izqu ie rd a )

(cen tro )

(derecha )

d: m utante 489C

m utante 26

h íbrido 489C/26

(izqu ie rd a )

(cen tro )

(derecha )

and pods on the stem, reduced plant height, and earliness are combined. Earliness derives in this recombination from mutant 46. It could be shown in biochemical investigations that the seed proteins of this mutant show a striking increase of all essential amino acids [18]. Therefore, the strain just mentioned would be very promising for breeding purposes. A few single plants of this phenotype were selected in segregating Б4 -families, but it is not yet clear whether they are homozygous, and no details can yet be given concerning their yielding capacity.


In general, it can be stated that some advantageous results could be obtained by combining some of our useful mutants. The combination of high number of ovules per ovary with stem bifurcation for instance did not result in any negative effects. On the contrary, it can be expected that the respective double recessive strains will show an increased yielding capacity in comparison with their parental mutants and the initial line.In 19 71, enough material of this genotypic constitution will be available for performing yield analyses. The combination of high number of ovules per ovary with earliness or with reduced seed size, however, was not successful because of some negative interactions between the mutant genes in question. Furthermore, some deleterious properties of the high-yielding mutant 489C, such as lateness and long internodes, could be removed by crossing it with other mutants and by selecting recombinants better suited for utilization in pea breeding. Consequently, our results show that a useful and valuable material can be obtained by combining specific mutant genes.

4. COMBINED ACTION OF MUTATED GENES IN THE HETEROZYGOUSCONDITION

Considering the Fj-plants of a relatively large number of different crossings between different mutants of our collection, no hybrid vigour could be observed in general. But there is an interesting exception: a clear heterosis effect occurs regularly if the fasciated mutant 489C is used as one of the parents. Some examples of our results concerning this phenomenon are given diagrammatically in Fig. 9. The stem length as well as the seed production of the parental mutants and their hybrids were used as a parameter for illustrating this effect. The situation of the crossing 489C (stem fasciation) X 176A (narrow leaves) can be taken as an example. In 1967, the mean value for the stem length of the hybrids 489C/176A was markedly above the corresponding means of the parental genotypes. The effect was even more marked if we consider the seed production of the three genotypes in question. Mutant 489С reached a mean of 154% of the corresponding control value of the initial line; the mean of mutant 176A was 117%. The seed production of the Fj -hybrids, however, was 180%. A corresponding situation was observed in 1970 in Fj-plants of the crossings mutant 489C X initial line, mutant 489C X mutant 189 (divergent shape and colour of the flowers, reduced fertility), mutant 489C X mutant 26 (extremely shortened internodes, very low seed setting) studied by my co-worker Milutinovic. The high capacity of mutant 489C is due to the stem fasciation resulting in a considerable increase in the number of pods per plant. The Fj-hybrids, however, are non-fasciated. Consequently, the astonishing improvement of their physiological efficiency can be regarded as an impressive example of heterosis in a selffertilizing species. Details can be seen in Fig. 9. Other results concerning this problem have recently been published [ 39 ].

5. DISCUSSION

Two problems should be discussed with regard to the results obtained in our crossing experiments. It is possible now to give an interpretation

214 GOTTSCHALK

concerning the complicated genetic situation of the faciated pea mutant 489C. The second problem of interest is the phenomenon of monohybrid heterosis in self-fertilizing species.

5.1. Genetic situation of mutant 489C

It is well known that not only one but several mutational events are often induced by the action of a mutagenic agent in the same initial cell of the growing point of an embryo. The mutated genes commonly present in the respective Mj-plant can be separated from one another in the following generations and pure lines can be developed, each of them containing only one gene out of the whole group. The same principle was used in our experiments. We crossed the genetically simple mutants 176A and 1201A with the very complicated mutant 489C in order to clarify how many genes mutated in the X-irradiated embryo 489C six years ago and which characters they control. The morphological and physiological peculiarities of this mutant are late flowering and ripening time, extreme stem fasciation, and relatively long internodes. In the F3- and F4- generations of the hybridizations mentioned above the following characters divergent from 489C were isolated in the form of homozygous strains: normal flowering and ripening time, relaxed stem fasciation, and at least five different degrees of internode length divergent from that of the initial line resulting in at least five different classes of plant height. There is no doubt that these characters, except the first one, have not been transmitted by the second mutant of the respective hybridizations but by mutant 489C. Furthermore, it cannot be assumed that some of these characters are due to the action of a single pleiotropic gene, because they could be separated from one another. Consequently, we have to assume that at least six different genes have mutated in that embryo, five of them influencing the length of the internodes.

This is a very unusual situation not comparable with other cases. Fasciated pea mutants have been known for a long time, most of them having spontaneously arisen [ 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ]. It is not known whether these mutants are identical with 489C because no hybridizations were made. However, a second fasciated mutant arose in our own mutagenic treatments following neutron irradiation [31]. It corresponded exactly with 489C. Crossings between these two mutants resulted in fasciated Fi-hybrids likewise completely similar to their parents. The mutants in question are obviously identical. It is impossible that the same six or even more specific genes, of a genome are mutating in the same way in different embryos during different mutagenic treatments if gene mutation is regarded to be a random effect. ' In this particular case there must be something like a mutual interdependence between the genes involved, resulting not in the occurrence of independent and random mutational events, but in the mutation of all genes belonging to this group.

5.2. Phenomenon of monohybrid heterosis

The phenomenon of heterosis is commonly connected with a high degree of heterozygosity; therefore, it is preferably studied in cross-fertilizing species such as maize. Only a few cases of monohybrid heterosis have as


yet been observed, for instance, in mutants of Sorghum [32], Antirrhinum [33, 34], and Hordeum [ 35, 36, 37, 38]. The results given in the present paper are obviously also due to heterosis on a mono- or dihybrid basis.The hybrid vigour observed in Pisum sativum is strongly dependent upon a specific genotypic constitution connected with the fasciated mutant 489C.As mentioned above, this mutant differs from the initial line in at least five or six mutant genes. Nevertheless, the heterosis effect is probably not due to a heterozygous condition of all genes of the whole group but to heterozygosity in one single gene of this group. Investigations concerning this question are in progress, and a further discussion of the whole problem will be given when more information is available.

Heterosis in a self-fertilizing species, such as Pisum, cannot be used for breeding purposes, because the proportion of natural cross-fertilization by insects is extremely low, reaching 0. 5 - 1. 5% at the most. Therefore, pea mutants showing a transition from self- to cross-pollination would be of great interest. If such a material were available, the heterosis effect caused by mutant 489C could be utilized commercially. So far, however, no mutants of this type are known in this species, but our results may help to interpret and to understand the heterosis phenomenon.

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va r ia tion in peas, Proc. A m . ph il. Soc. 56 (1917) 487.

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[2 4 ] W INGE, O ., Linkage in Pisum , C .r .L a b . Carlsberg 21 (1936) 271.

[2 5 ] LU T K O V , A . , R ec ip roca l translocations and gene m utations in Pisum sativum induced by X -rad ia tion

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[2 7 ] GELIN, O .E ., X -ra y mutants in peas and vetches, A c ta A g r ie , scand. 4 (1954) 558.

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exp er im en ta l m utagenesis". Induced Mutations and their U tiliz a t io n , Proc. Sym p. Erwin-Baur-

G edâchtnisvorlesungen IV , Gatersleben, 19B6, A k a d em ie -V e r la g , Berlin (1967) 123.

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U n ivers ity o f Bonn (1969).

[3 2 ] KARPER, R. E . , T h e e ffe c ts o f a s in g le gene upon d eve lop m en t in the h e te ro zygo te in Sorghum,

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[3 3 ] STUBBE, H . , P1RSCHLE, K . , Über e in en m onogen bed ingten F a ll von Heterosis von Antirrhinum majus L . ,

Ber. dt. bot. Ges. 58 (1940) 546,

[3 4 ] STUBBE, H . , Über m on o - und d igenbed ingte Heterosis b e i Antirrhinum m ajus L . , Z . VererbLehre 85

(1953) 450. ~

[3 5 ] GUSTAFSSO N, A . , T h e e f fe c t o f h eterozygos ity on v ia b il ity and v igou r, H ered itas 32 (1946) 263.

[3 6 ] GU STAFSSO N , Â . , Th e advantageous e f fe c t o f d eleterious mutations, H ered itas 33 (1947) 573.

[3 7 ] GUSTAFSSO N, A . , M utations, v ia b ility , and popu lation structure, A c ta A g r ie , scand. 4 (1954) 601.

[3 8 ] G USTAFSSON, A . , NYB O M , N ., T h e v ia b il ity reac tion o f some induced and spontaneous mutations

in barley, H ered itas 36 (1950) 113.

[3 9 ] G O T T S C H A L K , W . , T h e p rodu ctiv ity o f some mutants o f the p ea ( Pisum sativum L . ) and their hybrids.

A con tribu tion to the heterosis p rob lem in s e lf- fe r t i l iz in g species, Euphytica 19 (1970) 91.

DISCUSSION

H. GAUL: Your mutant 489C has a yield increase of 30 to 100%.Should not this mutant be put into official yield trials of the Federal Variety Office, so that the farmers could grow it?

W. GOTTSCHALK: A good mutant is not yet a good variety. But we were able to increase the breeding value of the fasciated mutant by removing its tallness and its lateness and some of our recombinants should be tested for release as varieties. We have already established contact with a German breeder, who was to use some of our strains for breeding purposes.

H. GAUL: Combination of mutations is, I think, a very important line of research and little has been done in this field. The reduction of vitality in double mutants, which you usually observed, may be expected. I would


like to suggest that you cross these double mutants with genetically unrelated peas in order to build up new systems and in this way to eliminate the deleterious effects, but to maintain the interesting character.

W. GOTTSCHALK: Thank you for this suggestion; we shall do it.A. ASHRI: In your work you produced mutants with profound contributions.

These mutations must affect the growth hormones of the plants (gibberellins, auxins); do you have data on this point?

W. GOTTSCHALK: One of my co-workers, Dr. Müller, is studying this problem and related ones such as the activity of certain enzymes in different dwarfy mutants of our collection, but we have not yet started this kind of work in the useful genotypes discussed in this paper.

B. SIGURBJÔRNSSON: In your discussion of the combination of two mutant genes you described very different sets of events. I wonder why you used the terms "additive" and "cumulative" because to me they both mean about the same thing. Would not some other term, such as "epistatic" or "complementary", be better than "cumulative" to describe this phenomenon?

W. GOTTSCHALK: The "additive" and "cumulative" action of mutant genes cannot be regarded to be the same phenomenon. If all single morphological effects of two mutant genes appear together in the double recessive plants you can interpret this behaviour as an "additive" gene action. However, if the common action of two or more mutant genes results in a completely new, unexpected character of the plant you cannot understand it as a mere additive effect of the respective genes; we call this behaviour a "cumulative" gene action. I would not use the term "epistatic" for characterizing this behaviour, because epistasis is a specific genetic situation not agreeing with that just mentioned.

A. GROBMAN: I would like to comment further on Dr. Gaul's remarks.Not reflecting on the excellent quality of Dr. Gottschalk's work, I would like to bring to the attention of this group what is normally a very common case in regard to the incorporation of new genes into basic adapted genotypes.In many cases, after incorporation of such genes, breeders tend - out of haste to achieve successful results - to start selection after too few generations of recombination, usually two to four. Among others,W. L. Brown showed that by allowing up to six or seven generations for recombination after incorporating exotic genes into adapted germplasm (maize), much greater variability from which to select and better results were obtained, than if selection proceeded at earlier stages. We have confirmed this point with similar introduction of Argentinian maize germplasm into tropical maize where transgressive segregates appeared only after four generations of backcross and selection had taken place, while they were not apparent initially.

I. RAMIREZ- ARA YA: The high Fi heterotic responses reported by you, were they measured with respect to the mid-parent value or compared to the highest-yielding parent entering the cross?

Were these Fj heterotic responses measured in isolated plants, space- planted seeds, or in plots with a rate of seeding approximately near commercial seeding rates?

W. GOTTSCHALK: The different criteria which we have used for demonstrating the heterosis effect of our material are related in the figures to the corresponding values of the initial line. All empirical

218 GOTTSCHALK

data from both parental mutants are available, however, and we could relate the Fj -values also to the values of the higher-yielding parents.

All Fj -hybrids, parental mutants and the initial line were cultivated in a similar manner on wire fences. Therefore, all data obtained are comparable.

We are now studying a comprehensive material not only morphologically but also using physiological and biochemical methods in order to obtain as much information as possible of this kind of heterosis.

C. PANTON: You mentioned that the extremely low degree of natural crossing in Pisum restricts the use of heterosis in breeding. I am wondering what are the possibilities of making artificial hybrids which could then be easily multiplied, the species being a highly self-fertile one.

W. GOTTSCHALK: We could do this for scientific purposes but not for practical purposes. Crossing by hand would be much too expensive in Europe.

A. MICKE: I would like to comment on your finding of strong heterosis effects in Fi after crossing induced mutants with the original strain and with other mutants of the same origin.

From my mutation breeding program with Melilotus albus at the Institute of Agronomy and Plant Breeding in Gottingen (Federal Republic of Germany)1, aimed at the production of non-bitter mutants, we have selected a series of mutants that are essentially free of the bitter principle (i. e. the glucoside of the ortho-oxy-cinnamic acid). Backcrossing of these mutants with the original bitter strain led in some instances to doubling of the yield, in other combinations to a yield increase of up to 50% above the better parent. Since these mutants originated from the same inbred line, the yield increase has to be ascribed to the induced genetic variability. In contrast to Dr. Gottschalk's opinion regarding his pea mutants, we do not assume that the heterosis is caused by a single mutated factor such as, e. g. the non-glucoside gene. From the crosses with the bitter original strain and the other non-bitter mutants we could not ascertain any correlation between the bitter principle and the degree of heterosis. It therefore seems more likely to us that a number of "background"- mutations are responsible for the yield increase. These background- mutations are usually considered deleterious and some mutation breeders try to avoid them as much as possible by applying lower mutagen doses or try to eliminate them by back-crossing mutants and reselecting for the desired mutated character. Our findings, however, seem to indicate that such "undesired" mutations might be very beneficial in heterozygous condition. On the basis of this, mutation breeding of cross-pollinating crops might be greatly facilitated.

1 M IC KE , A . , "Im p rovem en t o f low y ie ld in g sw eet c lo v e r mutants by heterosis breed ing"

Induced M utations in Plants (P roc . Sym p. Pullm an, 1969), IAEA , V ienna (1969) 541.

MUTATION BREEDING IN VARIOUS CROPS

MUTATION BREEDING FOR YIELD AND KERNEL PERFORMANCE IN SPRING BARLEY

H. HANSEL, W. SIMON, K. EHRENDORFER Institute for Crop Husbandry and Plant Breeding,Hochschule für Bodenkultur, Vienna,Probstdorfer Saatzucht, Probstdorf, N. Ô ., Austria

Abstract-Resumen

M U T A T IO N BREEDING FOR Y IE LD A N D KERNEL PERFORMANCE IN SPRING BARLEY.

A n app lied m icrom u ta tion experim en t in b a r ley is presented. It was planned as a part o f a p ra c tica l

b reed in g program at a c o m m erc ia l b reed in g station, w ith the a im to produce a new va r ie ty o f brew ing

barley . One lo t o f seeds o f a cross-bred strain o f spring barley was trea ted w ith th erm al neutrons and in the

fo llo w in g yea r another lo t o f the sam e l in e w ith EMS. Positive s e le c tion for y ie ld and kernel w idth

( 'g ra d in g ' > 2. 5 m m ) was carried out from M 2 to M 8 w ith vary ing s e le c tion pressure. T h e s e le c tion response

was measured by com paring the M -lin e s (a ) w ith the untreated strain (con tro l I ) and (b ) w ith the four best

lin es s e le c ted out o f con tro l I (c o n tro l I I ) . In ea r ly generations s e le c tio n fo i y ie ld was m o re e f fe c t iv e a fter

neutron irrad ia tion than a fte r EM S-treatm ent. In la te r generations the best lines o f both treatm ents g a ve

s im ila r results surpassing the best l in e o f con tro l I I in y ie ld or in ’ grad in g ’ by 3-5% . S evera l o f the m utant

lin es are now be in g tested in State tria ls.

M EJORAM IENTO F IT O T E C N IC O POR M U T A C IO N DE L A CEBADA DE PRIM AVERA C O N M IRAS AL

REND IM IENTO Y C A R A C TE R IST IC A S DEL GRANO .

Los autores describen un experim en to de m icrom u tac ión ap licada en la cebada. Se p royec tó com o

parte de un p rogram a p rá c tico de m e jo ram ien to en una estación fito té cn ic a c o m e rc ia l, con m iras a

ob tener una nueva variedad d e cebada ce rvece ra . Se trató con neutrones térm icos un lo te de sem illas

de una variedad cruzada de cebada de p rim avera y , a l sigu ien te año, otro lo te de la m ism a lín ea con

MSE (m etanosu lfonato de e t i lo ) . La s e le c c ió n pos itiva en cuanto a ren d im ien to y anchura de grano

(« g r a n u lo m e t r fa » >2 ,5 m m ) se e fe c tu ó de la gen era c ión Мг a la M 8, con un grado va r ia b le de rigor

s e le c t iv o . La respuesta a la s e le c c ió n se m id ió com parando las líneas M : a) con la variedad sin tratar

(c on tro l I ) y b ) con las cuatro m ejores líneas se lecc ionadas a partir d e l con tro l I (c on tro l I I ) . En las

prim eras generaciones, la s e le c c ió n 'en lo r e la t iv o a l rend im ien to fue más e fe c t iv a tras la irrad iac ión

neutrón ica que después d e l tra tam ien to con MSE. En las generacions posteriores, las líneas óptim as de

ambos tratam ien tos d ieron resultados sem ejan tes, que superaron a los de la lín ea óptim a d e l con tro l II,

en ren d im ien to o en «g r a n u lo m e t r fa » , en un 3-5% . A ctu a lm en te , se están som etiendo a ensayos o fic ia le s

varias de las supuestas líneas mutantes.

1. PROJECT

Micromutations (according to the definition given by Gaul [1]) have been found in different plant species, when the treated material was investigated thoroughly. This "was the case in Arachis hypogaea (Gregory [2]), in Soja hispida (Rawlings et al. [3]), Oryza sativa (Oka et al. [4]),, Trifolium subterraneum (Brock and Latter [5]), Triticum aestivum (Borojevic [6]), Triticum durum (Scossiroli [7]), Hordeum sativum (Gaul [8,9], Gaul and UlonskallO], Aastveit [11]), and in Arabidopsis thaliana (Brock [12]). Further literature dealing with induced micromutations is given by Scossiroli [13].

The first mutation experiments in barley carried out at our breeding station, Probstdorfer Saatzucht, date back to 1951 (Hânsel [ 14]). The

221

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M UTATIO N BREEDING IN BARLEY 2 23

more recent mutation work presented here should give some information about the usefulness of the induction of micromutations for the practical breeding of brewing barley at a medium-sized commercial plant breeding station and should result in an improved new barley variety.

In such a project some premises and some intentions may differ from those of model experiments which are designed first of all to solve fundamental questions of mutation breeding. Some of these differences may be summarized as follows:

(a) In model experiments and especially in micromutation experiments, a genetically homogeneous strain or variety should be used as motherline in order to maintain the highest possible probability that a variant, selected after the mutagenic treatment, represents an induced mutant.In our work, however, a young cross-bred strain was used which certainly was not genetically 'pure'. This was done to prevent the possibility that the variety type and the resistance type of the mother line might have been surpassed by a new variety type, when after about ten years of selection and field trials, a somewhat higher-yielding micromutant might be ready for release.

(b) In model experiments it can be of interest to apply the same artificial selection pressure in successive generations and after different mutagenic treatments. In practical mutation breeding the selection pressure would depend on various factors, e. g. climatic conditions ina given year, significance of the results of field experiments in a. given year, and average performance of the lines tested. This average performance may depend on the type of mutagenic treatment.

(c) In a model experiment for micromutations for yield, each mutant of higher yield than the mother line is of interest though other characters, suchas 1000-grain weight, 'grading', malting characters, standing capacity, time of ripening, etc. , might be altered in a 'negative' direction. However, in order to have a chance to produce a new variety, a higher- yielding mutant should surpass the mother variety in at least one important character, e. g. yield, without displaying even small defects in other characters.

(d) In practical breeding of a species only a part of the overall breeding program can be given to mutation breeding. At our station, for example, the mutation work had to be restricted to 5-10% of the area reserved for barley breeding. In order to perform a more extensive mutation programit was therefore necessary to start the different variants of the experiment in successive years. These variants were selection within the untreated strain P 6013, neutron irradiation of P 6013, and EMS-treatment of P 6013 (Table I).

2. MATERIAL

The barley line used was the F4 -plant progeny P 6013 derived from the cross Carlsberg II X Piroline. P 6013 was of homogenous appearance and proved its high yielding capacity, good kernel performance and good malting quality in three successive years. By reselection of the untreated strain P 6013, the variety Eura II has been developed, which is now composed of four lines each of them derived from one Fg -plant. Over a six-year average (State trials 1964-1969), Eura II equals the variety

224 HÂNSEL et al.

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Union in 'grading' and surpasses Union in yield by 5. 3% in the eastern part of Austria. Eura II is now the most widely grown brewing barley in the country. Before this reselection was finished another sample of the original strain P 6013 was irradiated with thermal neutrons and in the next year a new sample treated with EMS.

3. NEUTRON IRRADIATION (N) AND EMS TREATMENT (E)(see Hansel [13])

Dry grains (150 g) of P 6013 with a moisture content of 12. 0% were irradiated at the Reactor Centre at Seibersdorf, Austria, by thermal neutrons (1013 nth cm"2). In the year of irradiation (1962) the plants of the F4 -plant progeny P 6013 stood in the tenth selfing generation. The Ni-grains1 were sown in an isolated field plot. Only a small percentage of the Ni-seedlings survived, producing a total of about 1000 seeds. These were sown in pots in a glasshouse and gave 755 N2-seedlings. Of these,31 (4. 1%) were chlorophyll mutants.

Seeds from 1963 of the strain P 6013 were treated with 0 (= control),0. 5, 1. 0, 1. 5 and 1. 8°]o EMS aqueous solution for 4f h at 24-26°C and washed under running water (4 h). The seeds of the different treatments were divided into two lots, one planted out in a glasshouse and the other in the field. The mutation rates for chlorophyll déficiences are given in Table II. The M2-mutant percentage due to the 1% EMS treatment (6. 5%) came near to that observed after the neutron irradiation (4. 1%) when the glasshouse results are compared.

4. SELECTION

Positive selection was carried out for two quantitative characters, grain yield, and 'grading' (grain width as measured by the percentages of seeds remaining above a sieve with apertures of 2. 5 X 25. 0 mm. The breeding aims were a simultaneous improvement of both characters or at least an increase in one character without decrease of the other.

We are aware that for a statistical treatment the character 'grading' has disadvantages and that from this point of view the 1000-grain weight would have been preferable as a measure of grain performance. In the semi-arid area of eastern Austria, however, the grain performance of malting barley is measured by the nitrogen content and by the 'grading' percentage >2.5 mm. Though the intervarietal correlation between 1000-grain weight and 'grading' is strong, marked deviation from the regression line may occur depending on the degree of plumpness of the grains. The combination of high 'grading' with medium grain weight is of interest for the breeder. Therefore, selection was applied for 'grading' though the 1000-grain weight of the N-line was determined from the N3-generation onwards.

1 M t - genera tion trea ted w ith a m utagen .

N i = genera tion treated w ith neutrons,

Ei = genera tion treated w ith EMS,

226 HÀNSEL et al.

In the selection procedure we had to deal with two quantitative characters of different heritability. That of yield is low, especially when the selection is based on yield results of single plants or plant progenies, while that of grain dimensions is comparatively high. Therefore in the M2(N2 and E2) generations, the selection was based almost exclusively on the kernel performance. In the M3 and M4 generations also the 'grading' results were weighted more than the yield results with respect to the higher heritability of the grain dimensions. This preference of 'grading' in the selection procedure in early generations could have had an influence on yield in cases where a phenotypic correlation between 'grading' and yield existed. However, no correlation between these characters was found between the M-lines, or within the control (P 6013),i. e. between the different control-plots, in the different M-generations, with the exception of one year when the correlation was positive due to considerable soil differences between the blocks.

4. 1. Selection after neutron irradiation

The N2-plants were grown in a glasshouse and exposed to heat and to restricted water supply with the view to screening for mutant types able to develop well-filled grains under these stress conditions. After visual examination of the grains produced on normal-appearing N2-plants, about two-thirds of the progenies were discarded, because of their insufficient seed performance (slender seeds). The remaining ^-progenies (= N3-lines) were handsown in the field (per line two 1 m rows with 21 grains), every 11th number being the untreated control (P 6013).Due to the unequal germination in the field only 11 extremely low 'grading' lines of the 211 N3-lines were discarded. The N4-lines were machine- sown in 0. 5 X 5. 0 m plots and without replication, every sixth plot being a control plot of P 6013. From N4 onwards the plot size was 10 m2 and every fifth or sixth plot was a control plot.

In the N4 and following generations, both selection criteria, yield and 'grading', were equally weighted, resulting in a rather mild selection pressure as seen in Table I. In the N6, N7 and Ns generations, a part of the lines were included in trials with three or four replications and the less-hopeful lines replicated once only. Each of the N-lines tested for one or several years represents the progeny of a single N2-plant.

4. 2. Selection after EMS-treatment

The E2-generation was grown in the field in 1964 under very dry growing conditions. In the following E-generations the lay-out of the experiments was the same as in the corresponding N-generations. In the E3 generation, out of normal-appearing Expiant progenies de novo single plants were selected and their progenies (E3-plant progenies) tested in the following years. This was done under the assumption that especially in the seeds treated with higher concentrations of EMS multiple mutations might have been induced more frequently, and consequently the chance for the occurrence of 'desirable' segregates would be higher in later generations (Hansel [15, 16]).

The percentages of selected E2- and N2-plants out of the corresponding M2-generations were approximately the same. In the following generations,

M UTATIO N BREEDING IN BARLEY 227

however, a higher percentage of lines was selected out of the N-material, since in the E-material relatively few lines surpassed the control in one or both selection characters.

5. CONTROLS

When the F4 -progeny of P 6013 was treated by neutrons in the F10 generation and by EMS in the Fn generation, the seed material of P 6013 consisted most probably of a number of different, highly homozygous genotypes. This means that the lines selected after the mutagenic treatment of P 6013 and surpassing it in yield and/or grading may represent micromutants or non-mutated variants of P 6013.

The M-lines were compared with two different controls: Control I is the untreated and unselected F4 -plant progeny P 6013 reproduced each year in the same trials as the M-lines. Assuming that natural selection occurring within this genetically narrow population does not shift the population means of yield and 'grading' in a certain direction, the untreated strain P 6013 can serve as a control for the selection response in the irradiated material.

Control II should give information on the potential positive selection response within the untreated strain P 6013. As seen from Table I, the strain P 6013 underwent reselection in two successive years. Out of the total of 2440 single plants, the five best progenies were selected in the course of five and six years respectively, and finally reduced to four variety-lines. The breeding aims and the methods of selection were the same as in the treated material with the exception that the N2-plants were grown under artificial stress conditions as mentioned before. These four reselected lines (- Control II) were grown together with the mother strain P 6013 (Control I) and the M-lines in field trials since 1966. As seen in Table III, the average of the four lines (Control II) surpassed P 6013 in the mutation trials as well as in a set of State trials by 1 and 1. 5% in yield and by 2. 6 and 4. 5% in 'grading' respectively. The 1:1:1:1 mixture of the same four lines (= variety Eura II) gave similar results. As seen in Fig.l, the highest yielding line of Control II out-yielded Control I by about 2% and the highest 'grading' line surpassed Control I by about 4% in 'grading' in the average of the mutation trials.

TABLE III. YIELD AND 'GRADING' >2.5 mm OF CONTROL II AND EURA. II AS PERCENTAGES OF CONTROL I (P 6013)(T A B L A II I . REND IM IENTO Y ''G R A N U LO M E TR IA " > 2, 5 m m DEL CO N TRO L II Y DE EURA II,

EXPRESADOS EN PORCENTAJES DEL CO N TRO L I (P 6013))

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d o )

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№

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C on tro l I I = four lines m ean (m u ta tion exp. ) 6 101 102. 6

Contro l I I = four lines m ean (S ta te tria ls) 10 101.5 104.5

Eura I I = four lin es m ixtu re 1:1:1:1 (S ta te tria ls) 9 101. 9 102. 5

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M U TATIO N BREEDING IN BARLEY 229

T A B L E V. SPRING B A R LE Y P 6013 IRRADIATED WITH THERM AL NEUTRONS. PERCENTAGES OF N -L INES SURPASSING CONTROL I IN GRAIN YIELD , IN 'GRADING' >2. 5 mm AND IN BOTH CHARACTERS IN SUCCESSIVE N-GENERA.TIONS, W HEN POSITIVE SELECTION FOR BOTH CHARACTERS WAS A P P L IE D

(TABLA V. CEBADA DE PRIMAVERA P 6013 IRRADIADA CON NEUTRONES TERMICOS. PORCENTAJES DE LINEAS N QUE SUPERAN AL CONTROL I EN RENDIMIENTO EN GRANO, EN "GRANULOMETRIA" >2, 5 m mY EN AMBOS CARACTERES EN GENERACIONES SUCESIVAS N, AL PROCEDER A UNA SELECCION POSITIVA DE AMBOS CARACTERES)

Generation Number of N-lines

Percentages of N-lines surpassing Control I Yield 'Grading' Yield and 'grading'

N4 200 36 55 22

N5 106 57 49 26

Ns 78 53 60 38

N7 45 71 67 47

n 8 26 65 77 53

GRAIN YI ELD ( 100 = CONTROL I )

• = 11 comparisons at 10 m 2, + = 6 comparisons at 10 m 2, О = the four lines of Control II.

FIG. 1. Four-year (N5-N8) means of grain yield and 'grading' > 2.5 m m of the 26 N-lines tested up to Ns as compared with the untreated strain P 6013 (= Control I = Ю М ) and the four best lines selected out of Control I (= Control II).

(FIG. 1. Valores medios en cuatro affcs (N5-Ne) del rendimiento en grano y «granulometría» > 2,5 m m de las 26 líneas N ensayadas hasta la generación NB, en comparación con la variedad no tratada P 6013 (Control I = 100%) y las cuatro líneas óptimas seleccionadas a partir del Control I (= Control II).)

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6 . SELECTIO N RESPONSE

6 . 1 . N -lines

The selection response of the N -lines from N3 to Ng is summarized in Tables IV and V. The data for yield and 'grading* are given as p e r centages of the untreated Control I. The yield mean of the N -lines increases from 96% in N3 , to about 102% in N? and N8. The corresponding 'grading' data are about 100% in N4 and N5, and about 102% in N 7 and Ns . W e assume that the high 'grading' means in early N-generations may be due to the selection method applied in N 2 and N3 , based almost exclusively on the grain performance, as mentioned in 4. 1. Taking the number of surviving N 2- plants as 100%, the selection of plant progenies resulted in a reduction to 2 7. 9 % in N 3 and to 3. 6 % in Ns of the original number of N 2 plants.

To test the selection response in relation to the breeding aim of a combined increase of yield and 'grading', the percentage of N -lines su rpassing the mother line P 6013 in one of the two characters and in both of them were determined in the different N-generations. As shown in Table V, the percentage of the higher-yielding lines increased from 36 to 65%, the percentage of better 'grading' lines from 55 to 77% and the percentage of higher-yielding plus better 'grading' lines from 2 2 to 53% from N 4 to Ng respectively.

In F ig. 1, the four-year averages of the 26 N -lines tested up to Ng are compared with the untreated mother line (= Control I) and the four best lines selected out of the untreated mother line (= Control II). The data are weighted means and related to plots of 10 m2. The graph indicates that under the given circumstances a number of positive variants for yield, 'grading' and yield plus 'grading' were selected from the irradiated material. Those which surpass the best lines of Control II might be assumed to be micromutants.

6 . 2. E -lines

The selection response of the E -lines is summarized in Tables VI and VII. Although the number of E2-plant progenies was about seven times the number of the N2-plant progenies and the selection pressure applied in the E -m aterial was higher from N 3 onwards, the response to positive yield selection in the E -m aterial was lower than in the N-m aterial, especially in the first two generations. Consequently, the average yield of the selected E2 -plant progenies reached that of the untreated Control I two generations later (in E7) than the average yield of the N2-plant progenies. This holds true also for the E3-plant progenies. The reasons for this comparatively delayed selection response in the E -m aterial are not known. An analysis of the yield data of the different EMS-doses may help to clarify this problem. The final effectiveness of the selection is demonstrated by the data given in Table VII. In E 7 38% of the -plant progenies and 54% of the E3-plant progenies surpass the untreated Control I in both selection characters. These percentages are very near to those of the N -lines in N7 and Ng.

M UTATION BREEDING IN BARLEY 233

This applied mutation experiment w ill not be considered finished until the best of the presumed mutant lines have been tested for three years in State tria ls, and we can decide whether one mutant line or a combination of mutant lines w ill replace Control II (= Eura II) in multiplication and marketing or not. From the data available up to now, we think it reasonable to conclude that by the mutagenic treatment some positive variants in yield and 'grading' were induced.

R E F E R E N C E S

[ 1] GAUL, H., "The concept of macro- and micro-mutations and results on induced micro-mutations in barley", The Use of Induced Mutations in Plant Breeding (Rep. FAO/IAEA Tech. Meeting, Rome, 1964), Pergamon Press, Oxford (1965) 408.

[2] GREGORY, W.C., X-ray breeding of peanuts(Arachis hypogaea L.), Agron. J. £7 (1955) 396.[3] RAWLINGS, J. O. f HANWAY, D. G . , GARDNER, С. O., Variation in quantitative characters of soybeans

after seed irradiation, Agron. J. 50(1958) 524.[4] OKA, H.I., HAYASHI, J., SHIOJIRI, I., Induced mutations of polygenes for quantitative characters

in rice, J. Hered. 49 (1958) 11.[5] BROCK, R. D., LATTER, B. H. D., "Radiation-induced quantitative variation in subterraneum clover".

Proc. 3rd Australas, Conf. Radiobiol., Butterworth, London (1961) 205.[6] BORQfEVIC, K., "Studies on radiation-induced mutations in quantitative characters of wheat (Triticum

vulgàre)", Mutations in Plant Breeding (Proc. Panel Vienna, 1966) IAEA, Vienna (1966) 15.[7] SCOSSIROLI, R. E., "Wheat mutagenesis in quantitative traits", Proc. 2nd. Int. Wheat. Genet. Symp.

Lund, 1963, Hereditas Suppl. 2 (1966) 85.[8] GAUL, H., Untersuchungen zur Selektion von Kleinmutationen bei Gerste, Z. PflZücht. 45 (1961) 300,[9] GAUL, H., Zûchterische Bedeutung von Kleinmutationen I. Durch Rôntgenstrahlen induzierte

Variability von Kornertrag, Koingïôsze und Vegetationslânge bei dei Getste Haisa II, Z . PflZücht.55 (1966) 1.

[ 10] GAUL, H., ULONSKA, E., Zûchterische Bedeutung von Kleinmutationen II. Durch Rôntgenstrahlen induzierte Variabilité von Kornertrag, Korngrôsze und Vegetationslânge bei den Gersteu Volla und Wisa, Z. PflZücht. 58 (1967) 343.

[11] AASTVEIT, K., "Use of induced barley mutants in a cross-breeding program", Mutations in Plant Breeding (Proc. Panel Vienna, 1966) IAEA, Vienna (1966) 7.

[12] BROCK, R. D., Quantitative variation in Arabidopsis thaliana induced by ionizing radiations, Radiat.Bot. 7 (1967) 193,

[ 13] SCOSSIROLLI, R. E,, "Mutations in characters with continuous variation", Manual on Mutation Breeding (Tech. Rep. Ser. No. 119) IAEA, Vienna (1970) 117.

[14] HANSEL, H., "Induction of mutations in barley: Some practical and theoretical results”, Mutations in Plant Breeding (Proc. Panel Vienna, 1966) IAEA, Vienna (1966) 117.

[ 15] HÂNSEL, H., Untersuchungen über die Hàufigkeit induzierter Chlorophyllmutationen nach AMS- Behandlung, Neutronen- und RÔntgenbestrahlung von Gerstensamen, Bodenkultur 16 (1965) 325.

[ 16] HANSEL, H., "Model for a theoretical estimate of optimal mutation rates per Mi-nucleus with a view to selecting beneficial mutations in different M-generations", Induced Mutations and Their Utilization, Proc. Erwin-Bauer-Gedáchtnisvorlesungen IV, Gatersleben, 1966. Akademie-Verlag, Berlin (1967) 79.

D IS C U S S IO N

G. DE A LBA : Why does the number of control II progenies decrease from 1770 to 4 over a seven year period?

H. HÂNSEL: The selection pressure eliminated them since they were poor in yield or in weight per 1 0 0 0 grains.

K. BOROJEVIC: Did you find the increase of the mean in later generations (Ms, Мб) over the control?

7. CONCLUSION

23 4 HÀNSEL et al.

H. HANSEL: In the neutron-treated m aterial the mean yield of the selected N -lines surpassed control II not before N7 and the mean "grading" not before Ng. In the EM S-treated m aterial the yield and the mean "grading" of the selected E -lines surpassed control II not before E7 . In these late generations only a sm all percentage of the original M 2 and M3

progenies was tested and we assume that the increase of the M -lines means was due to the continuous positive selection.

H. G AU L: I am against the use of low doses. With high doses, I agree, there are all indications that the Mg plant you are starting with may contain several mutations, most of them being in the heterozygous condition. But with high doses the genetic variability is increased and consequently the chance to select positive mutants is increased.

H. HANSEL: The chance to select 'positive ' mutants depends not exclusively on the amount of induced variability. Since the expected ratio of ' positive' to ' negative' mutations is very small, the frequency of ' useful' and ' desirable ' M ^-segregates decreases rapidly with increasing number of mutant alleles per genotype. I remember that I defined a 'useful1 genotype as being a plant in which 'positive' mutations are either in a homozygous mutated or in a heterozygous state and none of the 'negative' mutations is in a homozygous mutated state, whereas ' desirable' genotypes are homozygous for 'positive' mutations and all of the additionally induced negative mutant alleles are replaced by normal alleles as a consequence of the segregation p rocesses.

I proposed therefore the use of high doses when intending to select for a certain type of macromutation, in order to obtain the highest possible number of this mutant type, being aware that more or less all of them w ill be loaded with induced 'negative' alleles. These could become diluted or lost by a following back-crossing to the mother line or out-crossing to other varieties. Otherwise when intending to select 'positive' m icromutants, I proposed to use lower doses, because micromutants cannot be recognized as single plants and consequently cannot be used as parents in early crossing.

H. G A U L : What I suggested is to use the micromutation technique for maintenance breeding. In maintenance breeding with top varieties, at least in Europe, the breeder conducts reselection every year within the strains. Using high doses in maintenance breeding with micromutations, reselection in the M -strains has of course to be done. I cannot see why there w ill be a delay of breeding as compared with the traditional maintenance breeding method; at least the delay w ill not be long.

H. HÂNSEL: There are different methods used for maintaining a barley variety. One of them is (a) to sow each year a high number of ear progenies of the variety, or of each of the variety lines, (b) to discard aberrant progenies in the field and/or laboratory, and (c) to pool the remaining progenies to a stock seed. In this procedure ear progenies derived from seeds treated with mutagens cannot be used.Another method consists of a continuous pedigree breeding, with progeny testing up to yield tria ls. By this within-variety selection the breeder hopes to improve rather than to maintain the variety. And I agree with D r. Gaul that for suchan ' improvement' breeding of a variety a mutagenic treatment may be useful. When a variety is on the market its maintenance breeding for stock seed production and its 'improvement' breeding after a mutagenic treatment have to be separate procedures.

M U TATIO N BREEDING IN BARLEY 2 3 5

R .D . BROCK: The dose at which a mutagen should be applied is related to the efficiency of selection which can be applied for the character to be improved. At high doses multiple mutations occur with high frequency. The desirable mutants must be separated from the undesirable mutants by segregation in later generations. At low doses the desired mutation will occur at lower frequency but it w ill be less liable to be associated with undesirable mutations. Hence if ease of recognition and selection efficiency is high, low doses are preferable. However, if efficiency of selection is low, higher doses should be used, and selection maintained for all agronomically important characters throughout successive generations.

H. HÂNSEL: I think that for reasons I have given before the induction of more than two mutations per Mj-nucleus is of no advantage when we intend to select micromutants.

M UTATION BREEDING IN W HEAT*

Katarina BOROJEVIC, S. BOROJEVICÍ Department of Genetics, Faculty of Agriculture,Novi Sad, Yugoslavia

Abstract-Resumen

MUTATION BREEDING Ш WHEAT.Mutation work in Triticum species is reviewed. A number of examples of induced mutations having

practical value in wheat breeding are presented. Such mutations involve spike characteristics, winter hardiness, root development, disease resistance, grain quality, lodging resistance, and yield. It is further discussed where to use mutation breeding. In particular it is pointed out that mutation breeding can only be carried out successfully if it is incorporated into a sound breeding program. Some guidelines on how to design and organize a mutation breeding project for wheat are given.

MEJORAMIENTO DEL TRIGO POR MUTACION.Los autores pasan revista a los trabajo? de mutación realizados en especies Triticum. Citan buen número

de ejemplos de mutaciones inducidas que tienen valor práctico en el mejoramiento fitotécnico del trigo. Estas mutaciones afectan a las características de la espiga, la resistencia al frío, el desarrollo de la raíz, la resistencia frente a las enfermedades, la calidad del grano, la resistencia al encamado y el rendimiento. A continuación examinan cuándo conviene recurrir a las mutaciones. Señalan en particular que el mejoramiento por mutación sólo puede tener éxito cuando es parte integrante de un programa fitotécnico bien concebido. Formulan directrices sobre la manera de preparar y organizar un proyecto de mejoramiento del trigo por mutación.

1. INTRODUCTION

The wheat plant became a research object in radiogenetics as soon as M üller discovered the mutagenic effects of X -rays in 1927. In 1929 Stadler [1 ] treated different Triticum species with X -rays, and after treatment he isolated chlorophyll mutations. The Russian investigators Delaunay [2, 3,4,5] and Sapehin [6 ] treated Triticum with X -rays and isolated awned, speltoid, dense eared, point glume, dwarf and other mutations which were clearly different from the mother variety.

Unfortunately, most isolated mutations have very weak vitality and very poor production of kernels per spike. Therefore, the use of induced mutations in wheat breeding was not much considered until Gustafsson and FrS ier obtained mutants in straw strength and height, earlines>s, and lodging resistance [7 ].

The excellent studies of MacKey [8,9, 10, 11] on basic and applied aspects of mutagenesis in Triticum species encouraged many research workers to use the wheat plant as an object for investigation. Wheat being a polyploid, the duplications of genes allow a greater number of prim ary induced changes to be preserved and transmitted to the next generation. The neutralizing effect of chromosomal duplications diminishes the degree of phenotypic penetration of every induced mutational event.

A new concept of the use of induced mutations in plant breeding developed from Gregory 1 s [12] discovery that normal appearing plants in irradiated

* Part of the research reported in this paper has been carried out under Research Agreement with the International Atomic Energy Agency No.360/CF.

23 7

238 BOROJEVld and BOROJEVIC

populations may be variously mutated with many small individual changes which form a sound basis for artificial and natural selection. Gregory compared his data with F ish e r 's adaptation sphere and found that, if mutations with great phenotypic deviation are removed from the population, the distribution of frequencies becomes more symmetrical and the probability of im provement approaches 0.5. This means that after removal of mutations with great phenotypic deviation the number of plus and minus variants becomes equal. It has thus been recognized that micromutations may play an im portant role in plant breeding, and studies of quantitative characters in irradiated wheat populations have become more frequent.

Scossiroli et al. [13, 14], Gaul [15, 16], Swaminathan [17], Borojevic.K. [18, 19, 2 0 ] and others [2 1 , 2 2 , 23] have studied changes in quantitative characters after mutagenic treatments of wheat. It has been found that mutagenic treatments decrease the mean value and increase the variability of quantitative characters in M 2 and M 3 generations. The changes in variability occur through an increase in the genetic components. With irradiated populations bred to later generations, it was found that the mean value gradually increased in later generations without any artificial selection, reaching the control mean around M 5 [24, 25]. In further generations the population exhibited a stabilization, staying on a plateau from M 5 to M 10. For some characters this plateau was 3-5% lower, for others 5-7% higher, than the mean control values. On the other hand, the variability, which increased up to four times in M2 and M 3 , gradually decreased in later generations and stabilized around M 5 [2 6 ].

The response of treated populations to selection was rather high in early generations, being sm aller in generations having reached the plateau. Therefore, the results of selection in early generations were not in full agreement with the expected response, though later generations did show a good agreement between expected and observed results. The extreme selected lines for high and low mean values showed significant differences between each other and also in relation to the control [25].

Examples of induced mutations which have or may have practical value in wheat breeding w ill be discussed together with the genetic aspects of the changes involved. Mutagens, treatment, and effect in M j w ill not be discussed in this paper as they are all well summarized in the FAO/ IAEA Manual on Mutation Breeding (1970) [27].

2. INDUCED M UTATIONS OF PR ACTICAL VA LU E IN W HEAT BREEDING

2.1. Spike mutations

The most frequently studied spike mutations in hexaploid wheat were those involving chromosome 5A. This chromosome is very well marked by four genes: Q speltoid inhibitor and squarehead promotor; В г awn suppressor; V pubescent nodes; and Sk spring habit. The locus Bj is closer to the centromere than Q, which is about 30 cross-over units distal from B-[[8 , 28]. Most frequent are speltoid mutations with a frequency of 30-50% per plant progeny.

The majority of speltoids and compastoids are very poor in agronomic traits and are little used in breeding program s, but may be of help in genetic studies.

M UTATION BREEDING IN WHEAT 239

Mutations which include only locus В! are less frequent than those involving locus Q, but they are more useful. In our experiments 1.29] the mutation frequency of B j locus ranged from 0 . 2 to 0 . 8 in comparison to the total frequency of mutations in chromosome 5A, which ranged from 7.50 to 12.82. Most of the B j mutations after mutagenic treatments are likely to be deficiencies, ratherthan intragenic changes. Therefore, the yielding capacity of these mutations is expected to reflect the degree of deficiency involved in each case; other characters of the plant could also be more or less influenced, partly due to the size of the deleted segment, partly due to additional changes.

MacKey [8 ,9 ] reported a spontaneously arisen awned mutant in the Scandia III variety which outyielded the parent strain in three years of trials. Jagathesan et al. [30] and Swaminathan [17, 31] gave data showing that awned mutants of the varieties M. P. 799 and N. P. 809 outyielded the parental strains in two years of trials. Borojevic, K. [29, 32] gave a survey of the quantitative characters of bearded mutants in M 7, M 8 and M g , selected after treatment with X -rays and thermal neutrons from the awnless varieties Campodoro and Mara. The bearded mutants differed from the original variety not only in their morphological appearance, but also with respect to the mean values of their quantitative characters, such as plant height, tillering, and vegetation period. None of these bearded lines had a lower yield than the non-irradiated control, and five of them, called MR 2, 4, 7, C T B 5,CTNjo> yielded up to 55 q/ha, which in some years was significantly higher than the mother varieties.

Some of the bearded lines can be considered isogenic, supporting the findings that awns contribute to photosynthetic' efficiency.

2.2. Winter hardiness

MacKey [8 ] denoted factor Sk located on chromosome 5A, as regulator of the spring habit in Kolben wheats. The location of this gene is given by the sequence S^-Bj-Q , from the centromere outwards. Later, MlacKey used the spring variety Rival in mutation experiments involving the genic constitution S^-S^-B i-B i-Q -Q . In this variety he found that a small deficiency including only factor 0 w ill cause homozygous speltoid, which is beardless and of spring habit. If the Bj is included in deletion, the homozygote w ill be a bearded spring wheat, but if Sk is included in deletion the homozygous speltoid will appear as a bearded winter wheat. Such a type of transition from spring to winter wheat included 17 mutations in the Rival X -ray and neutron experiments, and their frequency averages 0. 7% in M 2 . Transmission from spring into winter habit was monofactorially regulated in that material.

Khvostova [33] cited winter resistant mutants obtained in the Soviet Union after different mutagenic treatments. In the Bashkir Institute for A g r icultural Research, a winter-hardy, high-yield mutant, Lut. 6455, was obtained after gam m a-ray treatment. Belicheva in Rostov-on-Don selected the winter-hardy lines 30, 21, 31 and 39 from durum wheat; over-wintering was 69. 1%, 70.9%, 6 6 . 6 % and 6 6 . 6 %, respectively, in comparison with 23% in the control. Skhvarnikov also obtained winter-hardy mutants which had 85% to 75% over-wintering in comparison with 33% in the mother variety.

Induced mutants resistant to winter may be of particular importance for varieties which are otherwise outstanding but their spring habit limits their cultivation to certain areas, as, for example, is the case with some new sem i-dw arf and dwarf varieties.

2 4 0 BOROJEVICÍ and BOROJEVIC

2.3. D isease resistance

In some species of cultivated plants, sources for resistance are no longer available, or due to difficulties in species, crosses cannot be used. Therefore, the induction of resistance to diseases has a very great importance in plant breeding.

Konzak [34], summarizing the early studies on this problem, presented data indicating that disease resistance in higher plants may be obtainable via induced mutations. Favret and Ryan released a variety, Silvalocho Gamma, in 1962 resistant to leaf and black stem rust [31]. Favret et al. [35] also produced a mutation in Silvalocho that has modified its reaction to several pathogenic biotypes Pucc. recóndita and Pucc. gram inis.

So far, mutagenic treatments have contributed more indirectly than directly to disease resistance in plants. A classic example is the vulgare variety Transfer produced by Sears [36]. In order to incorporate resistance to leaf and stem rust into vulgare, Sears crossed T r . vulgare X Aegilops umbellulata and after a series of backcrosses obtained one line with 42vulgare chromosomes and 1 chromosome from A e . umbellulata. This line possessed rust resistance but also other undesirable characters. By means of irradiation Sears succeeded in obtaining a translocation between a wheat chromosome and a segment of the Aegilops chromosome carrying rust resistance without other negative characters.

Knott [37] applied irradiation to transfer the stem and leaf rust r e sistance of Agropyrum to common wheat. For each type of resistance one translocation showed irregularities in transmission through the gametes, particularly through the pollen.

D risco ll [38] used a combination of the 5B chromosome pairing method and irradiation, and succeeded in obtaining a translocation between chromosome 4A of wheat and a segment of chromosome 2R of rye. This strain, called Transec,possesses resistance to leaf rust and powdery mildew.

2.4. Male sterility

Male sterility, which is certainly biologically a very negative phenomenon, may serve as a useful property in a crossing program, particularly for the utilization of the heterotic effect.

Spontaneously occurring m ale-sterile mutants have been observed in several wheat varieties [39,40]. However, very few studies are reported on induced male sterility in wheat, while in other plants, like barley, onions, cabbage and tomatoes the phenomenon has been much more elaborated [41, 42, 43].

In a treated wheat population, we have observed male sterility in later generations (M 5 -M 7) after treatment with gam m a-rays, El, and EMS [44].In most cases, m ale-sterile plants occurred in segregating progenies together with speltoid, compactoid, bearded, and short straw mutations. Therefore, it may be assumed that the observed male sterility was a consequence of chromosomal aberrations. The frequency of male sterility was 7-13% per progeny. Very rarely was the total progeny sterile. Further studies are necessary to determine its inheritance and possible value in hybrid wheat breeding.

M U TATIO N BREEDING IN WHEAT 24 1

2.5. Quality

Spontaneous mutants opaque-2 and floury-2 in corn, possessing a higher lysine content, seem to have directed our attention towards chemical changes after mutagenic treatment. At the same time, new screening methods for non-destructive analysis in a single kernel are being developed, which are going to have a tremendous impact not only on screening previously undiscovered mutants but also on breeding, and which w ill permit testing of all kernels in segregating generations.

Of particular interest for us are mutants with higher protein content and better amino-acid composition in small grains, since the diet of the great majority of the human population depends largely on these crops.

By X -ray treatment Swaminathan [45] obtained in the Mexican wheat variety Sonora-64 several mutants which have a 1-2% higher protein content and 0. 5 - 1% more lysine. One of these mutants, called Sharbati Sonora, has been put into production.

Using the DBC method, Denic et al. [46] studied protein content in the M 3 generation after mutagenic treatment with El, EMS, and gam m a-rays.They found significant increase in the variability of the protein content, the standard deviation being 1. 18 and 0. 98 for treated material and control respectively. Dumanovic et al. [47] found a higher correlation coefficient for X 3 /X2 and X4 /X3than for the same generations in the control, indicating that increased variability was inheritable. The efficiency of inducing an inheritable variation in protein content increases in the order X -rays < EMS < E l.

Solari and Favret [48] studied the genetic control of protein constitution in the endosperm of four wheat mutants by electrophoresis. They found that the electrophoretic spectra of the mutants were identical to those of the corresponding mother lines, except for one mutant induced in the variety Silvalocho, which was characterized by a loss in baking quality in comparison with the mother variety, and also showed a different electrophoretic spectrum.

Changes in baking quality after different mutagenic treatments were studied in the USSR by Cherny, who analysed, using the Pelschenke method,622 mutants which were morphologically different from the mother varieties, by Maystrenko and Polychova, who studied the quality of gluten in spring wheat, and selected forms with better quality gluten than in the control, and by many others [33].

Some of the results obtained regarding induced changes in quality have to be regarded cautiously. After mutagenic treatment the size of the kernels is usually sm aller than in the control. Smaller kernels of the same variety contain a higher percentage of protein than larger kernels. This happens not only in the M j, where we found an increase of 50 - 67% in wet gluten and16 - 23% in dry gluten, and an increase in bread volume and improvement of bread shape over the control [49], but also it may be valid for later generations or for mutant lines [47]. Therefore, quality alone, independent of other kernel characteristics, may be misleading.

2.6. Resistance to lodging

Resistance to lodging is a complex character which depends on the shortness and stiffness of straw, and the development of the root system. Therefore, breeding for lodging resistance must include all these characters.

2 4 2 BOROJEVI¿ and BOROJEVIC

2.7. Stem shortening

2.7 .1 . Induction of short-straw mutants

Stem shortening in wheat appears to be a common phenomenon after mutagenic treatment, very often, but not always, associated with straw stiffness [50].

Short-straw and dwarf-stature mutants in wheat were found very early.In the first experiments with X -rays performed by Delaunay [3] and Sapehin[6 ], dwarf mutants in tetraploid wheat were obtained. Gustafsson [7] and F rô ier isolated short-straw mutants in the hexaploid variety Pudel. MacKey [8 ] selected several short-straw mutants from the common wheat variety Scandia III. Unfortunately, most of these early induced short-straw mutants show a negative pleiotropic effect on yielding ability. Since the short stature in vulgare could be rather easily obtained as a result of transgressive segregation after hybridization, not much attention was paid to the induction of short-straw mutants in vulgare after these first mutants.

However, in durum wheat, where dwarf stature has not been found so far in existing varieties, the induction of short and stiff-straw mutants became of great importance. Therefore, the success of Italian scientists in obtaining short-straw durum mutants has particular value. Their results are extensively summarized by Scarascia-Mugnozza [50], and Scarascia- Mugnozza et al. [51] . Some of these mutants proved superior in yield not only over the mother varieties, but also over the best durum varieties tested over several years in the Middle and Near East durum wheat yield trials [52]. Three of them have been released as varieties: Castelporzianoand Castelfusano in 1968 from the variety Cappelli, and Castel del Monte in 1969 from the variety Griffoni [53].

Khvostova [33] reported that Russian investigators also obtained a short stem after muta!genic treatments. Mozhawvoy produced a mutant resistant to lodging which had a 2 0-30% better yield in some years than the mother variety. Eiges, Shkvarnikov and Cherny also created short forms in wheat.

2 .7 .2. Inheritance of short-straw mutants

Morphological and anatomical studies of the short-straw durum mutants revealed that shortening was achieved through shortening of basal internodes and lengthening of the uppermost internode. Some short-straw mutants are characterized by a completely solid culm, or by various degrees of solidness of the uppermost internode [54].

The short straw in durum was often associated with chromosomal aberrations or other changes which exhibit a pleiotropic effect, such as vaviloid bradhytic, and sphaerococcoid type of spike [55], or with the compactoid type of spike in vulgare [29, 56].

Because of different genetic changes which cause or are associated with the occurrence of short straw, the inheritance of this induced trait is also different. For example, in the du rum mutant variety Castelporziano [51] with stem reduced to 70-80% of the control the shortness is controlled by one dominant factor, while in the mutant variety Castelfusano, control is due to one recessive factor. Both varieties were obtained from the durum variety Cappelli and both carry reciprocal chromosomal translocations.


FIG.l. Population means (x) and standard deviations (s) for the character height of plants in generations after mutagenic treatment (control 100%).

The mode of inheritance of the short straw induced by EMS in the vulgare variety M arfeld was somewhat different, according to Woo and Konzak [56] . Their analyses suggested that the short-straw character in some of the mutants was dominant, in some, partially dominant, and in others, single recessive .

Many authors found that short-straw mutations were polygenic, and showed a decrease in the mean plant height in M 2 and M3 from 3-20% after different mutagenic treatments [14,57, 58]. Borojevic, [18,25,26,44] found in later generations (after M 3) that the mean increased gradually without artificial selection towards the control mean, stabilizing on a plateau from M5to M 12 which was about 3 to 7% lower than the control (F ig. 1).

Contrary to the mean value, variability for this character was increased in M2 and M3 by about 10-15%, but in later generations variability decreased and stabilized in M 5 -M 6 and kept at this level until M 1 0 (F ig . 1). The increased variability was due to an increase of the genetic components. Therefore, many populations showed a response to selection.

In our experiments [26], the expected response to selection was greater in early generations (M 2 and Mg) than in later generations, but in later generations the expected response was in much better agreement with that observed. The lines selected for extreme high and low mean value in later generations showed significant difference between each other and in relation to the control. These results indicate that the greater part of induced variability in early generations was due to its dominant components. This is also supported by the analyses of diallel crosses in experiments of Bagnara [59] .

From all these results it can be said that mutations in shortening of straw, which are frequently obtained after mutagenic treatment, are different in their genetic nature. Therefore, their isolation w ill depend on their genetic constitution. If shortening is the result of a single gene function, isolation in early generations may give results. If shortening is a result of

244 BOROJEVIC and BOROJEVIC

changes in polygenes, selection in later generations may give better results. So far, numerous short-straw and lodging-resistant lines have been obtained, with different genetic backgrounds.

Of seven released varieties produced via induced mutations, six had short, strong straw. Sharbati Sonora has short straw, Lewis is lodging resistant, Stadler has short straw, and Zenkouzi-Komugi has straw 10-15% shorter than its mother variety [53].

2.8. Development of root system

The ability of a plant to make more efficient use of fertilizers, steadily increasing yield with increased fertilization, depends upon the uptake e fficiency of the root system and indirectly on resistance to lodging.

Tavcar and Kendjelic [60] studied the roots of 25 genetically different wheat mutants, and found that root length and percent of rootlets are positively correlated with plant height and kernel yield per plant. From these results it seems that dwarf stature is negatively associated with the development of the root system, and that therefore tillering ability, which is frequently associated with short mutants, shouldbe studied as a compensating character, or the possibility of breaking down this linkage should be investigated.

2.9. Higher photosynthetic efficiency

Due to dwarf stature and intensive production technology, the canopy of wheat fields has very much changed. In order to have higher efficiency of photosynthesis, which is particularly dependent on the upper-leaf activity and lea f-a rea duration, it is necessary to breed for more efficient leaf area. Also, disease damage is changed in such a field, and a more erect leaf position may minimize such damage.

There are already some good examples of changes in leaf position and leaf area obtained by induced mutations, but there has not been sufficient study on them and more attention should be given to these characteristics in treated m aterial.

2.10. Yielding ability

Although many plant characteristics and environmental factors are involved in yielding ability, breeding for yield should be directed towards final yield components. With wheat, the final components for yield can be taken as: 1 ) number of spikes per unit area, 2 ) number of kernels per spike,and 3) absolute kernel weight.

The number of spikes per unit area depends prim arily on the resistance to lodging and to diseases, but may be regulated by sowing rate, fertilizers, water and other environmental factors.

The production per spike is strictly speaking a function of number of kernels and kernel weight. In wheat a high correlation exists between these two characters of about r = 0.94 [19], which did not change appreciably in populations after irradiation [57]. Therefore, either character is a good indicator for yielding ability.

A more valid measure of yielding ability is given by the number of kernels, through which all genetic changes are transmitted to the next gener-

M U TATIO N BREEDING IN WHEAT 245

G é n é r â t ¡ o n s

FIG.2. Population means (x) and standard deviations (s) for the character number of kernels in generations after mutagenic treatment for the wheat variety Mara (control 100%).

ation. The frequency of induced changes in the next generation w ill depend on the number of kernels which transmit them. Therefore, the number of kernels may have an advantage in studying changes in quantitative characters, if all estimations are made on the basis of individual plants.

It has been found that after different mutagenic treatments the mean value for number of kernels decreases, while the variability increases in M 2 and M 3 [14, 19, 23, 57]. Heritability estimates for this character increase from one to four times in comparison to the control; therefore, most treated populations responded to selection for this character.

According to our results [19, 24], when selection for the spike with the greatest number of kernels was applied in every progeny of M 2 and M 3, the mean value increased up to 30% over control in M 4, but when selection ■ceased, the mean value declined sharply around the control value in M 5

and stayed at this level until M 10 (F ig. 2) [19, 24, 25, 44]. These results also indicate that the greater part of induced variability was dominant. Therefore the selection for this character is much more effective if it is applied in later generations on the basis of the progeny mean. In our experiment this was M 5.

Many mutations in other characters may influence the yield, and some were mentioned earlier. Mutations in qualitative characters, such as awned spikes, may also exhibit a positive pleiotropic effect on yielding ability. Therefore, the yielding value of a mutant w ill depend on the performance in several characters. Yield improvement may depend on decreasing or increasing a single character, or several. With mutation breeding it is easier to decrease one character than to increase several, for example, it is easy to shorten the straw, as mentioned earlier. If straw shortening occurs without changes in yield components, the new mutant may have an advantage in being able to use greater amounts of fertilizer and water, and to take a higher sowing density, which would influence yielding ability positively.

246 BOROJEVIC and BOROJEVIC

3. SCREENING METHODS AND ISOLATION OF M UTANTS

The screening method w ill depend on the nature of the treated organism, on the mode of inheritance of induced changes, as well as on the aim of plant breeders. Many of these methods were evolved by Gustafsson, and readers interested may consult his paper [61], and also the FAO /IAEA Manual on Mutation Breeding [27].

In the wheat embryo which is multicellular, diplontic selection operates in somatic tissue, as well as haplontic selection between gametes [62] . Changes which pass these two processes may appear as mutations, although many of them are hidden because of the polyploid buffering of wheat [ 1 0 ] .With self-fertilization, which is natural to wheat, many heterozygous lethal changes become homozygous. Therefore, after a few cycles of selfing, many prim ary induced mutations are lost. The loss of lethal and low-viability mutants in a treated population is expressed by increasing the mean, and decreasing the variability of quantitative characters [26]. A ll these processes, which lead to a decrease of primary induced changes, may be called re covery mechanisms in a treated population.

On the other hand, as a consequence of many prim ary induced changes, secondary mutations may arise, as is the case, for example, with the compactoid mutation (where the prim ary changes are deficiencies while the secondary are duplications with compactoid expression). However, the frequency of secondary mutations is never as great as is the loss of prim ary mutations [8 , 29] .

Having all this in mind and considering discussions about screening and isolation of mutants in treated populations, the following combined pedigree method may be convenient for wheat:

Mj regular sowing (from every Mj plant, one spike for M 2 line),M 2 spike per row (from every M 2 line, a few main spikes for M3 line),M 3 line per plot (a few main spikes for line of M4),M 4 line per plot (a few main spikes for sub-lines in M 5),M 5 branching line to sub-lines. Selection is possible between lines,

between sub-lines, and between plants within sub-lines.In this way the variability between lines may be saved until M s . This

method has advantages for selection of quantitative characters. Of course,it can be modified by branching in earlie r generations, depending on the variability of the selected trait.

The amount of material is always a very important question for plant breeders. There are not many data in the literature on this problem, but because the probability for improvement in sm all changes of quantitative character is about 0. 5 [63] and the frequency of induced mutation not high, the greater number of M 2 lines has to be saved until later generations. One of the weaknesses of many mutation experiments was the small number of M2 lines in generations where selection was applied.

Lines with undesirable characteristics may be discarded in earlier generations, in which macro-mutational selection may also be done. Selection on these traits may help in handling sm aller amounts of material.

4. W H EN TO USE M UTATION BREEDING

Mutation breeding is a new field of human endeavour which opens new possibilities for breeding work, but it can be successfully carried out only


if it is incorporated into the breeding program of a crop. The procedure is the same as in conventional breeding: to fix breeding objectives, then tomake a breeding program for a new variety. To make the program more successful, a model variety should be worked out on the basis of environmental conditions and market requirements. To give an example, the general breeding objectives for high-yielding wheat varieties of 60-80 q/ha w ill be mentioned [64]: a) production over 1 g per spike in a stand of 6 0 0 spikesper m 2, b) stiff straw imparting resistance to lodging and permitting the application of high fertilizer rates, c) winter hardiness with no plant mortality at the minimum temperature which may appear during a winter without snow (e .g . -15°C), d) maturity, an optimal period of five days earlier or later than the best local variety, e) genetic resistance to the prevalent races of stem and leaf rust, and f) quality, depending on the market requirem ents.

In order to carry out the designed program, it is important to see what is possible to do via induced mutations. From the data described earlie r we can say that by mutation it is possible to a) increase production per spike, b) obtain a lodging-resistant type, c) select a winter-hardy mutant, d) p ro long the vegetation period (which is very easy by mutation breeding, although there are not many results on shortening the vegetation period of wheat), e) obtain genetic resistance to disease, directly or indirectly, and f) change quality in a positive direction.

Thus, the creation of a new variety may be accomplished via induced mutations. Mutation breeding is not of course the only method for creating new varieties, and may form only a part of it.

Mutation breeding proves particularly valuable in cases when in a variety fitting in our model only one character has to be changed and the others kept unchanged, for example, the shortening of straw, which is done in the durum mutation program . Increasing the protein content will also mean a great improvement for many varieties, as it was in Sharbati Sonora. Increasing the winter hardiness in Mexican wheat w ill be of great interest for continental Europe.

4. 1. Combinations of genes

Induced mutations represent new combinations of genes, which if not of direct use may be utilized in crossing program s. From the changed genetic background, new interactions and combinations of genes may be expected. Sears [36], Knott [37], D risco ll [38] and others have given excellent examples along this line. By this method, new combinations of genes are brought together, which could otherwise not be achieved.

4.2. Choice of material for treatments

It is well known that choice of parents for crossing must be given the most careful attention if new, more promising varieties are to be created. This is even more true for mutation breeding, when it is based upon the gene-character concept. From varieties with many negative characters, a variety with good agronomic characteristics cannot be expected.

Regarding the choice of material for treatment, local varieties are very often over-estimated. Nowadays, due to the extensive exchange of breeding material and divergent crossing program s, it has been shown that new lines

248 BOROJEVld and BOROJEVld

may have better adaptability than local varieties. This may apply even more for new induced mutations, and some mutants may prove more useful in regions other than the region where they have been obtained (as for example, with Palas barley and durum wheat mutants).

It would be desirable therefore to establish an international wheat mutation nursery in which most prim ary induced mutations already extant could be tested. This would not only help in finding the most suitable mutants for a certain region, but also ensure that more consideration be given to the choice of mother varieties.

Another possibility would be to choose the best high-yield varieties, subject them to mutagenic treatment in selected centres in the world, and then distribute the treated populations in M 2 or M 3 to different regions for selection on the basis of local environmental conditions and requirements.

R E F E R E N C E S

[1] STADLER, L.J., Chromosome number and the mutation rate in Avena and Triticum, Proc.natn.Acad.Sci.USA 15(1929) 876.

[2] DELAUNAY, L., Die Chromosomen Aberranten in der Nachkommenschaft von rôntgenisierten Àhren einer reinen Unie von Triticum vulgare albidum, Allg.Z.ind.Abst.Vererb. 55 (1930) 352.

[3] DELAUNAY, L., Resultate eines dreijâhrigen Rôntgenversuches mit Weizen, Züchter 3 (1931) 129.[4] DELAUNAY, L., The X-mutations in wheat, Trudy Lab.Genet. 9 (1932) 173.[5] DELAUNAY, L., Experimenten erzeugte Mutationen bei Weizen, Res. Genet. 10(1934) 164.[6] SAPEHIN, A. A., X-ray mutants in soft wheat, Trudy prikl.Bot. Genet.Selek.Ser. 2.9 (1935) 3.[7] GUSTAFSSON, A., Mutations in agricultural plants, Hereditas 33 (1947) 1.[8] MacKEY, J., Neutron and X-ray experiments in wheat and a revision of the speltoid problem, Hereditas

40 (1954) 65.[9] MacKEY, J., "Mutation breeding in Europe", Genetics in Plant Breeding, Brookhaven Symp.

Biol. 9(1956) 141.[10] MacKEY, J., "Methods of utilizing induced mutation in crop improvement", Mutation and Plant Breeding,

NAS-NRC Publ. 891, Washington, D.C. (1961) 336.[11] MacKEY, J., Mutagenesis in vulgare wheat, Hereditas 59(1968) 505.[12] GREGORY, W.C., "Induction of useful mutations in the peanut”, Genetics in Plant Breeding,

Brookhaven Symp.Biol. 9 (1956) 177.[13] SCOSSIROLI, R.E., PALENZONA, D., RUSMINI, B., "Radiation experiments on Triticum durum and T.

vulgare”, Effects of Ionizing Radiations on Seeds (Proc.Conf. Karlsruhe, 1960), IAEA, Vienna (1961) 373.[14] SCOSSIROLI, R.E., PELLEGRINI-SCOSSIROLI, S., "Use of radiation applied to seed to induce new genetic

variability for quantitative traits in durum wheat" ( Symp. Genetics and Wheat Breeding, Martonvásár, Hung.), Agrie.Res.Inst., Hung. Acad.Sci. (1962) 231.

[15] GAUL, H., MITTELSTENSCHEID, L., Untersuchung zur Selektion von Kleinmutationen bei Gerste, Z. PflZiicht. 45(1961) 300.

[16] GAUL. H., "Induced mutations in plant breeding", Genetics Today (Proc.XI Int.Congr.Genet., TheHague, 1963) (1964) 689.

[17] SWAMINATHAN, M.S., "Evaluation of the use of induced micro-and macro-mutations in the breeding of polyploid crop plants", Symp. Applic.Nucl. Energy in Agrie. Rome, 1961(1963) 241.

[18] BOROJEVIC, Katarina, "Genetic advance in the height of plants induced by irradiation in wheat".Genetics Today (Proc.XI Int.Congr. Genet., The Hague, 1963) (1964).

[19] BOROJEVIC, Katarina, "The effect of irradiation and selection after irradiation on the number of kernels per spike in wheat", The Use of Induced Mutations in Plant Breeding (Rep.FAO/IAEA Tech. Meeting,Rome, 1964), Pergamon Press, Oxford (1965) 504.

[20] BOROJEVIC, Katarina, "Studies on radiation-induced mutations in quantitative characters of wheat (Triticum vulgare)" Mutations in Plant Breeding (Proc. Panel. Vienna, 1966), IAEA, Vienna (1966) 15.

[21] GOUD, J. V., Selection experiments for quantitative characters in wheat after treatment with mutagens, Genet.Agr. 22(1968) 2.

[22] DUMANOVIC, J., DENIC, М., EHRENBERG, L., BERGSTRAND, K.G., Radiation-induced heritable variation of quantitative characters in wheat, Hereditas 62(1969) 221.

M UTATION BREEDING IN WHEAT 2 4 9

[23] TRUJILLO, F.R., Indirekte Friihselektion auf induzierte genetische Variabilitát in Ertragsmerkmalen nach EMS-Behandlung von Weizen, Z.PflZiicht. 60 (1968) 327.

[24] BOROJEVlC, Katarina, BOROJEVIC, S., "Response of different genotypes of Triticum aestivum ssp. vulgare to m utagenic treatments", Mutations in Plant Breeding II(Proc.Panel Vienna, 1967), IAEA,Vienna (1968) 15.

[25] BOROJEVIC?, Katarina, Genetic changes in quantitative characters of irradiated population, Jap. J. Genetics, Suppl. 1. 44(1969) 404.

[26] BOROJEVIC, Katarina, BOROJEVIC, S., "Stabilization of induced genetic variability in irradiated populations of vulgare wheat", Induced Mutations in Plants (Proc.Symp.Pullman, 1969), IAEA, Vienna(1969) 399.

[27] FAO/IAEA, Manual on Mutation Breeding (Tech. Rep. Ser. No. 119), IAEA, Vienna (1970).[28] SEARS, E.R., "The systematics, cytology and genetics of wheat”, Handbuch der Pflanzenziichtung,

2nd Edn, Parey, Berlin (1959) 164.[29] BOROJEVIC, Katarina, Genetic changes induced by irradiation in the Triticum species, Zbom.Matice

Srp. 26 (1964) 41.[30] JAGATHESAN, D.C., BHATIA, C., SWAMINATHAN, M.S., Effect of induced awn mutations on yield in

wheat, Nature 190(1961) 468.[31] SWAMINATHAN, M.S., "Mutational analysis of the hexaploid Triticum complex", Proc. 2nd Int. Wheat

Genet. Symp., Lund (1963) 418.[32] BOROJEVIC, K.. "Study of quantitative characters of bearded mutation in wheat induced by irradiation",

Induced Mutations and their Utilization, Proc. Symp.Erwin-Baur-Gedâchtnisvorîesungen IV, Gatersleben, 1966, Akademie-Verlag, Berlin (1967) 199.

[33] KHVOSTOVA, V.V., MOZHAEVA, V.S., CHERNY, I.V., Experimental mutagenesis in wheat,Genetics (USSR) 5 11 (1969) 178.

[34] KONZAK, C.F., "Induction of mutations for disease resistance in cereals", Genetics in Plant Breeding, Brookhaven Symp.Biol. 9 (1956) 157.

[35] FAVRET, E.A., CENOZ, H.P., SILVERO SANZ, O.I., SOLARI, R.M., MUJíCA, F.L., "Efecto de posicion inducido en trigo para reacción a las royas", Induced Mutations in Plants (Proc. Symp. Pullman, 1969), IAEA, Vienna (1969) 123.

[36] SEARS, E.R., "The transfer of leaf-rust resistance from Aegilops umbellulata to wheat", Genetics in Plant Breeding,Brookhaven Symp.Biol. 9 (1956) 1.

[37] KNOTT, D.R., "The inheritance of stem rust resistance in wheat", Proc.2nd Int. Wheat Genet.Symp., Lund (1963) 156.

[38] DRISCOLL, C.J., "Alien transfer by irradiation and meiotic control',' Proc.3rd Int. Wheat Genet. Symp.,Canberra (1968) 196.

[39] SAVCENKO, N.T., LASTOVlC, A.S., Morphological and cytological features of wheat with cytoplasmic male sterility, Ukr.bot.Zh. jï2 1 (1965) 35.

[40] KRUPNOV, V.A., Genetic male sterility in common wheat, Genetics (USSR) 4 10 (1968).[41] EKBERG, I., Different types of sterility induced in barley by ionizing radiations and chemical mutagens,

Hereditas 63 (1969) 257.[42] GAUL, H., BENDER K., ULONSKA, E., SATO, М., "EMS-induced genetic variability in barley; the

problem of EMS-induced sterility; and a method to increase the efficiency of EMS treatment”, Mutationsin Plant Breeding (Proc. Panel Vienna, 1966), IAEA, Vienna (1966) 63.

[43] YAMAKAWA, K., Induction of male-sterile tomato mutants by gamma-ijradiation, Tech.News, No.3, Inst.Radiat.Breeding, Japan(1969).

[44 ] BOROJEVIC?, Katarina, Rep.Dep.Genet., F a c .A g rie ., Novi Sad, Yugoslavia, 1969(unpublished).[45] SWAMINATHAN, M.S., "Role of mutation breeding in a changing agriculture". Induced Mutations in

Plants (Proc. Symp. Pullman, 1969), IAEA, Vienna (1969) 719.[46] DENICÍ, М., DUMANOVlC, J., EHRENBERG, L., Induced variation of protein content and composition in

wheat, Contemp.Agrie., Novi Sad 11 (1969) 85.[47] DUMANOVIC, J., EHRENBERG, L., DENICÍ, М., "Induced variation of protein content and composition

in hexaploid wheat", Improving Plant Protein by Nuclear Techniques (Proc. Symp.Vienna, 1970), IAEA, Vienna (1970) 107.

[48] SOLARI, R.M., FAVRET, E.A., Genetic control of protein constitution in wheat endosperm and its implication on induced mutagenesis, Mutations in Plant Breeding II (Proc. Panel Vienna, 1967), IAEA,Vienna ( 1968) 219.

[49] SENBORN, B., BOROJEVIC Katarina, The possibility of improving flour quality by irradiating wheat grain and of inducing changes in biological properties in the Rx generation, Contemp. Agrie., Novi Sad 5(1963) 349.

250 BOROJEVl(j and BOROJEVIC

[50] SCARASCIA-MUGNOZZA, G.T., "Induced mutations in breeding for lodging resistance", The Use of Induced Mutations in Plant Breeding (Rep.FAO/IAEA Tech. Meeting, Rome, 1964), Pergamon Press,Oxford (1965) 537.

[51] SCARASCIA-MUGNOZZA, G.T., BAGNARA, D., BOZZINI, A., "Mutations induced in durum wheat and their significance in genetics and breeding”, Proc.3rd Int. Wheat Genet. Symp., Canberra (1968) 357.

[52] BOGYO, T.P., SCARASCIA-MUGNOZZA, G.T., SIGURBJORNSSON, B., BAGNARA, D., "Adaptation studies with radiation-induced durum wheat mutants", Induced Mutations in Plants (Proc.Symp.Pullman, 1969), IAEA, Vienna (1969) 699.

[53] SIGURBJORNSSON, B., MICKE, A., "Progress in mutation breeding". Induced Mutations in Plants (Proc. Symp.Pullman, 1969), IAEA, Vienna (1969) 673.

[54] BOZZINI, A., AVANZI, S., Solid stem: a radiation-induced mutation in Triticum durum.Desf, Caryologia 25 3(1962) 525.

[55] BOZZINI, A., "Sphaerococcoid, a radiation-induced mutation in Triticum durum", The Use of Induced Mutations in Plant Breeding (Rep.FAO/lAEA Tech. Meeting, Rome, 1964), Pergamon Press, Oxford (1965) 374.

[56] WOO, S.C., KONZAK, C.F., "Genetic analysis of short-culm mutants induced by ethyl methane sulphonate in Triticum aestivum L.", Induced Mutations in Plants (Proc. Symp.Pullman, 1969), IAEA, Vienna (1969) 551.

[57] SCOSSIROLI, R.E., Use of induced genetic variability for quantitative traits after seed irradiation in Triticum durum, Contemp.Agrie., Novi Sad 14(1966) 191.

[58] GAUL, H-, AESTVEIT, K., Induced variability of culm length in different genotypes of hexaploid wheat following X-irradiation and EMS-treatment,Contemp.Agrie., Novi Sad 14(1966) 227.

[59] BAGNARA, D.A., A diallel analysis of quantitative characters in varieties and radio-induced mutant lines of Triticum durum, Genet.agr. 21 (1967) 313.

[60] TAVCAR, A., KENDJELIC, V., Use of valuable radiation mutations in some ecotypes of Triticum turgidum for combination breeding with Triticum aestivum ssp. vulgare, Contemp.Agrie., Novi Sad 14(1966) 175.

[61] GUSTAFSSON, A., GADD, I., Mutations and crop improvement. VII. The genus Oryza L. (Gramineae), Hereditas 55(1966) 273.

[62] GAUL, H., Über die Chimárenbildung in Gerstenpflanzen nach Rontgenbestrahlung von Samen, Flora147 (1959) 207.

[63] GREGORY, W.C., "Mutation frequency, magnitude of change, and the probability of improvement in adaptation", The Use of Induced Mutations in Plant Breeding, Suppl.Radiat.Bot. 5(1965) 429.

[64] BOROJEVIC, S., POTOCANAC, J., The development of the Yugoslav Programme for creating high-yield wheat varieties, Contemp.Agrie., Novi Sad 14(1966).

D IS C U S S IO N

M .S. SWAMINATHAN: It seems to me that one important lesson weshould learn from the lack of practical utility of very interesting lines like "T ran sfe r" is that Chinese Spring, in the genetic background of which such translocation stocks were developed, is characterized by a very poor combining ability. It would be advisable to use varieties known to have a good combining ability in the development of such chromosomal and genetic breeding stocks.

K. BOROJEVIC: I completely agree.G .T . SCARASCIA-M UGNOZZA: With reference to the studies carried

out by Prof. TavSar on the root system of mutants from Triticum turgidum1, I would like to know whether he used a random sample of induced mutants or not. I think that in radiogenetical studies the use of large samples taken at random is necessary; on the contrary, in mutation breeding studies we should analyse an induced change in agronomically important traits in a

1 Ref. [60] of the paper.

M UTATION BREEDING IN WHEAT 2 5 1

group of mutants which are "useful" or which have a potential agronomical value, that is, free from detrimental mutations or chromosomal changes, e .g . monosomy.

K. BOROJEVIC: Prof. TavCar took mutant lines from collections whichwere isolated from different varieties.

I. RAM IREZ ARAYA: With respect to the choice of parents to start amutation breeding project, I agree that one has to start with the best germ - plasm available, be it of local origin or introduced. But for regions where highly specialized varieties or genotypes are needed to achieve maximum productivity, one has to test first the introduced material for adaptability and response to specific characteristics (like disease resistance, quality, etc. ) before deciding if they are suitable as parents to initiate a mutation breeding project.

K. BOROJEVIC: Yes, because the mother variety has to be chosen according to the aim of the plant breeders.

G. T. SCARASCIA-M UGNOZZA: I would like to ask you if you haveisolated completely or partially m ale-sterile individuals and, in the case you have found complete m ale-sterile mutants, what was the genetical behaviour of such mutations? Are they always associated with chromosomal aberrations?

K. BOROJEVIC: They were associated with chromosomal aberrations.W . GOTTSCHALK: You mentioned male sterility in wheats as a con

sequence of chromosomal aberrations. Is this an assumption or could it already be evidenced? In tomatoes and some other species (for instance in peas), a large number of m ale-sterile mutants have been carefully analysed cytogenetically, and in many cases male sterility was due to single recessive genes.

K. BOROJEVIC: Most of the male sterility I mentioned was due to chromosomal aberrations.

MUTATIONS AND PHYSIOLOGICAL REACTION TO SEVERAL CHEMICAL MUTAGENS IN PEANUTS, Arachis hypogaea L.

A. ASHMThe Hebrew University, Faculty of Agriculture,Rehovot, Israel

Abstract-Resumen

MUTATIONS'AND PHYSIOLOGICAL REACTION TO SEVERAL CHEMICAL MUTAGENS IN PEANUTS.Arachis hypogaea L.

Studies with chemical mutagens in several varieties of peanuts are described. Diethyl sulphate induced single-trait and pleiotropicmutations, but at a higher rate in the variety that was less sensitive physiologically. The macromutants were monogenic; one was dominant and all others were recessive or partially dominant.In some cases mutations in the background polygenic genotypes were apparently induced. Physiological sensiti' vity studies showed that one variety was consistently more sensitive than the other varieties to the chemical mutagens tested: EMS, EB, acriflavine, chloramphenicol, erythromycin, 5-BU, and MNNG. The utilization of chronic chemical mutagenesis in peanuts for the induction of nuclear and plasmon mutations is discussed.

MUTACIONES Y REACCIONES FISIOLOGICAS FRENTE A DIVERSOS MUTAGENOS QUIMICOS EN LOS CACAHUETES, Arachis hypogaea L.

El autor describe estudios efectuados con mutágenos químicos en distintas variedades de cacahuetes.El sulfato de dietilo induce mutaciones de un solo carácter y pleótropas, pero la proporción más elevada se da en la variedad menos sensible fisiológicamente. Los macromutantes son monogénicos; uno dominante y los restantes recesivos o parcialmente dominantes. En algunos casos, se han inducido al parecer mutaciones en los genotipos poligénicos de fondo. Los estudios de la sensibilidad fisiológica ponen de manifiesto que una variedad es regularmente más sensible que las otras a los mutágenos químicos ensayados: MSE, BE, acriñavina, cloranfenicol, eritromicina, 5-BU y MNNG. El autor examina el empleo de la mutagénesis química crónica en el cacahuete para inducir mutaciones en el núcleo y en el plasma.

1. INTRODUCTION

Plant breeding is concerned with the creation, identification, isolation, multiplication and management of genetic variability towards improved cultivars. Mutation breeding is an integral part of plant breeding and is another method for the creation of genetic diversity for further selection and hybridization. Plant breeding efforts have made in recent years very significant contributions to the "green revolution". This was done partly by emphasizing the comprehensive approach, encompassing the plant and its environment in a closely knit system [1]. Mutation breeding which has become of age in recent years has yielded increasing numbers of desirable cultivars as shown by several recent reviews [ 2-5] . This was at least partly due to the fact that mutation breeding became m ore closely integrated with plant breeding efforts as a whole.

In Latin Am erica lie the centres of origin and variability of many of our most important crops, e .g . maize, potatoes and peanuts, A rachis hypogaea L. Peanuts probably originated in Bolivia and the Guarani region including north-east Argentina [ 6 ]. The crop is an important source of oil, protein-rich meal, peanut butter, edible nuts, etc. The w orld 1 s production of peanuts in 1968 came to 15 million tons, grown on 17. 6 million hectares

25 3

25 4 ASHRI

[7 ]. Thus by their importance they qualified for intensified breeding and mutation breeding. The peanut is one of the few crops which is a dicot and which was exposed extensively to mutation breeding.

Peanuts are suitable for mutation breeding studies for several reasons: they are practically self-pollinated (although instances of cross-pollination have been noted [ 8 ]); the embryos are easily exposed to mutagens; the embryos have six to eight leaf primordia with buds in their axils [9], making several potential targets available. The disadvantages are that the peanut is an amphiploid having 2n=40 chromosomes which are very small and lack suitable m arkers.

The work of Gregory and his associates with radiation [10-15] exemplifies well the value of integrating mutation research and plant improvement. They have extended the experience with mutation work aimed at plant im provement by: being among the first to search for and find mutation in polygenic systems which are so important for breeding better cultivars; being able to obtain a variety of macromutants and study their inheritance; obtaining a successful selection released as N. C .4 -X . Prom ising results were also obtained from irradiating peanuts in West A frica [16].

With this background and certain breeding needs in the peanut varieties grown in Israel, it was decided in 1961 to employ mutation breeding in addition to more classical methods. In view of reports [17] on the mutagenic efficiency of diethyl sulphate (dES), it was decided to use it instead of radiations.

2. M UTATION RATES WITH dES

Some of the findings were published [18] and therefore only a summary of the salient points w ill be given here. The local varieties "V irginia Sihit Meshubahat" (VSM) and "Dixie Anak" (DA) were utilized. The first re presents the Virginia type and the second the Spanish type of peanuts [9].The mutagen solution was prepared according to the information then available [18]: 15 m l dES per litre of distilled water were hydrolyzed for 90 min at 20°C and then the seeds were submerged in it. Lots of 100 seeds were soaked for 5, 15 and 25 min, rinsed and space planted in the field. P r e liminary studies showed that the longer treatments utilized in barley [18] were lethal.

Even at the low exposures employed there was a difference in survival (emergence) between the two varieties: 80-90% in VSM and 30-60% in DA. The difference was rea l as shown by further tests (see below). Furtherm ore, in VSM, a high rate of mutations was found which increased with longer treatments. However, even these were short — 25 min only. The results are shown in Table I. It is noteworthy that the frequency of pleiotropic mutants increased with treatment time. These mutants could be due to single gene mutations affecting a complex pathway or hormone balance, or to chromosomal deletions which can occur following treatment with alkylating agents [19-21]. The single trait mutants affected various traits are shown in Table II. There were many mutations affecting plant habit and size, only few chlorophyll mutations and some sterility mutations. The frequency of the mutations was much higher than would have been estimated from chlorophyll mutations. Two beneficial, thin-shell mutants were found. One of them was high-yielding, not only in Israe l but also in tests in North

M UTATIONS IN PEANUTS 255

T A B LE I. NUM BER AND FREQUENCY PER BUD (%) OF SINGLE TRAIT AND PLEIO TR O PIC MUTATIONS INDUCED BY dES IN THE P E A N U T VARIETY VSM

Mutationsaffecting 5

Mutations/treatment (min)

15 25Total

Single trait:Number 20 17 28 65

Frequency 3.48 3.24 5.50 -

Pleiotropic:2 traits 3 10 4 17

3 traits 2 5 6 13

4 traits 0 2 2 4

Total 5 17 12 34

Frequency (°jo) 0.87 3.24 2.35 -

Grand totalNumber 25 34 40 99

Frequency (%) 4.35 6.48 7.85 -

Carolina, USA [Em ery, personal communication]. However, this selection was not released because a variety with a shorter growing period was produced through classical breeding methods and it superseded VSM and its selections. Mutants which could be beneficial are those with sm aller plants in which the pods retain their normal size. By cultivating such plants in denser stands, yields could probably be improved.

In DA there were fewer segregating mutants (Table III) and a ll but one were pleiotropic. There was a higher frequency of mutants in the lighter (5 min) treatment. These two observations, coupled with the higher physiological sensitivity of DA (see below), indicate that mutations were probably lost in the more severe treatments.

3. INHERITANCE OF M ACROMUTANTS

Many of the VSM and DA mutants which were followed in the M3 and M 4 generations gave monogenic segregation ratios. The inheritance of eight VSM macromutants (Table IV) was studied also by crossing them with the original parent variety [22]. The studies showed that dES induced polygenic mutations simultaneously with macromutations. Five mutants were inherited mono geni cally with the mutant allele being fully recessive (Table IV). In the F 2 of open habit! there were extreme, stunted and sterile plants which could have resulted perhaps from segregation of mutated m odifiers in the background genotype [12,13]. In the spherical mutant the intermediate F 2

plants could also result from mutated background genotypes. In the F 2

2 5 6 ASHRI

T A B LE II. SINGLE TRAIT MUTATIONS INDUCED B Y dES IN THE PE A N U T VAR IETY VSM CLASSIFIED BY THEIR EFFECTS



15 25Total

Leaflets 2 1 - 3

Chlorophyll 2 3 5 10Habit and size 13 6 14 33Fertility 1 2 2 5Pods 2 5 5 12Testa - - 2 2

Total 20 17 28 65

T A B L E III. NUM BER AND FREQUENCY PER BUD (%) OF SEGREGATING SINGLE TRAIT AND PLEIO TRO PIC MUTATIONS INDUCED B Y dES IN THE PEANUT VAR IETY DA



15 25Total

1 trait 0 0 1 1

2 traits 1 1 1 3

3 traits 5 1 0 6

4 traits 2 0 0 2

4+ traits 0 1 0 1

Total 8 3 2 12

Frequency (°Jo) 2.00 0.88 1.02 -

of dwarf, shortage of homozygous mutant and heterozygous plants could have resulted from reduced transmission of the dw3 allele through the pollen only or through both the pollen and the eggs. This could arise from chromosome aberrations associated with the mutant, as shown by Gaul [23].

A most interesting dominant mutation with variable penetrance and expressivity was found in the Mi of DA [24]. Heterozygous plants were either extremely diminutive, dwarfed and leafletless or intermediate or mixed, i .e . they started as diminutive and then produced some perfectly normal branches. It was shown that this change was not accompanied by a genotypic change, thus it was not a paramutation [25]. The phenotypic reversion to normal also did not fit all the requirements for "phase change" [26] . The mutation which probably affects the gibberellin levels in the plant can be used to study the environmental factors affecting hormone balance and output in the plant, as was done by Favret et al. in barley [27].


T A B LE IV. F 2 SEGREGATIONS AND GOODNESS OF F IT IN CROSSES OF dES-INDUCED M UTANTS X VSM PAR ENT

MutantNormal

Number of F2

Intermediate

plants

Mutant TotalP

for 3:1

Two phenotypic classes:Virescent 64 - 14 78 0.20 - 0.10Xanthamaculata, 2 57 - 20 77 0.90 - 0.80

л a Dwarf, 61 - 16 77 0.50 - 0.30Dwarf? 59 - 22 81 0.70 - 0.50Open habit? 35 - 19 54 0.10 - 0.05

Three phenotypic classes:Open habit, 62 - 18b 80 0.70 - 0.50Dwarf,, 88 88 20 196 -Spherical 48 17° 16 81 0.30 - 0.20

a Dihybrid ratio of xa, and dwt, P(9'.3: 3:1) = 0.95 - 0.90.13 Includes three extreme plants which produced only a few aborted seeds.c These plants were spherical but intermediate in plant and pod sizes. In x2 calculation grouped

with normal.

4. D IFFERENCES IN PHYSIOLOGICAL SENSITIVITY BETW EEN VARIETIES

With a view toward prolonged chronic treatments with chemical mutagens and in order to better understand mutation rate differences between varieties, the differences in physiological sensitivity between varieties were studied.In repeated tests with dES it was shown that DA was markedly more sensitive than VSM and Congo (a local variety of the Valencia type). The results of one test are shown in Table V. The dES solution was freshly prepared and used without prior hydrolysis. DA was extremely sensitive and even the relatively more tolerant Congo was far more sensitive than barley.The fact that in peanuts the mutagen solution can reach the embryo directly very rapidly probably leads to the higher sensitivity. There were two sharp drops in survival in both varieties but in Congo they occurred after longer treatments. The more severely affected samples also germinated more slowly. However, leaf flecking was not observed. Because up to approximately 21 days after planting there is only cell enlargement [9] , chlorophyll mutations induced should have been seen, although single cell mutants could not be detected visually.

Presoaking increased the sensitivity of both varieties (Table VI) but very markedly so in DA. The 6 h presoak period was apparently more critical to survival than longer periods and could probably be related to a specific metabolic phase.

In tests with EMS again DA was more sensitive than Congo and VSM. EMS produced leaf flecks in both varieties. DA was far more sensitive than Congo, also to ethidium bromide (EB) and acriflavine (Tables VII,VIII).

258 ASHRI

T A B LE V . MEANS (%) AND STANDARD ERRORS FOR GERMINATION OF SEEDS OF TWO PE A N U T VARIETIES SOAKED FOR VARIOUS PERIODS IN dES SOLUTION, 17 DAYS A FT E R PLA N T IN G 3

VarietiesSoaking period

0 96.6 ± 3.5 a 99.3 ± 3.5 a5 93.3 ± 8.0 a 99.3 ± 3.5 a15 58.0 ± 16.0 b 85.3 ± 5.3 b30 16.6 ± 14.0 с 60.6 ± 11.3 с45 4.0 ± 6.0 d 23.3 ± 16.0 d60 3.3 ± 4.5 d 10.0 ± 8.1 e75 2.0 ± 3.3 d 4.6 ± 3.9 e90 0 d 2.0 ± 2.3 e

a Solution freshly prepared and not hydrolysed, 15 ml dES/1 litre H20; changed every 30 min, unbuffered.In each treatment 5 replications of 25 seeds each,

k Within varieties, values differing at the 0.1 °}o level are designated by different letters. Between varieties significantly different (O.l o) values are not underlined.

T A B LE VI. MEANS (%) AND STANDARD ERRORS FOR GERMINATION,17 DAYS A FT E R PLANTING , OF SEEDS OF TWO PEANUT VARIETIES SOAKED IN dES FOR 15 min AFTER VARIOUS PERIODS OF PRESOAKING IN DEIONIZED W A T E R 3

Presoak(h) dES

DA

Varieties

Congo

0 - 92.8 i 5. 5 a 94.0± 5.0 a0 + 83.6 ± 5.2 b 92. 0 ± 8.3 a6 + 29.6 ± 3.2 с 78.8± 8.6b12 + 40. 8 i 7.0 c 84.4 ± 10.8 ab18 + 44. 4 ± 5. 5 с 77.6± 7.6b24 + 44.6 ± 6.6 с 78.0 ± 22.4 b

a Solution as in Table V. In each treatment 5 replications of 50 seeds each. b Significance level 0.1 °jo, letters and underlining as in Table V.


260 ASHRI

T A B LE V n i. M EAN GERMINATION, 17 DAYS AFTER PLANTING , OF SEEDS OF THE DA VAR IETY OF PEANUTS, TREATED WITH EB AND AC R IFLAVINE a

Mean germination No. of {% of respective water soaking control)seedlings,

water control EB Acriflavine(20 mg/1) (20 mg/1)

12 41 80.4 9.7

24 53 73.5 18.8

48 25 88.0 16.090 39 69.2 23.0

144 45 82.2 57.7

a In each treatment there were 5 replications of 25 seeds each.

T A B LE IX. A COMPARISON OF SURVIVAL OF OVARIES A TD IFFER ENT STAGES OF M ATURITY AFTE R TREATM ENTSWITH SEVER AL M UTAGENS (POOLED DATA OF SEVERAL VARIETIES)

Chemical

Treatment

Concentration No. of days Maturity stage a

OvariesNumber

Treated Survived

Erythromycin 500 mg/1 12 1 40 14

500 mg/1 12 4 73 72

Ethidium bromide 20 mg/1 7 1 33 32

50 mg/1 10 1 52 34

50 mg/1 15 1 27 16

Chloramphenicol 500 mg/1 8 1 50 26500 mg/1 14 1 60 21

500 mg/1 14 4 12 12

EMS 0.4 vol.°Jo 2 1 34 3

0.4 vol. o 2 4 14 5

0.2 vol.°}o 6 1 33 9

Acriflavine 30 mg/1 13 1 117 64

30 mg/1 13 3 31 30

30 mg/1 15 1 43 31

30 mg/1 15 3 12 12

Rated on a scale of 1 * 5, with 1 being the youngest and 5 being a mature pod.

Soaking period (h)

M UTATIONS IN PEANUTS 26 1

The consistently higher sensitivity of DA to dES, EMS, 5-BU, N -m ethyl- N' -nitro-nitrosoguanidin (M NNG), chloramphenicol, erythromycin, EB, and acriflavine could result from differences in permeability between varie ties. However, the underlying mechanism responsible remains tobe studied. Breeding experiments a re now under way in order to study their genetic control. It is hoped that findings at the varietal level w ill assist in c la r ifying the causes for differences in sensitivity at the species and higher levels.

5. CHRONIC TREATM ENTS WITH CHEM ICAL MUTAGENS

Chronic mutagenic treatments have been employed with radiation [28] but with chemical mutagens their utilization was limited. Developing peanut ovaries are ideally suited for such studies. The ovules and ovaries begin to develop only after they are inserted into the ground by the gynophore. Development lasts six to eight weeks and during that period there is some direct mineral uptake by the ovaries (pods). These attributes can be used to advantage in subjecting the developing ovaries and embryos to prolonged mutagenic treatments. Prelim inary sensitivity tests were conducted in 1969 with EMS and EB and further tests in 1970. A summary of the 1970 findings is shown in Table IX. The youngest ovaries (stage 1) were far more sensitive than the more mature ones. A lso, some mutagens (e .g . EMS) were m ore severe than others. It is hoped that mild, prolonged, chronic treatments w ill produce higher yields of mutations and possibly modify their spectrum and induce also plasmon alterations.

T A B LE X . SCHEME FOR SCREENING FOR POSSIBLE PLASM ON AND N U C LE A R GENE M UTATIONS IN THE PEANUT GROWTH HABIT SYSTEM

Gene mutation:M t [ 0] Hbx Hbj Hbg Н1%* Runner

;M 2 Variability, runner vs. bunch (... ht hbg)

IM 3 Mendelian segregation ratios from heterozygous M 2 plants, 3 runner: 1 bunch

Plasmon mutation:Mi [0*] HbiHbiH^Hbj Runner

;M 2 [mx ] Hbj Hbj Hbz Hbg bunch, some or all

IM 3 No further segregation from bunch and runner M 2 plants

Plasmon mutation:Mi [ 0*] hbx hbj НЬг Bunch

IM 2 [m2 ] hbjhbi Hbj НЬг Runner, some or all of the plants

;M 3 No further segregation from bunch and runner M 2 plants

262 ASHRI

Growth habit in peanuts (whether erect or trailing) is controlled by nuclear genes interacting with each other and with the plasmons [29, 30].This mechanism operates through modification of gibberellin inhibitors [31] which require light for induction. This peanut system is well suited for testing for plasmon mutations: the growth habit phenotype can be determined at the age of five to seven weeks, the differences in growth habit do not affect seed set, and self-fertilization is the rule in peanuts. A scheme for identifying such mutants is shown in Table X.

Encouragement is derived from the fact that three plasmons have a lready been identified in a limited survey of the germ plasm ([30], and Ashri, unpublished data). A la rge -sca le search for additional plasmon types is being conducted at present. Such natural divergence, which is known also in other crops [32], might have arisen through plasmon alterations. It is hoped that sim ilar alterations can be induced by prolonged treatments at low concentrations of developing embryos and seeds. Mayer and Simpson [33] showed in rat liver that mitochondrial DNA polymerase is much more sensitive to ethidium bromide and acriflavine than nuclear DNA. They advocated using low concentrations for differential mutagenic action on the cytoplasmic components. Prolonged treatments with low concentrations of chemical mutagens can also expose the nuclear DNA to their action during one complete replication cycle or more. Peanut embryos are ideally suited for prolonged chronic chemical mutagenesis. However, it seems worthwhile to extend evaluation of this approach to other crops. Chronic treatment with low concentrations may lead to higher mutation yields with fewer undesirable side effects.

R E F E R E N C E S

[1] BORLAUG, N.E., Wheat, rust and people. Phytopathology 55 (1965) 1088.[2] SIGURBJORNSSON, B., MICKE, A., "Progress in mutation breeding". Induced Mutations in Plants

(Proc. Symp. Pullman. 1969), IAEA. Vienna (1969) 673.[3] SIGURBJORNSSON, B., "Mutations in plant-breeding programs", Manual on Mutation Breeding (Tech.

Rep. Ser. 119), IAEA, Vienna (1970) 1.[4] SWAMINATHAN, M.S., "Role of mutation breeding in a changing agriculture". Induced Mutations

in Plants (Proc. Symp. Pullman, 1969), IAEA, Vienna (1969) 719.[5] ASHRI. A., "Recent progress and future perspectives in the utilization of induced mutations”, Proc.

Seminario Avanzado en Genetica Agricola para America Latina, Maracay, Venezuela, 1969 (in press).[6] KRAPOVICKAS, A., "The origin, variability and spread of the groundm/t (Arachis hypogaea)'',

The Domestication and Exploitation of Plants and Animals (UCKO, P.J., DIMBLEBY, G.W., Eds), Gerald Duckworth, London (1969) 427.

[7] FAO Production Yearbook 1969. 23 (1970) 233.[8] HAMMONS, R.O., Krinkle, a dominant leaf marker in the peanut, Arachis hypogaea L., Crop Sci. 4

(1964) 22.[9] GREGORY, W.C., SMITH, B.W., YARBROUGH, J.A., "Morphology, Genetics and Breeding", The

Peanut — A Symposium, The Natn. Fertilizer Ass., Washington, D.C. (1951) 28.[10] GREGORY, W.C., "Induction of useful mutations in the peanut", Genetics in Plant Breeding, Proc.

Brookhaven Symp. Biol. 9 (1956) 177.[11] GREGORY, W.C., "Mutation breeding". Plant Breeding (FREY, K.J., Ed.), Iowa State Univ. Press,

Ames, Iowa (1966) 189.[12] GREGORY, W.C., "A mutation breeding experiment with peanuts", Radiat. Bot. 8 (1968) 81.[13] EMERGY, D.A., GREGORY, W.C., LOESCH, P.J., Jr., Breeding value of the X~ ray induced macro~

mutant. I. Variations among normal appearing F2 families segregated from crosses between macromutants of peanuts (Arachis hypogaea L.), Crop Sci. 4 (1964) 87.


[14] LOESCH, P.J., Jr., Effect of mutated background genotype on mutant expression in Arachis hypogaea L., Crop Sci. 4 (1964) 73.

[15] GUSTAFSSON, À., GADD, I., Mutations and crop improvement. V. Arachis hypogaea L. (Leguminosae), Hereditas 53 (1965) 143.

[16] BILQUEZ, A.F., MAGNE, C.t MARTIN, J.P., "Bilan de six années de recherches sur l'emploi des rayonnements ionisants pour 1' amélioration des plantes au Sénégal", The Use of Induced Mutations in Plant Breeding, Suppl. Radiat. Bot. _5 (1965) 585.

[17] HEINER, R.E., KONZAK, C.F., NILAN, R.A., LEGAULT, R.R., Diverse ratios of mutation to chromosome aberration in barley treated with diethyl sulfate and gamma rays, Proc. natn. Acad. Sci. U.S.A.46 (1960) 1215.

[18] ASHRI, A., GOLDIN, E., The mutagenic activity of diethyl sulfate in peanuts, Radiat. Bot. j> (1965)431.

[19] LIFSCHYTZ, E., Gene structure and chromosome organization in Drosophila melanogaster, Dissertation submitted in partial fulfilment of the requirements for the Ph. D. degree to the Hebrew University, Jerusalem, Israel (in Hebrew , English summary) (1968).

[20] LOVELESS, A., Genetic and Allied Effects of Alkylating Agents, Butterworth, London (1966).[21] GAUL, H., "Cytological effects", Manual on Mutation Breeding (Tech. Rep. Ser. 119), IAEA, Vienna

(1970) 90.[22] SHCHORY, Y., ASHRI, A., Inheritance of several macromutations induced by diethyl sulfate in peanuts,

Arachis hypogaea L., Radiat. Bot. _10 (1970) 551.[23] GAUL, H., Mutations in plant breeding, Ra.diat. Bot. 4 (1964) 155.[24] ASHRI, A., A dominant mutation with variable penetrance and expressivity induced by diethyl sulfate

in peanuts, Arachis hypogaea L., Mutation Res. 9 (1970) 473.[25] BRINK, R.A., Genetic repression in multicellular organisms, Am. Nat. j)8 (1964) 193.[26] BRINK, R.A., "Genetic repression of R action in maize", The Role of Chromosomes in Development,

Academic Press, New York (1964) 183.[27] FAVRET, E.A., RYAN, G.S., MALVAREZ, E.M., "Mutaciones inducidas que afectan al crecimiento

inicial de la cebada", Induced Mutations in Plants (Proc. Symp. Pullman, 1969), IAEA, Vienna (1969) 109.

[28] BRIGGS, R.W., "Objects and methods of treatment", Manual on Mutation Breeding (Tech. Rep.Ser. 119), IAEA, Vienna (1970) 37.

[29] ASHRI, A., Intergenic and genic-cytoplasmic interactions affecting growth habit in peanuts, Genetics 50(1964) 363.

[30] ASHRI. A.. Genic-cytoplasmic interactions affecting, growth habit in peanuts, A. hypogaea. II. A revised model, Genetics 60 (1968) 807.

[31] HALEVY, A.H., ASHRI, A., BEN-TAL, Y., Peanuts; Gibberellin antagonists and genetically controlled differences in growth habit, Science 164 (1969) 1397.

[32] DUVICK, D.N., "Influence of morphology and sterility on breeding methodology", Plant: Breeding (FREY, K.J., Ed.), Iowa State Univ. Press, Ames, Iowa (1966) 85.

[33] MAYER, R.R., SIMPSON, M.V., Differential inhibition of mitochondrial and nuclear DNA polymerases by EB and acriflavine, Biochem. biophys. Res. Commun. 34 (1969) 238.

D I S C U S S I O N

R .D . BROCK: Does the differential sensitivity to alkylating agents you have demonstrated between the varieties DA and Congo apply with ionizing radiations?

A . ASHRI: We are about to try gam m a-rays.H. SMITH: In the case where a branch of a dwarfed mutant reverts

to a more normal phenotype — but shows the same heredity in progeny from mutant versus normal — would not the simplest explanation be that some change in gene activation has occurred?

A. ASHRI: It is possible.J. EREJOMOVICH: If, in the case of the dwarf plants which produce

highly developed branches, you plant seeds from the two types of branches,

264 ASHRI

what behaviour do you find in the following generation? Are there any genetic differences?

A . ASHRI: The differences are not genetic. Such plants are heterozygous for the mutation. From diminutive and from normal branches segregation of the same types and in the same frequencies is obtained. The types obtained are: normal, intermediate, diminutive and diminutive with normal branches.

W . GOTTSCHALK: Can you give us some details concerning the genetic background of the different sensitivity of some of your varieties against chemical mutagens? Is it due to a single gene comparable with the situation in the Pisum genome studied by Blixt in Sweden? Did you investigate this behaviour genetically?

Did you make any meiotic investigations in the giant sterile mutant you mentioned? What kind of sterility is involved?

A . ASHRI: We are going to examine the F 3 of the relatively tolerant Congo X the sensitive DA in the coming season. We hope to get an idea about the genetic mechanism then. It may be a single gene or two as you mention or a polygenic system.

Meiotic investigations have not been made yet. Pollen staining studies showed that the grains were viable.

G. DE A LB A : The percentage germinations given for the firstthree soaking periods in treatment EB (Table VII) are greater than for the control. How do you explain this?

A . ASHRI: F o r very short periods some mutagens have stimulating effects. This is very often found. We did not check if the stimulation is inherited. It doubt it.

G. DE A LB A : Sim ilar effects have been noted on germination andplant height of cereals when gam m a-rays were used.

STUDIES OF COMBINED TREATMENT OF Acer negundo L. SEEDS WITH GROWTH REGULATORS AND MUTAGENS*

G .F . PRIVALOV

Institute of Cytology and Genetics,Siberian Branch, USSR Academy of Sciences,Novosibirsk, USSR

Abstract-Resumen

STUDIES OF COMBINED TREATMENT OF Acer negundo L. SEEDS WITH GROWTH REGULATORS AND MUTAGENS.

The application of induced mutagenesis in forest tree breeding is seriously limited by a variety of factors, the main obstacle being the perennial developmental cycle of woody plants. Therefore, it would be of particular importance to have mutations induced preferably in economically-important characters like growth rate. An experiment was started in 1969 to test the hypothesis that active genes are more sensitive to mutagenesis than inactive ones. Various phytohormones supposedly affecting growth rate were applied to seeds in combination with various mutagens. First data on survival and seedling growth are presented.

ESTUDIOS SOBRE EL TRATAMIENTO COMBINADO DE LAS SEMILLAS DE Acer negundo L. CON AGENTES MUTAGENOS Y REGULADORES DEL CRECIMIENTO.

La aplicación de la mutagénesis inducida en fitotecnia forestal resulta gravemente limitada por una serie de factores, siendo el principal obstáculo el ciclo perenne de desarrollo de las plantas leñosas. Por consiguiente, tendrfa especial importancia inducir las mutaciones de preferencia en caracteres de importancia económica como es la velocidad de crecimiento. En 1969 se ha puesto en marcha un experimento al objeto de someter a prueba la hipótesis de que los genes activos son más sensibles a la mutagénesis que los inactivos. Se han aplicado a las semillas, en combinación con diversos mutágenos, diversas fitohormonas que se supone afectan a la velocidad de crecimiento. Se presentan los primeros datos relativos a supervivencia y crecimiento de las plántulas.

1. INTRODUCTION

The high efficiency of induced mutagenesis has been demonstrated in many species of woody plants [1, 2, 3]. However, the perennial developmental cycle in woody plants and a number of specific factors limit the application of this method in the selection of these plants for practical purposes.

It is known that in most of the cases, due to statistical regularities, mutations arise only at one of the two loci of the homologous chromosomes of diploid plants. As a result, mutations of two types arise:AA ** Aa or Aa?* aa. The simultaneous occurrence of mutations at two loci of the same gene (AA <2 aa) is an exceedingly rare event. However, the genetics of the characters of most woody plants of practical significance has not been studied so far and there are no reliable methods for differentiating heterozygous forms (Aa) from the homozygous (AA or aa)

* Part of the research reported in this paper has been carried out under Research Agreement withthe International Atomic Energy Agency No. 774/CF.

265

266 PRIVALOV

ones. It is impossible to develop a clear and adequate program of mutational selection in woody plants without data on the genetic structure of the initial material.

As a result of the treatment of seeds and other vegetative parts of plants (buds, cuttings), different mutations arise in various cells.

A complex structure of tissues and organs is formed, which makes it difficult to identify and isolate mutations in their pure line. The absence of rapid and dependable methods of detection of many somatic mutations make plant breeders uncertain whether they have finished creating a variety.

One of the means of overcoming the problems mentioned above in mutational selection of woody plants may be the regulation of the function of genes controlling characters of importance in selection. It has been suggested that actively functioning genes may be more sensitive to the effect of chemical mutagens than non-functioning or repressed genes [9]. Thus, the use of specific inductors capable of transferring certain genes from an inactive state to an active one, and conversely, from an active state to an inactive one might permit the inclusion of corresponding genes into the mutational process or to limit the emergence of undesirable mutations by repressing them.

To date four main groups of natural regulators of plant growth (phytohormones) have been identified, which play the role of inductors in target cells [4, 5, 6 , 7]. They include auxins, gibberellins, cytokinins and growth inhibitors (abscisin and others). The aim of the present work, started in 1969, was to study the frequency and spectra of somatic mutations brought about by the combined treatment of seeds of woody plants by growth regulators and mutagens. This communication concerns the first results obtained on the ash-leaved maple (Acer negundo L.) which in Siberian conditions is suitable for experimental mutagenesis studies.

2. M ATERIALS AND METHODS

The initial m aterial for this study was seeds of the ash-leaved maple (Acer negundo L .), from the chlorophyll mutant obtained in 1960 as a result of X -ray treatment of seeds. The fertility and viability of the seeds of this mutant were nearly normal. Since the plant was c ro ss - pollinated, the seeds chosen, for this study were probably heterozygous for the induced chlorophyll mutation, the genetic nature of which is still unknown. The following chemicals were used as mutagens: ethyl methane sulphonate (EMS) at a concentration of 0.5%; dimethyl sulphate (dMS),0.05 and 0.1%; X -ray s (5 kR). The following growth regulators were used: indolyl-3 acetic acid (IAA, auxin) at concentrations of 1.0, 0.1, 0.01 and 0.001%; gibberellin (GA), 1.0 and 0.1%; and adenine (Ad), 0.9%.Before treatment the seeds were soaked in water for 24 hours. The seeds were then soaked in mutagen or growth regulator solution for 6 hours. A fter each treatment with mutagen or growth regulator, the seeds were washed with tap water. In the field experiments, 400 seeds were used in each variant, and in greenhouse conditions 2 0 0 seeds in each variant. Seeds were sown in the fields on 15 May 1969, and in the

GROWTH REGULATORS AND MUTAGENS 267

TABLE I. SEEDLING SURVIVAL AFTER SINGLE AND COMBINED TREATM ENTS OF SEEDS WITH MUTAGENS (EMS, X -RAYS) AND AUXIN (IAA)

Treatment Concentration C7o) and dose (kR)

Number of seeds

Number of seedlings

Survival6o)

IAA 1. 0 - 0. 001 1600 541 33. 8EMS 0.5 400 165 41.2

EMS + IAA 0.5 + 1.0 - 0.001 1600 490 30.6IAA + EMS 1. 0 - 0. 001 + 0.5 1600 468 29.2X-rays 5 400 183 45.7

X-rays + IAA 5 + 1. 0 - 0. 001 1600 574 35.8

IAA + X-rays 1. 0 - 0. 001 + 5 1600 483 30.2

Control 0. 0 1200 621 51.7

greenhouse on 19 February 1970. The numbers of seedlings were periodically taken into account. Internode length of the seedlings was measured in September 1969 and in May 1970.

3. RESULTS

3.1. Viability

Plant viability in field conditions is given in Table I. From the Table it is evident that high concentrations of auxin decreased viability from 51.7% in the control to 33.8% (from 21.5% to 39.0% for different auxin concentrations). EMS treatment of seeds decreased viability by 1.0.5% and X -ray treatment by 6.0%. The combined treatment of seeds r e sulted in some decrease of plant viability in comparison with separate treatment with each of the agents; this effect was not, however, entirely additive. In both cases, plant viability was lower in those variants where mutagen treatment of seeds was preceded by their treatment with auxin. Difference in viability between IAA + EMS and EMS + IAA treatments was 1.4%; the difference between IA A + X -ra y and X -ra y + IAA was 5.6%. Interesting results were obtained in the greenhouse where seeds were used without preceding seed stratification necessary for breaking seed dormancy. In our field conditions at a soil temperature of 5 - 10°C, seedlings of the ash-leaved maple appeared 3 -4 weeks after the dry seeds were sown. In the greenhouse, however, at a soil temperature of about 15°C only 12.4% of the seeds grew. Based on the data available in the literature [4], it may be expected that gibberellin, and possibly adenine, may promote the breaking of seed dormancy. Actually, as a result of pre-sow ing treatment with gibberellin and adenine, seed germination rates were increased to 20.2 and 26.0% respectively (Table II). The high efficiency of the chemical mutagen dMS was unexpected in that it broke the dormant state in the seeds that did not undergo treatment at

2 6 8 PRIVALOV

TABLE II. SEEDLING SURVIVAL A FT E R SINGLE AND COMBINED TREATM ENTS OF SEEDS WITH M UTAGEN (dMS) AND GROWTH REGULATORS (GA, Ad)

Treatment Concentrationво )

Number of seeds Number of seedlings

Survival6o)

GA 1. 0; 0.1 400 81 20.2dMS 0. 05 200 118 59.0

dMS + GA 0. 05 + 1. 0 - 0. 1 400 277 69.2GA + dMS 1.0 - 0. 1 + 0.05 400 209 52.2

Ad 0. 09 200 52 26.0dMS 0. 1 200 104 52. 0

dMS + Ad 0.1 + 0.09 200 106 53.0Ad + dMS 0. 09 + 0.1 200 82 41.0Control 0. 0 450 56 12.4

decreased temperature. The pre-sowing treatment at a solution concentration of 0.05% increased germination rate up to 59% and at concentration 0.1% to.52%. The combined treatment of seeds with dMS + GA increased germination rate still more (up to 69.2%). These data demonstrate the capacity of dMS to substitute completely the pre-sowing treatment of seeds with low temperature (i.e. stratification). In greenhouse as well as field conditions the maximal decrease of plant viability was obtained when treatment was carried out in the following order: hormone + mutagen. Thus, the data obtained indicate that susceptibility of seeds of the ash-leaved maple to mutagenic agents rises considerably with increased content of growth regulators. The most efficient combinations of hormone + mutagen were as follows: G A+dM S, viability decreased by 17.0%; Ad + dMS (12%); and IA A + X -ra y s (5.6%). IA A +E M S were of low efficiency decreasing viability only by 1.4%.

3.2. Rate of seedling growth

According to their growth rate, two groups of seedlings of the ashleaved maple are distinguished in Novosibirsk conditions. Some of the plants formed short internodes in the beginning of summer, then they increased in length at the middle of the vegetation period and subsequently decreased again by autumn. The graph representing internode length in such plants formed a smooth curve of growth, with the peak located at about the middle of the shoot. This type of growth was arbitrarily designated "stab le". In the plants of the second group, short and long internodes alternate forming a curve with rises and falls irrespective of their location on the shoots. Such growth types were called "unstable". The ratio between plants with "unstable" and "stable" height growth in sem i-sibs progeny of one control tree and a chlorophyll


•/.

*/ . IA A 1,0 0.1 0.01 0001 X-R X-R X-R X-R 1.0 0.1 0.01 0.001 EMS EMS IEMSEMS

FIG. 1. Percentages of plants with "stable" growth after seed treatments with auxin (IAA) and mutagens (X-rays, EMS). (field experiment).

60

40

20— CONTROL

G A Ad dM S dMS GA dM S Ad

GA dMS Ad dMS

FIG. 2. Percentages of plants with "stable” growth after seed treatments with gibberellin (GA), adenine (Ad), and dimethyl sulphate (dMS), (greenhouse experiment).

mutant was 3:1. Although the genetics of this character is as yet unknown, the behaviour of these two plant groups in consequence of seed treatment with growth regulators and mutagens were so different, that it was decided to use this character in the study along with the other characters. The results of the registration of the "stable" and "unstable" plants are given in Figs 1 and 2.

The data of the field experiment presented in F ig .l show that:a) Seed treatment with auxin only or with X -rays alone did not lead to any change in the ratio between these two groups of plants. The number of "stable" plants remained the same as in the control (i.e. about 25%);b) Seed treatment with EMS resulted in an increase of the number of "stable" plants (up to 35.7%); and c) In the variants with combined auxin and mutagen treatment the number of plants with "stable" growth increased to 40 - 50%.

Experiments performed in the greenhouse (F ig .2) gave sim ilar r e sults. In a ll the variants with combined seed treatment the number of plants having "stable" growth rose considerably (from 25% in the control

27 0 PRI VALOV

UO 120 -

100

80

6 0 -

40JL— C ONTROL

E M S X - R A Y S E M S » I A A X - R A Y S . I A A

100

806 0 -

¿0 ДП П — C O N T R O L

I A A * E M S I A A ■* X - R A Y SI A A

FIG. 3. Percentages of seedlings with "stable" □ and "unstable" ■ growth in relation to control groups (100%) after seed treatments witft mutagens (X-rays, EMS) and auxin (IAA, 1 = 1. 0%, 2 = 0. 1%, 3 = 0. 0l%, 4 = 0. 001%), (field experiment).

*/.2 5 0

200

1 5 0

100

5 0

"U N S T A B L E " SEED LIN G S “ S T A B L E ’ SEED L IN G S

LDÏCONTROL

GA Ad dMS dM SG AdM SAd GA Ad dMS dMS GAdMS Ad • * ♦ ♦ ♦ ♦ ♦ ♦ ♦GA dMS Ad dMS GA dMSAd dMS

FIG. 4. Percentages of seedlings with "stable” and "unstable" growth in relation to control groups (100 ) after seed treatments with gibberellin (G A), adenine (Ad), and dimethyl sulphate (dMS), (greenhouse experiment).

to 57 - 77% in the experimental groups). However, in contrast to the field experiments, in greenhouse conditions some increase in the number of plants with "stable" growth under the effect of growth regulators (GA and Ad) was also observed.

It seemed interesting to determine which group of plants contributed to the changes in the established ratios. For this purpose, we calculated the absolute values of viability in relation to the corresponding group of control plants taken as a 100% reference. The results plotted in Figs 3 and 4 show that in most cases changes in the ratios between the plants were due only to the dying off of the plants with "unstable" growth. The


1.0 0.1 0.01 0.001

V. A U X I N ( IA A )

FIG. 5. Mean height of seedlings in relation to control groups (10Ctfo) after seed treatments with auxin (IAA) and mutagens (EMS, X -rays).

ш E M S » I A A X - R A Y S - I A A I A Ax

I A A - E M S I A A - X . - R A Y S C O N T R O L S

FIG. 6. Mean height of seedlings with "stable" □ and "unstable" ■ growth in relation to control groups after seed treatments with mutagens (EMS, X-rays) and auxin (IAA, 1=1. 0%, 2 = 0.1°/o, 3 = 0. 01%,4 = 0. 001%), (field experiment).

data show that the plants with "unstable" growth are more susceptible to treatment with chemical mutagens only and to combined treatment with chemical mutagens and growth regulators, whereas plants with "stable" growth show marked resistance to different effects in the initial developmental period from dormant seeds.

27 2 PRI VALOV

3.3. Plant height

The mean height of the plants after seed treatment with EMS or X -rays was approximately at the control level. This indicates that the EMS concentration and X -ray dose were relatively low for the ash-leaved maple. Auxin treatment of the seeds at concentrations of 0.1, 0.01, and0 .0 0 1 % stimulated growth of the seedlings and at a concentration of 1 .0 % inhibited it (F ig .5). The combined hormone and mutagen treatment strongly inhibited plant growth at different hormone concentrations both in field and greenhouse conditions. However, no marked height differences in seedlings after different sequences of seed treatment were observed.

An interesting dependence of the mean plant height on the type of growth, as estimated by internode length, was found. In all variants of the experiments, including the controls, the mean height of the plants with "unstable" growth considerably exceeded the mean height of plants with "stable" growth. However, the range of variability of plant height within these two groups was almost the same. The difference in plant height in the field was 2.7 - 14.9 cm (or 12 - 36% of the total length) and0.8- 14.7 cm (or 2 - 20%) in greenhouse conditions. As an example, data of one of the field experiments are given in F ig . 6 .

4. DISCUSSION

The present work was carried out with the purpose of studying the influence of different concentrations of natural growth regulators on the frequency and spectrum of somatic mutations arising in the ash-leaved maple. E arlie r, a wide spectrum of somatic mutations involving different characters had been produced in this species by ionizing radiation (gam m a-rays, X -rays and fast neutrons) [3, 8 ].

The high mutability of the ash-leaved maple, and the possibility of revealing mutations by relatively simple methods facilitate the study of the modifying influence of various factors on mutational variability in a relatively short time (four to five years). Here, the first results of studies on the effects of various sequences of combined treatments of seeds with hormones and mutagens on the viability and plant height as measured by internode length are reported. In a ll experiments where the seeds were treated first with hormones and then with mutagens, increased susceptibility to mutagens (EMS, dMS, X -rays ) was observed. The data suggest that phytohormones administered to the seeds at increased concentrations induce an activation of new chromosome loci which did not function before the treatment. It is possible that in the process of gene function (in the state of replication or transcription) they are less pro tected and mutate more often under the effect of mutagens. B rock 's results lend some support to this suggestion [9]. He showed with the example of the (3-galactosidase locus of E. coli that this gene in the active (induced) state mutates under the effects of certain mutagens (EMS, dMS) at higher frequencies than in the inactive (uninduced) state. E arlie r, Salganik [10] and Voronina [11] showed that DNA is more sensitive to chemical mutagens in the state of replication. The most efficient combinations of hormones plus mutagens with regard to de-


creased viability in our experiments were: GA + dMS, Ad + dMS, and IAA +X -ray s . Future observations w ill permit confirmation of the hypothesis after the detection of somatic mutations in the ash-leaved m aple.

Another characteristic change through which the efficiency of the combined mutagen plus growth regulator treatment of seeds could be judged was the growth rate estimated by internode length. Although the genetics of this character have not been studied as yet, it is quite p ro bable that changes in the growth rate of plants under the influence of drastic changes in environmental conditions are related to the adaptive characteristics of different genotypes. The strong reaction of plants of "unstable" growth to environmental conditions is related to their increased susceptibility to various treatments as early as at the seed stage. Viability was lower in plants from this group. At the same time, plants with "unstable" growth show a stronger reaction to optimal environmental conditions, and the longest internodes were noted among plants of this group. On the contrary, the "stable" plants exhibited more uniform internode length, and were more resistant to the combined seed treatments with growth regulators and mutagens.

An interesting observation made in the present work was the capacity of the chemical mutagen dimethyl sulphate to substitute low temperature treatment of the seeds (or stratification). This indicates that dMS can act as a specific inductor capable of breaking seed dormancy in the ashleaved maple. In this respect, dMS proved to be more efficient than the natural constituents of plants such as gibberellin and adenine. It s's possible that the establishment of the mechanism of the capacity of dMS to initiate the growth of seed germ s by breaking dormancy will help to understand the nature of some of the specificity of the effects of chemical mutagens on the mutational process as compared with the effects of ionizing radiations. If the relation between gene activity and their mutability is confirmed, then the search for and study of specifically- acting chemical mutagens w ill acquire a more meaningful direction. Chemical mutagens in this case should prim arily possess specificity in activation of the genetic apparatus of the cells.

The possibility should not be excluded, however, that higher concentrations of phytohormones decrease plant viability. They may not affect the genetic apparatus of the cells, but by inducing shifts in hormone balance impair the physiological processes. In such a case, they would fa il to exert a marked influence on the frequency and spectrum of induced mutations.

5. CONCLUSIONS

1. The effect of the combined treatment of the seeds of the ashleaved maple (Acer negundo L .) with hormones (GA, Ad, IAA) and mutagens (EMS, dMS, X -ray s ) are presented. It was found that seed treatment in the sequence hormone-mutagen results in considerable decrease of plant viability as compared to the mutagen-hormone treatment. Treatments with IAA + EMS, IA A + X -ra y s , Ad+dM S, a ndGA+ dMS resulted in decreases in plant viability by 1.4, 5.6, 12.0, and 17.0% respectively.

2 7 4 PRIVALOV

2. The sem i-sibs populations of ash-leaved maple seedlings were assigned to two plant groups on the basis of their reaction to drastic fluctuations of environmental conditions: a) Plants showing strong r e actions to such changes (mainly due to increase or decrease of temperature) by considerable and abrupt change in internode length. This group was termed plants with "unstable" growth; and b) Plants showing a weak response to drastic changes in environmental conditions, with "stable" growth. The progeny of the seeds from the two trees studied (chlorophyll mutant and control tree) have a 3 : 1 ratio between the plants with "unstable" and "stable" growth. As a result of combined seed treatment with growth regulators and mutagens only seedlings of the "unstable" group died.

3. In all the variants of the experiment, including the controls, the mean height of the plants with "unstable" growth exceeded considerably that of plants with "stable" growth.

'RE F E RE N C E S

[1] GUSTAFSSON, A., "Polyploidy and mutagenesis in forest-tree breeding", Proc. 5th World Forestry Congr. , Seattle (1960) 2,

(2] NYBOM, N. , KOCH, A., "Induced mutations and breeding methods in vegetatively propagated plants”, The Use of Induced Mutations in Plant Breeding (Rep. FAO/IAEA Tech. Meeting,Rome, 1964), Pergamon Press, Oxford (1965) 661,

L3] PRIVALOV, G. F. , Investigations of experimental mutagenesis in arboreous plants, Genetics,USSR 6 (1968) 144.

[4] LEOPOLD, A.C., Plant Growth and Development, McGraw-Hill, New York (1964). t5] OVERBEEK, I. , Plant hormones and regulators, Science 152 (1966) 721.L 6] ZEEVAART, I.A.D. , "Hormonal regulation of plant development", Proc. 4th Int. Symp. Cell

Differentiation Morphogenesis, Wageningen, 1965, North Holland, Amsterdam (1966) 144.L 7] KHVOSTOVA, V. V. , Mutation and growth substances of plants, Priroda 1_ (1970) 20.[8] PRIVALOV, G. F., "Experimental mutations in woody plants", Induced Mutations and Their

Utilization, Proc. Symp. Erwin-Bauer-Gedâchtnisvorlesungen IV, Gatersleben, 1966, Akademie- Verlag, Berlin (1967) 383.

[9] BROCK, R.D. , "Increasing the specificity of mutation", Induced Mutations in Plants (Proc. Symp. Pullman, 1969), IAEA, Vienna (1969) 93.

[ 10] SALGANIK, R. , "Application of radiation on dénaturation of DNA in combination with chemical mutagens to affect the mutational process", Genetics Today (Proc. XI Int. Congr. Genet.) 1_(1963) 55.

[11] VORONINA, E.N. , POSLOVINA, A.S., SALGANIK, R.I., The regulation of the mutation spectrum in Escherichia coli B. by treatments with chemical mutagens at different periods of the DNA replication, Genetics, USSR 4 (1968) 89.

D I S C U S S I O N

A. ASHRI: Of which generation were the pictures shown, M i or M 2 ?G.F. PR IVALOV: In M a only.H. SMITH: If the purpose of the hormone treatments was prim arily

to activate or “ open up" genes so that they are more susceptible to mutagenic treatments, would not an effective cytokinin (like kinetin) have been a most logical choice for use?

GROWTH REGULATORS AND MUTAGENS 2 7 5

G.F . PR IVALO V: The data available in the literature show that adenine acts just as cytokinin.

C. BROERTJES: If I have understood you well you have treated seeds with various combinations of chemicals and mutagenic agents in May 1969 and February 1970. You then measured the results of the treatment approximately six months later.

We all know, however, that any mutagenic treatment has two effects,1 ) permanent genetic effects, and 2 ) temporary physiological effects, which generally disappear in the next generation.

I consequently do not understand how you can conclude something about mutation percentage and spectrum from data obtained only six months after the treatment.

G .F. PR IVALOV: This is a mistake. In this paper, spectrum of mutations is not discussed. This data will be obtained after two to three years.

R.D. BROCK: Is the 3:1 segregation of "stable" : "unstable" growth habit a regular feature from a ll Acer negundo trees, or is this a special characteristic of your seed stock indicating that you commenced with heterozygous material.

G .F. PR IVALOV: The relation between seedlings with "unstable" and "stable" growth was studied in individual generations from three trees. In the report, results obtained from generations of two trees are given. Their ratio was 3:1. In the generation produced by the third tree the ratio after segregation was nearer 1 : 1 .

INVESTIGATION OF THE ECOLOGY OF THE M UTANT GENE*

К. K. SIDOROVA, V. V. KHVOSTOVA Institute of Cytology and Genetics,Siberian Branch, USSR Academy of Sciences,Novosibirsk, USSR

Abstract-Resu men

INVESTIGATION OF THE ECOLOGY OF THE MUTANT GENE.Induced mutants frequently differ from their parent varieties not only in morphological characters

but also in their reaction norm to environmental conditions. This might lead to a wider or smaller adaptability or to a shift of optimal adaptation to environmental conditions different from those of the place of origin.

The paper reports on results of two years’ experiments with mutants of Pisum sativum grown in different locations of the USSR. The aim of the work was to study the variability of single characters, mainly those of economic importance and to establish the agro-economical potential of the mutant lines. It was found that the mutants differ in their adaptability: some gave stable yields under the various conditions, other mutants showed a variable productivity depending on the particular environment.

ESTUDIOS SOBRE LA ECOLOGIA DE LOS GENES MUTANTES.Los mutantes inducidos difieren frecuentemente de las variedades progenitoras no sólo por sus caracteres

morfológicos sino también por su reacción a las condiciones ambientales. Esto puede provocar una mayor o menor adaptabilidad o una desviación de la adaptación óptima a condiciones ambientales distintas de las predominantes en el lugar de origen.

En la memoria se exponen los resultados obtenidos en 2 afios de experimentos con mutantes de Pisum sativum cultivados en distintos lugares de la Unión Soviética. La finalidad de estas investigaciones era estudiar la variabilidad de determinados caracteres, en especial aquellos de importancia económica, y juzgar et interés agroeconómico de las líneas mutantes. Se ha comprobado que los mutantes difieren en lo que respecta a la adaptabilidad. Algunos han dado rendimientos estables en distintas condiciones, mientras que en otros la productividad varía según el medio ambiente.

1. INTRODUCTION

Natural as well as induced mutants which arise from the treatment of seeds and other parts of plants with different chemical mutagens and ionizing radiation are new genotypes as compared with the initial variety and differ from it not only in morphological characters, but also by the "reaction norm " to the environmental conditions.

By "reaction norm" is meant the property of the given genotype to provide, within certain limits, ontogenetic variability depending upon the changing environmental conditions. In other words, the range of the variability in the realization of the genotype expresses the reaction norm.

Gustafsson et al. [ 1 , 2 ] studied the reactions of barley mutants to certain environmental conditions. The behaviour of X -ray induced mutants and of the initial variety was observed at different temperatures Sind day lengths in phytotron. As a result wider adaptability of the mutant to

Part of the research reported in this paper has been carried out under Research Agreement with the International Atomic Energy Agency No. 774/CF.

27 7

2 7 8 SIDOROVA and KHVOSTOVA

environmental conditions was established as compared with the initial va riety. In climatic conditions optimal for the initial variety the mutant was lower yielding, but it proved to have higher adaptability and in certain conditions it attained the yield of the initial variety and even surpassed it. Gustafsson also studied a bright green barley mutant which was higher yielding in the north of Sweden than in the south where it was originally produced.

Italian investigators obtained interesting results in their studies of induced wheat mutants in different ecological conditions [ 3 ]. Eight mutant lines as well as the two widely distributed wheat varieties in Italy, Capelli and Capeite, were sown in several countries with different climatic and soil conditions, including Italy, Tunisia, Syria, Arab Republic of Egypt, Libya, Turkey, Iran and India. The productivity of mutant lines was compared with that of the best varieties in each country, and the best conditions for the cultivation of each mutant were established.

Chlorophyll mutants are suitable for the study of the influence of environmental conditions on mutant characteristics. Hentrich [4 ] studied the effect of temperature on chlorophyll mutations in barley. In our studies (unpublished results), differences were found in the intensity of leaf colour in pea chlorophyll mutants depending upon whether they were sown in the field or in the greenhouse. The experiments of Elshuni et al. [5 ] with chlorophyll barley mutants gave sim ilar results.

Differences in mutation behaviour depending on environmental conditions have also been studied using other m aterials. Shmalhausen [ 6 ] has summ arized the results of analyses of mutations obtained in Drosophila melanogaster.

This paper reports the results of tests on induced pea mutants (Pisum sativum) carried out for two years (1968-1969) in different areas of the USSR. The aims of the work were 1) to study the variability of single characters, mainly those of economic importance, in mutants and in initial varieties depending on environmental conditions, and 2 ) to establish the potential of each mutant line for plant breeding.

2. M ATERIALS AND METHODS

The experiments were carried out on constant pea mutant lines obtained from the commercial pea varieties Torsdag and Falensky-42 widely distributed in our country. The seeds were treated with chemical mutagens (El, EMS) and gam m a-rays.

The mutant lines were studied for six to eight generations in Novosibirsk conditions. A morphological characteristic was set up for each line. The mutants differed from the initial variety in their productivity, length of the vegetation period, stem shape, branching, number of beans on the pedicle and by other characters. Genetical analysis has shown that these mutations are mainly monogenic and the characters are recessive [7] .The mutants crossed with the initial variety gave in F2 the 1:3 Mendelian segregation ratio. No meiotic disturbances were observed in the mutants.In addition, the mutants studied differed from the initial variety in a whole complex of characters which are a ll inherited together, i. e. their appearance was due either to the pleiotropic effect of a single gene or to the simultaneous mutation of closely-linked genes.

M U TAN T GENE ECOLOGY 27 9

The mutants were tested in a number of sites in the USSR differing in their soil and meteorological conditions, including the Moscow, Kalinin, Kirov, Omsk, Novosibirsk and Tomsk regions, the Bashkir ASSR, the Krasnoyarsk Territory and the Buryat ASSR. The mutants were also tested at different altitudes above sea level (800, 1100, 2500 m). In all the sites the same sowing technique was used. Konstantinov1 s widely applied paired method of comparison was used. The initial variety was sown as control.

Besides the phenology of each mutant line and initial variety, the following characters were determined: plant height, number of beans, number of seeds per plant, weight of 1 0 0 0 seeds, and vegetation period.In a number of sites were studied the total number of internodes, the number of branches, the size of leaflets and stipules, the size of the beans, and content of crude protein in the seed.


The results of the ecological studies of induced pea mutants have shown that mutants and initial varieties differ in their reaction norm to the environmental conditions: in certain conditions the mutant could be very tall and produce more grain compared with the initial variety, whereas in other conditions its productivity was inferior to that of the initial variety (Pig. 1). Thus, for example, the productivity of mutant No. 3 varied according to the growing conditions from 31 to 122%.

Moreover, it has been established that some mutants have relatively wide adaptability to environmental conditions, and when sown in different sites give stable yields. Other mutants, however, differed much in productivity, length of vegetation period and other characters when sown in different sites. To the group with stable yields belongs the productive early-ripening mutant No. 1 obtained from the fodder variety Falensky-42. Inl968, in five of six sites, it proved to be the most productive as compared with the initial variety, and in one site its yield was almost equal to it.This mutant gave sim ilar results in 1969.

The largest difference in productivity in different areas was observed in the compact mutant No.. 3 obtained from the grain variety Torsdag (Fig. 1). In the Moscow and Kalinin regions it gave a considerable increase in yield by 9 and 22. 5% respectively; in the Omsk region, the Buryat ASSR, and the Krasnoyarsk Territory its yield was at the level of the initial variety; in the Kirovsk and Tomsk regions and the Bashkir ASSR its productivity proved to be much lower than that of the initial variety.

The different reactions of mutants to changes in environmental conditions may be due to the specificity of the effect of the mutant gene itself, as well as to the peculiarities of the genotypical environment of the given line.

Along with mutant productivity, plant height, vegetation period, grain size, protein content in the grain and other characters were also observed to change depending on the environmental conditions. Thus, the productive early-ripening mutant No. 1 sown in the Novosibirsk region ripened six to seven days earlier than the initial variety, in the Moscow region four to five days earlier, and in the Omsk region the vegetation period was shorter by ten to eleven days. In the compact mutant No. 3, on the other hand, deviations in vegetation period in different sites were not significant.

280 SIDOROVA and KHVOSTOVA

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l/ttra ear/у ripening m utant Afc2

Hi

Com pact m utant № 3

Product/ re eartu ripening mutant №1

ûescgna 11 on •

I / / / / I M o s c o w пиши Xa/inin foSMj Xirotv И И Bashkiria I I Omsk(ШШ1И NovosiSirsk- fietd fesi Nowsi6irsk-gre/>r> house KW\\\4 7omsk Cu'i'iM /frasnoyarsk IÓ&9I Buri/atia

FIG. 1. Productivity of pea mutants in different geographical areas (% of initial variety, 1968).

M U TAN T GENE ECOLOGY 28 1

Protein content of the grain of the mutants also changed depending on the sowing site. Thus, in the Novosibirsk region in 1968, crude protein in the grain of the early-ripening mutant No. 2 (obtained from the variety Torsdag) was 23. 6 %, i. e. 2. 7% less than the initial variety. The protein content of the grain of this mutant in the Moscow region was 27. 9%, i. e.0. 4% less than the initial variety. In the other mutants, protein content in the grain sown at various sites changed quite insignificantly.

When the mutants were tested at three sites of Tadjikistan situated at different heights above sea level, a more distinct difference in the reaction norm to environmental conditions was found in the mutants than in the initial variety (Table I).

At altitude I the productive mutant No. 3 did not differ in height from the initial variety. At altitude III, however, the mutant proved to be higher. The mutant No. 9 with fasciated stem at altitude I was shorter than the initial variety and most of the mutants. At altitude III, however, this mutant was the highest.

Thus, it may be considered that a mutant has relative economic value in respect to definite envirónmental conditions. We determined some of the features of the mutants to be taken into account in their use for plant breeding purposes. Thus, the early-ripening mutant No. 2 ripens earlier in the Omsk region than in the Moscow or Novosibirsk regions. The compact mutant No. 3 proved to be better in the Moscow and Kalinin regions than in the Novosibirsk region where each year it gives lower yield than the initial variety. The productive early-ripening mutant No. 1 is characterized by increased productivity in different years and areas. Thus, the mutant can be valuable in a very wide distribution area.

Our studies showed that the different characteristics of a particular mutant were not always consistent with one another. Thus, for example, a mutant differing in increased productivity in certain environmental conditions did not always have a higher stem than the initial variety. In other words, each character of the mutant changed to some extent independently of the other characters. This can be illustrated by the following example. The mutant No. 9 with the fasciated stem sown at altitude III in Tadjikistan was the tallest compared to the initial variety; but with respect to seed productivity it was much inferior to most of the mutants and to the initial variety since it had very sm all seeds.

Our observations also showed that under certain environmental conditions various characters of the mutant may change in different directions,1. e. some characters, in particular deleterious ones arising from the mutation effect, may be extinguished or pass into a latent state, while other, more favourable characters are retained. Such behaviour is of particular interest, since in general, due to the pleiotropic effect of a mutant gene, several characters are changed, one or two of them favourably, the others unfavourably.

It is extremely difficult to use such mutants in plant breeding, since it is impossible to isolate favourable characters from the unfavourable ones by segregation in progenies from crosses between à mutant and the initial variety (or some other mutant). When growing such mutants in different sites, the choice of environmental conditions is possible: under which the unfavourable characters are not shown, while the favourable ones are pronounced.

28 2 SIDOROVA and KHVOSTOVA

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M U TA N T GENE ECOLOGY 283

The compact mutant No. 4 gives an example of such a mutation differing in the exhibition of a number of characters. Studies of this mutant in the Novosibirsk region and at altitudes II and III in Tadjikistan have shown that along with its main character, compact stem, other characters among them seed productivity are also changed. However, when this mutant was grown in other conditions, e. g. at altitude I in Tadjikistan, as well as in the Moscow and Kalinin regions, its productivity increased with stem compactness remaining at the same level. In other words, the deleterious character of the mutant was not expressed.

The results of the ecological tests of pea mutants have shown that the occurrence of mutations leads to changes in the reaction norm to environmental factors. Thus, in response to different environmental conditions a certain autonomy of the reaction norm of characters is observed.

It was inferred that changes in the reaction norm are due to the mutation of a single gene. The reaction of mutants to ionizing radiation confirm this conclusion [ 8 ]. Differences in radiosensitivity between pea. mutants and the initial variety were observed under X -ray treatment at 5 kR. Radiosensitivity was estimated by the frequency of chromosomal rearrangements of the first mitoses of the meristematic cells of seedling rootlets.In nine of the eleven mutants, radiosensitivity proved to be much higher than in the initial variety and in two of them it was lower. The initial Torsdag variety had 19. 6 % cells with chromosomal rearrangements (the mean for the first cell cycle), in nine radiosensitive mutants the number of chromosomal rearrangements varied between 29. 6 and 46. 8 %, and in the radioresistant mutants between 17 and 17. 8 %.

The differences we have found between the reactions of the mutants and the initial varieties to their environment as well as the dependence of certain mutant characters on the environmental conditions permit the assignment of an important role to ecological tests in the understanding of the genetic nature of induced mutants, as well as in developing efficient methods of using them in plant breeding.

The practical problem of increasing plant productivity is being solved, first, by creating the required genotypes and, second, by the development of methods controlling ontogenesis of the organism providing the best development of characters inherent to the genotype.

4. SUMMARY

1. Based on the results of ecological tests of a number of mutants, differences in the reaction norm to environmental conditions were found between the mutants and the initial varieties.

2. Mutant lines differ in their adaptability to various environmental conditions. Some of the mutants sown in different sites gave stable yields; other mutants, however, differ in their productivity.

3. The degree of exhibition of certain characters of a particular mutant in different conditions is not always positively correlated. In certain conditions, changes in different mutant characters are possible in different directions.

4. Ecological tests of mutants are shown to be an efficient method for the use of mutants for breeding purposes, since they permit the determination of conditions under which the genotypical possibilities of the organism are fully realized.

284 SIDOROVA and KHVOSTOVA

R E F E R E N C E S

[1] GUSTAFSSON, Â., Sel', -khoz. Biol. 3 1 (1968).[2] DORMLING, I., GUSTAFSSON, A., JUNG, H.R., von WETTSTEIN, D., Hereditas 56 (1966) 222.[3] TESSI, J., SCARASCIA-MUGNOZZA, G.T., SIGURBJORNSSON, B., BAGNARA, D., Mutations

in Plant Breeding II (Proc. Panel Vienna, 1967), IAEA, Vienna (1968) 251.[4] HENTRICH, W., Induced Mutations and their Utilization, Proc. Erwin-Bauer-Gedachtnisvorlesungen IV,

Gatersleben, 1966, Akademie-Verlag, Berlin (1967).[5] ELSHUNI, K. A., KHVOSTOVA, V.V., STOLETOV, V.N., Genetika 3 (1965) 70.[6] SHMALHAUSEN, 1.1., Factors of evolution, Nauka Publishing House, (1968).[7] SIDOROVA, K.K., Genetika 6 (1968).[8] SIDOROVA, К. K.. KHVOSTOVA, V.V., KALININA, N.P., Genetika 3 (1969).

M UTATION BREEDING IN SOYBEANS

F.к . s. к о оTropical Agro-Sciences Division,Puerto Rico Nuclear Center*Río Piedras, Puerto Rico

Abstraer-Resumen

MUTATION BREEDING IN SOYBEANS.Reviewed in this paper are the accomplishments of the past 20 years and the new investigations in

progress on the subject of soybean mutation breeding in the world. Topics scrutinized include yield, maturity, disease resistance, quantity and quality of oil and protein, heat tolerance, photorespiratory deficiency, etc. As evident in some of the studies presented here, this additional breeding tool has enabled the breeders to attack the problems of importance in soybean improvement.

MEJORAMIENTO DE LA SOJA POR MUTACION.En la memoria se pasa revista a los progresos logrados durante los últimos 20 años en el mejoramiento

de la soja por mutación, a las nuevas investigaciones en curso sobre esta materia en todo el mundo. Entre los temas analizados figuran el rendimiento, la madurez, la resistencia a las enfermedades, la cantidad y calidad del aceite y de las proteínas, la resistencia al calor, la deficiencia fotorrespiratoria, etc. Como puede verse por algunos de los estudios resellados por el autor, este nuevo instrumento de selección ha permitido a los fitotécnicos abordar los problemas de importancia para el mejoramiento de la soja.

1. INTRODUCTION

Mutation breeding research in crop plants was pioneered some forty years ago in Sweden by N ilsson-Ehle and Gustafsson soon after the publications of the findings on radiation induction of mutations in Drosophila by M uller and in barley and maize by Stadler in the late 1920's. Sim ilar investigations on plant mutation breeding were also initiated in Russia, Germany and other countries during the 19301 s and 1940, s. Since the end of World W ar II, a number of workers in the United States and Canada, and also in various countries of Europe and Asia have begun research on induction of beneficial mutations by radiations and chemicals in sn increasing variety of crop plant m aterials. These developments have coincided with the general expansion of nuclear energy program s, availability of facilities and promotion of nuclear energy for peaceful uses. In Latin Am erica and the Caribbean area some research effort has been directed towards crops such as coffee, rice, beans, cotton, bananas, coco, sugarcane, soybean, yam, sweet potatoes, wheat, oats, etc. [7, 10, personal communications with several investigators].

Soybean mutation breeding investigation was begun in the early 19501 s and four varieties improved by this method have been released to date [14] . The main objective of soybean breeding varies, of course, with the needs of the breeder, yet the ultimate objective inescapably centers on yield and quality. This review summarizes the present status of various soybean mutation breeding program s as well as the past achievements in this field.

Operated by the University of Puerto Rico under Contract No. AT-(40-l)-1883 for the US Atomic Energy Commission.

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286 коо

Humphrey [3 ] and Zacharias [cf. 5, 15] reported yield improvement in soybeans by mutation breeding but no new varieties were released from their investigations. Ishikawa et al. [4 ] recently published the detailed mutation breeding results of two varieties. The variety Raiko was selected from the progenies of the gam m a-irradiated Nemashirazu variety and released in 1969. The new variety has higher yielding potential than the parental variety even though it is about 15 d earlier in maturity. The other improved variety Raiden, released earlier in 1966, was developed from the same irradiated m aterial from which Raiko was extracted. This new variety, 25 d earlier in maturity, yields less than Nemashirazu but outyields other varieties with comparable maturity. Cheng in Taiwan also reported successful breeding of two varieties, namely Tainung No. 1 and No. 2, by application of thermal neutron and X -ray treatments [cf. 14]. Both varieties showed improvement over the original variety in yield and other characteristics including shattering resistance.

In the irradiated populations, one almost invariably finds considerable increase in genetic variance for the quantitative traits including yield. Rawlings et al. [12] observed a significant increase in variability in yield in the M 2 generation of the varieties Adams and Hawkeye treated with X -rays and thermal neutrons but the increased variance was largely in the negative direction. Papa et al. [11 ] studied the third generation of the same two irradiated populations and found that selection for high yield was effective in the irradiated Hawkeye. However, the amount of gain through selection was of no practical value as the genetic gain through selection of the upper yielding group was hardly enough to compensate for the initial decrease in the mean due to irradiation.

2. YIELD

3. M ATURITY

Soybean is very sensitive to the length of dark period to which it is subjected each day. Different varieties may differ greatly in their response to the daily dark period for inflorescence formation, and their specific response determines their adaption to a particular latitude. This character has been used as a basis for classification of varieties into various maturity zones. In the North American Continent, for instance, there are ten maturity zones covering the regions from the southern part of Canada to the Gulf Coast area of the USA.

Induction of mutations for different maturity has been reported in many crop plants including soybeans. Ishikawa et al. [ 4 ] , as mentioned above, obtained mutants which were much earlier than their parental variety. Zacharias also found early mutants in the X -irradiated variety Kermkraft I; some of them were five to seven days earlier than the parental variety [ c f. 5, 15]. In Germany, earliness in the soybean crop is important for the increase and reliability of the yield [15] .

The soybean has been grown in many parts of the tropical countries including Latin Am erica although its adaption to these regions is marginal. Varieties cultivated in the USA do not yield to capacity when grown in the tropics because the crop growing cycle is shortened. For instance, when H ill and Lee varieties, belonging to the maturity groups 5 and 6 , respectively,

M U TATIO N BREEDING IN SOYBEANS 287

are grown in Puerto Rico (lat. 18° N) they tend to mature in about three months, thus reducing the yield. If the mutants with proper maturity, delayed maturity in this case, could be obtained, the yield of these varieties might be increased as it is generally believed that late maturity contributes to higher yield. In 1968 a mutation breeding program was initiated at the Puerto Rico Nuclear Center to induce proper maturity in these two varieties as one of the objectives. In the M2 -M 4 generations of the gam m a-irradiated m aterials, many late-flowering and/or late-maturing mutants were selected; some of these bred true. At present the late variants are being grown under short-day conditions for selection for the day-length neutral type. P re sumably any late variants which are sensitive to short photoperiod would flower and mature early while those non-sensitive types would not. It is our intention to isolate such mutants to be grown for higher yield during the winter season under conditions of reduced heat stress. Late mutants were also obtained by Santos et al. [13] in M 4 of the irradiated Lincoln variety.

4. DISEASE RESISTANCE

Breeding for disease resistance is considered as important as for yield itself. D isease resistance insures the yield potential of a variety. Dunleavy attempted to induce disease-resistant mutants in Hawkeye soybean by X -rays [cf. 5] . Apparent resistance to bacterial blight caused by Pseudomonas glycinea and to stem canker was observed in early generations following irradiation but the resistance to stem canker was lost in later generations. Also the resistance to bacterial blight became increasingly diluted in succeeding generations. Nevertheless, some plants in the M 7

generation were less susceptible than others. Recently, Lu [ 8 ] used radiation to induce rust resistance in soybeans after failing to find any good resistance source in over 300 varieties he had screened. This disease, commonly found in spring and fall in Taiwan, is caused by a fungus Phakopsora pachyrhizi Sydow. In the M 2 m aterials of three varieties treated with gamma-radiation, he found many plants with no disease lesions. Presum ably some of these were escapes as a result of low spontaneous epidemics in the field during the winter season. Further tests in M 3 also revealed some true resistant lines. In a co-operative study conducted by the soybean breeders in the USA with the aim of induçing disease re sistance, certain variant types have been obtained with less susceptibility to diseases than the parental varieties [cf. 5] . Unfortunately no final results have been made public.

5. OIL CONTENT

In the Far East, the soybean is used as a high protein food as well as for producing oil. The bean meal after oil extraction is used for livestock feeding or as a fertilizer. In the Western World, soybeans are produced mainly for oil.

The feasibility of increasing oil content by mutation induction has been investigated by W illiam s and Hanway [17] using the same m aterials

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which were evaluated for other characters by Rawlings et al. [12] . E stimates of genetic variance for oil content in the M 2 populations were six or more times greater than in the controls. Heritability (70. 3%) for oil content in the irradiated m aterial was five times as great as that in the controls. Selection gain expressed in per cent of the mean averaged 1. 9 and 2. 8 % for high and low oil, respectively. A ll these values were comparable in magnitude to those observed in segregating populations derived from hybridization. In the M 3 generation, Papa et al. [11] carried out selections for both high and low oil and found no gain for high oil.

6 . O IL QUALITY

Linolenic acid is believed to be partly related to the development of non-agreeable flavour in the refined soybean oil. If this fatty acid could be reduced or even eliminated from the seed, the quality of the extracted oil in storage might be maintained. The survey made by White et al. [ cf. 5 ] indicates there is a wide range in content of this fatty acid among varieties. Conceivably this variation would provide a sound genetic basis for breeding new varieties with lower linolenic acid. However, the lower limit of linolenic acid (0. 5%) as reported by the earlie r workers was not observed when refined analytical methods were employed in the determination [cf. 5, Fehr by personal communication]. Recently, Fehr at Iowa State University initiated a breeding program with the m ajor objective of reducing linolenic acid to the lowest possible level (personal communication). One of his procedures involves mutation induction by gamma-radiation in varieties with low linolenic acid content, F2 1 s and advanced selections from the crosses involving these varieties. Determination of linolenic acid in these m aterials is in progress. The other fatty acid, linoleic acid is a valuable component of soybean oil and any reduction of this would be highly undesirable. In view of the reported correlations between linoleic and linolenic acids, Johnson and Bernard [5 ] questioned the desirability of breeding for lower linolenic acid. However, there is hope that such close associations may be broken by genetic manipulation or mutagen treatment.

7. PROTEIN CONTENT

Basically the soybean is a protein crop. The present-day varieties in the USA contain an average of 40. 5% protein on a dry matter basis even though these varieties were originally bred for higher oil content. Recently, however, more attention has also been paid to the protein content im provement [1 ]. Protein content in soybeans is known to be negatively correlated with oil content. Therefore, improvement in protein content might adversely affect the oil content. However, improvement in protein content by breeding still holds promise since two to three units of protein could be gained for the loss of only one unit of oil [ 5] . Santos et al. [ 13 ] searched for protein mutants in the later generations of Lincoln variety following thermal neutron treatment and found the content ranging from 36. 5 to 53. 4%. Enken also observed increased variability in protein content in M 3 in the gam m a-irradiated series [cf. 6 ].

M U TATIO N BREEDING IN SOYBEANS 2 8 9

W illiam s and Hanway [17] reported estimates of genetic variance for protein content were six or more times greater in the X -ray and neutron treated progenies than in the controls. Heritability (71. 8 %) obtained for protein in the irradiated population was ten times as great as that in the controls. Selection gain expressed in per cent of the mean averaged 3. 2

and 1. 9% for high and low protein, respectively. When selection was practised for both high and low protein considerably more gain was made for high than for low protein [ 1 1 ].

8 . PROTEIN Q UALITY

Methionine is the most serious limiting essential amino acid in soybean protein. Results of the analysis of several thousand collections by the USDA show that this amino acid has a very narrow range of variation [ Bernard by personal communication]. Sim ilar results were reported earlier by Kuiken and Lyman [ cf. 5 ] with limited material. In view of lack of natural variability, it seems desirable to resort to the mutation induction technique to bring about the increase of methionine content. Often the mutant screening is the bottle neck to the progress of this type of mutation breeding. However, it appears promising to utilize the isotopic dilution principle to develop a convenient, rapid, economical mass screening method for methionine determination in the seed protein [9] . The Puerto Rico Nuclear Center has been engaged in the development and improvement of such a technique which involves hydrolysis of pulverized seeds, addition of 1 4C-labelled methionine to the hydrolyzate, thin-layer chromatographic separation, and liquid scintillation counting. This technique obviates the limitations of microbiological and colorimetric methods for methionine determination.As a further refinement, thin-layer electrophoresis has been used for amino-acid separation. Further experiments to standardize and perfect the method are in progress.

9. HEAT TOLERANCE

Varieties developed in the temperate zones may be grown in the tropics if their tolerance to higher temperatures is improved. As indicated byHowell [2 ] in a review paper, the soybean plant is fairly resistant to injurycaused by high temperature extremes although the growth rate declines at temperatures above 100°F. Temperatures over 100°F early in the season may cause reduction in rate of node formation and growth of the internodes. During flowering and pod-setting periods, high temperature may increaseshedding, thus reducing yield. It has been shown that high temperatures may also have an adverse effect on quality of seed. There is some evidence that varieties differ in tolerance to high temperatures [ cf. 2, 7 ].

In our program , the hot water dipping method of Yarwood [ 18 ] has been tested for suitability as a m ass screening technique for selecting heat- tolerant mutants. The double-dipping method requires first dipping the whole plant in water at 50°C for 30 sec as the pre-conditioning treatment, and 24 h later the same m aterial is given the challenge treatment in hot water at 55°C for various durations. For the paired first leaf test with the double-dipping method, the procedure is modified somewhat. One of

29 0 коо

the leaves o f the f ir s t pa ir is dipped in w ater at 50°C fo r 30 sec, and 24 h la te r both leaves o f the pa ir a re dipped in w ater at 55°C fo r various durations. The single-d ipp ing method consists o f only the challenge treatm ent at 55°C fo r various durations. F rom a series of studies, Koo et al. [7 ] found va r ie ta l d iffe ren ces and also established two types o f heat to lerance, the inherent and the acquired types. The acquired to lerance apparently resu lts from protection provided by the pre-condition ing treatm ent. It is conceivable that both types o f to lerance are important fo r crop production in the humid trop ics . In the actual screen ing for heat-to leran t mutants in M 2's o f H ill and L ee , plants that withstood the heat treatm ent w ere obtained [7 and unpublished].

10. PH O TO R E SPIR A T IO N

Photoresp ira tion m ay a ffect crop y ie lds adverse ly because the C 0 2 lo s t in photoresp iration serves no known function and is a to ta l waste of energy. Soybean is a photoresp iratory species and it is estim ated that about 15-50% o f the to ta l C 0 2 fixed in soybean photosynthesis is lost through photoresp iration [ Ogren by personal com m unication]. In a p re lim in a ry study, W idholm and Ogren [1 6 ] screened 300 soybean va r ie t ie s and found not a single one with photoresp iratory-defic iency. A s a fo llow up, they have in itiated a mutation p rogram with the sole purpose o f inducing photoresp ira to ry -d e fic ien t mutants. A lso they have developed a p rac tica l mass screen ing technique [O gren by personal com m unication ]. The test involves grow ing soybean and corn plants in a certa in proportion under continuous illum ination in a closed chamber. Since photosynthesis reduces the C 0 2

concentration in the cham ber the soybean plants with photorespiration w ill senesce in a week as a resu lt o f net C 0 2 loss but the mutants with low ered o r no photoresp iration w ill survive. I f photoresp iration can be spared without d e le teriou s ly affecting the l i fe cyc le o f the soybean, this energy now lost in photoresp iration w ill presum ably go into increased growth and y ie lds.

11. OTHERS

In the soybean mutation induction work, many other mutant types o f agronom ica l value have a lso been encountered, such as in crease in seed s ize [3 ,1 2 , cf. 5, 6 ] and in the number o f pods per plant [ 13,15, cf. 5 ].In many instances, mutants with increased v igou r [3 , cf. 14] o r changes in plant height [1 2 ] w ere a lso observed. Zacharias a lso found mutants which germ inated at low er tem perature (4. 5°C) in the va r ie ty Heim kraft I. Stubbe [1 5 ] v e r i f ie d that the co ld -res is tan t varian ts a lso germ inated about s ix days e a r lie r than the parental line and had an advantage in growth that p ers is ted to m aturity. A lso as mentioned e a r lie r , Ishikawa et al. [4 ] obtained lodging res is tance, and Cheng [c f. 14] and Humphrey [3 ] shattering res is tance in th e ir mutation breeding program s.

The achievem ents o f soybean mutation breeding during the past 20 years have been v e ry lim ited , but the prospect fo r new advancement in va r ie ta l im provem ent through the uses o f this spec ia l too l appears bright. As indicated in this rev iew , the mutation breed ing technique m ay be u tilized

M U T A T IO N BREEDING IN SOYBEANS 291

to our advantage in the im provem ent o f d isease res is tance and methionine content o r in the elim ination o r low ering o f linolen ic acid and photorespiration .

In the experim ents conducted by various investiga tors , X -ra y s , gam m a- rays and neutrons o f various types have been used. A lso chem icals such as E l and EMS have been tested [c f. 6 ]. The X -ra y doses used range from6 to 60 kR with 10-12 kR being the optimum doses fo r mutation induction.The gam m a-doses used range from 5 to 30 kR. Our experience indicates 15 kR is the appropriate dose fo r mutation work. Th erm a l neutron doses used va ry from 2 X 10u to 7.5 X 1013 N th/cm 2 but the p roper dose is in the range o f 3 - 7 X 1012 N th/cm 2.

A C K N O W L E D G E M E N T

The author is gratefu l to the fo llow ing persons fo r provid ing invaluable in form ation that made this rev iew possib le: R. L. Bernard , W. L . Ogren,W. R. Fehr, M. Ishikawa, S. Matsumoto, Y. C. Lu, I. S. Santos, C. A. Panton,S. N. Deshpande and J. Cuevas-Ruiz.

R E F E R E N C ES

[1] HARTWIG, E.E., "Breeding soybeans for high protein content and quality", New Approaches to Breeding for Improved Plant Protein (Proc. Panel, Rostânga, 1968), IAEA, Vienna ( 1969) 67.

[2] HOWELL, R.W., "Physiology of the soybean", The Soybean (NORMAN, A.G., Ed.), Academic Press, N.Y. (1963) 75.

[3] HUMPHREY, L. M., Effects of neutron irradiation on soybeans. II., Soybean Digest 14 (1954) 18.[4] ISHIKAWA, M. et al., The New Soybean Varieties "Raiden" and "Raiko" Induced by Gamma-ray

Irradiation, Natn. Reg. Tohoku Exp. Station, Japan, Res. Rep. 40 (1970) 65.[5] JOHNSON, H, W., BERNARD, R. L., "Soybean genetics and breeding", The Soybean (NORMAN, A.G.,

Ed.), Academic Press, N.Y. (1963) 1.[6] KHVOSTOVA, V. V., The present state of mutation breeding in USSR, Gamma Field Symp. No. 7,

Inst. Radiat. Breeding, Japan (1968) 123.£7] KOO, F.K.S., CUEVAS-RUIZ, J.( GUTIERREZ-COLON, G., "Gamma-ray induction of mutations

in soybeans for environmental adaptation", Improving Plant Protein by Nuclear Techniques (Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 367.

[8] LU, Y .С., "Mutation breeding for rust resistance in soybeans", Improving Plant Protein by NuclearTechniques (Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 185.

[9] LUSE, R.A., "Mass screening for specific amino acids by nuclear chemical, biological and enzymatic techniques", Improving Plant Protein by Nuclear Techniques (Proc. Symp. Vienna, 1970), IAEA, Vienna(1970) 237.

[ 10] MOH, C.C., The use of radiation-induced mutations in crop breeding in Latin Americé. and some biological effects of radiation in coffee, Int.J. appl. Radiat. Isotopes 13 (1962) 467.

[11] PAPA, К. E., WILLIAMS, J.H., HANWAY, D. G., Effectiveness of selection of quantitative charactersin the third generation following irradiation of soybean seeds with X-rays and thermal neutrons, CropSci. 2U961) 87.

[12] RAWLINGS, J.O., HANWAY, D. G., GARDNER, C.O., Variation in quantitative characters of soybeans after seed irradiation, Agron. J. £0 (1958) 524.

[13] SANTOS, I. S., FUKUSAWA, C.A., ELEC, J.V., DELAROSA, A.M., "Acclimatization andimprovement of a Lincoln variety soybean through mutation breeding", Improving Plant Protein byNuclear Techniques (Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 189.

[14] SIGURBJORNSSON, B., MICKE, A, "Progress in mutation breeding", Induced Mutations in Plants(Proc. Symp. Pullman, 1969), IAEA, Vienna (1969) 673.

[15] STUBBE, H., "Advances and problems of research in mutations in the applied field", Proc. 10th Int.Congr. Genet. 1(1958) 247.

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[16] WIDHOLM, J.M., OGREN, W. L,, Photorespiratory-induced senescence of plants under conditions of low carbon dioxide, Proc. natn. Acad. Sci. U.S.A. 63 (1969) 668.

[ 17] WILLIAMS, J. H., HANWAY, D. G., Genetic variation in oil and protein content of soybeans induced by seed irradiation, Crop Sci. 2(1961) 34.

[18] YARWOOD, C.E., "Adaptation of plants and plant pathogens to heat", Molecular Mechanisms of Temperature Adaptation (PROSSER, C.L,, Ed.), AAAS Publ. 84(1967) 75.

IMPROVEMENT OF VEGETATIVELY PROPAGATED PLANTS BY IONIZING RADIATION

C. BROERTJES

Institute for A tom ic Sciences in Agriculture,

Wageningen, The Netherlands

Abstract-Resumen

IMPROVEMENT OF VEGETATIVELY PROPAGATED PLANTS BY IONIZING RADIATION.The main advantage of mutation induction in vegetatively propagated plants is the ability to change

one or a few characters of an outstanding cultivar without altering the remaining genotype. It must therefore be considered as an obvious means to perfect the leading products of conventional plant breeding.

Special attention is paid to the adventitious bud technique, which makes use of the formation of adventitious buds on isolated leaves. These buds originate from single epidermal cells and thus the consequences of chimera formation and diplontic selection are avoided. This has been proven in Achimenes, Streptocarpus. Begonia. Peperomia and Lilium, and is under investigation in a number of other crops.Factors which influence the development of the technique are discussed, such as age, size and part of the leaf, as well as the use of modified leaves. In addition, a few general points are discussed.

MEJORAMIENTO DE LAS PLANTAS DE MULTIPLICACION VEGETATIVA MEDIANTE LAS RADIACIONES IONIZANTES.

La principal ventaja que ofrece la inducción de mutaciones en las plantas de multiplicación vegetativa es la posibilidad de modificar uno o varios caracteres de una variedad excelente de cultivo, sin alterar el genotipo restante. Debe, pues, considerarse como un medio ideal para mejorar los productos más destacados de la fitotecnia clásica.

Se presta especial atención a la técnica de las yemas adventicias, basada en la formación de éstas en hojas aisladas. Estas yemas provienen en definitiva de células epidérmicas particulares, con lo que se evitan las consecuencias de la formación de quimeras y de la selección de diplontes. Ello se ha demostrado en los géneros Achimenes. Streptocarpus, Begonia. Peperomia y Lilium y se encuentra en estudio en una serie de otras plantas de cultivo. Se examinan diversos factores que influyen en el desarrollo de esta técnica, tales como la edad, tamaño y parte de la hoja, así como el empleo de hojas modificadas. Se discuten además ciertos puntos de carácter general.

1 . INTRO D U CTIO N

Mutation breed ing o ffe rs la rge poss ib ilit ies in vege ta tive ly propagated plants, such as ornam entals, numerous fru it crops, potatoes, sweet potatoes, sugar cane, cassave and grapes. The main advantage is the p o ss ib ility to im prove one o r a few characters o f an o therw ise excellen t cu ltivar, without a lte r in g the rem ain ing genotype. Thus the outstanding cu ltivars, often the resu lt o f a tim e-consum ing and painstaking c ro s s breeding p rogram , can be further p erfected within a reasonable tim e.

In ornam entals an additional advantage is the fact that se lection o f v is ib le changes g en era lly o ffe rs no prob lem s and a favourable change m ay soon lead to the com m erc ia liza tion o f the mutant. Th is holds true also fo r v is ib le changes in other crops, like fru it colour and spurtype in apples and pears, skin colour o f potato tubers, growth pattern, s ize , fo rm and many other d ire c tly percep tib le characters.

293

294 BROERTJES

It is th ere fo re not su rpris ing that the m a jo r ity o f the alm ost 170 p ractica l mutation breed ing p ro jects , started since 1960 as co -opera tive program s between the A ssoc ia tion Euratom - ITAL, at Wageningen and plant b reeders o r institutions, deal with vege ta tive ly propagated plants, m ostly ornamentals, but also fru it crops and potatoes.

M ost attention is paid in this paper to mutation breeding o f ornamentals because it s e rves as an excellen t m odel to dem onstrate techniques and p oss ib ilit ies and because m ost experience has been obtained using these plants.

2. PROCEDURES

A l l kinds o f plant parts a re being used fo r the irrad ia tion technique, nam ely bulbs, tubers, rh izom es, cuttings, g ra fts o r (sm all) plants, which a ll ca rry buds with multicellula-r apices, which gen era lly a re composed of a number o f fa ir ly autonomous ce ll la yers .

Since a mutation is a on e-ce ll event the irrad ia tion o f such a com plex system leads to the form ation o f ch im eras. The mutated ce ll, m oreover, is subjected to the so -ca lled diplontic selection , which is the com petition between this c e ll and the surrounding non-mutated ones. Both circum stances resu lt in a low mutation frequency o f (p eric lin a l) ch im eras (sports ). Th is ch im era form ation is the m ain stum bling-block, which can only be o v e r come by grow ing plants from single ce lls , in v itro o r in v ivo , resu lting in a h igh (er) frequency o f solid, non-ch im eral mutants.

F o r the p rac tica l plant b reeder the m ost im portant method fo r the tim e being is the adventitious bud technique, which makes use o f the phenomenon that adventitious buds, form ed at the base o f the petio le o f separated leaves , o rig inate from a s ingle ep iderm al ce ll. Th is has a lready been proven in Saintpaulia [ 1 ], Ach im enes, Streptocarpus [ 2 ], Begonia, P eperom ia and L iliu m [B ro e r t je s , unpublished], and is under investigation in other crops, such as Chrysanthemum, Endymion, Kalanchoe, M uscari, Ornithogalum and Sc i l ia .

Without anticipating the resu lts, it seem s ju stified on the basis o f data a lready accumulated to conclude that the adventitious bud technique is an im portant one, and that consequently e v e ry b reed er should apply or try to develop it, when he wants to optimiz;e the chances o f mutation breed ing in vege ta tiv e ly propagated plants. In Streptocarpus, fo r instance, com m erc ia l resu lts w ere obtained using this method in an ex trem e ly short period , less than three years from the f ir s t irrad ia tions to determ ine the rad iosen s itiv ity until the com m erc ia liza tion o f five mutants [ 3 ] .

M any plants can be propagated from adventitious p lantlets on separated leaves ; B ro e r t je s et al. [4 ] lis t over 350 species, coverin g a va r ie ty o f fam ilies , reported in the lite ra tu re to belong to this group. Th is does not mean that plants not lis ted cannot be propagated in this way; many have been tr ied without success, but many m ore have never been tr ied and the b reed er should always make an attempt with the cu ltivar in which he is in terested . He should consider variab les such as rooting-m edium (sand, peat, so il, v e rm icu lite o r m ixtu res) and environm ental conditions (tem perature, humidity, ligh t), but a lso various lea f- fa c to rs should not be overlooked.

The age o f the leaves proved to be dec is ive in Kalanchoe [ B roertjes and L e ffr in g , unpublished]; old leaves , just m ature leaves and young

T A B L E I. PRO D U CTIO N O F AD VEN TIT IO U S P L A N T L E T S ON Kalanchôe cv L E A V E S 3

V E G E T A T IV E L Y PROPAGATED PLANTS 295

(CUADRO 1. PRODUCCION DE PLANTULAS ADVENTICIAS EN HOJAS DE Kalanchoe)

cv.With petiole

AnnetteWithout petiole

cv.With petiole

JosineWithout petiole

Young leaves 22 - 68 19

Almost mature leaves 9 - 38 5

Old leaves - 1 28 13

aIn all cases 20 leaves were used. All leaves were irradiated with 2 krad X-rays. The number of plantlets was determined seven months after the start of the experiment (March 1970 - October 1970).

T A B L E II. PRO D U C TIO N O F AD VE N TIT IO U S BU LB ILS ON LE AVE S AND L E A F -P A R T S O F Ornithogalum thyrsoïdes

(CUADRO 2. PRODUCCION DE BULBILOS ADVENTICIOS EN HOJAS Y EN PARTES DE HOJAS DE Ornithogalum thyrsoïdes)

No. of leaves Length No. of bulbils(cm) per leaf

Whole leave 10 20 16.2 ±1.4

Base 20 10 6.7 ± 0.4 (larger bulbils)

Mid-piece .20 10 8. 4 ± 0.6

Top 20 10 8. 8 ± 0. 5

leaves w ere com pared. Although a ll leaves rooted read ily , young leaves produced many m ore adventitious plantlets and produced them sooner at the base o f the petio le as com pared to o ld (e r ) leaves .

The le a f petio le a lso seem s to p lay a ro le : leaves o f the K alanchoe cu ltivar 'Annette ' produced alm ost no plantlets when the petio le was cut o ff, whereas the cv, 'J os in e ' produced som e plantlets on leaves o f a ll ages, though much few er on those without a petio le (Tab le I). Th is experim ent a lso shows that adventitious plantlet production can va ry g rea tly among d ifferen t cu ltivars . One should th e re fo re not conclude that the technique w ill not work in a given species when only one va r ie ty o r cu ltivar has been tested.

The s ize o f the le a f as w e ll as the le a f part is an im portant fac to r for the number o f adventitious buds form ed in m onocotyledons. In Ornithogalum, leaves about 20 cm in length w ere com pared with lea f-tops , lea f-bases

296 BROERTJES

and m id - le a f p ieces o f approxim ately 10 cm length. F rom Tab le II it can be seen that whole leaves produce tw ice as many adventitious bulbils as any le a f p iece; bases produced the low est number, but the bulbils w ere la rg e r .

In plants with compound leaves the question a r ises whether whole leaves o r lea fle ts a re to be p re fe rred . In roses en tire leaves rooted with much m ore d ifficu lty than lea fle ts , and o f the d ifferen t lea fle ts the top lea fle t gave the best resu lts . H ow ever, neither leaves nor le a f parts form ed adventitious buds, although they rooted . When one learns how to induce bud form ation on such rooted leaves and le a f parts, these factors m ay a lso in fluence the quantitative production o f buds.

The number o f adventitious plantlets is a lso influenced by the way the leaves are used: in Streptocarpus the g rea tes t number is obtained when the two le a f halves a re planted a fter taking out the m idrib as com pared to the whole le a f with petio le .

Fu rtherm ore the use o f m odified leaves should not be overlooked. In L iliu m , bu lb-scales a re w ide ly used as propagation m ateria l. A fte r irrad ia tion and mutation induction on ly solid , non -ch im era l mutants have thus fa r been obtained [ 5 ] . The use o f these sca les, o r o f a r t if ic ia l scales (by cutting bulbs in p ieces ), o ffe rs p oss ib ilit ies in other crops, espec ia lly since the detrim enta l e ffec t o f fungi can be v e ry e ffe c t iv e ly prevented by new fungicides. M oreove r the use o f wounded bulbs, a norm al procedure to propagate Hyacinth fo r instance, looks a lso prom ising.

Another consideration is the use o f chem icals; rooting is often prom oted by the application o f a plant horm one but the factors which control adventitious bud form ation a re as yet unknown. Cyto-kin ins and other plant regu la tors certa in ly p lay a ro le but so fa r no p ractica l application is in sight.

3. O TH ER METHODS

If the adventitious bud technique is not ava ilab le o r cannot be developed, other plant parts ca rry in g m u ltice llu la r apices have to be used. But even then it is im portant to irrad ia te these at the ea r lie s t possib le developm ental stage in o rd er to g ive the mutated c e ll the g rea test possib le chance to take part in the form ation o f the shoot or plant.

In Dahlia, fo r instance, tubers should be irrad ia ted im m ed ia te ly a fter h arvest: the (in v is ib le ) buds a re in a v e ry prem ature stage and probably consist o f only a few ce lls since irrad ia tion resu lts in a certa in percentage o f com plete, so lid mutants [ 6 ] . Th is could be explained by assuming that in a certa in number o f cases a ll but one (mutated) ce lls a re k illed or inactivated, o r have lost the diplontic se lection with the rem ain ing mutated ce ll.

In A ls tro e m e r ia , stolons o f ac tive ly grow ing young plants are the best m a ter ia l; so fa r so lid mutants have been obtained alm ost exc lu s ive ly o f which two w ere recen tly com m erc ia lized [unpublished].

I f sec to rs cannot be avoided, p roper m easures should be taken to get as la rg e a sec to r as possib le by the irrad ia tion o f young(er) buds, eventually fo llow ed by frequent pruning. Th is assures em ergence o f a x illa ry buds within the secto r which then lead to ' com p lete ' mutants, in many cases being p e r ic lin a l ch im eras o r sports.

VE G E T A T IV E LY PROPAGATED PLAN TS 297

Although these methods g ive m ore d ifficu lties com pared to the adventitious bud technique, they have been successfu lly applied in many species, such as Chrysanthemum, potato, fru it trees [ 7 ] , and a few bulb crops [ 8 ].

F rom the d iscussion above one should not conclude that a so lid mutant is always the optim al endpoint. In m ost cases a mutation a ffects m ore than one character, e ither because it is accom panied by other mutated genes (linkage), o r because o f p le io trop ism . It is th ere fo re conceivab le that the favourable change is accom panied by one o r m ore unfavourable ones which would lead to an unacceptable cu ltivar. H ow ever, when the favourable m utation exp resses it s e l f in a d iffe ren t c e ll- la y e r than the unfavourable one(s), a p e r ic lin a l ch im era would be better than a so lid mutant. F o r instance, thorn less (which is expressed on ly when in L J accom panied by reduced growth (exp ressed only when in L 3) would p rov ide a usefu l mutant i f these mutated characters a re only present in the L j, whereas a solid mutant, in which the reduced growth would be in a ll la yers and th ere fo re could express its e l f in L 3, would be w orth less.

One can im agine s im ila r situations with flow er and fru it colour (exp ressed alm ost exc lu s ive ly in L j o r L 2) and a lso in the case o f cyto- ch im eras (m ixtures o f la yers o f d ifferen t p lo id y ).

4. M ISC ELLAN EO U S REM ARKS

The poss ib ilit ies o f any mutation breed ing p ro jec t depend on many m ore factors than have been d iscussed above, such as the mutagenic agent: rad iation is to be p re fe r red because chem icals gen era lly produce a much low er mutation frequency, probably as the resu lt o f poor penetration into the vegeta tive m ateria l. Neutrons produce m ore drastic chrom osom e aberrations as com pared to X -ra ys o r 7 -rays . The genotype o f the starting m a ter ia l can a lso determ ine success: number o f genes (dominant o r re c ess iv e ) con tro lling the character involved, the h eterozygos itj' and the p lo idy. Fu rtherm ore the ava ilab ility o f a d iscrim ina tive se lection method is d ec is ive fo r the success o f any breeding program and consequently also o f a mutation breed ing p ro ject. Selection fo r v is ib le characters (ornam entals) is not d ifficu lt but the se lection o f quantitative tra its (quality, y ie ld , res is tan ce ) needs a good selection method. F o r further in form ation the FAO/LAEA Manual on Mutation B reed ing [9 ] should be consulted.

5. P R A C T IC A L RESU LTS

Although mutation breed ing is a young science, the number o f p ractica l resu lts is a lready fa ir ly la rg e : Sigurbjôrnsson and M icke lis t 77 mutants in 1968 o f which 59 w ere re leased between 1962 and 1968 [ 10 ] . The actual number must, how ever, have been considerab ly h igher. The lis t is incom plete since many com m erc ia l ornam entals, fo r instance, have never been reported .

The grea t sc ien tific e ffo rt throughout the w orld as w e ll as the la rge number o f p ractica l mutation breeding p ro jects under way w ill soon provide us with a much g rea te r number o f im proved va r ie tie s .

2 9 8 BROERTJES

R E F E R E N C E S

[1] SPARROW, A.H., The use of X-rays to induce somatic mutations in Saintpaulia. Afr. VioletMag. 13fl960 .[2] BROERTJES, C., "Mutation breeding of vegetatively propagated crops", Proc. 5th Eucarpia Congr.,

Milan, 1968, 139.[3] BROERTJES, C., Mutation breeding of Streptocarpus. Euphytica 18 (1969) 333,[ 4 ] BROERTJES, C., HACCIUS, B., WEIDLICH, S., Adventitious bud formation on isolated leaves and its

significance for mutation breeding, Euphytica 1Л (1968) 321.[5] BROERTJES, C., ALKEMA, H. Y., "Mutation breeding of flowerbulbs", 1st Int. Symp. Flowerbulbs,

Noordwijk/Lisse, 1970 (in press).[6] BROERTJES, C., BALLEGO, J.M., Mutation breeding of Dahlia variabilis, Euphytica 16 (1967) 171.[7] VISSER, T., VERHAEGH, J.J., De VRIES, D.P., Pre-selection of compact mutants induced by

X-ray treatment in apple and pear, Euphytica 20 (1971) (in press).[8] HEKSTRA, G., BROERTJES, C., Mutation breeding in bulbous Iris. Euphytica 17 (1968) 345.[9] F AO/IAEA, Manual on Mutation Breeding, Tech. Rep. Ser. No. 119, IAEA, Vienna (1970) 134, 141.

[10] SIGURBJORNSSON, B., MICKE, A., "Progress in mutation breeding", Induced Mutations in Plants(Proc. Symp. Pullman, 1969), IAEA, Vienna (1969) 673.

D I S C U S S I O N

R. S . LOOM IS: What potential do tissue and single c e ll cultures have fo r m utagenesis in vege ta tiv e ly propagated species?

C. BRO ERTJES: A s I mentioned in m y paper, one o f the poss ib ilit ies to avoid the consequences o f ch im era form ation and part o f the diplontic se lection is to use in v itro methods. H ow ever, the number o f species which can be grown from single c e ll culture is v e ry res tr ic ted at present. As soon as m ore is known about the factors which control regenera tion to com plete plantlets from tissue cultures, this method opens w ide perspectives fo r p rac tica l mutation breeding. But we have to start from single ce lls , since the irrad ia tion o f a group o f ce lls w ill a lso lead to diplontic se lection and ch im era form ation .

G . T . SC AR ASC IA -M U G N O ZZA : I would like to know i f you are try in g to induce spur mutants in other species than apples o r pears, as you said. A t the C asaccia labora tory m y co lleague D r. Donini has been able to obtain a lso som e spur mutants in ch erry trees , and at the present tim e an im portant task in m y country is the induction o f spur mutations in o liv e trees . M oreo ve r , accord ing to your knowledge, in what la y e r do you think that spur mutations are induced?

C. BROERTJES: The spur types shown w ere obtained by D r. T. V is s e r at W ageningen and so fa r as I know he has not yet obtained s im ila r mutants in ch erry . Thank you fo r your in form ation about ch erry spur types; I w ill pass it on. I do not know in which la y e r the spur type expresses its e lf but by com paring with other crops it w il l v e ry lik e ly be the L 3.

R . N IL A N : I ob serve from your m anuscript that you have a penetration p rob lem with chem ica l mutagens when used on vegeta tive m ateria l. There are now reports indicating that the penetrating agent dim ethyl sulphoxide (dMSO) has helped in crease the mutation e ffec tiven ess o f chem ical mutagens.I would lik e to know i f you have used this agent along with the chem ical mutagens you have tried .

C .B R O E R TJE S : We have tr ied other chem ical mutagens only v e ry occas iona lly ; our experience as w e ll as reports from other scien tists have discouraged the use o f them so fa r . M oreo ve r , the treatm ent with ion izing

V EG E T A T IV E LY PROPAGATED PLANTS 299

rad iation o f often bulky vegeta tive m ateria l is much ea s ie r and less tim e- consuming. But i f dMSO or other chem icals have a better penetration we certa in ly m ight consider its use.

B. SIGURBJORNSSON: In connection with D r. S carasc ia 's question on ch err ies I have in form ation about two mutant ch erry va r ie t ie s . One, ca lled Compact Lam bert, was re leased in Canada in 1964. It resu lted from X -irra d ia tion o f Lam bert scions. The mutant is a true dwarf, quarter to fifth the s ize o f a norm al ch erry tre e , while fru it s ize rem ains the sam e. The other, named Stella, was re leased in 1968, a lso in Canada. It resu lts from a cross between Lam bert and a radiation-induced mutant o fJohn Innes Seedling 2420. The b reeder, D r. Lapins at Summerland, B . C . , has told m e that Stella is the f ir s t s e l f- fe r t i le sweet ch e rry va r ie ty named.

I read ily adm it that our lis t o f mutant va r ie t ie s re leased last yea r is defic ien t, e sp ec ia lly with regard to ornam entals. Could you te l l us approxim ately how many induced mutants o f ornam entals have been re leased in total?

C. BRO ERTJES: Thank you fo r your in form ation concerning the ch erry mutants.

Concerning your question, I do not have the s ligh test idea how many mutants have been com m erc ia lized , but it must be hundreds. In England many Chrysanthemum cu ltivars w ere obtained by irrad ia tion , which is also the case in the USA. The p ractica l plant b reeders to not advertise such facts fo r two reasons, f ir s t ly to protect the ir com m erc ia l in terests , and secondly because som e clients m ay be scared by a descrip tion such as "X - r a y induced sport o f . . . . " . Some people s t ill think that such m ateria l is dangerous.

C .F . K O N Z A K : R egard ing the point made by D r. N ilan that chem ical agents m ay have use in vege ta tive ly propagated species i f suitable penetrating agents a re used. A word o f caution should be expressed ,how ever, to those considering use o f d im ethyl sulphoxide or s im ila r po lar solvents, since there is a lso a v e ry serious health hazard possib le with the ir use and extrem e care should be taken in handling such chem icals. Th ere has been evidence o f uptake through human skin of m ateria ls that o rd in a r ily would not penetrate.

I m ight a lso mention as a further point about dw arf o r spur types in ch err ies that I recen tly learned from D r. Toyam a at the Washington State Experim ent Station that they have a dwarf Bing ch erry induced by irrad ia tion o f L . scions which m ay soon be re leased .

SPECIFIC OBJECTIVES OF MUTATION BREEDING

BREEDING AND SCREENING TECHNIQUES FOR SEED PROTEIN IMPROVEMENT IN CROP PLANTS

O .P . KAMRA

Laboratory o f Radiation B iology, Dalhousie University,

H a lifax , Nova Scotia, Canada

Abstraa-Resumen

BREEDING AND SCREENING TECHNIQUES FOR SEED PROTEIN IMPROVEMENT IN CROP PLANTS.The problem of protein improvement by breeding is by no means new. However, little progress

was achieved in the first half of this century in restructuring grains of crop plants for chemical composition. Increasing awareness of the nutritional requirements of monogastric animals and man, together with the development of fairly simple and cheap chemical screening methods.have created a new interest in the last decade and have stimulated a world-wide research activity. The paper attempts to summarize the remarkable progress achieved by plant breeders in recent years in improving protein composition of staple food and feed grain.

METODOS FITOTECNICOS Y SELECTIVOS PARA MEJORAR EL CONTENIDO PROTEINICO EN LAS SEMILLAS DE LAS PLANTAS DE CULTIVO.

El mejoramiento protemico por vía fitotécnica no constituye en modo alguno un problema nuevo.Sin embargo, en la primera mitad de este siglo se han conseguido pocos progresos en la labor de reestructurar los granes de las plantas de gran cultivo en cuanto a su composición química. El mejor conocimiento de las necesidades en materia de nutrición de los animales monogástricos y del hombre, junto con el desarrollo de métodos químicos de selección relativamente baratos y sencillos, ha despertado un nuevo interés en el último decenio y estimulado las actividades de investigación en todo el mundo. En la memoria se resumen los considerables progresos logrados por los fitotécnicos, en los últimos años, en el mejoramiento de la composición proteica de los alimentos básicos y de los cereales de consumo animal.

1. INTRO D U CTIO N

Although m odern agricu ltu ra l produce has been characterized by a surplus o f starch and a defic it in protein, sp ec ific chem ical substances in the seeds o f c e rea ls , legum es, c ru c ife rs and other crops (o il, fats, starch, sugar, proteins and som etim es noxious and to x ic substances), a re o f considerab le im portance to the diet and health o f man and animals'. In this respect plant proteins are o f specia l im portance to a m a jority o f the inhabitants o f the earth who ra re ly or never have access to anim al proteins, and fo r others who fo r re lig iou s o r other reasons p re fe r to obtain the n ecessary p rote in from plant sources alone.

The prob lem s o f p rote in im provem ent by b reed ing and selection are by no means new. The c la ss ica l experim ents in m a ize started by Hopkins [12] at Illin o is in 1896 have now gone through some 68 generations. It was c lea r b e fo re many yea rs had elapsed that one could indeed se lect v e ry e ffe c t iv e ly in re la t iv e ly sm all b reed ing populations fo r high and low p ro tein content as w e ll as o i l content by making successive se lec tive matings o f parents. L a te r Scholz [28] in Gatersleben, Germany, succeeded a fter repeated irrad ia tion and se lection in iso lating barley mutants with a v e ry high protein content, high d igestib ility and nutritional value. How ever,

303

3 0 4 KAM RA

litt le p rog ress was ach ieved in the f ir s t half o f this century in the re s tru c turing o f crop plants fo r phys ico-chem ica l com position through breeding. W hile there may be se ve ra l reasons fo r this lack o f p rogress , the two main reasons appear to b e ( l ) a genera l lack o f awareness o f nutritional requ irem ents o f m onogastric anim als, and ( 2 ) non -ava ilab ility o f sim ple, accurate, cheap and non-destructive m ass screen ing methods to aid in the se lection o f the d es ired genotypes.

The re a l break-through came in 1963 when the ly s in e -r ich mutants o f m a ize 1 opaque-2 ' iso la ted by Jones and Singleton in the twenties and ' flou ry -2 ' by Mumm in the th irt ie s w ere analysed chem ically by Nelson and co -w ork ers [25 ]. M ore recen tly H agberg et al. [8 ] have identified 1 H ip ro ly 1 gene in b a r ley fo r high lysine. Op-2, fl-2 and h ily a re m a jor genes with a c lea r segregation pattern; they are easy to use in recom bination work and distinct with rega rd to m orphology and am ino-acid pattern. Not only lys in e but seve ra l other des ired am ino-acid changes are also brought about through the action o f these genes. These d iscover ies have ra ised hope that additional genetic varia tion necessary to im prove protein quality w ill be found. Th is paper attempts to sum m arize the rem arkab le p rog ress ach ieved by plant b reed ers in recen t y ea rs in im proving protein com position o f seed. F o r detailed analysis o f a ll aspects o f plant protein im provem ent and the developm ent o f appropriate screen ing methods re fe ren ce is made to the P roceed in gs o f two recen t FAO /IAE A m eetings [14 ,26 ].

2. BREED ING AN D SCREENING FO R SEED PR O TE INIM PR O V E M E N T

2.1 . Seed p rote in fractions

Seed proteins consist o f s e v e ra l fractions o f d iffe r in g am ino-acid com position. The Osborne method o f fractionation [27] based upon solub ility in (a ) w ater (album in), (b) 0. 5M N aC l (g lobu lin ), (c ) 50% propanol/water m ixture (p ro lam in ), and (d) 0. 1M K O H (g lu te lin ), r e flec tin g ex trem e ly d ifferen t ph ys ica l-ch em ica l ch aracteris tics o f the fractions, is s t i l l in genera l use. M ost o f the p rote in in the ce rea l seed is stored in the endosperm (up to 80% in m a ize ) and to a sm a ller extent in the em bryo. The em bryo proteins a re r ich in albumin and globulin fractions having a balanced am ino-acid pattern, presum ably requ ired during seed germ ination and growth o f the ea r ly seedling.On the other hand, endosperm proteins, which a re inert m etabolica lly , have v e ry poor am ino-acid pattern. P ro lam in s which constitute nearly 50% o f the seed p rote in a re defic ien t in m ost amino acids, containing only n eg lig ib le amounts o f essen tia l amino acids, such as lysine and tryptophane, while glutelins contain somewhat la rg e r proportions o f essen tia l amino acids [29 ]. In the developm ent o f endosperm tissue, glutelins predom inate at f ir s t while prolam ins increase during gra in maturation. It is possib le to a lte r the seed p rote in quantity and quality through cu ltural p rac tices and genetic manipulation. H igher protein le v e ls , ach ieved by increas ing fe r t i l i z e r application, invariab ly y ie ld increased p ro lam in fra ction [4 ], without increas ing the content o f m ore des irab le globulin and albumin fraction [29 ], resu lting in proteins o f

SEED PROTEIN IMPROVEMENT 3 0 5

low er b io lo g ica l value. Genes op-2 , fl-2 and h ily b ring about th e ir desirab le e ffec ts ch ie fly through m odifications o f the re la t iv e amounts o f endosperm protein fractions, i. e. a partia l suppression o f the p ro lam in and its rep lacem ent by other fractions which are r ich e r in lys ine and tryptophane. Significantly, no new o r d ifferen t proteins are synthesized. Hence the m ost e ffe c tiv e way o f a lte r in g am ino-acid com position o f seeds with a high concentration o f a lcoho l-so lub le proteins (co rn , sorghum, m ille t, wheat, b a r ley and rye ) is through mutations which reduce the concentration o f synthesized p ro lam in and at the same tim e a llow increased synthesis o f other protein fractions. In instances where no such m ajor genes a re known, as in hexaploid wheat o r crops with a lim ited natural va ria tion in p rote in (e . g. C ic e r arietinum , Cajanus ca jan ), the induced m utagenesis approach has to be attempted. Bhatia et al. [2] and Dumanovic et al. [3] w ere able to detect varia tion in m a jor protein fractions and to ta l p rote in as w e ll as lysine in M 2, M3 and M 4 s e g re gating populations o f hexaploid wheat a fter gam m a-ray, E l and EMS treatm ents.

Once they a re recogn ized , the m a jor genes such as op-2 and h ily can be incorporated in h igh ly-b red stock, i. e. in new genetic backgrounds, producing m arked im provem ent in protein [8 , 25, 32 ]. It is in teresting to note that fo r both op - 2 and f l - 2 mutations in a number o f d ifferen t backgrounds, the em bryo s ize is in creased s ign ifican tly o v e r that in com parable strains ca rry in g the norm al a lle le s [25 ].

2.2. Seed em bryo

Another im portant change would be to in crease in cerea ls the s ize o f the em bryo re la tiv e to the s ize o f the endosperm , since in a ll seeds the em bryo p rote in is o f high nutritive value. Incidentally, the question o f whether the em bryo too, and not only the endosperm , can be strik ing ly changed in its am ino-acid com position is s t ill open, and is one o f fundam ental im portance. A certa in amount o f varia tion in the protein content and com position may be to lera ted in the aleurone la ye rs as w e ll as in the scutellum . W hether there is some storage p rote in in the scutellum o r whether it is a ll o f a functional nature is not yet known. L a rg e r s ize o f the em bryo is particu la rly im portant where whole seed is consumed in the diet o r feed , fo r exam ple, consumption o f unleavened whole wheat bread (chapattis) by a la rge population in India, feed gra in (b a r le y and m a ize ), etc.

2.3. A leu rone la yers

The seed aleurone is a p ro te in -r ich la ye r o f good nutritional quality.The aleurone la ye r is one c e ll thick and bounds the endosperm in ce rea l seeds. H ow ever, va ria tion o f this character is known. W o lf et al. [36] found in an investigation o f Am y lose extender stocks that the apparent basis o f th e ir high protein content was the existence o f seeds with m ultiple aleurone la ye rs . B a r ley seeds may have up to th ree la ye rs o f c e lls in the aleurone w hereas in r ic e the number va r ie s from one to fiv e c e ll la yers .It certa in ly seem s possib le to se lect fo r v a r ie t ie s with a spec ific number o f c e ll la yers in the aleurone.

306 KAM RA

Another re la ted approach to th is prob lem is to increase genetica lly the surface area o f seed which should lead to an increase in aleurone la yer , resu lting in increased content o f good quality protein . N elson [25] is in the p rocess o f combining m ultiple aleurone la yers with fa c to rs which condition increased surface area (p is tilla te in flo rescen ce ) o r in trin s ica lly high p rote in content ( op-2 ) in m aize. P lant b reed ers should note that in situations where m a jor am ino-acid shifts im pose drastic res tr ic tion s on p erm iss ib le changes, it is s t ill possib le to find in teresting and somewhat ingenious means o f circum venting these res tr ic tion s .

2.4. Sub-aleurone la ye rs

In r ic e there is a considerable concentration of protein in the aleurone and sub-aleurone la yers o f the grain. Because o f this pecu liar distribution a m a jor portion o f the protein is lost through the rem ova l o f outer la yers o f the endosperm and com plete loss o f em bryo during m illin g and polish ing [17 ]. By a combination o f m icroscop ic scoring o f sectioned r ic e gra in and m icro -D B C tests on part o f the grain, a la rge co llection o f strains has been screened and a great amount o f natural varia tion found by Kaul et al. [17 ]. The restructu ring of r ic e gra in to obtain a m ore uniform distribution o f p rotein in the endosperm through genetic means may soon be rea lized . Since the protein e ffic ien cy ra tio is high in r ic e , an increase in the to ta l protein content coupled with a better distribution would be of m a jor s ign ificance in enhancing the nutritional value o f this important crop.

2. 5. F re e amino acid in the seed

M ost seeds contain v e ry litt le fr e e amino acid. The production o f any amino acid is regu lated to correspond c lo se ly to the demand fo r it in protein synthesis and other reactions. Hardly anything is known about the regulation o f am ino-acid synthesis in h igher plants, an aspect which has been studied in some deta il in m icro -o rgan ism s [34 ]. И the gene-cod ing fo r the production o f a key regu la tory enzym e was to mutate in such a way that it no longer recogn ized the usual cu t-o ff signals, it could resu lt in over-production o f a particu lar amino acid. No c lea r-cu t mutation o f this type has been recogn ized in h igher plants, though the poss ib ility cannot be ruled out.

2.6. Selection c r ite r ia

The weight and volum e o f the grain are subject to strong environm ental in fluences and are negatively corre la ted with the percentage o f protein , whereas absolute values va ry to a le s s e r degree. T h ere fo re ' protein content p er seed 1 is a m ore stable c r ite r ion and less influenced by the environm ent than 1 p rote in per unit dry w eigh t’ [5] . W here the se lection is based upon the amount o f to ta l n itrogen (K je ldah l) p er unit dry weight, se lection may take p lace fo r decreased synthesis o f non-nitrogenous com pounds (v iz . carbohydrates) and not n ecessa rily fo r a high protein leve l.In fact this is the lesson to be learned from the lon g-term selection ex p e r iments leading to the Illin o is High P ro te in stra in o f m a ize [25 ]. Selection

. on the basis o f N/seed (p e r plant) is not only reasonable but necessary. O therw ise high p rote in percentage may be ach ieved at the expense o f y ie ld .

SEED PROTEIN IMPROVEMENT 307

Screen ing fo r seed protein also invo lves the prob lem o f co rre la tion between whole gra in test and part grain test, and protein ch aracteris tics in the flou r and in the polished grain, particu larly in single seed selection .In the case o f r ic e , se lection values should be determ ined on brown r ic e since degree o f lo ss upon polish ing may not be con tro lled accurately from one experim ent to another.

V aria tion to the extent o f 2. 5% in protein content was found between t i l le r s o f the same plant in wheat [2] . Late t i l le r s genera lly show re la t iv e ly h igher protein and gliad in content and should be avoided. S im ila r ly , the number o f seeds p er spike is found to influence the p rote in content, and even the position o f seeds on the spike may a ffect the values. It may be n ecessary to d iscard the top and bottom seeds in the spike to obtain m ore uniform analysis.

We should consider adopting a concept o f 1 crop p rote in y ie ld ', e x pressed , fo r exam ple, as kg protein/ha, to determ ine the value o f mutant lin es in provid ing p rote in to the food supply. Using the data o f Tanaka and Takagi [33 ], the prom is ing r ic e mutant No. 5 with approxim ately 90% h igher p rote in content', even a fte r correc tion fo r re la t iv e y ie ld com pared with its parent, gave a re la tiv e protein y ie ld o f 180% in com parison to the o r ig in a l va r ie ty . H ere re la tiv e protein y ie ld as m g protein/plant was calculated from (num ber o f panicles/plant) X (num ber o f grains/panicle) X (w eight/gra in ) X (p ro te in content/unit weight).

2.7. M aternal influence

One point o f considerab le in terest in choosing se lection procedures is the question o f whether the amount o f p rote in synthesized by a seed is conditioned by its own genotype o r by the genotype o f the plant on which it is borne. Influence o f m aternal genotype on p rote in content o f seed in m aize [25] and b a r ley [5] has been indicated.

In leguminous species, such as peas, beans and vetches, the re s e rv e food m a teria ls are stored in the cotyledons. The genotype o f the seed tissue is exactly like the genotype o f the plant develop ing from the seeds. Hence m a jor changes in the chem ical com position o f cotyledons are apparently unaffected by the m other plant, as evidenced by segregation fo r starch versu s am ylodextrin storage in d ifferen t seeds o f the same pod on a heterozygous pea plant. D rastic changes in the proportions of the storage proteins in legum es (cotyledons) may noj be v iab le to the same extent as changes in endosperm protein o f cerea ls . N everth e less m a jor sh ifts in am ino-acid pattern have been detected in induced mutants o f P isum by Gottschalk et al. [7] , and Zokhrabian and S idorova [31 ].

In ce rea ls , the situation is m ore complex. In diploid species (e . g. m aize, r ic e , barley), the endosperm is tr ip lo id and the em bryo is diploid. Both tissu es can be genetica lly d ifferen t. The phenotypic expression in the endosperm w ill depend on the exp ress iv ity o f the particu lar gene. In addition, dosage e ffec ts w ill be observed . M aternal in fluences on endosperm com position w ill be m ore frequent than on the com position o f the em bryo. In spite o f th is expectation, Munck [24] observed c lea r s eg rega tions fo r h ily character in b a r ley endosperm with no suggestion o f m aternal influences.

In a llo tetrap lo id plants such as durum wheat o r an a llohexaploid bread wheat it can be assumed that basic genes fo r p rote in quality and quantity

3 0 8 KAM RA

are present in a ll genom es o f an allopolyp lo id . T h e re fo re single gene mutations with drastic e ffec ts on am ino-acid com position would be r a re r as com pared to diploid species. H ere the solution may lie in a r t if ic ia lly inducing m icro-m utations fo r re la t iv e amounts o f endosperm protein as dem onstrated recen tly by Bhatia et al. [2 ] and Dumanovic et al. [3] .

2 .8 . M acro-m utations versu s m icro-m utations

Which type o f mutations, m acro-m utations o r m icro-m utations, o ffe r the best chance o f im proving protein quality in crop plants? We can only say that there appears to be a reasonable chance o f producing protein im provem ents in mutants which have undergone d rastic m orphologica l changes. Th is approach has been successfu l in r ic e [ 9,33] , peas [7 ,3 1 ], V ic ia (Â k erb e rg , unpublished) and ba rley [16 ]. Related experim ents on m icro-m utations in wheat a re reported by Bhatia et al. [2] and Dumanovifc et al. [3] . Long range m odel experim ents are in p rog ress at the IA E A Labora to ry at S e ibersdorf [16 ], designed to shed some light on the re la t iv e ro le o f m acro - versus m icro-m utations in breed ing fo r quality in durum and bread wheats.

3. SCREENING METHODS

Rapid m ass screen ing techniques fo r analyses o f protein and amino acids which determ ine nutritional value ( e. g. lys ine, methionine, tryp to phane), as w e ll as fa c to rs which a ffect th e ir u tiliza tion (e . g. tox ic substances, d igestib ility ), a re urgently needed. F o r the determ ination o f am ino-acid com position, ord inary methods are fa r too elaborate, tim e-consum ing and, as a consequence, expensive.

3 .1 . Crude protein

Crude protein has been conventionally determ ined using the K je ldah l method, and a m ic ro -K je ld ah l method can be em ployed fo r single grain analysis. The Dumas combustion method has a lso been em ployed, but the Low ry method appears to be unsuitable (Lu se , personal communication). C o lo r im e tr ic methods include the Udy procedure and dye binding capacity (D BC ). H ow ever, DBC appears to be negative ly corre la ted to crude protein .

3.2. Dye binding methods

The DBC methods fo r bulk sam ples [23, 24] and m icro -D B C fo r single g ra in o r part o f a gra in [17] have been fu lly described . DBC has the added advantage that the values are highly co rre la ted to basic am ino-acid content and consequently lys ine content. The methods have been successfu lly em ployed with ba rley , wheat, oats, rye , rye-w heat, m aize, sorghum, Phaseolus aureus, Cajanus cajan, and m ille ts (Penn isetum typh iodes). H ow ever, they have som e inherent e r r o r s which must be kept in mind:( 1 ) values depend upon the weight of the sam ple used, necessitating the use o f a co rrec tion fac tor, ( 2 ) values a re strongly a ffected by heat, and (3) by the content o f fat and o il (e . g. in soybeans, m a ize ). The methods


are ex trem e ly sim ple, accurate, rapid, inexpensive and non-destructive.They a re recom m ended to plant b reed ers fo r in itia l screen ing together with the K je ldah l o r m ic ro -K je ld ah l methods.

The DBC method em ploys the azosulphonic dye, A c ilane Orange G.The use o f A c ilan e V io le t has been suggested [29] since it binds m ore to the g lu telin frac tion o f the endosperm protein. Since it is considered des irab le to reduce the content o f the a lcohol-so lub le p ro lim ine, a dye method to screen against prolam ine (r ic h in glutam ic acid) should also fa c ilita te the se lection work.

Another method under investigation fo r lys ine estim ation is based on the use o f lys ine decarboxylase on protein hydro lysates and the conversion o f the amine into the corresponding coloured com plex with l- f lu o ro -2 , 4- din itrobenzene [29 ].

3.3. M icroscop ic methods

Kaul has successfu lly used se ve ra l conventional h istochem ical tech niques to study the d istribution o f p rotein and amino acid in the sub-aleurone and in te r io r o f the r ic e gra in [17 ]. F o r the estim ation o f the to ta l protein, brom ophenol blue was most suitable but Orange G, Sakaguch i's reagent. A lloxan , Xylid ine, N inhydrin, Am ido-b lack and N igros in e w ere also tried . S ites fo r the accumulation o f lys in e w ere stained redd ish -purp le by d in itro- fluorobenzene, those fo r tryptophane, purple by N (l-n a p th y l) ethylene diam ine, and s ites containing sulphur (m ethionine and cystine) w ere detected by a m ixture o f DDD (2, 2' -d ih yd roxy-6 , 6 ' dinaphthyl disulphide) and 95% ethanol. A pos itive and lin ear co rre la tion between the section sco re and the p rote in o f gra in (DBC) was found [17 ]. Unfortunately, these techniques m ay be o f lit t le use in wheat, b a r ley and m aize where our own w ork indicates that p rote in distribution is m ore o r less uniform in the endosperm .

Munck [24] has developed a quick and sim ple m icroscop ic technique to study the adhesion o f starch to protein . The sam ple is taken from an incision at the end o f the endosperm , placed on a slide and stained with one drop o f A c ilan e Orange solution. Since in h ily ba rley adhesion o f protein to starch appears to be the m ost c r it ic a l character, he was able to c la ss ify segrega tin g progeny ea s ily w ith the aid o f the m icroscope. It is a lso im portant to note that va ria tion in gross m orphology, as has been found by section ing dry seeds from the w orld barley co llec tion with an ord inary m icrotom e, is not always accom panied by va r ied am ino-acid com position [24 ].

One way to quantify and im prove the h is to log ica l techniques is to use p ro te in -sp ec ific rad ioactive dyes, study the p rote in d istribution pattern under the m icroscope and at the same tim e m easure the rad ioactiv ity in the un iform ly cut c ro ss -sec tion s by means o f a conventional liquid s c in tilla tion counter. Th is approach has not been tr ied as yet.

3.4. M orph o log ica l methods

In soybeans, on the basis o f the d ifference in density o f the o il and nono il portions o f the seed, it has been possib le to c la ss ify seed from single plants in a segrega tin g population and thereby se lect plants with seed rich in o i l o r in p rote in [10 ]. The density separation can be e ffe c t iv e ly u tilized

3 1 0 KAM RA

by the soybean b reed er as a coarse screen ing to increase the frequency o f plant p rogen ies with high protein since the method is also non-destructive.

Screen ing fo r b igge r em bryo and a lteration in the em bryo-endosperm ratio as w e ll as fo r the number o f c e ll la y e rs in the aleurone are other m orpho log ica l c r ite r ia .

3. 5. Ph ys ica l methods

Nuclear m agnetic resonance (N M R ) spectroscopy has been u tilized e ffe c t iv e ly to c la ss ify seeds o f single plants o f ea rly -gen era tion s e g re gating populations fo r o il in soybean [10 ]. The use o f NM R o f hydrogen fo r the estim ation o f the o il content o f o il- r ic h gra in has been developed by the Yu goslav group under the d irection o f P ro fe s s o r B lincs. Th is favourable situation occurs because o i l is highly m obile and thus the proton N M R lin es are v e ry narrow , y ie ld in g la rge signals, and may be ra ther eas ily quantified. In o il- r ic h gra in there is a negative corre la tion between o il and p rote in content. In n on -o il-r ich seeds, NM R o f n itrogen appears to o ffe r litt le prom ise fo r determ in ing protein quantity and quality [18 ].

Many elem ents are cu rren tly determ ined by neutron activation analysis. K osta et al. [19] have shown that activation analysis o f n itrogen in wheat gra in with 15-M eV neutrons (14N (n , 2n)13N reaction ) g ives resu lts in ve ry good agreem ent with the standard K je ldah l procedures.

N itrogen determ ination in seed by d irect nuclear reaction (14N(p, d)13N) has also been studied [15 ].

X -ra y photoelectron spectroscopy o ffe rs a method o f high potential fo r estim ating the quantity and quality o f gra in protein. By this method a ll e lem ents across the p eriod ic table may be estim ated. P re lim in a ry experim ents [18] indicate that ( 1 ) to ta l p rote in may be estim ated by quantitative determ ination o f the n itrogen peak, ( 2 ) am ide n itrogen in lys in e- and a rg in in e -r ich p rote in may be distinguished from amino nitrogen, and (3) sulphur content may be estim ated by observ ing the sulphur photoele ctrons.

3.6. O ther methods

S evera l other analytica l p rin c ip les are applicable to the analysis of p rotein quantity and quality but none o f these has been applied to the p ra c t ica l prob lem o f plant seed analysis. These have been discussed by Luse [20] and include: (1) double isotop ic labelling and T L C separation o f spec ific amino acids, ( 2 ) rad io iso top ic assay o f carbon dioxide from the action o f decarboxylase on spec ific amino acids, and (3) u tiliza tion o f a 1 4C -labe lled energy source in the m icrob io lo g ica l determ ination o f sp ec ific amino acids.

It should be rem em bered that s e ve ra l screen ing methods discussed above need fu rther developm ent and standardization. It would certa in ly be worth while to have seve ra l o f the m icroscop ic and dye binding methods com pared and standardized fo r routine plant breed ing work, fo r m arketing and control. A s fa r as is known no labora tory is engaged in this important endeavour.


S evera l plant species which a re otherw ise good as p rote in sources, produce and store tox ic, noxious o r bad-tasting substances in th e ir seeds, leaves o r other organs, rendering the ir consumption harm ful to man and anim al. The p rote in in other species (e. g. Paspalum , Setaria , E leucine) has low d iges tib ility due to a fibrous envelope in the seed [32 ]. Reducing fib re content in the seed o f such species should th ere fo re constitute an im portant goal o f breeding.

The cruciferous and leguminous fam ilies often contain noxious substances such as g lucosinolates, alkaloids and glucosides, which can be elim inated through genetic means. Lupinus species produce severa l alkaloids and the u tiliza tion o f these p ro te in -r ich plants was not at a ll feas ib le until Von Sangbusch ( 1942) perfo rm ed successfu l screen ing e x perim ents fo r spontaneous mutants, fr e e o f a lkaloids. M elilo tus albus, the white sweet c lo ver , contains a glucoside o f the о -oxycinnam ic acid which converts into coum arins and the tox ic dicoum arol. Through induced m utagenesis with chem icals and ion izing radiation it has been possib le to se lec t fo r non-b itter mutants [2 2 ].

In India, the consumption o f Lathyrus sativus has been associated with the d isease, la thyrism . Lathyrism , which cripp les the a ffected individuals and renders walking im possib le, is now known to be caused by a neurotoxin, (3 - (N )-oxa ly lam in o -L -a lan in e , present in the seeds o f L . sativus. The pulse is cu ltivated on nearly five m illion acres because o f its high drought res istance. Success in iso la ting induced mutants p ra c t ica lly devoid o f the neurotoxic p rin c ip le has been reported by Swaminathan et al. [32] . The incidence o f p e llagra in populations consuming sorhum has been co rre la ted to the abnorm ally high leu c in e-iso leu c in e ratio in the protein . C lea r ly the p ro lam in frac tion is responsib le fo r the serious am ino-acid im balance in th is m ille t.

B ra ss ica napus, B. cam pestus, B. júncea and E ruca sativa are w idely cu ltivated o il crops. T h e ir seed m eal and green m atter are valuable protein sources fo r anim al feed but have not been used because o f the high concentra tion o f g lucosinolates, which y ie ld degradation products with toxic e ffec ts on anim als and, through the m ilk, on children. Rapid and accurate se lection methods based on spectrophotom etry o r gas chrom atography o f degradation products are now on hand to aid in the im provem ent o f these species.

A m a jo r protein o f the so lven t-extracted cotton seed protein concentra te is unfit fo r consumption due to the p resence o f the toxin gossypol.The introduction o f ' g landless ' genes, found in some w ild va r ie t ie s of cotton, into com m erc ia l v a r ie t ie s has helped to produce go ssyp o l-free protein concentrate.

E ffo r ts are a lso being made to develop va r ie t ie s o f peanuts res istan t to the mould A sp erg illu s flavus, which is responsib le fo r the production o f aflatoxin. These toxins are m ost potent in low (com pared to high) protein diets fed to monkeys [ 2 1 ].

In o rd e r to obtain non-toxic va r ie t ie s a mutation breed ing p rogram has been started in A u stra lia on trop ica l fo rage legum es, Leucaena leucocephala, which contains delipatory amino acid m im o sine, and Ind igo fera spicata, which contains the live r-d am ag in g amino acid indo- spicine [ 1 1 ] .

4. SOME DELETERIOUS SUBSTANCES IN PLA N T SPECIES

3 1 2 KAM RA

It is known that la rge amounts o f rye in the diet depress growth o f anim als (p ig , chicken, hen, rabbit, cow, horse, sheep). Growth depression in ra ts and swine fed on high le v e ls o f rye has been attributed to the presence o f 5 -a lky l reso rc in o ls in the aleurone la ye r [35 ]. Wheat and ryewheat have low er amounts o f the sam e group o f compounds, the sign ificance o f which fo r humans has not as yet been evaluated. Dr. B resan i, IN C A P , Guatemala, has reported the ex istence o f an anti-enzym e fac to r in black seed-coated Phaseolus vu lgaris with deleterious e ffec ts on the growth o f m ice. The anti-enzym e fa c to r is p a rtia lly o r com pletely e l i m inated by cooking the beans and is not present in beans with white seed coat.

The present paper rev iew s some o f the aspects and p rov ides a number o f exam ples o f the u tility o f plant breed ing in im proving the nutritional quality o f food crops in the past few yea rs . It is c lea r that genetic and b iochem ica l means are at hand to ach ieve the im portant ob jectives .

A C K N O W L E D G E M E N T S

Financia l assistance o f the National R esearch Council o f Canada and the Facu lty of Graduate Studies, R esearch Developm ent Fund, Dal- housie U n ivers ity , is gra te fu lly acknowledged in the preparation o f this manuscript.

R E F E R E N C E S

[1] BÁLINT, A ., MENYHÉRT, Z . , SUTKA, J,, KOVACS, Magda, KURNIK, E., "Increasing the protein content of maize by means of induced mutations". Improving Plant Protein by Nuclear Techniques (Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 77.

[2] BHATIA, C. R., JAGANNATH, D.R., GOPAL-AYENGAR, A.R., "Induced micro-mutations for major protein fractions in wheat", Improving Plant Protein by Nuclear Techniques (Proc. Symp.Vienna, 1970), IAEA, Vienna (1970) 99.

[3] DUMANOVIC, J., EHRENBERG, L ., DENIC, M ., "Induced variation of protein content and composition in hexaploid wheat, Improving Plant Protein by Nuclear Techniques (Proc. Symp. Vienna, 1970),IAEA, Vienna (1970) 107.

[4] EWALD, E., WENZEL, G., Effects of nitrogen fertilization on protein composition and amino-acid content of wheat grain, Qualitas PI. Mater, veg. 14 (1967) 98.

[5] FAVRET, E. A., MANGHERS, L ., SOLARI, Rut, AVILA, A ., MONESIGLIO, J. C. , "Gene control of protein production in cereal seeds", Improving Plant Protein by Nuclear Techniques (Proc. Symp.Vienna, 1970), IAEA, Vienna (1970) 87.

[6] GOLOVCHENKO, V. I . , SOLONENKO, L. P., CHERNY, I. V., KHVOSTOVA, V. V ., TROFIMOVA, O.S., "Amino-acid composition of protein in radiation-induced varieties of Lupin for fodder and in economically valuable mutants of spring wheat", Improving Plant Protein by Nuclear Techniques (Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 149.

[7] GOTTSCHALK, W ., MÜLLER, H ., "Monogenic alteration of seed protein content and protein pattern in X-ray induced Pisum mutants", Improving Plant Protein by Nuclear Techniques (Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 201.

[8] HAGBERG, A., KARLSSON, K.-E., MUNCK, L ,, "Use of Hiproly in barley breeding", Improving Plant Protein by Nuclear Techniques (Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 121.

[9] HAQ, M.S., CHOUDHURY, N., RAHMAN, М. М., "Breeding for high protein content and qualityof rice through induced mutation", Improving Plant Protein by Nuclear Techniques (Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 63.

SEED PROTEIN IMPROVEMENT 3 13

[10] HARTWIG, E.E., "Breeding soybeans for high protein content and quality", New Approaches to Breeding for Improved Plant Protein, (Proc. Panel, Rôstânga, 1968), IAEA, Vienna (1969) 67.

[11] HEGARTY, M. P., Personal communication.[12] HOPKINS, C.G., Improvement in the chemical composition of corn kernel, Univ. 111. Agrie.

Exp. Station Bull. 55 (1899).[13] HUELSEN, W.A., GILLIS, M .C., Inheritance of kernel arrangement in sweet corn, Univ. 111.

Agrie. Exp. Station Bull. 320 (1929) 299.[14] FAO/IAEA, Improving Plant Protein by Nuclear Techniques (Proc. Symp. Vienna, 1970), IAEA,

Vienna (1970).[15] JOHANSSON, A., LARSSON, B», TIBELL, G. , EHRENBERG, L ., "Possibilities of nitrogen deter

mination in seeds by direct nuclear reactions", New Approaches to Breeding for Improved Plant Protein (Proc. Panel, Rôstânga, 1968), IAEA, Vienna (1969) 169.

[16] KAMRA, O.P., Unpublished.[17] KAUL, A.K., DHAR, R.D., SWAMINATHAN, M.S., "Microscopic and other dye-binding techniques

of screening for protein in cereals", Improving Plant Protein by Nuclear Techniques (Proc.Symp.Vienna, 1970), IAEA, Vienna (1970) 253.

[18] KLEIN, M.P,, KRAMER, L.N ., "Estimation of protein quantity and quality by X-ray photoelectron spectroscopy", Improving Plant Protein by Nuclear Techniques (Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 243.

[19] KOSTA, L ., RAVNIK, V., DUMANOVlé, J., "Determination of nitrogen in plant seeds by fast neutron activation analysis", New Approaches to Breeding for Improved Plant Protein (Proc. Panel, Rôstânga, 1968), IAEA. Vienna (1969) 161.

[20] LUSE, R. A ., "Mass screening for specific amino acids by nuclear chemical, biological and enzymatic techniques", Improving Plant Protein by Nuclear Techniques,(Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 237.

[21] MADHAVEN, M.F., Effects of dietary protein level on susceptibility of monkeys to aflatoxin injury, Indian J. med. Res. _53 (1965) 984.

[22] MICKE, A., Eine bitterstoffreine Mutante bei Melilotus albus nach Bestrahlung von Semen mit thermischen Neutronen, Naturwissenschaften 49 (1962) 332.

[23] MOSSBERG, R., "Evaluation of protein quality and quantity by dye-binding capacity: A tool in plant breeding", New Approaches to Breeding for Improved Plant Protein (Proc. Panel, Rôstânga, 1968) IAEA, Vienna (1969) 151.

[24] MUNCK, L ., "Increasing the nutritional value in cereal protein: Basic research on the hily character", Improving Plant Protein by Nuclear Techniques (Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 319.

[25] NELSON, O.E., "The modification by mutation of protein quality in maize", New Approaches to Breeding for Improved Plant Protein (Proc. Panel, Rôstânga, 1968), IAEA, Vienna (1969) 41.

[26] FAO/IAEA, New Approaches to Breeding for Improved Plant Protein (Proc. Panel, Rôstânga, 1968),IAEA, Vienna (1969).

[27] OSBORNE, T.B. , The Vegetable Proteins, 2nd Edn, London (1924).[28] SCHOLZ, F ., Versuche zur zuchterischen Steigerung des Eiweissgehalts der Gerste mit Hilfe der

experimentellen Mutationauslosung, Qualitas PI. Mater, veg. 6 (1960) 276.[29] SCHON, W.J., Analyses of proteins and amino acids in protein-rich barley strains, Improving

Plant Protein by Nuclear Techniques (Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 265.[30] SENGBUSCH, R. von, SÜsslupinen und Ôllupinen, Landw. Jbr 91 (1942) 720.[31] ZOKHRABIAN, R, P., SIDOROVA, К. K ., "The use of stimulating and mutagenic does to increase

protein content and improve protein quality in maize and peas", Improving Plant Protein by Nuclear Techniques (Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 217.

[32] SWAMINATHAN, M .S., NA1K, M.S., KAUL, A .K ., AUSTIN, A., "Choice of strategy for the genetic upgrading of protein properties in cereals, millets and pulses", Improving Plant Protein by Nuclear Techniques (Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 165,

[33] TANAKA, S., TAKAGI, Y ., "Protein content of rice mutants", Improving Plant Protein by NuclearTechniques (Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 55,

[34] VOGEL, H.J., VOGEL, R. H., Regulation of protein synthesis, A. Rev. Biochem. 36 (1967) 519.[35] WIERINGA, G. W., On the Ocurrence of Growth Inhibiting Substances in Rye, Weenman, Wageningen

(1967).[36] WOLF, M.J., ZUBER, M.S., HELM, J.L., Multiple aleurone layering in maize, Maize Genet,

Co-op. Newsl. 43 (1969) 121.

3 14 KAMRA

H. D O LL: You said that DBC is often negatively co rre la ted with n itrogen content, I think this is only the case when DBC is expressed on a protein basis ; i f DBC is expressed on a dry m atter basis one finds a strong pos itive corre la tion .

You fu rther mentioned that m orphologica l mutants often have a higher p rote in content. I would like to ask you whether you know some particu lar mutants which norm ally have a h igher protein content.

O. P. K AM R A : It would be d ifficu lt to say i f one m orpholog ica l type is m ore associated with higher protein than the other. However, we know o f s e v e ra l exam ples ( see text o f the paper) where scientists have checked th e ir co llections o f m orpholog ica l mutants fo r th e ir protein o r lys ine content and found rather high frequency o f m orpholog ica l mutants with a higher protein quantity o r quality. In our own work we found five mutants with s ign ifican tly h igher protein content in a rep lica ted test and four mutants with quite high DBC values. In the fo rm e r case four o f the five mutants w ere c la ss ified as erec to id es by P ro fe s so r M ikaelsen at Se ibersdorf, IAEA . O f course from this sm all sample we should not say that there is any particu la rly strong co rre la tion between high protein and any specia l m orpholog ica l characters.

A . HAGBERG: I would like to mention that at Svalôf, Dr. Sjodin has obtained a la rge s e r ie s o f mutants se lected fo r m orphologica l characters; some o f these have proved to be grea tly im proved with regard to protein having about 5%-units h igher protein content.

O. P. K A M R A : Thank you ,M r. C hairm an ,for this inform ation.G. T . SC AR ASC IA -M U G N O ZZA : I would like to add to the numerous

re fe ren ces g iven in your v e ry useful rev iew paper, in form ation regard ing a m acro-m utation found in the co llection o f our durum mutants. Six o r seven y ea rs ago, A van zi and B ozzin i studied a mutant line, which was ca lled "d e fec tive endosperm " since it showed a notched caryopsis , in which there was a change in protein content and in lip id content, but with a s ign ifican tly low er gra in y ie ld .

O .P . K A M R A : Thank you, P r o f . Scarascia . Yesterday, Dr. Swaminathan a lso mentioned in th e ir high protein barley the ' notched' characteris tic .I f you w ill re ca ll, sh rive lled seeds seem to have higher protein content.Even in h ily I am not too sure if shrinking o f the gra in is in any way re la ted to h igher protein in a ll these cases.

R. D. BROCK: W ith rega rd to the suggestion you make in your paper regard in g the poss ib ility o f increasing the fr e e am ino-acid com position o f the seed. In our labora tory we a re attempting to se lect mutants which are insensitive to the norm al control system . Th is is a p a ra lle l study o f lys ine b iosynthesis con tro l system s in E sch erich ia co li and higher plants.We a re attem pting to u tilize the technique o f se lectin g in the p resence o f a growth inhibiting lys ine analogue. We have concentrated upon the E^ co li phase and have obtained lys ine secreting strains but so fa r these have only been obtained as the resu lt o f two successive mutational events. U ntil we obtain m ore knowledge o f the control system in E_. c o li and the mutation prospects we w ill not com m ence screen ing o f h igher plants.

O. P . K A M R A : Th is is potentially a most im portant means o f in creasing the content o f a particu lar fr e e amino acid in the seeds and we should look fo rw ard to new developm ents in the study o f control system s in higher plants.

D I S C U S S I O N

SEED PROTEIN IMPROVEMENT 31 5

H. SM ITH : Th ere is a grow ing lite ra tu re on the mutation and in h er itance o f iso zym es which, by definition, are changes in proteins and enzym es. Hopefully, there m ay be some in terre la tions developed between this l i t e r a ture and that d iscussed here on protein im provem ent in plants. We find, fo r exam ple, that neutron induced mutations fo r plant height in tobacco are in som e cases associated with a lterations in prote ins that can be repeated e lec trop h ore tica lly and dem onstrated by appropriate staining.

A. GROBMAN: No mention was made in your su rvey o f screen ing methods to b ioassay techniques fo r the determ ination o f various com position le v e ls fo r sp ec ific amino acids. A t least two such methods have been reported in the lite ra tu re ; A sp erg illu s nidulans and Leuconostoc m esen tero ides. The la tte r one in particu lar may be adapted to quick determ inations o f lysine with a high e ffic ien cy fac to r in term s o f number o f sam ples studied p er week o f work.

O. P . K A M R A : S evera l such b ioassay techniques have been discussed at the 1970 Vienna Symposium1, and are potentia lly v e ry in teresting. However, they are not quite as rapid and as easy to handle fo r a plant b reed er fo r routine screen ing as m icro -D B C o r regu lar DBC methods. I also want to mention that I did not discuss nutritional evaluation which in the fina l analysis is the most useful criterion . Th is has a lso been covered in the 1970 Symposium by Dr. Eggum.

A . HAGBERG: Dr. Kam ra, you quite c o rre c t ly stated that we should be concerned about the protein quantity and quality produced p er hectare.Th is w ill requ ire a system to pay the fa rm er not only by quantity d e livered but a lso by quality o f his product.

O. P. K A M R A : That is v e ry true, and I think methods such as DBC w ill be useful also in trade and m arketing.


INDUCED VARIATION IN QUANTITATIVELY INHERITED CHARACTERS*

R. D. BROCK, H .F . SHAW, D .F . CALLEN

Com m onwealth S c ien tific and Industrial

Research Organization (CSIRO),

Canberra, Australia

Abstract-Resumen

INDUCED VARIATION IN QUANTITATIVELY INHERITED CHARACTERS.The plant breeder who uses induced mutations in his breeding program is frequently faced with the

problem of whether he can make better or faster progress towards a particular breeding goal by crossing his mutants or by creating the necessary variability for further improvements by a second mutagenic treatment. Therefore, in this experiment the additional variation obtainable from a second mutagenic treatment was compared with that generated by a single cycle of recombination, using extremely early and late flowering mutants and mutants with increased plant weight of Arabidopsis thaliana, selected after one mutagenic treatment. The results indicate that a second mutagenic treatment is able to create more additional variation than crossing of mutants. The further selection response depends, however, on the character under study.

VARIACIONES INDUCIDAS EN LOS CARACTERES HEREDADOS CUANTITATIVAMENTE.El fitotécnico que utiliza mutaciones inducidas en su programa de mejoramiento a menudo se

enfrenta con el problema de si puede conseguir progresos mis importantes o mis rápidos hacia una meta determinada cruzando sus mutantes o creando la variabilidad necesaria para nuevas mejoras mediante un segundo tratamiento mutagénico. Por consiguiente, en este experimento se ha comparado la variación adicional que permite un segundo tratamiento mutagénico con la producida por un solo ciclo de recombinación, utilizando mutantes de floración extremadamente temprana y tardía, y mutantes de mayor peso de planta de Arabidopsis thaliana, seleccionados después de un solo tratamiento mutagénico. Los resultados indican que un segundo tratamiento mutagénico puede crear más variaciones adicionales que el cruzamiento de mutantes. Sin embargo, el resultado definitivo de la selección depende del carácter estudiado.


The resea rch reported h ere deals with th ree populations of A rab idopsis thaliana which w e re generated fo llow ing a f ir s t cyc le o f mutation and se lection [1 ]. One selection from each group was g iven a second mutagenic treatm ent. The additional va ria tion and the response to se lection fo llow ing the second m utagenic treatm ent has been com pared with that generated by recom bination w ithin the ex trem e selections. D eta ils o f some parts o f this w ork have a lready been published [2 ].

2. P A R E N T A L PO PU LA T IO N S

The f ir s t population com prised ea r ly - flow erin g mutants, the second population com prised la te - flo w e r in g mutants, and the th ird population

* Part of the research reported in this paper has been carried out under Research Contract with the International Atomic Energy Agency No. 563/RB.

317

3 1 8 BROCK e t al.

T A B L E I. M EAN FLO W ERIN G TIM E (DAYS) AND P L A N T W EIGHT (LOG mg FRESH W EIGHT A T D A Y 11) OF M 5 M U TAN TS FROM A F IR ST C Y C LE OF M U TA T IO N AND SE LE C TIO N WHICH W ERE USED AS PA R E N TS FOR RE M U TA T IO N AND H YB R ID IZA T IO N

Mutant No. Flowering time Plant weightInheritance of mutation

Early mutants

7 a 16. 00 ± 0.33 1. 236 ± 0.014 Quantitative

8 17.12 ± 0.35 1.163 ± 0. 013 Quantitative

6 17.26 ± 0. 33 3. 270 ± 0. 013 Quantitative

10 17.36 ± 0. 39 1. 126 ± 0.01.5 Quantitative

1 17.50 ± 0.32 1. 220 ± 0. 013 Quantitative

Late mutants

21 a 34.41 ± 0,35 1.273 ± 0.013 Single recessive

22 32. 59 ± 0. 35 1.260 ± 0.015 Single recessive

33 25.47 ± 0.37 0.946 ± 0.015 Quantitative

24 22. 90 ± 0.46 1.004 ± 0.015 Quantitative

35 21. 18 ± 0.35 1. 076 ± 0. 013 Quantitative

34 20. 50 ± 0. 36 1. 152 ± 0.015 Quantitative

28 20.42 ± 0. 33 1. 063 ± 0.016 Quantitative

Estland 18.44 ±0.34 1.235 ± 0.014

a Mutant chosen for remutation. * Standard error.

com prised se lections fo r increased plant weight. F low erin g tim e and plant weight data of the parental populations a re given in Tab le I. A l l mutants w e re se lected from the race Estland a fter treatm ent with ion izing radiations or chem ical mutagens [ 1 ].

3. E A R L Y P O P U L A T IO N

Mutant 7 (T ab le I) was given a fu rther mutagenic treatm ent with EMS (approx im ate ly LD50) and crossed with the other ea r ly - flow er in g mutants and with Estland. Mutagenic treatm ent resu lted in an in crease in variance and a shift in mean o f the M 2 population towards lateness. C ross ing of the e a r ly - flow e r in g mutant with other ea rly mutants resu lted in a somewhat sm a lle r in crease in variance and a g rea te r shift in the mean o f the F2 population towards lateness. C ross ing with Estland resu lted in an even sm a lle r in crease in va rian ce and a la r g e r shift in mean towards lateness [ 2 ]. Selection at an intensity o f approxim ately 2% fo r earlin ess in the R M 2 F2 , R M 3 and R M 4 F4 generations,resu lted in only sligh t responses in both

INHERITED CH ARACTER VA R IA TIO N 319

T A B L E II. M E AN FLO W E R IN G TIM E (DAYS) AND P L A N T W EIGHT (LOG m g FRESH W EIGHT A T D A Y 11) OF E A R L Y SELECTIO NS IN THE R M 5 and F 5 G E N E R ATIO N OF THE E A R L Y P O P U L A T IO N

Population or family Flowering time Plant weight

RMS Mean 15. 75 ± 0.09 1. 179 ± 0.007

Earliest family 14. 83 ± 0. 23 1. 206 ± 0.018

F= Mean 15. 88 ± 0. 07 1. 189 ± 0. 005

Earliest family 14. 92 ± 0.22 1. 232 ± 0. 017

rc5 Mean 16. 03 ± 0.12 1. 212 ± 0. 016

Earliest family 15. 50 ± 0.33 1. 204 ± 0. 028

Estland Mean 18.88 ±0.15 1.184 ± 0.010

Earliest family 18.33 ± 0.23 1.230 ± 0.017

the RMg and F5 populations. Somewhat la rg e r responses occurred in the ea r lie s t fam ilie s (T ab le II). Thus, although the response to se lection a fte r the second mutagenic treatm ent was sm a lle r than a fter the f ir s t mutagenic treatm ent, it was as la rg e o r la rg e r than that achieved a fter a cyc le o f recom bination.

4. L A T E P O P U L A T IO N

Mutant 21 (T ab le I) which ca rr ied a s ingle r e c e s s iv e gene fo r lateness induced by the f ir s t mutagenic treatm ent was g iven a fu rther mutagenic treatm ent with EMS (approxim ately LD50). It was a lso crossed with a s is te r - l in e ca rry in g the sam e mutant gene fo r lateness (2 2 ), with two independent quantitatively inherited mutants (24,28) and with Estland. Fou r additional c rosses a ll invo lv ing independently iso la ted quantitatively inherited la te mutants (24, 28, 33, 34, 35) w e re made.

As with the ea r ly population the second mutagenic treatm ent induced a substantial in crease in variance. A sligh t sh ift in mean towards lateness indicated a co rre la tion between la te flow erin g and reduced plant weight, con firm ing the co rre la ted response reported by B rock [1 ]. C ross ing generated less varia tion than mutation. The F2 fam ily means w ere somewhat below the m id-paren t values indicating the p resen ce of d ifferen t mutant lo c i and a degree o f non-additivity o f gene action [2]. Selections w e re made fo r la teness at a se lection intensity of approxim ately 2 % in each of the R M 2 F2 , R M 3 F3 , and RM 4 F4 generations. P lant weight and flow erin g tim e w ere m easured in the R M 6 F6 generation (Tab le III).

The response to se lection a fter the second mutagenic treatm ent (20 days) was somewhat la r g e r than the response achieved a fter the f ir s t mutagenic treatm ent (16 days). W hether this additional response is re la ted to the p resen ce o f another gene of m a jo r e ffec t is not yet known.

3 2 0 BROCK e t a l.

T A B L E III. M E A N FLO W E R IN G T IM E (D AYS ) AND P L A N T W EIGHT (LOG m g FRESH W EIGHT A T D A Y 11) OF L A T E SELECTIO NS IN TH E R M 6 AND F 6 G E N E R ATIO N OF TH E L A T E P O P U L A T IO N

Population Flowering time Plant weight

RMfi 56. 05 ± 0. 68 1 .174 ± 0. 004

F6

S X S (21 X 22) 43. 65 ± 1. 36 1. 216 ± 0. 007

Fertile plants only 38.52 1.250

S X Q (21 X 24) 33. .74 ± 1.36 1.218 ± 0.007

S X Q (21 X 28) 38.44 ± 1.36 1. 266 ± 0.007

S X EST (21 x Estland) 35. 79 ± 1.36 1.216 ± 0. 007

All crosses involving single gene for latenessa 37.82 ± 0. 68 1.224 ± 0. 004

Q X Q (34 x 33) 35.72 ± 1.36 1,210 ± 0.007

Q x Q (35 x 24) 32.96 ±1.36 1.201 ± 0.007

Q X Q (33 X 24) 26.62 ± 1.36 1.012 ± 0,007

Q X Q (28 X 33) 25.20 ± 1.36 1.124 ± 0. 007

All crosses not involving single gene for lateness b 30.12 ± 0. 68 1. 137 ± 0. 004

RC6 35. 77 ± 1.36 1.269 ± 0.007

Estland 19.08 ± 1.36 1. 233 ± 0. 007

a S Parents carry single recessive gene for lateness, b Q Parents do not carry single recessive gene for lateness. ± Standard error.

The response to se lection fo llow ing hybrid ization va r ied accord ing to the parents that w e re involved, but in no case was the response as la rg e as that ach ieved a fte r mutation. In addition to the s in g le gene of m a jor e ffec t fo r la teness, p rev iou s ly identified in parents 21 and 22 (Tab le I), s e v e ra l other genes affecting m orphology, fe r t i l i t y and flow erin g tim e w ere iden tified in this population.

T h e .c rosses involving the two s is te r lines (21 X 22) segregated fo r a gene inducing s te r ility . P lants homozygous fo r this gene w ere squat, la te - flo w e r in g and s te r ile . R em ova l o f these plants reduced the mean flow erin g tim e o f the la te se lected F6 fam ily from 43. 65 days to 38. 52 days. Th is represen ts a response to se lection of only 2. 7 days and is o f com parable magnitude to the response ach ieved when the sam e parent (2 1 ) was hybrid ized with the mutant 28 (2. 6 days). The response when mutant 21 was crossed to mutant 24 was negative (-2 . 0 days) and when crossed to Estland it was zero .

Th ese sm a ll responses contrasted with those achieved from som e of the c rosses invo lv ing parents which did not ca rry a s ingle m a jo r gene fo r lateness. Com paring the F6 mean flow erin g tim es (Tab le III) with the m id

INHERITED CH ARACTER VA R IA TIO N 321

paren t values (T ab le I) fo r these crosses , the se lection responses from two o f the crosses (35 X 24 and 34 X 33) w ere 12. 1 and 12. 7 days resp ec tive ly . H ow ever, the se lection response from the other two crosses w e re only2. 4 and 2. 3 days. As no fu rther mutagenic treatm ent had been applied to these parents, it is un likely that the la rg e responses w e re due to new mutations. Hence, they a re presum ed to rep resen t favourab le recom binations o f mutations se lected a fte r the f ir s t mutagenic treatm ent.

The la rg e response in flow erin g tim e in the R M g se lection was associated with a sign ificant drop in plant weight. Some o f the reduction in plant weight associated with the quantitatively inherited parents (Tab le I) was recove red in the se lected progeny from the two best crosses (34 X 33, 35 X 24). Th is suggests the loss o f deleterious genes during recom bination and selection .

5. INCREASED P L A N T W EIGHT

Attem pts w e re m ade to se lec t fo r increased growth ra te by se lectin g the h eaviest plants at the end o f the log phase o f growth (day 11 at 25°C).Tw o mutant lin es TW -1 and T W - 6 , orig inating from the f ir s t cy c le of mutation and se lection , w ere con firm ed as having increased plant weight at day 1 1 . N e ith er of these had increased growth rate. The h eav ier plant weight in the case of TW -1 was associated with fa s te r germ ination and establishment.In the case of mutant T W - 6 it was not associated with fa s te r germ ination o r establishm ent. In both mutants the increased plant weight m ay be associated with la r g e r seeds but this has ye t to be confirm ed.

Selection based on 11-day plant weight a fter e ither recom bination o r hybrid iza tion did not resu lt in fu rther increases in plant weight.

R E F E R E N C E S

[1] BROCK, R.D., Quantitative variation in Arabidopsis thaliana induced by ionizing radiation, Radiat. Bot. 7 (1967) 193.

[2] BROCK, R. D., SHAW, H.F., "Response to a second cycle of mutagenic treatment in Arabidopsis thaliana", Induced Mutations in Plants (Proc. Symp. Pullman, 1969), IAEA, Vienna (1969) 457.

D I S C U S S I O N

G. DE A L B A : Why is it not possib le to ach ieve fu rther p rog ress with respec t to earlin ess once the 16-day m ark has been reached?

R. D. BROCK: Apparen tly som e low er lim it o f developm ent com es into play; with less tim e the plant could not develop to the minimum n ecessary fo r flow ering.

The asym m etry o f the se lection response is due in part to the fact that the orig inating race , Estland, is a v e ry ea r ly flow erin g genotype of A rab id op s is . Hence I would expect a la rg e r response to se lection fo r lateness than to se lection fo r earlin ess. It is a lso due to physio log ica l lim its a ffecting sealing. The plant requ ires a certa in m in im al tim e fo r germ ination and growth to the stage w here it can produce a flow er. Th is im poses a ph ysio log ica l lim it on earlin ess. T h e re is no such lim it on lateness.

3 2 2 BROCK e t a l.

H. HANSEL: I would lik e to ask you under which light conditions the plants w e re grown and whether the extrem e lateness o f some o f your se lected lines could be due to a change in the type o f photoperiodic reaction.

R . D . BROCK: Plants w ere grown under continuous light. The ex trem ely la te flow erin g mutants have not yet been checked to see i f we have photop eriod mutants.

R. T R U J ILLO F IG U ERO A: Dr. B rock, in your w ork published in 1966 you form u late that the induced va r iab ility a fter mutagenic treatm ent has a reduction towards the previous selection . Now in you r present work you show that a fter se lection fo r lateness in flow ering tim e o r a fte r a second irrad ia tion cyc le , this character increased. I would lik e to ask you, which is the explanation in this case?

R. D. BROCK: Th is sligh t shift in M 2 mean flow erin g tim e o f the la te population, which a lso has reduced plant weight, con firm s the co rre la ted se lection response which was discussed in deta il in my e a r lie r rep o r t1.


INDUCED MUTATION RESEARCH IN WHEAT

C .F . K O N ZAK

Department o f Agronomy and Program in Genetics,

Washington State University,

Pullman, Wash., United States o f Am erica

Abstract-Resumen

INDUCED MUTATION RESEARCH IN WHEAT.The mutation program on wheat at Pullman, Wash. (USA) is described. A part of it is concerned with

increase in efficiency of mutagen treatments using radiations as well as chemicals. A major problem to be solved was also the management of large numbers of mutagen-treated populations and mutant progenies necessary to demonstrate unequivocally the usefulness of induced mutations in wheat breeding. Many of the induced wheat mutants have been analysed genetically and are now incorporated in breeding experiments. About 80°jo of the crosses made in the regular spring wheat breeding program in 1970 used induced mutants. "Direct” mutation breeding has so far not led to any new variety. There are m a n y mutant lines selected which yield significantly more than their mother variety, but none of these have exceeded competing strains bred by conventional methods. More success is expected from recent work on induction of mutations in modern wheat varieties and from the incorporation of various useful mutant genes in such varieties by cross breeding.

INVESTIGACIONES SOBRE MUTACIONES INDUCIDAS EN TRIGO.El autor describe el programa de mutaciones del trigo ejecutado en Pullman, Washington (Estados Unidos

de América). Partedelmismo versa sobre el aumento de la eficacia de los tratamientos mutagénicos utilizando radiaciones y productos químicos. Uno de los principales problemas que ha habido que resolver es el relativo a la capacidad de manipular gran cantidad de colectividades tratadas con mutágenos y de progenies mutantes necesarias para demostrar de manera inequívoca la utilidad de las mutaciones inducidas en el mejoramiento del trigo. Muchos de los mutantes inducidos del trigo se han analizado genéticamente y se utilizan ahora en experimentos fitotécnicos. En 1970, se emplearon mutantes inducidos en un 80 0, aproximadamente, de los cruzamientos realizados como parte del programa regular de mejoramiento del trigo de primavera. Hasta la fecha, el mejoramiento por mutación «directa» no ha permitido obtener ninguna nueva variedad. Hay muchas líneas mutantes seleccionadas cuyo rendimiento es mucho mayor que el de la variedad madre, pero ninguna de ellas ha superado a las variedades competidoras obtenidas por métodos tradicionales. Se espera tener más éxito con los recientes trabajos sobre inducción de mutaciones en variedades modernas de trigo y con la incorporación por cruzamiento en dichas variedades de distintos genes mutantes útiles.

1. INTRO D U CTIO N

Our orig in a l aim s in mutation research w ith wheat w ere to con firm and in vestigate the cause o f d iffe ren tia l responses o f wheat va r ie tie s to mutagens, and to obtain and study the inheritance o f mutations useful fo r breeding. H ow ever, the f ir s t o f these aim s proved u n rea lis tic since our exp lora tory studies showed that our methods o f mutagen treatm ent did not p rov ide su ffic ien t control o ve r the m ajor va riab les a ffecting the mutation response. In addition, until now we could not e ffic ien tly manage la rge

* Scientific Paper No. 3595. College of Agriculture, Washington State University, Pullman, Project 1568. Supported in part by grants from the Washington State Department of Agriculture and the Washington Wheat Commission. Part of the research reported in this paper has been carried out under Research Agreements with the International Atomic Energy Agency Nos 321/CF and 615 /CF.

323

3 2 4 K O N Z A K

populations o f m utagen-treated m ateria ls from which v e ry la rg e numbers o f usefu l mutants m ight be recovered . The evaluation o f la rge numbers of a spectrum o f usefu l mutations by extensive se lec tion and breeding ex p e r iments m ay be requ ired to dem onstrate unequ ivocally the usefulness o f induced mutations and induced mutation techniques in p ractica l breeding. Consequently, we p laced our m ajor emphasis f ir s t on labora tory studies to ach ieve better control o ver the e ffects o f mutagen treatm ents while a lso continuing fie ld tests to develop better fie ld management techniques.M o re recen tly we have increased our e ffo r t on the genetic analyses o f mutants and have begun to extensive ly use s e ve ra l new sem i-d w arf (erecto id ) mutations in our p ractica l combination breeding p rogram s.

Some o f the highlights from our various study areas are described below .

2. E F F IC IE N C Y OF M U TAG E N TR E A TM E N TS

2.1. Radiation

We found that the control o f seed m oistu re content and exposure to oxygen fo llow ed by post-treatm ent hydration and red ry in g m arked ly im proved the consistency and reprodu cib ility o f b io log ica l e ffects induced by gamma irrad ia tion [ 1 ].

2.2. Chem ical agents

Our g rea test d ifficu lty with the chem ical mutagens has been with ethyl methane sulphonate (EM S), one o f the m ost e ffec tive mutagens we have tr ied . P ost-trea tm en t e ffects can be so seve re that a ll m ateria l m ay be lost, the m a ter ia l m ay be v iab le but s te r ile or, depending on conditions, only m ild e ffects m ay be induced [ 2 ] . The basis fo r this prob lem with EMS (less so with re la ted chem icals) seem s traceab le to active mutagen which is tenaciously retained by the tissue o f the treated seeds. Retention appears to be increased by increased mutagen concentration and to some extent by increased treatm ent tim e. P ost-trea tm en t washing at room tem peratu re o r above appears to concurrently stim ulate m etabolic a c tiv ity which probably in creases mutagen sensitiv ity . Post-trea tm en t washing in running water fo r as long as 72 hours at low tem perature (approxim ately3 ± 1°C) p r io r to red ry in g seem s most e ffe c tiv e . The low tem perature during washing retards m etabolism , lim itin g in ju ry caused by the long soaking tim e. The p rocess does perm it red ry ing and storage without any fu rther in crease in damage (we have d ried up to four w eeks), but there s t ill is som e in crease in damage as com pared with the non-dried seeds.No post-treatm ent devised so fa r has enabled us to avoid this part o f the damage. An experim ent with ba rley in which post-washing was ca rried out fo r 48 hours w ill be analysed this fa ll. Th is should provide an indication o f the e ffects o f the post-wash treatm ent, but the longest wash period did not com p lete ly stop damage from occurring during storage. H ow ever, indications from other labora tories are that some post-washing treatm ents m ay im prove the re la tion [ 3 ].

Studies conducted by M . J. DeKock as part o f a graduate research p ro jec t m ay extend beyond recen t reports [ 4, 5, 6 , 7, 8 ] that the n itrosom ethyl and ethyl ureas m ay be ex trem e ly e ffec tive mutagens causing somewhat d ifferen t

M U T A T IO N RESEARCH IN W HEAT 3 2 5

mutation spectra than obtained with e ither EMS or radiation . In teresting ly , the mutation spectra appear to d iffe r m arked ly o ve r a range o f p re-soak tim es, probably corresponding to stages in the m ito tic cyc le . The evidence supports recen t d iscove r ies particu la rly by Natarajan, Swaminathan and others in India and in F rance, who reported that b r ie f, h igh ly concentrated EMS treatm ents to seeds during various p re-germ in a tion stages could a lter the spectrum o f ch lorophyll mutations induced in b a r ley [9 , 1 0 , 1 1 , 1 2 ].Th ese new resu lts o ffe r p rom ise fo r obtaining quantitatively sign ificant numbers o f the r e la t iv e ly ra re kinds o f mutations.

3. P O P U L A T IO N M AN AG E M E N T

W e have stead ily im proved our ab ility to e ffic ien tly manage la rg e r quantities o f m ateria ls w ith decreas ing inputs o f labour and funds. The M2

populations a re managed e ith er as spike p rogen ies o r bulk populations from the treatm ents. P lan ting e ffic ien cy is in creased g rea tly through the use o f O. A . V o g e l's sem i-au tom atic p lan ter. The treated and red r ied М х seeds are planted th ick ly spaced at approxim ately one-ha lf inch in terva ls o r less to reduce t ille r in g to a maximum o f about three per M] plant. Th is procedure helps to assure that only those p rim ord ia fu lly form ed in the em bryo at the tim e o f treatm ent w ill contribute to the M 2 population since secondary t i l le r s m ay tend to have low er mutation rates [ 13] . M a ter ia l fo r M 2 bulk handling was cut by plot and ca re fu lly threshed by treatm ent lo t. The M 2 population fo r the spike progeny analysis is sown in rows one foot apart and fiv e feet in length, o r fo r the bulk study the M 2 is sown in d r i l l plots, with e ither s ix o r tw elve inch spacing but spaced thinly to im prove the ease with which individual varia tions can be distinguished. Spacing o f the M 2 plants is im proved through the incorporation o f about 50% or m ore o f hea t-k illed seed among the m utagen-treated seed lots.

W eed control in both types o f experim ents is achieved by the use o f a contact type h erb ic ide such as b rom oxyn il1 fo r spring grains and T erb u tryn 2

fo r winter, wheats. The herb ic ide 2, 4-D is not acceptable because it has a system ic action. A lle y s between plots can be e ffic ien tly made using a spray o f paraquat3 chem ical. F ie ld labels and reco rd books a re produced using the com puter, and a fter notes have been recorded , the data are ' com puter p rocessed . P lan ts o f poss ib le agronom ic value o r o f in terest fo r M 3 evaluation and genetic studies a re tagged and harvested for m easurem ent and/or p rocess in g fo r the progeny tests . With the bulk populations, sam ple counts can be made o f the M x seed lings, М х spikes and o f M 2 plants to p rov ide a basis fo r the M 2 analysis. These procedures seem to o ffe r the hope fo r maximum e ffic ien cy in population management wh ile a lso p rovid ing usefu l data. Th is year we have evaluated m ore than seven acres o f m a ter ia l as spike p rogen ies and about'three acres of exp lora tory bulks; how ever, the la tte r populations w ere grown only to iso la te usefu l mutants fo r plant breed ing and genetic analyses and to test the bulk method. W e hope to p rocess even m ore m a ter ia l v ia bulk means next yea r in spite o f few er funds now ava ilab le fo r the studies.

1 3,5-dibromo-4-hydroxy benzonitrile.2 2-tert, butylamino-4-ethylamino-6-methylthio-s-triazine.3 1,1' -dimethyl-4,4' -bipyridinium ion.

326 K O N Z A K

4. M U TA T IO N BREEDING E X PER IM EN TS

The useful mutants iden tified to date, including Luther, the w inter b a rley mutant va r ie ty described by N ilan [14] , have encouraged us to put even m ore e ffo rt into p ractica l applications and mutation breeding.Our main c r ite r ia a re to start with a new, m odern or potential va rie ty , screen la rge populations fo r mutants that would co rrec t a weakness, and subject these mutants o r the ir cross progeny to se lection p rocesses consistent with current breeding practice . Im proved straw and em ergence ch aracteris tics appear among the easiest to obtain and are at the same tim e among som e o í the m ost urgently needed fo r e ffic ien t culture of wheats at high fe r t i l i ty le v e ls . Currently, we a re placing emphasis on the induction o f sem i-dw arf, stronger straw mutations in durum and soft white wheats. S evera l p rom is ing strains are now in advanced evaluation tests.

5. G ENETIC A N A LYSE S AND NEW M U TAN TS INDUCED

5.1. E recto ids

O f the m any wheat mutants induced in the experim ents conducted to date, we have genetica lly analysed seve ra l p rom is ing induced sem i- dwarfing mutations and have begun to incorporate them in breeding experim ents. Our genetic studies have been concentrated on the isolation and analysis o f sem i-d w arf mutations ( e rec to id ) . Numerous erecto ids have been induced in both durum and common wheats. Genetic studies o f only a few o f these erecto ids have been com pleted, though many other genetic analyses a re cu rren tly in p rog ress . We have p a rtia lly characterized two mutants o f Burt, each o f which c a rr ie s a d ifferen t single re c ess iv e gene fo r sem i-dw arfin g . Both genes appear to be re c ess iv e and d ifferen t from those o f N orin 10, and the re la tion between the plant height and co leoptile length o f the mutants and recombinants appears to d iffe r from those o f the N orin 10 sources ¡ 15] (F ig . 1). In the mutants, co leop tile length appears to be inherited independently from plant height. B iochem ical studies o f these mutants indicate that high peroxidase ac tiv ity is associated with shorter plant height. The m illin g and baking ch aracteris tics o f the Burt mutants appear to be s im ila r to their parent. One o f these, WA5435, has been added to the USDA W orld Wheat C o llection and as such is availab le to anyone on request.

Another esp ec ia lly in teresting and apparently new, fu lly dominant single fac to r s em i-d w a rf (e rec to id ) mutant, WA5519, was recove red from mutagen treatm ents o f M arfed spring wheat [ 16] . The height o f this mutant is about tw o-th irds norm al, and it tends to produce an Fj about tw o-th irds the height o f the ta ll parent. The mutant gene appears to be com pletely dominant, thus d iffe r in g from the sem i-dom inant va r ie t ie s Tom Thumb, the O leson dwarf, andperhaps from the induced durum mutant B-132. The new dominant erec to id mutant has som e undesirable features such as sm all seed s ize , excess ive t il le r in g and sm all spike s ize under crowded conditions. Its y ie ld ing ab ility , however, seem s about equal to its parent. It is cu rren tly being evaluated w ide ly in breeding experim ents with m ateria ls o f d ifferen t background in an e ffo r t to overcom e som e o f the weaknesses. The new dominant sem i-dw arfin g gene should be valuable in hybrid wheat developm ent.

M U T A T IO N RESEARCH IN WHEAT 32 7

C O LEO PTILE

Г-------1 P LA N T

SI>5493

FIG. 1. Relation of plant height and coleoptile length (10-12 d, 15 ± 2°C) for backcross-derived "isogenic”(Ml = 1 gene, SI = 2 genes, MM and SM each 1 gene) mutant strains and recombinant (SM/MM = 2 genes) in the same genetic background. Positions of culm nodes ( 1 = peduncle)are shown by horizontal bars.

(FIG.l. Relación entre la altura de la planta y la longitud del coleóptilo (10-12 d, 15 ± 2*C), en el caso de cepas mutantes «isogénicas» obtenidas por retrocruzamiento (Ml = 1 gene, SI = 2 genes, ММ у SM cada uno 1 gene), y de un recombinante (SM/MM = 2 genes) con el mismo fondo genético. Los trazos horizontales indican la posición de los nodulos del culmo (1 = pedúnculo). )

A stock o f WA5519 has been made availab le to others v ia the USD.A W orld Wheat C o llection as C l 13988.

Genetic analyses o f s e ve ra l other sem i-d w arf mutants a re cu rren tly in Fj and F2 . The segregations indicate the p oss ib ility that additional useful sem i-dw arfin g factors w ill be iden tified . C erta in o f the mutants a re being used as genetic te s te r stocks in tests fo r a lle lism .

In addition, s e ve ra l other possib le new sources o f sem i-dw arfin g in which the co leop tile length appears to be norm al w ere iso la ted from mutagen treatm ent o f both T\ aestivum va r . vu lgare and va r. compactum wheats, and o f T . turgidum va r. durum. S em i-dw arf ( e rec to id ) mutants having shorter co leop tile length a re also found.

5.2. M a rk er mutants

A number o f mutants having potential value as genetic m arkers have been iso la ted . M ost o f these have been d istributed to other w orkers fo r analysis. These include v iab le chlorina, stria ta , and ecer ife ru m types,

80

о2

70

60

50

40

30

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/

IN T E R N O D E

BURT MM

Cl J3736 W A 4564

SM

W A 5435

SM/MM

2 8 *1 0 5

MlW A 5492 W,

32 8 K O N Z A K

with e ither increased or decreased wax, and an "on ion " le a f fo rm we ca lled fistu losum , accord ing to the uniform system used by Scandinavian sc ien tists [ 17] .

5.3. D isease res is tance

We a re p reparing to in tensive ly study the induction o f res is tance to s tr ip e rust. We a lready have isolated som e stripe rust res istan t probable mutants, and s e ve ra l res istan t strains from the club wheat Om ar a re now being analysed genetica lly and are being characterized v ia the International Stripe Rust N u rsery . We have had d ifficu lty establish ing e ffic ien t fie ld screen ing conditions to obtain la rge numbers o f these mutants. H ow ever, the situation m ay soon im prove through acqu isition by Washington State U n ive rs ity o f bench land near the Snake R iv e r . . Th is area has m ild w inter tem peratures fac ilita tin g developm ent o f the strip e rust organ ism and is w e ll iso la ted from m a jor wheat production areas. Evidence fo r the ready induction o f mutations fo r d isease res is tance w ill be an im portant test o f the ab ility o f thê mutagens and techniques we have developed at this point to create the kinds o f genetic d ifferen tia tion upon which m an's su rv iva l m ost depends. F o r the m ost part, d isease res is tance can be obtained e ffe c t iv e ly now only from natural sources. Th is is not at a ll to sligh t the strong evidence from our own and other labora to ries fo r the induction of d isease res istan t mutations, but rather to point out that such mutations are yet ra re , and useful ones even m ore so.

5. 4. F e r t i l ity res tora tion in cy tosterile wheat

Recen tly , we have becom e in terested in the poss ib ility to induce better re s to re r genes. F o r these studies, conducted under m y d irection by a graduate student, C . M . Huang, over 50 lb o f A line cy tos te r ile was treated using a number o f mutagens and the M i generation grown on about four acres th in ly spaced in genetic iso lation from other wheat. A number of М г plants with fe r t i le sectors have been iso la ted . P rogen y from the fe r t ile sectors and any other fe r t i le plants from seeds set on the rem ain ing population w ill be te s t-c rossed in 1971.

6 . BREEDING STUDIES

The use o f mutants in wheat breeding at Washington State a lready shows an im pact o f this p rogram . About 80% o f the crosses made in our regu lar spring wheat breeding p rogram in 19 70 used induced mutants to im prove straw ch aracteris tics . D irec t mutation breed ing has not so fa r y ielded any new wheat va r ie t ie s even though we have obtained wheat mutants y ie ld ing s ign ifican tly h igher than their m other line (from 10-30% depending on the tes t). H ow ever, none o f these have so fa r exceeded competing strains bred by conventional methods. New mutants recen tly obtained in m ore m odern wheat va r ie tie s may a lte r the situation. Even so, the new sources o f sem i-dw arfin g when incorporated with the y ie ld -im p rov in g factors from the Suwon 92 and Norin 10 deriva tives m ay have an even g rea te r im pact in our wheat breeding p rogram than would the d irec t use of a g iven mutant.

M U T A T IO N RESEARCH IN W HEAT 3 2 9

R E F E R E N C E S

[1] KONZAK, C. F. , BOTTINO, P.J., NILAN, R. A., CONGER, B.V., "Irradiation of seed;:: A review of procedures employed at Washington State University", Neutron Irradiation of Seeds II, Tech. Rep.Ser. No. 92, IAEA, Vienna (1968) 83.

[2] FROESE-GERTZEN, Edith E., KONZAK, C.F., NILAN, R. A., HEINER, R. E., The effect of ethyl methanesulfonate on the growth response, chromosome structure and mutation rate in barley, Radiat.Bot. 4 (1964) 61.

[3] BENDER, K., GAUL, H., Variierung der AMS-Wirkung bei Gerste durch Anwendung verschiedener Behandlungs-und Nachwaschtemperaturen, Radiat. Bot. 7 (1967) 289.

[4] GICHNER, T., GAUL, H., OMURA, T., The influence of post-treatment washing and redrying of barley seeds on the mutagenic activity of N-methyl-N-nitroso-urea and N-ethyl-N-nitroso-urea,Radiat. Bot. 8 (1968) 499.

[5] EHRENBERG, L., GICHNER, T., On the mutagenic action of N-alkyl-N-nitrosoamides in barley,Biol. Zbl. Suppl. 86 (1967) 107.

[6] GICHNER, T., VELEMINSKŸ, J., The mutagenic activity of 1-alkyl-l-nitrosoureas and l-alkyl-3- nitro-l-nitrosoguanidines, Mutation Res. 4 (1967) 207.

[7] GICHNER, T ., "The mutagenic activity of nitrosoamides with methyl and ethyl groups", Arabidopsis Research (Proc. Symp. Gottingen, 1965) 200.

[8] HESLOT, H., FERRARY, R., TEMPE, J., The relative mutagenic effects of some nitrosoamines on barley seeds, Mutation Res. 13(1966) 354.

[9] NATARAJAN, A. T., SHIVASANKAR, G., Studies on modification of mutation response of barley seeds to ethyl methanesulfonate, Z. Vererblehre 96 (1965) 13.

[10] SAVIN, V.N., SWAMINATHAN, M. S., SHARMA, B., Enhancement of chemically-induced mutation frequency in barley through alteration in the duration of pre- soaking of seeds, Mutation Res. 6 (1968) 101.

[11] SWAMINATHAN, M.S., SARMA, N. P., Alteration of the mutation spectrum in barley through treatments at different periods in the S phase of DNA synthesis, Curr. Sci. 37 24 (1968) 685.

[12] GRANT, C.J., HESLOT, H., FERRARY, R., "The effects of chemical mutagens in relation to the chromosome cycle” , Chromosomes Today (Proc. 2nd Oxford Chromosome Conf., 1967)2, (1967) 75.

[13] GAUL, H., "Studies on diplontic selection after X-irradiation of barley seeds”, Effects of Ionizing Radiations on Seeds (Proc. Conf. Karlsruhe, 1960), IAEA, Vienna (1961) 117.

[14] NILAN, R. A., "Mutagenic specificity in flowering plants: Facts and prospects", these Panel Proceedings.[15] KONZAK, C.F., SADAM, М., RAMIREZ.-ARAYA, I., "Identification of semi-dwarfing genes in

wheat", Agron. Abstr. Univ. Arizona, Tucson (1970) 14.[16] WOO, S.C., KONZAK, C.F., "Genetic analysis of short-culm mutants induced by ethyl methane

sulphonate in Triticum aestivum L.". Induced Mutations in Plants (Proc. Symp. Pullman,, 1969),IAEA, Vienna (1969) 551.

[17] GUSTAFSSON, Â., HAGBERG, A., LUNDQVIST, U., PERSSON, G., A proposed system of symbols for the collection of barley mutants at Svalov, Hereditas 62 (1969) 409.

D I S C U S S I O N

C. K R U L L : You mentioned the dominant dw arf se lection CI 13988.Could you te l l us i f it is hard or soft grained, its gra in colour and how its m aturity under your conditions would com pare with, e. g. Sonora 64.

C . F . K O N ZA K : CI 13988, M a r fed -E R T 1 is a soft white spring wheat.It seem s to be la te r in m aturity than M arfed and has somewhat sm a lle r seeds. C oleop tile length appears only s ligh tly shorter ( i f at a ll d ifferen t) than M arfed . The plant type is agronom ica lly v e ry good, however.

C. K R U L L : You a lso state in the text that this line is m ore com plete ly dominant than the Tom Thumb dw arf gene(s ). Would you g ive us an idea as to how much m ore dominant it is , and whether you a re com paring it with Tom Thumb d ire c t ly o r a d er iva tive of Tom Thumb which m ay a lso have some N orin genes?

C . F . K O N ZA K : D r. A llan has conducted the studies with Tom Thumb deriva tives in the Burt background. He says Tom Thumb is a sem idominant, and I b e lieve it has been reported so a lso from the Netherlands.

VARIATION IN PROTEIN QUANTITY AND QUALITY INDUCED IN BARLEY BY EMS TREATMENT *

H. DOLL

Agricultural Research Department,

Danish AEC Research Establishment Riso,

Roskilde, Denmark

Abstract-Resumen

VARIATION IN PROTEIN QUANTITY AND QUALITY INDUCED IN BARLEY BY EMS TREATMENT.Seeds of the barley variety Carlsberg II were treated with EMS. A yield trial was carried out with

92 randomly selected mutant lines and 8 untreated control lines, and grain yield, seed size, nitrogen content, and DBC were measured.

A large induced variation was found in dry matter yield, but none of the mutant lines had a significantly higher yield of protein or non-protein dry matter per unit area than the control lines. The non-protein yield of the mutant lines was generally reduced more than the protein yield. The unequal reduction of the two dry matter components resulted in an increased percentage of protein in most of the mutant lines.

Of the 92 mutant lines, two fell outside the general relation found between percentage of protein content and DBC of the lines. Analyses for lysine showed that the two DBC-deviating lines had considerably more lysine in their protein than either the control lines or other mutant lines. Compared with other mutant lines with the same protein content as the DBC-deviating lines, the increase in lysine content averaged IÇPjo.

VARIACIONES CUANTITATIVAS Y CUALITATIVAS INDUCIDAS EN LAS PROTEINAS DE LA CEBADA MEDIANTE EL TRATAMIENTO CON MSE.

Se han tratado semillas de cebada de la variedad Carlsberg II con MSE (metanosulfonato de etilo).En el mareo de un ensayo de rendimientos se han cultivado 92 líneas mutantes seleccionadas al azar y 8 líneas testigo (no tratadas), midiéndose el rendimiento en grano, el tamaño de las semillas, el contenido de nitrógeno y la CFC (capacidad de fijación de colorante).

Se ha observado una fuerte variación inducida del rendimiento en materia seca, pero ninguna de las líneas mutantes presentó, por unidad de superficie, un rendimiento en materia seca protefnica o no proteínica sensiblemente superior al de las líneas de control. El rendimiento no protefnico de las líneas mutantes resultó en general más reducido que el rendimiento proteínico. Consecuencia de la reducción desigual de los dos componentes de la materia seca fue un aumento de la proporción de proteína en la mayoría de las líneas mutantes.

De las 92 líneas mutantes, dos se apartaron de la relación general observada entre el contenido porcentual de protefna y la CFC. Por determinación de la lisina se puso de manifiesto que las dos líneas discrepantés contenían mucha más lisina en su protefna que las líneas testigo o que las demás líneas mutantes. En comparación con las demás líneas mutantes que poseían el mismo contenido protefnico que las líneas discrepantes en cuanto a la CFC, el aumento del contenido de lisina fue por término medio del 16°¡o,

1. IN TRO D U C TIO N

In feeding o f m onogastric anim als, ba rley has so fa r been used m ainly as a carbohydrate source. The content of protein and esp ec ia lly the nutritional value o f the protein is so low in barley that it is not suitable fo r use as the only component in the diet.

* Part of the research reported in this paper has been carried out under Research Agreement with the International Atomic Energy Agency No. 769/CF.

33 1

3 32 DOLL

Although the concept o f nutritional value is v e ry com plex [1 ], much seem s to be gained i f the content o f protein and the amount o f some of the essen tia l am ino acids, p r im a r ily lys ine, could be increased. V a r ie ties w ith a h igher percentage o f protein in their seeds a re known [2, 3], and it hks been shown that this tra it can be increased by means o f mutations [4 ].

The poss ib ility o f im proving the nutritional value o f ce rea ls was f ir s t dem onstrated by the finding o f the two m aize mutants opaque- 2

and flo u ry -2 [5 ], which have a considerab ly h igher lysine content than norm al m aize. The finding o f s im ila r types in b a r ley [ 6 , 7] indicates that it should a lso be possib le to im prove the protein quality and th e re by the nutritional value o f barley .

This paper presents the resu lts of a study o f the induced varia tion in d ifferen t characters related to percentage content, y ie ld and quality o f protein in barley .

2. M A T E R IA LS AND METHODS

Seeds from a single plant o f the spring b a r ley v a r ie ty C arlsberg II w ere treated with a 0.10 M solution of ethyl methane sulphonate (EMS) fo r 7 h at 20°C. The treated seeds and a number o f untreated seeds from the sam e m other plant w ere grown to m aturity. On the basis o f an o ffspring test, a s ing le , ch lorophyll-m u tan t-free line was made from each surviv ing treated and untreated seed. The lines w ere made homozygous fo r the induced mutations by grow ing one plant per line in a greenhouse until the tenth generation . No conscious se lection was applied during this grow ing, and the spikes w ere iso lated at flow erin g to prevent c ro s s fe rtiliza tion .

The lines orig inating from the EMS treatm ent a re ca lled mutant lines in the fo llow ing. When the m ateria l was propagated in the fie ld , a few o f .the mutant lines w ere d iscarded on account o f v e ry low v ita lity .The 92 mutant lines and 8 con tro l lines w ere random ly d ivided into4 s e r ie s each containing 23 mutant and 2 control lines. Each s e r ie s was grown in a y ie ld t r ia l in the fie ld in 1969, using a la ttice square design with three rep lica tes and a plot s ize o f 5.5 m2 .

The gra in y ie ld , seed weight, n itrogen content, and dye-binding capacity (DBC) w ere determ ined fo r each plot. The n itrogen was measured in about 150 mg flour by the standard K je ldah l procedure. The DBC was determ ined as described by M ossberg [ 8 ]: a flou r sam ple containing 120 mg crude protein was shaken fo r 1 h with 40 m l o f a 0.2% solution of A cilane Orange G at pH2.5, and the dye-binding capacity was m easured with a co lo r im e te r .

The lys ine content was determ ined fo r some of the lines on a Beckman 120C automatic am ino acid analyser.


The resu lts a re g iven in Tab les I to IV fo r the four s e r ie s . The f ir s t two lines in each table a re the con tro l lines. The protein content is the n itrogen percen tage o f d ry m atter m ultip lied by 6.25, and the DBC values a re exp ressed as mg dye bound per g d ry m atter.

PROTEIN IN BARLEY 333

Analyses o f varian ce on the resu lts showed that the d ifferen ce between the lines within s e r ie s was h igh ly sign ificant fo r a ll the characters in Tab les I - IV . The resu lts o f these tests a re not g iven in detail. Taking an average fo r the four s e r ie s , the mean e r ro rs fo r d ry m atter y ie ld , kerne l s iz e , protein content, and DBC w ere 14.3, 0.93, 0.25, and 0.65 resp ec tiv e ly . Since the d ry m atter y ie lds o f the con tro l lines did not va ry s ign ifican tly between s e r ie s , the lines in the four s e r ie s w ill be evaluated together in the fo llow ing.

T A B L E I. D RY M A T T E R Y IE LD , K E R N E L SIZE, P R O T E IN CO NTEN T AND DBC OF THE L IN ES IN SERIES I

Line No.Dry matter yield

(g/m2)Kernel size

(mg)Protein content

Cfc)DBC

(mg/g dry matter)

I a 541 42.5 10. 3 42.4

2a 532 39. 7 9.8 42. 0

3 527 38. 7 9. 8 41,4

4 513 40. 0 9.9 41. 9

5 479 39.2 10.8 44. 0

6 466 43. 6 9.9 41. 8

7 483 40. 7 10.5 43. 7

8 422 38. 9 11.4 47. 1

9 476 35. 7 10.1 43. 0

10 515 43.0 10.1 42. 1

11 289 36. 6 14. 8 55. 8

12 298 39.4 14.9 55. 6

13 413 36. 6 11. 0 45. 7

14 527 43. 3 9.5 40.5

15 407 37. 3 11.1 45,4

16 403 47.5 11. 9 47. 3

17 536 40. 0 9.5 40. 6

18 472 40. 2 10.6 43.4

19 517 45. 3 10.3 42.5

20 443 36.5 10.9 44.5

21 389 36.2 11. 0 45.3

22 326 ■ 42. 7 12. 0 47.9

23 321 46. 1 12. 9 50. 9

24 500 36.6 10.5 43. 0

25 459 37.3 10.6 43.6

a Control line.

3 3 4 DOLL

TABLE II. DRY M ATTER YIELD , KERNEL SIZE, PROTEIN CONTENTAND DBC OF THE LINES IN SERIES II


Cg/m2)Kernel size

(mg)Protein content

d o )DBC

(mg/g dry matter)

26a 540 41.1 9. 9 42.2

27a 537 42. 1 9.8 41. 6

28 427 38. 0 9.9 42. 0

29 445 37.5 11.4 49. 6

30 443 39.5 10. 0 42. 6

31 373 34. 0 11.6 45. 9

32 525 39. 6 9.6 41.3

33 413 38. 9 10. 7 44.3

34 418 38.2 11.4 46. 1

35 485 34. 2 10. 9 45.2

36 417 37.5 10.5 44. 0

37 474 38. 0 9. 9 42. 0

38 497 41.1 9.6 40. 7

39 466 43. 3 10.4 43. 0

40 531 46. 0 9. 7 42. 0

41 501 35. 9 10.1 43.2

42 454 37. 6 10. 8 44.3

43 489 40.2 10. 7 44.5

44 519 38.3 10.2 43.3

45 329 29.3 10.4 43.6

46 532 41.5 10.4 42. 3

47 530 39. 9 9.3 40.5

48 476 37.5 9. 8 41.2

49 96 42. 2 14.8 56. 3

50 363 35. 9 12.3 48. 9

a Control line.

F o r the evaluation o f the induced varia tion in y ie ld it seem s convenient to look at the y ie ld o f protein and the y ie ld o f other d ry m atter components separate ly . The protein y ie ld was calculated from the total d ry m atter y ie ld and the protein content (Tab les I - IV ). Th is was subtracted from the tota l y ie ld , g iv ing what is term ed non-protein y ield .The y ie ld s of p rote in and of non-protein of a ll the lines have been plotted against each other in F ig . l , where the absolute y ie ld values are expressed in percent o f the mean of the eight control lines.


TABLE III. DRY M ATTE R YIELD , KERNEL SIZE, PROTEIN CONTENTAND DBC OF THE LINES IN SERIES III


(g/m2 )Kernel size

(mg)Protein content

m

DBC(mg/g dry matter)

51a 545 41.6 9. 7 41.5

52a 550 41. 6 9.9 41.6

53 429 42.5 12. 1 48.1

54 441 38.0 10.4 43.5

55 356 31. 7 11.3 45. 7

56 394 35.3 11. 1 46. 8

57 517 40.1 10. 0 43. 1

58 488 38.2 10.4 43.4

59 450 37. 1 11.1 44.8

60 * 528 40.3 9.9 41.6

61 517 40. 8 10.2 42. 9

62 275 29.2 12.2 48.6

63 400 47. 1 11.1 45.6

64 535 40.5 10. 0 42.4

65 478 40.2 10. 8 45. 0

66 301 35.3 11. 9 47.9

67 444 34.2 11.2 45. 8

68 547 40. 1 9.8 41. 8

69 546 40. 0 9. 7 42. 0

70 413 32.2 11.3 45. 8

71 542 39.4 10.4 43.2

72 450 37.0 11.1 46.4

73 391 38. 1 12.4 49. 7

74 416 38.4 10. 7 44. 0

75 524 37.4 9.9 42.0

aControl line.

One mutant line (N o .49, Tab le II) y ie lded only 18 and 27% non-protein and protein resp ec t iv e ly (F ig . l ) . The re la tive y ie lds o f the rem aining mutant lines varied from 50 to 102% fo r non-protein, while the protein y ie ld va ried from 63 to 107% o f the mean o f the con tro l lines. None of the mutant lines had a s ign ifican tly higher y ie ld o f protein o r non-protein per unit area than the con tro l lines.

It appears from F ig . l that the y ie lds o f protein and non-protein w ere rather strongly co rre la ted in the mutant lines. A dotted diagonal through

336 DOLL

TABLE IV. DRY M ATTE R YIELD, KERNEL SIZE, PROTEIN CONTENTAND DBC OF THE LINES IN SERIES IV


Cg/mz)Kernel size

(mg)Protein content

C7°)DBC

(mg/g dry matter)

76a 517 42. 7 9.9 41.7

77a 526 42.4 9. 7 41.3

78 490 41.8 9.9 42.3

79 453 41.0 10.8 44. 8

80 481 38.4 10.4 43.1

81 429 37.2 10.2 43.5

82 499 39.8. 10. 0 41. 7

83 391 32.4 11.2 46.5

84 427 39.7 10.4 43.5

85 526 38.3 9.7 41.5

86 388 33. 0 12.0 52. 1

87 499 41.3 10. 0 42. 0

88 433 43.5 10. 8 44.5

89 383 36.3 11. 7 47.3

90 425 35.6 10.2 43.0

91 485 39.8 9.9 42.4

92 396 35. 7 11,2 45.1

93 345 35.5 11.5 47.0

94 530 40.6 10. 0 42.1

95 507 41. 1 9.8 41.6

96 451 41. 7 11.0 45.2

97 422 34.6 11.3 47.3

98 499 38.8 9.8 41. 8

99 410 38.4 10. 7 44.5

100 426 34.8 11.2 45.6

aControl line.

the mean o f the controls is superim posed on F ig . l . By com parison with this diagonal it is seen that the non-protein y ie ld o f the mutant lines was gen era lly reduced m ore than the protein y ie ld . Th is unequal reduction o f the two d ry m atter components has resu lted in an increased percentage o f p rotein content in m ost o f the mutant lines (Tab les I - IV ). Other in vestigations in b a r ley [4] and r ic e [9, 10] have shown that mutants with a h igher percentage o f protein in the d ry m atter can be obtained. H ow eve r , since a reduced y ie ld o f protein per unit area cannot be to lerated in feeding barley , it is n ecessary to know not only the percentage of


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•

/•/

//

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о c o n t r o l l i n e • m u t a n t l i n e

10 20 30 A0 50 60 70 80 90 100 110

R E L A T I V E N O N - P R O T E I N Y I E L D ( % )

FIG. 1. Relation between yield of non-protein and of protein per unit area of the control and mutant lines. The yield values are expressed in percent of the mean of the control lines.

(FIG. 1. Relación entre el rendimiento no protefnico y protefnico, por unidad de superficie, de las lfneas testigo y de las mutantes. Los valores del rendimiento se expresan en porcentaje del valor medio correspondiente a las lfneas testigo. )

protein , but a lso the d ry m atter y ie ld o f a particu lar mutant b e fo re its agricu ltu ra l value can be judged.

A considerab le varia tion was found in the seed s iz e o f the mutant lin es , and som e lines had s ign ifican tly la rg e r seeds than the con tro l lines. A s seen in F ig .2 there was no c lea r relation between the seed s ize , i.e . k ernel weight, and the protein content of the mutant lines.

The dye-b inding capacity (DBC) was used as a f ir s t estim ate o f the lysine content o f the lines. The relationsh ip between protein content and DBC, i.e . mg dye bound per g d ry m atter, is shown in F ig .3 . The figu re shows that there is in gen era l a strong, pos itive co rre la tion between percentage of p rotein and DBC. Varia tion in the re la tiv e amount of lys ine is expected to appear as deviations from this gen era l co rre la tion .It is seen from F ig .3 that two mutant lines (Nos 29 and 8 6 , Tab les II and IV) c le a r ly deviate from the genera l re la tion between protein content and DBC. This indicates that these two lines had a re la t iv e ly h igher content of lysine in their protein .

3 3 8 DOLL

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10

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> c o n t r o l t i n e

m u t a n t l i n e

30 5034 36 42 46

KERNEL WEIGHT (m g)FIG. 2. Relation between kernel weight (mg) and protein content fflo) of control and mutant lines.

(FIG. 2. Relación entre el peso por grano (en mg) y el contenido protefnico (en porcentaje) de las lfneas testigo y de las mutantes. )

The protein quality of the two mutant lines with deviating DBC was investigated fu rther by d irec t determ ination of the lys in e content. F o r com parison two con tro l lin es , two mutant lines with medium and two mutant lines with high protein content w ere a lso analysed. The results are g iven in Tab le V , which a lso shows the y ie ld of protein and o f lysine p er unit area.

A com parison o f the two control lines with the four mutant lines with norm al DBC revea ls that the percentage of lys ine in the protein has decreased with increasing protein content (Tab le V ). The two DBC- deviating mutant lin es, Nos 29 and 8 6 , had a considerab ly h igher lysine content in the protein than both the control and the four non-DBC-deviating mutant lines. Since the percentage o f lysine in protein and protein content a re negative ly co rre la ted , it seem s m ost appropriate to com pare the deviating lines with the two lines with about the sam e protein content as the deviating ones. On the average, percent lysine was 0.56 h igher in Nos 29 and 86 than in Nos 34 and 83 (Tab le V ). The increase amounts to 16% re la t iv e ly . This is somewhat low er than what is reported fo r the b a rley lin e H ip ro ly found in the W orld B a rley C ollection [ 6 ], and in another line iso lated in Denmark [7]. H ow ever, since our two mutants w ere induced in a h igh -y ie ld ing va r ie ty , they may probably be u tilized m ore ea s ily in plant breeding.

PROTEIN IN BARLEY 33 9

5 0 r

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10 11 12 13

P R O T E I N C O N T E N T ( % )

15

FIG. 3. Relation between protein content fío ) and DBC (mg dye bound/g dry matter) of the control and mutant lines.

(FIG. 3. Relación entre el contenido protefnico (en porcentaje) y la CFC (en mg de colorante fijado por g de materia seca) de las lineas testigo y de las mutantes. )

T A B L E V . PE R C E N TAG E S AND Y IE LD S OF P R O T E IN AND LYSINE IN SELEC TED LINES

Line

Type No.

Protein content in dry matter

(%)

Lysine content irf protein

Cft)

Yield (g/m2 )

Lysine- Protein

Г 29 11.4 4.17 2.12 50. 9DBC -deviating ja e 12. 0 4. 07 1. 89 46.5

ControlsГ 26 9.9 3.82 2. 04 53.51 76 9. 9 3. 77 1.92 51. 0

f 34 11.4 3.59 1. 71 47.5Medium protein content 1 83 11.2 3.54 1.55 43.8

Г” 14.8 3.35 1.43 42. 7High protein content

1 12 14.6 3.39 1.48 43.6

3 4 0 DOLL

1 1 0 г

>100 ■

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90 ■

80’

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70 ■

60

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X d e v i a t i n g D B C

'60 70 80 90 100RELATIVE PROTEIN Y IELD (°W

110

FIG. 4. Relation between yield of protein and of lysine per unit area of two control lines, two lines with medium protein content, two lines with high protein content, and two DBC deviating lines. The yield values are expressed in percent of the mean of the control lines.

(FIG. 4. Relación entre el rendimiento en protefna y en lisina, por unidad de superficie, de dos lfneas testigo, dos lfneas con un contenido protefnico intermedio, dos lfneas de elevado contenido protefnico y dos lfneas discrepantes en cuanto a la CFG. Los valores del rendimiento se expresan en porcentaje del valor medio correspondiente a las lfneas testigo. )

A m ore quantitative evaluation o f the deviating lines can be obtained on the basis o f th e ir y ie ld o f protein and o f lysine p er unit area (Tab le V ). The re la tion between protein and lysine y ie ld s o f the eight lines investigated further is given in F ig .4, where the absolute y ie lds a re expressed in percent o f the mean o f the two controls. Th ree dotted lines a re superim posed on the figu re , a v e r t ic a l and a horizon ta l quantity line showing the average y ie ld s o f protein and lysine resp ec tiv e ly o f the two con trols, and a diagonal quality line indicating the re la tive content of lys ine in the protein o f the con tro l lines.

The ultim ate aim in quality breeding is types that not only have a h igher lys in e content, i.e . are above the diagonal quality line in F ig .4, but that are a lso equal to o r better than the con tro l w ith respect to protein and lys ine y ie ld s . The four mutant lines with norm al DBC values a ll belong to an undesirable category, which besides the y ie ld ing o f less protein and lys ine per unit area, also has a poorer protein quality than the con tro ls . These four lines are assumed to represen t the m a jo rity

PROTEIN IN BARLEY 3 41

o f the mutant lin es . The two D B C -deviating lin es. Nos 29 and 8 6 , are c le a r ly situated above the diagonal quality line in F ig .4. L ine N o .29 is the m ost p rom ising o f the two dev ia tors , s ince it y ie lded n early as much protein as the con tro l lines and had an absolu tely h igher lysine y ie ld than the con tro ls. Fu rth er tr ia ls , which are not yet com plete, have so fa r con firm ed these p roperties o f the two deviating mutants Nos 29 and 8 6 .

The two mutants with a h igher lysine content w ere found among a tota l o f only 92 mutant lin es . Th is is a v e ry high frequency o f mutations in the d es ired d irection , and it is natural to ask whether the two lines originated from induced mutations, o r could be due to contaminations from other sources o f barley . A study o f the m orphology, the DDT reaction , the am ylase [ 1 1 ] and the es terase [ 1 2 ] isozym es has so fa r not revea led any d iffe ren ces between the mutants and the assumed m other va r ie ty , C a rlsb erg II. F u rth erm ore, contamination is unlikely because no other va r ie t ie s with so high a lys ine content w ere grown in Denmark when the present m ateria l was established. It.is th ere fo re concluded that the two lys ine dev ia tors originated by mutation induced by the EMS treatm ent.

R E F E R E N C E S

[1] MUNCK, L. , Plant breeding and nutritional value in cereals, Hereditas 52 (1964) 151.[2] MUNCK, L. , The variation in nutritional value in barley. I. Variety and nitrogen fertilizer effects

on chemical composition and laboratory feeding experiments, Hereditas 52_ (1964) 1.[ 3 ] VIUF, B . T . , "Breeding of barley varieties with high protein content with respect to quality". New

Approaches to Breeding for Improved Plant Protein (Proc. Panel, Rostânga, 1968), IAEA, Vienna (1969)23.[4] FAVRET, E. A ., SOLARI, R., MANGHERS, L. , AVILA, A., "Genetic control of the qualitative

and quantitative production of endosperm proteins in wheat and barley", New Approaches to Breeding for Improved Plant Protein (Proc. Panel, Rostlnga, 1968), IAEA, Vienna (1969) 87.

[5] MERTZ, E. T. , BATES, L.S., NELSON, О. E. , Mutant gene that changes protein composition and increases lysine content of maize endosperm, Science 145 (1964) 279.

[6] MUNCK, L. , KARLSSON, K. -E., HAGBERG, A., EGGUM, B., Gene for improved nutritional value of barley protein, Science 168 (1970) 985.

[ 7] VIUF, B. T ., Unders gelser vedr rende sortsvariation i kvælstofindhold og proteinkvalitet hos byg,Ph. D. Thesis, К. V. L., Copenhagen (1970).

[8] MOSSBERG, R., "Evaluation of protein quality and quantity by dye-binding capacity.: A tool inplant breeding", New Approaches to Breeding for Improved Plant Protein (Proc. Panel, Sostlnga, 1968), IAEA, Vienna (1969) 151.

[9] HAQ, M.S., CHOUDHURY, N. , RAHMAN, М. M. , "Breeding for high protein content and quality of rice through induced mutation", Improving Plant Protein by Nuclear Techniques (Proc. Symp.Vienna, 1970), IAEA, V ie n n a (1970) 63.

[10] TANAKA, S., TAKAGI, Y. , "Protein content of rice mutants", Improving Plant Protein by Nuclear Techniques (Proc. Symp. Vienna, 1970), IAEA, Vienna (1970) 55.

[11] FRYDENBERG, O. , NIELSEN, G., SANDFAER, J. , The inheritance and distribution of cc-Amylase types and DDT response in barley, Z. PflZticht. 61_ (1969) 201.

[12] NIELSEN, G. , FRYDENBERG, O. , "The inheritance and distribution of esterase isozymes in barley",Proc. 2nd Int. Barley Genet. Symp., Pullman (1969).

D I S C U S S I O N

O . P . K A M R A : I do not re c a ll i f you mentioned any m orphologica l ch aracteris tics o f your two high lys ine barley mutants.

H. D O LL : The two mutants a re a litt le ta lle r and a few days la te r than the m other va r ie ty . H ow ever, I do not think these characters are

3 4 2 DOLL

associated with the increased lysine content o f the mutants, since there was in genera l a v e ry high induced varia tion in the m ateria l.

A . HAG BERG : D r. D oll, which is the m other va r ie ty used in your treatm ents?

H. D O LL : A s mentioned in my paper the m other line was the va r ie ty C a rlsb erg II.

MUTAGENESIS OF A FLUCTUATING CHARACTER: GRAIN DORMANCYIN KRISTINA BARLEY*

Â . GUSTAFSSON, U. LUNDQVIST, J. KUCERA, J. GHATNEKAR

Institute o f Genetics, Lund,

Swedish Seed Association, Svalôv,

Sweden

Abstract-Resumen

MUTAGENESIS OF A FLUCTUATING CHARACTER: GRAIN DORMANCY IN KRISTINA BARLEY.Kristina barley is a variety obtained by crossing the X-ray-induced Swedish Mari variety with the Norwegian

Domen variety. It gives a high yield, shows good resistance to lodging, and its malting properties are excellent. It has, however, one drawback, its short dormancy period, on account of which it has a marked tendency to germinate on the culm in years when the harvest period is particularly rainy. It has proved possible to reduce this tendency by mutagenic treatment (with radiation and chemical agents) and rigorous selection in special fog chambers over three successive generations (M2-M4). Different mutagenic treatments have produced different results as regards the selection efficiency: it was maximal after treatment with EMS, El and neutrons, and minimal after treatment with MNU and gamma-radiation. Prolongation of dormancy is often accompanied by changes in morphology, maturation period (early or late), and other physiological properties.

MUTAGENESIS DE UN CARACTER FLUCTUANTE: LETARGIA SEMINAL EN LA CEBADA KRISTINA.La cebada Kristina es una variedad obtenida por cruzamiento de la variedad Mari sueca inducida por

rayos X y la variedad noruega Domen. Es de elevado rendimiento, muy resistente al encamado y de excelentes propiedades para el malteado. Sin embargo, tiene una desventaja: su breve letargo seminal, por lo que en años de elevadas precipitaciones en la época de la cosecha, tiene una elevada tendencia a germinar. Por tratamiento mutagénico (con radiaciones y agentes químicos) y procediendo con gran rigor selectivo en cámaras especiales de niebla durante tres generaciones sucesivas ( M2 - M4) ha sido posible reducir esta tendencia. Los distintos tratamientos mutagénicos han dado resultados diferentes en cuanto a la eficiencia de selección: ésta fue máxima tras el tratamiento con MSE, El y neutrones, y mínima tras el tratamiento con MNU y rayos gamma. La prolongación del letargo seminal va a menudo acompañada por cambios de la morfología, de la época de la maduración (temprana o tardía) y de otras propiedades fisiológicas.

In a paper o f 1941 the sen ior author (Gustafsson) reported the y ie ld behaviour o f some induced mutations in barley . They w ere d ivided into two groups: m orphologica l and physio log ica l (qualitative versus quantitative).The re la t iv e ly h igh -y ie ld ing mutants w ere analysed a couple o f months a fter harvest fo r th e ir d egree o f germ ination ripeness and gra in dorm ancy. The parent, Gull barley , was (and is ) characterized by a short period o f inhibited germ ination. M orpholog ica l mutants of the erecto ides type conspicuously delayed germ ination . The well-known erecto ides mutant 1 (e r t - c 1), however, was alm ost iden tica l in this respect with Gull.

In the "p h ys io lo g ica l" group, one mutant o f s traw -stiffn ess sign ifican tly d iffe red from Gull barley , having a low rate o f germ ination in O ctober (1940) and a norm al germ ination in N ovem ber. O f four mutants in the physio log ica l group two increased and two decreased the rate o f kernel r ipeness.

* Part of the research reported in this paper has been carried out under Research Agreement with the International Atomic Energy Agency No.358/CF.

343

344 GU STAFSSON e t a l .

Another feature concerning germ ination ab ility was analysed by Ehrenberg et al. (1965) and Jansson and Ehrenberg (1970) dealing with the so -ca lled w ater sensitiv ity . By this feature is meant a particu lar kind of dorm ancy m anifesting its e lf by making it im possib le fo r the barley grains to germ inate p rop er ly in an excess of w ater. The last-m entioned authors indicated by th e ir s ta tis tica l analysis that EM S-treated m ateria ls gave a h igher amount o f genetic va r iab ility leading to an increase in w ater sensitivity, whereas neutron-treated m ateria ls approached con tro l values. H owever, a certa in mutagenic e ffec t o f neutrons was a lso indicated. The authors concluded that the se lection fo r a changed w ater sen s itiv ity should p re ferab ly be done on an M3 plant b a s is .

Another p roperty has been studied in extensive experim ents started in1967 with the K ristina va r ie ty o f barley (Hagberg and Persson 1968), a c ro ss - product o f the Norw egian Domen barley and the Bonus mutant M ari (Gustafsson 1969). K ristina is an ex trem ely lodging-resistant and h igh-yield ing va rie ty , esp ec ia lly useful in productive a reas. The m alting quality is h igher than in M ari (o f average quality) and even h igher than in Domen (o f high quality).Th is goes p a ra lle l to an important p roperty that to a certa in extent hampers the cu ltivation o f the va r ie ty in regions with wet and rainy harvest periods.It has a rapid, often im m ediate germ ination a fter the ripening of the grains ("sh o rt dorm ancy"). The content o f a -am ylase, the diastatic power, the Kolbach index, as w e ll as the malt and extract production are high. The enzym atic a c tiv ity causes a malting quality su perior to that o f the control m ateria ls tested but, in addition, leads to an extrem e germ ination energy.

Th is highly m od ifica tive property o f germ ination rapid ity (short dormancy) was e lected fo r mutagenic treatm ent. W hether numerous genes with sm all e ffec ts o r a few genes with conspicuous e ffec ts (o r a m ixture o f both types of genes) are responsib le fo r germ ination behaviour in this specia l case, is not known. Germ ination energy and rapidity show a high degree o f fluctuation depending on the e x te r io r conditions, as w e ll as on the vary ing kernel m etabolism influencing germ ination rate. The possib le induction o f mutants with a delay in germ ination would involve numerous problem s of th eoretica l and p rac tica l in terest.

In 1967, kernels o f K ristina barley (11% w ater) w ere treated with d ifferen t doses o f gamma and neutron radiation, o f EMS, ethylene im ine (E l) and m ethyl n itroso urea (M NU ) solutions. The M 2 generation was sown according to routine methods, spike progeny a fter spike progeny. Chlorophyll and other lethal mutations w ere recorded, together with other in teresting mutant deviants, including those o f both m orphologica l and physio log ica l characters.

Fog chambers have recen tly been arranged at the Swedish Seed A s s o c iation fo r exam ining seed dormancy and germ ination rapid ity in rye, wheat and barley.. A descrip tion o f Finnish fog cham bers (greenhouses) was presented by K iv i and Ram m -Schm idt (1969) fo r se lection o f res istance to sprouting in 6QC o-irrad ia ted wheat. In M 2, M 3 and M 4 o f the K ristina m ateria ls four plants from each spike progeny w ere y ea r ly analysed, being w e ll- t ille re d , fe r t ile and fa ir- look in g to avoid an unnecessary accumulation o f sem i-le th a l o r v ita lity -d ec rea s in g mutants. The se lection was thus defin ite ly on the pos itive side with regard to genera l progeny v igour.

Germ ination o f fog -trea ted spikes (tem perature: 20°C; a ir humidity:fu lly saturated) was determ ined a fter five o r seven days, depending on the clim ate of the y ea r and the degree o f dorm ancy. Individual plants (sp ikes ) w ere given values from 1 to 1 0 , 1 being v is ib ly ungerm inated, 1 0 fully

F L U C T U A T IN G CH ARACTER M UTAGENESIS 345

1 2 3 A 5 6 7 8 9 10

FIG. 1. Germination rate ( dormancy), 1-10, in fog chamber material of Kristina barley. (S days, room temperature, saturated humidity; 1 + 2-germination type A¡ 5+6 = B; 9 + 10 = C).

germ inated (w ith long roots and w e ll-d eve loped plumules) (F ig . 1). Th ree groups o f m ateria ls w ere sp ec ia lly selected in M 2: С -m a te r ia l with g e rm ination values o f 9 and 10, B -m a te r ia l with values o f 5 and 6 , and A -m a te r ia ls with values o f 1 and 2. The M 3 p rogen ies w ere analysed in a s im ila r way, w ith four individual plants from each selected M 2 row . In the M 4 generation (1970) m ateria ls o f E A A (E =con tro l 1968), CA, BA, and AA w ere sown, with new con tro l m ateria ls o f K ristina in every tenth row (E ). Each row consisted o f 2 0 sown kernels resu lting in a sligh tly va ry in g number o f mature plants.In M 4 , as in the M 3 generation, only rows with at least 12 plants w ere se lected , and in each row four "n o rm a l-lo ok in g ", w e ll- t i l le r e d and fe r t ile plants. The sum values o f germ ination fo r individual M4 rows could a fter analysis v a ry from 4 (4 X 1) to 40 (4 X 10).

Corresponding to the А -m ateria ls in 1968 and 1969 (w ith slow sprouting ab ility ) a re the 1970 rows with average values o f 4-8. On the whole, the sum values o f 4-8 denote a slow sprouting ab ility.

The tota l m a ter ia l se lected fo r final analysis in 1970 com prised (see Tab le I) 4567 rows (to which could be added 520 rows o f the so -ca lled DA,i . e . a K ristina con tro l 1969, se lected fo r A plants only in 1969. Owing to its d iffe ren ce from other m ateria ls , which have been selected tw ice, DA is ' excluded from this ana lysis ).

It is evident from Tab le I that there is a steady in crease in the proportion o f 4-8 row s (as com pared to the 1970 con tro l E ) ranging from E A A o ve r CA and BA to A A . Simultaneously there is a steady increase in s ta tis tica l s ign ificance, determ ined on the basis o f ch i-square va lues. EAA , i . e . the1968 con tro l tw ice se lected , has a value s ligh ly h igher than E but without s ign ificance. In CA, se lected in opposite d irections in 1968 and 1969, there is a certa in indication o f an in crease . BA, i . e . medium m ateria l in 1968, then selected fo r A in 1969, has a c lea r ly sign ificant in crease . W ith the m a ter ia l o f AA , se lected tw ice fo r A , the in crease is evident.

In this summing-up o f the m ateria ls distinct d ifferences between the individual mutagenic treatm ents occur. Taking the unselected con tro l o f 1970 as base m ateria l the proportions between EMS, E l, N, MNU and 7 , in

346 G USTAFSSO N et a l .

T A B L E I. SE LE C TIO N IN M 4 (1970) FOR P L A N T ROWS W ITH G E R M IN ATIO N VALU ES OF 4-8 (SUM OF VALUES FOR FOUR A N A LYS E D P LA N T S PER ROW)

No. of rows4-8

Sum of rows °]o rows 4-8

Ratio Significance

E 19 575 3.30 1

EAA 17 390 4.36 1.320 0.5>P>0.3

CA 37 704 5.26 1.59<*) 0.10>P>0.05

BA 51 878 5.80 1.76*(*) 0.05>P>0.01

AA 141 2020 6.98 2.12*** P=0.001

TA B L E II. RATIO S OF GROUPS 4-8 IN AA : E AND EAA: E USING D IF F E R E N T M UTAGENS

Ratio AA : E (4-8)

Ratio EAA : E (4-8)

EMS 1.50°

El 3.71**(*) 2.11°

N 2.78° 0.94°

MNU 1.65» 1.38°

У 1.07“ 1.06»

Sum 2.12*** 1.32°

com parison with the tw ice -se lec ted con tro l o f 1968 (EAA) , are shown in Tab le II.

The average ra tio o f the 4-8 groups in 1970 o f EAA: E amounts to 1.3, an indication o f a slight se lection e ffect. The ra tio va r ie s in d ifferen t se r ies between 2. 1 and 0.9, in no instance sign ificant.

In the mutagenic treatm ents the average ratio o f the 4-8 groups in AA: E is 2. 1 with a high degree o f s ign ificance ( * * * ) . The individual values va ry from 5. 6 w ith EMS ( * * * ) , through 3. 7 with E l ( * * ) and 2. 8 with neutrons (no s ign ificance ), down to 1. 7 and 1. 1 with MNU and y resp ec tive ly . In fact the trea ted gamma m ateria l does not deviate from the untreated corresponding control m a ter ia l in mean ratio (1. 07 against 1. 06). Th is does not im ply, of course, that gam m a-rad ia tion cannot produce this type of mutation, only that the frequency is so low that no sign ificant average d ifferences are found. In fact, there seem to occur rea l dorm ancy mutants in the gamma m ateria l too.

What a re then the mutual e ffects o f se lection and mutation in the m ateria ls analysed? I f we assume that the E rep resen tatives o f group "4 -8 " in 1970 are gen era lly due to a phenotypic fluctuation, there is a certa in increase of 4-8 groups in EAA (the tw ice -se lec ted 1968 con tro l). Th is in crease, i f rea l, should be due to a heterogeneity in the o rig in a l K ris tina e lite o f 1967/ 6 8 .

F L U C T U A T IN G CH ARACTER MUTAGENESIS 347

T A B L E III. M O D IFICATIO N , SE LE C TIO N AND M U TA T IO N IN M U TAG ENIC T R E A TM E N TS OF KR ISTINA B A R L E Y (CO NSTRU CTED FRO M THE BEHAVIOUR OF E AND E A A )a

Ratios of groups

1. Modificative fluctuation

(<7°)

"4-8" (1970) depending

2. Selection

(%)

on

3. induced mutation

(%>

E 100 - -

EAA 76 24 -

CA 62 22 16

BA 57 18 25

AA 48 14 38

a Note that these percentages are fictive. The ratios vary with the type of mutagen applied; EMS gives the highest-values of induced mutation, ythe lowest values. Individual mutagens are not recorded in this Table.

With E and EAA fo rm in g the basis fo r calculation we may then decide how much o f the 4-8 groups o f each set o f m utagenically treated m a teria l is due to ( 1 ) environm ental fluctuation, ( 2 ) se lection within an o r ig in a l h etero geneity, and (3) induced mutation. By this we assume that no spontaneous mutations to dorm ancy have occu rred in the (E and) EAA m ateria l. The figu res obtained a re g iven in Tab le III.

These average figu res are uncertain but g ive some idea about the r e la tive proportions o f se lection and mutation in the mutagenesis m ateria l s t ill rem ain ing in the fourth generation. Probably much o f the m ateria l o f type 2 ("s e le c t io n " ) w ill in fu rther analysis show its e lf to have been m od ifica tive,i . e . belonging to type 1 ("m od ifica tiv e fluctuation").

T ru ly induced mutations o f dormancy, esp ec ia lly a fter EMS, E l and neutron treatm ent, do occur and w ill be studied in fu rther progeny and cross ing experim ents, invo lv ing a lso b iochem ical tests . It is a lready evident that se lection fo r gra in dormancy, in mutagenic treatm ents, often influences p roperties as fo r instance m orphologica l appearance, ch lorophyll pigment and plastid form ation , heading tim e and m aturity, as w e ll as genera l y ie ld ing ab ility . M oreover, certa in resu lts in the M 2 - M 4 generations indicate that the se lection e ffic ien cy was m ore pronounced in the M 3 than in the M 2 generation.

R E F E R E N C E S

EHRENBERG, L., EKMAN, G., GUSTAFSSON, A., JANSSON, G . , LUNDQVIST, U., 1965, "Variation in quantitative and biochemical characters in barley after mutagenic treatments”. The Use of Induced Mutations in Plant Breeding (Rep.FAO/IAEA Tech.Meeting, Rome, 1964), Pergamon Press, Oxford: 477.GUSTAFSSON, A., 1941, Preliminary yield experiments with ten induced mutations in barley, Hereditas 27; 337.GUSTAFSSON, A., 1969, "Positive Mutationen und ihre Verwendung in der Ziichtung hochleistender Gersten- Sorten", Ber.Arbeitstag.1969, Arbeitsgemeinschaft Saatzuchtleiter, Gumpenstein: 63.HAGBERG, A., PERSSON, G., 1968, Induced mutations in barley breeding, Hereditas 59: 3915.JANSSON, G., EHRENBERG, L., 1970, On the mutability of water-sensitivity in barley, Hereditas 64 : 181. KIVI, E.I., RAMM-SCHMIDT, C., 1969, "Selection for resistance to sprouting in 60Co-irradiated wheat". Induced Mutations in Plants (Proc.Symp.Pullman, 1969), IAEA, Vienna: 535.

3 48 GU STAFSSON e t a l .

D I S C U S S I O N

H. HANSEL: A s you know, we b reeders are always a fra id that a"p o s it iv e " change in one character could be associated with a "n ega tive " change in another im portant character, esp ec ia lly i f there is a tendency o f a negative physio log ica l corre la tion between these two characters . F o r this reason I would like to ask you whether you have a lready obtained resu lts of m icro -m a ltin g tests o f your mutant lines, resistant to sprouting.

A. GUSTAFSSON: Such malting studies are on the im m ediate program .Lack o f tim e has prevented us from ca rry in g them out this year.

C. K R U LL ; These resu lts are v e ry encouraging and you mention in the text o f your paper that the fog chambers are a lso being used fo r rye and wheat. Could you te l l us i f a para lle l p ro jec t is being ca rr ied out by you o r others at SvalSv in wheat, and i f the resu lts are a lso encouraging?

Â. GUSTAFSSON: Y es , profound resu lts have been obtained in rye andbarley , and to some extent a lso in wheat. F og chambers are a regu lar instrum ent both in recom bination and mutation work. In both fie ld s encouraging se lections w ere made.

A. HAGBERG: Sprouting is one o f our main quality prob lem s in Sweden.F o r the last 12-15 yea rs we have been working m ainly on sprouting resistance in rye and wheat. In rye we have just re leased two va r ie tie s which w ill g ive the fa rm ers about 1 0 days longer period during harvest tim e to find a suitable day to combine a quality crop suitable fo r baking. In wheat this work is also advanced and new va r ie t ie s with im proved sprouting res istance w ill be re leased soon.

A . ASHRI: You mentioned possib le natural va r iab ility in the controls.Can you elaborate on it and on its orig in in the K ristina barley?

Â. GUSTAFSSON: K ristina barley, as w e ll as most other modernva r ie t ie s , is ch aracterized by a certain sm a ll genetic va r iab ility within the fram ew ork o f its m orphologica l and agronom ic ch aracteris tics .

A . HAGBERG: K ris tina is a plant se lection in F3 repeated in F4.Severa l lines a re se lected in Fg -F g and those s im ila r enough are bulked — thus the va r ie ty is a m ultiline va rie ty .

A . GROBM AN: In both your papers, D r. Gustafsson, as w e ll as inDr. G ottscha lk 's paper, mention was made to mutants with prom ising agronom ic ch a racteris tics , which could not be u tilized because o f lateness. Lateness in Northern Europe is a re s tr ic t iv e ch aracteris tic , but this would not be lim itin g at a ll in other latitudes. Does a system ex ist fo r listing these useful mutants and making them available to b reeders in d ifferen t areas?I f not, may I suggest that F A O take up this subject, and include it in its v a r ie ta l lis ts o f m a ter ia l available to b reeders around the w orld .

A. GUSTAFSSON: We have at SvalOv (Sweden) a mutation assortm entwhich is ava ilab le fo r any scientist the w orld o ver . This assortm ent was p a rtia lly described in a paper in Hereditas 1969 by Gustafsson, Hagberg, Lundqvist and Parsson . In the present investigation o f dorm ancy we shall keep ea rly , norm al and late variants and these also w ill be ava ilab le to anybody in terested .

INDUCED MUTATIONS AND WINTER BARLEY IMPROVEMENT*

R .A . N ILAN

Department o f Agronom y and Program in Genetics,


Pullman, W ash., United States o f Am erica

Abstract-Resumen

INDUCED MUTATIONS AND WINTER BARLEY IMPROVEMENT.A barley mutant selected in 1962 at Pullman (Washington) has been developed into a new variety named

"Luther" which was officially released in 1966. It has short straw, good lodging resistance and improved winter-hardiness and is now the highest-yielding winter barley grown in the Pacific Northwestern States of the USA, the acreage being 120000 in 1970, only three years after release. The increase in farmers’ income in the states of Washington, Idaho and Oregon, due to the new variety amounts to more than US$ 1 million annually. The variety is also used extensively in cross breeding and newly developed strains promise another 10°Jo yield increase over "Luther" barley.

LAS MUTACIONES INDUCIDAS Y EL MEJORAMIENTO DE LA CEBADA EN INVIERNO.Un mutante de cebada seleccionado en 1962, en Pullman (Washington) se ha desarrollado hasta

convertirlo en una nueva variedad denominada « Luther>> que fue autorizada oficialmente en 1966. Es de tallo corto, buena resistencia al encamado y mejor aguante al frío; actualmente, es la cebada de invierno de mayor rendimiento en los Estados del noroeste de los Estados Unidos, ascendiendo la superficie cultivada en 1970, sólo tres aííos después de su comercialización, a 120 000 acres. El aumento de los ingresos de los agricultores de los Estados de Washington, Idaho y Oregon, gracias a esta nueva variedad, es superior a un millón de dólares anuales. Esta variedad se utiliza también extensamente en cruzamientos y las variedades recientemente obtenidas parece que tendrán un rendimiento superior en su lO o al de la cebada « Luther».

In 1966 and 1967 [ 1, 2] the developm ent by induced mutation o f a new w inter b a r ley v a r ie ty ca lled "L u th e r" was described . The chem ica lly - induced mutant was se lected in 1962 and re leased as a v a r ie ty in 1966.

Luther, in com m erc ia l production since 1967, has been grown ch iefly in w in ter b a r ley reg ions o f the States o f Washington, Oregon and Idaho.Its short straw , good lodging res is tance, high y ie ld s and im proved w in ter- hardiness have made it appealing to fa rm ers in this reg ion . It is the highest y ie ld in g w inter b a r le y now grown in the P a c ific Northwest.

Luther has re c e iv ed extensive agronom ic testin g and m ore recen t data a re presen ted in Tab le I along with s im ila r data o f the parental va r ie ty , A lp ine. The resu lts o f seven y e a rs ' testing c le a r ly dem onstrate its agronom ic su perio rity to A lp ine. M ore important, we have now obtained com m erc ia l y ie ld data from many grow ers and it appears that this va r ie ty outyields A lp ine and other cu rren tly grown w in ter b a r ley va r ie t ie s by about one quarter o f a ton (500 pounds o r 10 bushels) p er acre .

P r im a r ily because o f its high y ie ld it has en joyed a ra ther rapid acreage in crease . In 1968, 1969 and 1970 it was produced on 10 000,42 000 and 120 000 acres resp ec tiv e ly , in the th ree P a c ific Northwest States. The 1971 acreage could w e ll exceed 200 000 acres .

* Part of the research reported in this paper has been carried out under Research Agreements with the International Atomic Energy Agency Nos 321/CF and 615/CF.

3 4 9

3 5 0 NILAN

T A B L E I. SEVEN Y E A R S ' AGRONOM IC D A T A (1964-70) CO M PARING LU TH E R AND A L P IN E

°Io Plant height Lodging ‘Vo Yield (bu/ac)Survival

NFa Fb NF F NF F

Luther 97 35 39 0 4 102.5 116.4

Alpine 95 42 45 9 35 90.3 97.3

a NF = Regular fertilization, b F = 50 lb nitrogen added.

T A B L E II. TW O Y E A R S ' AGRONOM IC D A T A (1969-70) CO M PARING LU TH E R AND TW O NEW SELECTIO NS C O NTAIN ING LU TH E R

% Plant height Lodging °Jo Yield (bu/ac)

Survival NFa Fb NF F NF F

Luther 90 37 39 0 4 108.2 106.8

Luther X 1255-60 Selection 1094-67 95 35 36 0 0 115.1 119.0

Luther x Hudson Selection 2116-67 98 34 36 0 0 104.1 126.6

a NF = Regular fertilization, b F = 50 lb nitrogen added.

The com m erc ia l production o f this va r ie ty is now becom ing a considerab le econom ic fa c to r in the agricu ltu re o f the States in which it is grown. W ith cu rren t feed b a r ley p r ices and its y ie ld advantage o ver A lpine and other w in ter b a r ley v a r ie t ie s , Luther is return ing o ve r $9 m ore p er acre . In the th ree States o f Washington, Idaho and Oregon this means an increased incom e in 1970 (120 000 acres X 9) o f $1 080 000 because o f the production o f Luther barley . During this com ing yea r this figu re should be considerab ly h igher.

Th is va r ie ty a lso appears to be one o f the best parents fo r fu rther in creas ing b a r ley y ie ld s and production in our w in ter b a r ley areas. Th is is revea led in Tab le Ы. F rom two y ea rs ' data it can be seen that certa in se lections from c rosses invo lv ing Luther are considerab ly h igher-y ield ing than Luther, pa rticu la rly under fe r t i l iz e d conditions. F u rth erm ore, they are shorter, m ore lodging res is tan t and w in ter-hardy, and possess plum per

W INTER BARLEY IMPROVEMENT 351

kernels than Luther. It is expected that one o f these new selections w ill be re lea sed within two o r th ree yea rs , g iving another 10% in crease in y ie ld o ve r cu rren tly grown Luther, and in turn p rovid ing many m ore do lla rs to the econom y o f this and other States.

C hem ica lly and radiation-induced mutations are also helping in the im provem ent o f other types o f barleys that are grown in the P a c ific Northwest o f the USA, In fact, m ore and m ore mutants are appearing in the p ed ig rees o f many p rom is in g selections in our tw o -row m alting, s ix -ro w m alting and s ix -ro w spring feed barley p rogram s.

R E F E R E N C E S

[1] NILAN, R.A., "Barley cytogenetics and breeding (A progress report)", Mutations in Plant Breeding (Proc. Panel, Vienna, 1966), IAEA, Vienna (1966) 177.

[2] NILAN, R.A., MUIR, C.E., Registration of Luther barley, Crop Sci. 7 3 (1967) 278.

MUTATION RESEARCH AND UTILIZATION OF INDUCED MUTANTS IN LATIN AMERICA

REACCION FRENTE A Puccinia recóndita, tritici DE LAS LINEAS DERIVADAS DEL CRUZAMIENTO ENTRE LA VARIEDAD DE TRIGO SINVALOCHO MA Y SU MUTANTE INDUCIDA"

F. L. MUJICA, E. F. ANTONELLI, H. P. CENOZ

Centro de Investigaciones en Ciencias Agronómicas,Castelar, Argentina

Abstract-Resumen

REACTION AGAINST Puccinia recóndita tritici OF LINES DERIVED FROM A CROSS BETWEEN SINVALOCHO M A WHEAT VARIETY AND ITS INDUCED MUTANT.

In previous work the possibility of changing the rust reaction of cereals by means of artificially induced position effects-was emphasized. The mutant induced in wheat by means of y-ray treatment showed a modification in reaction against some physiological races of Puccinia recóndita tritici, as well as in other characters, thus being considered a multi-site mutation. The reaction of lines derived by recombination from the cross of the mutant and the original variety revealed the presence of plants resistant to the international groups 2, 20, 77 and 144 of Puccinia recóndita tritici, Sinvalocho being resistant only to the races of group 20. Lines susceptible to all races were also found.

Recombinants which either gained or lost resistance to some races, as well as some other lines in which a simultaneous gain and loss of resistance took place when compared with Sinvalocho MA, were obtained. These results would suggest that the correspondence between the host-pathogen genetic systems would constitute only a part of a more complicated process. It would rather appear that resistance and susceptibility to a specific race are correlated with metabolic changes that might or might not be utilized by the pathogen.

REACCION FRENTE A Puccinia recóndita tritici DE LAS LINEAS DERIVADAS DEL CRUZAMIENTO EMTRE LA VARIEDAD DE TRIGO SINVALOCHO M A Y SU MUTANTE INDUCIDA.

En trabajos anteriores se ha destacado la posibilidad de producir cambios en la reacción de los cereales a las royas por medio de la inducción artificial de efectos de posición. La mutante inducida en trigo por medio del tratamiento con rayos y presentaba una modificación de su reacción frente a algunas razas fisiológicas de P. recóndita tritici, además de otros caracteres, tratándose por lo tanto de una mutación múltiple. El estudio de la reacción, frente al mismo patógeno, de las líneas derivadas por recombinación del cruzamiento entre la mutante y el material original reveló la aparición de formas resistentes a las razas internacionales 2, 20, 77 y 144 (siendo Sinvalocho sólo resistente a las razas del grupo 20), además de otras susceptibles a todas ellas.

Se obtuvieron también otras formas, algunas de las cuales ganaron y otras perdieron resistencia con respecto a la variedad original Sinvalocho M A y recombinantes que mostraron una pérdida y ganancia simultánea de resistencia frente a algunas razas de P. recóndita tritici. Estos resultados revelarían que la correspondencia entre los genes del huésped y del patógeno puede ser sólo una parte de un proceso mucho más complicado. Parecería más bien que la resistencia y susceptibilidad a una determinada raza está correlacionada con cambios metabólicos que pueden ser o no aprovechados por el patógeno.

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* Publicación Técnica Gen. №447 del Centro de Investigaciones en Ciencias Agronómicas, CICA, INTA, Castelar.

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TRIGO SINVALOCHO Y SU M UTANTE 3 5 7

c ió n a r t if ic ia l c o n la r a z a 15. E n la g e n e r a c ió n M 2 la m is m a s e g r e g ó

p la n t a s r e s is t e n t e s y s u s c e p t ib le s a aque l p a r á s i t o , p e r o en fo r m a i -

n e s p e r a d a s e o b s e r v ó a c a m p o la p r e s e n c ia de p la n t a s m u y s u s c e p t i b le s a P u c c in ia re c ó n d ita t r i t íc i . c a r a c t e r í s t ic a é s ta q u e no p r e s e n t a b a la v a r ie d a d o r ig in a l [ \ J . E n e fe cto , S in v a lo c h o M . A . , p o r su

r e s is t e n c ia a la r a z a in te rn a c io n a l 2 0 de r e c ó n d ita t r i t íc í . q u e e r a la q u e e n to n c e s p r e d o m in a b a , p r e s e n t a b a un b u e n c o m p o r ta m ie n to a

c a m p o a u n q u e e r a su s c e p t ib le a o t r a s r a z a s p o c o d ifu n d id a s (1 4 4 , 2 y 7 7 ) , a lg u n a s de la s c u a le s s o n a c tu a lm e n te m u y c o m u n e s [ i j , r a

z ó n p o r la c u a l s e la u tiliza c o m o v a r ie d a d d ife re n c ia l r e g io n a l.

D e la d e s c e n d e n c ia de l c ru z a m ie n to de la m utan te o r ig in a l G a m a 4 p o r s u lín e a m a d r e , s e p u d ie r o n a i s l a r n u e v o s g e n o t ip o s q u e d if ie re n

d e S in v a lo c h o M . A . en s u r e a c c ió n a la s d is t in ta s r a z a s d e P u c c in ia

q r a m in is tr it ic i y _ P . r e c ó n d ita t r i t ic i .

E l a g e n te m u ta g é n ic o h a b r ía c a u s a d o u na t r a n s lo c a c ió n r e c íp r o c a con

el s u b s é q u e n te c a m b io de p o s ic ió n de un g e n e , m a n ife s ta d o p o r la

g a n a n c ia y p é r d id a s im u ltá n e a d e r e s is t e n c ia a d o s r a z a s de JP. r e c ó n d ita t r i t ic i . a d e m á s de la a lte ra c ió n de o t r o s c a r a c t e r e s , tratándos e p o r lo tanto de u n a m u tac ió n m ú ltip le 3J ,

E n e s te t ra b a jo s e a n a liz a la r e a c c ió n , fre n te a n u m e r o s a s r a z a s d e £ . r e c ó n d ita t r i t ic i . de la s l ín e a s d e r iv a d a s p o r re c o m b in a c ió n del c r u z a m ie n to e n tre la v a r ie d a d o r ig in a l y s u m utan te G a m a 4 , c o n el objeto de in d a g a r s o b r e la f r e c u e n c ia y m a gn itu d de aqu e l fe n óm e n o y e v a lu a r s u s p o s ib i l id a d e s t e ó r ic a s y p r á c t i c a s .

M a te r ia l y m é to d o s

S e e n s a y a r o n s ie te r e c o m b in a n te s s e le c c io n a d a s p o r s u r e a c c ió n d ife re n c ia l fre n te a a lg u n o s b io t ip o s de P . g r a m in i s t r it ic i y P_. r e c ó n

d ita t r i t ic i .

L_as p r u e b a s s e e fe c tu a ro n al e s ta d o d e p lá n tu la c o n 17 d is t in ta s

r a z a s d e lo s g r u p o s in te r n a c io n a le s 2 , 1 2 , 2 0 , 77 y 1 44 . l_ a identi

f ic a c ió n d e la s d is t in ta s s u b r a z a s s e r e a l iz ó m e d ian te el u so de u n a

s e r ie d e v a r ie d a d e s d i f e r e n c ia le s r e g io n a le s ( C u a d r o 1 ). E s t a s v a r ie d a d e s s o n u t i l iz a d a s p a r a in d iv id u a liz a r la s d is t in ta s f o r m a s f is io ló

g i c a s to m a n d o en c u e n ta a lg u n a s de s u s c a r a c t e r í s t ic a s p a t o g é n ic a s

m á s c o n s p ic u a s . A s í , p o r e je m p lo , la r a z a 2 T i R ( s ig n if ic a n d o K le in

T it á n R e s is t e n t e ) e s la ú n ic a d e n t ro del g r u p o 2 , a v iru le n ta s o b r e

K . T it á n ; la r a z a 2 0 T ( N R ) a ta c a a B u c k T a n d i l s ie n d o N o r m a n d ie r e s is te n te ; e tc .

A lo s e fe c to s de u n a m e jo r v is u a l iz a c ió n de lo s r e s u l t a d o s , s e ind ic a la r e a c c ió n d e l a s l ín e a s c o n lo s s ím b o lo s R ^ M R , M S y S q ue

s e c o r r e s p o n d e n c o n lo s s ig u ie n te s t ip o s de in fe c c ió n : 0 , 1 a 2 + ,2 + + a 3j y 4 r e s p e c t iv a m e n te .

3 5 8 MUJIC A et al.

R e s u lt a d o s

C o m o p u e d e a p r e c i a r s e en el C u a d r o I , to d a s la s re c o m b in a n te s

e n s a y a d a s d if ie re n de la v a r ie d a d o r ig in a l, Sinvalocho M.A. en su r e a c c ió n a p o r lo m e n o s 4 r a z a s . E n a lg u n o s c a s o s ( G a m a 1 R ,

G a m a 4 y G a m a 6 ) s e r e g is t r ó u n a g a n a n c ia d e r e s is t e n c ia . G a m a 1 R fue r e s is te n te a t o d a s la s r a z a s d e lo s g r u p o s 2 , 144 y 77 . G a m a

4 g a n ó r e s is t e n c ia al g r u p o 144 y a a q u e lla s r a z a s d e lo s g r u p o s 2

y 7 7 , a v ir u le n t a s s o b r e B u c k T a n d i l . G a m a 6 fue r e s is te n te al g r u p o

77 y a la r a z a 2 T a S , v iru le n ta s o b r e B u c k T a n d il; lo s t ip o s de in fe c c ió n r e g i s t r a d o s fre n te al g r u p o 2 0 h a c e n s u p o n e r la a c c ió n de

f a c to r e s de r e s is t e n c ia d is t in to s a lo s de S in v a lo c h o M . A . fre n te a

e s e g r u p o .

U n c a s o de p é r d id a total de r e s is t e n c ia con r e s p e c t o a la s r a z a s

e n s a y a d a s lo representó la re c o m b in a n te G a m a 3 , p e r o el h e c h o m á s c o n s p ic u o lo c o n st itu y ó el c a s o de la s r e c o m b in a n te s G a m a 1 S y

G a m a 5 , d o n d e s e - o b s e r v ó una g a n a n c ia y p é r d id a s im u ltá n e a de r e s is t e n c ia . G a m a 1 S re s u ltó s u s c e p t ib le a las d o s r a z a s del g r u p o 20

v ir u le n ta s s o b r e B u c k T a n d i l , p e r o g a n ó r e s is t e n c ia a la s t r e s r a z a s

del g r u p o 2 , a v ir u le n t a s s o b r e e s ta ú ltim a d i f e r e n c ia l, co m o a s í tam b ién fre n te al g r u p o 144 y a la s d o s r a z a s del g r u p o 77 a v ir u le n ta s

tam b ién s o b r e B u c k T a n d i l . P o r s u p a r te G a m a 5 in c o r p o r ó r e s i s

te n c ia al g r u p o 77 y a la r a z a 2 T a S , p e r d ie n d o r e s is t e n c ia a la s r a z a s 2 0 P R , 2 0 P S ( N S ) y 20 P S ( N R ) . E n el C u a d r o II s e in

d ic a la g a n a n c ia o p é r d id a de r e s is te n c ia o b s e r v a d a p a r a c a d a r e c o m b in an te .

CUADRO II. GANANCIA O PERDIDA DE RESISTENCIA A ALGUNAS RAZAS DE Puccinia recóndita tritici DE LAS RECOMBINANTES DEL CRUZAM IENTO GAMA 4 X SINVALOCHO MA, CON RESPECTO A ESTA ULTIM A VARIEDAD

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G a m a 1 R 1 0 0 + 10

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G a m a 5 4 3 + 1

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TRIGO SINVALOCHO Y SU M OTANTE 3 5 9

D i s c u s ió n

l_ a v a r ie d a d o r ig in a l S in v a lo c h o M . A , p o s e e un g e n e A ( ) q u e le

c o n f ie r e r e s is t e n c ia a la r a z a 20 L d e P , r e c ó n d ita t r i t ic i , el c u a l, a c o n s e c u e n c ia de u n a t r a n s lo c a c ió n r e c íp r o c a fue t r a n s fe r id o a una

n u e v a p o s ic ió n A i (e n la m utan te G a m a 4 ) en o tro c r o m o s o m a , m o d if ic a n d o su r e a c c ió n a r o y a p u e s to q ue g a n ó r e s is t e n c ia a la r a z a 144

/ 3 7 .

l_ a d e s c e n d e n c ia del c ru z a m ie n to e n tre la m utan te o r ig in a l G a m a 4 y S in v a lo c h o M . A . p e rm it ió a i s l a r n u e v o s g e n o t ip o s ( G a m a I R y

G a m a 6 ) en lo s c u a le s p o r r e c o m b in a c ió n , el g e n e A-¡ v o lv ió a una

p o s ic ió n m u y c e r c a n a a la o r ig in a l A o , d e s ig n á n d o s e la c o m o A 2 / ï J[ * J .

L a r e c o m b in a n te G a m a I R fue la q u e p r e s e n t ó el e s p e c t r o m á s

a m p lio de r e s i s t e n c ia , s ie n d o s ó lo a ta c a d a p o r la r a z a 12. E n t r a b a

jo s a n t e r io r e s su resistencia a la r a z a 2 0 L fue atribuida a lo s g e n e s

A i У A 2 / 3 7 . E l a n á l i s i s de la s e g r e g a c ió n F 3 de l c ru z a m ie n to e n tre

G a m a 1 R y A x m in s t e r fre n te a e s a r a z a , c o n f ir m a la p r e s e n c ia de

d o s f a c to r e s q u e tam b ié n c o n d ic io n a r ía n la r e s is t e n c ia a la r a z a 77 N S

( C u a d r o 111) ,d a d a la c o r r e s p o n d e n c ia o b s e r v a d a en la r e a c c ió n de fam il ia s R 3 fre n te a a m b a s r a z a s . L a p r e s e n c ia d e n tro del tipo r e s i s ten te , de fa m il ia s h o m o c ig o ta s y h e te r o c ig o ta s p a r a lo s t ip o s de in fe c

c ió n 0 , y 1 a 2 = in d ic a r ía la p r e s e n c ia d e f a c to r e s d e d ist in ta e x p r e s iv id a d . E l g e n e d e s ig n a d o c o m o A ^ d a r ía el n ive l m á s a lto d e r e s i s te n c ia y s e r ía e p is tá t ic o s o b r e el s e g u n d o .

CUADRO III. CLASIFICACION DE LAS FAM ILIAS F3 DEL CRUZAM IENTO GAMA 1RX AXMINSTER, FRENTE A LAS RAZAS 20 L Y 77 NS DE Puccinia recóndita tritici

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( R y M R )1 03 9 2 ,3 1 1 ,2 3 7 9

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( R , M R / M S , S )99 1 0 5 ,5 0 0 ,4 0 0 4

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( M S y S )9 1 3 ,1 9 1 ,3 3 1 0

T o t a l 21 1 2 1 1 2 ,9 6 9 3

R = 0 , 2 - 0 , 3 .R e a c c ió n de lo s p a d r e s fre n te a a rr ib a s r a z a s :

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TRIGO SINVALOCHO Y SU MUTANTE 3 6 1

C o n r e s p e c t o a la r a z a 2 T a R ( N S ) la s e g r e g a c ió n fue m o n o g é -

n ic a , s ie n d o uno de lo s d o s a n t e r io r e s el fa c to r q u e c o n d ic io n a r ía la

r e s is t e n c ia de G a m a 1 R fre n te a e s a r a z a ( C u a d r o I V ) .

E s t e h e c h o a b r e n u e v a s p e r s p e c t iv a s en lo s t r a b a jo s de m e jo ra m ie n to

y a q u e s e r í a fac tib le la o b te n c ió n de n u e v a s fu e n te s d e r e s is t e n c ia a t r a v é s de v a r ie d a d e s s u s c e p t ib le s .

E l g e n e A 2 p r e s e n t e en la r e c o m b in a n te G a m a 6 , r e g u la r e s i s

te n c ia a la s r a z a s 2 0 L y 2 T a S , p e r o n o a la r a z a 144 ,

E l e s p e c t r o d e r e a c c io n e s de G a m a '5 fre n te a la s r a z a s del g r u p o 2 0 d if ie re de lo o b s e r v a d o c o m u n m e n te , p u e s to q u e , de p r u e b a s

r e a l i z a d a s s o b r e n u m e r o s a s v a r ie d a d e s de m u y d ist in to o r ig e n s e ha

p o d id o c o m p r o b a r q u e , en la to ta lidad de lo s c a s o s , la s v a r ie d a d e s

r e s is t e n t e s a la s r a z a s 2 0 T ( N R ) , 2 0 T ( N S ) y 20 L tam b ién lo

e r a n a la s r a z a s 2 0 P R , 2 0 P S ( N R ) y 2 0 P S (A n to n e ll i y M u j ic a ,

d a to s n o p u b l ic a d o s ) . E s t e h e c h o la h a c e d e s u m a u tilid ad en la s e p a r a c ió n d e r a z a s f i s io ló g ic a s .

A g r u p a n d o la s r a z a s u t i l iz a d a s en g r u p o s de v ir u le n c ia s im i la r e s c o n r e s p e c t o a la s r e c o m b in a n te s e n s a y a d a s , y de l a n á f is is d e lo s

d a to s d is p o n ib le s , s e p u e d e n p o s t u la r lo s g e n o t ip o s q u e s e in d ic a n en

el C u a d r o V , c o n s u s r e s p e c t iv o s e fe c to s y h o s p e d a n te s p o r t a d o r e s .

A lo s e fe c to s de s im p l i f ic a r la in te rp re ta c ió n de lo s d a to s s e a g r u p a r o n la s r e a c c io n e s R y M R , y M S y S , b a jo lo s s ím b o lo s R y S ,

re s p e c t iv a m e n te .

S e s u g ie r e c o m o h ip ó te s is d e t ra b a jo q u e el g e n e A , p o r t r a n s lo c a c ió n a p o s ic io n e s A 3 y A 4 p r o v o c ó , p o r n u e v o s e fe c to s d e p o s ic ió n ,

e s p e c t r o s de r e a c c io n e s p a r t ic u la r e s t r a d u c id o s en la s im u ltá n e a p é r d id a y g a n a n c ia d e r e s is t e n c ia a P . r e c ó n d ita t r i t ic i . e je m p lif ic a d o s

en G a m a 1 S y G a m a 5.

I_ a in d u c c ió n d e e fe c to s de p o s ic ió n d e te r m in a r ía la p r o d u c c ió n de

m u ta c io n e s m ú lt ip le s d e b id a s a un s o lo e v e n to m u ta g é n ic o , c o m o p o d r ía s e r el c a s o o b s e r v a d o p o r H o ffm a n ( c f . K o n z a k , 1 9 5 9 ) / 4 / , q u ién

o b tu vo m u tan te s r e s is t e n t e s s im u ltá n e a m e n te a v a r i a s r a z a s de P u c c i n ia s t r i i f o r m is y £ . r e c ó n d ita t r it ic i.

T a n t o la p r e s e n c ia d e m u ta n te s a p a re n te m e n te e x t r a ñ a s en la n a

tu r a le z a , c o m o la p é r d id a y g a n a n c ia s im u ltá n e a de r e s is t e n c ia fren te a un p a tó g e n o n o s o b lig a a un r e p la n te o , p o r lo m e n o s p a r c ia l , de a lg u n o s c o n c e p to s d e la r e la c ió n h u é s p e d -p a t ó g e n o , y a q u e la c o r r e s p o n d e n c ia e n tre a m b o s s i s t e m a s g e n é t ic o s s e r í a s ó lo u n a p a r te d e un

p r o c e s o m u c h o m a s c o m p lic a d o .

T r a b a j o s r e c ie n te s s o b r e la s im ilitu d d e lo s s is t e m a s g e n é t ic o s de lo s m ic r o o r g a n i s m o s y lo s o r g a n is m o s s u p e r io r e s s u g ie r e n la p o s ib i

lid a d d e e x p lic a c ió n su p o n ie n d o al g e n e c o m o un ente c o m p le jo fo r m a do p o r s u b - u n id a d e s p r o d u c t o r a s d e d is t in to s m e ta b o lito s n e c e s a r io s

p a r a a lg ú n c ic lo m e ta b ó lic o .

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TRIGO SINVALOCHO Y SU M UTANTE 3 6 3

L a r e s is t e n c ia o su s c e p t ib il id a d a u n a d e te rm in a d a r a z a , e s t a r ía

e n to n c e s c o r r e la c io n a d a c o n c a m b io s m e ta b ó lic o s q u e p u e d e n o no s e r a p r o v e c h a d o s p o r el p a tó g e n o .

A d e m á s d e s u s im p lic a n c ia s t e ó r ic a s , r e s u lta o b v ia la u tilidad

p r á c t ic a d e m e c a n is m o s c o m o el d e s c r ip to , y a q u e p o r m e d io de

e fe c to s d e p o s ic ió n s e r ía fac tib le a u m e n ta r la v a r ia b i l id a d g e n é t ic a .

R E F E R E N C I A S

[ 1] FAVRET, E. A., «Induced mutations in breeding for disease resistance», The Use of Induced Mutations in Plant Breeding (Rep. FAO/IAEA Tech. Meeting, Rome, 1964), Pergamon Press,Oxford (1965) 522.

[2] ANTONELLI, E. F. , MUJICA, F. L. , FRECHA, J.H. , R. AMIEVA, P., HOROVITZ, N. , CENOZ, H. P. , Fuentes de germoplasma de resistencia a enfermedades y plagas, Rev. Inv. Agrop., Serie 2 73 (1970) 133.

[3] FAVRET, E. A. , CENOZ, H. P. , SILVERO SANZ, O.I. , SOLARI, Rut M. , MUJICA, F. L. , «Efecto de posición inducido en trigo para reacción a las royas», Induced Mutations in Plants (Actas Simp. Pullman, 1969), OIEA, Viena (1969) 123.

[4] KONZAK, C. F., «Induced mutations in host plants for the study of host-parasite interactions»,Plant Pathology Problems and Progress 1908-1958, Univ. of Wisconsin Press, Madison, Wis. (1959) 202.

D IS C U S S IO N

C. KRULL: This group of mutant selections is interesting in that changes were encountered that are both mor'e resistant and more susceptible to different races. Frequently resistance to the rusts is dominant to susceptibility. Have you analysed among the new mutant types and in comparison with Sinvalocho to see if these new types of reaction are dominant or recessive to the original Sinvalocho reaction and in comparison with one another?

F .L . MUJICA: When crossing has been effected with the parental line, Sinvalocho MA, or with other susceptible varieties the resistance character has always shown dominant expression.

E. GIANDANA: I should like to ask whether studies were carried out with a view to observing the behaviour of P. graminis in the mutants studied with respect to P. recóndita, in other words whether there was any change in P. gram inis.

E .A . FAVRET: In the case of the reaction to P. graminis tritic i, strains 15, 17 and 11 in the various biotypes present in Argentina, the resistance is complete and is never lost. The reason for this is that the original line is susceptible to all of them. Two genes are involved in the reaction, but they are not the ones that regulate the reaction to P. recóndita tritici.

ASOCIACION ENTRE LA CONDICION HARINOSA Y LOS COMPONENTES PROTEICOS DEL ENDOSPERMA EN LA MUTANTE OPAQUE-2*

J. CORRENTI, Rut M. SOLARICentro de Investigaciones en Ciencias Agronómicas,Castelar, Argentina

Abstract-Resumen

ASSOCIATION BETWEEN THE FLOURY CONDITION AND THE PROTEIC COMPONENTS OF THE ENDOSPERM IN MUTANT OPAQUE-2.

On account of its high protein quality, mutant opaque-2 is being intensively used in corn breeding. Studies regarding its chemical as well as its nutritional properties are also frequent. Seeds from opaque-2, two flint lines (A j and P21)p and the Fx of Aj X opaque-2 and P2i X opaque-2,'and their reciprocal, as well as 34 F2 seeds from A± X opaque-2 were analysed. The isoelectric behaviour of the analysed material allowed the detection of a proteic component easily identifiable and associated with the character of opaque-2. This association would allow the characterization of the product of the gene responsible for the alteration observed in the prolamine fraction of opaque-2.

ASOCIACION ENTRE LA CONDICION HARINOSA Y LOS COMPONENTES PROTEICOS DEL ENDOSPERMA EN LA MUTANTE OPAQUE-2.

Debido a la elevada calidad de sus proteínas, el mutante opaque-2 se utiliza intensamente en el mejoramiento del maíz. Igualmente, se efectúan estudios frecuentes sobre sus propiedades químicas y nutritivas. Se han analizado semillas opaque-2, dos lfneas tipo flint (Ai y P21), y las Fi de los cruzamientos А г y P2i con opaque-2 y sus recíprocos, asf como 34 semillas F2 provenientes del cruzamiento entre las lfneas A x X opaque-2. El comportamiento isoeléctrico del material analizado permitió detectar un componente proteico fácilmente identifiable y asociado al carácter opaque-2. Esta asociación permitiría caracterizar el producto del gene causante de la alteración observada en la fracción prolamfnica de opaque-2.

E l maíz ha sido considerado esencialmente un alimento energético. Su valor nutritivo es escaso por el bajo contenido de algunos aminoácidos esenciales en las proteínas del endosperma, especialmente lisina, methionina y triptofano.

Con el reciente descubrimiento de las propiedades de opaque-2 y floury-2 por Mertz, Bates y Nelson [1, 2] se ha demostrado que la composición de las proteínas del endosperma del maíz podrían ser alteradas por cambios genéticos. Estas mutantes presentan una composición proteica más adecuada desde el punto de vista nutritivo, debido a una alteración en el balance de las distintas fracciones proteicas, d is minuyendo la correspondiente a la soluble en alcohol (zeínas) y aumentando relativamente las solubles en soluciones salinas e hidróxidos [ 3 ], estas últimas poseyendo mayor cantidad de los aminoácidos lisina y triptofano, que las zeínas.

* Publicación Técnica Gen. №449 del Centro de Investigaciones en Ciencias Agronómicas, CICA, INTA, Castelar.

3 6 5

3 6 6 CORRENTI y SOLARI

Por otro lado, la disponibilidad de métodos electrocromatográficos de fraccionamiento de proteínas perm itiría identificar las modificaciones cualitativas y cuantitativas de las mismas.

En esta comunicación se presentan los resultados logrados por medio del análisis por electroenfoque de la zeína, basado en la separación de sus componentes de acuerdo a su punto isoeléctrico.

M ATER IAL Y METODOS

Se han utilizado para el análisis sem illas de la mutante opaque-2, de las líneas argentinas tipo flint A i y P 2 1 , las Fj de los cruzamientos ^ 1 У P 2 1 con opaque - 2 y sus recíprocos, y 34 sem illas F 2 provenientes del cruzamiento entre las líneas А г X opaque-2.

E l análisis se limitó a una sem illa, considerando a esta última como la unidad biológica que permite establecer parámetros genéticos, indeterminables cuando se analizan conjuntos de sem illas [4].

La proteína a ser analizada fue obtenida del endosperma previa separación del embrión.

E l endosperma fue molido en un mortero y la harina se maceró con etanol al 6 8 % durante toda la noche; la relación harina-etanol fue de 1 : 5 (p/v). Luego se centrifugó y el sobrenadante se liofilizó.

Las proteínas fueron fraccionadas de acuerdo a su punto isoeléctrico sobre gel de acrilam ida [5].

RESULTADOS Y DISCUSION

E l empleo del método por electroenfoque que separa las proteínas de acuerdo a su punto isoeléctrico, ha permitido detectar en la zeína,

Diagrama isoeléctrico de las proteínas solubles en etanol del endosperma de mafz de opaque-2 y las líneas A! y P21 tipo flint utilizando anfolitos de un rango de pH 3-10. La flecha indica la banda proteica estudiada.(Isoelectric diagram of the ethanol-soluble proteins of the endosperm of opaque-2 maize and the flint lines Ai and P21 ; the ampholytes used had a pH range of 3-10. The arrow indicates the protein band studied. )

M UTANTE OPAQUE-2 3 6 7

entre otras, una proteína con un punto isoeléctrico de alrededor de 6 .2 .La misma se presenta como una banda brillante en opaque-2 y de menor intensidad en las líneas flint. E l diagrama isoeléctrico de las líneas flint y opaque- 2 puede ser observado en la figura, indicando la flecha la banda en estudio que diferencia ambos fenotipos.

Los espectros de las sem illas F^ no difieren del de las líneas flint cualesquiera sea el sentido del cruzamiento.

Se analizaron 34 sem illas F 2 , 24 con el fenotipo flint y 10 con el fenotipo opaque-2; los resultados se muestran en el cuadro. En éste se observa una asociación completa entre la expresión fenotípica del endosperma y la presencia o ausencia de la proteína citada en la generación segregante. Dicha asociación es, por consiguiente, genética y permite concluir que el mismo par génico es responsable de ambas expresiones.

Las ideas actuales sobre la regulación de la síntesis de las proteínas sugieren que el estado alélico de un gene productor o estructural es el responsable de la codificación de una proteína. A partir de este razonamiento es posible relacionar directamente un par génico con su expresión a nivel proteico y morfológico. E l análisis electro-crom atográfico permite estudiar tal situación. Se ha sugerido que las proteínas de reserva presentes en el endosperma responden al concepto anterior, por lo cual se puede asociar un mismo gene para el control de una proteína y la expresión propia del opaque-2 [4].

Jiménez (cf. Nelson) [3] ha observado que tres bandas de la zeína están ausentes en opaque- 2 y presentes en la línea normal, no encontrando diferencias en otras bandas ni en las correspondientes fracciones salinas y alcalinas. Como en este caso sólo una parte de la fracción prolamínica fue analizada, Nelson [3] acepta la posibilidad de que las diferencias entre los genotipos pueda ser debida a otras proteínas. Como no se registra que tales proteínas han sido analizadas genéticamente, la asociación podría ser, asim ismo, casual.

En el método de electroenfoque todas las proteínas presentes hasta cierta concentración son visibles. Además, nuestro análisis se realiza sobre generaciones segregantes, lo cual permite establecer una asociación de ligamiento entre los loci involucrados [4].

Según nuestros resultados, el fenotipo de opaque-2 estaría regulado por un alelo capaz de producir cantidades mayores de una proteína, que en el normal (flint) sólo se produciría en pequeña cantidad o no se produciría.

ASOCIACION ENTRE LA EXPRESION FENOTIPICA D E L ENDOSPERM A D EL M AIZ Y LA PROTEINA E N ESTUDIO (UNК BETWEEN THE PHENOTYPE EXPRESSIVITY OF THE MAIZE ENDOSPERM AND THE PROTEIN UNDER STUDY)

Flint Opaque -2

BandaSin brillo 24 -

Con brillo 10

3 6 8 CORRENTI y SOLARI

RE F E RE N C IA S

[ 1] MERTZ, E. T. , BATES, L.S., NELSON, O.E., Mutant gene that changes protein composition and increases lysine content of maize endosperm, Science 145 (1964) 279.

[2] NELSON, O.E., MERTZ, E. T., BATES, L.S., Second mutant gene affecting the amino-acid pattern of maize endosperm proteins, Science 150 (1965) 1469.

[3] NELSON, O. E. , "The modification by mutation of protein quality in maize", New Approaches to Breeding for Improved Plant Protein (Proc. Panel Rostânga, 1968), IAEA, Vienna (1969) 41.

[4] SOLARI, Rut M., FAVRET, E. A. , "Genetic control of protein constitution in wheat endosperm and its implication on induced mutagenesis", Mutations in Plant Breeding II (Proc. Panel Vienna, 1967), IAEA, Vienna (1968) 219.

[ 5] WRIGLEY, C. W. , Analytical fractionation of plant and animal proteins by gel electrofocusing,J. Chromât. 36 (1966) 362.

D IS C U S S IO N

H. SMITH: This paper represents a most interesting application of electrophoretic separation techniques to problems associated with objectives of protein improvement in plants. Do I understand the electro- pherograms correctly that the uniquely heavy band in opaque- 2 is also present in the flint lines but is much lighter? If so, is it not the simplest interpretation that activity of the opaque gene is to "turn on" or regulate the production of this protein rather than to synthesize a new protein? That is, is it a regulator gene rather than a structural gene?

E .A . FAVRET: The electrophoretic method can be used to study enzymes. In the case presented by Correnti it is a question of the alcohol-soluble fractions from the endosperm proteins.

The explanation in the case of opaque-2 may be that the gene pair involved can produce more protein than the normal alleles (flint) or that it may consist of a mixture of more than one protein. Either explanation may be valid at the present time.

J. EREJOMOVICH: Is the lysine content of the F 2 flint segregants of the opaque-2 X (A^ or P 2 1 ) cross sim ilar to that of opaque-2?

J. CORRENTI: This analysis was not effected.H. SMITH: Has any effort been made to associate the band in

opaque-2 , that shows up with a general protein stain, with some specific enzyme system by appropriate staining techniques?

J. CORRENTI: No.A. BLUM ENSCHEIN: In the F 2 generation from some crosses

between opaque- 2 and flint, canbe obtained kernels completely opaque or completely flint in appearance, but kernels showing opaque and/or flint spots can also appear. Did you consider this aspect in your analysis?

J. CORRENTI: What you say is certainly true of some lines, but in the m aterial used by us (line A J it was not the case.

RENDIMIENTO Y ESTABILIDAD EN MEZCLAS DE MUTANTES DE CEBADA*

A . von der PAHLENCentro de Investigaciones en Ciencias Agronómicas,Castelar, Argentina

Abstract-Resumen

YIELD AND STABILITY IN BARLEY MUTANT MIXTURES.The yield and stability pattern of one variety of brewing barley, five of its mutants, and mixtures of

these six strains was studied over a four-year period at five variability levels. Mixtures of the strains, assumed to be isogenic, showed the same properties as mixtures of varieties, i.e. a slightly higher yield than the average of the constituents in most media, and better stability. The mixtures of the most stable strain with the highest-yielding strains combined the desirable properties of both constituents. This "ecological" combination has the added advantage that in general these mutants show no differences as regards growth cycle and quality. They could therefore be used to form mixtures without prior selection for uniformity in these two characteristics.

RENDIMIENTO Y ESTABILIDAD EN MEZCLAS DE MUTANTES DE CEBADA.Se estudió el comportamiento del rendimiento y de la estabilidad de una variedad de cebada

cervecera, de cinco de sus mutantes y de las mezclas de estas seis lfneas, en cinco niveles de variabilidad, durante cuatro años. Las mezclas de estas líneas, supuestamente isogénicas, manifestaron las mismas propiedades que las mezclas de variedades: un rendimiento ligeramente superior a la media de los componentes en la mayor parte de los ambientes y una mayor estabilidad. Las mezclas de la línea más estable con las de mayor rendimiento aunaron las buenas características de los dos componentes. A esta combinación «ecológica» se une la ventaja de que estas mutantes no difieren generalmente entre sí en ciclo vegetativo ni en calidad, lo que permitiría emplearlas para formar mezclas sin necesidad de seleccionarlas para obtener uniformidad en estos dos caracteres.

Trabajos publicados en los últimos años [1] han indicado que las poblaciones de especies predominantemente autógamas poseen un grado de variabilidad mucho mayor que el supuesto anteriormente [ 2 ]. Aparentemente la variación observada permite a los diversos genotipos explotar más eficientemente las oportunidades ecológicas de los habitats heterogéneos en el espacio y en el tiempo. A pesar de ello, siempre existieron fitomejoradores interesados en form ar variedades compuestas, aún inmediatamente después de adoptado el método de obtención de líneas puras que descartaba las viejas variedades con alto grado de variabilidad [3 ,4 ].

La variabilidad encontrada en las poblaciones naturales y los experimentos de Sakai [5], que demostraron la importancia de la competición entre plantas, dieron un nuevo impulso a los experimentos con mezclas para determinar su comportamiento con respecto al rendimiento y su estabilidad en diferentes ambientes [ 6 , 7, 8 ]. Se comprobó que las mezclas adquieren propiedades inherentes a sí m ismas, diferentes de las de sus componentes, debido a que la productividad de determinados genotipos varía en presencia de otros. Los valores reproductivos de los componentes son una función de la asociación con otros genotipos [9, 10]. Los datos analizados hasta el

''' Publicación Técnica Gen. №446 del Centro de Investigaciones en Ciencias Agronómicas, INTA, Castelar.

3 6 9

3 7 0 von der PAHLEN

presente indican que existe una tendencia de las mezclas a tener rendimientos superiores a la media de sus componentes y a que este carácter sufra menos oscilaciones en diversos ambientes, o sea, a que tenga una mayor estabilidad o adaptabilidad. En contadas excepciones la m ezcla llegó a rendir más que el m ejor componente [ 1 1 ].

Con respecto a la estabilidad, algunos resultados sugieren que las m ezclas son iguales o superiores a sus componentes [ 6 , 7, 12, 13, 14] aunque este fenómeno no ocurre indefectiblemente [ 8 ].

Otra ventaja de las mezclas con respecto a las líneas puras estriba en una resistencia más amplia a los parásitos cuando sus componentes varían con respecto a la reacción a las diferentes razas fisiológicas de un parásito [3]. Estos estudios fueron efectuados con mezclas de variedadeso genotipos que diferían entre sí en numerosos genes. A fin de determinar si diferencias monogénicas podrían ser responsables del comportamiento de las mezclas, se planeó un experimento empleando en las mismas mutantes de una variedad de cebada, supuestamente de líneas isogénicas.

M ATERIALES Y METODOS

La variedad de cebada elegida fue Maltería Heda de dos hileras, y cinco de sus mutantes obtenidas por Favret, en el Instituto de Fitotecnia, en Castelar, que se presume diferían de la variedad original sólo en los genes mutados. Dos de las mutantes, М. C. 20 y laxa, la prim era de ellas resistente al oídio (Erysiphe graminis hordei), rinden generalmente más que la variedad original [15]; tres mutantes, erectoides 2a (ert-2a), erectoides 3a (ert-3a) y la doble mutante М. C. 20 erectoides 6 a (e r t - 6 a), rinden menos.

E l experimento se efectuó en el Instituto de Fitotecnia durante cuatro años (1964-1967) y consistió en 24 tratamientos en bloques al azar, con cuatro repeticiones. Los tratamientos fueron los siguientes:

Líneas puras Mezclas de dos(50%- 50%)

1. Maltería Heda2. М. C. 203. laxa4. ert-2a5. ert-3a6 . М .C. 20 e r t - 6 a

7. líneas 1 , 2

8 . líneas 2, 39. líneas 1, 3

10. líneas 1, 411. líneas 3, 4 1 2. líneas 4, 6

13. líneas 2, 6

Mezclas de dos (6 6 % - 33%)

Mezclas de tres (33%- 33%- 33%)

14. líneas 1 , 2

15 . líneas 2, 1

16. líneas 1, 4 17 . líneas 4 , 1

18. líneas 2 , 6

19. líneas 6 , 2

20. líneas 1, 2, 321. líneas 4, 5, 6

22. líneas 1, 2, 423. líneas 3, 5, 6

MUTANTES DE CEBADA 37 1

Mezclas de seis

24. líneas 1 , 6

Cada parcela consistió en siete hileras de 2, 50 m de largo donde se cosecharon las cinco hileras centrales. Dentro de cada hilera, las plantas estaban espaciadas a 0 , 1 0 m.

Con el objeto de obtener una medida de estabilidad en relación con el nivel de diversidad del tratamiento, se efectuó un análisis de variancia para cada nivel de diversidad y para cada tratamiento. Los datos originales fueron transformados en logaritmos naturales para detectar las diferencias. Este método [16] provee de una medida de variabilidad intrínseca que es invariable bajo un cambio multiplicativo de la media y es independiente de las unidades de medida, o sea que permite comparar variancias aunque las medias sean diferentes, como ocurre en el presente caso. Cuando se supone que el coeficiente de variación es una m ejor estimación que la variancia, se justifica una transformación de los datos a logaritmos y estas variables transformadas se pueden tratar como valores distribuidos normalmente. Las variancias transformadas fueron comparadas mediante la prueba de homogeneidad de variancias de Bartlett.

E l componente debido a años, variancia macroambiental, provee una medida inversa de la estabilidad relativa de una población con respecto a la variancia y al conjunto de ambientes en consideración. La variancia microambiental contiene todas las fuentes de variación debida a las repeticiones.

La estabilidad fenotípica también fue analizada por el método de la regresión del rendimiento de cada tratamiento sobre el rendimiento medio de todos los tratamientos [17, 18] mediante el siguiente modelo:

Y ij =Q,i +CTij

dondea es el parámetro que intercepta el eje de Y, /3 el coeficiente de

regresión de la iésima entidad y cr ij es la desviación de la regresión de la iésim a entrada en el ambiente j.

La aptitud combinatoria «ecológica», o sea la capacidad de favorecer la productividad de otros genotipos en una mezcla, fue analizada mediante un medio dialelo [19] adaptado al análisis de las mezclas de genotipos, como sugiere Harper [20], difiriendo un poco del análisis de Chalbi [21].Las líneas puras constituyeron los valores de la diagonal, y las mezclas, el resto de los casilleros, empleando las medias de parcela por año como repeticiones.

RESULTADOS

Los rendimientos se compararon mediante un análisis combinado de variancia (cuadro I) empleándose la prueba de Duncan para las comparaciones múltiples (cuadro II). E l gran efecto que tuvo el factor «a ñ o s » sobre la variancia se explica debido a la presencia de años extremadamente favorables, y desfavorables durante el curso de la experiencia.

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CUADRO II. COM PARACION DE LOS RENDIMIENTOS DE LAS M EZCLASY LINEAS PURAS DE CEBADA DE LOS RESULTADOS DE LOS ENSAYOS DE TRES Y CUATRO AÑOS

E n s a y o s de 1964 - 1967 E n s a y o s de 1964 - 1966

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9 573 1 00 7 651 1 05 1 04

8 573 1 03 9 641 1 00 95

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20 556 96 14 632 102 101

24 554 1 1 0 20 631 100 93

1 5 553 99 2 625 - -

1 552 - 15 623 1 00 1 00

13 546 1 06 1 618 - -

1 0 534 1 08 22 61 5 1 05 98

16 533 104 1 0 612 108 99

22 532 1 03 24 611 109 91

19 531 106 13 597 105 96

18 52 5 99 19 593 107 95

23 523 106 1 1 590 1 00 88

14 489 88 18 585 100 92

17 480 101 23 584 11 0 87

11 474 92 17 567 1 04 92

6 469 - 12 526 102 1 02

12 462 1 02 6 515 - -

21 439 1 00 4 512 - -

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En el año 1965, las precipitaciones fueron abundantes y se registraron en la época necesaria, mientras que 1966 fue un año muy seco.

A sí como se esperaba de los experimentos anteriores [4, 12] ninguna de las mezclas sobrepasó significativamente al m ejor componente, pero la mayor parte de ellas fueron numéricamente superiores a la media de sus componentes en cada uno de los tres prim eros años, y para el conjunto de los tres prim eros años como se puede observar en el cuadro III. El test no paramétrico del signo fue significativo, debido a que un número muy reducido de mezclas fue inferior a la media de sus componentes.En esos tres años, las mezclas rindieron en promedio un 4% más, con un rango de 2% menos a un 12% más de lo esperado. Sin embargo, en el cuarto año, las diferencias entre las mezclas y las medias de sus componentes se manifestaron en ambos sentidos. Por esta razón, la prueba del signo para el conjunto de los cuatro años no fue significativa y el rendimiento de las mezclas en promedio sólo fue superior al rendimiento esperado en 1%, con un rango de 12% inferior y un 10% superior. A pesar de ello, el ensayo de 1967 no llegó a alterar sustancialme.nte el orden de los rendimientos observados para el período 1964-1966 (cuadro IV).

Considerando los cinco niveles de diversidad estudiados, se trató de hallar alguna relación entre éstos y la ventaja de las mezclas con respecto a la media de sus componentes, con el objeto de averiguar si una mayor diversidad puede aprovechar mejor las oportunidades ecológicas [ 2 0 ].Según esta hipótesis, las mezclas de mayor número de componentes tendrían que poseer una diferencia mayor entre el rendimiento obtenido y el esperado, calculado en base a la media de sus componentes, que las mezclas con un pequeño número de ellos. Las diferencias de las mezclas de dos líneas, en la proporción de 2/3 : 1/3, de dos líneas en iguales proporciones, de las mezclas de tres líneas y mezclas de seis líneas, fueron respectivamente de - 0, 5%, 2%, 1, 25% y 10%, indicando que entre los niveles bajos de diversidad no hubo diferencias. La m isma conclusión se obtuvo observando la regresión de los rendimientos obtenidos sobre los rendimientos esperados. La regresión fue lineal, b = 0, 61 significativamente diferente de cero al nivel del 1 %, o sea que no muestra interacciones consistentes (figura 1 ).

Tampoco se encontró una relación entre el porcentaje desventaja de las mezclas con respecto al rendimiento esperado y la diferencia de rendimiento entre sus componentes, resultados que concuerdan con los de Clay y A llard [ 8 ].

L a mayor parte de las diferencias no fueron significativas, probablemente debido a que se usaron pocas repeticiones, lo que disminuyó la sensibilidad del ensayo. E l análisis dialélico con todas las combinaciones de las líneas 1 a 4, menos una (2 y 4) indicó que existe aptitud combinatoria «eco lógica» general [7, 20], pero no específica (cuadro V). La combinación que faltaba se reemplazó con valores hipotéticos suponiendo que e r t - 2 a se comportaría con М. C. 20 de manera análoga a la mezcla de ert-2a y M altería Heda, o sea, que en 1964, 1965 y 1967 la mezcla de ert-2a y М. C. 20 superaría levemente a ert-2a y Maltería Heda y sería superada en 1966, debido al m ejor comportamiento en ese año de ésta última.

E l Cuadro VI muestra los componentes de variancia m acro- y m icro- ambiental. La prueba de Bartlett aplicada a las dos estimaciones mostró que las variancias de algunos tratamientos diferían entre sí.Si el análisis se aplicó a todos los tratamientos conjuntamente, el resultado


440 460 480 500 520 540 560 S80 600

FIG.l. Relación entre el rendimiento obtenido de la mezcla y el rendimiento medio de sus componentes.

(FIG.l. Relationship between yield of the mixture and average yield of its components.)

CUADRO V. ANALISIS D IALELICO CON RESPECTO A LA APTITUD COMBINATORIA ECOLOGICA

P u e n t e s de

v a r ia c ió n3 .1 . Cuadrados

m e d io sF

a efecto g e n e r a l 3 6 1 9022 7 ,0 2 *

b efecto e sp e c íf ic o 6 4 8 3 9 0 1 ,1 7

A ñ o x a 8 88231

A ñ o x b 17 4 1 3 9 0

* S ig n i f ic a t iv o a l n ive l de l 0 ,0 2 5 % .

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CUADRO VI. COMPONENTES DE VARIANCIA D EL ERROR (MICROAM B IE N TA L ) Y DE AÑOS (M ACROAM BIENTA L ). ESTE ULTIM O SE COM PARA CON LA LINEA DE LA M E ZC LA DE MENOR VARIANCIA, LA M E ZC LA DE LA VARIANCIA DE LAS LINEAS COMPONENTES DE LA M EZCLA , LA COVARIANCIA Y E L VALOR DE VARIANCIA CALCULADO

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7 1 ,2 0,017 0,62 + 0,19 0,62 0 ,83 0 ,8 0 0,829 1 ,3 0 ,03 3 0 ,69 ± 0 ,22 0,62 0,72 0,71 0,72

14 1 .2 0,017 0 ,7 0 + 0,22 0,62 0,73 - -20 1 ,2 ,3 0,028 0,72 + 0 ,2 3 0,62 0,83 0,81 0,82

1 0 1 ,4 0 ,023 0 ,79 + 0 ,24 0,62 1 , 12 0 ,9 9 , 1 ,0524 1 ,6 0,020 0 ,79 + 0 ,25 0,62 1 ,07 0 ,9 8 0 ,99

3 3 0,01 0 0,82 + 0 ,26 - - - -

15 2 , 1 0,06 7 0,82 + 0,76 0,62 0 ,90 - -

8 2 ,3 0,026 0 ,84 + 0,27 0,82 0,93 0 ,93 0 ,9322 1 ,2 ,4 0,028 0 ,89 + 0,29 0,62 1 ,09 0,99 1 ,02

13 2 , 6 0 ,013 0,91 + 0 ,29 1 ,05 1 , 12 0,86 1 ,0218 2 , 6 0 ,0 5 4 0 ,9 5 ± 0,31 1 ,05 1 , 1 0 - -

5 5 0 ,03 5 0 ,98 + 0,31 - - - -

2 2 0,059 1 ,0 5 + 0 ,34 - - - -

19 6 , 2 0,027 1 ,0 5 + 0 ,34 1 ,05 1 ,24 - -23 3 ,5 ,6 0,019 1 ,08 + 0 ,34 0,82 1 ,04 1 ,01 1 ,02

17 4,1 0 ,03 4 1 ,0 9 + 0 ,3 5 0,62 1 ,27 - -

21 4 ,5 ,6 0 ,056 1 ,20 + 0,42 0,98 1 ,31 1,13 1 ,196 6 0 ,093 1 ,33 + 0,43 - - - -

12 4 ,6 0 ,006 1,59 + 0 ,5 0 1 ,33 1 ,47 1 , 00 1 ,244 4 0,041 1,61 + 0,51 - - - -

11 3 ,4 0 ,039 1,71 + 0 ,5 5 0,82 1 ,21 1 , 1 2 1,17

(1 ) C om p on en te de v a r ia n c ia m ic roam b ie n ta l,(2 ) C om p on en te de v a r ia n c ia m a cro am b ie n ta l.(3 ) M e d ia de la v a r ia n c ia m acroam bienta l de la s líneas com ponentes

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m e d ia , N núm ero de lín e a s de la m ezcla (M a r s h a ll , 1 9 6 8 ).

(4 ) х/ = V - + ( N ^ N


no es significativamente diferente de cero, debido a que muchas de las mezclas presentan variancias semejantes a uno de los componentes. Con el objeto de estudiar qué estimación puede predecir mejor la adaptabilidad de una mezcla, se compararon las variancias macroambientales de las mezclas con las del componente más estable y en los casos de igual proporción de componentes, con la variancia media macroambiental, la covariancia macroambiental y la variancia esperada, esta última en base a una ecuación calculada por M arshall [7] (cuadro VI). Se observa que las mezclas de mayor adaptabilidad tendieron a igualar al componente más estable de la mezcla, o sea que Maltería Heda le confirió estabilidad a las mezclas en las cuales intervino. Sobre 14 casos, cuatro se apartaron de este comportamiento; en éstos, todas las líneas involucradas en las mezclas fueron de baja estabilidad.

E l buen comportamiento de la variedad Maltería Heda y de las mezclas en las cuales intervino con respecto a estabilidad, se debió a su mayor resistencia a la sequía de 1966.

Las estimaciones de variancia microambiental fueron aparentemente más semejantes entre sí en los tratamientos más estables. Considerando los prim eros nueve tratamientos del Cuadro VI, el X 2 del test de Bartlett fue igual a 5, 43; en los nueve siguientes, 9, 00 y en los últimos nueve,17, 00. E l cuadro VII muestra que, en general, los diferentes componentes de variancia disminuyeron en magnitud a medida que aumentaba el nivel de diversidad.

CUADRO VII. COM PARACION DE LOS COMPONENTES DE VARIANCIAS MACRO Y MICRO AM BIENTALES PARA DIFERENTES NIVELES DE DIVERSIDAD

N iv e le s 0 ^ ( 1 )2

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M e z c l a s de t r e s J_ x 33

0 ,0 1 8 0 , 6 6 0 ,0 1 9 2

M e z c le s de s e i s 1 x 66

0 ,0 1 8 0 ,7 9

0^ C o m p o n e n te d e la v a r ia n c ia m ic r o a m b ie n ta l.

20 ^ C o m p o n e n te d e la v a r ia n c ia m a c ro a m b ie n ta l.

^2 C o m p o n e n te d e la v a r ia n c ia de in te ra c c ió n de a ñ o s con

A B tra ta m ie n to s .

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Los coeficientes de regresión del rendimiento de cada tratamiento sobre el rendimiento medio de todos los tratamientos [17] coincidieron en ordenar a las mezclas de igual manera que las estimaciones del componente de variancia de «años» (cuadro VIII). Muchos de los coeficientes de regresión no difieren significativamente de uno debido, probablemente, al número pequeño de ambientes en el cual se efectuó el experimento.

En general, no influyó que los componentes de una mezcla estuvieran en proporciones diferentes, con la excepción del tratamiento 17, donde la diferencia de estabilidad entre los dos componentes fue muy grande y el componente menos estable se encuentra en menor proporción. Es por ello que este tratamiento se encuentra alejado de los otros dos con los mismos componentes en el ordenamiento por estabilidad de los cuadros VI y VIII.Se calculó además la estimación Sy X que mide las desviaciones de la regresión del tratamiento i en el ambiente j, donde

2 = E (Y U - У ц )*y.* n - 2

y n es el número de ambientes pero ninguna de las estimaciones fue significativamente diferente de cero cuando se la comparó mediante la prueba de F con el error conjunto [18].

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0 , 7 6

i i i i i i i i i i l l ........................................... i i i i • 16i i_i. i i i i 1 1— 1 I 1

6 0 S 6 1 0 6 1 5 6 2 0 6 2 5 6 3 0 6 3 5 6 4 0

FIG.2. Comparación entre el rendimiento y la estabilidad de los tratamientos.

(FIG.2. Comparison of yield and stability after treatment.)

3 8 2 von der PA HLEN

En el cuadro VIII se incluyeron los rendimientos por parcela con el objeto de comparar rendimiento y estabilidad. Estos dos caracteres están, aparentemente, positivamente asociados (figura 2 ), o sea que el coeficiente de regresión b que estima la estabilidad está inversamente correlacionado con el rendimiento, con un coeficiente r = - 0,69 significativo al nivel del 1%.

DISCUSION

E l presente experimento indicaría que las mezclas de líneas isogénicas se comportan de la m isma manera que las mezclas de variedades: aumenta levemente el rendimiento y la estabilidad. Esta conclusión puede tener interés para fitomejoradores cuando se quieran producir variedades multilíneas que difieran solamente en genes de resistencia a parásitos, como se ha hecho en trigo, en Colombia, y cuyos componentes no difieren en el ciclo vegetativo ni en la calidad de su producto.

Considerando el hecho de que M altería Heda le confiere estabilidad a las mezclas en las cuales interviene, y que las mezclas con las mutantes laxa у М. C. 20 poseen el rendimiento de éstas, se puede decir que se produce una combinación «ecológica» . Estas mezclas presentan la adaptabilidad del componente más estable y el rendimiento del componente de mayor rendimiento, como es el caso de los tratamientos 7 (Maltería Heda у М. C. 20) y 9 (M altería Heda y laxa). Por lo tanto, adquirieron las propiedades de los dos componentes, o sea que es factible, empleando mutantes, aunar las buenas características de dos variedades en una m ezcla cuyos componentes tengan el mismo ciclo vegetativo y calidad.Este método podría reem plazar temporariamente, o complementar, la recombinación genética cuando es necesario adelantar un programa de mejoramiento, dado que Finlay [20] demostró que es relativamente fácil obtener líneas más adaptables mediante cruzamientos.

Los resultados de este estudio sugieren que se pueden elegir mutantes de una misma variedad, hacer mezclas entre ellas y superar a la variedad original en rendimiento, o por lo menos igualarla en adaptabilidad. Ilustra también el efecto pleiotrópico que tienen algunos genes sobre el rendimiento y la adaptabilidad de una línea.

La aparente demostración de la inexistencia de una relación entre nivel de variabilidad y ventaja de las mezclas con respecto a la media de los componentes es la misma que la observada por Clay y A llard [ 8 ], con una salvedad: en ambos experimentos se usaron muchos niveles bajos de diversidad y sólo una mezcla con un nivel de diversidad sustancialmente mayor que el resto. Así, en el trabajo de Clay y A llard se empleó una mezcla de diez líneas en lugar de una de cinco, y una de seis en lugar de una de tres, fue utilizada en el presente trabajo. Tanto la de diez líneas como la de seis, aventajan a la media de los componentes mucho más que los niveles bajos de diversidad. Los datos conjuntos de ambos trabajos permiten suponer que existe una relación entre variabilidad mecánica de la población y aumento del rendimiento, pero es preciso experimentar con mayores niveles de diversidad para obtener una conclusión valedera.

La correlación positiva encontrada entre el rendimiento y el coeficiente de regresión que mide la adaptabilidad, puede ser debida a la muestra particular de líneas usadas en el presente estudio, dado que no se encuentra

MUTANTES DE CEBADA 383

en otros experimentos donde intervienen variedades de cebada [15], poblaciones en F 2 de cebada [ 8 ] o híbridos de maíz [16]. Sin embargo, en las mezclas de variedades de Clay y A llard [ 8 ] se observa una corre la ción sim ilar al presente caso, que calculada con los datos que presentan los autores, da un valor de r = 0, 44, significativo al nivel del 1%. Es difícil comparar los dos experimentos para poder explicar este hecho debido a que en el presente estudio el componente de años fue el factor ambiental más importante y además se pudo deducir la causa de la mayor estabilidad de algunos tratamientos, mientras que en el otro, el componente ambiental principal fue localidades, sin que se explique a que se deben las variaciones en adaptabilidad. Además en un caso, con excepción de la variedad Maltería Heda, se puede considerar que las líneas isogénicas no tuvieron historia de selección, mientras que en el otro ensayo intervinieron variedades seleccionadas para líneas puras, lo que podría explicar la falta de coadaptación de esas mezclas, como lo destacan los autores.

Si se considera el comportamiento a largo plazo de una población natural que contendría las seis líneas isogénicas estudiadas, es probable que la variedad M altería Heda se impondría debido a su mayor adaptabilidad en condiciones desfavorables. Estaría menos sujeta a las fluctuaciones ambientales, ajustándose a la estrategia óptima «m axim in», según Lewontin [ 2 2 ], que consistiría en mantener la probabilidad local de supervivencia lo más alta posible bajo las peores condiciones de los estados de la naturaleza. Como en algunas de las mezclas estudiadas parece haber facilitación mutua o sobrecompensación [23, 24] sería posible el mantenimiento de un determinado número de polimorfismos con una carga menor que la de la selección individual independiente [25], de manera que no solamente habría combinaciones más productivas en ambientes desfavorables, sino que también las habría en condiciones favorables.


[1] ALLARD, R.W., JAIN, S.К., WORKMAN, P.L., The genetics of inbreeding populations, Adv. Genet. 14(1968) 55.

[2] STEBBINS, G.L., Variation and Evolution in Plants, Columbia Univ, Press, New York (1951).[3] BORLAUG, N.E., "The use of multi-lineal or composite varieties to control airborne epidemic diseases

of self-pollinated crop plants", 1st Int. Wheat Genet. Symp., Winnipeg, Man. (1959) 12.[4] SIMMONDS, N.W., Variability in crop plants, its use and conservation, Biol. Rev. 37 (1962) 422.[5] SAKAI, K., Competition in plants and its relation to selection, Cold Spring Harb. Symp. quant.

Bioi. 20 (1955) 137.[6] ALLARD, R.W., Relationship between genetic diversity and consistency of performance in different

environments, Crop Sci. 1 (1961) 127.[7] MARSHALL, D.R., Relationship between Levels of Genetic Diversity and Populational Buffering in

Mixtures of Grain Sorghum, Ph. D. Dissertation, Univ. Calif, Davis (1968).[8] CLAY, R.E., ALLARD, R.W., A comparison of the performance of homogeneous and heterogeneous

barley populations, Сюр Sci. 9 (1969) 407.[9] SAKAI, K., "Competitive ability in plants: its inheritance and some related problems", Mechanisms

in Biological Competition 15 (1961) 245.[10] SCHÜTZ, W.M., BRIM, C.A., Inter-genotypic competition in soybeans, I. Evaluation of effects

and proposed field plot design, Crop Sci. 2 (1967) 371.[11J ROY, S.K., Interaction between rice varieties, J. Genet. 57 (1960) 137,[12] JENSEN, N.F., Multiline superiority in cereals, Crop Sci. 5 (1965) 566.[13] PFAHLER, P.L., Genetic diversity for environmental variability within the cultivated species of Avena,

Crop Sci. 5 (1965) 47.


[14] PFAHLER, P.L., Environmental variability and genetic diversity within populations of oats (cultivated species of Avena) and rye (Secale cereale L.)t Crop Sci. 5 (1965) 271.

[15] GOLDENBERG, J.B., PAHLEN, A. von der, Heterosis por sobredominancia en varios genes de cebada, Bol. Genet. Inst. Fitotec. Castelar, 6 (1969) 27.

[16] LEWONT1N, R.C., On the measurement of relative variability, Syst. Zool. 15 (1966) 141.[17] FINLAY, K.W., " Breeding for yield in barley", Barley Genetics II, Wash. State Univ. Press(1971)

338.[18] EBERHART, S.A., RUSSELL, W.A., Stability parameters for comparing varieties, Crop Sci. 6

(1966) 36.[19] HAYMAN, B.I., The analysis of variance of diallel crosses, Biometrics 10 (1954) 235.[20] HARPER, J.L., A Darwinian approach to plant ecology, J. Ecol. 55 (1967) 247.[21] CHALBI, N., Biométrie et analyse quantitative de la competition entre génotypes chez la luzerne,

AnnlsAmêl. Pl. 3 (1967) 247.[22] LEWONTIN, R.C., Evolution and theory of games, J. Theor. Biol. 1 (1961) 382.[23] ALLARD, R.Wf, ADAMS, J., Population studies in predominantly self-pollinating species, XII Inter-

genotypic competition and population structure in barley and wheat, Am. Nat. 103 (1969) 621.[24] SEATON, A.P.C., ANTONOVICS, J,t Population interrelationships. I. Evolution in mixtures of

Drosophila mutants, Heredity 22 (1967) 19.[25] SVED, J.A., REED, T.W., BODMER, W., The number of balanced polymorphisms that can be

maintained in natural populations, Genetics 55 (1967) 469.

D IS C U S S IO N

W . GOTTSCHALK: You mention in your paper the double mutant М. C. 20 erectoides-6 a . What do you understand by the term "double mutant"? Have two different genes mutated in the treated embryo or is it a double recessive form obtained by crossing of two different mutants, or is it a matter of pleiotropic gene action?

A. von der PAH LEN : М. C. 20 was first obtained by E. A. Favret by irradiation and afterwards this mutant was treated with EMS, obtaining in this way the double mutant.

R. TRUJILLO FIGUEROA: Did you carry out any observations on yield components characteristic of the aerial and radicular parts that might explain the better yields under mixture conditions and individually?

A. von der PAH LEN : Studies have been carried out on the competitive relationships of M altería Heda and М. C. 20 ert 6 , but analysis of the results has not yet been completed.

A. ASHRI: Do you have studies in progress also on changes in gene frequencies in the mixtures of varieties and mutants?

A. von der PAH LEN : Yes, but the data are not yet available.C .F . KONZAK: Your adaptation analysis is very interesting,

especially since you obtained wide differences in nursery yields in different years. It would interest me to see your data evaluated by a variation of the Finlay-W ilkenson technique which we reported at the 1970 Meeting of the Am erican Society of Agronomy. This technique variation involves use of the nursery high yield (or high 1 0 %) rather than the mean as the environmental index. The basis for this variation is (1) the fact that as yield potential of the environment increases, the deviation of the high (maximum) yield within the environment (nursery/year) increases. This is easily demonstrated. (2) The regression values can more readily be interpreted in terms of the goals set by the plant breeder for a) ability to respond in yield (or in lack of a response in height) to improved environment potential, measured by the regression coefficient, and (b) stability


or consistency of the response — or deviation from the regression line (goodness of fit) as measured by r 2.

A. von der PAH LEN : It sounds a very interesting suggestion;I w ill analyse my data with this method.

C. KRULL: Before releasing the multiline variety "M iram ar" in Colombia that you mentioned, we made a sim ilar series of studies comparing the mixture with the yield of the component lines. Unfortunately, the data are not published. The studies are somewhat different from the type that you report in that the component lines had been selected for sim ilarity of agronomic type and phenotype and differed by source of stem rust resistance. These studies were made in regional yield trials throughout the Colombian wheat regions and in more than 20 sites. The mixtures markedly outyielded the average of the components in a few cases but not in others. It was never satisfactorily explained why the mixture was so much better in some conditions and not in others.

A . von der PAH LEN : In the present case, apparently the mixtures did not outyield that expected in the fourth year because the stand was not dense enough to provoke the necessary interactions.

H. HANSEL: Do you have an indication that the differences in plant height, in lodging resistance and in mildew resistance had an influence on yield, in pure stand and/or in line-mixtures?

A. von der PAH LEN : The erectoides mutants are lower in height but may have also a sm aller root development. The factors responsible have not been investigated. Mildew is always present in the field, but it apparently does not affect the yield. Nevertheless М. C. 20 slightly outyielded the original variety.

A . HAGBERG: P lease let me present one example showing the reason why we in Sweden want to use more heterogeneous varieties. In the middle of the 40's two oat varieties were released: Sun II was a "multiline" vkriety and Blenda was the best yielding homogeneous line out of Sun II. Blenda outyielded Sun II when it was released by 3 - 4% - we had had a number of fa irly dry seasons. Twenty-five years later Blenda has been dropped from the list of varieties and Sun II (the multiline variety) is still the dominating variety outyielding Blenda by about 4% for the 25-year period. Sun II is still the most widely adapted variety and the most re liab le . Many other examples could be quoted — but this demonstrates nicely our philosophy!

COMPARACION ENTRE LOS EFECTOS DEL METANOSULFONATO DE ETILO Y LOS RAYOS X EN LA INDUCCION DE MUTACIONES EN Capsicum annuum L.

H.M. ZUBRZYCKI, A. von der PAHLENCentro de Investigaciones en Ciencias Agronómicas,Castelar, Argentina

Abstract-Resumen

COMPARISON BETWEEN EFFECTS OF ETHYL METHANE SULPHONATE AND X-RAYS ON INDUCTION OF MUTATIONS IN Capsicum annuum L.

Results presented here were obtained from work aiming to produce genetic markers in the chromosomic map of pepper. Treatments were made on seeds of the California Wonder variety. It was determined that the lethal dose for X-rays was approximately 40 kR, while concentrations of EMS as high as 6 x 1()~3, during 20 h of soaking, did not reach a lethal dose.

In order to compare X-rays and EMS as chlorophyll and morphological mutation inductors, M 2 progenies were analysed. The chemical agent proved to be more efficient and effective in the induction of chlorophyll mutations, while no significant statistical difference in the induction of morphological mutations was observed between the two agents. It was established that the chemical agent provoked more damage on ovule viability than X-rays. Based on the number of chimeras obtained, it was concluded that at the time of the treatment, different meristems which originate fruits, existed in the seed.

COMPARACION ENTRE LOS EFECTOS DEL METANOSULFONATO DE ETILO Y LOS RAYOS X EN LA INDUCCION DE MUTACIONES EN Capsicum,annuum L.

Los resultados que aquí se presentan se obtuvieron mediante un trabajo planeado con el objeto de producir marcadores genéticos en el mapa cromosómico del pimiento. Los tratamientos fueron hechos sobre semillas de la variedad California Wonder. Se determinó que, para rayos-X, la dosis letal máxima fue de aproximadamente 40 kR, mientras que con concentraciones de hasta 6 X 10 “3 de metanosulfonato de etilo (EMS), durante 20 h de remojo, no se alcanzó la dosis letal máxima.

Con el fin de comparar los rayos X con el EMS como inductores de mutaciones clorofílicas y morfológicas, se analizaron progenies en M 2. El agente químico fue más eficiente y efectivo en la inducción de mutaciones clorofílicas, mientras que no se observaron diferencias estadísticamente significativas en la inducción de mutaciones morfológicas. Se comprobó que el agente químico provocó más darío sobre óvulos fertilizados que los rayos X. De acuerdo al número de quimeras obtenidas se dedujo que los distintos frutos del pimiento provienen de células diferentes, las que se encuentran preformadas en el tejido meristemático de la semilla, en el momento del tratamiento.

L_os r e s u l t a d o s q u e a q u í s e p r e s e n t a n s e o b tu v ie ro n c o m o c o n s e -

c u e n c ia de un t ra b a jo p la n e a d o c o n el objeto de o b te n e r m a r c a d o r e s

g e n é t ic o s en el m a p a c r o m o s ó m ic o del p im ie n to , u s a n d o d ife re n te s d o s i s de m e ta n o su lfo n a to de etilo ( E M S ) y R a y o s X .

S im u ltá n e a m e n te , s e tra tó de c o m p a r a r lo s e fe c to s de e s to s a g e n te s m u ta g é n ic o s y, a d e m á s , d e te r m in a r el n ú m e r o de c é lu la s de l tejido g e n e r a t iv o de lo s f r u to s .

* Publicación Técnica Gen. № 448 del Centro de Investigaciones en Ciencias Agronómicas, INTA. Castelar.

38 7

3 8 8 ZUBRZYCKI y von der PAHLEN

M a te r ia l y m é to d o s

t_ o s t ra ta m ie n to s fu e ro n h e c h o s s o b r e s e m i l la s , al e s ta d o d u r m ie n

te , d e la v a r ie d a d c o m e r c ia l C a l i f o r n ia W o n d e r , p r o c e d e n c ia I N T A ,

c o n h u m e d a d re la t iv a n o r m a l. l_ a s i r r a d ia c io n e s c o n r a y o s X se r e a l iz a r o n a 160 k V , 15 m A y a 10 c m del fo c o , c o n un flujo de a p r o

x im a d a m e n te 1 1 0 0 R / m in . P a r a c a d a c o n c e n t r a c ió n de E M S s e to m a r o n v o lú m e n e s ig u a le s de a g u a y s e m il la , s e t r a ta r o n d u ra n te 2 0 h o r a s

s o m e t id a s a a g ita c ió n . T r a n s c u r r i d o el t ie m p o in d ic a d o , el m a te r ia l s e

la v ó c o n a g u a y s e s e c ó a la s o m b r a .

l_ o s t ra ta m ie n to s c o n lo s a g e n te s m u ta g é n ic o s fu e ro n c o o r d in a d o s

en fo r m a tal de p e r m it ir la r e a liz a c ió n s im u ltá n e a d e la s s ie m b r a s .

E s t a s s e e fe c tu a ro n en in v e r n á c u lo , d o n d e s e c o m p le tó el c ic lo

del m a te r ia l t ra ta d o . D e c a d a p la n ta M^ s e c o s e c h a r o n d e u n o a t r e s

f r u t o s .

P a r a m e d ir le ta lid a d s e t r a ta r o n 100 s e m i l la s c o n c a d a u na de la s

s ig u ie n te s d o s i s : p a r a r a y o s X , 5 K r , 10 K r , 2 0 K r y 40 K r .P a r a E M S , c o n c e n t r a c io n e s en v o lu m e n d e 0 ,5 ; 1; 2 y 4 x 1 0“ 3 ,

c o n un te s t ig o en c o m ú n . L u e g o s e r e g is t r ó el n ú m e r o de p la n ta s M j

s o b r e v iv ie n t e s , d e s p u é s q u e e s t a s a lc a n z a r o n a d e s a r r o l l a r la 5 a . o

6 a . h o ja .

I_ a s e le c c ió n d e l a s m u tan te s erl la s p lá n tu la s M ¿ s e r e a l iz ó d e sd e

la a p a r ic ió n de lo s c o t i le d o n e s h a s ta la 5 a , o 6 a , ho ja fo r m a d a .

R e s u l t a d o s y c o n c lu s io n e s

S e d e te rm in ó q u e la d o s i s letal m á x im a p a r a r a y o s X fue de

a p r o x im a d a m e n te 4 0 K r y ia L . D 50 = 1 9 ,9 K r + 9 K r , c o m p r o b á n d o s e a d e m á s q u e la le ta lid a d fue fun c ión lin e a l c o n r e s p e c t o a la inte n s id a d ( F i g . 1 ) .

A la s c o n c e n t r a c io n e s u s a d a s , lo s t ra ta m ie n to s c o n E M S no p r o d u je ro n le ta lid a d e s ta d ís t ic a m e n te s ig n if ic a t iv a ( F i g . 2 ) .

C o n o c ie n d o lo s r e s u l t a d o s de la s f ig u r a s 1 y 2 y p a r t ie n d o de la

h ip ó te s is de q u e a m a y o r d o s i s p o r t ra ta m ie n to s e p r o d u c e u n a m a y o r

f r e c u e n c ia de m u ta c io n e s , s e e l ig ie r o n in te n s id a d e s de 10 K r , 2 0 K r y 30 K r p a r a r a y o s X y c o n c e n t r a c io n e s de 2 ; 4 y 6 x 1 0” ^ p a r a

E M S , t o m á n d o se 2 0 0 0 s e m i l la s p a r a c a d a d o s i s , c o n un te s t ig o c o m ú n

U n n ú m e r o v a r ia b le de p la n ta s M-j fu e ro n c o s e c h a d a s en c a d a t r a tam ie n to , c o m o p u e d e o b s e r v a r s e en el C u a d r o I II, 2 a . c o lü m n a .

D o s m u ta c io n e s en p la n ta s M } fu e ro n d e te c ta d a s d e n tro de l m a te r ia l t ra ta d o c o n E M S al 6 x IO -З ^ U n a de e l la s s e e x p r e s ó c o m o

p la n ta c o n ta llo s in r a m if ic a c io n e s y f lo r e s en r a c im o s , no d a n d o

d e s c e n d e n c ia . L a o t ra fue v a r ie g a d a y la p r o g e n ie d io 6 p la n ta s x a n ta s le ta le s .

EFECTOS DEL MSE Y LOS RAYOS X 3 8 9

FIG. 1. Regresión del número de plantas sobrevivientes, después de ser tratadas con distintas intensidades de rayos X.

FIG.2. Regresión del número de plantas sobrevivientes, después de ser tratadas con distintas concentraciones de EMS.


D e 946 p la n ta s M-| s e o b tu v ie ro n 2 1 7 1 8 d e sc e n d ie n te s M 2 , e n tre

lo s c u a le s fu e ro n identificadas 508 p lá n tu la s c o m o m u tan te s c lo r o f í l ic a s

y 200 c o m o p r e s u n t a s m u tan te s c lo r o f í l ic a s .

L a s p r o p o r c io n e s d e m u tan te s p o r fru to M i , s e o b tu v ie ro n de la

r e la c ió n del n ú m e ro de p lá n tu la s m u ta d a s s o b r e el n ú m e r o total de

p lá n tu la s y lo s r e s u l t a d o s h a l la d o s s e p r e s e n t a n en lo s C u a d r o s I y II y F i g s . 3 y 4 . D e a c u e r d o a la s p r o p o r c io n e s lo g r a d a s , s e c o m p r o bó q u e lo s t ra ta m ie n to s co n E M S p r o d u je r o n u n a s e g r e g a c ió n m e d ia

p a r a m u tan te s c lo r o f í l ic a s del 25 % , v a lo r e s p e r a d o p a r a s e g r e g a c io n e s m o n o g é n ic a s r e c e s iv a s . E n c a m b io , c o n rayos X s e ob tu vo un

v a lo r m u c h o m e n o r ( F i g . 3 ) . P a r a m u tan te s m o r f o ló g ic a s n o s e

a p r e c ia r o n d i fe r e n c ia s e n tre t ra ta m ie n to s y lo s v a lo r e s fu e ro n m u y

i n f e r io r e s al t e ó r ic o e s p e r a d o ( F i g . 4 ) .

CUADRO I. MUTANTES CLOROFILICAS POR FRUTOS Mj MUTADOS

TRATAMIENTOSN * TOTAL DE

PLANTAS

№ D EPLANTULAS

CLORO FIL ICAS

MUTANTES POR FRUTO

( * )

TESTIGO 2 5 1 0 4 0 ,0

10 K r R X 1 0 7 1 3 1 2,1

2 0 K r R X 2 0 3 3 2 1 0 , 3

3 0 КГ R X 2 0 1 1 9 1 0 ,1

2 % . E M S 2 1 6 7 3 3 3 ,7

4 % „ E M S 2 8 0 4 5 1 6 ,0

6 E M S 1227 3 1 6 25 ,7

CUADRO II. MUTANTES MORFOLOGICAS POR FRUTOS MUTADOS

TRATAMIENTOSN i TOTAL DE

PLANTAS

N i DE PLANTULAS

M O RFOLOG ICAS

MUTANTES POR FRUTO ( % >

TESTIGO 3 2 1 3,1 2

10 K r R X 2 3 2 9 3 ,8 7

2 0 K r R X 4 5 7 2 2 4 ,8 5

3 0 K r R X 3 1 3 2 9 9,2 6

2 % „ E M S 2 2 9 2 0 8 ,7 3

4 7o. E M S 1 9 5 1 4 7,17

6 lo o E M S 1.1 0 5 1 0 5 9 ,5 0

EFECTOS DEL MSE Y LOS RAYOS X 39 1

FIG.3. Proporciones.de mutantes clorofílicas por frutos mutados.

3 EMS _ R X

_J__30 Kr RX 6 3x>EMS

FIG.4. Proporciones de mutantes morfológicas por frutos mutados.

F u e in te re sa n te el h e c h o d e q u e la s s e g r e g a c io n e s p e r m a n e c ie r o n

c o n s ta n te s , p r in c ip a lm e n te c o n r a y o s X ( F i g . 3 ) y n o h a y a n s u f r id o

un in c r e m e n to al a u m e n ta r s e la d o s i s , c o m o o c u r r ió en c e b a d a , s e

gú n F a v r e t y R o d r í g u e z Л7.C o n el objeto d e c o m p a r a r lo s e fe c to s d e lo s r a y o s X con e l

E M S c o m o a g e n te s m u t a g é n ic o s , s e h a l la r o n la s f r e c u e n c ia s d e m u t a c io n e s p a r a p la n t a s y f r u to s M-| y p lá n tu la s M 2 , d e a c u e r d o a lo s

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s e u t i l iz a ro n la s t a b la s de K a s t e n b a u m y B o w m a n , d e te rm in á n

d o s e q u e el E M S fue m á s e fe ctivo en la in d u c c ió n d e m u ta c io n e s c lo r o f í l ic a s ( C u a d r o 111, 9 a . c o lu m n a ) . E n c a m b io a m b o s a g e n te s tu v ie r o n igu a l e fe c t iv id a d p a r a in d u c ir m u tan te s m o r f o ló g ic a s ( C u a d r o IV ,

9 a . c o lu m n a ) . E s t a m is m a c o n c lu s ió n s e p u d o o b s e r v a r en la s t r e s

ú lt im a s c o lu m n a s de lo s C u a d r o s III y I V , s e g ú n lo s m é to d o s de

S t a d le r y G a u l .

S e p u e d e c o n c lu ir , c o n s id e r a n d o la s f r e c u e n c ia s de m u ta c io n e s ,

q u e el E M S fue m a s e fe ctivo q ue lo s rayos X , y to m a n d o c o m o d a ñ o

b io ló g ic o la le ta lid a d p r o d u c id a en p la n t a s M i (F igs., 1 y 2 ) , el a ge n te

q u ím ic o fue m á s e fic ie n te C § J E l J l7 •

Q u e d ó c o m p r o b a d o , a d e m á s , q u e p a r a a m b o s a g e n te s m u ta g é n i- c o s , c o n in c r e m e n to s de d o s i s s e lo g r a m a y o r f r e c u e n c ia de m u ta c io n e s .

C o n r e s p e c t o al v a lo r del in c re m e n to d e la f r e c u e n c ia de m u ta c io n e s p o r d o s i s , s e o b s e r v ó u na d ife re n c ia de a c u e r d o al m é to do e m pleado. S e g ú n el s u g e r id o p o r G a u l ( C u a d r o III, ú lt im a c o lu m n a ) s e

a p r e c ió un in c re m e n to de f r e c u e n c ia p o r u n id a d de d o s i s al a u m e n ta r

la c o n c e n t r a c ió n de E M S d e 4 a 6 x 1 0 "^ , E n c a m b io s e o b s e r v ó lo

c o n t r a r io al a n a l iz a r s e p o r el m étodo t ra d ic io n a l ( C u a d r o III, 9 a . c o

lu m n a ) . E s t a d ife r e n c ia s e e x p l ic a r ía , s e g ú n G a u l £ 3J , p o r u n a s u b e s t im a c ió n de v a l o r e s , p r o v o c a d a p o r un e r r o r de m u e s t r e o , y a q ue

c o n el au m e n to d e d o s i s s e p r o d u c e u na d ism in u c ió n en el tamaño de

la p r o g e n ie . P e r o lo n o to r io fue q u e lo s in c r e m e n to s lo g r a d o s co n

rayos X d is m in u y e r o n al s e r a n a liz a d o s p o r el m é to d o de G a u l y a u

m e n ta ro n al u s a r s e el s i s t e m a t ra d ic io n a l ( C u a d r o I II, 1 0 a . y 9 a . c o lu m n a r e s p e c t iv a m e n te ) .

L_a d ism in u c ió n del n ú m e ro de ó v u lo s fé r t ile s e s u n a m e d id a del d a ñ o p r o v o c a d o p o r el E M S y lo s rayos X. I_ a r e la c ió n e n tre el n ú m e r o d e s e m i l la s p o r fru to y la d o s i s fue u n a fu n c ión linea l n e g a t iv a ,

FIG. 5. Regresión del número de semillas FIG. 6. Regresión del número de semillaspor frutos sobre tratamientos con rayos X. por frutos M : sobre tratamientos con EMS.


s ie n d o la r e g r e s ió n e s ta d ís t ic a m e n te s ig n if ic a t iv a p a r a el a ge n te q u ím ic o , p e r o no s ig n if ic a t iv o p a r a rayos X ( C u a d r o V , F i g s . 5 y 6 ) . A d e m á s , c o m p a r a n d o a m b a s r e g r e s io n e s e n tre s í , s e c o m p r o b ó q u e

é s t a s d ife r ía n s ig n if ic a t iv a m e n te ( P ¿ 0 ,0 5 ) , lo q u e p e rm it ió l l e g a r a

la c o n c lu s ió n d e q u e el E M S p r o v o c ó m a s d a ñ o s q u e lo s rayos X.

CUADRO V. DAÑO PROVOCADO POR TRATAM IENTOS CON RAYOS XY EMS EN OVULO FERTILIZADO

TRATAM IENTOS

№ TOTAL DE FRUTOS M 1

CON DESCEND.

N * TOTAL DE PLANTULAS M 2

№ S E M IL L A S

POR FRUTO

TESTIGO 6 4 1750 2 7,34

XceëoY— 1 1 6 3078 2 6,5 3

2 0 K r R X 1 4 2 4144 2 9,1 8

3 0 КГ R X 9 8 2 0 0 2 2 0 ,4 2

2 % , E M S 1 61 3867 2 4,01

4 % „ E M S 8 6 1689 1 6,6 3

6 У . , EMS 4 1 9 5188 1 2 ,3 8

CUADRO VI. NUMERO DE QUIMERAS LOGRADAS CON RAYOS X Y EMS CONSIDERANDO PLA N T A S M x CON DOS FRUTOS

AGENTES PLA N T A S CON DOS FRUTOS

MUTAGENICOSCON NINGUN

FRUTO MUTADO

CON UN

FRUTO MUTADO

SENDOS FRUTOS CON MUTACIONES

D IST INTAS

SEN D O S FRUTOS CON LA M IS M A

MUTACION

R AYO S-X 29 3 3 0

EMS 47 30 4 9

33 7

TO TAL 76 40 9

39 6 ZUBRZYCKI y von der PAHLEN

P a r a a n a l iz a r q u im e r a s , s e p a r t ió de l s u p u e s t o d e q u e s i en la s e m il la h a y un tejido m e r is te m á t ic o p r e f o r m a d o , constituido por una s o la c é lu la y é s ta e s a fe c ta d a , la m u tac ió n en a m b o s f ru to s d e b e rá

s e r la m is m a , y s i s o n d o s o m á s la s c é lu la s p r e f o r m a d a s y s ó lo u n a e s a fe c ta d a , o a m b a s , p e r o en lo c u s d is t in to s , o b te n d r ía m o s g e n o t ip o s d ife re n te s Ц.\ Qjf . P a r a c o m p r o b a r e s ta h ip ó te s is s e c o n s id e r a r o n ú n ic a m e n te p la n ta s M i c o n d o s f r u to s . S e o b tu v ie ro n 4'9

f ru to s m u ta d o s d e lo s c u a le s 4 0 fu e ro n q u im e r a s ( C u a d r o V I ) . S i

l a s p r e s u n c io n e s a n t e r io r e s s o n c ie r t a s , s e d e d u c e q u e lo s d is t in to s f ru to s de l p im ie n to p ro v ie n e n d e c é lu la s d ife r e n te s , l a s q u e s e en

c u e n t ra n p r e f o r m a d a s en el tejido m e r is te m á t ic o de la s e m il la , en el m o m e n to del tra ta m ie n to .

A g r a d e c im ie n t o

L o s a u t o r e s d e s e a n e x p r e s a r su a g r a d e c im ie n to a l In g . A g r . EEw ald

A . F a v r e t , D i r e c t o r de l C e n t r o de In v e s t ig a c io n e s en C ie n c i a s A g r o n ó m ic a s , p o r la s v a l io s a s s u g e r e n c ia s b r in d a d a s d u ra n te la r e a l iz a c ió n d e e s te t r a b a jo , y al In g . A g r . E n r iq u e F . A n to n e i l i , p o r la le c tu ra de l m a n u s c r i to .


[1] FAVRET, E. A., RODRIGUEZ, A. A,, Inducción de mutaciones con tratamientos agudos de radiaciones gamma de cobalto-60 en semillas de cebada, Revta Invest, agríe. 1Л (1957) 313.

[2] STADLER, L.J., Some genetic effects of X -rays in plants, J. Hered. 23 (1930) 3.[3] GAUL, H., Critical analysis of the methods for determining the mutation frequency after seed

treatment with mutagens, Genet, agr. 12 (1960) 297.[4] KASTENBAUM, M., BOWMAN, K. 0., Tables for determining the statistical significance of mutation

frequencies, Mutation Res. 9 (1970) 527.[5] EHRENBERG, L., Induced mutation in plants: Mechanisms and principles, Genet, agr. 12(1960) 364.[6] EHRENBERG, L., "Chemical mutagenesis: biochemical and chemical points of view on mechanisms

and action", Chemische Mutagenese, Akademie-Verlag, Berlin (1959) 124.[T] EHRENBERG, L. , UJNDQV1ST, u., AHNSTÏtOM, G., The mutagenic action oí ethylene imine in

barley, Hereditas 44(1958) 330.[8] KONZAK, C.F., NILAN, R.A., WAGNER, J., FOSTER, R.J., "Efficient chemical mutagenesis", The

Use of Induced Mutations in Plant Breeding (Rep. FAO/1AEA Tech. Meeting, Rome, 1964), Pergamon Press, Oxford (1965) 49.

[9] FAVRET, E. A., Somatic mutations of four genes for albinism in barley induced by X-rays and ethyl methane sulphonate, Hereditas 46 (1960) 622.

[10] HILDERING, G.L., VERKER, K. K., "Chimeric structure of the tomato plants after seed treatment with EMS and X-rays", The Use of Induced Mutations in Plant Breeding (Rep. FAO/IAEA Tech. Meeting, Rome, 1964), Pergamon Press, Oxford (1965) 313.

D IS C U S S IO N

G .T . SCAKASCIA -M UGNOZZA: Did you find differences in mutation frequencies and types among fruits according to the fruit position on the plant?

H .M . ZU BR ZYC K I: This study has not yet been carried out and the data presented are concerned only with mutation frequencies that appear in seedlings.

DEVELOPMENT OF GOSSYPOL-GLANDLESS STRAINS OF COTTON

M. GUTIERREZ, J. VRDOLJAK, A. RICCIARDI Regional Agricultural Experimental Station,Pcia, Roque Sáenz Peña,Instituto Nacional de Technologie Agropecuaria,Prov. del Chaco, Argentina

Abstract-Resumen

DEVELOPMENT OF GOSSYPOL-GLANDLESS STRAINS OF COTTON.Gossypol in cotton seed cake is toxic to non-ruminant animals. Furthermore, the glands which produce

gossypol contain a dark pigment which transmits an undesirable colour to cotton seed oil. Removal of gossypol by the oil industry is possible but costly. Development of glandless and gossypol-free cotton varieties would be of great economical importance. A spontaneous mutant has been found which is glandless and contains almost no gossypol. It is extensively used in breeding programs, but it has a serious drawback. Gossypol seems to be a component of the natural defence mechanism of the cotton plant against pests, gossypol-free plants being very susceptible. Glandless stocks developed so far, frequently exhibited poor agronomic and/or fibre characteristics. The paper describes a breeding program aimed at overcoming these unfavourable correlations.

DESARROLLO DE VARIEDADES DE ALGODON EXENTAS DE GLANDULAS PRODUCTORAS DE GOSIPOL.El gosipol de la torta de semilla de algodón es tóxico para los animales no rumiantes. Además, las

glándulas que lo producen contienen un pigmento oscuro que da un color poco agradable al aceite de semilla de algodón. Es posible la eliminación industrial del gosipol, pero resulta costosa. La obtención de variedades de algodón exentas de esas glándulas y de gosipol revestiría una gran importancia económica. Se ha encontrado un mutante espontáneo sin glándulas y que no contiene prácticamente gosipol. No obstante, si se utiliza ampliamente en los programas fitotécnicos presenta un serio inconveniente. El gosipol parece ser un elemento del mecanismo natural de defensa de la planta de algodón contra las plagas, siendo muy susceptibles las plantas que de él carecen. Las variedades exentas de glándulas desarrolladas hasta la fecha presentan con frecuencia malas características desde el punto de vista agronómico o de la fibra. En la memoria se describe un programa fitotécnico encaminado a soslayar estas correlaciones desfavorables.

1. INTRODUCTION

The glandless character is caused by a group of recessive genes gU» ë -2 > gl-з» gis» an<3 gig- This causes the norjnal characteristicwith glands of the species to disappear (F ig. 1). These glands are very sm all but are visible due to their dark colour. The glands produce gossypol which has the following formula: 1, 1' , 6 , 6 ', 7, 7* -hexahydroxy-5,5* -diisopropyl-3,3f-dimethyl-2, 21 -binapthalene-8 , 8 1 -dicarboxaldehyde. The mutant has been found in derivatives of crosses of strains of hirsutum with Hopi of the punctatum race of hirsutum.

The seeds of the glandless plants contain almost no gossypol. Gossypol is light-yellow in colour and in the glands there are also other dark pigments that give an undesirable colour to cotton seed oil. Stored oil rapidly takes on a dark colour. In addition, cotton seed cake containing gossypol is toxic for non-ruminant animals.

In the oil industry it is necessary to chemically remove the gossypol, a process which increases the cost of oil and cake production and which is unnecessary with the use of glandless seed. Glandless seed is a cheap

3 9 7

3 9 8 GUTIERREZ et a l.

CO TYLED O N

P H E N O T Y P E S

H YPO C O TYL STfPU LE

$C A P S U L E

Norm al p la n ts with g land sG Í2 G l2 GI3 GI3

и

(J

S egregating plants w ith fe w er g la n d s GI2 G l2 д!з g t3

g l2 gl2 GI3 GI3

G l2g l2 9(3 9 >э

gljglîGt-î дЦ

Double recessive p lants without g la n d s

g l2g l2 д1з д!з

FIG. 1. Segregation for the character gossypol-glandless.

source of protein for animals and its importance is even greater if used for human consumption. Cotton seed cake containing gossypol can only be used for feed for ruminants because the fermentation bacteria in the rumen synthesizes several vitamins and amino acids and inactivates the gossypol. This is not the case for non-ruminants whose rations should contain the nutrients required by the animal.

The mutant possesses an adverse character because when the normal characteristic with glands disappears the resistance to pests is diminished since gossypol is a component of the natural defense mechanism of the plant.

Some cotton research institutions in the United States are conducting work to increase the resistance of cultivated hirsutum by increasing the quantity of glands and consequently of gossypol. Various wild species and some primitive species of hirsutum have a larger amount of gossypol glands than the commercial upland varieties.

However, the importance of achieving a combination of glandless character in the seed with the presence of glands in the flower, fruit, and vegetative parts should be pointed out. This task would appear very difficult but perhaps not impossible. At least, the Cienfugosia related genera possesses these characteristics.

Three principal genes for the glandless condition can be identified: g li g l j , for stocks, stems, and carpel walls; g l2 and g l3, for cotyledons, leaves, stocks, stems, and carpel walls; g l 4 and g l5, modifiers having less effect; and g l6 g l6, which seems to be a homologue to glj glj . Consequently, in the cotton seed, the quantity of gossypol depends mainly on combinations of gl^ and g l3. The normal seed of G. barbadense has more gossypol than that of G^ hirsutum.

GOSSYPOL-GLANDLESS COTTON STRAINS 3 9 9

McMichael, in the U .S . Cotton Field Station at Shafter, California, worked patiently for many years, first with the character g lx and was later able to incorporate the genes gl2 and g l 3 of Hopi Moencopi in a strain of Acala. Hopi Moencopi cotton is a non-commercial type of the punctatum race of hirsutum.

The work of Lee on the inheritance of the glandless character indicates that in the F 2 of the glandless stock of McMichael, crossed with a double haploid normal with respect to glands, or in the first back-cross with the glandless material, neither exact genetic proportions nor a definite relationship between genotype and phenotype were obtained. The plants with glands presented a continuous distribution from one gland per cotyledon up to the normal quantity. Lee described four distinct types in the segregation:Type A — glands distributed in the entire sub-epidermic region; Type В — glands distributed more abundantly near the margins and principal vein of the leaf; Type С — glands near the margins of the leaves only; and Type D — without glands.

Lee found that when crossing glandless plants with a series of normal plants of commercial varieties, the majority segregated in a proportion of 15:1, corresponding to the original cross GI2 Gl2 Gl3 Gl3 X gl2 gl2 gl-з gl-з •The G I 4 GI5 genes are rare in common cottons and Lee suggests that they are relic genes, weakened by the process of functional diploidization of the tetraploid cottons. To confirm the presumptions with respect to the value of each of the genes mentioned, Lee analysed the gossypol of the four distinct genotypes (Table I). It is evident that G l2 is more potent than G l3 and that G I 4 has very little effect.

The pilot plant of the Oilseed Products Research Center of Texas A and M University has maintained the leadership for several years in the study of the possibilities of oil and meal of strains and varieties of cotton with gossypol-glandless seeds. This pilot process culminated in October 1969 when the first commercial extraction of oil from the meal of glandless cotton seed was carried out in a plant in Oklahoma City. The process was carried out with solvent without the use of steam which denaturalizes the protein and limits application as a food product. The prime material was the Watson GL 16 variety and the meal was slightly reddish, containing protein which was not denaturalized by the steam. It was classified as adequate for the food industry as a low cost, high quality protein source for human nutrition.

TABLE I. GOSSYPOL CONTENT OF GLANDLESS GENOTYPES OF COTTON (after Lee)

^ Gossypol contentGenotype

G12 G1 g G13 G13 G14 G14 1 .3 4 4

G12 G12 gl3 gh g U g U 1 .0 3 7

g* 2 gl 2 G13 G1 3 gl 4 gl 4 0 .3 0 6

g b g ^ g b g b g U g U 0 .0 0 6

g ^ g l z g b g U G l i G l * 0 .0 0 5

4 0 0 GUTIERREZ et al.

The Texas A and M Laboratory has also prepared a product called "Tamunuts" from glandless cotton seed kernels. This product is used as a snack just as peanuts. Another use of the flour without gossypol would be as a substitute for casein in, for example, baked products, powdered coffee "cream ", and in mixtures in frozen dairy products. Also the possibility of using this product in the preparation of a protein supplement drink is being, studied.

The Southern Utilization Research and Development Laboratory ARS, USDA, has produced protein concentrates of about 70% with glandless cotton seed meal and has separated different protein substances with revolutionary properties for the industry.

In summary, great interest has been awakened among many geneticists of both official and private organizations in the USA and other countries of the world, and in many sectors of the cotton seed industry. However, two important questions relative to the future of the cultivation of gossypol- glandless varieties are raised: (1) What w ill be the pest control situation and its cost upon diffusion of germ plasm not containing one of the most important resistant mechanisms? (2) If cotton is losing relative importance in world textile consumption as the proportion of synthetic fibres advances, would not these new possibilities of a sub-product be a means of salvation?We hope that we in Argentina w ill also be able to contribute to the resolution of these unknowns.

2. D EVELO PM EN T OF GLANDLESS COTTON SEED STRAINS INARGENTINA

2.1. Methods and materials

The Sáenz Peña Agricultural Experiment Stations received the basic stock, Glandless 23B from McMichael. In 1958/59 a plan of crossing, backcrossing, and selection on the Stoneville 7, INTA Sáenz Peña Toba I,Fox 4, Sáenz Peña M58 and La Banda 56 were initiated. This was the beginning of the long process of incorporating the glandless character but without the complex of poor agronomic and fibre characteristics, and of the Hopi Moencopi, in the adapted varieties.

In the first years a method of backcrossing sim ilar to that followed by various institutions in the USA was followed.

The Fx double heterozygote was obtained and backcrossed with the commercial recurrent parent. The segregation is 1; 1; 1; 1. The double heterozygotic plants were detected by their sm aller quantity of glands and were again backcrossed with the adapted variety. At the same time, all the plants were selfed and were planted to observe the presence of glands in the cotyledonar state. The plants segregated in the proportion of 15 with glands to 1 glandless, indicating the double-heterozygote. These were the only ones with which the backcrossing process was continued with previous selection of the most productive.

The system was as follows: P) g l2 gl2 gle g ^ X G I 2 GI2 GI3 GI3 ; Fj andBC j) G l 2 g l2Glg g l3X G l 2 G l2 Gig Gig .

BC i progenies

1/4 G12 G12 Gig Gig selfed -* all with glands 1 / 4 Gl2 G12G13 g l 3 selfed -» all with glands

GOSSYPOL-GLANDLESS COTTON STRAINS 40 1

1/4 G l 2 gl2 G l 3 G l 3 selfed - all with glands1 /4 G l 2 g l 2 G I3 g l 3 selfed - 15 with glands to 1 glandless

XG l 2 Gig GI3 Glg -> backcrossing process

repeated.

The purpose was to rapidly approach the germ plasm of the commercialstrains. However, this method was soon abandoned because it was notalways easy to distinguish the double heterozygote in spite of continual selection from the seedling to the flowering stages. Moreover, when the year for the first field comparisons of the glandless strains with the comm ercial varieties arrived, it was obvious that the objective of acquiring the germ plasm of the recurrent parent had not be§n achieved. It was evident that the glandless character is linked, or possibly there exist plei.otropic effects from the Hopi Moencopi, which results in strains of inferior agronomic and fibre properties to those of the recurrent commercial varieties.

Therefore, we opted for the alternative of planting the BCi seed in the greenhouse, and then obtaining the F2 from the backcrosses. The glandless F2 seedlings, gl2 g l2 g l3 g l3> were transplanted in the field. The plants showing the best agronomic behaviour were selected and again backcrossed.In addition, seed for F3 progenies rows from the selected plants were obtained by means of selfing. This composed a general block of strains in the process of development with different years of backcrossing and selfing under continuous selection pressure for economic characteristics.

The most undesirable characteristics were lateness, excessive vegetativeness, low ginning percentage, and short fibre. In addition, in Sáenz Peña, the susceptibility of these strains to pest attacks, especially aphids f Aphis gossypii), leafworms (Alabama argillacea) and bollworms fHelicoverpa gelotopoeon and Heliothis virescens) in field trials with strains with glands was observed. This situation was confirmed in laboratory trials.

Strains were distributed to the E l Colorado, Reconquista, and Corrientes Experiment Stations for regional trials and reselection. This procedure, moreover, permitted selection of strains with greater adaptability and facilitated the necessary screening that the incorporation of the glandless character to commercial varieties advanced strains requires.

At the present time, regional comparative trials are being conducted in Sáenz Peña, E l Colorado, Reconquista, and La Banda. In addition, in Sáenz Peña, a spectrum of different generations derived from the fourth to the eighth backcross are under development.

Three years ago material from the Bebedja Experiment Station, Chad, and supplied by Dr. J .B . Roux of the IRCTE, France, was also included as a glandless source. This stock is agronomically late for our region but the germ plasm is of interest to explore and it already has genes B 9 and Bio for resistance to angular leaf spot (Xanthoroonas malvacearum ). In addition, this biotype of germ plasm which is unrelated to our glandless strains is of interest for initiating crosses between glandless strains.

2.2. Results

In Table II, the results from two years of trials in Sáenz Peña of advanced strains with six backcrosses in their germ plasm can be seen. The Sáenz Peña Toba II variety used as a check is the one recommended for the

4 0 2 GUTIERREZ et al.

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GOSSYPOL-GLÀNDLESS COTTON STRAINS 4 0 3

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region because of its adaptability, agronomic properties, and fibre characteristics. The results indicate the attainment of acceptable agronomic and fibre levels in strains without gossypol in the seed.

In Table III, a spectrum of other strains without gossypol which are in development is presented. This is indicative of the variability attained for the more important economic characteristics.

In the 1970 spring plantings, several seed increase plots were planted.In addition they serve for complementary observations, reselection, evaluation of pest damage, and acquisition of seed for determination of gossypol content in the oil and cake.

The next step, after conducting all possible tests of agronomic characteristics and fibre properties, will be to release some of the strains without gossypol for la rge -sca le cultivation, wait for the reaction from farm ers and spinning m ills, determine the intensity of pest attacks and study the necessary management for their control. Finally, the reaction of the oil industry and the consumers of cake and meal for feeding poultry, ruminants, and swine will be evaluated. In addition, the human nutrition properties should also be analysed.

B I B L I O G R A P H Y

AGRICULTURAL EXPERIMENT STATIONS, ARS.USDA, Genetics and Cytology of Cotton, 1956-67, Sth.Co-op. Ser.Bull.139 (1968)."Cotton seed meal now used safely for non-ruminants", Cotton International, 1970.HYER, A.H., "Breeding for glandless cotton seed in California", Proc. 18th Cotton Improv. Conf., Memphis, 1966.LEE, J.A., Genetical studies concerning the distribution of pigment glands in the cotyledons and leaves of upland cotton, Genetics 47 (1962) 131.LEE, J.A., "Some prospects for breeding more glandular cottons", Proc. 18th Cotton Improv.Conf., Memphis. 1966.LEWIS, C.F., McMICHAEL, S.C..LEE, J.A., "Review of the genetics of glandlessness in cotton, Proc.Cotton seed Oual. Res.Conf. Greenville, Miss., 1962.LUKEFAHR, M.J., BOTTGER, G.I., MAXWELL, F., "Utilization of gossypol as a source of insect resistance", Proc.18th Cotton Improv.Conf., Memphis, 1966.McMICHAEL, S.C., "Selecting for glandless seed in cotton", Proc.lOth Cotton Improv.Conf., USA, 1957.McMICHAEL, S.C., "Recent developments on genetics of glandless seed of cotton", Proc. 11th Cotton Improv. Conf., USA, 1958.McMICHAEL, S.C., Hopi cotton, a source of cotton seed free of gossypol pigments, Agron.J. 51 (1960) 630.MIRAVALLE, R.J.,"Breeding for glandless cotton", Proc. 13th Cotton Improv.Conf., Greenville, S.C., 1961.MIRAVALLE, R.J.,"Glandless cotton research", Proc.20th Cotton Improv.Conf., Hot Springs, Ark., 1968.SMITH, K.J., "Potential unlimited for cotton seed as human food", cotton International, 1970.UNIVERSITY OF TEXAS, Cotton Economic Research, Summary Report of Texas Cotton and Related Data for the 1969-70 Season, Res.Rep.No.95.

ESTUDIO SOBRE LA VARIABILIDAD INDUCIDA POR ETILMETANSULFONATO (EMS) EN CARACTERES CUANTITATIVOS DE PLANTAS HERMANAS DE MUTANTES DRASTICOS EN TRIGO (T. vulgare)CON PROPOSITOS DE SELECCION

R. TRUJILLO FIGUEROA, M.J. RIOS BETANCOURT

Rama de Genética, Colegio de Postgraduados,Escuela Nacional de Agricultura,Chapingo, México

Abstract-Resumen

STUDY OF THE VARIABILITY INDUCED BY ETHYL METHANE SULPHONATE (EMS) IN QUANTITATIVE CHARACTERS OF SISTER PLANTS OF DRASTIC MUTANTS IN WHEAT (Triticum vulgarel FOR SELECTION PURPOSES.

A study was made of the genetic variability induced in quantitative characters in the second and fourth generations {M2 and M 4 ), and of the possibilities of selection for yield in sister plants of drastic mutants (speltoid, large spike with spaced spikelets, broadhead spike, compactoid, dwarf, awned, spherococcoid, waxless and other types), after the treatment of wheat with EMS.

In the M 2 generation it was found that in general the variances of the characteristics considered (main culm length, number of spikes per plant, length, number of spikelets and density of the main spike, and also the yield per plant) and determined in 152 families increased, and that the mean values decreased in comparison with the control. The third generation (Mg) served to increase the material considered in M 2 for the purpose of yield tests in the M 4 generation. Here only 18 of the 152 families considered in M 2 were tested, since the remainder had been eliminated in the Mg generation, either because the material had been segregated for drastic mutants and was eliminated, or because the families did not possess 10 lines or sufficient seed for the yield tests.

The results of the M 4 generation, determined on the basis of the variances and mean values of the characters (main culm length, number of spikelets, main spike length and 100-seed weight), .'¡how that the variances were even greater than in M 2, while the mean values were closer to the mean of the control. This is due to the improved homozygosis of the material and the elimination of lethal factors, because of self- fertilization and the selection process during the reduction of the material. The 100-seed weight character shows that there were values exceeding those of the control, although not significantly, in three different lines. This material requires further yield testing in order to better define the differences.

ESTUDIO SOBRE LA VARIABILIDAD INDUCIDA POR ETILMETANSULFONATO (EMS) EN CARACTERES CUANTITATIVOS DE PLANTAS HERMANAS DE MUTANTES DRASTICOS EN TRIGO (T_. vulgare) CON PROPOSITOS DE SELECCION.

Se estudian las características de la variabilidad genética inducida en caracteres cuantitativos, en la segunda y cuarta generaciones (M2 y M 4 respectivamente) y las posibilidades de selección para rendimiento, en plantas hermanas de mutantes drásticos (espeltoides, espigas grandes con espiguillas espaciadas, espigas cabeza ancha, compactoide,- enanos aristados, esferococoide, carentes de cera, entre otros) en trigo, después de un tratamiento con etilmetansulfonato(EMS).

En la segunda.generación (M2) se observa, en general, que las varianzas de los caracteres considerados (longitud del tallo principal, número de espigas por planta, longitud, número de espiguillas y densidad de la espiga principal, así como rendimiento por planta) determinados en 152 familias, aumentan y las medias disminuyen en comparación con el testigo. La tercera generación (M3) sirvió para aumentar los materiales considerados en la M 2, para conducir los ensayos de rendimiento en la cuarta generación ( M 4). En la M 4 solamente se probaron 18 familias de las 152 consideradas en la M 2, ya que el resto se había eliminado en la M 3, por una parte, debido a que los materiales segregaron para mutantes drásticos, y fueron eliminados, y.

4 0 5

40 6 TRUJILLO FIGUEROA y RIOS BETANCOURT

por otra parte, porque las familias no poseían un número de 10 lineas o bien suficiente semilla para los ensayos de rendimiento requeridos.

Los resultados de la M 4 determinados sobre las varianzas y medias de los caracteres (longitud del tallo principal, número de espiguillas y longitud de la espiga principal, así como peso de 100 semillas) muestran que las varianzas son aun mayores que en la M 2, mientras que los valores medios se aproximan más a la media del testigo. Esto se observa debido a la mayor homocigosis de los materiales, y a la eliminación de factores letales debido a la autofecundación y al mismo tiempo al proceso de selección al reducir los materiales. El carácter peso de 100 semillas muestra que hubo valores superiores a los del testigo aunque no significativos, para tres diferentes líneas. Estos materiales requieren ser estudiados posteriormente en ensayos de rendimiento para definir mejor esas diferencias.

INTRODUCCION

El mejoramiento en caracteres cuantitativos mediante la utilización de mutaciones requiere la utilización de métodos apropiados de selección para que tenga mayor éxito [ 1 ]. Para ello se requiere un conocimiento de la variabilidad genética inducida, en los caracteres de interés, después de un tratamiento mutagénico.

Numerosos han sido los investigadores que se han abocado al estudio de la variabilidad inducida utilizando tanto agentes físicos como químicos en diferentes caracteres de variadas especies vegetales (Gregory [2 ] en cacahuete; Gaul [3 -5 ], Gaul et al. [ 6 ], Bouma [ 8 ] y Scholz [9 ] en cebada; Bhatia y Swaminathan [10], Borojevic [11 y 12], Gaul [5] y Trujillo [1 ] en trigo; Krull y F rey [13 ], Frey [14], Abram s y Frey [15 ] en avena; Bhatia y Van der Veen [16] y Brock [17] en Arabidopsis; Rawlings et al.[18], Papa et al. [19] en frijo l soya; Porsche [20] en lupinus, entre otros). E l conocimiento de las características de la variabilidad inducida en las prim eras generaciones después del tratamiento mutagénico es de suma im portancia cuando se tienen propósitos de selección, como señalan los autores' arriba mencionados y las amplias discusiones de Scossiroli [2 1 ], Brock [17] y Gaul [4 ]. E llos señalan que en general las variaciones aumentan mientras que los valores de las medias disminuyen; lo cual se atribuye a la presencia de factores letales y subletales (Scossiroli [2 1 ]). En consecuencia estas poblaciones en las prim eras generaciones poseen un alto grado de hetero- cigocis y en gran parte genes letales, los cuales sólo podrán ser eliminados a través de las generaciones. En las generaciones avanzadas se dispondrá de una mayor variabilidad por población y estabilidad genotípica por individuos y en consecuencia la selección será de mayor éxito (T ru jillo [1 ]). Las poblaciones en generaciones avanzadas serán muy grandes y óptimas para rea lizar una selección, pero por razones prácticas serán difíciles de manejar por lo cual se ha recurrido a la selección de las mismas en una u otra forma. A sí se han hecho selecciones en M2 para rendimiento, ya sea al azar, seleccionando plantas aparentemente normales (Gregory [2] en cacahuete; Gaul[4] y Ehrem berg et al. [7] en cebada; Gaul [5] en trigo y cebada) o directas sobre caracteres de rendimiento (Gaul [3 ], peso de 1000 semillas en cebada, Rawlings et al. [18], número de granos en frijo l soya; Bouma [8 ], valores arriba de la media del testigo para diferentes caracteres del rendimiento en cebada; Scholz [9 ], rendimiento en cebada, entre otros). La selección directa generalmente se lleva a cabo a partir de la M3 (Gregory [2 ], producción de grano en cacahuete; Scossiroli [22], altura de la planta número espiguillas/espiga principal, número de tallos/planta en trigo ( T. durum y vu lgare ); Gaul et al. [6 ], rendimiento de grano en cebada; Papa et al. [19],

VARIABILIDAD INDUCIDA POR EMS 40 7

alto rendimiento en frijo l soya). Este proceso de selección más tardío es más recomendable ya que la selección temprana (M 2) directa puede dejar de incluir a todos los caracteres componentes de rendimiento (Brock [17]). Debido a esto se han hecho selecciones en generaciones tempranas, pero no a base de caracteres directos sino indirectos, que en algunos casos no tiene mucho que ver con el rendimiento, sino que solamente sirven de indicadores de variabilidad genética inducida (Gaul [23]). A sí Gaul y Mittelstenscheid [24 ], seleccionaron mutantes precoces en cebada; Bhatia y Swaminathan [10] y Borojevic [11], mutantes aristados en trigo; Trujillo [ l ] , basándose en la variabilidad de la espiga principal, entre otros.

En el presente trabajo se estudian en plantas aparentemente normales, hermanas de mutantes drásticos (espeltoide, espigas grandes con espiguillas espaciadas, espigas cabeza ancha, compactoide, enanos, aristados, esferococoide, carentes de cera y otros; véase Trujillo [1 ]) las características de la variabilidad genética inducida en caracteres cuantitativos y las posibilidades de selección para rendimiento en generaciones avanzadas.

M ATERIALES Y METODOS

E l material empleado en el presente estudio fue un trigo de prim avera de Alemania (variedad 388 0/48) correspondiente a fam ilias 1 en las generaciones M 2 y M 4 (provenientes de material tratado con etilmetansulfonato EMS CH 3 S0 2 0C 2 H 5) de las cuales se habían separado las plantas que m ostraban mutantes drásticos, tales como los tipos espeltoides, compaetoides aristados, etc., citados por Trujillo [1 ]. En su trabajo Tru jillo solamente consideró aquellas fam ilias aparentemente normales, mientras que aquellas donde aparecieron mutantes drásticos se separaron, destinándose solamente las plantas hermanas aparentemente normales para este estudio Esobre la variabilidad de algunos caracteres cuantitativos.

E l trabajo se condujo hasta la tercera generación (M3) en el Institut für Pflanzenbau u. Pflanzenzüchtung de la Universidad de Gotinga, Alemania, y en Chapingo, México, se estudió la cuarta generación (M 4).

La siem bra de la segunda generación se hizo en surcos por espiga (la cual provenía del tallo principal de plantas M j), con una longitud de 1 m y los granos espaciados a 4 cm en el surco; la distancia entre surcos era de 2 0 cm .

Del material sembrado en la M 2 se pudieron separar 152 familias que mostraron mutantes drásticos. E l número de plantas normales fue variable para cada fam ilia haciendo un total de 1716. Cada una de estas plantas se cosechó individualmente sembrándose en la M3 en surcos por planta. Solo se sem braron aquellas fam ilias que tenían un número igual de 1 0 plantas cada una; cada surco constituía una línea. En la cuarta generación (M 4) se hizo un ensayo de rendimiento con 18 familias, las cuales no habían segregado mutantes drásticos y poseían un número igual a 1 0 líneas con cantidad de sem illa suficiente para establecer el experimento. Del testigo se tomaron 10 hipotéticas líneas al azar. Para la siembra se utilizó un arreglo de parcelas divididas en un diseño de bloques al azar con cuatro repeticiones,

1 Familia: Plantas provenientes de una espiga Mi. La descendencia de cada una de las plantas formaron una línea en las generaciones siguientes.

4 0 8 TRUJILLO FIGUEROA y RIOS BETANCOURT

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donde las parcelas principales estuvieron constituidas por fam ilias y las subparcelas por líneas.

La variación de los diferentes caracteres se analizó bajo el siguiente modelo.

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i = 1, 2, . . . , 19 familiasj = 1, 2, 3, 4 bloquesК = 1, 2, . . . líneas

E l experimento estuvo constituido por 190 subparcelas por repetición haciendo un total de 760 subparcelas. La subparcela constó de 5 surcos de1 m de largo separados de 2 0 cm. Las semillas se sembraron a una distancia de 5 cm .

La fertilización se hizo a razón de 100 kg de N (sulfonato de amonio) y 60 kg de P2 O s(superfosfato de calcio simple) por hectárea.

Durante el ciclo vegetativo y después de la cosecha, se tomaron datos para diferentes caracteres de la planta (longitud del tallo principal, longitud y número de espiguillas de la espiga principal, número de tallos con espiga por planta, rendimiento y peso de 1 0 0 granos) en cada una de las generaciones, los cuales se analizaron estadísticamente.

RESULTADOS

1. Segunda Generación (M 2)

Los resultados del análisis estadístico de los caracteres considerados en la segunda generación (M 2) para medir el efecto del etilm.etansulfonato (EMS) en plantas normales hermanas de mutantes drásticos, se pueden observar en el cuadro I. En general se observa, en relación al testigo, que la variabilidad ha sido mayor para todos los caracteres excepto para número de espigas por planta y rendimiento por planta y las medias fueron menores comparativamente a las del testigo como se muestra, por ejemplo para algunos caracteres, en las figuras 1 y 2. A sí de las 152 familias estudiadas para los caracteres longitud del tallo principal, númef-o de espigas por planta, longitud de la espiga principal, número de espiguillas de la espiga principal, rendimiento por planta y densidad de la espiga principal, 73, 36, 92, 72, 35 y 81 familias, respectivamente, presentaron varianzas mayores a la varianza media del testigo, siendo el resto iguales o inferiores a las del testigo. Respecto a las medias, se encontraron 12, 32, 24, 7, 20 y 40 familias con medias superiores a las del testigo respectivamente, para los caracteres mencionados.

Debido a la diferente segregación que se presenta para diferentes caracteres considerados componentes del rendimiento y además por la disminución en vigor de muchas de las plantas o la desaparición de otras por la presencia

2 Nótese que el término L.„ K (líneas dentro de familias) está reemplazando a los términos L^(FL)^ del modelo clásico, ya que se trata de líneas diferentes en cada una de las familias consideradas.


FIG. 1. Distribución de frecuencias para el carácter longitud del tallo principal en la M 2.

de factores letales y subletales, se observó una amplia diferencia en el desarrollo de las plantas dentro y entre las familias propiciando esto una mayor o menor competencia entre las plantas y desarrollándose unas m ejor que otras, por la acción diferencial del medio ambiente. Estos factores genéticos y ambientales que actúan en la segunda generación (M 2) no pueden sin embargo separarse y debido a ello, cualquier propósito de selección a este nivel de generación (Mg) basado en un rendimiento global por planta sería incorrecto, puesto que se estaría arrastrando la influencia del medio ambiente. Sin embargo, con el propósito de hacer una observación prelim inar a este respecto, se compararon plantas individuales en cuanto al número de tallos con espiga y al rendimiento, encontrándose que a medida que aumentó el número de tallos por planta aumentó también el rendimiento tanto como en el testigo, siendo siempre superior para igual número de tallos el rendimiento del testigo. Por otra parte, las varianzas fueron en general mayores en el m aterial tratado (cuadro II y figura 3). A l observar el rendimiento promedio por planta se encuentra que los incrementos de rendimiento son mayores cuando las plantas poseen menor número de espigas hasta un número de 7; sin embargo, después de ese número aunque los rendimientos por planta siguen siendo mayores, los incrementos de rendimiento tienden a ser uniformes y decrecen paulatinamente. De tal forma que se podría plantear una consideración que sería ideal enlos program as de mejoramiento en el sentido de seleccionar aquellas plantas con un número intermedio de tallos


CUADRO II. VALO R DE MEDIAS Y VARIANZAS EN IVt, , PARA EL CARACTER RENDIM IENTO POR PLA N T A CUANDO SE T IENE EL NUMERO DE ESPIGAS FIJO POR LINEA.

(TABLE II. MEAN VALUES AND VARIANCES IN M 2 FOR THE CHARACTER PLANT YIELD WHEN THERE IS A FIXED NUMBER OF SPIKES PER LINE)

Núm erode

espigas

M aterial tratado (M 0) J TestigoRendimiento Rendimiento

Númeroindividuos

X S2Media por es piga

Número indivi—

duosX S2

Media por e s piga.

1 37 1,27 0,19 1,27 11 1,39 0,17 1,392 235 2,27 1,14 1,13 40 3,00 2,97 1, 50

3 394 3,34 1,34 1,11 95 4,04 1,45 1,34

4 351 4,79 2,06 1,19 149 5, 53 1,03 1,38

5 281 6,15 2,30 1,23 126 7,08 2,00 1,42

6 174 7,59 3,52 1,26 70 8,40 3,59 1,40

7 105 9, 13 5,28 1,30 48 9,35 3,85 1,33

8 69 10,22 5,69 1,27 I 32 11, 57 4,65 1,44

9 27 11,64 9,28 1,29 15 12,99 3,83 1,44

10 13 12,90 16,73 1,29 14 14,13 9,87 1,41

U 10 13,45 19,87 1,22 2 13,20 25,92 1,20

12 7 14,34 7,30 1,19 5 16,86 2,48 1,40

13 6 16,47 7,39 1,26 4 16,97 4,11 1,30

14 4 20,13 3, 58 1,43 Л 1 18,60 - 1,32

15 1 16,40 - 1,09 I 2 21,70 40,99 1,44

16 1 16,90 - 1,05 I

17 -I "

18 - 1 -

19 - 1 15,70 - 0,82

20 1 3,60 - 0,18 -


FIG.2. Distribución de frecuencias para el carácter longitud de la espiga principal en la M,, .

pero con altos rendimientos promedios por espiga, en lugar de tener plantas con elevado número de tallos con mayor rendimiento por planta, pero no sería por unidad de superficie.

E l mayor rendimiento presente en aquellas plantas con mayor número' de tallos trató de explicarse sacando las correlaciones existentes entre diferentes caracteres y el rendimiento total. A sí se estimaron co rre la ciones entre longitud de la espiga principal, longitud del tallo principal, número de espiguillas por espiga y rendimiento (cuadro III), encontrándose que el número de espiguillas está correlacionado significativamente con longitud de tallo y rendimiento desde las plantas con una sola espiga, hasta con 8 espigas, mientras que longitud de espiga está correlacionada a partir de las plantas con 2 hasta las que tienen 6 tallos. Cuando el número de espigas es muy alto no se presentan correlaciones positivas en el rendimiento, lo cual se puede atribuir a una mayor competencia entre órganos de la planta.

2. Cuarta generación (M^)

E l estudio de la M 4 se llevó a cabo en un ensayo de rendimiento de aquellas fam ilias que en la M 3 no habían segregado mutantes drásticos y que


FIG. 3. Rendimiento medio en gramos y desviación estándar (s) de plantas Mj con diferente número de espigas.

tenían por lo tanto un número de 1 0 líneas cada una con suficiente cantidad de sem illa (mínima 400) para el diseño empleado. Por tal motivo solamente se pudieron probar 18 fam ilias con 1 0 líneas cada una o sea un total de 180 líneas.

Estos m ateriales y aún el testigo (1 familia con sus 10 líneas) mostraron en general un desarrollo precario en comparación a como se observó en Alemania. Por otra parte la germinación fue en general baja, presentándose desigualdad en la competencia individual de las plantas, razón por la cual los caracteres número de espigas/planta y rendimiento por superficie no se consideraron. Solamente se tomaron en cuenta para evaluar la variación de estos m ateriales la longitud del tallo principal y la longitud de la espiga del tallo principal. E l cuadro IV muestra diferencias altamente significativas entre los cuadrados medios de los caracteres considerados tanto entre familias como entre líneas dentro de algunas familias del material tratado.E l testigo (fam ilia 19) no presentó ninguna variación significativsi, lo cual pudo deberse a su homogeneidad genética. A l obtenerse las varianzas de las líneas dentro de cada fam ilia (cuadro IV) se comprobó que éstas son más grandes en un mayor número de fam ilias para el carácter peso de 1 0 0 sem illas, comparándolas con el testigo, que para aquellos otros caracteres considerados, lo cual se puede atribuir a una mayor variabilidad genética inducida y

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CUADRO IV. CUADRADOS MEDIOS P A R A LOS C ARACTERES LO NG ITU D DE LA E S P IG A /TA LLO P R IN C IP A L , LO NG ITU D D EL T A L L O P R IN C IP A L , NUMERO ESPIG U ILLAS/ESPIG A P R IN C IP A LY PESO DE 100 SE M ILLAS EN M 4 .

(TABLE IV. MEAN SQUARES OF THE SPIKE LENGTH/MAIN CULM, MAIN CULM LENGTH, NUMBER OF SPIKELETS/MAIN SPIKE AND WEIGHT OF 100 SEEDS, IN M4)

F.V. G.L.Long. E s piga principal

# Espigui

llas/ espiga principal

Long, tallo principal

Peso 100 semillas

Total 759

Repeticiones 3 19,396** 20,833 725,000* 2,527**

Familias 18 18,422** 27,552** 1067,528** 1,292**

Error (a) 54 4,256 7,813 210,778 0,165

Líneas / familias 171 1,420** 3,486** 58,056** 0,116**

Líneas/familia 1 9 2,260** 2,566 83,615** 0,186**

Líneas/familia 2 9 2,033** 3,779* 56,659** 0,159**

Líneas/familia 3 9 3,288** 6,411** 149,097** 0, 085*

Líneas/familia 4 9 1,834** 3,479 85,562** 0,288**

Líneas / familia 5 9 3,254** 3,391 41,913* 0,037

Líneas/familia 6 9 0,820 1,959 153,462** 0,269**

Líneas/familia 7 9 1,676* 7,219** 30,031 0,201**

Líneas / familia 8 9 0,769 2,194 40,007* 0,074

Líneas/familia 9 9 1,184 0,894 33,823 0,058

Líneas / familia 10 9 0,748 5,2 59** 104,618** 0,167**

Líneas/familia 11 9 0,717 1,714 28, 653 0,085*

Líneas / familia 12 9 2,604** 4,196* 31,260 0,113**

Líneas/familia 13 9 1,993** 4,092* 21,142 0, 049

Líneas/ familia 14 9 1,382 5,546** 61, 909** 0,079*

Líneas/ familia 15 9 0,582 5,059** 38,031* 0, 179**

Líneas/ familia 16 9 0,811 2, 863 75,819** 0,070

Líneas/familia 17 9 0,299 2, 574 26,788 0,043

Líneas/familia 18 9 0,323 1,538 24, 559 0,067

Líneas/familia 19 9 0, 532 1,769 18,826 0,015

Error (b) 513 0,732 1,975 18,496 0,040

C.V. 6,22% 6,45% 5,89% 7,30%

416 TRUJILLO FIGUEROA y RIOS BETANCOURT

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a la vez más fija en com paración con los otros ca racteres que son más dependientes de la acción am biental. A l hacer un desglosam iento de las líneas dentro de cada fam ilia respecto a los va lo res m edios, se encuentran líneas con va lo res superiores a la m edia del testigo , aunque no s ign ifica tivos , para e l c a ra c te r peso de 100 sem illas (cuadro V ). Esto requ iere p osterio res estudios para d e fin ir claram ente estas d iferencias.

E l cuadro V I nos m uestra los componentes de la varianza genética para los ca rac te res considerados. D el m ism o se desprende que con propósitos de se lecc ión los ca rac te res longitud del ta llo principal y peso de 1 0 0 sem illas serían los más recom endables por e l m ayor número de fam ilias cuyas líneas presentan una m ayor varian za genética.

DISCUSION

Muchos de los estudios sobre variab ilidad inducida en ca rac te res cuantita tivos se han hecho sobre plantas de apariencia norm al, es d ec ir, sin lle ga r a considerar las varian tes o mutantes d rásticos de las plantas (G rego ry [2 ]; Gaul [4 ], Ehrenberg et al. [7 ]; F re y [14 ], entre o tros); sin em bargo, no se encuentran re feren c ias respecto a si las plantas s e le c cionadas, aparentem ente norm ales, provenían de descendencias de plantas o espigas M;t en las cuales no aparecieron mutantes d rásticos o tam bién se incluyeron plantas de entre las descendencias donde aparec ieron dichos mutantes, en cuyo caso quedarían m ezcladas. La u tilización de los mutantes d rásticos ha sido principalm ente en program as de cruzam ientos tratando de in troducir las ca ra c te r ís tica s deseables de los mutantes en m ateria les de d iferen te base genética, donde su expresión fenotíp ica puede s e r m ayor (H agberg [2 5 ]) o bien cuando en los mutantes drásticos se presentan además de un ca rá c te r deseable muchos otros indeseables, en cuyo caso mediante cruzam ientos pueden separarse en m ateria les con otra base genética (Hesem ann y Gaul [2 6 ]). P o r otra parte tam bién se re fie ren estudios r e s pecto a la variab ilidad genética inducida en ca rac te res cuantitativos para mutantes drásticos ta les como precocidad, enanismo, tamaños gigantes, hojas pequeñas, sem illas grandes, ta llos y hojas sin cera , hojas enrolladas, etc. (Gaul et al. [ 6 ] ) , encontrándose una m ayor variab ilidad para d iferentes ca racteres como rendim iento, fecha de flo rac ión por fam ilia y una m ayor dism inución de las m edias com parativam ente a los m ateria les provenientes de plantas aparentem ente norm ales. Muchos de estos m acromutantes pueden ser de in terés agronóm ico y s e r u tilizados d irectam ente, otros pueden u tiliza rse en program as de cruzam iento como se indica a rriba . A s í también o tros autores han seleccionado mutantes drásticos y estudiado su variab ilidad con propósitos de se lecc ión (Gaul y M ittelstenscheid [24 ] se leccionaron mutantes p recoces en cebada; Bhatia y Swaminathan [10] y B o ro jev ic [11] se lecc ionaron mutantes aristados en tr ig o ), encontrando m ejoras en e l rendim iento. En ‘muchos casos los mutantes drásticos como ta les no tienen im portancia agronóm ica ya que van unidos a otros ca racteres indeseables; así, por e jem plo , los mutantes com pactoides presentan un tamaño de paja muy bajo que será deseable para ev ita r e l acam e, aunque sus espigas son muy pequeñas; los mutantes de cabeza ancha presentan espigas muy densas, pero e l grano es muy pequeño o no se d esarro lla . La p resencia de estos ca racteres es, sin em bargo, un indicador de que se indujeron cambios genéticos mediante e l tratam iento mutagénico y que por lo tanto pueden

4 18 TRUJILLO FIGUEROA y RIOS BETAN COU RT

CUADRO VI. VA.RIANZA.S GENETICAS DE LAS L IN EAS DENTRO DE F A M IL IA S P A R A LOS C ARACTERES PESO DE 100 SEM ILLAS , LO NG ITU D DE E S P IG A /TA LLO P R IN C IP A L , NUMERO DE ESPIG U ILLAS/ESPIG A P R IN C IP A L Y LO NG ITU D D EL T A L L O P R IN C IP A L , E N M 4 .(TABLE VI. GENETIC VARIANCES OF LINES WITHIN FAMILIES FOR THE WEIGHT OF 100 SEEDS,SPIKE LENGTH/MAIN CULM, NUMBER OF SPIKELETS/MAIN SPIKE AND MAIN CULM LENGTH, IN M4)

Línea s/ F a m ilia sPeso 100

semillasLongitud

espiga

N ú m e r o es

piguillas

Long, tallo

principal

/ F 1 0,037 0,382 0,148 16,279

"ч to

0,030 0,325 0,451 9,541

/F 3 0,011 0,639 1,109 32,650

/F 4 0,062 0,275 0,376 16,767

/F 5 0,000 0,631 0,354 5,854

/ F 6 0,057 0,022 -0,004 33,742

/ f 7 0,040 0,236 1,311 2,884

/ f 8 0,008 0,009 0,548 5,378

/f 9 0,005 0jll3 - 0,270 3,832

/F 10 0,031 0,003 0,821 21,531

/ F ll 0,011 - 0,003 - 0,065 2,539

H-‘

to

0,018 0,468 0,555 3,191

/ F 13 0,002 0,315 0,529 0,662

/ F l4 0,009 0,162 0,893 10,853

/ F 150,034 -0,037 0,771 4,884

/F 160,008 0,019 0,222 14,331

/ F 170,000 -0,108 0,149 2,073

/f i8 0,006 -0,102 - 0,109 1,441

/F l9 - 0,006 -0,050 - 0,052 0,083

VARIABILIDAD IND UCID A POR EMS 4 1 9

presen tarse cambios también a n ive l cuantitativo. En nuestro estudio con plantas herm anas, aparentemente norm ales, de estos mutantes drásticos tam bién se observa variab ilidad genética inducida en los ca racteres cuantitativos. A s í las varianzas de los ca racteres estudiados en la M í;, longitud del ta llo principal, longitud, número de esp igu illas y densidad de la espiga p rincipal, número de espigas y rendim iento por planta, fueron generalm ente m ayores, m ientras que los va lo res medios fueron in fe r io res con respecto al testigo . Estos resultados son ca rac te rís ticos , en genera l, en las p rim eras generaciones después de un tratam iento mutagénico; as í nuestros resultados concuerdan con los obtenidos por S coss iro li [21 ], B rock [17 ], Gaul [4 ], B o ro jev ic [11, 12, 27], Abram s y F re y [15 ], T ru jillo [ i ] . E l aumento de la variab ilidad se atribuyó al efecto del agente mutagénico, pero la reducción de la media está indicando que dichos efectos se deben a la inducción de m icrom utaciones en sentido negativo (T ru jil lo [ l ] ), a s í com o a la presencia de factores leta les y subletales (S coss iro li [21 ]). B rock [1 7 ] atribuye la desviación de las medias en sentido con trario a la se lecc ión p rev ia , m ientras que Gaul y A astve it [28] form ulan la hipótesis de que los cambios de la media son independientes de los genotipos usados y que s iem pre tendrán una d ir e c ción asociada con la reducción de vita lidad. E l conocim iento de estas ca ra c te r ís tica s de las generaciones tempranas es de gran im portancia ya que para se lecc ion ar m ateria les con estas ca racterís ticas puede caerse en e l e r r o r de no considerar todos los ca racteres de rendim iento (B rock [17 ]), com o lo dem uestra B o ro jev ic [2 7 ], quién después de una se lecc ión para m ayor número de granos en la M 2 halló que e l am acollo resu ltó bajo en generaciones avanzadas.

Como consecuencia de esas ca racterís ticas de los m ateria les en tem pranas generaciones y debido a la acción del m edio ambiente, e l d esa rro llo de las plantas no solam ente entre las fam ilias sino dentro de las m ism as es poco uniform e, propiciando esto m ayor o m enor com petencia. Estos fa c to res genéticos am bientales que actúan en la segunda generación (M 2) no pueden, sin em bargo, separarse y en consecuencia una se lecc ión a este n ive l sobre ca racteres espec íficos del rendim iento o rendim iento global por planta ser ía in correc to . En nuestro estudio, aunque un gran número de fam ilias 12, 32, 24, 7, 20 y 40 presentaron m edias superiores con respecto a la m edia del testigo , para los ca racteres longitud del ta llo principal, número de espigas por planta, longitud de la espiga principal, número de esp igu illas de la espiga principal, rendim iento por planta y densidad de la esp iga principal, respectivam ente, no se consideró ninguna de ellas en particu la r para se lección , sino que se aumentó todo e l m ateria l hasta la cuarta generación (M 4).

Con e l propósito de no perder las ca racterís ticas del rendim iento al se lecc ion ar tempranamente sobre un ca rác te r espec ífico , se recom ienda que la se lecc ión a ese n ivel, la cual s iem pre se hace por razones prácticas, sea solam ente con e l propósito de se lecc ion ar variab ilidad genética inducida a base de indicadores (Gaul [2 3 ]) de variab ilidad inducida y que solamente en generaciones avanzadas se lleve a cabo una se lecc ión d irecta (Gaul y M itte lstenscheid [2 9 ] sobre ca racteres espec íficos del rendim iento.Gaul y M ittelstenscheid [24 ], Bhatia y Swaminatan [10 ], B o ro jev ic [12 ],L i et al. [30] se lecc ionaron basándose en un ca rác te r indicador para ren d im iento con buen éxito . T ru jillo [1 ] m uestra que la variab ilidad de la longitud de la esp iga es un buen ind icador para variab ilidad inducida en otros ca ra c te res del rendim iento. E l encuentra que las fam ilias seleccionadas bajo

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VARIABILIDAD IND UCID A POR EMS 421

m ayores v a r ia r la s de la longitud de la espiga (x 2 s de sus varian zas ), tam bién presentaban m ayores varianzas en otros ca racteres cuantitativos com parativam ente a las fam ilias del testigo y a las seleccionadas a lrededor de las m edias de las varianzas (x ) del testigo , pudiéndose encontrar en la M4 líneas con m ayores rendim ientos.

En la M 4 las varianzas de algunas fam ilias y líneas dentro de fam ilias para los ca racteres considerados: longitud de la espiga, número de esp igu illas de la espiga principal, longitud del ta llo p rincipa l y peso de 1 0 0

granos, presentaron m ayores varianzas (cuadro IV ) y las m edias, aunque m enores a la del testigo , fueron m ayores que en la M 2 .

En generaciones avanzadas se tiene por lo tanto una m ayor variab ilidad y a l m ism o tiem po una m ayor hom ocigocis para cada uno de los m ateria les y a la vez com o lo señala S coss iro li [21 ] ya se han elim inado por la autofecundación los fac tores le ta les y subletales teniéndose por lo tanto más éxito en la se lecc ión (Gaul [31 y 32 ], Abram s y F re y [15 ], S coss iro li et al.[33 ], Bhatia y Swaminathan [10 ], Gaul y M ittelstenscheid [24 ], G regory [2 ] ) .

E l ca rá c te r peso de 100 sem illas se tomó com o uno de los caracteresmás estab les genéticam ente (F re y [14 ] para peso de 100 sem illas , HSnsel[34] para peso de 1000 sem illa s ) en los cuales e l m edio ambiente tiene m enor e fecto que sobre los otros ca rac te res . En e l cuadro V se puede observar que las líneas 8 , 8 y 10 correspondientes a las fam ilias 4, 15 y 8 , re sp ec tiva mente, presentan va lo res m edios p o r encima de la media del testigo aunque no s ign ifica tivas .

Con la m ayor cantidad de sem illa de que se dispone ahora de esas líneas se podrán conducir nuevos ensayos de rendim iento, con m ayores p robab ilidades de com parar adecuadamente las d iferencias ya que se presentará un m e jo r d esa rro llo de los m a teria les .

EXPRESIO N DE AG RAD E C IM IE N TO

D eseo exp resar m i agradecim ien to al P ro f. D r. Gerhard RCbbelen, del Institut fü r Pflanzenbau und Pflanzenzüchtung, Universidad de Gotinga, p o r las sugerencias sobre e l tem a y por haberm e perm itido e l traslado de los m ateria les (G eneración M 4 ) a Chapingo, M éxico, y a l S erv ic io de In te rcam bio Académ ico Alem án, DAAD, por haberme otorgado una beca que p e rm itie ra re a liz a r m is estudios y parte de este trabajo en A lem ania.


[1] TRUJILLO, F.R., Indirekte Frühselektion auf índuzierte genetische Variabilitat in Ertragsmerkmalen nach EMS-Behandlung von Weizen, Z.PflZücht. 60(1968) 327.

[2] GREGORY, W.C., X-ray breeding of peanut (Arachis hypogaea L. ), Agron.J. 47 ( 1955) 396.[3] GAUL, H., "Use of induced mutants in seed-propagated species". Mutation and Plant Breeding,

NAS-NRC Publ. No. 891 (1961) 206.[4] GAUL, H., «The concept of macro-and micro-mutations and results on induced micro-inutations in

barley», The Use of Induced Mutations in Plant Breeding (Rep.FAO/IAEA Tech.Meeting, Rome, 1964), Pergamon Press, Oxford (1965) 407.

[5] GAUL, H., «Studies on populations of micro-mutants in barley and wheat without and with selection», Induzierte Mutationen und ihre Nutzung, Erwin-Baur-Gedà'chtnisvorlesungen IV, Gatersleben, 1966, Akademie-Verlag, Berlin (1967) 269.


[6] GAUL, H., BENDER, K., ULONSKA, E., SATO, M., «EMS-induced genetic Variability in barley ; the problem of EMS-induced sterility; and a method to increase the efficiency of EMS treatment», Mutations in Plant Breeding (Proc.Panel, Vienna, 1966), IAEA, Vienna (1966) 63.

[7] EHRENBERG, L., EKMAN, G., GUSTAFSSON, A., JANSSON, G., LUNDQVIST, U., «Variation in quantitative and biochemical characters in barley after mutagenic treatments'», The Use of Induced Mutations in Plant Breeding (Rep.FAO/IAEA Tech. Meeting. Rome, 1964), Pergamon Press, Oxford (1965) 477.

[8] BOUMA, J., «New variety of spring barley’ Diamant* in Czechoslovakia», Induzierte Mutationen und ihre Nutzung, Erwin-Baur-Gedachtnisvorlesungen IV, Gatersleben, 1966, Akademie-Verlag, Berlin (1967) 177.

[9] SCHOLZ, F., «Utility of induced mutants of barley in hybridization», Induzierte Mutationen und ihre Nutzung, Erwin-Baur-Gedáchtnisvorlesungen IV, Gatersleben, 1966, Akademie-Verlag, Berlin (1967) 161.

[10] BHATIA, C.R., SWAMINATHAN, M.S., Induced polygenic variability in bread wheat and its bearing on selection procedures, Z.PfIZücht. 48 (1962) 317.

[11] BOROJEVIC, Katarina, «Study of quantitative characters of bearded mutation in wheat induced by irradiation», Induzierte Mutationen und ihre Nutzung, Erwin-Baur Gedáchtnisvorlesungen IV, Gatersleben, 1966, Akademie-Verlag, Berlin (1967) 199.

[12] BOROJEVIC, Katarina, «Studies on radiation-induced mutations in quantitative characters of wheat (Triticum vulgare)» , Mutations in Plant Breeding ( Proc.Panel, Vienna, 1966), IAEA, Vienna (1966) 15.

[13] KRULL, C.F., FREY, K.J., Genetic variability in oats following hybridization and irradiation, Crop Sci. 1(1961) 141.

[14] FREY, K.J., «Mutation breeding for quantitative attributes». The Use of Induced Mutations in Plant Breeding (Rep. FAO/IAEA Tech, Meeting, Rome, 1964), Pergamon Press, Oxford (1965) 465.

[15] ABRAMS, R., FREY, K.J., «Variation in quantitative characters of oats (Avena sativa L.) after various mutagen treatments, Crop Sci. 4(1964) 163.

[16] BHATIA, C.R., VAN DER VEEN, J.H., «Two-way selection for EMS-induced micromutations in Arabidopsis thaliana (L.) Heynh» , The Use of Induced Mutations in Plant Breeding (Rep.FAO/IAEA Tech. Meeting, Rome, 1964), Pergamon Press, Oxford (1965) 497.

[17] BROCK, R.D., «Induced mutations affecting quantitative characters», The Use of Induced Mutations in Plant Breeding (Rep.FAO/tAEA Tech. Meeting, Rome, 1964), Pergamon Press, Oxford (1965) 451.

[18] RAWLINGS, S.O., HANWAY, D.G., GARDNER, C.O., Variation in quantitative characters of soybeans after seed irradiation, Agron.J. 50 (1958) 524.

[19] PAPA, K.E., WILLIAMS, J.H., HANWAY, D.G., Effectiveness of selection for quantitative characters in the third generation following irradiation of soybean seeds with X-rays and thermal neutrons. Crop Sci. 1(1961) 87.

[20] PORSCHE, W., «Results of X-irradiation in breeding of Lupinus albus» , Induzierte Mutationen und ihre Nutzung, Erwin-Baur-Gedáchtnisvorlesungen IV, Gatersleben, 1966, Akademie-Verlag, Berlin (1967)241.

[21] SCOSSIROLI, R.E., «Value of induced mutations for quantitative characters in plant breeding»,The Use of Induced Mutations in Plant Breeding (Rep.FAO/IAEA Tech. Meeting, Rome, 1964), Pergamon Press, Oxford (1965) 433.

[22] SCOSSIROLI, R.E., «Induction of mutations for quantitative characters», Induzierte Mutationen und ihre Nutzung, Erwin-Baur-Gedachtnisvorlesungen IV, Gatersleben, 1966, Akademie-Verlag, Berlin (1967) 283.

[23] GAUL, H., Stand der Mutationsforschung und ihre Bedeutung fur die praktische Pflanzenziichtung, Arb. dt.LandwGes. 44(1956) 54.

[24] GAUL, H., MITTELSTENSCHEID, L., Untersuchungen zur Selektion von Kleinmutationen bei Gerste, Z.PflZiicht. 45(1961) 300.

[25] HAGBERG, A., «The use of induced mutations in practical barley breeding at Svalôf», Induzierte Mutationen und ihre Nutzung, Erwin-Baur-Gedachtnisvorlesungen IV, Gatersleben, 1966, Akademie- Verlag, Berlin (1967) 147.

[26] HESEMANN, C.V., GAUL, H., Ziichterische Bedeutung von Grossmutationen. П .. Beispiel fur die unabhángige Variation von Teilmerkmalen einer Sommergersten-Mutante im veranderten genetischen Hintergrund, Z.PflZiicht. 58 (1967) 1.

[27] BOROJEVIC?, Katarina, «The effect of irradiation and selection after irradiation on the number of kernels per spike in wheat», The Use of Induced Mutations in Plant Breeding (Rep.FAO/IAEA Tech. Meeting, Rome, 1964), Pergamon Press, Oxford (1965) 505.

[28] GAUL, H., AASTVEIT, K., Induced variability of culm length in different genotypes of hexaploid wheat following X-irradiation and EMS-treatment, 5th Jugoslav Symp.Res.Wheat(1966) 263.

VARIABILIDAD IND UCID A POR EMS 423

[29] GAUL, H., MITTELSTENSCHEID, L., Hinweise zur Herstellung von Mutationen durch ionisierendeStrahlen ín der Pflanzenzüchtung, Z.PflZücht. 43(1960) 404.

[30] LI, H.W., HU. C.H., CHANG, W.T., WENG, T.S., «The utilization of X-radiation for riceimprovement», Effects of Ionizing Radiations on Seeds (Proc. Conf. Karlsruhe, 1960), IAEA, Vienna(1961)485.

[31] GAUL, H., Present aspect of induced mutations in plant breeding, Euphytica 7 (1958) 275.[32] GAUL, H., Zûchterische Bedeutung von Kleinmutationen. I. Durch Rôntgenstrahlen induzierte Variabilitat

von Kornertrag, Korngrosse und Vegetationslânge bei der Gerste Haisa II, Z.PflZücht. J55 (1966) 1.[33] SCOSSIROLI, R.E., PALENZ.ONA, D.L., SCOSSIROLI-PELLEGRINI, S., «Studies on the induction of new

genetic variability for quantitative traits by seed irradiation and on its use for wheat improvement», Mutations in Plant Breeding (Proc. Panel, Vienna, 1966), IAEA, Vienna (1966) 197.

[34] HANSEL, H., Physiologie der Ertragsbildung und die Züchtung auf Ertrag bei Getreide, Z.PflZücht, 54 2 (1965) 97.

D I S C U S S I O N

M .S . SW AM INATH AN : Your data on the mean length o f the main t i l le rin the M 2 and M 4 generations revea l v e ry wide d iffe ren ces . W ere there any d ifferences e ith er in the grow ing conditions o r seasonal ch aracteris tics during the yea rs when the M2 and M 4 populations w ere ra ised? I presum e the so il fe r t ility and irr iga tion conditions must have been identical.

R. TR U J ILLO FIG U ERO A: As I said at the beginning o f my paper, theM 2 was ra ised at the E xperim ental Station o f the Agronom y and P lant B reed ing Institute, Gottingen, F ed era l Republic of Germ any, and the M4 at the E x p e r im ental F ie ld of the Escuela Nacional de Agricu ltura, Chapingo, M exico . So the c lim atic conditions w ere v e ry d ifferen t. The d ifferences in each eco log ica l m edia fo r the character main spike length w ere increased e ith er in the trea ted m a ter ia l o r in the control.

MUTACIONES INDUCIDAS Y PROGRAMA DE MEJORAMIENTO DEL TRIGO EN CHILE

I. RAMIREZ ARA Y A , C . SANZ DE CO RTAZAR

Instituto de Investigaciones Agropecuarias,

Santiago, C h ile

C .F . K O NZAK


Pullman, Wash., Estados Unidos de Am erica

Abstract-Resumen

INDUCED MUTATIONS AND THE WHEAT IMPROVEMENT PROGRAM IN CHILE.The objectives of the wheat improvement program are the development of better varieties for the different

zones of Chile, having high yields, disease resistance, and adaptation to high fertilizer levels. Various mutation projects have been carried out so far. In 1958, seeds of 10 varieties belonging to 7 species were irradiated at the Brookhaven National Laboratory. The objectives were mainly to induce stem rust resistance in spring wheat, earliness in Phaseolus vulgaris, resistance against seed shattering in Phalaris tuberosa, better storage quality in Dactylis glomerata, longer persistance in Trifolium pratense and resistance against Coryne bacterium insidiosum in Medicago sativa. However, no useful mutants were obtained from that program. Further mutation experiments with Zea mays, bread wheat and durum wheat using gamma irradiation were initiated in 1962/63, but the expected disease resistant mutants have not been found. From an experiment with various chemical mutagens on spring wheat, three mutants were selected with resistance to race 15B of Puccinia graminis. At present further experiments are being carried out with bread wheat and durum wheat using EMS and dES to obtain mutants more-resistant to P.graminis.

MUTACIONES INDUCIDAS Г PROGRAMA DE MEJORAMIENTO DEL TRIGO EN CHILE.Los objetivos del programa de mejoramiento de trigo son la formación de variedades mejoradas para cada

zona de cultivo en Chile, que posean elevado rendimiento, resistencia a las enfermedades, y adaptación a la fertilización intensiva. Hasta la fecha se han ejecutado varios proyectos en materia de mutaciones. En 1958, en el Laboratorio Nacional de Brookhaven se irradiaron semillas de 10 variedades pertenecientes a 7 especies.Los objetivos eran, en especial, inducir la resistencia a la roya del tallo, un período vegetativo más corto en Phaseolus vulgaris, la resistencia al desgrane en Phalaris tuberosa, un mayor valor forrajero en Dactylis glomerata, una mayor duración de la pradera en Trifolium pratense y resistencia a la bacteriosis Coryne bacterium insidiosum en Medicago sativa. No se obtuvieron mutantes utiles con este programa. En 1962-63, se iniciaron nuevos experimentos sobre mutaciones por irradiación gamma de Zea mays, trigo de pan y tritio durum, pero no pudieron conseguirse mutantes resistentes a las enfermedades. Como resultado de un experimento con distintos mutágenos químicos sobre trigo de primavera, se seleccionaron tres mutantes resistentes a la raza 15 В de Puccinia graminis. Actualmente, se efectúan nuevos experimentos con trigo de pan y trigo durum utilizando metanosulfonato de etilo y sulfato dietílico a fin de obtener mutantes más resistentes a P.graminis.

IM PO R TA N C IA Y C AR AC TE R IST IC AS G ENERALES D E L C U LT IV O

E l tr ig o ocupa a lred edor de un 60% de la superfic ie que cada año se destina a los cu ltivos en Chile.

E l v a lo r de su producción es cercano al 15% de la producción agríco la chilena, incluyendo cu ltivos y ganadería, y exceptuando los productos fo res ta les . E ste c e rea l proporciona e l 40% de las ca lo rías y más del 15% de las proteínas que consume e l chileno medio (cuadros I y II [1, 2] ).

4 2 5

4 26 RAMIREZ ARA Y A e t a l .

CUADRO I. SU PE R FIC IE C U LT IV A D A , REN D IM IEN TO PROM EDIO POR H E C TA R E A Y PRODUCCION DE TR IG O E N SECANO Y R IEG O 3

Superficie Rendimiento Producción(ha) (kgM) (ton)

Secano 536 900 1310 701 200

Riego 190 000 2180 414 600

Total 726 000 1540 1 115 800

a Fuente: IV Censo Nacional Agropecuario, Dirección de Estadísticas y Censo, Tomo 1, 1968, Santiago, Chile. Datos del año agrícola 1965.

CUADRO II. D AM AND A G LO B A L DE TR IG O EN C H ILE 3

Demanda, en miles de toneladas

Consumo humano, total 1203, 7

Consumo per cápita^ 137,0

Alimentos para ganado 71, 5

Semillas0 108, 2

Otros usos y desperdicios 37,9

Total 1403,4

a Fuente: «Programa de producción de trigo», ODEPA, Ministerio de Agricultura, Santiago, Chile (en mimeógrafo, no publicado) 1970. Datos del año agrícola 1965.

En kg por habitante. c Estimados en base a una dosis de 160 kg/ha.

Hasta e l año 1940 Chile producía tr ig o suficiente como para tener un pequeño saldo exportab le. Desde 1941 en adelante e l dé fic it de la producción tr igu era ha ido aumentando. En la actualidad es n ecesario im portar anualmente cerca del 15% de las necesidades de consumo, es d ec ir , a lrededor de 300 000 toneladas [1 ].

En e l país se cu ltiva tr ig o de pan ( T riticum aestivum L . em . T e ll ) en m ayor proporción que tr ig o «can d ea l» o durum ( Triticum durum Desf. ) (cuadro III ). Se em plean variedades de hábito de crec im ien to p r im avera l e inverna les . Recientem ente se han entregado al cultivo algunas variedades de hábito de crecim ien to in term ed io o a lternativas. Las variedades de tr igo durum son todas de hábito p r im avera l (cuadro III).

Con respecto al cu ltivo de este ce rea l, en e l país pueden d istinguirse tres reg iones. La Zona N orte , com prendida entre las provincias de Tarapacá y Curicó, contiene la casi totalidad de los suelos regados en que se cultiva e l tr ig o . La p lu viom etría es escasa — 200 a 600 mm — siendo casi in existente en e l ex trem o norte de esta región . Se cultivan sólo variedades de p rim avera y la totalidad del tr ig o durum se produce aquí (cuadro IV ).

MEJORAMIENTO DEL TRIGO EN CHILE 427

CUADRO III. TR IG O DE P A N Y durum: SU PERFIC IE C U LT IV A D A , RE N D IM IE N TO PROM EDIO POR H E C TAR E A Y PR O D U CCIO N 3

Superficie(ha)

Rendimiento(kg/ha)

Producción(ton)

Trigo de pan 681 800 1510 1028 700

Trigo durum 45100 1930 87100

a Fuente: IV Censo Nacional Agropecuario, Dirección de Estadística y Censo, Tomo 1, 1968, Santiago, Chile. Datos del año agrícola 1965.

CUADRO IV . SU PE R FIC IE SEM BRADA, PRODUCCION, REN D IM IEN TO PROM EDIO Y PO RC EN TAJES D E L T O T A L , CORRESPONDIENTES A LAS TRES ZONAS TR IG UERAS D EL P A IS 3

Zona ProvinciasSuperficiesembrada

(ha)

Producción(qqm)b

Rendimientopromedio(kg/ha)

Norte Tarapacá a Curicó

180 0 59 (24, 5%

3450 248 (30, 8%)

1660

Centro-Sur Talca a Bío-Bío

265435 (36, 2°¡o)

3333054 (29, 8%)

1180

Sur Malleco a Magallanes

288 544 (39,3%)

4415 885 (39,4%)

1720

a Fuente: IV Censo Nacional Agropecuario, Dirección de Estadística y Censo, Tomo 1, 1968, Santiago, Chile. Datos del año agrícola 1965.

° 1 qqm = 100 kg.

La reg ión situada entre las p rovincias de Ta lca y B ío -B ío se ha denominado Centro-Sur o In term edia. En esta zona, si se la com para con la Zona Norte, se advierte un aumento de la p luviom etría , la que va ría entre 8 00 y 1300 mm anuales. E l tr ig o se cu ltiva en secano y en riego , pero en este ú ltim o caso se trata só lo de r iego suplem entario pues por lo genera l, en años de condiciones c lim áticas norm ales, las p recip itaciones proporcionan la m ayor parte del agua necesaria a l cultivo. Las variedades em pleadas en esta reg ión son tr igos inverna les típ icos de la Zona Sur o bien trigos de p rim avera de la reg ión N orte . Como es lóg ico suponer, estas variedades no rinden en estas p rovincias interm edias como en aquellas para las cuales están más específicam en te adaptadas. Se ha estim ado necesario fo rm ar variedades m ejoradas o in troducir nuevos cu ltivares m ejor adaptados a las ca ra c te r ís tica s de la zona [3] .

4 2 8 RAMIREZ ARA Y A e t a l .

La Zona Sur comprende la región situada entre las provincias de M alleco y M agallanes. E l tr ig o se cu ltiva exclusivam ente de secano, ocasionalm ente con r iego suplem entario. La p luviom etría de esta zona alcanza 800 a 2000 y más mm anuales, es decir, hay humedad más que suficiente para suplir las necesidades del cu ltivo de tr igo . Las variedades predom inantes son de hábito invernal, aunque en los últim os diez años se ha extendido e l em pleo de variedades p r im avera les e in term edias, respondiendo a necesidades de rotación y uso más intensivo de la t ie r ra con cu ltivos como rem olacha azu carera [4] .

Si se consideran las condiciones am bientales para e l cu ltivo del tr ig o en e l país, es posib le conclu ir que Chile tiene condiciones excepcionales para e l cu ltivo intensivo de este ce rea l. Más del 85% de la su perfic ie cu ltivable con tr ig o rec ibe suficiente cantidad de lluvia, o tiene r iego . Sin em bargo, los rendim ientos unitarios del país son bajos. Esto se debe, entre otros factores , a que se cu ltiva tr ig o en zonas m arginales, como en algunas áreas del Norte del país en que la escasa p luviom etría es e l fac to r lim itante para los terren os de secano, o en partes de las zonas Centro Sur y Sur donde la baja calidad de los suelos conduce a rendim ientos muy pobres.

PR O B LE M AS Y OBJETIVOS EN M A TE R IA DE M EJO RAM IENTO

En genera l, se considera que para lo g ra r e l m ejoram iento de la p ro ducción nacional de tr ig o es n ecesario preocuparse de aspectos fundamentales com o investigación , extensión, industria lización y com erc ia lizac ión . En este trabajo sólo nos ocuparem os de los prob lem as relacionados con la investigación .

La institución que tiene a su cargo las investigaciones en m ateria de m ejoram ien to del tr ig o es e l Instituto de Investigaciones Agropecuarias, dependiente del M in is ter io de Agricu ltu ra . Hay algunas organ izaciones privadas que rea lizan investigaciones en m ateria de m ejoram iento de v a r ie dades, com o la Sociedad Nacional de Agricu ltu ra , y otras que se dedican a la producción de sem illa genética y certificada , pero la extensión de estos traba jos es lim itada.

E l program a de m ejoram ien to de ce rea les pequeños del Instituto tiene com o ob jetivos r e a liz a r investigaciones sobre los siguientes aspectos:1. F o rm ac ión de variedades m ejoradas para cada zona, de altos rend i

m ientos, res is ten tes a las enferm edades, con buena res is ten c ia a la tendidura y adaptadas a las condiciones de alta fe rtilid ad de una a g r icultura intensiva.

2. D eterm inación de las dosis óptimas de abonaduras económ icas para e l em pleo de las variedades m ejoradas e introducidas de buena adaptación.

3. Métodos de con tro l de m alezas.4. P rob lem as fitopato lóg icos del tr ig o .5. P rob lem as entom ológicos del cu ltivo y granos alm acenados.6 . P reparac ión de suelos y rotaciones.7. P rob lem as de r iego en ce rea les .

No se ha logrado abordar todos estos aspectos con la m ism a intensidad. Se han rea lizado investigaciones, principalm ente en la form ación de v a r ie dades m ejoradas en re lac ión con su res istenc ia a las royas. Algunos de los prob lem as que deben considerarse para la introducción y m ejoram ien to del m a ter ia l genético en Chile se resum en brevem ente a continuación.


PR O B LE M AS F ITO PATO LO G IC O S

Entre las en ferm edades de m ayor im portancia económ ica y que afectan seriam ente los rendim ientos del tr ig o en e l país se encuentran los «p o lv il lo s » , o royas: Puccin ia gram in is P e r s . f . sp. t r it ic i E ricks y Henn. , «p o lv il lo dela cañ a »; Puccin ia recóndita f. sp. t r it ic i Rob, e x D e s m ., «p o lv il lo de la h o ja »; y Puccin ia s tr iifo rm is W est, «p o lv il lo e s tr ia d o » [5 ]. Tam bién son im portantes los patógenos que causan la putrefacción de la ra íz y e l tallo, como Linocarpum ca r ic e t i (B .y B. ) Petrack , «m a l del p ie » y hongos del género Fusarium causantes de la fu sarios is . L es siguen en im portancia dos especies del género U stilag ina les, T ille t ia ca r ies (P C ) Tu l. y U stilago t r it ic i (P e r s . ) R o s t r . , causantes del «carbón hediondo» y «carbón v o la d o r » respectivam ente. E spec ies fungosas como Septoria t r it ic i Rob. y Septoria modorum Berk , que orig ina la isep torios is de la hoja y del nudo, resp ec tiva mente, son de m enor im portancia económ ica, como tam bién E rysiphe gram in is t r it ic i E . M archai, causante del oíd io [6 ].

PR O B LE M AS ENTOM OLOGICOS

Entre los insectos que producen daños de c ie rta im portancia económ ica se encuentra H ilam orpha elegans B u rn ., cuyas la rvas atacan ra íces y ta llos , Leucania unipuncta Haw. y L . inpuncta Haw., «cu ncu n illas» que atacan las hojas y esp igas. Ocasionalm ente causan daños de alguna consideración los «gusanos a lam b re » de la famili-a E la teridae y la rva s de T ipu la sp p ., que atacan e l cuello y r a íc e s 1. Recientem ente (1968) ha aparecido un áfido, Metapolophium dirhodum W . , que afecta severam ente a las sem enteras entre los períodos de encañado y grano lechoso. No se ha determ inado, todavía, su im portancia económ ica pero se estim a que puede ser un problem a serio . En tre los insectos que m ayores pérdidas económ icas ocasionan en granos almacenados se han determ inado, en orden de im portancia; Sitophilus o ryza (L ) , «g o rg o jo del a r r o z » ; S. granarius (L ) , «g o rg o jo del t r ig o » ; Rhysoperta dom inica (F), «pequeño b a rren ad o r»; y S itotroga c e rea le lla (O liv . ), «p o lilla de los c e r e a le s » [6 ] .

PR O B LE M AS E N C ALID AD M O LIN ERA Y PAN AD E R A

Hasta hace poco, las variedades que se cultivaban com ercial.m ente eran de in fe r io r calidad industria l en com paración con e l tr ig o que se im portaba para sup lir las necesidades del consumo. En cuanto a las propiedades nutritivas del tr igo , expresadas desde e l punto de v is ta de su contenido p rote ico , las variedades com erc ia les eran defic ien tes. En genera l los ni-veles de proteína observados fluctuaban entre e l 8 y 9%. En los ú ltim os 8 años se han entregado a los agricu lto res variedades m ejoradas cuyo contenido es bastante más alto, entre 11 y 14%. E l program a de m ejoram iento considera que las nuevas variedades deben poseer además del potencial genético para alto rendim iento, una calidad m olinera y panadera ajustada a las necesidades de la industria nacional y un contenido p ro te ico que perm ita e le v a r e l n ive l nutricional de los productos elaborados con tr igo .

1 Comunicación personal de C. Caballero V. y H. Dell*Orto T., Instituto de Investigaciones Agropecuarias.

4 30 RAMIREZ ARA Y A e t a l .

En tr ig o durum e l blanqueamiento del grano (ye llow b erry ) es un factor que lim ita geográ ficam ente e l cultivo de durum a la Zona N orte. Como ex iste demanda crec ien te de este tipo de grano, hay in terés en extender su cu ltivo a las zonas Sur y Centro-Sur, pero no se dispone hasta hoy día de germ oplasm a adaptado que pueda producir grano de la calidad requerida.

E M P L E O DE LAS M UTACIONES INDUCIDAS EN E L M EJO RAM IENTO D E L TR IG O E N CHILE

L a u tilizac ión de irrad iac iones y agentes mutagénicos quím icos en los program as de m ejoram ien to vegeta l en nuestro país ha sido bastante lim itada y de rec ien te data. Hasta ahora, en genera l, han habido sólo intentos a is lados, no s istem áticos, para tra tar de inducir variab ilidad genética u tilizab le en e l m ejoram ien to de algunas especies de in terés agríco la . Con los p ro ce dim ientos em pleados no se han obtenido form as mutantes de utilidad práctica o que hayan dado, origen a nuevas variedades cultivadas.

Uso de irrad iac iones

En 1958 e l M in is ter io de Agricu ltura, mediante la colaboración del Labora to rio Nacional de Brookhaven, envió sem illa de 10 variedades de7 espec ies d iferen tes para que fuese irrad iada . Los ob jetivos de este trabajo eran los siguientes:- Obtener res is ten c ia a la roya del ta llo , P . gram in is en las variedades de

tr ig o de p rim avera M enflo, V ilu fén y Capelli.- P eríodo vegeta tivo más corto en Phaseolus vu lgaris L . cult. Coscorrón .- R es istenc ia a l desgrane en Phalaris tuberosa L . cult. Is ra e l P. F . 1580.- M ayor va lo r fo r ra je ro en D actylis g lom erata L . cult. Is ra e l P. F . 1580 y

Centifén P . F . 1571.- M ayor duración de la pradera en T rifo liu m pratense, cu lt. C orrien te de

Teño.- R es is ten cia a la b acterios is , Coryne bacterium insidiosum (M e C all) H L.

Jens. en M edicago sativa L . , cult. Peruana P . F . 28 08 .Las dosis em pleadas com o tratam ientos con rayos X y neutrones t e r

m ales fueron las siguientes:

Phaseolus sp :

T riticu m sp. :

Ph a la ris sp. ;

D actylis sp. :

M ed icago sp. :

T r ifo liu m sp.:

5, 10, 15*, 20 kR

20*, 25 kR

20*, 25, 30, 40 kR

20*, 30 kR

50, 60*, 70, 80 kR

30*, 40 kR

4, 6 , 8 10, 12 h n .t

4, 5, 5. 5 h n .t

10*, 15, 20, 25 h n. t

10, 15, 20*, 25, 30 h n .t

10, 15, 20*, 25, 30 h n .t

25*, 30 h n. t

E l m ateria l irrad iado fue sembrado en form a aislada en 1959. De acuerdo a las observaciones rea lizadas, las dosis m arcadas con un asterisco fueron las más e fectivas en la producción de form as mutantes. De este m a ter ia l no se obtuvieron mutantes para u tilizac ión en los program as de m ejoram ien to respectivos .

MEJORAMIENTO DEL TRIGO EN CHILE 431/

En 1962 se efectuó en Santiago la exposición «A to m o s en A c c ió n », de la Com isión Atom os para la Paz. A l l í se irrad ia ron sem illas de m aíz, variedades C hoclero, Cam elia y Eureka con dosis de 5 a 25 kR. Se observó en R2 un aumento de la frecuencia de mutaciones en ta llos, hojas y órganos flo ra le s , de acuerdo con e l aumento de la dosis.

En 1963 se estab lece una fuente de 60Co en la Escuela de Ingen iería de la U n iversidad de Chile. Se irrad ia a llí ese año sem illa de las variedades de tr ig o de pan Cappelle D esprez y durum Candealfén, con dosis de 15 y 20 kR. E l ob jetivo era obtener plantas de baja estatura y res is ten c ia a P . g ra m in is . En se lecciones p os te r io res de este m ateria l no se consigu ieron las form as mutantes deseadas.

E M P L E O DE M UTAGENOS QUIMICOS

En 1957 la sección de C ito log ía del Departamento de Investigaciones A g r íc o la s , M in is ter io de Agricu ltu ra , com ienza a probar algunos mutágenos qu ím icos. Se tra taron d iferen tes especies vegeta les de in terés a g r íco la con compuestos como form aldehido, hidrato de d o r a l y colch icina.

En 1967 se em plea e t il metano sulfonato (EM S), etilen im ina ( E l ) e h idracida m ale ica (H M ), además de colchicina [7] para tra ta r de inducir res is ten c ia a P . gram in is en la variedad de tr ig o de p rim avera Orofén . Los tratam ientos fueron: colchicina, en emulsión con lanolina, al 2 %, sobreco leop tilos de 2 a 5 mm de d esarro llo ; E l en solución acuosa a l 0,03%; EMS en solución acuosa al 0,5%; y HM en solución acuosa a l 0,012%. Estos tres ú ltim os tratam ientos se h ic ieron a la sem illa sin h idratarla previam ente, a tem peratura de 27°C. Después de tratada, se lavó la sem illa en agua destilada por 30 y 60 min; la sem illa tratada y las generaciones M¡ y M2 se sem braron en invernadero. Las plántulas M 2 se probaron con un biotipo de la ra za 15B viru len to en Orofén. Se obtuvieron dos plantas res is ten tes en e l m a teria l derivado de los tratam ientos con colchicina y una de aquel tratado con E l. No se observó inducción de mutantes res isten tes en los tratam ientos con EMS o HM. Debe hacerse notar que e l volumen de m ateria l tratado fue bastante reducido y será necesario v e r if ic a r estos resultados usando poblaciones más numerosas y condiciones de tratam iento más p rec isas .

Recientem ente se ha em pleado EMS y sulfato d ie tílico (DS) para tra ta r de obtener mutantes res is ten tes a P . gram in is en la variedad invernal de tr ig o de pan Capelle D esprez y en la variedad durum Candealfén 5. Los tratam ientos em pleados fueron 0,02 M EMS durante 24 h a 20°C y 0,01 M DS durante 2 h a 20°C en la variedad Cappelle D esp rez. La variedad Candealfén 5 se som etió a 0, 2 M EMS y 0,012 ]V[ DS durante 2 h a 20°C.

La sem illa tratada proven ía del stock Fundación, de alta pureza va rie ta l. Fue cosechada en Enero 1970 y tratada en Mayo 1970, habiéndose probado previam ente su poder germ inativo y uniformado e l contenido de humedad a 14% mediante vac ío pa rc ia l bajo campana de v id r io sobre solución de g ly c e ro l a l 60%. Lotes de 8000 sem illas de tamaño uniform e se rem ojaron , antes del tratam iento, en agua destilada a 0°C durante 16 h. Pa ra todos los tra ta m ientos se mantuvo un pH 8 a 9. Después de tratada, la sem illa se lavó en chorro continuo de agua corrien te a 25°C, por 6 h2. Posterio rm en te se mantuvo en re fr ig e ra c ió n a 2-3°C durante la noche, y se sem bró al día sigu ien te .

2 Comunicación personal del Dr. C.F. Konzak.

432 RAMIREZ ARA Y A e t a l .

E l m ateria l tratado se ha sembrado con la a is lac ión adecuada en la Estación Experim en ta l Cen tra l La P latina, de la zona N orte , y en la Estación Experim en ta l Quilamapu en la zona Centro-Sur. E l ob jetivo es obtener mutantes de Cappelle D esprez que sean res isten tes a la roya de la caña y en consecuencia am p liar e l area de cultivo de esta variedad a toda la región cen tra l de Chile. Esta variedad tiene excelen tes rendim ientos en las regiones Centro-Sur y Sur pero no puede cu ltivarse en la región Norte por su extrem a susceptibilidad a P . g ra m in is . En la variedad Candealfén 5 se persigue obtener plantas de baja estatura y ta llos cortos y fuertes con res is tenc ia a P . g ra m in is . Esta variedad du rum es de buen rendim iento y produce grano de gran calidad con muy bajo porcentaje de blanqueamiento.


[1] Plan de Desarrollo Agropecuario 1965-1980, Ministerio de Agricultura, Oficina de Planificación Agrícola, Imp.Camilo Henríquez Ltda., Santiago, Chile(1968) 30-34.

[2] Programa de Producción de Trigo, Ministerio de Agricultura, ODEPA (en mimeógrafo, no publicado), Santiago, Chile (1970) 1-6.

[3] RAMIREZ, I., AGUAYO, L., Etoile de Choisy trigo de buena adaptación para la zona Centro-Sur, Agrie, y Gan. 1X31 (1965) 25-28.

[4] Guía de Producción de Trigo para la Zona Sur, Circular Informativa N*22, Estación Experimental Carillanca, Instituto Йе Investigaciones Agropecuarias, Ministerio de Agricultura, Santiago, Chile(1969) 62.

[5] PARODI, P., Nombres comunes para los polvillos del t r i g o : Puccinia graminis f.sp. tritici Eriks. y Henn. Puccinia recóndita f.sp.tritici Rob.ex Desm, ; Puccinia striiformis West, Agrie, técn. 25(1965) 175.

[6] Informe Quinquenal 1964-1969, Instituto de Investigaciones Agropecuarias, Ministerio de Agricultura, Santiago, Chile (1970).

[7] SANZ, С., HACKE, E., Resistencia al polvillo de la caña (Puccinia graminis f.sp. tritici Eriks. y Henn.) inducida por mutagénicos en trigo (Triticum aestivum L. em.Tell), variedad Orofén, Agrie.técn- 30(1970) 27-30.

D I S C U S S I O N

M .S . SW AM INATH AN: Can you g ive m ore in form ation on the virusdisease you a re finding on wheat? Is the d isease confined to any particu lar va r ie ty o r is it o f genera l occurrence?

I. R A M IR E Z A R A Y A : As I said be fo re , this is a v e ry recent problemin Ch ile. Though we have had in the last three yea rs a w idespread in festation with aphids throughout the wheat-grow ing areas, the presence o f BYD virus d isease symptoms has not been observed yet to be genera lized . We are exam ining a ll the germ plasm availab le in our Wheat Im provem ent P rogram . W e know there is genetic resistance in other species like oats and barley, and a lso in wheat. S evera l "in d ica tor v a r ie t ie s " w ere introduced in 1970 to check the poss ib ility o f BYD virus being present and its transm ission by aphids in Chile, but we have not yet determ ined the res istance o r suscep tib ility o f any particu lar va rie ty .

E . A . OSORIO: I should like to in form you that we have now dem onstratedthe existence o f the b a r ley ye llow dw arf v iru s in B ra z il. We have shown its harm ful e ffec ts and have isolated some foc i o f resistance.


Katarina BOROJEVIC: Do you plan to work on y ie ld components o fwheat such as number and weight o f kernels, and lodging resistance?

I. R A M IR E Z A R A Y A : Yes, we have a lready in itiated a y ie ld componentsstudy using se ve ra l h igh -y ie ld ing va r ie tie s which d iffe r in straw -height, number o f t i l le r s per plant, spike length and kernel s ize and weight, as w e ll as in industria l quality (m illin g and baking ch a ra c te r is tics ). These va r ie tie s have been entered in a d ia lle l-c ro s s in g scheme where it should be possib le to obtain in form ation on F j heterosis , combining ab ility both GCA. and SCA (G r iff in 1 s methods), and gene action (through Haym an 's analysis) fo r any and a ll o f the characters mentioned above, besides investigations on the inheritance o f these genetic tra its .

EL MEJORAMIENTO GENETICO DEL TRIGO EN EL BRASIL Y LAS POSIBILIDADES DE UTILIZACION DE MUTACIONES INDUCIDAS

E.A. OSORIO

Facultad de Agronomía Eliseu Maciel,

Universidad Federal de Pelotas,

Rio Grande do Sul, Brasil

Abstract-Resumen

GENETIC IMPROVEMENT OF WHEAT IN BRAZIL AND WAYS OF USING INDUCED MUTATIONS.Because its internal production is inadequate, Brazil has become a wheat importer and approximately

10°Jo of its foreign exchange has to be spent for this purpose. The low crop yield, on average below 900 kg/ha, is due to climatic conditions unfavourable to this crop and also to the insufficient fertility of the soil.

The Brazilian program on wheat improvement has led to the production of varieties which, by reason of their increased resistance to diseases and their adaptation to the conditions of low-grade, acidic soils with high percentages of aluminium, have stabilized the average productivity of wheat crops and have enabled the area under cultivation to be increased. The methods of genetic improvement used were the traditional ones of hybridization and selection.

Induced mutations were used as a method of improvement in 1960 and again in 1968 and 1969, and favourable mutations were obtained. This method, which originally was not considered very advisable given the rather undeveloped status of the Brazilian improvement program, should now be encouraged because the traditional methods of hybridization and selection are being used to a sufficient degree.

EL MEJORAMIENTO GENETICO DEL TRIGO EN EL BRASIL Y LAS POSIBILIDADES DE UTILIZACION DE MUTACIONES INDUCIDAS.

La insuficiente producción interna transformo al Brasil en un importador de trigo, ocasionando gastos aproximados del 10°Jo de sus divisas. El bajo rendimiento de los cultivos, en promedio inferior a los 900 kg/ha, se deben a las condiciones climáticas desfavorables a este cultivo y, por otro lado, a la deficiente fertilidad de los suelos.

El programa brasileño de mejoramiento del trigo creó variedades que, por su acentuada resistencia a las enfermedades y adaptación a las condiciones de suelos pobres, ácidos y con altos porcentajes de aluminio, estabilizaron la productividad media deloscultivos trigueros, permitiendo un aumento del área cultivada. Los métodos de mejoramiento genético usados fueron los tradicionales de hibridación y selección.

La inducción de mutaciones fué utilizada en el mejoramiento en 1960 y nuevamente en 1968 y 1969, con la obtención de mutantes ventajosas. Este método, anteriormente poco aconsejado por la situación poco desarrollada del programa brasileño de mejoramiento, debe ser actualmente estimulado puesto que los métodos tradicionales de hibridación y selección están siendo suficientemente utilizados.

1. S ITU AC IO N DE L A PRODUCCION TR IG U E R A E N E L B R A S IL

L a producción tr igu era b ras ileña es insu ficiente para atender e l consumo interno. P o r esta razón este ce rea l está siendo im portado anualmente en cantidades crec ien tes (cuadro I).

E l tr ig o es e l único alim ento im portado en gran esca la , disputando anualmente con e l p e tró leo la incóm oda posición de p rincipa l producto adquirido en e l ex te r io r .

No se ju stifica que un país considerado com o fundamentalmente agríco la , con m ás de 8 , 5 m illones de km2, continúe gastando aproxim adamente 1 0 % de sus d iv isas importando tr igo .

43 5

436 OSORIO

CUADRO I. CANTIDADES Y VALO RES DE LAS IM PO RTAC IO N ES B R AS ILE Ñ AS DE TR IG O Y PE TR O LE O (1965 - 1968)

AnosCantidad de

trigo importado (1000 t)

Valor de la importación (US$1000 000)

Trigo Petróleo

1965 1876 136 156

1966 2381 168 165

1967 2429 178 172

1968 2614 181 199

Fuente: Serviço de Estatistica Económica e Financeira do Ministério da Fazenda, Brasil.

CUADRO П. AR E A , PRODUCCION T O T A L Y REND IM IENTO MEDIO D E L C U LT IV O DE TR IG O (1962 - 1969)

AñosArea

cultivada (1000 ha)

Producción total

(1000 t)

Rendimientomedio(kg/ha)

1962 258 303 1175

1963 302 116 383

1964 301 250 833

1965 355 257 724

1966 385 333 866

1967 562 406 721

1968* 815 730 895

1969* 1182 1088 920

* Estimación.Fuente: Comissao de Levantamento e Fiscalizaçao da Produçao Tritfcola.

E l cu ltivo está situado en la reg ión sureña del país. Más del 70% de la producción es cosechada en e l Estado de R io Grande do Sul, estando e l res to radicado en los Estados de Santa Catarina (16%) y Paraná (12%).

L os datos dem ostrados en e l cuadro П, ilustran l a evolución de los tr ig a le s bras ileños en las ú ltim as cosechas.

L a osc ilac ión de las cosechas en los años 1962, 1963 y 1964 evidencia la inestable situación por la que pasaba este cu ltivo desde la época de su resu rg im ien to . Debido a una gran inestabilidad c lim ática , que fa vo rec ía a veces e l d esa rro llo de enferm edades, y a la inex istencia de variedades con acentuado grado de res is ten c ia a las m ism as, e l prom edio de rendim iento de los cu ltivos oscilaba enorm em ente. V er ificam os a s f que e l prom edio de la cosecha de 1962, debido a condiciones c lim áticas favorab les , alcanzó la c ifra de 1175 kg/ha, m ientras que la cosecha del año siguiente bajó

MEJORAMIENTO GENETICO DEL TRIGO 4 37

a 383 kg/ha debido a la elevada humedad a tm osférica y tem peratura, excepcionalm ente favorab les al d esa rro llo de las en ferm edades fúngicas, en la fase que antecedió la espigazón hasta la m adurez.

Las p rincipa les en ferm edades que atacan a l tr ig o en la reg ión tr igu era trad ic iona l son: « fu s a r io s is » ( G ibberella zeae (Schw. ) Petch . ); «s e p to r io s is de las g lu m as» (Septoria nodorum Berk . ); «s e p to r io s is de las h o ja s »(Septoria t r it ic i R ob .); « r o y a del ta l lo » (Puccin ia gram in is t r it ic i E r ik s . y Henn. ); « r o y a de la h o ja » (Puccin ia recóndita Rob. ); « r o y a l in e a r »(Puccin ia s tr i i fo r m is W est. ); « o íd io » (Erysphe gram in i t r i t ic i M archai); « c a rb ó n » (Ustilago t r it ic i (P e rs . ) R ostr. ); «h e lm in to sp o r io s is » (Helm inthos porium sativum P . K. et В. ) y v ir o s is (B a rley Y e llow Dwarf, p rincipalm ente).

Adem ás de las en ferm edades, otro factor lim itan te de la triticu ltu ra b ras ileña lo constituyen los suelos pobres, ácidos y con elevados tenores de alum inio.

En v irtud de los altos p rec io s de los co rrec tivo s y abonos, este cu ltivo está siendo efectuado con variedades capaces de producir aún en esas condiciones desfavorab les de fe rtilid ad .

La suma de estos prob lem as (condiciones favorab les al d esa rro llo de las en ferm edades, suelos pobres con elevados tenores de aluminio y p rec ios altos de los co rre c t iv o s y abonos), ob ligaron a los técn icos b rasileños a c re a r variedades muy rústicas, pero de baja productividad. Estas variedades, en genera l, responden muy poco a la e levación de la fe rtilidad , son muy altas, poseen s ign ifica tiva re lac ión hoja:grano, pocos granos por esp igu illa , y débil calidad panificadora.

2. PROGRESOS DE L A IN VESTIG AC IO N G E N E T IC A D EL TR IG O ENE L B R A S IL

E l m a ter ia l p rim itivam ente cultivado en R io Grande do Sul, según S ilva [ 1], tenia como ca ra c te r ís tica s principa les un alto porte , la rgo c ic lo vegeta tivo , elevada susceptibilidad a la « r o y a del t a l lo » , y grano de m ala form ación . Estaba, en cam bio, muy bien adaptado a los suelos pobres y ácidos.

Con la finalidad de sumar a la adaptación reg ion a l de la variedad F ron te ira la ex traord inaria capacidad de adaptación a condiciones variadas que posee Mentana, fueron cruzadas estas variedades y seleccionada Frontana que vino a constitu irse en un hito básico de la triticu ltu ra nacional. Esta nueva variedad, colocada a d isposición de los agricu lto res en 1940, represen taba un cambio fundamental del tipo de tr ig o que hasta entonces se cultivaba. E ra muy p recoz , de porte más bajo y poseía granos b ien form ados. Sin em bargo, parte de la excelen te adaptación a los suelos ácidos y pobres que e l m a ter ia l p r im itivo poseía , había sido perdida.

Las variedades P re lu d io , Carazinho y F o rta leza , creadas a p a rtir de Frontana, reunían las cualidades de ésta y la excelen te adaptación del m a te r ia l p r im itivo a los suelos ácidos.

De un program a que intentaba in corporar a Frontana res is ten c ia a las razas de « r o y a del t a l lo » presen tes en B ras il, se orig inaron las variedades P ira t in i, Sao B orja , IAS 13-Passo Fundo, IAS 28-Iju i e IAS 27-Itapeva,

Con posterio ridad se obtuvo la variedad IAS 20-Iassul, considerada otro hito en e l cu ltivo del tr ig o en e l pa ís. Esta nueva variedad, entregada a los agricu lto res en 1963, rápidam ente alcanzó notables producciones, llegando a rep resen ta r en 1967 m ás del 70% del área to ta l sem brada con este ce rea l.

OSORIO

CUADRO HI. DATOS C O M PAR ATIVO S DE PRODUCCION DE LAS VARIEDADES IAS 20-IASSUL Y F R O N TA N A . PROM EDIOS G ENERALES DE LOS ENSAYOS SA -IPE A S EN RIO GRANDE DO SUL

Año

Promedio de la producción (kg/ha)

IAS 20-Iassul Frontana

°Io de IAS 20-Issul sobre Frontana

№ de locales

№ de experimentos

1960 1396 774 80,4 5 10

1961 705 363 94,2 10 17

1962 1719 1485 15,7 13 20

1963 811 459 76,7 11 17

1964 2200 1585 38,8 13 22

1965 1515 1098 38,0 10 19

1966 1583 1217 30,0 15 26

1967 974 768 26,8 21 36

1968 1166 963 21,1 21 33

Promedio 1341 968 46,7 13 200*

* Total.Fuente: Datos de los archivos del Setor de Fitotecnia del Instituto de Pesquisas e Experimentado

AgropecuSrias do Sul, Pelotas, Brasil (no publicados).

Es im portante destacar que la variedad IAS 20-Iassu l producía por su extraord inaria rusticidad cerca de 47% más que la variedad Frontana, especia lm ente en los años considerados adversos como e l 1961 y e l 1963, cuando las producciones decayeron bastante en función de las condiciones c lim áticas adversas, pero la variedad IAS 20-Iassul aún mantuvo una productividad razonable (cuadro III).

En térm inos genera les puede a firm a rse que e l program a de m ejoram ien to genético desarro llado en e l B ra s il condujo a la creac ión de variedades que, por su acentuado grado de res is ten c ia a las en ferm edades p reva lec ien tes y por su adaptabilidad a suelos pobres y ácidos, garantizan a los agricu ltores una re la tiva estab ilidad en la producción, ca ra c te r ís tica esta que no se ve r ificab a an teriorm ente.

Esta situación p erm itió un continuo ascenso en la producción b rasileña de tr ig o , a p a rtir de 1964, m ás en función del aumento del área de cu ltivo, que de la producción por área (cuadro П).

P o r lo tanto, la difusión de las nuevas variedades desempeñó un papel fundamental y d ec is ivo en este increm ento, las cuales, por su rusticidad, garan tizaron en los años en que las condiciones c lim áticas fueron desfavorab les, una cosecha razonable. De esta form a, m erced a una po lítica favorab le de p rec io y asistencia técn ica, pudo e l agricu ltor con seguridad extender su área de cu ltivo con tr ig o .

Se estab ilizó la productividad del cu ltivo, pero en n ive les muy bajos (menos de 900 kg/ha). P o r este m otivo, desde entonces, la preocupación de los fitom ejo radores ha res id ido en la creac ión de variedades que, sin

MEJORAMIENTO GENETICO DEL TRIGO 4 3 9

redu c ir sus n ive les de res is ten c ia a las en ferm edades, fuesen capaces de producir altos rendim ientos cuando son aumentados los n ive les de fe rt ilid ad del suelo.

Otro punto que v iene preocupando bastante a lo s fito técn icos es la capacidad m o lin era y panadera. De modo genera l las variedades b rasileñas de tr ig o poseen débil capacidad de panificación. Hasta hace cuatro años, cuando e l B ra s il producía apenas e l 10% de su consumo de tr igo , este aspecto no e ra m otivo de consideración , ya que esta pequeña producción e ra m ezclada al tr ig o im portado, de excelente calidad panificadora, resultando una harina de superior calidad. Sin em bargo, en la actual cosecha de 1970 es esperada una producción de aproxim adam ente 1 500 000 t, o sea la m itad del tr ig o consumido internam ente. P o r esta razón, no es más aceptable la idea de continuar produciendo tr ig o de m ala calidad m olin era y de panificación, teniendo en cuenta la tendiente dism inución en las im portaciones de tr ig o .

3. U T IL IZ A C IO N DE L A INDUCCION DE M U TACIO NES COMO UN METODODE M E JO RAM IENTO D E L TR IG O EN E L B R A S IL

En e l d esa rro llo del p rogram a brasileño de m ejoram ien to del tr igo , pocas veces ha sido usada la inducción de m utaciones.

A p a rtir de 1960, en la E sco la de Agronom ia E liseu M ac ie l y en e l Instituto de Pesqu isas e Experim entaçao A gropecuárias do Sul (R io Grande do Sul), se se leccionó m a ter ia l proveniente de la irrad iac ión de tres variedades, Frontana, IAS 13-Passo Fundo e IAS 20-Iassu l, con dosis de10, 20 y 30 kR, teniendo com o ob jetivo prin c ipa l la reducción de la altura de las plantas. S ilva [2 ] c ita no haber alcanzado este ob jetivo , habiéndose conseguido, en cam bio, en la variedad IAS 20-Iassu l mutantes m ás productivas y m ás res isten tes a la « r o y a del ta llo » . Se pudo constatar que esta re s is ten cia adicional de las mutantes seleccionadas e ra debida al gen Sr 5, ausente en la variedad irrad iada . En los descendientes de cruzam ientos entre las mutantes res is ten tes fueron seleccionadas dos lineas que, en la cosecha 1969, produjeron, en un prom edio de 11 experim entos efectuados, 15% y 13% más que la variedad orig in a l IAS 20-Iassul.

En la EstaçSo E xperim en ta l F ito técn ica Julio de Castilhos (R io Grande do Sul), se están ensayando mutantes seleccionadas de s ie te variedades irrad iadas . M iranda [ 3] re la ta haber enviado en 1968 al Centro de Energia N uclear da A gricu ltu ra (Sao Pau lo ), sem illas de los cu ltivares Torop i,Giruá, Nobre, Nova P ra ta , J. 9160-67, S 28 у В 8 para irrad iac ión , con rayos gama, por un período de una hora, en dosajes de 20 kR. P rincipa lm en te se deseaba la obtención de mutantes más p recoces y de porte bajo. En e l m a ter ia l segregante fueron seleccionadas mutantes con las ca ra c te r ís tica s de ta llo m acizo , c ic lo de 1 0 días más corto y espigas sin la es ter ilid ad de las esp igu illas de la base, hecho norm al en las variedades orig in a les .

En 1969 se efectuó una nueva irrad iac ión , usando dosajes de 25 kR.Las variedades irrad iadas fueron E rex im y Frontana, con e l objeto de obtener mutantes bajas y sin f lo re s e s té r ile s en las esp igu illas de la base, y d iversas líneas m exicanas del cruzam iento Blue B ird , a fin de lo g ra r mutantes adaptadas a las condiciones de bajo pH y altos tenores de aluminio, ca ra c te r ís tica , com o se d ijo de los campos de cu ltivo en R io Grando do Sul. Este m a ter ia l está siendo som etido durante e l transcurso de l co rrien te año a su p r im era se lección .

4 4 0 OSORIO

Gomes [4 ] de la Estaçâo Esperim enta l de Passo Fundo, del Instituto de Pesqu isas e Experim entaçSo Agropecuárias do Sul, envió al O rganism o Internacional de E nerg ía A tóm ica, Viena, sem illas de cinco cu ltivares bras ileños y cinco extran jeros para irrad iac ión . Los cu ltivares brasileños irrad iados fueron IAS 52, IAS 54, Lagoa Verm elha, P e l 13180-65 y P e l 13494-64, E l p rincipa l ob jetivo sobre este m ateria l, consiste en se lecc ion ar las mutantes que, conservando las ca rac te r ís ticas de los cu ltivares o rig in a les , tengan sus alturas reducidas. Los cu ltivares extran jeros enviados fueron Gabo, P it ic 62, Nainari 60, N P 881 y [Son 64 X T z P P -N a i 60 (A )]. En este m ateria l se espera se lecc ionar principalm ente mutantes res is ten tes a las en ferm edades.

Las sem illas irrad iadas de estos 10 cu ltivares están sem bradas y en observaciones experim enta les.

4. PO SIB ILID AD ES DE USO DE L A INDUCCION DE M UTACIONES EN E LPR O G R A M A B RASILEÑO DE M E JO RAM IENTO D EL TR IGO

L a e fic ien c ia de la inducción de mutaciones com o un m étodo de m ejoram ien to de las plantas, se ha constituido en uno de los asuntos más polem izados entre los m ejoradores de plantas.

A l con sidera rse la va lid ez o no del uso de mutaciones inducidas en un program a de m ejoram ien to del tr igo , los argumentos de esta discusión vuelven a re su rg ir .

S ilva [ 1] consideró, en aquella época, e l m ejoram ien to del tr ig o en e l B ra s il por e l uso de m utaciones como poco indicado. Justificó su punto de v is ta basado en la entonces insuficiente exp loración de la variab ilidad genética existente por e l uso de los m étodos trad ic iona les de h ibridación y se lecc ión .

Desde entonces, e l program a brasileño de crianza de cu ltivares de tr ig o aumentó considerablem ente. E sto puede v e r if ic a rs e analizando e l hecho de que, en la ú ltim a cosecha tr igu era (1969), se hayan rea lizado en e l B ra s il aproxim adam ente 10 000 cruzas, sumando las de las estaciones experim enta les del M in is terio de Agricu ltu ra , de la S ecretaría de Agricu ltu ra y las rea lizadas por particu lares.

Se p revé para la actual y próxim as cosechas aumentos en e l número de cruzas a e fectuar, com o a s í también increm entos en e l número de m a ter ia l segregante de cruzas introducidas de otros centros de creación de variedades de tr igo .

P o r lo tanto, los m étodos trad ic iona les de m ejoram ien to de tr ig o ya están siendo su ficientem ente u tilizados. Nuestro punto de v is ta es que en e l actual estado del p rogram a brasileño de m ejoram ien to genético del tr igo , e l m étodo de inducción de mutaciones debe s e r estim ulado, complementando a los m étodos convencionales.

Uno de los posib les problem as de solución por e l uso de inducción de mutaciones, res id e en e l aparente ligam ien to genético existente entre los genes determ inantes de la res is ten c ia de las variedades b rasileñas a las en ferm edades de la esp iga («s e p to r io s is de las g lum as» y « fu s a r io s is » ) y los genes que regulan las ca racterís ticas de porte bajo.

Estudios rea lizad os en e l Japón por Nakagawa et al. [5 ] dem ostraron la ex is tenc ia de co rre la c ión positiva entre las ca ra c te r ís tica s de porte bajo y susceptib ilidad a la « fu s a r io s is » . Existen d iversas variedades brasileñas

M EJORAMIENTO GENETICO DEL TRIGO 441

de tr ig o poseedoras de un apreciab le grado de res is ten c ia a « fu s a r io s is » , m ostrando por otro lado la ca ra c te r ís tica de porte muy alto, bas variedades ex tran jeras bajas, de alto rendim iento, en cruzam iento con las variedades b rasileñas res is ten tes producen descendientes que, cuando son de porte bajo, son susceptib les a la re fe r id a enferm edad. Posib lem en te, irrad iando o tratando con m utagénicos qu ím icos sem illas F j de estos cruzam ientos, se consiga rom per este aparente ligam iento genético.

O tra posibilidad res id e en la esperanza de que, por inducción de m utaciones en plantas altas, res is ten tes a la s en ferm edades, se consigan plantas de portes bajos, tam bién res is ten tes , o por lo menos plantas altas res is ten tes al vuelco .

Otra justificac ión para e l uso de inducción de mutaciones en e l p rogram a b ras ileño de m ejoram ien to del tr ig o es la posib ilidad de aparición de nuevos genes, d iferen tes de lo s ya existentes y capaces de p roporc ionar ventajas ad icionales a los cu ltivares que los posean.

. R E F E R E N C I A S

[13 SILVA, A.R., Melhoramento das Variedades de Trigo Destinadas ás Diferentes Regioes do Brasil,SIA-MA, Rio de Janeiro (1966),

[2] SILVA, A.R., MARINHO, V.P., Experimento de induijao de mutaçao em trigo pela energía nuclear,O Agropecuário, Boletim Técnico do DANV, FAEM, Pelotas (1962).

[3] MIRANDA, H.V., comunicación personal.[4] GOMES, E.P., comunicación personal.[5] NAKAGAWA, М.. GOCHO, H., NISHIO, K ., WARANAGE, S., Nature and Inheritance of Ear-scab

Resistance in Wheat. 1. Heritability Estimates and Heritable Relationship of Ear-scab Resistance and Some Agronomic Characters in F2 of the Cross Shinchu-naga x Norin 12, Bull. Tokai Kinki Nat. Agrie. Exper. St., 15, Tsu-City, Japan (1966) 43.

D I S C U S S I O N

C. K R U LL : You mentioned a group o f s e v e ra l d iseases that m ay lim it wheat production in B ra z il. Do you have sources o f res is tance to a ll o f them at present?

E. A . OSORIO: Y e s , but in d ifferen t va r ie t ie s .M .S . SW AM INATH AN : How do the M exican sem i-dw arf va r ie tie s

behave under your conditions? Can you cu ltivate any o f them? A lso , do the Norin dwarfing genes show any association w ith 's te r ility under your environm ent?

E . A . OSORIO: G enera lly they are susceptible to Septoria and G ibberella . We have tr ied these va r ie t ie s many tim es, but due to th e ir susceptib ility to these d iseases they have not yet produced m ore than the B raz ilian va r ie t ie s . W ith rega rd to the s te r ility in va r ie t ie s with N orin dwarfing genes, we do not have this problem .

INFORMACIONES SOBRE LA INDUCCION DE MUTACIONES EN TRIGO MEDIANTE LA IRRADIACION DE SEMILLAS

E. PEIXOTO GOMES

Estación Experimental de Passo Fundo,

IPEAS, M inisterio de Agricultura,

Río Grande del Sur,

Brasil

Abstract-Resumen

INFORMATION ON THE INDUCTION OF MUTATIONS IN WHEAT BY SEED IRRADIATION.In 1960, seeds of three wheat varieties were irradiated with 10 - 30 kR, with the objective of shortening the

straw without altering other important characteristics. Short straw mutants have not been found, but mutants with improved resistance to Puccinia graminis tritici have been found. By crossing these resistant mutants, two new wheat strains have been developed which yield 13 - 15% more than the parent variety IAS 20. This higher yield is probably due to the resistance against the stem rust race 17/63, which is at present very common in the south of Brazil.

INFORMACIONES SOBRE LA INDUCCION DE MUTACIONES EN TRIGO MEDIANTE LA IRRADIACION DE SEMILLAS.En 1960, semillas de tres variedades de trigo fueron irradiadas con dosis de 10 a 30 kR, operación cuyo

objetivo fue producir plantas de tallo más corto, sin alterar las demás características importantes. No se han obtenido mutantes de tallo corto, pero sf mutantes con mayor re s is te n c ia a P u c c in ia g ra m in is t r i t i c i . Por cruzamiento de estos mutantes resistentes, se han obtenido dos nuevas variedades de trigo, cuyo rendimiento es del 13 al 15% más elevado que el de la variedad progenitora IAS 20. Este rendimiento más elevado se debe probablemente a una mayor resistencia a la raza de la roya del tallo 17/63, que actualmente está muy extendida en el sur del Brasil.

En 1960, sem illas de tres variedades de tr ig o denominadas Fontana,IAS 13 e IAS 20, fueron irrad iadas en e l Instituto de Energía N uclear de la Universidad de SSo Paulo con las siguientes dosis: 10 000, 20 000 y 30 000 R[1 ]. Este trabajo fue rea lizado bajo los auspicios de la E sco la de Agronom ia E liseu M ac ie l y del Instituto de Pesquisas e Experimentaçéio Agropecuarias do Sul, en Pelotas, R ío Grande del Sur, B ras il.

E l ob jetivo de este proyecto de irrad iac ión fué producir plantas de ta llo más corto, a través de m utaciones, pero sin a lte ra r las demás c a ra c te r ís ticas agronóm icas y fis io ló g ica s de las variedades orig in a les . Las tres variedades u tilizadas en este experim ento son buenas productoras de grano, pero tienen la lim itación de s e r muy altas y por lo tanto de acam arse, e s pecia lm ente bajo un abonamiento desbalanceado en donde e l N es exces ivo .

A continuación se presentan los resultados con la variedad IAS 20 ya que las otras variedades no produ jeron mutaciones utiles.

E l ob jetivo o rig in a l de consegu ir plantas de ta llo corto no fué alcanzado; sin em bargo, s í se logra ron plantas con m ayor res is ten c ia a la roya del ta llo (Puccin ia gram in is t r i t ic i ) y m ayor producción de grano.

Con la aparición de nuevos biotipos de la raza 17 (17/61 17/63) capaces de atacar la variedad IAS 20, con solam ente e l gene Sr 6 , fue posib le se lecc ion ar mutantes res is ten tes .

« L a descendencia de las plantas res isten tes a la roya del ta llo fue inoculada con las razas 11, 11T, 15, 15/65 17, 17/61, 17/63 y 17T, en-

443

4 44 PEIXOTO GOMES

contrándose plantas res is ten tes a todas las razas antiguas 1 1 , 15 y 17 y tam bién a las razas nuevas. Como resultado del cruzam iento entre las mutantes y las variedades que contienen e l gene Sr 5 y Sr 11, hubo una fuerte indicación de que la res istencia es debida a l gene Sr 5, y ninguna de las variedades que entraron en la form ación de IAS 2 0 contienen e l gene Sr 5 » [2] .

De un total de 42 891 plantas en e l I2( 2— generación ) proveniente de IAS 20 irrad iada , 37 plantas sin roya del ta llo fueron seleccionadas, todas ellas muy sem ejantes a IAS 20 norm al [ l ].

Estas plantas res is ten tes fueron cruzadas entre s í y con otras variedades, inclusive IAS 20 norm al. A s í, mediante e l cruzam iento de dos líneas p ro ven ientes de IAS 20 irrad iadas, las de número 19 906 - 62 y 18 102 - 62, las líneas P e l 21 382 - 66 y P e l 21 383 - 66 fueron obtenidas.

Estas líneas están en la fase final de experim entación y su producción, com parada con la de IAS 20 norm al, se encuentra en e l cuadro I, y sus reacciones con las d iferen tes razas de roya del ta llo , se presentan en e l cuadro II.

Las dos líneas mutantes produjeron 15 y 13% más que IAS 20 (cuadro I) lo cual es probablem ente debido a su m ayor grado de res is ten c ia a la roya del ta llo . A pesar de que la d iferenc ia en producción con LAS 20 es r e la t iv a mente pequeña, estas líneas mutantes pueden ser ú tiles en e l m ejoram iento de la res is ten c ia a la roya del tallo.

Se pudo v e r i f ic a r (cuadro II) que m ientras las dos líneas mutantes son res isten tes a la ra za 17/63, la variedad IAS 20 es muy susceptible.

Es im portante destacar que la raza 1 7/63 es la más difundida en e l sur del B ra s il, representando un 82, 1% de las m uestras estudiadas en 1,969.

CUADRO I. PRODUCCION MEDIA DE LAS L IN E AS M U TAN TES E IAS 2 0 E N LOS 11 ENSAYOS DE PRODUCCION CONDUCIDOS E N 1969 [3]

Variedad y lfneas mutantes

Producción de grano ( kg/ha)

Producción relativa aUS 20

IAS 20 1556 100

Pel 21 382/66 1790 115

Pel 21383/66 1759 113

CUADRO II. RESULTADOS DE LAS PRUEBAS CON P L A N T U L A S SEGUN LAS P R IN C IP A L E S RAZAS DE L A RO YA D E L T A L L O Puccin ia gram in is t r it ic i [4]

Variedad y lfneas mutantes 11/65 15

Razas de la roy 15/65

a del tallo17 17T 17/63

IAS 20 4 0 0 0 0 4

Pel 21 382/66 3 0 0 0 0 0

Pel 21 383/66 з ■ 0 0 1 0 0

M U TA C IO N ES EN TRIGO 445

Las observaciones de campo hechas durante la flo rac ión en dos ensayos com parativos de rendim iento, conducidos en Passo Fundo, -en 1969, m os traron tam bién que estas dos líneas mutantes tuvieron mucho menos roya en e l ta llo , que la variedad paterna IAS 20. Esto parece ind icar que e l gene Sr 5 es también resisten te a 17/63 durante la fase adulta del cu ltivo.

CONCLUSIONES .

1. Las nuevas líneas proven ientes de IAS 20 irrad iadas, produ jeron 13 y 15% más que IAS 20 norm al.

2. Las nuevas líneas fueron res isten tes a la raza 17/63 (nota cero ) m ien tras que IAS 2 0 fue muy susceptib le (nota 4).

3. Siendo 17/63 la raza de roya del ta llo que ocurre con m ayor frecuencia en e l sur del B ra s il, las m utaciones conseguidas son altamente s ign ifica tivas .


[1] SILVA, A.R., MARINHO, V.P., Experimento de Inducâo de mutacáo em trigo pela energía nuclear, O agropecuario, Boletim Técnico de DANV, EAEM(1962).

[2] SILVA, A.R., Melhoramento das variedades de trigos destinadas ao Sul do Brasil, SIA-MA ( 1966).[3] Diversos autores. Relatónos experimentaçâo com trigo em 1969, IPEAS e SA-RS (no publicado).[4] COELHO, Elisa T., Relatório técnico da secçSo de fitopatologia do IPEAS ( 1969) (no publicado).

D I S C U S S I O N

M. S. SW AM INATH AN : Do you know how the 15% increased y ie ld in the mutants is brought about? Is the m aturity period in the mutants and IAS 20 s im ila r? Can the increased y ie ld be attributed so le ly to the resistance to the race 17/63 o f stem rust?

E. PE IX O TO GOMES: The reason why the two mutant lines y ie ld ed 13% and 15% m ore than the o rig in a l va r ie ty (IAS 20) was probably th e ir im proved resistance to Puccin ia gram in is t r it ic i since they have agricu ltu ra l characte r is t ic s which are otherw ise v e ry s im ila r to those o f IAS 20.

FRECUENCIA DE MUTACIONES INDUCIDAS POR RADIACION GAMMA Y METANOSULFONATO DE ETILO EN DIFERENTES ESTADOS DE GERMINACION DE LAS SEMILLAS DE FRIJOL (Phaseolus vulgaris L.)

F. DELGADO DE LA FLOR B.

Universidad Nacional T écn ica del A ltip lano,

Puno, Perú

Abstract-Resumen

FREQUENCY OF MUTATIONS INDUCED BY GAMMA-RADIATION AND ETHYL METHANE SULPHONATE IN DIFFERENT STAGES OF GERMINATION OF BEAN SEEDS ( Phaseolus vulgaris L.).

Seeds of two varieties of. Phaseolus vulgaris were soaked for 12 to 72 h and were then treated with either3 kR gamma-rays or with 0.04 M EMS for 6 h at 20°C. The frequency of mutant seedlings was up to 58% after EMS treatment but only up to 21% after gamma-irradiation. No apparent differences were observed between the two varieties. Pre-soaking for 24 h prior to irradiation yielded the highest mutation frequency, whereas no consistent effects of different pre-soaking times between 24 and 72 h were observable with EMS treatment.

FRECUENCIA DE MUTACIONES INDUCIDAS POR RADIACION GAMMA Y METANOSULFONATO DE ETILO EN DIFERENTES ESTADOS DE GERMINACION DE LAS SEMILLAS DE FRITOL( Phaseolus vulgaris L.).

Semillas de dos variedades de Phaseolus vulgaris se pusieron en remojo por períodos de 12 a 72 h, tratándose a continuación con rayos gamma (dosis de 3 kR) o con 0, 04 M de EMS durante 6 h a 20°C. La frecuencia de las plántulas mutantes fue de hasta un 58% después del tratamiento con EMS, pero sólo de un 21% después de la irradiación gamma. No se observaron diferencias apreciables entre ambas variedades. Con un remojo previo de 24 h se obtuvo la frecuencia más elevada de mutaciones después de la irradiación, mientras que no se observaron efectos definidos de la puesta en remojo previa durante 24 a 72 h, en el caso del tratamiento con EMS.

INTRO D UCCIO N

La frecuencia de mutaciones espontáneas es generalm ente baja, pero mediante e l em pleo de agentes mutagénicos es posib le increm en tarlas. En los ú ltim os 50 años, exhaustivas investigaciones sobre mutaciones inducidas han m ostrado que algunos agentes fís icos com o los rayos gamma y quím icos como e l metanosulfonato de etilo (EM S), son potentes mutagénicos.

Si la ap licación de estos agentes mutagénicos se hace a d iferen tes estados de germ inación de las sem illas , la frecuencia de mutaciones puede aumentar [9Î porque las célu las a l ten er m ayor actividad a-umentarán la sensib ilidad de los te jidos a la acción de los agentes mutagénicos.

Es necesario in ves tiga r cual es e l estado de d esa rro llo ce lu lar en e l que se deben ap lica r estos agentes mutagénicos, para obtener una m ayor f r e cuencia de mutaciones.

En este estudio se ha u tilizado e l fr i jo l ( Phaseolus vu lgaris L . ) como m ateria l experim enta l por su gran im portancia como cu ltivo y por sus espec ia les condiciones m orfo lóg icas que lo hacen especia lm ente apto para e l estudio de mutaciones inducidas.

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M U TAC ION ES IN D U CID A S EN SEMILLAS DE FRIJOL 449

M A T E R IA L E S Y METODOS

Los agentes mutagénicos u tilizados en este experim ento fueron rad ia ciones gamma y EMS. Se u tiliza ron dos [2] variedades de fr i jo l, las que previam ente habían sido autopolinizadas por dos o más generaciones y almacenadas en una estufa a 34°C a fin de es ta b iliza r su contenido de humedad, que fue 8 , 9% en la variedad «T u rr ia lb a - 1» y 12,7% en la variedad « P o r r i l lo » .

P rim eram en te se determ inó la dosis le ta l m edia (LD 50 ) para cada variedad y agente mutagénico, a las 24 h de in iciada la germ inación. La LD 50 fue de 3 kR de radiaciones gamma y 0,04 M de EMS.

Antes de tra ta r las sem illas con los agentes m utagénicos se les puso en agua por períodos de 0, 12, 24, 36, 48, 60 y 72 h. Pa ra cada tratam iento se u tiliza ron 200 sem illa s . En los tratam ientos con EMS las sem illas perm anecieron en la solución durante 6 h y a tem peraturas constantes de 20°C, durante las cuales se les agitó continuamente y luego se lavaron rep e tidas veces con agua destilada.

P osterio rm en te se sem bró en los invernáculos una sem illa por m aceta.La cosecha se re a liz ó planta por planta separadamente y las sem illas de una m ism a planta se sem braron en una m ism a h ilera en una cama de a lm acigo . Cuando las plántulas tenían dos semanas de edad, se h ic ieron observaciones sobre los distintos tipos de mutaciones que se habían producido y su frecuencia .

Se calcu ló la frecuencia de mutaciones con respecto a variedades, agentes mutagénicos y tratam ientos a distintos grados de germ inación de las sem illas para encontrar e l estado de germ inación y tratam iento más mutagénico. (P a ra com parar los d iferen tes e fectos se u tilizó la prueba de partic ión ortogonal de los componentes de va riac ión por x 2 . )

E l tratam iento y estado de germ inación más mutagénico se determ inó por la frecuencia de mutaciones.

RESULTADOS

F recuencia de mutaciones

En e l cuadro I se presenta la frecuencia de mutaciones inducidas por EMS y radiación gamma en los d iferen tes estados de germ inación de las sem illa s en las variedades de fr i jo l .

La prueba de partic ión ortogonal de los componentes de va riac ión por X2 (cuadro II) m ostró que no existen d iferencias en la frecuencia de mutaciones tota les entre las dos variedades.

Entre los agentes m utagénicos se encontraron d iferencias en la f r e cuencia de mutaciones entre e l EMS y la radiación gamma. En estos casos la frecuencia de mutaciones inducidas por EMS fue tres veces m ayor que la inducida por radiación gamma.

La frecuencia de mutaciones en re lac ión con la ap licación de los agentes m utagénicos a d iferen tes estados de germ inación de la sem illa de fr i jo l sigue una tendencia curva. Esta tendencia expresa que la frecuencia de mutaciones asciende conform e aumenta e l estado de germ inación de las sem illas , p r im ero bruscam ente y luego paulatinamente hasta lle g a r a un punto donde se es tab iliza o d ecrece . La frecuencia de mutaciones asciende

4 50 DELGADO DE LA FLOR В.

CUADRO И. PRU EBA DE PA R T IC IO N O RTO G O NAL DE LOS COM PONENTES DE VA R IA N C IA POR x 2 E N LA FREC U EN C IA DE M UTACIONES

Fuentes de variación G1 X2

Turrialba 1 VS Porrillo 1 1, 04 NS

EMS VS Radiación gamma 1 151, 10 * *

Interacción 1 0, 25 NS

Testigo VS Tratamientos 1 121, 87 * *

Tendencia lineal 1 8, 59 * *

Tendencia cuadrática 1 25, 70 * *

Otros 25 58, 81 * *

Total 31 367,36

hasta las 24 h de in iciada la germ inación y luego aumenta paulatinamente hasta es tab iliza rse a las 72 h de in iciada la germ inación.

Esta tendencia ocu rre más o menos regu larm ente para las dos v a r ie dades y los dos agentes mutagénicos.

DISCUSION

P o r la sim ilitud de sus frecuencias de m utaciones, tanto c lo ró ticas como m orfo lóg icas las variedades «T u r r ia lb a -1» y « P o r r i l l o » aparentemente no d ifieren en su contenido genético en lo que respecta a las ca racterís ticas tomadas en cuenta en este estudio.

Investigaciones p re lim in ares han m ostrado que e l EMS y la radiación gamma producen una alta frecuencia de mutaciones [5, 9] y que estas tienen posib ilidades de aumentar aún más, si la ap licación de los agentes mutagénicos ocu rre durante e l p roceso de germ inación [1, 2, 4, 9] .

La m ayor frecuencia de mutaciones inducidas por EMS con re lac ión a la radiación gamma podría deberse: 1) a la acción a lcalin izante del EMS quepuede inducir más mutaciones al actuar directam ente sobre los componentes genéticos de las célu las; 2 ) a l m ayor rango de supervivencia de las mutaciones inducidas por EMS a pesar de que puede inducir un m enor o igual núm ero de mutaciones en la célu la que las radiaciones.

L a tendencia cuadrática de los efectos de los agentes m utagénicos con re lac ión al p roceso de germ inación de las sem illas , se debió al aumento de la cantidad de agua del te jido . E l alto contenido de agua de los te jidos ce lu lares determ ina que se encuentran más daños y m ayor cantidad de aberraciones crom osóm icas y de mutaciones en las plantas [1, 7 ]. (En algunos casos la frecuencia de mutaciones inducidas puede s e r 6 a 8 veces m ayor que en e l estado de latencia [3] . )

La frecuencia de mutaciones tam bién puede s e r afectada por e l d esa rro llo ce lu la r [7] . Investigaciones en la m itosis de los m eris tem as ap icales del

M U TACION ES IN D U CID AS EN SEMILLAS DE FRIJOL 4 5 1

f r i jo l m ostraron que las configuraciones m itoticas pueden observarse 36 h después de term inado e l estado de latencia [6 ] . Según Savin et al. [8 ] , la s ín tesis de DNA en las célu las tiene lugar antes de ese tiem po y ésta es sensib le a los tratam ientos con EMS.


[1] EHRENBERG, L., Induced mutations in plant mechanisms and principles, Genet.agr. 12 (1959) 369.[2] EHRENBERG, L. etal., Irradiation effects on seed soaking and oxygen pressure in barley, Hereditas 39

4 (1953) 493.[3] EHRENBERG, L.. GUSTAFSSON, A., LUNDQUIST, U., Viable mutants induced in barley by ionizing

radiations and chemical mutagens, Hereditas 47 20 (1961) 243.[4] GUSTAFSSON, Á ., «The induction of mutations as a method in plant breeding», Mechanisms in Radio-

biology (ERRERA, М., FORSSBERG, A., Eds), J, Academic Press, New York, (1961) 477.[5] HESLOT, H., <<The nature of mutations The Use of Induced Mutations in Plant Breeding(Rep. FAO/ÏAEA

Tech. Meeting, Rome, 1964), Pergamon Press, Oxford (1965) 3.[6] МОН, C.C., The effect of low temperature on mitosis in meristematic cells of the shoot apex of beans,

Caryologia 19 4(1906) 413.[7] NILAN, R.A., Factors governing plant radiosensitivity, Conf.Radioactive Isotopes in Agrie., Michigan State

Univ.,East Lansing, Mich. (1956) 151.[8] SAVIN, V.N., SWAMINATHAN, M.S., SHARMA, B., Enhancement of chemically-induced mutation

frequency in barley through alteration in the duration of pre-soaking of seeds, Mutation Res. ^ 1 (1968) 101.[9] STADLER, L.J., Some genetic effects of rays in plants, J.Hered. 21 1 (1930) 3.

D I S C U S S I O N

R. T R U J ILLO FIGUEROA: Could you te ll me what types o f m orpholo g ica l change occurred? A lso , w ere there any indications of mutations o f econom ic in terest?

F . D ELG AD O D E L A F LO R В. : The main types o f m orphologica lchange com prised the production o f dwarf, sem i-d w arf and ta ll plants and deform ations in the shape and texture o f the leaves . None o f these mutations was o f econom ic in teres t. Those exhibiting s ize d ifferences died within a short tim e, and those having deform ations o f the leaves died o r w ere s te r ile o r produced few pods.

R. D. BROCK: What was the p re-soak ing tem perature?F . DELG AD O DE LA FLO R В .: The pre-soak ing tem perature was 20°C.

IRRADIACION DE SEMILLA DE SORGO (Sorghum vulgare) Y DE TRIGO (Triticum vulgare) CON RAYOS GAMMA

G. de ALBA, N . REYES, A . HERN ANDES

Instituto T ecn o ló g ico y de Estudios Superiores

de Monterrey, Departamento de Agronomía,

Monterrey, M éxico

Abstract-Resumen

IRRADIATION OF SEEDS OF SORGHUM ( Sorghum vulgare) AND WHEAT (Triiicum vulgare) WITH GAMMA-RAYS.Seeds of Sorghum vulgare and Triticum vulgare were irradiated with gamma-rays in the dose ranges

5-25 and 5-45 krad respectively. Observations were made on survival, fertility and various yield characters in order to assess the radiation effects in the M: generation.

IRRADIACION DE SEMILLA DE SORGO ( Sorghum vulgare) Y DE TRIGO (Triticum vulgare) CON RAYOS GAMMA.Se han irradiado semillas de Sorghum vulgare y Triticum vulgare con rayos gamma, en dosis comprendidas

entre 5-25 krads y 5-45 krads, respectivamente. Se han efectuado observaciones sobre la supecvivencia, fertilidad y vigor a fin de evaluar el efecto de las radiaciones en la generación M i.

I. IR R AD IAC IO N DE S E M ILLA DE SORGO

Con e l propósito de conocer los efectos de d iferen tes dosis de irrad iac ión en e l sorgo, se irrad ia ron cuatro variedades de sorgo para grano. Las dosis de irrad iac ión usadas fueron: 0, 5, 10, 15, 20 y 25 krads; la fuentede irrad ia c ión fue 6Cfco.

La sem illa irrad iada se sem bró en e l campo en un a rreg lo de parcela dividida, poniendo en parce la grande a las variedades y en parcela chica a las dosis; e l diseño usado fue bloques al a za r con cuatro repetic iones y 1 0 plantas por parce la chica.

T A B L A I. E FECTO S DE SEIS DOSIS DE IR R AD IAC IO N E N SORGO PA R A GRANO (PROM EDIOS DE CUATRO VARIEDADES POR CUATRO BLOQUES, O SEA 16 R E PE TIC IO N E S )

Dosis ( krads)

SupervivenciaProducción de grano

Cg)

Polen viable(°¡o)

Peso seco planta(g)

0 68 220 98 420

5 68 210 94 420

10 62 190 89 385

15 48 195 88 470

20 45 165 84 430

25 62 130 77 445

453

4 5 4 de ALBA et a l .

Las va riab les estudiadas fueron: fertilidad , supervivencia y v ig o r . Lafertilid ad se estudió mediante e l porciento de polen v iab le y la producción de grano; la supervivencia por medio del número de plantas que v iv ie ron hasta la m adurez del grano; finalm ente, e l v ig o r se estim ó por medio del peso seco prom edio por planta.

Los datos recog idos para las cuatro va riab les se consignan en la tabla I. Los anális is de varianza indicaron que no hay efecto de irrad iac ión en supervivencia y v ig o r , pero sí lo hay en fertilidad ; las cuatro variedades se com portaron en form a s im ila r. E l e fecto de la irrad iac ión en la fe rtilidad es negativo, reduciéndose la viabilidad del polen y la producción de grano a medida que se aumenta la dosis.

Se calcu laron los coe fic ien tes de reg res ión y co rre la c ión entre las dosis de irrad iac ión com o variab le fija y e l porciento prom edio de polen viable, ignorando variedades, como variab le ligada. Los va lo res obtenidos fueron: b = -0,70, r = - 0 , 95, r 2 = 0,90; todos altamente s ign ifica tivos.

CONCLUSIONES

1. A las dosis estudiadas, e l sorgo es inmune a la irrad iac ión en superv iven c ia y v igo r .

2. Los e fectos de la irrad iac ión en fe rtilid ad son negativos y proporcionales a las dosis.

3. Las variedades muestran una respuesta uniform e.

II. IR R AD IAC IO N DE SE M ILLA DE TRIGO

Con e l propósito de conocer los efectos de una am plia gama de irrad ia ciones en tr igo , se rea lizó un experim ento con 1 0 dosis de irrad iac ión , de

T A B L A II. V ALO RES OBSERVADOS BAJO CONDICIONES DE C AM PO P A R A D IFE R E N TE S C ARAC TE R IST IC AS FE N O T IP IC A S E N TRIGO (T . vu lgare ) E N 10 DOSIS DE IRRAD IAC IO N

Dosis(krads)

Supervivencia(%)

Alturaplanta(cm)

Granos por espiga

Espiguillas por espiga

Peso 100 granos(g)

Días de madurez

0 92 62 45 21 2, 6 128

5 92 64 47 21 3,2 126

10 92 62 44 21 2, 8 125

15 98 56 42 20 3,4 125

20 70 54 37 20 2,7 128

25 68 46 24 19 3,0 130

30 38 40 19 18 2,6 137

35 10 Pérdida total de plantas por ataque de roedores

40 0

45 0

IRRADIACION DE SORGO Y TRIGO 455

О a 45 krads a in terva los de 5 krads; la fuente de irrad iac ión fué 60Co. E l experim ento se rea lizó en e l campo en una distribución bloques al a za r con cuatro repetic iones. Los datos se concentran en la tabla II.

Se h ic ieron anális is estad ísticos para las variab les ; supervivencia , a ltura de planta y granos por espiga. En los tres casos se encontró que las dosis de irrad ia c ión tienen un e fecto de tipo lin ea l negativo; para granos por espiga se calcu laron los coefic ien tes, de reg res ión b = -0,85 y co rre la c ión r = -0,78..

CONCLUSIONES

1. La dosis le ta l m edia DL50 está entre 25 y 30 krads, y la DL100 entre 35 y 40 krads.2. Dosis de 20 krads o más son perjud ic ia les para todos los fac tores

estudiados, excepto peso de 1 0 0 granos.3. Dosis entre 5 y 15 krads muestran estím ulos para los fac tores estudiados,

excepto esp igu illas por espiga.

D I S C U S S I O N

R. LASSO G UEVARA: Can you explain the d ifference in the duration ofthe life cyc le produced when changing from a low radiation dose to a high one?

G. DE A LB A ; These d ifferences are the e ffec t of the radiation dose on the Mj generation . High doses upset the physio log ica l balance o f the plants and lengthen the life cyc le , whereas m oderate doses act as a stimulant and shorten the life cyc le without harm ing the other ch aracteris tics o f the plant.

R. LASSO G UEVARA: Does the e ffect disappear in the second generationa fte r irrad ia tion? What happens a fte r irrad iation?

G. DE A LB A : In genera l I have noticed that v e ry pronounced e ffec ts inthe generation tend to disappear in subsequent generations, so that the plants re v e rt to a certa in extent to the norm al p re-trea tm en t types. The return to norm al is not com plete, however, particu la rly where high radiation doses are concerned.

M .S . SW AM INATH AN: Since Sorghum is a polyploid, what type ofhandling procedure do you propose to adopt in the M2 and la te r generations?It is im portant that the procedure perm its the identification o f re c e ss iv e s o f po lysom ic or duplicate lo c i.

G. DE A LB A : I w ill handle that population in two system s: the bulk andm ass se lection system fo r genera l purposes and the se lfing and ped igree system in a lim ited number o f lines.

INDUCCION ARTIFIC IAL DE MUTANTES EN PAPA CRIOLLA (Solanum phureja Juz. et Buk. )

P. L. G O M E Z C U E R V O

Departamento de Biología, Universidad del Valle,

Cali

N. E S T R A D A R A M O S

Instituto Colombiano Agropecuario,

Bogotá, Colombia

Abstract-Resumen

ARTIFICIAL INDUCTION OF MUTANTS IN CREOLE POTATO (Solanum phureja luz, et Buk.).Solanum phureja presents some problems to farmers because it lacks a dormancy period, and also to

the geneticist and breeder because of its gametophytic self-incompatibility. With the aim of altering both characters, several mutation experiments hâve been carried out since 1967. Seeds as well as tubers of several clones were irradiated and observations were made on various characters in subsequent generations.Seeds of the different clones showed different radiosensitivity. Phenotypical changes observed in subsequent generations were mostly deleterious. However, one plant was selected, the tubers of which did not sprout for seven months. Another probable mutant gave a fairly high seed setting after pollination within the clone (125 seeds in two berries with about 6№> germination).

INDUCCION ARTIFICIAL DE MUTANTES EN PAPA CRIOLLA (Solanum phureja Juz. et Buk.).La papa Solanum phureja presenta algunos problemas a los agricultores por no poseer período de reposo.

Igualmente plantea dificultades al genetista y al fitotécnico a causa de su autoincompatibilid&d gametofítica. A fin de alterar ambos caracteres, se están efectuando experimentos de mutación desde 1967. Se han irradiado semillas y tubérculos de distintos clones, efectuándose observaciones con respecto a los diversos caracteres aparecidos en generaciones subsiguientes. Las semillas de los diferentes clones mostraron una radiosensibilidad diferente. Los cambios fenotípicos observados en generaciones subsiguientes fueron en su mayoría perjudiciales. Sin embargo, se seleccionó una planta cuyos tubérculos no germinaron durante siete meses. Olio mutante probable dio un número relativamente elevado de semillas tras cruzamiento dentro del mismo clon (125 semillas en dos bayas con una germinabilidad aproximada del 60%).

INTRO D UCCIO N

Uno de los prob lem as que presentan las papas cultivadas a los a g r icu lto res , es la conservación de los tubérculos por períodos más o menos la rgos una vez cosechados.

E l aumento del período de reposo no se ha logrado por los sistem as más comunes de m ejoram ien to como son cruzam ientos entre variedades, cruzam ientos in te resp ec ífico s , retrocru zam ien tos , autofecundación (para obtener hom ozigosis ) y se lecc ión clonal. Las mutaciones naturales o las inducidas pueden s e r v ir eventualmente en e l m ejoram ien to.

E l p r im er intento en la inducción de cam bios som áticos en papa por m e dio de rad iación ionizante fué rea lizada por A ssey e ra y B lagovidova en 1935, quienes dem ostraron que los rayos X podían causar perm anentes a lteraciones vegetativas produciendo en la planta estructuras qu im era les. H eiken en 1961, investigó los efectos som áticos causados por rayos X en cinco variedades de papa cultivada (Solanum tuberosum L . 2n = 48); tra tó 3420 tubérculos divid idos, en dosis que va ria ron de 250 a 8000 R [ 1 ] .

457

4 5 8 G O M EZ CUERVO y ESTRADA RAMOS

En Colom bia, P erdom o y Sanin (1963) estudiaron e l efecto causado por los rayos gamma en la brotación de los tubérculos de las variedades Tuquerrena y Parda Pastusa. Las dosis que em plearon fueron: 0, 5000,7 500, 10 000 y 15 000 R ; además analizaron e l contenido de proteína del tubérculo. Concluyeron que la radiación en las condiciones estudiadas no produce cam bios desfavorab les en la calidad del producto, y la dosis más adecuada para la conservación hasta los ocho m eses era de 10 000 R en ambas variedades [ 2 ].

L a papa c r io lla (Solanum phureja Juz. et Buk. 2n = 24) no sólo presenta prob lem as para e l agricu lto r por no poseer período de reposo — en muchas ocasiones en e l momento de la cosecha los tubérculos ya están brotados — sino tam bién para e l genetista ya que presenta autoincompatibilidad gam eto fítica entre e l polen y e l estigm a de una m ism a planta o clon y, por lo tanto, d ificu lta los análisis genéticos.

Aunque, como se v io anteriorm ente, se ha intentado la inducción de mutantes en Solanum tuberosum L . (2n = 48), no se tiene ninguna in form ación de trabajos al respecto en S. phureja Juz. et Buk. (2n = 24). E l objeto de este estudio fue ir ra d ia r sem illa sexual y asexual (tubérculos) de papa c r io lla (Solanum phureja) con rayos gamma, con e l propósito de obtener mutaciones favorab les ta les como aumento de la fe rtilid ad por la form ación de otros a le los que conduzcan a la elim inación total o pa rc ia l de la autoincom patib ilidad. Tam bién observar e l efecto sobre la precocidad de brotación en los tubérculos.

M A T E R IA LE S Y METODOS

E l estudio se in ic ió en ju lio de 196 7 y se u tilizó un rea c to r de rad ia ción gamma 60Co. E l m ateria l empleado proven ía de ocho clones de Solanum phureja Juz. et Buk. de la CCC (C o lecc ión C entra l Colombianad e P a p a s ): 1, 10, 81, 118, 1386, 1388, 1222 y 1449.

In icia lm ente se trataton yemas de tubérculos que aún no habían brotado, empleando cuatro yem as por tratam iento. Las dosis de rad iación empleadas fueron 0, 465, 930, 1960, 3920 y 5880 R .

En e l momento de la flo rac ión se h ic ieron cruzam ientos dentro de un m ism o clon empleando como m adre la planta irrad iada y como padre el testigo , con e l objeto de observar e l efecto de la rad iación en la com patibilidad del polen y del estigm a.

En una rad iación pos te rio r, se trató la totalidad del tubérculo em pleando dos por cada tratam iento. Las dosis empleadas fueron las m ism as que se u tiliza ron para la radiación de las yem as.

En la te rc e ra rad iación de sem illa asexual, se tra taron yemas del clon 81 de S. phureja, empleando 15 por tratam iento. Las dosis empleadas fueron 9, 465, 930 y 1960 R.

L a cuarta rad iación se h izo en la sem illa sexual de los siguientes c lo nes de S. phureja: 10, 118, 1222, 1386, 1388 y 1449; además se irrad iósem illa sexual de la variedad Tocana N egra (Solanum tuberosum L . con 2n = 48). Se em plearon 50 sem illas para cada tratam iento y las dosis utilizadas fueron 0, 465, 930, 1960, 3920, 5880 y 8370 R.

Se anotaron tanto para e l m a ter ia l proven iente de sem illa sexual como para e l de asexual los sigu ientes datos: porcenta je de germ inación, período vegeta tivo , número de bayas y número de tubérculos producidos, tamaño

M U TA N TE S EN PAPA CRIOLLA 4 59

y form a de los tubérculos, peso de los tubérculos en k ilos por planta, co lo r de la carne, co lo r de la p ie l, p resencia o ausencia de an illo en e l tubérculo, form a de los o jos, fecha de brotación y co lo r de los brotes. Los an teriores datos se tom aron por cada planta cosechada, con el objeto de observar los cam bios producidos por los rayos gamma.

RESULTAD O S Y DISCUSION

Los cambios efectuados por acción de los rayos gamma se determ inaron m ediante la observación d irecta de los tubérculos. En algunos casos, como en e l de las plantas proven ientes de sem illa sexual, no se pudo determ inar si los cambios observados se debían al efecto de las rad iaciones o a segregac iones.

En la p r im era radiación, como resultado de todos los cruzam ientos que se h ic ieron dentro de un m ism o clon, se obtuvo en una planta del clon 118 que proven ía de una yem a que rec ib ió 465 R dos bayas que produjeron un to ta l de 125 sem illa s , las cuales fueron sem bradas dos m eses después de reco lectadas y de e llas un total de 60 germ inaron.

Dentro del m a ter ia l cosechado se observó un mutante que proven ía del clon 118 con una dosis de 930 R; las ca rac te r ís ticas que se observaron fueron las sigu ientes: período vegetativo de 1 2 0 días,que es in fe r io r al obtenido con e l resto del m a ter ia l irrad iado que fue de 180 días; produjo un tota l de 13 tubérculos pequeños, los cuales se encontraron en la base del m atero ; la altura de la planta fue de 1 0 cen tím etros; hojas anchas, gruesas pubescentes y loca lizadas en form a de roseta a lrededor del ta llo , y no produjo flo res a pesar de haberse estim ulado la flo rac ión sum inistrándole luz a r t if ic ia l durante va ria s noches. La p rincipa l ca ra c te r ís tica observada en este mutante fue que los tubérculos producidos brotaron a los s ie te m eses de s e r cosechados. En la segunda siem bra, e l período vegeta tivo se redujo en 30 días com parado con e l an terior, es dec ir em plearon sólo 90 días de

T A B L A I. NUM ERO DE TUBERCULO S QUE BRO TARO N EN LOS OCHO CLONES DE Solanum phureja Juz. et Buk. DESPUES DE SER SOMETIDOS A D IFEREN TES DOSIS DE RAYOS GAM M A, EN L A SEGUNDA RAD IAC IO N

Dosis en RClon Ne

0 465 930 1860 3720 5580Total

1 1 1 1 1 0 0 4

10 1 1 2 1 0 0 5

81 1 1 1 1 0 0 4

118 1 2 1 0 0 0 4

1222 1 1 1 1 0 0 4

1386 1 2 1 1 0 0 5

1449 1 1 1 0 0 0 3

7 9 8 5 0 0

460 G O M EZ CUERVO y ESTRADA RAMOS

FIG. 1. Plantas provenientes de tubérculos del híbrido 55-300-1; la de la izquierda fue irradiada con 465 R y la otra es el testigo. Se puede observar el ensanchamiento del pecíolo en la planta tratada.

(FIG. 1. Plants grown from tubers of the hybrid 55-300-1; the one on the left was irradiated at 465 R, the other being the control. Enlargement of the leaf-stalk in the irradiated plant can be observed. )

la fecha de brotación a la cosecha. Las ca rac te r ís ticas de la planta y de los tubérculos observadas en la p r im era generación se conservaron en la segunda.

En la segunda rad iación se trataron tubérculos com pletos de los ocho clones, empleando dos por tratam iento; e l número de plantas producidas por tratam iento aparece en la tabla I. Se observa que las plantas que germ inaron rec ib ie ron una dosis de 0 a 1860 E . Se notó que los p rim eros tubérculos en b rotar fueron aquellos que no rec ib ie ron rad iación y los ú ltim os en brotar fueron los que rec ib ie ron 1860 R , a un m es de d iferencia .

L o s tubérculos producidos no m ostraron reposo respecto a la brotación.Se observaron cam bios en la form a de las hojas y ta llos. En la figura 1

se puede v e r e l ensanchamiento del p ec ío lo de las hojas pertenecientes al h íbrido 55-300-1, proveniente del cruzam iento 159 a 25 (S. tuberosum 2n = 48) x E cuatoriana (S. andigenum 2n = 48), cuyo tubérculo rec ib ió una dosis de 465 R ; estructuras sem ejantes se presentaron en los clones Solanum phureja.

En la te rc e ra rad iación a sem illa asexual se tra taron yemas del clon 81 empleando cuatro dosis : 0, 465, 930 y 1860 R , utilizando quince yemas por tratam iento. Todas las yem as produ jeron plantas que se cosecharon excepto en e l tratam iento de 1860 R, donde se cosecharon sólo once plantas.

M U TA N TE S EN PAPA CRIOLLA 461

E l período vegeta tivo prom edio fue de 180 días. Se observó que en todos los tratam ientos va ria s plantas form aron gran cantidad de fru tos. Las ca ra c te r ís tica s de los tubérculos producidos no fueron alteradas;.

En la tabla II se observa e l número de sem illas sexuales germ inadas y plántulas transplantadas a l campo, provenientes de la rad iación de sem illa sexual; en cada tratam iento se em plearon 50 sem illas . En la p rim era columna se puede ob serva r e l bajo porcentaje de germ inación de los testigos, siendo e l clon 1449 e l que presentó m ayor porcen ta je con 72% y e l de menos e l 118 con 28%; una de las causas del bajo porcen ta je de germ inación fue e l que las sem illas llevaban la rgo tiem po de alm acenam iento. L a reducción de la germ inación en e l clon 1 0 ocu rre a p a rtir de exposiciones superiores a 5580 R . E l clon 118, con exposiciones de 465, 930 y 1860 R , su frió una dism inución en la germ inación de un 50% com parado con e l testigo ; de 3720 R en adelante es mucho más drástica hasta l le v a r a 0 con 8370 R.Las sem illas de los clones 1222, 1386 y 1388 tuvieron un com portam iento s im ila r . En e l caso de la Tocana N egra su germ inación , aparentemente, no se ve muy afectada por la radiación, aunque una dosis como de 1860 R puede a fec ta rla . Se observó que la germ inación de la sem illa del clon 10 es estim ulada con exposiciones de 930, I860 y 3720 R, es d ec ir , en lugar de inducir le ta rgo , com o en los tubérculos, lo rom pe trayendo como consecuencia una m ayor germ inación.

Las figu ras 2, 3 y 4 m uestran los clones 118, 1222 y 1449 respectivam ente, donde se observa e l e fecto de la rad iación en e l porcenta je de germ inación y d esa rro llo de las plántulas con los d iferen tes tratam ientos. E l d esa rro llo de las plántulas se ve afectado por la radiación; algunas se m ueren antes de transplantarlas, como se puede observar en la tabla II, y un número m ayor después de transplantadas.

En la tabla III se encuentra e l número de plantas cosechadas; se observa que e l clon 1388, que tuvo uno de los más altos porcen ta jes de germ inación , con 57%, fue en e l que se cosecharon m enor número de plantas; en este clon, aunque la germ inación no se ve muy afectada por la radiación , casi todas las dosis de exposición em pleadas inciden en el d esa rro llo de las plántulas. En genera l las dosis que más afectan a los demás clones son las de 3720 R o más.

En la c las ificac ión de las ca rac te r ís ticas de los tubérculos no se pudo determ inar con exactitud el e fecto de las radiaciones en e llo s , pues provenían de sem illa sexual y las anorm alidades observadas podían deberse a segregac ión y no a l e fecto de los rayos gamma, debido a que la papa es altam ente heterocigota .

Los tubérculos del clon 10 poseen una buena form a, tamaño mediano, o jos medianamente profundos, p ie l ro ja y carne am arilla . L os testigos sexuales de este clon m ostraron como ca racterís ticas p rincipa les form a m ala y c o lo r de la p ie l am arilla . L os provenientes de sem illa tratada presen taron toda c lase de form as posib les desde la buena hasta tubérculos con configuraciones sem ejantes a muñecos.

L a figu ra 5 m uestra e l producto de cuatro plantas del clon 10; en la parte in fe r io r están los dos testigos , e l sexual (S) y asexual (A ), pudiendo ob serva rse la d ife ren c ia ex istente en cuanto a form a y co lo r . L a parte superior de la m ism a figura corresponde a los tubérculos producidos por dos plantas proven ientes de sem illa sexual que fueron som etidas a un p e ríodo de exposición equivalente a 930 R ; los de la derecha son parte del producto de una planta que se ca rac te r iza ron por su tamaño grande superior


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M U T A N T E S EN PAPA CRIOLLA 463

FIG. 2. Plántulas que germinaron del clon 118 (Solanum phureja Juz. et Buk. ), en los diferentes tratamientos de exposición a los rayos gamma. Las dosis aumentan de derecha a izquierda, siendo la primera el testigo.

(FIG.2. Seedlings which germinated from clone 118 of Solanum phureja. Juz, et Buk. (various doses of gamma-radiation). The doses were progressively higher from right to left, the first row being the control.)

FIG. 3. Plántulas que germinaron del clon 1222 de Solanum phureja Juz. et Buk., en los diferentes tratamientos de exposición a los rayos gamma. Las dosis aumentan de derecha a izquierda, siendo la primera el testigo.

(FIG. 3. Seedlings which germinated from clone 1222 of Solanum phuiej.a Juz. et Buk. (various doses of gamma-radiation). The doses were progressively higher from right to left, the first row being the control.)

464 G O M EZ CUERVO y ESTRADA RAMOS

FIG. 4. Plántulas que germinaron del clon 1449 de Solanum phureja Juz. et Buk., en los diferentes tratamientos de exposición a los rayos gamma. Las dosis aumentan de derecha a izquierda, siendo la primera el testigo.

(FIG. 4. Seedlings which germinated from clone 1449 of Solanum phureja Juz. et Buk. (various doses of gamma-radiation). The doses were progressively higher from right to left, the first row being the control.)

T A B L A III. NUM ERO DE P L A N T A S COSECHADAS PR O V E N IE N TE S DE S E M ILLA SEXU AL, EN L A RAD IAC IO N R E A L IZ A D A A SEIS CLONES DE Solanum phureja Juz. et Buk. Y A TO C AN A NEG RA (Solanum tuberosum L . )

Clon0 465

Dosis en R

930 1860 3720 5580 8370Total

10 3 3 8 6 6 3 0 29

118 10 3 5 5 0 1 0 24

1222 8 8 6 8 6 4 3 43

1388 8 1 3 0 0 2 0 14

Tocana Negra (2n = 48)

1 6 6 4 8 1 0 25

1386 12 12 19 23 8 13 2 87

1449 20 17 18 18 10 3 0 36

M U TA N TES EN PAPA CRIOLLA 4 6 5

LiÇJFIG. 5. Tubérculos del clon 10 de Solanum phureja Juz. et Buk. provenientes de semilla sexual que recibieron diferentes dosis de rayos gamma. Se puede observar la diferencia existente entre el testigo sexual (S) y el asexual (A).

(FIG. 5. Tubers of clone 10 of Solanum phureja Juz. et Buk. grown from sexual seed (various doses of gamma-radiation). The differences between the sexual (S) and asexual (A) controls can be seen.)

a cualquiera de los testigos sexuales o asexuales; además se puede observar lo profundo de los ojos, as í como tam bién e l g roso r de los brotes que es superior a l de los tes tigos . En los tubérculos de la izqu ierda se puede notar la configuración de éstos y e l número de brotes que salen de cada o jo. Estos dos tipos de tubérculos podrían considerarse como mutantes, ya que las ca ra c te r ís tica s an teriorm ente descritas no se han presentado en plantas provenientes de sem illa sexual ni asexual.

L a form a del tubérculo del clon 118 varía de regu la r a buena, su p ie l y carne son am arilla s y su tamaño es mediano; los testigos sexuales presentaron todo tipo de form as, tamaño pequeño, ojos medianamente profundos, p ie l ro ja en su m ayoría y una planta presentó en los tubérculos p ie l ro ja y an illo ro jo ; todas produ jeron bayas. Este clon fue uno de los

4 66 G O M EZ CUERVO y ESTRADA RAMOS

FIG. 6. Tubérculos del clon 118 del Solanum phureja Juz. et Buk. provenientes de semilla sexual que recibieron diferentes dosis de rayos gamma. Obsérvese la diferencia existente entre los dos testigos.

(FIG. 6. Tubers of clone 118 of Solanum phureja Juz. et Buk. grown from sexual seed (various doses of gamma-radiation). The difference between the two controls can be seen.)

que m ostró m ayor susceptibilidad a la rad iación en cuanto a l numéro de plantas cosechadas; en los tubérculos producidos no se observaron cam bios a l com pararlos con los testigos. P a re c e que en este clon las plantas que logran s o b re v iv ir no han sido afectadas por la radiación. La figura 6 , m uestra tubérculos provenientes de los testigos y de un tratam iento de 465 R.

La variedad Tocana N egra (2n = 48) pertenece a la especie S. tuberosum L . , posee tubérculos de buena form a, tamaño mediano a grande, p ie l negra y carne blanca; e l testigo sexual presentó las m ism as ca rac te r ís ticas an terio res , a excepción de su tamaño que fue más pequeño. L a form a de los tubérculos proven ientes de sem illa tratada va r ió de regu la r a buena, predominando la form a buena; e l tamaño fue en su m ayoría pequeño; e l

M U TA N TES EN PAPA CRIOLLA 467

T O C A N A N E G R A

FIG. 7. Tubérculos de la variedad Tocana Negra de Solanum tuberosum L. provenientes de semilla sexual que recibieron diferentes dosis de rayos gamma.

(FIG. 7. Tubers of the Tocana Negra variety of Solanum tuberosum L. grown from sexual seed (various doses of gamma-radiation).)

co lo r de la p ie l, junto con la profundidad de los ojos y la form a de las cejas fueron los ca racteres más afectados por la radiación . La p ie l en algunos casos fue ro ja , en otros negra y en algunos presentó m ezc la de los dos co lo res . La figu ra 7 m uestra e l efecto ocasionado por la rad iación en los tubérculos de Tocana N egra . En la parte in fe r io r izqu ierda se observa e l testigo sexual que presenta buena form a, co lo r negro y tamaño pequeño; además se m uestran tubérculos provenientes de dos tratam ientos de 1860 y 3720 R, en los que se ha cambiado su co lo r a ro jo . F inalm ente, en los tubérculos de la parte superior izqu ierda, se observa que e l co lo r de la p ie l es más c la ro que e l de los otros tubérculos de la figura, la form a de las cejas es prolongada a m anera de dientes de s ie r r r a que cubren los ojos y se puede notar, además, la form a alargada de los tubérculos.


De los resu ltados obtenidos se pudo conclu ir lo sigu iente:Una vez irrad iados, los tubérculos o yem as su frieron un período de

retardo en la brotación; este retardo aumentó a medida que aumentaba la dosis de exposición a los rayos gamma. En la m ayoría de los casos la germ inación de la sem illa sexual disminuyó cada vez más a medida que aumentaba la dosis, hasta lle g a r a 8370 R donde prácticam ente ya no hubo germ inación.

Las sem illas que germ inaron no a ltera ron su período de germ inación, es d ec ir , no hubo inducción de le ta rgo como en e l caso de los tubérculos.En algunos casos se estim uló la germ inación de la sem illa con determ inadas dosis, como en e l clon 1 0 .

La brotación temprana de los tubérculos de papa c r io lla , Solanum phureja Juz. et Buk., se a lteró obteniéndose una planta que produjo tubérculos que m ostraron reposo por siete m eses.

L a incom patibilidad entre e l polen y e l estigm a fue otro ca rác te r que se cam bió mediante e l em pleo de rayos gamma.

L a dosis de rad iación que se debe em plear depende de cada clon; a lgunas dosis no afectan a la germ inación pero s í al d esa rro llo de las plántulas y en otros clones es lo con trario . L a sem illa asexual res is te dosis más altas que la sexual.

Es más fá c il ob servar los cambios en tubérculos provenientes de s em illa asexual que los procedentes de sem illa sexual, porque en estos últim os los cambios que se presenten se pueden deber a segregac ión y no a afectos de la radiación .

A pesar de que la m ayoría de los cambios observados fueron des fa vora b les, tam bién se encontraron plantas con ca racteres deseables que pos ib lem ente en un futuro podrán usarse para cruzam ientos o análisis genéticos.


[1] HEIKEN, A., Induction of somatic changes in Solanum tuberosum by acute gamma irradiation, Hereditas 47 (1961) 606.

[2] PERDOMO, M.A., HERNANDEZ, J.A., SANIN, J., Preservación de tubérculos de papa por medio de radiaciones gamma, Instituto de Asuntos nucleares. Bogotá (1963) 15.

D I S C U S S I O N

H. SM ITH : I w ish to ca ll attention to a m ost unusual mutation in Solanum tuberosum produced with ion izing radiation by D r. O. Chita, U n ivers ity o f C ra iova , Rom ania. Th is mutant is ch aracterized by producing potato tubers on the a e r ia l parts o f the plants. It was reported in a Romanian publication in 1969 o r 1970.

P . L . GOM EZ CUERVO : I did not know aboutthat mutant, but as I have stated in m y paper, no in form ation is ava ilab le on e ffo rts to induce mutations in Solanum phureja Juz. et Buk.

M .S . SW AM IN ATH AN : In your seed treatm ent, you obtained mutants re la tin g to tuber colour and shape. Did they occur in the tubers set in the irrad ia ted seeds, i . e . in the M i generation?

P . L . GOM EZ CUERVO : Yes, they occurred in the Mj, generation o f treated sam ples o f both sexual and asexual seeds. In the mutant produced

M U TA N TE S EN PAPA CRIOLLA 4 6 9

in o rd e r to in crease the dorm ancy period this character did not change until the M 4 generation , which was the last generation studied.

M .S . SW AM IN ATH AN : You reported that treatm ent o f tubers with doses above 1800 rad resu lted in lack o f germ ination. One prob lem in potato tuber irrad ia tion is the radiation-induced sprout inhibition. You can g ive higher doses by keeping the irrad ia ted tubers fo r a few months until they sprout o r by trea tin g the tubers a fter irrad ia tion with dorm ancy - breaking chem icals.

C. BROERTJES: I noted quite a number o f skin colour mutants in your m ateria l. What was the percentage o f such events?

P . L . GOM EZ CUERVO : It is true that one o f the characters m ost a ffected by gam m a-rad iation during this work was the colour o f the skin and flesh , and another was the shape o f the tubers. The percentage o f colour mutants was not recorded , however, as the object o f the work was to obtain mutations resu lting in e a r lie r sprouting o f the tuber and to observe the behaviour o f the pollen and stigm ata with rega rd to se lf-in com patib ility .

OBTENCION DE GIRASOLES ANDROESTERILES

C. REMUSSI, H. SAUMELL, G. VIDAL

Facultad de Agronomía y Veterinaria,

Universidad de Buenos Aires,

Buenos Aires, Argentina

Abstract-Resumen

DEVELOPMENT OF MALE STERILE SUNFLOWERS.During the spring of 1966 seeds of the sunflower varieties Precoz FAV and Vinnik 1646 were irradiated

with gamma-rays from a 60Co source. The doses applied were 6, 12 and 18 kR. Detailed study of these seeds in the following years allowed the identification of male sterile plants, which were crossed with sister lines, lines with high male sterility and commercial varieties. In the sowing of 1969/70, 17 lines were found with high male sterility, one reaching 70%. The male sterile lines originate from one of the inflorescences harvested in 1967 from seeds of the Vinnik variety which received 6 kR. Work to meet the objectives of this experiment will follow during the present year's crop.

OBTENCION DE GIRASOLES ANDROESTERILES.Durante la primavera de 1966 fueron irradiadas con rayos gamma semillas de girasol de las variedades

Precoz FAV y Vinnik 1646. Se aplicaron dosis de 6000, 12000 y 18000 R. El estudio detallado de estas semillas en los años siguientes permitió encontrar plantas macho estériles. Estas fueron cruzadas con polen de plantas hermanas, lineas con alto grado de androesterilidad y con variedades comerciales. En la siembra de 1969/70 se encontraron 17 lfneas con alto grado de androesterilidad, alcanzando una de ellas más del 70%.Las líneas macho estériles se originaron de uno de los capítulos cosechados en 1967 de semilas de la variedad Vinnik, que recibió 6000 R. Durante la cosecha de este año se proseguirá con esta experiencia..

La m anifestación de la heterosis o v ig o r h íbrido en g ira so l ha sido dem ostrada, tanto en experiencias ex tran jeras com o en las de nuestro país.

Unrau y White [1] obtuvieron un híbrido sim ple con un rendim iento que supero en un 40% a l de una variedad com erc ia l u tilizada como testigo , y en un 50% al de la m e jo r de las líneas em parentadas. Kinman y E a rle [2] estudiaron e l com portam iento de cuatro híbridos in terlíneas en cuatro es ta ciones experim enta les de los Estados Unidos; a l com parar los rendim ientos con la variedad Vinnik 1646 utilizada como testigo , hallaron que en algunos híbridos los rendim ientos duplicaron a los del testigo . En Chile, Bugueño V a ld iv ia [3 ], ensayó híbridos sim ples y dobles encontrando que gran cantidad de los m ism os superaban a la variedad K lein u tilizada com o testigo ; los porcentajes de aumento llega ron a un 59% del rendim iento en sem illa y, además, algunos contenían un m ayor porcentaje de aceite .

En la A rgentina, Kugler, Luciano y Davreux [4 ], en la Estación E x p e r im ental de Pergam ino, en ensayos efectuados durante s iete años, hallaron que e l rendim iento de los h íbridos duplicaron en algunas ocasiones e l del testigo , en este caso Selección K lein .

P e ro la producción com erc ia l de sem illa híbrida en g ira so l trop ieza con e l inconveniente que al aparear dos líneas endocriadas, no sólo se produce cruzam iento entre e llas , sino que tam bién ocurren cruzam ientos entre h e r manos y autofecundaciones. E llo hace que e l porcentaje de sem illa híbrida obtenida sea muy ir re g u la r y va riab le de año a año. '

La obtención de un macho e s té r il genético no lle g a r ía a solucionar in te gra lm ente e l problem a, ya que en e l m ateria l segregante habría que e lim in ar

4 7 1

4 7 2 REMUSSI e t a l .

a campo las plantas macho fe r t ile s antes de la flo ración , basándose en algún ca rác te r cualitativo ligado a la macho esterilidad , ta l como fue hallado en F ran c ia por Mme L e c le rc q [5] .

L o idea l s e r ía ha llar un citoplasm a que actuara sobre los factores genéticos que condicionan la androesterilidad , ta l como se ha conseguido en otras espec ies . Otra posibilidad ser ía e l aprovecham iento de la androesterilid ad parc ia l, que presenta las siguientes ventajas; facilidad de h ibri- darse en parcelas de cruzam iento natural; posibilidad de se lecc ion ar líneas con muy baja proporción de polen norm al; fá c il propagación y, por último, la Fx de los h íbridos que incluyen este ca rác te r en genera l producen so la mente polen norm al, ta l como fue dem ostrado por Putt y H e iser [6 ] .

Com penetrados de la im portancia que podría tener para e l país una contribución a la solución de este problem a, en la p rim avera de 1966 la Cátedra de Cultivos Industria les hizo ir ra d ia r sem illas de g ira so l de las variedades Vinnik 1646 y P re co z F A V , con intensidades de 6000, 12 000 y 18 000 R en e l Centro de Radiobiología de la Facultad.

D icho m ateria l junto con otro sin tratam iento, considerado como testigo , se sem bró en e l campo experim ental que posee dicha Cátedra durante e l mes de noviem bre de ese año en parcelas apareadas.

Las plantas as í obtenidas quedaron durante todo su período de flo rac ión en estado de fecundación lib re , por cuyo m otivo los an droestérilés hallados, como se v e rá más adelante, pueden o no haber sido provocados por la irrad ia c ión . Las observaciones se h ic ieron durante todo e l período vegeta tivo , siendo identificadas aquellas plantas que presentaban ca rac te r ís ticas m o rfo lóg icas a lteradas, ya sea én ta llos, hojas y especia lm ente en deform aciones de sus capítulos.

A cosecha se reco lec ta ron así 62 capítulos de las parcelas tratadas y algunas de las no tratadas para su pos te r io r com paración, desgranándose individualm ente y conservando hasta la siem bra.

La m ism a se rea lizó a princip ios de noviem bre de 1967, y durante e l c rec im ien to y d esa rro llo de las plantas, especia lm ente en estado de florac ión , se proced ió a e fectuar detenidas observaciones identificándose solam ente 9 plantas, cuyos capítulos presentaban ca ra c te r ís tica s que en ta l oportunidad fueron consideradas de im portancia, y por lo tanto se controló su fecundación. Seis de e llos poseían anteras sin polen y f lo re s de co lo r norm al.

Es im portante destacar que de un capítulo v io lá ceo de poco polen to ta lmente autofecundado, se cosecharon 1063 granos vanos bien form ados y ninguno fé r t il; en e l resto de los capítulos los porcentajes de fertilid ad va ria ron según e l tratam iento, pero siem pre fueron bajos, notándose alta producción de frutos vanos bien desarro llados.

Cosechados, trillados individualmente y lim pios se obtuvieron unos 6000 aquenios fé r t ile s .

Se p roced ió a su s iem bra en la p rim avera de 1968, agregando algunas parcelas con sem illas de plantas hermanas autofecundadas, de aquellas que habían dado bajo porcentaje de fertilidad .

E l estudio detenido en e l momento de la flo rac ión y a medida que ésta se producía, p erm itió ind iv idua lizar en las distintas parcelas plantas con las siguientes ca ra c te r ís tica s flo ra les :

- capítulos sin polen, 63 c o lo r norm al y 6 c o lo r v io láceo ;- capítulos con muy poco polen, 2 co lo r norm al y 1 co lo r v io láceo ;- capítulos con poco polen, 9 c o lo r norm al y 14 co lo r v io láceo ;

OBTENCION DE GIRASOLES ANDROESTERILES 473

- capítulos con m u c h o polen,pero com pletam ente aglutinado, 14 co lo r norm al y 3 co lo r v io láceo .

A la apertura de cada capítulo y luego de eva luar sus ca ra c te r ís tica s se proced ió a l control de su fecundación, optándose tam bién este año por re a liz a r la m ayor cantidad de cruzam ientos por herm anos, y e l resto por macho fé r t ile s segregantes de «m acho e s té r ile s de T e x a s » , por líneas determ inadas, y/o por fecundación lib re . Se p roced ió así pensando en la m ultip licación del m ateria l.

Cosechados, c las ificados y lim pios los frutos en a b ril de 1968, nos hallam os con e l hecho de haber logrado poco m a teria l fé r t i l , proven iente éste , en genera l, de los sec tores de capítulos donde se p erm itió fecundación lib re , o de algún cruzam iento controlado entre líneas, muy escaso o nulo en las autofecundaciones o cruzam ientos por hermanos, y nulo com pletamente cuando e l padre fue polen originado por segregación del «m acho e s té r il de T e x a s » .

Un p r im er análisis de lo acontecido en este últim o año, p erm itir ía suponer que podríam os hallarnos con líneas de m ateria l altamente autoincom patib le .

La ausencia de fe rtilid ad se presentó, com o en e l año an terior, de distinta m anera: fru tos vanos bien form ados (sem ejan tes a los fé r t ile s perosin sem illas o con rudimentos de e lla ), y luego decrecientem ente fru tos vanos regu la res y pequeños, y en casos particu lares, f lo res sin fecundar. Existen parce las que dan una tendencia p re fe ren c ia l a cada una de estas m odalidades.

En la campaña 1969-70 se sem bró parte del m ateria l cosechado, en contrándose 17 líneas con alto porcentaje de androesterilidad , una de las cuales alcanzó a l 70%. Este m ateria l fue cruzado con polen de plantas herm anas, de líneas con alto grado de androesterilidad y con variedades com erc ia les . Adem ás, se u tiliza ron en los cruzam ientos polen de capítulos de c o lo r norm al y ro jo v io lá ceo .

Es im portante m encionar que las líneas an droestériles provienen, en su totalidad, de uno de los capítulos cosechados en 1967, originado de sem illa de la variedad Vinnik 1646 irrad iada con 6000 R.

En posesión de este m ateria l para e l corrien te año se proyecta:

a) T ra ta r de lo ca liza r algún ca rác te r cualitativo ligado a la androesterilidad , que perm ita e lim in a r en e l campo los individuos macho fé r t ile s en el m a ter ia l segregante.

b) E fectuar cruzam ientos controlados con herm anos, g ira so les s ilv e s tre s y variedades, tratando de ha llar un citoplasm a que actúe sobre los factores genéticos que condicionan la androesterilidad.

c) S e leccionar individuos con androesterilidad parcia l, y probar su aptitud com binatoria a campo con variedades com erc ia les de reconocido rend im iento y calidad industrial.


[1] UNRAU, I., WHITE. W.J., The yield and other characters of inbred lines and single crosses of sunflower, Scient.Agrie. 24(1944) 11.

[2] KINMAN, M.L., EARLE. E.R., Agronomic performance and chemical composition of the seed of sunflower hybrids and introduced varieties. Crop Sci. 4(1964) 417.

4 74 REMUSSI et a l.

[3] BUGUEÑO VALDIVIA, V., PUTT, E.D., Métodos de mejoramiento en maravilla, Bol.Técn.№7, Ministerio de Agricultura, Santiago de Chile (1944).

[4] KUGLER, W.F., LUCIANO, A., DAVREUX, M., Mejoramiento del girasol en Pergamino, Comunicaciónpresentada a la IV Reunión Latinoamericana de Fitotecnia, Santiago de Chile (1958).

[5] LECLERCQ, P., Une stérilité utilisable pour la production d'hybrides simples de tournesol, Annls Amél.Pl.16(1966) 134.

[6] PUTT, E.D., HEISER, C.B., Male sterility and partial sterility in sunflower. Crop Sci. 2(1966).

D I S C U S S I O N

H. SMITH: It was not c lea r to me i f you had any in form ation on whetherthe induced m ale s te r ility is controlled by factors in the nucleus o r in the cytoplasm .

C. REMUSSI: The m ale s te r ile s obtained so fa r are o f the genetic typeand depend on a re c ess iv e gene. I am not aware that a cytop lasm ic m ale s te r ile has yet been d iscovered .

C .E . M UHLENBERG: What w ere the rea l causes o f fa ilu re o f gam etesto produce m ale s te r ility in the sunflower?

C. REMUSSI: Sm all doses had little e ffec t but la rge doses a ffected bothm ale and fem ale organs.

G. DE A L B A : Was there any evidence o f fem ale s te r ility associatedwith the m ale s te r ility?

C. REMUSSI: No. The fem ale organ was com pletely norm al: in everycase where the in flo rescence o f the m ale s te r ile sunflower was allowed to pollinate fr e e ly , norm al fe rtiliza tion occu rred and the seed produced was fe r t i le .

MUTACIONES INDUCIDAS POR IRRADIACION EN EL PERAL PACKHAM1 S TRIUMPH

F. ROBYInstituto Nacional de Tecnología Agropecuaria (IN TA ),Mendoza, Argentina

Abstract-Resumen

MUTATIONS INDUCED BY IRRADIATION IN THE PEAR PACKHAM'S TRIUMPH.In 1959, a given quantity of twigs separated from the mother plant were treated with X-irradiation

of 2000, 4000, and 6000 R; the scions were then grafted on French rootstocks. In 1961, the 2-65 samples thus obtained were planted. When these plants began their fructification, nine mutants could be selected; eight of these arose from the 6 0 0 0 R irradiation, which, of the three applied doses, was the optimum and the nearest to that considered lethal. The mutants obtained show alterations in the tree, and fundamental changes in the fruits. The undesirable induced changes always surpass the favourable ones. Only one of the mutants obtained deserve our special attention.

MUTACIONES INDUCIDAS POR IRRADIACION EN EL PERAL PACKHAM'S TRIUMPH.En el afio 1959 se procedió a tratar con rayos X una determinada cantidad de ramitas separadas de

la planta madre, las cuales fueron sometidas a irradiaciones de 2000, 4000 y 6000 R, y posteriormente injertadas de púa sobre patrones francos. En 1961, se plantaron los 265 ejemplares así obtenidos. Al entrar éstos en fructificación, se lograron seleccionar nueve mutaciones; ocho de ellas provinieron de la irradiación de 6000 R que, de las tres ensayadas, resultó ser la dosis óptima de aplicación y más próxima a la considerada letal. Las mutantes conseguidas muestran alteraciones en el árbol y fundamentalmente sobre los frutos. Los cambios indeseables originados siempre superan en mucho a los favorables. Solamente una de las mutaciones logradas merece especial interés.

INTRODUCCION

E l p era l Packham ' s Trium ph se ca ra c te r iza por las excelentes cualidades y buena aceptación com erc ia l que poseen sus fru tos. Es, después de W illiam s ' Bon Chrétien , uno de los cu ltivares más difundidos en e l país, habiendo ya encontrado una favorab le aceptación entre los fru ticu ltores del A lto V a lle del R ío N egro y Mendoza [1 ].

En 1955 nos propusim os la tarea de obtener una m ejora para este cu ltivar. Nuestro ob jetivo consistía en reun ir las buenas ca rac te r ís ticas que poseen los fru tos de Packham 1 s Trium ph, con una época convenientemente más tem prana de maduración, suprim iendo, de s e r posib le, ese leve dejo o sabor am argo que tiene su pulpa. Pers igu iendo esos propósitos, se efectuó entonces un cruzam iento de W illiam s con Packham 1 s Trium ph. De los num erosos individuos obtenidos con posterioridad - ahora ya en plena fru ctificac ión - , se ha seleccionado uno que parece poseer esas ca rac te r ís ticas deseadas y, en particu lar, porque la época de maduración de sus fru tos es mucho más tem prana. Se lo está ahora propagando, con e l fin de som eterlo luego a un estudio más profundo antes de darlo a conocer como nuevo cu ltivar.

En 1959, la Com isión Nacional de E nerg ía A tóm ica , por in term ed io del Ing. A g r . Santos Soriano, o fre c ió e l uso de sus instalaciones destinadas

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MUTACIONES EN EL PERAL 477

a fines pac íficos para p rom over e l m ejoram ien to de las plantas cultivadas. Aprovechando esta oportuna invitación se p roced ió a ir ra d ia r ram itas de Packham ' s Trium ph para in je r ta r , con e l propósito de inducir mutaciones favorab les en este pera l.

Una labor s im ila r a la que aquí se expone, pero buscando otros obje tivos d istin tos, ha rea lizad o e l autor con e l cu ltiva r W illiam s ' Bon Chrétien [2].

M A T E R IA L Y METODO

A fines de ju lio de 1959, en las instalaciones que la Com isión Nacional de E n erg ía A tóm ica posee en Buenos A ir e s , se p roced ió a tra ta r con rayos X 150 ram itas de p era l Packham ' s Trium ph, proven ientes de plantas de la co lecc ión pom ológica que e l autor había implantado con an terioridad en dependencias de la Estación E xperim en ta l Agropecuaria de Mendoza. Las ram ita s -in je rto s fueron som etidas a irrad iac ion es de 2000, 4000 y 6000 R, e n tr e s grupos de 50 unidades, respectivam ente.Se m antuvieron luego convenientemente p reservadas en cám aras fr ig o r íf ic a s , hasta e l m omento oportuno para su in jertación .

A princip ios de septiem bre de 1959, en e l V iv e ro F ru tíco la de Rama Caída (San R a fael, M endoza) se p roced ió a in je rta r , en ta lle r y sobre p ies francos, púas de esas ram itas irrad iadas. P o r im pedim ento de orden c lim ático , las plantas in jertadas as í obtenidas rec ién estuvieron en condiciones de co loca rse defin itivam ente en e l te rren o a princip ios de septiem bre de 1961. En e l campo de la Estación E xperim en ta l A g r o pecuaria de Mendoza, en Luján de Cuyo, se plantaron 100 e jem p lares que correspondían al tratam iento de 2000 R, 90 a l de 4000 R y 75 a l de 6000 R. Cabe señalar que, del to ta l de 265 e jem p la res plantados, sólo se obtuvieron nueve mutaciones; una de e llas procede del tratam iento de 2000 R , y las ocho restantes provienen de los que fueron som etidos a la dosis de 6000 R, que es la más próx im a a la considerada leta l.

Las mutaciones as í obtenidas se las designa con un número de orden, que corresponde a l de la h ile ra seguido por e l re fe ren te a la planta.

V a rios e jem p la res de cada una de estas mutaciones han sido ya propagados por in jertac ión y se encuentran defin itivam ente implantados en la Estación E xperim en ta l A gropecuaria de Mendoza.

RESULTADOS

Una m inuciosa descripc ión de las ca ra c te r ís tica s propias del cu ltivar Packham ' s Trium ph fue publicada ya por e l autor [1 ]. En consecuencia, sólo corresponde exponer seguidam ente, a l d esc r ib ir cada una de las mutaciones obtenidas, los cambios que éstas experim entaron por e fecto de la ap licación de rayos X.

Mutación 56-2 (figu ra 1)

La púa in jertada p roviene de una ram ita som etida a un tratam iento con rayos X de 2000 R. E l á rbo l es pequeño y débil.

478 ROBY


Los frutos experim entaron considerab le reducción de tamaño y percep tib les deform aciones; m uestran surcos y protuberancias en la su perfic ie com prendida entre la cavidad ca lic ina l y e l cuello; e l sabor de la pulpa no su frió varian te.

Las ca ra c te r ís tica s no deseables que se mencionan, son causas su fic ientes para rech azar esta mutación.

Mutación 59-2 (figu ras 1 y 2)

Se orig inó de un tratam iento efectuado con 6000 R de rayos X. La planta tiene m ediano v igo r ; su flo rac ión se produce con algún re traso respecto a l cu ltivar que le dio origen , lo que resu lta ventajoso, dada la flo rac ión algo anticipada que posee esta últim a.

Los fru tos conservan su form a norm al, notándose que se ha producido alguna reducción de tamaño; la ep iderm is se ca ra c te r iza por poseer un c o lo r de fondo más verd e y estar cubierta de puntos y manchas de co lo r b erm ejo , estas ú ltim as más notables hacia la cavidad del cá liz y e l ped icelo ; la pulpa m e jo ró en jugosidad y sabor, no presentando la aspereza propia de la Packham ' s Trium ph.

Los frutos tienen la m ism a gran res is tencia en la conservación que los del cu ltivar que le dio o rigen , y se ha conseguido tam bién m antenerlos en buenas condiciones hasta se is m eses y m edio en cám aras fr ig o r íf ic a s .

Esta mutación, que es la más interesante lograda hasta ahora, no llega a m e jo ra r la o rig in a l, sa lvo en ese re tra so de su época de f lo ra ción y en las ca ra c te r ís tica s de la pulpa ya m encionadas.


P rov ien e de la aplicación de 6000 R de rayos X . E l á rbol es de v ig o r m edio y su flo rac ión se lle va a cabo con un leve re tra so sobre la época norm al.

Los frutos m od ificaron su form a y la co lorac ión de la p ie l, pero mantienen e l tamaño natural. Son a lgo a largados, con cuello más cónico y menos pronunciado que en los testigos.

La ep iderm is tiene, en la superfic ie expuesta a l so l, una mancha de co lo r b erm ejo sobre la que se destaca un leve tinte ro jizo . La pulpa es jugosa, agradable y sin e l sabor áspero típ ico de la pera Packham ' s Trium ph.

Se observa que ha dism inuido sensiblem ente la res is ten c ia en la conservación fr ig o r íf ic a de los frutos.


Originada por la ap licación de 6000 R de rayos X . La planta es pequeña y débil.

Los fru tos, que son de tamaño mediano a grande, denotan un leve a largam ien to de l cuello. La ep iderm is está poblada de num erosos puntos pardos en toda la su perfic ie , y sólo ocasionalm ente presenta manchas de co lo r b erm ejo . La pulpa tom ó un sabor más áspero que en la pera o rig ina l, vo lviéndose desagradable.

480 ROBY

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La yem a in jertada que indujo esta mutación proviene de una ram ita som etida a una irrad iac ión de 6000 R. E l á rbo l es muy pequeño y débil.

Sus fru tos, que tienen tamaño chico, son de fo rm es , con su perfic ie protuberante y acanalada. E l c o lo r de la ep iderm is y e l sabor del m esocarp io no su fr ie ron m od ificaciones.

Los frutos de esta mutación se han vuelto com ercia lm en te inaceptables por pérdida de calidad.


Se obtuvo mediante una irrad iac ión de 6000 R. La planta se m uestra v igo rosa .

Los fru tos, que tienen form a alargada, tamaño considerablem ente reducido y ep iderm is parcia lm ente cubierta de manchas pardas, su frieron c ie r to retardo en su época de maduración.

Esta mutación perd ió su calidad y e l va lo r o rig in a l como consecuencia de las ca ra c te r ís tica s re g re s iva s adquiridas.


Se orig inó con una irrad iac ión de 6000 R. E l á rbo l es de mediano v ig o r , y las ram itas de un año tom aron una co lorac ión parda más am arillen ta que en la Packham 1 s Trium ph.

En los frutos se observan m od ificaciones de tamaño, form a, c o lo ra ción de la p ie l y pecu liaridades de l ped ice lo , que no contribuyen a m e jo ra r las ca ra c te r ís tica s propias del cu ltiva r que le dio origen . E l tamaño es más reducido; su form a más achatada, con cuello corto ; la ep iderm is tomó una co lorac ión más am arillen ta , siendo las len tice las grandes y b erm e jiza s ; e l ped ice lo - por ú ltim o - es notablemente más grueso y corto que en los fru tos o rig in a les . T ienen, adem ás, buena conservación fr ig o r íf ic a .


Se orig inó mediante una irrad ia c ión de 6000 R. La planta es v igorosa .Los frutos no su frieron cam bios en e l sabor de la pulpa y ca racterís ticas

del ped ice lo , pero experim entaron m odificaciones de form a, tamaño, y co lorac ión de la p ie l. Su fo rm a es a lgo alargada; e l tamaño no un iform e - con frutos ch icos, medianos y grandes - pero con tendencia a reducirse; la ep iderm is se cubrió de puntos y'm anchas b erm e jiza s . Los cam bios que se han producido en esta mutación, como consecuencia de la ap licación de rayos X a la yem a in jertada que la engendró, no m ejoran las ca ra c te r ís tica s de los fru tos de origen .


P rov ien e de la ap licación de un tratam iento de 6000 R de rayos X.E l árbo l, que es de tamaño mediano a chico, f lo re ce a lgo más tardíam ente que e l cu ltivar que le dio origen ; sus frutos tienen, tam bién, una época más tard ía de m aduración.

482 ROBY

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Se observan en los frutos las m odificaciones sigu ientes: dism inución de tamaño; form a redondeada, con cuello menos prom inente; ped icelo más corto; ep iderm is con puntos y manchas berm e jizas preferen tem ente dispuestas hacia la cavidad del c á liz y e l ped ice lo , y, a v eces , con vetas de co lo r b erm ejo que se extienden a lo largo del fruto.

Esta mutación, tampoco aporta m ejora alguna para e l cu ltivar de origen .

CONCLUSIONES

Nueve mutaciones de Packham 1 s Trium ph se orig inaron mediante la ap licación de rayos X a ram itas u lteriorm en te in jertadas. Sólo una mutante se obtuvo del tratam iento de 2000 R; no se alcanzó ningún cam bio percep tib le con la irrad iac ión de 4000 R; las ocho mutaciones restantes fueron inducidas por ap licación de 6000 R, que, para e l caso particu lar, resu ltó s e r la dosis óptim a, aunque peligrosam ente próx im a a l punto le ta l.

Las mutantes as í obtenidas muestran a lteraciones re feren tes a tamaño y v ig o r del árbo l, co loración de las ram itas de un año y retardo en la época de florac ión ; pero los cambios fundamentales se advierten sobre d iferen tes ca ra c te r ís tica s de los fru tos, a saber: no uniform idad y va r ia c ión del tamaño respecto a l considerado norm al; transform aciones y deform aciones varias ; d iversas m odificaciones concern ientes si c o lo ra ción de la ep iderm is y sabor del m esocarp io; retardo en la época de maduración, y dism inución del período de conservación . Se advierte tam bién aquí que las ap licaciones rad iactivas provocan muchos más cam bios de ca ra c te r ís tica s indeseables que favorab les . E sto confirm a la aprec iac ión del autor, ya expuesta en otro trabajo s im ila r a éste pero re fe ren te a l p era l W illiam s [2 ].

De las nueve mutaciones logradas, solam ente la que lleva e l número de orden 59-2 m erece algún in terés especia l. Esta conserva la m ayor parte de las ca rac te r ís ticas propias de la Packham 1 s Trium ph, y, aunque perd ió parte de la natural lim p idez que tiene la ep iderm is del fru to, m e jo ró en cambio la calidad de su pulpa, vo lviéndose más jugosa y menos am arga que en la pera o rig in a l. M an ifiesta , a la v e z , un lim itado pero ventajoso re tra so en su época de floración .

E X P R E S I O N D E A G R A D E C I M I E N T O

E l autor desea exp resar su agradecim iento al Ing. A g r . Rodolfo A .G h e lfi, por la aplicación de rayos X efectuada a l m ateria l o r ig in a l en la Com isión Nacional de E n erg ía A tóm ica , a l Ing. A g r. Julio C ésar Gatica, por la conducción de las labores de in jertac ión rea lizadas en e l V iv e ro F ru tíco la de Rama Caída, y al técn ico fo tógra fo Hugo N. Rabino, por las tomas fo tográ ficas que ilustran este trabajo.


[ 1] ROB Y, F. , Las principales variedades de peral cultivadas en Argentina, Rev. Inv. Agrie. V I I I (1953) 52-55.

[2 ] ROB Y, F ., Doce mutaciones en el peral Williams obtenidas por injertación de ramitas irradiadas,VIII Reunión Latinoamericana de Fitotecnia, Bogotá, Colombia (1970),

484 ROBY

G. DE A L B A : Have subsequent mutations been observed in the mutants obtained?

F . ROBY: No, not so fa r .G. DE A L B A : One of the mutants that you considered could be d is

carded seem ed to me to be v e ry a ttractive in colour, shape and s ize . Could you say something about the quality o f this mutant.

F . ROBY: You a re re fe r r in g to mutant 69-52. The taste o f the flesh was s im ila r to that of the orig ina l. In my view the mutant was in fe r io r in com m erc ia l quality with respect to s ize and shape, but not as regards skin colour.

F .R . D IAZ : With re fe ren ce to mutant 59-2, is fru it shape uniform or does it show fluctuations? Is y ie ld acceptable? W ere there d ifferences in se lf-com p a tib ility between pollen and ovum?

F . ROBY: F ru it shape is uniform and does not show fluctuations.Up to now we have observed that productivity has not varied with respect to the o rig in a l va r ie ty . Th is mutant does not show prob lem s of s e l f com patib ility .

C. BROERTJES: The last sentence o f the English sum m ary reads: "O n ly one of the mutants ... " , and I would like to object to the word "o n ly " . Considering the res tr ic ted number of scions irrad ia ted as w e ll as the few mutants obtained, having one p rom ising one is not bad. Fu rth erm ore , I would advise you to en large the s ize of your e x p e r im ents, because we know that one needs hundreds o f mutants in order to im prove the chance to find one o r a few mutants with im proved characters without a lterin g the rem aining combination of favourable characters .

F . ROBY: I mention the word "on ly " because I consider that the o rig in a l va r ie ty has been im proved exc lu s ive ly in taste while at the same tim e other characters lost quality.

It would have been m y wish to work with a h igher number o f plants, but as you know resea rch in fru it tre e s ca lls fo r quite a number of years to reach fru ctifica tion and g ive resu lts . A ls o it must be considered that a g rea t extension o f land is requ ired to maintain a high number of individuals.

D I S C U S S I O N

EL USO DE RADIACIONES EN EL MEJORAMIENTO DEL BANANO (Musa sapientum L. )

J. VELEZ FORTUNO Departamento de Genética,Estación Experimental Agrícola, Río Piedras

A . CEDEÑO MALDONADO Subestación Experimental Agrícola,Fortuna, Puerto Rico

Abstract-Resumen

USE OF RADIATION IN BREEDING BANANA ( Musa sapientum L. )In spite of its high fruit quality, the banana variety "Gros Michel" has been rapidly replaced by varieties

of the "Cavendish" type, due to its great susceptibility to Fusarium oxysporum cubensis and Cercospora musae. With the aim of developing resistant types with high fruit quality, rhizomes of the variety "Gros Michel" were treated with 2.5 - 40 kR gamma-rays. Germination occurred only up to 5 kR, the surviving plants showing some growth reduction but developed flowers and fruits. From the M2 generation, two mutants were selected, one showing drastic leaf deformations, the other characterized by a more intensive pigmentation and small morphological differences, but comparable to the control in height, vigour and fruit characters. This latter mutant will now be tested for disease resistance.

EL USO DE RADIACIONES EN EL MEJORAMIENTO DEL BANANO( Musa sapientum L .).A pesar de la elevada calidad de su fruto, la variedad de banano Gros Michel está siendo desplazada

rápidamente por las variedades del tipo Cavendish, debido a su gran susceptibilidad al Fusarium oxysporum cubensis y a la Cercospora musae. A fin de obtener tipos resistentes con frutos de elevada calidad, se han tratado rizomas de la variedad Gros Michel con rayos gamma, con dosis totales de 2, 5 a 40 kR. Sólo hubo germinación hasta 5 kR, presentando las plantas supervivientes cierta disminución del crecimiento, si bien florecieron y fructificaron. A partir de M2, se seleccionaron dos mutantes, uno con hojas deformes y el otro caracterizado por una pigmentación más intensa y ligeras diferencias morfológicas, pero semejante al testigo en altura, vigor y caracteres de la fruta. Este último mutante se ensayará ahora para determinar su resistencia a las enfermedades.

En Puerto R ico, las investigaciones sobre e l m ejoram ien to de plantas mediante mutaciones inducidas han sido muy lim itadas. Los recu rsos d is ponibles se han asignado principalm ente a l program a de fitom ejoram ien to por los m étodos convencionales.

Sin em bargo se han rea lizado estudios en esca la lim itada sobre e l f i to m ejoram ien to mediante mutaciones inducidas desde e l año 1957. En dicha fecha se in ic iaron estudios para e l m ejoram ien to del gandur ( Cajanus cajan) y la guayaba ( Psidium guajaba) utilizando radiaciones gamma y se logra ron muy buenas se lecc iones tanto en producción como en calidad.

A l obtenerse resu ltados muy prom etedores con estos cu ltivos, en 1964, e l difunto D r. H. A zzam [ l ] , in ic io estudios lim itados sobre la in ducción de mutaciones en e l banano ( Musa sapientum L . ) u tilizando la v a r ie dad Gros M ichel. Desgraciadam ente e l D r. A zzam fa lle c ió en e l año 1965 y no pudo v e r e l resu ltado de su labor. No obstante, continuamos e l estudio del cual presentam os en esta comunicación algunos de los resultados obten idos.

485

486 VELEZ FORTUNO y CEDEÑA MALDONADO

E l G ros M ichel fué la variedad de banano de m ayor im portancia y a base de e lla fue que se d esa rro lló la gran industria bananera en A m erica Central, e l nordeste de A m er ica del Sur y las A n tilla s . Su im portancia en Puerto R ico y demas países productores ha decaído rápidam ente siendo desplazada por variedades del tipo Cavendish, debido a su gran susceptibilidad a l Mal de Panamá (Fusariurn oxysporum cubensis) y a la Sigatoka ( C ercospora musae) .

Las variedades del tipo Cavendish, aunque res isten tes a l M al de Panamá, no se com paran con e l G ros M ichel en cuanto a calidad de la fruta.

E l estudio se lle vó a cabo con e l propósito de exp lora r la posib ilidad de d esa rro lla r mutaciones y subsiguientemente clones de banano de esta v a r ie dad res is ten tes a dichas en ferm edades.

L IT E R A T U R A REVISADA

Hay num erosos casos en que se ha dem ostrado la e fectiv idad de las radiaciones en la inducción de mutaciones con res is ten c ia a enferm edades.

Entre estos pueden c ita rse uno de los trabajos de Konzak [2] en e l que se obtuvo mutantes de avena resisten tes a la roya ( Puccin ia g ram in is ) . Tam bién G regory [3] obtuvo líneas de maní (A rachys hypogaea) res isten tes a la mancha de la hoja ( C ercospora personnata).

Adem ás e l banano, com o cualquier otro organism o, está sujeto a v a r ia ción natural com o resultado de mutaciones som áticas. Siendo las variedades de banano de naturaleza clonal, e l p roceso de mutación es de suma im p ortancia, pues es la única fuente de va riac ión genética natural disponible, ya que la propagación asexual im posib ilita la recom binación genética.

P o r consiguiente, la inducción de mutaciones puede s e r e fec tiva en el m ejoram ien to del banano. De hecho, esto se ha com probado en estudios rea lizados en flo re s , habiéndose producido nuevas variedades [4 ], y en fru ta le s .

M A T E R IA LE S Y METODOS

Los r izom as se tra taron con distintas dosis de rayos gamma en e l Centro N uclear de Mayaguez, Puerto R ico . Cada rizom a se colocó en una bolsa de p lástico con pesas de plomo y se depositaron en e l centro del irrad iad o r.Las cápsulas de 60Co se colocaron en posición para fo rm ar un cilindro hueco de un d iám etro de 20 cm que em itía una dosis de 500 R por minuto. La dosis to ta l para cada rizom a se reguló por e l tiem po que perm aneció en e l campo rad iactivo .

Los tratam ientos consistieron en dosis de 2500, 5000, 10 000, 20 000 y 40 000 R, a cada uno de los cuales se expusieron 5 r izom as.

RESULTADOS

La s iem bra de los rizom as tratados y rizom as sin tra tar, para u tiliza rlos como testigos , se efectuó en la Subestación Experim enta l A g r íc o la de F o r tuna e l 24 de enero de 1964. E l 4 de m arzo del m ism o año se efectuó la siguiente observación:

MEJORAMIENTO DEL BANANO 487

D osis (R ) Germ inación (N !B)

1- 0 (T e s t ig o ) 52- 2500 53- 5000 24- 10 000 05- 20 000 06- 40000 0

Los rizom as que rec ib ieron más de 5000 R no germ inaron .Los tratam ientos con 2500 y 5000 R dism inuyeron la altura de las plantas

en com paración con las plantas testigo . A los cinco m eses las plantas del tratam iento con 2500 R tenían una altura de 1, 2 - 1, 8 m y las del tratam iento con 5000 R 0, 45 - 2, 13 m, en com paración con la altura del testigo , que va r ió entre 1,8 y 2, 7 m. Aparentem ente los tratam ientos retrasaban e l c r e cim iento de las plantas. Se observó, además, que e l ahijam iento era menos profuso que en e l testigo , y la fru ctificac ión más lenta.

En d ic iem bre de 1965 se sem braron las progen ies M 2 de los tratam ientos con 2500 y 500 R, y del testigo .

Los rizom as presentaban las siguientes ca racterís ticas :

Tratam ien to Núm ero de rizom as D iám etro m edio Peso medio_________ _________________ (cm) (kg)

T es tigo 10 14 2,362 500 R 21 10 1,005000 R 10 11 1,41

Durante e l d esa rro llo vegeta tivo de las plantas se observaron más variac ion es en las plantas del tratam iento con 2500 R.

A los 11 m eses de edad, cuando las p rim eras plantas em pezaron a inducir flo re s , la altura y e l número m edios de hojas de las plantas eran los siguientes:

Tratam ien to Num ero de hojas A ltu ra (m )

T es tigo 13,3 2,772500 R 12,0 2, 165000 R 12, 5 2, 31

Todas las plantas flo re c ie ro n y fru ctificaron . E l número m edio de manos por rac im o fue e l siguiente:

T es tigo 7, 1 manos (prom ed io de 8 rac im os)2500 R 6,64 manos (prom ed io de 11 rac im os)5000 R 7, 1 manos (prom edio de 6 rac im os)

Debido a la intensa sequía y a la desaparición de algunos racim os no se obtuvieron datos represen ta tivos en cuanto a peso, tamaño y otros atributos de las fru tas.

D el tratam iento con 2500 R se han aislado dos m utaciones. Una de ellas es una mutación drástica , pues las hojas son deform es, pequeñas y de poca

488 VELEZ FORTUNO y CEDEÑA MALDONADO

adherencia al ta llo ; además ahija muy poco. Los racim os son pequeños al com pararse con los del testigo , aunque las frutas individuales (dedos) son de buen tamaño.

La otra mutación tiene buenas ca racterís ticas y podría con vertirse en un nuevo tipo de G ros M ichel. En altura y v ig o r se asem eja al testigo pero tiene una pigm entación en e l ta llo muy intensa y oscura, en contraste con e l c o lo r c la ro del testigo . E l racim o es muy parecido al del testigo , pero la in flo rescen c ia masculina, cuando joven, es algo distinta pues las brácteas no cubren totalm ente e l ápice flo ra l. Las ca ra c te r ís tica s de la fruta madura son s im ila res a las del testigo .

Actualm ente se propaga esta mutación para estudiar su reacción a en fe rmedades, M al de Panamá (Fusarium oxysporum cubensis) y Sigatoka (C ercosp o ra m usae).

CONCLUSIONES

A pesar del número lim itado de rizom as tratados, se logró a lte ra r la uniform idad clonal del banano Gros M ichel para inducir mutaciones de posible u tilidad mediante tratam iento de rizom as con dosis de 2500 R.

Es de conocim iento genera l e l alto costo de program as de m ejoram iento genético del banano por e l método convencional, com o se rea liza en Trin idad y o tros pa íses. La obtención del germ oplasm a de otros países y de los centros de origen es d if íc i l y costoso, además de a rr iesgado debido al p e lig ro de introducción de insectos y enferm edades perju d ic ia les . Más aún, e l p ro cedim iento de c r ía es muy com plicado y tarda un sinnúmero de años en producir resultados e fec tivos .

En problem as como e l que plantea la variedad de banano Gros M ichel, la inducción de mutaciones represen ta una técn ica muy ventajosa para su m ejoram ien to. Esta variedad supera a todas las variedades del tipo Cavendish en cuanto a calidad de la fruta. Su p re feren c ia en los m ercados es unánime. Sólo adolece de su alto grado de susceptibilidad al M al de Panamá, además de la Sigatoka. De ahí que la inducción de rad iaciones puede ser la form a más rápida y económ ica para su m ejoram ien to en cuanto a los únicos defectos de que adolece. E l gran número de éxitos obtenidos mediante esta técnica en e l d esa rro llo de variedades res isten tes a enferm edades, ju stifica su ap licación en e l caso del G ros M ichel.

Esto resa lta e l v a lo r de esta técnica, en e l m ejoram ien to vegeta l, ya que donde existen facilidades de irrad iac ión acces ib les e l costo de las in vestigac iones es re la tivam ente m enor.


[1] AZZAM, H., LINDEN, D.B., Radiation effects on banana corms, Musa sapientum, J. Agrie. 49 2(1965) 270.

[2] KONZAK, C .F., Radiation-induced mutations for stem rust resistance in oats, Agron. J. 51 (1959) 118.[3] GREGORY, W .C ., Induction of useful mutations in the peanut, Genetics in Plant Breeding, Brookhaven

Symp.Biol. 2(1956) 177.[4] SIGURBJORNSSON, B., MICKE, A ., «Progress in mutation breeding», Induced Mutations in Plants

(Proc.Symp.Pullman, 1969), OIEA, Viena (1969) 673.

MEJORAMIENTO DEL BANANO 489

D IS C U S S IO N

С. PAN TO N : I am in terested to know the degree o f im provem entobtained in e ither diploid, tr ip lo id , o r tetrap lo id bananas by the use o f ion iz ing radiation.

J. V E L E Z FO RTU ÑO : The in form ation from this study is just p re lim in a ry . W e obtained the mutaint re fe rred to in my paper which we w ill test fo r r e sistance to C ercospora le a f spot and to Panama disease, which is the obje c t iv e o f our studies. The reduction in height o f the mutant is an advantage o v e r the o r ig in a l va r ie ty as presum ably it can res is t strong winds much better. I b e lieve that radiations can be e ffe c tiv e in im prov ing other banana types.

SOME BIOLOGICAL EFFECTS OF POST-TREATM ENT WITH CYSTEINE ON GAMMA-IRRADIATED RICE SEEDS*

A. ANDODepartment of Genetics, ESALQ,University of S3o Paulo,Piracicaba, Brazil

Abstract-Resumen

SOME BIOLOGICAL EFFECTS OF POST-TREATMENT WITH CYSTEINE ON GAMMA-IRRADIATED RICE SEEDS.

Post-treatment o f dry rice seeds with cysteine for 45, 30 and 24 h was compared with gamma-ray treatment. The experimental data and statistical analyses indicate the following: (1) Post-treatment for 45 h had adverse and mutagenic effects under the given conditions. (2) Post-treatment for 30 h showed a protective effect on various Rx characters and increased chlorophyll mutation frequency in the R2 though this increase was significant only in the post-treatment after 20 kR of gamma-irradiation.(3) The effect of the post-treatment for 24 h was not significant either in the protection of the Ri characters or in the increase of chlorophyll mutation frequency. (4) Frequencies of albina, v iiidis and other types of chlorophyll mutations in the gamma-ray treatment were influenced neither by the posttreatment with cysteine, nor by different times of treatment. (5) Only the post-treatment for 30 h increased the average segregation ratio of the K l panicles.

ALGUNOS EFECTOS BIOLOGICOS DEL TRATAMIENTO POSTERIORCON CISTEINA DE LAS SEMILLAS DE ARROZ SOMETIDAS A IRRADIACION GAMMA.

El autor compara el tratamiento posterior de semillas secas de arroz con cistefna durante 45, 30 y 24 horas y el tratamiento por irradiación gamma. Los datos experimentales y los análisis estadísticos consecutivos indican lo siguiente: (1) El tratamiento posterior durante 45 horas tiene efectos perjudiciales y mutagénicos en las condiciones dadas. (2) El tratamiento posterior durante 30 horas tiene efectos protectores sobre distintos caracteres RA y aumenta la frecuencia de mutación clorofílica en R 2, aunque tal aumento es sólo significativo si dicho tratamiento se efectúa tras una irradiación gamma de 20 kR.(3) El tratamiento durante 24 horas no tiene un efecto significativo, ya sea en cuanto a la protección de ios caracteres Rj o al aumento de la frecuencia de mutación clorofílica. (4) En la frecuencia de mutación clorofílica albina, viridis y de otros tipos por irradiación gamma no influye e l tratamiento posterior con cisteína; dicha frecuencia tampoco varía con la duración del tratamiento. (5) Sólo el tratamiento posterior durante 30 horas aumenta la razón media de segregación de las panículas R :.


It is w e ll known that cyste ine, one of the sulphydryl compounds used as chem ical protectants, shows its p rotective e ffec ts only in the p re treatm ent o f the irrad ia ted organ ism . It is a lso w e ll known, how ever, that the rad iosen s itiv ity of m a ter ia l soaked in w ater is g rea te r than that of d ry m ateria l. Thus, in irrad ia tion experim ents w ith plant seeds, it is gen era lly recom m ended to use dry m ateria l.

* Part of the research reported in this paper has been carried out under Research Contract with the International Atomic Energy Agency No. 476/RB.

491

492 ANDO

On the other hand, accord ing to Z im m er et al. [1 ] and Conger and Randolph [2 ], fr e e rad ica ls form ed by ion izing radiations rem ained fo r a re la t iv e ly long period in the em bryos o f the irrad ia ted d ry seeds.These rad ica ls m ay fo rm peroxides which subsequently cause physio log ica l damage in the irrad ia ted seeds. Thus, i f we can e lim inate these rad ica ls a fte r irrad ia tion we can expect less phys io log ica l damage and, consequently, m ore gene mutations. In fact, Yamaguchi [3] reported that post-treatm ent with cysteine o f X -irrad ia ted d ry seeds of r ic e considerab ly reduced the adverse e ffec t of X -ra y treatm ent on seed ling height and fe r t i l ity o f the R a plants.

The p resen t experim ents w ere ca rr ied out in o rd er to gain m ore in form ation on the post-treatm ent e ffec ts o f cysteine on various characters in the R j and R 2 o f gam m a-irrad ia ted r ic e seeds.

2. M A T E R IA LS AND METHODS

D ry seeds o f the B raz ilian r ice va r ie ty "Dourado P r e c o c e " , which w ere kept in a d es icca to r with СаС1г fo r m ore than one month, was the m a ter ia l used. The average w ater content o f the m a teria l was 8.1%.

P r io r to the experim ent, a p re lim in ary investigation was ca rried out in o rd er to determ ine the relation between treatm ent tim e and cysteine consumption. Cysteine solution (100 m l, 10"3 M) was introduced into 18 E rlen m eyer flasks o f 250 m l capacity, and to each of nine w ere added 400 selected r ic e seeds. A l l flasks w ere kept closed and maintained at 25 ± 1°C. A fte r g iven tim es , two flasks, one with seeds and another without seeds, w ere taken together, and cysteine consumption due to the presence of the seeds was estim ated by the c o lo r im e tr ic method developed by L id d e ll and Saville [4].

G am m a-irrad ia tion was ca rried out in the rea c to r o f the A tom ic E nergy Institute o r with the 60Co source of the N uclear E nergy Centre in A gricu ltu re , both of the U n ivers ity of Sao Paulo. Doses applied w ere 10, 20 and 30 kR, with the varied dose rate from 350 to 1000 R/min. Im m ediate ly a fte r irrad ia tion , the m a ter ia l was soaked in w ater, or in cysteine solution o f volume 0.125 m l/seed. Treatm ent tim es w ere 45 h at 27 ± 2°С in 1965, 30 h at 27 ± 2°C in 1966, and 24 h at 25 ± 1°C in 1967 and 1968.

G erm ination and seed ling height w ere m easured one and two weeks a fte r sowing, resp ec tiv e ly . A fte r transplantation, su rv iva l rate at harvest tim e was estim ated on the basis of sown seed number, and three f ir s t panicles per plant w ere co llected fo llow ing the flow erin g order. F e r t i l ity was calculated on about 100 panicles which w ere taken at random in each treatm ent. A l l panicles w ere sown separa te ly in the greenhouse in o rd er to observe ch lorophyll mutations.


Tab le I shows the resu lts o f the p re lim in a ry experim ent on cysteine consumption. Apparently, cysteine consumption is tim e-dependent.F ig . l shows the cysteine consumption curve obtained from the above data; the w ater absorption curve at 25 ± 1°C fo r the same m ateria l is

CYSTEINE EFFECTS ON RICE 493

T A B L E I. R E L A T IO N B ETW EEN T R E A T M E N T T IM E AND CONSUM PTION OF CYSTE INE B Y RICE SEEDS(TABLA I. RELACION ENTRE EL TIEMPO DE TRATAMIENTO Y EL CONSUMO DE CISTEINA POR LAS SEMILLAS DE ARROZ)

Treatment time (h)

Weight o f seeds

Cg)

Consumption of cysteine by rice seeds 6 ° ) (Pg/g seeds)

0. 00 0. 00 0. 00

5. 00 14.2845 1. 00 8.47

9. 75 14.1724 5. 11 43.61

18. 75 14. 0690 7. 98 68.66

23. 75 14. 0380 9. 33 80.42

28. 75 14.3664 10. 20 85. 89

33, 75 14.3580 17. 78 149. 81

43.25 14.3292 44. 82 378.46

48.25 14.0095 35. 32 305.08

53.25 14.2306 43.45 369.42

FIG. 1. Cysteine consumption and water absorption curves.

(FIG. 1. Curva de consumo de cistefna y curva de absorción de agua. )

shown fo r com parison. The two curves are approxim ately p a ra lle l up to about 26 h, indicating that se le c tiv e absorption o f cysteine by the r ic e seeds occurred or that cysteine decom position took p lace due to s e c r e tion o f substances from the m a ter ia l o r to som e other unknown factors . F ro jn about 30 to 45 h, cysteine consumption acce lera ted , and a fter about 50 h slowed down again. F rom these data, th ree treatm ent tim es, 45, 30, and 24 h w ere chosen fo r our experim ent.

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T A B L E III. S IG N IF ICANCE TE ST OF P O S T -T R E A T M E N T E F F E C T OF C YSTE INE ON VARIOUS C H ARACTERS IN R x(TABLA III. PRUEBA DE SIGNIFICACION DEL EFECTO DEL TRATAMIENTO POSTERIOR CON CISTEINA SOBRE DISTINTOS CARACTERES R:)

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1966 (C, 30 h) 12 3 4. 26 0. 02 - 0. 05

1967 (C. 24 h)1968 v

12 10 0.045 0. 8 - 0. 9

a X z = ((nt - n2) - l )V (n ! + n2). k С = Post-treatment with cysteine (10 " 3M).

T A B L E IV . F O U R -Y E A R AVERAG ES OF G E R M IN ATIO N , SEEDLING H EIG H T, S U R V IV A L AND F E R T IL IT Y OF RICE SEEDS AS A. FU N C TIO N OF G A M M A -R A Y DOSE(TABLA IV. VALORES PROMEDIOS (DURANTE CUATRO AÑOS) DE LA GERMINACION, LA ALTURA DE LAS PLANTULAS, LA SUPERVIVENCIA Y LA FERTILIDAD DE LAS SEMILLAS DE ARROZ EN FUNCION DE LAS DOSIS DE RAYOS GAMMA)

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Seedling height

б°)Survivala

Cft)Fertility

(%)

Chlorophyll mutations per 100 Rj panicles

0 100. 0 100. 0 100.0 100. 0 0.39

10 100.6 95.4 81.0 82.2 4. 82

20 98. 8 78.3 77.2 62.1 6. 67

30 89.3 61.4 39.3 38.5 8.50

a ~Two-year average.

Tab le II shows the resu lts o f a ll treatm ents in the experim ents on germ ination , seed ling height, su rv iva l, fe r t i l ity and ch lorophyll mutation frequency. F o r the experim ents ca rr ied out in 1967 and 1968, su rv iva l was not calculated because of partia l damage o f the fie ld by heavy rain . The post-treatm ent e ffects of cysteine on the various R j characters a re somewhat confused in this Tab le. The sign ificance test th ere fo re was u tilized in ord er to assess these e ffec ts : when the posttreatm ent e ffe c t was pos itive , compared to the non-cysteine treatm ent, the (+) sign was g iven , and when negative, the (- ) sign was given. When there was no d iffe ren ce , these treatm ents w ere excluded from the test. Since it is recom m ended to apply this test only when the tota l number of signs is m ore than 15, the com parisons between the con tro l and the treatm ent with cysteine only w ere added to the data fo r 1965 and 1966. F rom the resu lts shown in Tab le III, it can be concluded that post-


Gamma - ray dose (kR)

FIG. 2. Four-year averages of germination, seedling height, survival and fertility as a function of gamma-ray dose.(FIG. 2. Valores medios de la germinación, altura de la plántula, supervivencia y fertilidad en varios experimentos durante varios afios.)

■p H 00 O •P -H

0 a) P<

>»«AА Оo oU r-ioИ hA <uO Q,

Gamma - ray dose (kR)

FIG. 3. Post-treatment effect of cysteine on chlorophyll mutation frequency in rice seeds (* , * * and denote significant at levels of 5, 0, 1, 0 and 0 .1?jo respectively, compared with corresponding

gamma-ray treatment alone).

(FIG. 3. Efecto del tratamiento posterior con cistefna sobre la frecuencia de mutación clorofílica. )

498 ANDO

T A B L E V. FR E Q U E N C Y AND S T A T IS T IC A L A N A LYS IS OF D IF F E R E N T T Y P E S OF C H LO R O P H Y LL M U TATIO N(TABLA V. FRECUENCIA Y ANALISIS ESTADISTICO DE LOS TITOS DE MUTACION CLOROFILICA)

Treatmenta albina viridis Others Total

1965 1 30 6 10 462 28 7 11 463 22 8 10 40

(C, 45 h) 1 +C 33 7 12 522 +C 43 18 13 743 +C 27 5 14 46

1966 1 30 11 13 542 35 14 20 693 12 6 11 29

(C, 30 h) 1 + c 24 12 14 502 + С 33 9 14 563 + С 28 8 14 50

1967 1 7 4 7 182 25 5 13 433 42 7 16 65

(C, 24 h) 1 +C 35 12 10 572 + С 53 18 25 963 +C 20 2 6 28

1968 1 2 0 7 92 3 1 1 53 4 1 3 8

(C, 24 h) 1 + C 21 8 15 442 + С 38 13 10 613 +C 20 14 3 37

Treatmenta X 2 d. f. P

1965(C, 45 h)

RR + C

0.10 2 0.9<

1966(C, 30 h)

RR + C

Q.45 2 0.8

1967(C, 24 h)

RR + C

2.22 2 0. 3 - 0. 5

1965 R1966 R 4. 86 4 0.3 - 0.51967 R

C, 45 h R + CC, 30 h R + C 1.41 4 0. 8 - 0. 9C, 24 h R + C

a1 ,2 ,3 = treatment with 10, 20 and 30 kR of gamma-rays, respectively; С = post-treatment with cysteine (10~8 M ); R = treatment with gamma-rays.


treatm ent with cysteine fo r 45 h has an adverse e ffe c t on the R j characte rs . On the other hand, post-treatm ent fo r 30 h is p ro tec tive or stim ulating not only to the irrad ia ted but a lso to the non-irrad iated seeds. P ost-trea tm en t fo r 24 h is not sign ificant.

R ad iosensitiv ity o f the r ic e seeds was estim ated by taking the average of four experim ents ca rr ied out during four yea rs . The data in Tab le IV fo r germ ination , seed ling height, su rv iva l and fe r t i l ity as a function of gam m a-ray dose a re plotted in F ig .2. Germ ination appears to be the character least sensitive to irrad ia tion and seem s not to be appropriate to assess rad iosen s itiv ity within the dose lim it used in our experim ent. The m ost sensitive character is fe r t i l ity fo r which the d o se -e ffe c t curve is a lm ost lin ear.

Regarding ch lorophyll mutation frequency, the resu lts a re shown in Tab le V and F ig .3 . As mentioned above, post-treatm ent with cysteine fo r 45 h was p re ju d ic ia l or toxic to the R ! characters . This treatm ent was, how ever, revea led to be mutagenic when com pared with the gam m a- ray treatm ent alone.- The sta tis tica l analysis (Tab le V) shows that the ch lorophyll mutation frequencies fo r treatm ents with cysteine only,10 kR + cyste ine, and 20 kR + cysteine are s ign ifican tly higher than those fo r the corresponding gam m a-ray treatm ents. The frequency fo r the 30 kR + cysteine treatm ent was reduced but not s ign ifican t when com pared with the treatm ent with 30 kR gam m a-rays. Post-trea tm en t fo r 30 h gen era lly resu lted in h igher ch lorophyll mutation frequency fo r a ll doses used, com pared with the gam m a-ray treatm ent. H ow ever, on ly the treatm ent with 20 kR + cysteine gave a sign ifican t increase in ch lorophyll mutation induction. S tatistica lly , post-treatm ent with cysteine fo r 24 h gave the sam e mutagenic e ffec t as gam m a-ray treatm ent.

Com paring ch lorophyll mutation frequencies o f the post-treatm ents w ith cysteine on the basis o f the sam e fe r t i l ity (around 40% of the control in Tab le II ), it seem s that post-treatm ent fo r 30 h is m ore e ffe c tiv e than those fo r 45 or 24 h.

T A B L E V I. P O S T -T R E A T M E N T S W ITH C YSTE INE AND SEGREGATION RATIOS(TABLA VI. TRATAMIENTO POSTERIOR CON CISTEINA Y RAZON DE SEGREGACION)

TreatmentaNo. of chlorophyll

mutantsTotal No. of R2 seedlings

Segregationratio

X 2 P

1965 R 1731 11451 0.15120.38 0.5 - 0. 7

(C, 45 h) R + C 1751 11808 0.1479

1966 R 1077 9 762 0.11035. 19 0. 02 - 0. 05

(C, 30 h) R + C 1394 11584 0.1203

1967, 68 R 821 7 021 0.11711. 11 0.2 - 0.3

(C, 24 h) R + C 2042 18 189 0.1123

aR = treatment with gamma-rays; С = post-treatment with cysteine (10* 3 M).

500 ANDO

A l l ch lorophyll mutations observed in the greenhouse w ere c la ss ified into three types: a lb ina, v ir id is and others. Tab le V shows the frequencies of these th ree types during four years and the s ta tis tica l analysis of various treatm ents. The data o f the experim ent ca rried out in 1968 w ere e x cluded from the analysis because of v e ry low ch lorophyll mutation frequencies in the gam m a-ray treatm ent. The resu lts show that the frequencies o f the three types a re not influenced by d ifferen t environ ments nor by d iffe ren t cysteine treatm ent tim es.

Tab le V I shows a com parison o f the segrega tion ratios fo r the gam m a-ray treatm ents and the corresponding post-treatm ents with cyste ine. F rom the resu lts , it is c lea r that only the treatm ent with cysteine fo r 30 h s ign ifican tly increased the average segregation ratio as com pared with that fo r the gam m a-ray treatm ent alone. In the trea tments with cysteine fo r 45 and 24 h, the average segrega tion ra tios decreased in com parison with those o f the gam m a-ray treatm ents, but the d iffe ren ces w ere not sign ificant. Apparently, the three segregation ratios fo r the treatm ents w ith gam m a-rays only in d ifferen t years are d ifferen t (X 2 = 89.71, h ighly s ign ifican t), indicating that the segregation ra tio is subject to the influence of environm ental fac to rs .

R E F E R E N C E S

[1 ] ZIMMER, K.G. , EHRENBERG, L. , EHRENBERG, A ., Nachweis langlebiger magnetischer Zentren in bestrahlten biologischen Medien und deren Bedeutung fur die Strahlenbiologie, Strahlentherapie 103 (1957) 3.

[2 ] CONGER, A. D ., RANDOLPH, M. L. , Magnetic centers (free radicals) produced in cereal embryos by ionizing radiation, Radiat. Res. 11 (1959) 54.

[3] YAMAGUCHI, H ., The effects of post-treatments with cysteine and sodium hydrosulfite on the radiation-induced injury and mutation in rice, Jap. J. Breed. 12 (1962) 8.

[4] LIDDEL, H. F. , SAVILLE, B. , Colorimetric determination o f cysteine, Analyst, Lond. 84 (1959) 188.

D IS C U S S IO N

G. DE A L B A : W ith respect to the data presented in F ig . 3,I would suggest calcu lating the response components (lin ear, quadratic, etc .) fo r the post-treatm ents fo llow ing the various treatm ents.

H. D O LL : How do you explain the increased segrega tion frequency a fte r the 30 h post-treatm en t?

A . ANDO: I think that the increase o f segrega tion ra tio in the posttreatm ent fo r 30 h was p rin c ipa lly due to the recuperation o f fe r t ility .

H. D O LL: Have you tr ied to calculate the mutation frequency accord ing to the m easure proposed by Gaul, i.e . the frequency of mutant seedlings among a ll R 2 seed lings. Since this estim ate often is a m ore va lid one, it m ay lead to another conclusion in your investigations.

A . ANDO: No, I did not. Although G au l's method is m ore co rrec t, the d iffe ren ce between the resu lts by the two methods seem s not to be so grea t within the dose lim it used in our experim ent.

EFECTO DEL DIMETIL SULFOXIDO SOBRE LA RADIOSENSIBILIDAD DEL ESPERMA DE Drosophila melanogaster EN DOS ESTADOS DE SU MADURACION

Beatriz MAZAR BARNETTComisión Nacional de Energía Atóm ica,Buenos Aires, Argentina

Abstract-Resumen

EFFECT OF DIMETHYL SULPHOXIDE ON THE RADIOSENSITIVITY OF THE SPERM OF Drosophila melanogaster AT TWO STAGES OF ITS MATURATION.

The paper presents data on differences in the action of ionizing radiations observed at two very slightly separated stages in the gametogenesis of Drosophila melanogaster, when a radioprotective compound is present and absent. The last two stages o f spermiogenesis were studied (immobile sperm and mature, mobile sperm).The genetic effect of the treatments was investigated at the level of chromosome breakage and recombination in I I - I I I translocation experiments. X-radiation was employed, the dose being 1000 rad in all cases. The radioprotective agent, 10 % dimethyl sulphoxide, was administered by intra-abdominal injection before irradiation. Pre-treatments were carried out at various times. The experimental procedure followed for crossings-over permits adequate sampling of the germinal cells at the stages mentioned. The results obtained in the translocation tests do not indicate differences in radiosensitivity between the two stages of sperm maturation. On the other hand, these cells exhibit a distinct response to dimethyl sulphoxide, which has a radioprotective effect on immobile sperm without modifying the frequency of translocations induced in mobile sperm. In the case of males treated 30 min before irradiation, the differences are very significant, indicating that this fact must be taken into account.

EFECTO DEL DIMETIL SULFOXIDO SOBRE LA RADIOSENSIBILIDAD DEL ESPERMA DE Drosophila melanogaster EN DOS ESTADOS DE SU MADURACION.

La memoria presenta datos sobre diferencias a la acción de las radiaciones, observadas en dos etapas muy próximas de la gametogénesis de Drosophila melanogaster, con y sin la presencia de un compuesto radioprotector. Se estudiaron los dos últimos estados de la espermiogénesis: esperma inmóvil y esperma maduro móvil. El efecto genético de los tratamientos se investigó a nivel de ruptura y reunión de cromosomas, en experimentos de translocaciones II-III. Se utilizó radiación X, siendo de 1000 rads la dosis suministrada en todos los casos. El agente radioprotector usado, dimetil sulfóxido al 10°Jo, se administró por inyección intraab- dominal, previamente a la irradiación. Se hicieron pretratamientos a distintos tiempos. El procedimiento experimental seguido para los cruzamientos, permite el adecuado muestreo de las células germinales en los estados mencionados. Los resultados obtenidos en las pruebas de translocación no indican diferencias de radiosensibilidad entre los dos estados de maduración del esperma. Existe en cambio una respuesta distinta de esas células al dMSO, que ejerce un efecto radioprotector sólo en esperma inmóvil, sita modificar la frecuencia de translocaciones inducidas en esperma móvil. En el caso de machos pretratados 30 min antes de la irradiación, las diferencias son altamente significativas, lo que indica que este hecho debe ser tenido en cuenta.

En e l estudio de la inducción de cambios en e l m ateria l h ered ita rio por m edio de rad iaciones y compuestos mutagénicos, se ha com probado que las célu las germ ina les m uestran variac iones en su respuesta a esos agentes durante e l transcurso de su d iferenciación . Esta respuesta se designa c o rrien tem ente com o sensib ilidad. Sin em bargo, esas va riac iones podrían deberse, más que a una sensib ilidad d ife ren c ia l, a una distinta capacidad para rep a ra r e l daño inducido. Este trabajo se ha encarado como una c on tr ibución al conocim iento de ese problem a.

501

502 MAZAR BARNETT

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EFECTO DEL DIMETIL SULFOXIDO 503

E l d im etil sulfóxido ha dem ostrado propiedades rad ioprotectoras en va r io s sistem as b io lóg icos: ratón, rata, célu las de m am íferos cultivadasin v itro , Pseudomonas sp. [1 -6 ] . Se consideró de in terés estudiar s i esa acción se m anifiesta en D rosophila m elanogaster ya que este organism o perm ite extender las investigaciones a dos n iveles: som ático y genético.E xperiencias rea lizadas en nuestro laboratorio nos perm itie ron com probar que e l dMSO proporciona una s ign ifica tiva protección a ambos n ive les, es d ec ir , que aumenta la supervivencia de machos y hem bras som etidos a dosis altas de irrad iac ión gamma (64 000 rads) y dism inuye la frecuencia de mutaciones le ta les reces ivas ligadas al sexo, inducidas por 800 rads de radiación X, en esperm a.

En este trabajo se presentan datos sobre las frecuencias de tran s loca ciones I I - III inducidas por rayos X en esperm a maduro, con y sin la p resencia de dMSO. Se estudiaron los dos últim os estados de la esperm iogénes is de D rosophila: esperm a maduro inm óvil y esperm a maduro m óv il. A pesar de que se trata de estados muy próxim os, ya que sólo la m otilidad los distingue, se ha observado que presentan d iferencias de rad iosensib ilidad (por ejem plo en la frecuencia de mutaciones leta les reces ivas [7 ,8 ] ) .

Se tra taron machos adultos de la cepa sa lva je Canton S, que se cruzaron por hem bras v írgen es adultas en bw, e, m arcadores adecuados para la detección de translocaciones entre los crom osom as mencionados, y los machos de la f i l ia l p r im era se re trocru zaron por hem bras v írgen es en bw, e. En la f i l ia l segunda se recobran los casos de translocación; todos los casos observados se vo lv ie ron a probar. Se siguió un proced im iento experim enta l de cruzam ientos que perm ite e l adecuado m uestreo de las célu las germ ina les en los estados mencionados. E l d im etil sulfóxido se u tilizó en solución a l 10% en Na C1 al 0,4%, isotón ica para Drosophila. Se adm in istró por inyección intraabdom inal, con ayuda de una m icro je r in ga A G LA , en dosis de 0,4 /ni, con in terva los de 30 min o de 20 h antes de la irrad iac ión . Las irrad iac ion es se lleva ron a cabo con un equipo para terap ia intensiva, operando a 150 kV,10 m A, 1 mm A l y 1 mm Cu, con 43 R/min. La dosis en todos los casos fué de 1000 rads. Los resu ltados obtenidos pueden v e rs e en la tabla . E l p r im er subcultivo corresponde a esperm a m óvil y e l segundo subcultivo corresponde a esperm a maduro, aunque no m óvil, en e l momento de la irrad iac ión .

La progen ie del p r im er subcultivo de los machos som etidos a los tres tratam ientos no m uestra d ife ren c ias en cuanto a la frecuencia de tran slocaciones inducidas. Tam poco se ven d iferencias entre las frecuencias de translocación inducidas por 1000 rads entre las progen ies correspondientes a ambos subcultivos. Se observa en cam bio una m arcada dism inución en las frecuencias de translocaciones inducidas por la rad iación en la progen ie de los machos pretratados con dMSO, del segundo subcultivo. E l va lo r de P hallado indica que en e l caso de los machos inyectados 30 m in antes de la irrad ia c ión la d iferenc ia es altamente s ign ifica tiva .

La falta de d iferenc ia entre las frecuencias de translocaciones inducidas por la radiación entre esperm a m óv il y esperm a inm óvil, concuerda con los resultados de otros investigadores [7 ], aún cuando se observaron d iferenc ias en las frecuencias de mutaciones inducidas. Es d ec ir que la radiosensib ilidad del esperm a no d ifie re , aparentem ente, en los estados de m aduración considerados, en cuanto a la ruptura y reunión de crom osom as. Considerando e l e fecto producido por e l dMSO, podría pensarse que e je rc e su acción p ro tec to ra perm itiendo de alguna m anera que tenga lugar una reparación , o au-

504 MAZAR BARNETT

mentando la capacidad de reparación de l esperm a que es menos sensib le, о más factib le de repa ra r. Es decir, que, si la s im ila r radiosensib ilidad m ostrada por e l esperm a en esos dos estados de maduración respondiese por igual a la acción del agente rad ioprotector, no cabría duda de que estam os realm ente ante iguales sensib ilidades. P a rece razonable suponer que existen d iferencias que ser ía conveniente in ves tiga r más a fondo. Están en marcha otros experim entos para estudiar la inducción de mutaciones leta les r e c e s ivas ligadas al sexo, del m ism o modo en que se rea liza ron éstos, para tra ta r de es tab lecer si responden de m anera igual al dMSO.

L os aportes sobre sensibilidad a mutágenos y capacidad de reparación a los cam bios inducidos de las célu las germ ina les en d esa rro llo , constituyen una contribución ú til para e l m e jo r conocim iento de los m ecanism os básicos de la herencia. Con este sentido se ha presentado este trabajo aquí.


[1] MOOS, W .S., KIM, S.E., Radioprotective effect of topically applied dMSO on mice, Experientia 22 12(1966) 814.

[2] MOOS, W .S., LeVAN, H., MASON, H .C ., Radioprotective effects of dMSO vapour on mice, Experientia 23 11 (1967) 923.

[3] DOD, J.L., SCHEWELL, J., Local protection against X-irradiation by dimethyl sulphoxide, Br.J.Radiol. 41 492 (1968) 950.

[4] ASH WOOD-SMITH, M.J., The radioprotective action of dimethyl sulphoxide and various other sulphoxides, Int.J.Radiat.Biol. 3 1(1961)41.

[5] VOS, O., KAALEN, M .C ., Protection against ionizing radiation at the cellular level assessed by various parameters, Int.J.Radiat.Biol. 14 2 (1968) 107.

[6] BRIDGES, B .A ., Protection of Pseudomonas sp.against gamma-radiation by dMSO, Int.J.Radiat. Biol. 5 1 (1962) 101.

[7] MOSSIGE, J.C., «D ifferen tia l yields of mutations from the first and second matings after irradiation of mature sperm in Dresophila melanogaster», Repair from Genetic Radiation Damage and Differential Radio- sensitivity in Germ Cells ( SOBELS. F.H ., Ed.), Pergamon Press, New York (1963) 253.

[8] LEFEVRE, G., Jr., JONSSON, U.B., X-ray induced mutability in male germ cells of Drosophila melanogaster, Mutation Res. 1_( 1964) 231.

D IS C U S S IO N

R. N ILAN : W e have analysed irrad ia ted b a r ley seeds fo r a protectivee ffec t o f dMSO. This chem ica l exerts a v e ry strong p rotective e ffec t, but only fo r the oxygen-dependent damage. It had no e ffec t on the oxygen- independent damage. We have concluded along with Kaul (who has published his resu lts i'n Radiation Botany, 1969, 1970) that in seeds dMSO influences the in itia l radiation interactions and does not operate on reco ve ry p rocesses .

INVESTIGATION OF A POSSIBLE MUTAGENIC EFFECT OF JUICE FROM IRRADIATED BANANA *

A. BLUMENSCHEIN, M .M . IGREJA, M .A . VALERIO Department o f Genetics, ESALQ,

University o f S3o Paulo,Piracicaba, Brazil

Abstract-Resumen

INVESTIGATION OF A POSSIBLE MUTAGENIC EFFECT OF JUICE FROM IRRADIATED BANANA.Contradictory reports can be found in the literature regarding the mutagenicity of irradiated food and

food products. The paper presents first results from an experiment, where com seeds were treated with juice from gamma-irradiated bananas. The effects observed from treatment with irradiated juice were inconsistent.

ESTUDIOS SOBRE EL PROBABLE EFECTO MUTAGENICO DEL ZUMO DE BANANAS SOMETIDAS A IRRADIACION.

En la literatura especializada se encuentran afirmaciones contradictorias en lo que concierne a la mutagenícidad de los alimentos y productos alimenticios irradiados. La memoria expone los primeros resultados de un experimento en el que se han tratado semillas de maíz con zumo de bananas sometidas a irradiación gamma. Los efectos observados tras el tratamiento con zumo irradiado carecen de uniformidad.

1. IN TRO D U CTIO N

Changes in the chem ical com position and physio logy w ere observed in irrad ia ted fru its by M assey and Bourke [1 ]. Chopra et al. [2] reported an in crease o f chrom osom e breakage in rye and onion developed on ju ice from irrad ia ted orange and apple. Swaminathan [3 ], and R inehart and Ratty [4] observed an increase in the frequencies o f r e c e ss iv e sex linked lethals and v is ib le dominant o r re c ess iv e mutants when Drosophila m elanogaster was grown on irrad ia ted culture medium. H ow ever,Chopra [5 ] and Reddi et al. [6 ] d id not ob serve any change in the mutation frequency o r chrom osom e breakage in Drosophila grown on irrad ia ted medium. S evera l other con trad ictory resu lts can be found in the litera tu re .

In this paper we rep ort the f ir s t resu lts observed when corn seeds w ere trea ted with ju ice from gam m a-irrad ia ted bananas.

2. M A T E R IA LS AN D METHODS

G reen bananas (v a r ie ty "nan ica") w ere gam m a-irrad ia ted (200, 400,6 00 and 800 kR) using a 60Co source. U n irrad iated and irrad ia ted fru its w ere tr itu ra ted with pure water and the ju ice was obtained by filtra tion . M a ize seeds (v a r ie ty "ca te to ” ) w ere soaked in the ju ice fo r four days, and the same number (500) w ere soaked in w ater as a control. F rom

=!= Supported by CIEN and developed in the IGen and CENA, ESALQ, USP, Brazil.

505

506 BLUMENSCHEIN et al.

T A B L E I. PE R C E N TAG E S OF SEED G ERM IN ATIO N

Treatments(kR)

Mi Experiment 1

M 2

Experiment 2

Control (HzO) 86 94 90

0 62 98 96

200 43 97 99

400 62 99 91

600 19 98 98

800 52 97 95

each treatm ent 36 0 seeds w ere germ inated under greenhouse conditions and the percen tages o f seed germ ination w ere determ ined. A fte r 13 and 24 days the height o f 50 seedlings was m easured. One hundred seeds from each treatm ent w ere grown under regu la r fie ld conditions and the plants obtained w ere se lf-po llinated . F rom each treatm ent ten good ears w ere obtained.

The M2 seeds w ere also germ inated under greenhouse conditions, using a random ized b lock design. In a f ir s t experim ent four repetitions w ere made, and in a second experim ent, 30 days la te r , fiv e repetitions w ere ca rr ied out. Twenty M 2 seeds from each o f the ten selfed ears from each treatm ent w ere germ inated but the height m easurem ents w ere made in only ten seed lings, 13 and 24 days old.

3. RESULTS

3.1 . Seed germ ination

A s shown in Tab le I, germ ination percentage in the Mi generation o f seeds trea ted with irrad ia ted banana ju ice was low er than that o f nontrea ted seeds. The 600 kR treatm ent resu lted in a particu la rly low percentage o f seed germ ination . The resu lts on the germ ination o f M2 seeds did not indicate any re la tion with treatm ent.

3. 2. Seed ling height

3 .2 .1 . Mi seedlings

In genera l the average height of Mj seed lings, 13 and 24 days old, developed from seeds trea ted with banana ju ice , was low er than that o f Mx seedlings from non-treated seeds (T a b le II). The value o f F re la ted to the lin ear re g re ss io n between treatm ents and seed ling height was s ta tis tica lly s ign ificant as shown in Tab le III.

IRRADIATED BANANA JUICE 507

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T A B L E III. REGRESSION B ETW EEN SEED LING HEIGHT AND TR E A T M E N T S (F V ALU ES)

Regression M iExperiment 1

M 2

Experiment 2

(13 days old)

Linear 7 .31 *# 0.70 1.46

Quadratic 0.01 13 .4 5** 8 .4 7 **

(24 days old)

Linear 4 .41 * 0.22 0.24

Quadratic 0.02 7 .9 7 ** 11 .4 7**

3 .2 .2 . M2 seedlings

The resu lts observed fo r the M2 seed lings w ere d ifferen t from those fo r the Mi generation. In experim ent 1 the ta lles t seedlings d erived from treatm ent at 400 kR and in experim ent 2 from treatm ents at 400 and 600 kR (T ab le II). The seed ling height increased from treatm ent at 0 kR to tre a tments at 400 and/or 6 00 kR and decreased in treatm ent at 800 kR.

The F value re fe r r in g to the lin ear re g ress ion between seed ling height and treatm ent was not sign ificant; however, the F value re fe r r in g to the quadratic re g re ss io n was significant in both experim ents (T ab le III).

The values observed in the H20 contro l w ere not as high as those fo r treatm ents at 400 and/or 600 kR.

T A B L E IV . VAR IAN C E W ITH IN M2 PROGENIES

TreatmentsExperiment

13 days

1 Experiment 2 Experiment 1

24 days

Experiment 2

Control (H20) 12.12 31.75 34. 52 36.45

0 6.84 19.80 20.20 31.77

200 7.31 9.78 21. 80 24. 58

400 9.13 23.82 20.12 40.28

600 11.07 30.61 32.62 31.63

800 6.05 19. 58 22.95 31.87

IRRADIATED BANANA JUICE 509

3 .2 .3 . Variance within M2 progen ies

A s indicated in Tab le IV , the variance approxim ately fo llow ed the resu lts fo r the means. The highest variances w ere found in the p rogen ies derived from treatm ents at 400 and 600 kR. These va riances w ere s im ila r o r somewhat low er than those observed in the p rogen ies d erived from the H20 control.

4. DISCUSSION AND CONCLUSIONS

The resu lts showed that banana ju ice treatm ent a ffected the Mi seeds, reducing germ ination and seed ling development. The e ffec t was even ob served when ju ice from non -irrad ia ted fru its was used. Banana ju ice probably contains substances which in te r fe re with the physio logy o f corn seeds. In som e cases the e ffec t in creased with the use o f irrad ia ted fru it ju ice. Th is could indicate that irrad ia tion can change the ju ice com position, in creas ing the amount o r developing new substances which in te r fe re with the seed physiology.

Am ong the M2 seedlings it was observed that the m ore v igorous p rogen ies w ere a lso the m ore va riab le ones. These resu lts cannot be explained on the basis o f the data obtained so fa r in our work. The banana ju ice could perhaps act as a se lec tive agent on the corn seeds. The resu lts observed in the germ ination o f Mi seeds could support this suggestion. A s a resu lt o f this selection , the m ore genera l un iform ity (in re la tion to the H20 control) observed in m ost o f the M2 progen ies could be expected. On the other hand, assum ing that high rad iation doses cause the form ation o f m utagenic substances in the fru it ju ice, such substances could have induced a h igher va ria tion in some o f the M2 progen ies. The un iform ity found in the progen ies derived from treatm ent at 800 kR then could be due to a se lection at gam etic le v e l as a consequence o f the accumulation o f mutations and harm ful e ffec ts at high doses.

H ow ever, the m odel d iscussed here would not explain the h igher v igour shown by the M2 seed lings derived from the treatm ents at 400 and 6 00 kR.

It must be recogn ized that the corn va r ie ty used had a high genetic va ria tion and that the number o f M2 progen ies tested was too low to draw genera l conclusions from th is experim ent.

R E F E R E N C E S

[1 ] MASSEY, J.L.M. , BOURKE, J. B ., "Some radiation-induced changes in fresh fruits and vegetables", Proc. Symp. Radiat. Preservation Foods. Atlantic City, 1965, ACS, Washington, D.C. (1967).

[2 ] CHOPRA, V .L . , NATARAJAN, A .T . , SWAMINATHAN, M. S ., Cytological effects observed in plant material grown on irradiated fruit juices, Radiat. Bot. 3 (1963) 1.

[3 ] SWAMINATHAN, M .S ., Incidence of mutations in D. melanogaster fed on irradiated medium,Science 141 (1963) 637.

[4 ] RINEHART, R. R ., RATTY, F .J ., Mutation in D. melanogaster cultured on irradiated food, Genetics 52 (1965) 1119.

[5 ] CHOPRA, V .L . , Tests on Drosophila for the production of mutations by irradiated medium or irradiated DNA, Nature, Lond. 208 (1965) 699.

[6 ] REDD1, O .S ., REDDY, G.М ., RAO, J.J., EBENEZER, D. N ., RAO, M .S ., Lack of mutagenic effect of irradiated Drosophila medium, Nature, Lond. 208 (1965) 702.


D IS C U S S IO N

B. SIGURBJORNSSON: I would be v e ry care fu l in drawing some o f the conclusions you have done in your paper. F ir s t o f a ll the number o f seeds is v e ry low and you used no rep lica tions fo r the germ ination tests.The only conclusion I can draw, looking at your data, is that banana ju ice seem s to d ecrease germ ination and reduce, at least in itia l seed ling growth.I see no va lid d iffe ren ce due to irrad iation . Furtherm ore, in d iscussing your Tab le IV you state that the variance was highest in treatm ents at400 and 600 kR. In fact your table shows that the highest variances occu rred in the w ater treatm ent consistently.

A . BLUM ENSCHEIN : I agree the d iffe ren ces are not strik ing; however, the rep rodu cib ility o f the tendencies did im press me. On the other side, the sta tis tica l resu lts seem highly significant.

In re la tion to Tab le IV , i f you consider the d ifferen ce between H20 treatm ents and 400 and 6 00 kR (in genera l) to be sign ificant, the other d iffe ren ces re la ted to the height means a re m ore sign ificant. I recogn izedI should repeat the experim ent using an isogen ic line instead o f a highly va riab le va r ie ty as I did. Th is w ill cut some corners.

G. T . SC AR ASC IA -M U G N O ZZA : B esides the resu lts you have presented now, have you observed chrom osom e breakage?

A . BLUM ENSCHEIN : We have not checked that yet.M. S. SW AM IN ATH AN : Did you prepare the ju ice im m ediately a fte r the

bananas w ere irrad ia ted o r did you store them fo r some tim e? I f you stored them a fter irrad ia tion was the storage tim e constant in the d ifferen t treatm ents?

A . BLUM ENSCHEIN : Y es , the ju ice was prepared right a fter irrad iation , and was stored fo r two days under re fr ig e ra t io n (ca . 5°C). The storage tim e was constant fo r a ll treatm ents.

C. PA N TO N : Could you te l l me whether the bananas used w ere seeded o r parthenocarp ic types and a lso at what stage o f "g reen -n ess " w ere they used? A s I r e c a ll certa in m edicinal p roperties are attached to the banana, and I th e re fo re wonder about the e ffec t o f this prin c ip le on seeds o f germ inating m aize.

A . BLUM ENSCHEIN : They w ere parthenocarpic types. The stage was the right one fo r harvest (as defined by the banana grow ers ). I know banana has been used in some cases, m ain ly by poor people, fo r treatm ent o f wounds; how ever, I do not know m ore about the m ed icinal e ffec ts .

C. F . K O N ZA K : I would suggest that such an exp lora tory study should rather be done on m icro -o rgan ism s firs t . F ew i f any higher plant m ateria ls o ffe r a su ffic ien t d egree o f resolu tion a fter studying such few numbers as you did. It would be better to do such a study on bacteria o r yeast firs t , then test any in terestin g treatm ents on a plant system , and in this regard cy to log ica l studies would be appropriate.

A . BLUM ENSCHEIN : P a ra lle l to the banana experim ent,other c o lleagues in our Institute are developing experim ents using A sperg illu s strains. And as I r e c a ll they a re getting some e ffec t with 400 kR doses.The cy to log ica l studies w ill be ca rr ied out. Thank you fo r the suggestion.

GENERAL DISCUSSION

THE ROLE OF INDUCED MUTATIONS IN PLANT IMPROVEMENT

R.D. BROCKCommonwealth Scientific and Industrial Research Organization (CSIRO), Canberra, Australia

Abstract-Resumen

THE ROLE OF INDUCED MUTATIONS IN PLANT IMPROVEMENT.Induced mutations are considered as an alternative to naturally occurring variation as the source of

germ plasm for plant improvement programs, and as an alternative to hybridization and recombination in plant breeding.

While there is no theoretical need to preserve naturally occurring variation as a source of germ plasm for traits controlled by single genes, it is often more economical to transfer available genes than to induce new mutations. Until there is better understanding of the role of linked complexes of genes in determining quantitatively inherited traits, it w ill not be possible to fully delineate the relative roles of induced mutations and recombination for these traits.

The relative advantages of mutation and recombination in plant breeding have been considered in relation to the genetic nature o f the trait to be improved and the breeding system of the species. The cost of mutation plant breeding has been assessed in terms of the effort required (population sizes) and the effect on the background genotype.

New concepts, which suggest that mutations are neither rare events nor largely deleterious in their effects, have been reviewed. The relevance of these concepts to some aspects o f the use of induced mutations in plant improvement have been considered.

FUNCION DE LAS MUTACIONES INDUCIDAS EN EL MEJORAMIENTO DE ESPECIES VEGETALES.El autor considera las mutaciones inducidas como fuente de plasma germinal para programas de

mejoramiento de especies vegetales, en comparación con la variación natural; también las considera como método aplicable en fitotecnia, en comparación con la hibridación y la recombinación.

Si bien teóricamente no hay ninguna necesidad de conservar la variación natural como fuente de plasma germinal para los caracteres dependientes de genes particulares, a menudo es más económico transmitir los genes disponibles que inducir nuevas mutaciones. En tanto no se conozca mejor e l papel de los complejos enlazados de genes en la determinación cuantitativa de los caracteres hereditarios, no será posible delimitar plenamente las funciones respectivas de las mutaciones inducidas y de la recombinación en lo que respecta a dichos caracteres.

El autor examina las ventajas relativas de la mutación y la recombinación en fitotecnia en relación con la naturaleza genética del carácter que se pretende mejorar y las modalidades de reproducción de la especie. Evalúa el costo de la fitotecnia por mutaciones en función del trabajo requerido (magnitud de la población) y del efecto sobre e l genotipo base.

Pasa revista a nuevas teorías según las cuales las mutaciones no son sucesos raros ni de efectos grandemente perjudiciales. Estudia la aplicabilidad de estas teorías a algunos de los aspectos relativos al empleo de mutaciones inducidas en el mejoramiento de las plantas.

1. IN TRO D U CTIO N

Plant im provem ent in vo lves f ir s t , the assem bly o f an adequate gene pool and the se lection o f d es irab le genes o r genotypes either as parental m a ter ia l fo r further im provem ent, o r fo r im m ediate testing as potential cu ltivars . Secondly, manipulation o f the se lected genes o r groups o f genes to generate m ore favourable combinations fo llow ed by further selection . Th ird ly , com parative tests to dem onstrate the su perio rity o f the se lected genotypes, culm inating in the re lea se o f im proved cu ltivars.

513

514 BROCK

P lan t introduction is the conventional source o f genetic varia tion fo r the f ir s t phase. Recom bination fo llow ing hybrid ization (conventional plant breeding) is the usual source o f varia tion fo r the second phase. How ever, the induction o f mutation p rov ides potentia l a lternatives to both plant introduction and hybrid ization . W hile it is s t ill not possib le to define p re c is e ly the re la t iv e m erits o f the a lternative system s, a rev iew o f some o f the basic features o f induced mutations establishes prin c ip les fo r choosing between inducing mutations and the m ore conventional methods o f plant im provem ent,

2. INDUCED M U TATIO N S AS A SOURCE O F GERM PLA S M

Plant b reeders a re w e ll aware o f the need fo r la rg e and d iverse sources o f germ plasm to m eet the demands o f current and future plant breed ing p rogram s. So fa r these needs have been la rg e ly supplied from the re s e rv o ir s o f the natural gene pools.

In v iew o f the great advances in our knowledge o f the nature o f genes and genetic varia tion , and our ab ility to create varia tion by the induction o f mutations, is there any need to p re se rv e natural gene pools?

Natural varia tion is s im p ly the product o f spontaneous mutation, moulded by recom bination and natural selection . W e can induce any mutation that has occu rred naturally, and probably many which have either never occu rred spontaneously, o r have been los t from the natural populations. We can, by applying the appropriate se lection techniques, reta in those mutants suitable fo r m odern agricu ltu ra l system s rather than be dependent upon those that have su rvived natural and p r im itiv e se lection . Thus at the le v e l o f the individual gene there seem s lit t le doubt that we can induce the fu ll range o f genetic varia tion that ex ists in the natural gene pools. The choice o f the m ost appropriate system to be used then becom es la rg e ly a question o f the e ffic ien cy o f se lection that can be applied fo r the mutant gene com pared with the ease o f tran s fer o f the mutant character to other genotypes. Th ese are la rg e ly questions o f working econom ics and are determ ined by the genetic nature o f the character under study and the breed ing system o f the spec ies . F o r characters determ ined by the action o f s ing le genes there is no th eoretica l need fo r the p reserva tion o f natural germ plasm but it m ay be m ore econom ical to tran s fer ra ther than to induce mutations. W hile this is undoubtedly true at the le v e l o f the individual gene, does it apply at the le v e l o f the genotype phenotype?

The extent to which induced mutations p rovide a usefu l a lternative to natural varia tion as a source o f germ plasm fo r the im provem ent o f such tra its , w ill be la rg e ly determ ined by the im portance o f linked groups o f genes, and the degree to which natural se lection has built up linked gene com plexes o f adaptive sign ificance in naturally occu rring genotypes. W here such linked sets o f genes occur they can be tran s fe rred to other genotypes, but they are un likely to be produced as the resu lt o f random mutations.

W hile it is gen era lly assumed that linked com plexes o f genes are im portant fo r plant im provem ent there is su rp ris in g ly lit t le evidence to support this assumption. Until the true ro le o f gene com plexes in plant im provem ent is adequately determ ined, the p lace o f induced mutations as a source o f germ plasm fo r quantitatively inherited tra its must rem ain uncertain.

INDUCED MUTATIONS IN PLANT IMPROVEMENT 515

3. INDUCED M U TATIO N S IN P L A N T BREEDING

Any proposa l to use induced mutations in plant breed ing must consider the likelihood o f success when com pared with conventional techniques and the e ffo r t requ ired to obtain the des ired genotype. Th is has been discussed in deta il in the Manual on Mutation B reed ing [ 1 ] and only a sum m ary w ill be presented here. The likelihood o f success is considered in re la tion to the breeding system o f the species and the genetic control o f the character to be im proved .

3 .1 . S e lf- fe r t iliz in g species

3 .1 .1 . S im ply inherited tra its , e .g . d isease res istance

The choice between induced mutations and gene tran s fe r in the case of s im p ly inherited tra its is la rg e ly determ ined by the ease with which the gene can be mutated, com pared with the ease with which it can be in corporated from another genotype.

The induction o f a re c e s s iv e mutation is a much m ore lik e ly event than the induction o f a dominant mutant. Th is fact, plus the ease o f in tra sp ec ific tran s fer o f a dominant gene by conventional genetic methods, favours hybrid ization breed ing where the d es ired ch aracteris tic is known, o r presum ed to be conditioned by a dominant gene.

In the case o f re c e s s iv e genes the induction o f mutations is m ore worthy o f consideration. I f the gene is ava ilab le in a genotype c lose ly re la ted to the agricu ltu ra l cu ltivar, tran s fer o f the gene by back-cross ing would norm ally be favoured as the method m ore lik e ly to p rove successfu l.I f c lose linkage with undesirable characters o r undesirable p le io trop ic e ffec ts a re found to be involved in a gene-tran s fer p rogram , o r i f tran s fer o f the d es ired gene invo lves in te r -sp ec ific o r in te r-g en e r ic crossing, induced mutations m ay be the p re fe rab le technique. I f there is no known source from which the gene can be tran s ferred , induced mutations are, o f course, the only p o ss ib ility and the decision to in itia te a program based on induced mutations depends la rg e ly upon econom ic considerations such as the im portance o f the ob jective and the cost o f screen ing fo r the desired mutation.

Mutations a re p a rticu la r ly e ffic ien t aids to the dom estication o f natural spec ies . R em ova l o r suppression o f undesirable features such as a toxin, spines o r thorns, is the f ir s t step in the adaptation o f a plant to m an's use. With a ll o f our o ld er agricu ltu ra l species this has a lready been accom plished but it rem ains o f im portance fo r the introduction o f new species into agricu ltu re.

3 .1 .2 . Quantitatively inherited tra its , e .g . y ie ld , m aturity tim e, adaptability

These characters a re con tro lled by the in teraction o f many genes each o f sm a ll e ffect. In these situations the e ffic ien cy o f se lec tin g the desired mutant is gen era lly low er than fo r sp ec ific characters which are con trolled by a single gene, but this is la rg e ly o ffse t by the increased frequency o f mutants resu lting from the g rea te r number o f genes invo lved . A ltern a tive sources o f va r iab ility , and the p r ic e to be paid in term s o f a ltera tion to

516 BROCK

the background genotype a re the m ost im portant considerations to be taken into account in deciding whether to use induced mutations to im prove quantitatively inherited characters.

3 .2 . C ro s s - fe r t i l iz in g species

In genera l, induced mutations o ffe r less p rospect fo r the im provem ent o f c r o s s - fe r t i l iz in g spec ies . Th is is p a rtly because o f the d ifficu lty o f se lecting, incorporating and maintaining re c ess iv e mutations in such populations and partly because the plant breeding prob lem s in cross - fe r t iliz in g species a re m ore often prob lem s o f handling the existing v a r ia b ility than lack o f v a r ia b ility p er se. The use o f induced mutations in these situations cannot be gen era lly recom m ended unless it can be c lea r ly established that naturally occu rring va r ia b ility has been fu lly u tilized .

3. 3. V ege ta tive ly propagated species

Plan t im provem ent has depended v e ry la rg e ly upon the se lection o f naturally occu rring mutants (sp o rts ). Consequently, techniques which in crease the frequency o f mutations should be o f grea t value. Th is is a lready p roving so [ 2, 3, 4 ], and a number o f new flo r icu ltu ra l va r ie tie s , produced as the resu lt o f the induction o f mutations, have been re leased .

4. COST O F M U TA T IO N BREEDING

The cost o f mutation breed ing must be assessed both in term s of a lterations to the background genotype o f the parental va rie ty , and in term s o f the e ffo r t which w ill have to be expended to obtain the des ired mutant.

4 .1 . P lan t genotype

A common m isconception about induced mutations is that they occur without any a ltera tion to the background genotype. Consideration o f the e ffec t o f mutagenic treatm ents on quantitatively inherited characters shows that this is not so. A l l o f the mutagenic treatm ents at our command induce essen tia lly random changes to the genotype, and at the treatm ent le v e ls used to g ive an appreciab le amount o f v is ib le mutations, considerable varia tion in quantitatively inherited characters is a lso induced [5 , 6 ].Hence, i f se lection is applied only fo r a spec ific mutant phenotype, the se lected mutant is v e ry lik e ly to be changed in a number o f other subtle but nevertheless im portant ways. Random mutation in creases the variance fo r a ll quantitatively inherited characters and, in the absence o f any se lection o r co rre la ted response, the mean o f the character w ill shift away from the d irec tion o f the previous se lection [7 ] . A s m ost agricu ltu ra l species have a lready been selected fo r high perform ance and adaptability, random mutation w ill on the average be deleterious and reduce the o ve ra ll perform ance. Th is trend can, of course, be re ve rsed by applying se lection fo r a ll im portant characters o r by incorporating the se lected mutant into a breed ing p rogram w here the desired mutant gene can be separated from the undesirable mutations.


T A B L E I. NUM BER O F C E L L PROGENIES TO BE EXAM INED FOR VARIOUS M U TA T IO N R ATE S AND P R O B A B IL IT IE S O F OCCURRENCE

(TABLA I. NUMERO DE PROGENIES CELULARES QUE HAN DE EXAMINARSE PARA DISTINTAS FRECUENCIAS DE MUTACION Y PROBABILIDADES DE OBTENCION)

Mutation frequency

(V)

No. of cell

p: = 0.90

progenies (n)

p: = 0.99Type of mutation

1 X lC f2 233 465 Chromosome changes and quantitatively inherited variability

1 X 10"S 2 326 4 652 Several recessive genes

1 X 10"4 23 260 46 520 Single recessive gene

1 X 10"5 232 600 465 200 Single dominant gene

T A B L E II . M 2 F A M IL Y SIZES FOR D IFFE R E N T SEG REG ATIO N RATIOS AND L E V E LS OF P R O B A B IL IT Y O F OCCURRENCE OF THE HOMOZYGOUS M U T A N T .

(TABLA II. MAGNITUD DE LA FAMILIA M2 PARA DISTINTAS RAZONES DE SEGREGACION Y GRADOS DE PROBABILIDAD DE OBTENCION DEL MUTANTE HOMOCIGOTICO)

Segregationratio

M2 family size (m)

p2 = 0.90 p2 = 0.99

a = 1/4 8. 0 16.0

1/8 17.2 34.5

1/12 26.3 52.6

1/16 35.7 71.4

1/20 45.5 91.0

4.2 . E ffo rt

An assessm ent o f the com parative e ffo rt, in term s o f plant m ateria l, labour and tim e, o f producing a new and useful genotype by mutation breed ing or by conventional breed ing is , unfortunately, not often possib le. H ow ever, b e fo re a mutation breeding program is in itiated som e assessm ent should be made of the plant population that w ill have to be exam ined to obtain the d es ired mutation.

518 BROCK

F o r single gene mutations, i f we assume a mutation rate (/u) and set a le v e l o f p robab ility fo r the occurrence of at least one mutation (PjJ, then the number o f treated ce lls that have to be exam ined (n) can be calculated from the form u la: n = log (1 -p ^ / lo g (1 - д ) .

By assum ing an expected mutation frequency fo r d ifferen t classes of mutations the number o f treated ce ll progen ies to be examined can be calculated (Tab le I ). W ith the uncertainty involved in estim ating the lik e ly mutation rate the application o f these figu res is n ecessa r ily approxim ate.

Mutations a re detected in the progeny o f the treated ce lls and re cess iv e mutations a re not expressed until at least the second generation (M 2) in diploid organ ism s. Hence, the values o f n given in Tab le I rep resen t the number o f M 2 fam ilies that have to be exam ined. Assum ing the mutagenic treatm ent to have a 50% lethal e ffec t (LD50 dose), the number o f f irs t generation (M i) plants requ ired to p rov ide these M 2 fam ilies is represen ted by the value 2n.

The number o f plants that have to be exam ined per M 2 fam ilies (m) can a lso be sim ply calculated fo r d ifferen t segrega tion ratios in the M 2 generation (Tab le II ).

Combining these two estim ates the population s izes can be calculated. F o r exam ple, accepting 90% probab ility o f occurrence o f the mutant and its presence as a detectable hom ozygote in the M2 generation, and assuming an induced mutation frequency o f 1 X 10'3, an Mj population o f 5000 plants (o r spikes) y ie ld ing 2500 fe r t i le progeny would be su ffic ien t. The mutant would have to be detected in an M 2 population o f approxim ately 50 000 (2500 X 20). H ow ever, i f the mutation frequency is 1 X 10"5, these populations would have to be 250 000 fo r the M j generation and 5 000 000 for the M 2 population.

Estim ates o f population s ize requ ired fo r detecting useful quantitatively inherited varia tion a re m ore d ifficu lt because neither the number o f genes involved, nor the m inimum s ize of e ffec t detectable in the M2 generation, can be determ ined. In m y own experience, where flow erin g tim e varia tion has been assessed in a number o f d ifferen t species, M 2 population s izes of approxim ately 1000 plants and the application o f 5% se lection intensity has always resu lted in quite substantial se lection responses [8 -1 2 ].

These calculations o f population s ize have been based la rg e ly upon mutation rates induced by gam m a- and X -ra ys because these data are m ore read ily ava ilab le and m ore eas ily com pared than the data from chem ical mutagens. The m ore e ffe c tiv e chem ical mutagens induce h igher mutation frequencies than do ion izing radiations [ 13] and to this extent the population s izes a re overestim ates .

5. NEW CO NCEPTS IN M U TATIO N

5. 1. Mutation rates and se lec tive e ffects

It is gen era lly be lieved that mutation rates are low and that m ost mutations a re de leterious. These b e lie fs have g rea tly influenced thinking about the ro le o f induced mutations in plant im provem ent and spontaneous mutations in evolution. Because spontaneous mutation ra tes have appeared to be v e ry low and mutations la rg e ly letha l o r deleterious, evolu tionary p rog ress has been be lieved to be p r im a r ily dependent upon recom bination


and segrega tion o f genetic d ifferences a lready present in the population. S im ila rly , because mutations have been believed to be la rg e ly towards a loss o f function, the ir ro le in plant im provem ent has been considered lim ited . Recent studies in m olecu lar genetics and esp ec ia lly on the rates o f evolution o f m olecu les suggest that spontaneous mutations m ay be neither ra re nor m ain ly deleterious.

Calculations o f expected rates o f nucleotide base in corporation e r ro rs during DNA rep lica tion a re at least two o rders o f magnitude h igher than the observed spontaneous mutation ra tes . These d iffe ren ces m ay be accounted fo r by e r ro r -c o r r e c t in g mechanism s in the intact c e ll o r they m ay indicate the presence o f many nucleotide base changes which do not have read ily observab le e ffects on the phenotype o f the whole organism .Such mutations would be neutral, o r nearly neutral to the e ffects of s e lec tive fo rc es .

The presence o f a high frequency o f s e le c t iv e ly neutral mutations has been postulated by K im ura [1 4 ] and K ing and Jukes [1 5 ] fo llow ing estim ations o f the ra tes o f amino acid rep lacem ent in d ifferen t species o f m am m als, and the extent o f nucleotide d ivergence estim ated by in v itro DNA hybrid ization . Other evidence fo r the presence o f a high frequency o f s e le c t iv e ly neutral o r n early neutral genetic changes com es from various sources [1 6 -2 3 ].

These findings tend to refu te the old concept that mutation rates are low and that m ost mutations a re deleterious. Th ere is lit t le doubt that ra tes o f nucleotide base change a re v e r y much higher than the rates suggested by phenotypic changes. S im ila r ly it is v e ry lik e ly that the great m a jo r ity o f these mutations have on ly a sm all e ffec t o r no e ffec t on the phenotype.K ing and Jukes [1 5 ] estim ate that the frequency o f s e le c t iv e ly neutral mutations is approxim ately equal to the frequency o f r e c ess iv e lethals, and that the frequency o f mutations o f sm all e ffec t is approxim ately ten tim es that o f the re c es s iv e lethals. Th is constitutes a tremendous store o f genetic va r ia b ility which has obvious evolu tionary consequences. Even g rea te r amounts o f genetic v a r ia b ility w ill be generated by mutagenic agents.

A n appreciab le frequency o f s e le c t iv e ly neutral nucleotide base changes would provide the opportunity fo r the occu rrence o f mutational events requ irin g seve ra l independent changes to the DNA. These new concepts p erm it the poss ib ility o f sequential mutational changes culm inating in new o r drastic a lterations to function o r form . The o ld er concepts, o f low er mutation rates and se lec tion tending to elim inate o r f ix each mutant genotype b e fo re it could be m od ified by further mutation, favoured recom bination as the m a jor m echanism fo r the evolution o f new function. Induced mutations should th ere fo re be ser iou s ly considered as a possib le source o f novel genetic change. These new concepts necessitate a re-eva luation o f the re la t iv e ro le s o f mutation and recom bination in evolution and as plant im provem ent is on ly a m od ified form o f evolution, they a lso requ ire a re-eva lu ation o f the re la t iv e ro les o f mutation and hybrid ization in plant im provem ent.

R E F E R E N C E S

[1 ] BROCK, R. D., "When to use mutations in plant breeding", Manual on Mutation Breeding, Tech. Rep.Set. No. 119, IAEA, Vienna (1970) 183.

[2 ] BROERTJES, C ., ’’Mutation breeding of vegetatively propagated'crops", Proc. 5th Eucarpia, Congr.,Milan (1968) 139.

520 BROCK

[3 ] BROERTJES, С ., Mutation breeding of Streptocarpus. Euphytica 18 (1969) 333.[4 ] BROERTJES, C. , HACCIUS, B. , WEIDLICH, S., Adventitious bud formation on isolated leaves and

its significance for mutation breeding, Euphytica V]_ (1968) 321.[5 ] BROCK, R.D., "Induced mutations affecting quantitative characters” , The Use of Induced Mutations

in Plant Breeding (Rep. FAO/IAEA Tech. Meeting, Rome, 1964), Pergamon Press, Oxford (1965) 451.[6 ] EMERY, D .A ., GREGORY, W .C ., LOESCH, P.J., "Breeding value of the radiation-induced macro-

mutant. II. Effect of mutant expression and associated backgrounds on selection potential in Arachis hypogaea L. ", The Use of Induced Mutations in Plant Breeding (Rep. FAO/IAEA Tech. Meeting,Rome, 1964), Pergamon Press, Oxford (1965) 339.

[7 ] BROCK, R.D., Quantitative variation in Arabidopsis thaliana induced by ionizing radiation. Radiat.Bot. 7 (1967) 193.

[8 ] BROCK, R.D., ANDREW, W .D ., KIRCHNER, R ., CRAWFORD, E.J., Early flowering mutants of Medicago polymorpha var. polymorpha. Aust. J. agrie. Res. 22^<1971) (in press).

[9 ] BROCK, R.D., ANDREW, W .D., X-ray-induced variation in Medicago polymorpha var. vulgaris.Aust. J. biol. Sci. 18 (1965) 1119.

[10 ] BROCK, R.D., Early maturing tomato mutants, J. Aust. Inst, agrie. Sci. 32 (1966) 136.[11 ] BROCK, R. D ., Quantitative variation in Arabidopsis thaliana induced by ionizing radiations, Radiat.

Bot. 7 (1967) 193.[12 ] BROCK, R. D ., LATTER, B .D.H., "Radiation-induced variation in subterraneum clover", Proc. 3rd

australias. Conf. Radiobiol., Butterworfh, London (1961) 205.[13 ] KAWAI, T ., "Relative effectiveness of physical and chemical mutagens", Induced Mutations in Plants

(Proc. Symp. Pullman, 1969), IAEA, Vienna (1969) 137.[14 ] KIMURA, М ., Evolutionary rates at the molecular level, Nature 217 (1968) 624.[15 ] KING, J .L ., JUKES, Т .Н ., Non-Darwinian evolution, Science 164 (1969) 788.[16 ] MUKAI, T ., The genetic structure of natural populations of Drosophila melanogaster. I. Spontaneous

mutation rates of polygenes controlling viability, Genetics 50 (1964) 1.[17] HUBBY, J .L ., LEWONTIN, R.C., A molecular approach to the study of genic heterozygosity in

natural populations, I. The number of alleles at different loci in Drosophila pseudobscura. Genetics 54 (1966) 577.

[18] LEWONTIN, R.C ., HUBBY, J .L ., A molecular approach to the study of genic heterozygosity in natural populations, II. Amount of variation and degree of heterozygosity in natural populations of Drosophila pseudobscura, Genetics 54 (1966) 595.

[19 ] HARRIS, H .f Enzyme polymorphisms in man, Proc. R. Soc. В 164 (1966) 298.[20 ] SICK, K .. BEALE, D ., IRVINE, D., LEHMANN, H ., GOODALL, P .T . , MacDOUGALL, S.,

Haemoglobin GCopenhagen ancl haemoglobin J Cambridge* Two new 13-chain variants of haemoglobin A, Biochim. biophys. Acta^.40 (1967) 231.

[21 ] MARGQLIASH, E., FITCH, W .M ., DICKERSON, R. E., Molecular expression of evolutionary phenomenain the primary and tertiary structures of cytochrome c, Brookhaven Symp. Biol. 21^(1969) 259.

[22 ] YANOFSKY, C ., Amino acid replacements associated with mutation and recombination in the A gene and their relationship to in vitro coding data. Cold Spring Harb. Symp. quant. Biol. 28 (1963) 581.

[23] LANGRIDGE, J., CAMPBELL, J-H., Classification and intragenic position of mutations in the ô-galactosidase gene of Escherichia coli, Molec. gen. Genet. 103 (1969) 339.

D IS C U S S IO N

H. G A U L : I.d isa g ree with many o f your points. I w ill mention only a few points. T o me it appears m ore econom ical to induce a mutation in an adapted va r ie ty (top va r ie ty ) instead o f tran s fe rr in g the sam e or a s im ila r character from a p r im itiv e o r w ild form .

Changes o f so -ca lled quantitatively inherited tra its like y ie ld , etc., m ay be ca rr ied by single factor mutations. The e ffic ien cy (e fficacy ) of using m icro-m utants is higher than that o f m acro-m utations.The vast m a jo r ity o f mutations is deleterious.

R . D. BRO CK: In the f ir s t p lace you r e fe r to m y statement that fo r characters determ ined by the action o f single genes, there is no th eoretica l


need fo r the p reserva tion o f natural germ plasm , nevertheless it m ay be m ore econom ical to tran s fer ra ther than to mutate. Th is statement is ju stifica tion fo r p reserva tion o f natural germ plasm and its re levance to any particu lar plant breed ing situation has to be considered separate ly .

A s I pointed out when d iscussing the use o f induced mutations in plant breed ing, the best s tra tegy fo r any particu lar plant breeding situation w ill be determ ined by the breeding system o f the species, the genetic control o f the character and re la tiv e costs o f achieving the ob jective by mutation o r by hybrid ization . The cost must be assessed both in econom ic term s and in the extent o f the a lteration to the background genotype o f the va r ie ty to be im proved.

A s fa r as I understand your second point you a re r e fe r r in g to the d ifferen t se lection e ffic ien c ies that apply fo r tra its o f la rg e and sm all phenotypic e ffec ts . Undoubtedly, la rg e phenotypic e ffec ts , such as res is tan ce to d isease, can be m ore read ily detected than the sm all phenotyp ic e ffec ts , such as sm a ll y ie ld in creases. But i f sm a ll phenotypic e ffects occur with a v e ry much h igher frequency, response to se lection w ill be ach ieved in re la t iv e ly sm all populations; whereas v e r y much la rg e r populations w ill be requ ired fo r even the single occu rrence o f a d isease res is tance mutant.

Your th ird point is a repetition o f the old concept that m ost mutations a re deleterious. Th is is undoubtedly true i f you re s tr ic t your concept o f • a mutation to a heritab le change which has a detectable e ffec t on the phenotype o f the whole organ ism . The point I was try in g to make was that, at the le v e l o f DNA there a re m any nucleotide base changes which do not have any detectable e ffec t on the phenotype o f the whole organ ism . Because these a re h eritab le a lterations to the genetic m a ter ia l they are by defin ition mutations. Because they have no e ffec t on the phenotype o f the whole organ ism they a re s e le c t iv e ly neutral. These new concepts have im portant im plications fo r the way we think about mutations and the ro le they m ay have in evolution and plant im provem ent. They do not a lte r the p ra c tica l fact that most mutations which a ffec t the phenotype o f the whole organ ism are deleterious.

A. GUSTAFSSON: The f ir s t X -ra y v a r ie ty re leased into the m arket was the "P r im e x " s tra in o f Sinapis alba. The o r ig in a l v a r ia b ility o f the population ava ilab le was quite low and by irrad ia tion ал enormous increase o f genetic va r ia b ility was obtained. A fte r "s e v e r e se lection " this resu lted in a new highly im portant va r ie ty , la te r on giving r is e to even further im provem ent. Thus a lready in the 1940's induced varia tion was accom plished in h igh ly c r o s s - fe r t i l iz in g species. Your statements in section 3. 2. o f your paper a re th e re fo re in a sense m islead ing.

R .D . BROCK: I do not agree that m y statement was m islead ing. WhatI said was that the use o f induced mutations cannot be gen era lly recom m ended fo r c r o s s - fe r t i l iz in g species unless it can be c le a r ly established that naturally occu rrin g v a r ia b ility has been fu lly u tilized . A s you pointed out, the natural va r ia b ility in the population o f Sinapis alba was low . Th is va r ia b ility was increased by the induction o f mutations and a new va r ie ty resu lted .

SUMMARY OF GENERAL DISCUSSION

On the last day o f the Study Group M eeting a gen era l d iscussion was held fo llow ing the in troductory rev iew paper o f B rock (A u s tra lia ). Some o f the highlights o f the d iscussion are sum m arized below.

Among the f ir s t questions to be asked w ere whether the use o f induced mutations should be ca lled an "advanced plant breed ing technique" com pared with se lection and hybrid ization , and whether it should be p re fe r red to these "conven tional techn iques". T h ere was fu ll agreem ent that a good plant b reeder w ill make use o f a ll p rom ising techniques fo r crop im provem ent w henever appropriate, no m atter whether they have been known fo r 100 years o r 10 yea rs . A l l plant breeding techniques a re com plem entary to each other and mutation breed ing like other breed ing schem es w ill always include se lection and w ill a lso frequently in vo lve hybrid ization .

Another question was concerned with the re la tiv e value o f natural versus induced genetic va r iab ility . It was asked whether natural genetic va r ia b ility could be d iscarded because o f the p oss ib ility o f re -es tab lish in g such va r ia b ility by mutagen treatm ents. S evera l speakers com m ented that although natural v a r ia b ility is bas ica lly not d ifferen t from induced v a r ia b ility it has its own unique value. Th is derives not from a d ifferen t mutation spectrum but from a long se lection p rocess which has elim inated the m a jo r ity o f u se less fac tors and accumulated those leading to m ore b io log ica l fitness. Furtherm ore, as the genotype o f a crop plant is made up o f s e v e ra l thousand genes which can be a ltered one by one, the r e - induction o f lo s t genotypes appears p ra c tica lly im possib le w ithin a reasonable tim e lim it. Experts w ere convinced that a ll lo s t a lle le s o f e v e ry gene can be re-induced, but that th e ir combinations cannot, e ither by mutagen treatm ent o r by extensive cross ing experim ents. T h ere fo re , p reserva tion o f ex isting genotypes is o f fundamental im portance. H ow ever, since losses o f genetic v a r ia b ility w ill never be fu lly avoidable, the induction o f mutations was considered as the on ly re a lly e ffec tive means to p rovide future plant b reeders with the building blocks fo r new im proved va r ie tie s . Induction o f mutations would not be som ething unnatural, but ra ther help to maintain and re -es tab lish the w ide natural base fo r crop plant im provem ent.

In fo llow ing these thoughts, it was suggested by som e participants to have g e rm p lasm co llections system atica lly trea ted with mutagens in o rd er to counteract the continuous leakage o f genes from such co llections. Th is suggestion did not find genera l approval, how ever, since it would entail duplication o f the a lready la rg e assortm ents, because the untreated orig in a l populations would need to be maintained as w ell.

Em phasizing the rem arkab le success in recen t yea rs o f plant b reeders using induced mutations in th e ir p rogram s, one participant expressed his view that this success is p a rtly due to the fact that mutation techniques have attracted many b rillian t,sc ien tis ts , while the m a jo r ity o f plant b reeders

523

524 SUMMARY OF GENERAL DISCUSSION

"d id not use a ll the ir brain pow er", but rather fo llow ed trad itiona l lines.It was fu rther stressed that mutation resea rch on various le ve ls has contributed v e ry valuable knowledge about the plant organ ism , its genetics, its ph ys io log ica l m echanism s, its d isease reactions, e tc ., but that so fa r lit t le o f this in form ation has been used in plant breeding. The hope was expressed that the w ider d issem ination o f sc ien tific knowledge by various means, including m eetings lik e the present one, would stim ulate plant b reeders to apply a ll th e ir sk ill and would encourage them to fo llow non- trad itional sc ien tific trends.

Turning to m ore spec ific subjects, the questions w ere discussed whether so -ca lled m ic ro - o r m acro-m utations a re o f g rea te r im portance fo r plant breeding and whether the use o f d ifferen t mutagens in fact o ffe rs d ifferen t chances fo r the se lection o f useful mutants. The f ir s t question was answered in the sense that both m ic ro - and m acro-m utations are o f value fo r plant breed ing. The term s as such w ere re je c ted by some participants as being s c ien tifica lly m isleading. H ow ever, it was agreed that they a re usefu l fo r the p ractica l b reeder with regard to the d ifferen t se lection techniques to be applied. It was suggested that, to prevent m is understanding, it is better to ta lk in term s o f "mutations with la rg e r o r sm a lle r phenotypic exp ress ion ". R egard ing the question o f mutagenic spec ific ity , the participants w ere r e fe r re d to a number o f recen t rev iew a rtic les which g ive a fa ir ly la rge number o f exam ples o f d iffe r in g mutation spectra aris ing from treatm ents with d ifferen t types o f rad iation and other mutagens (see FAO /IAE A Manual on Mutation Breeding, Tech . Rep. Ser. No. 119, IA E A , Vienna, 19 70). It was a lso commented that so fa r in h igher plants we are probably s t i l l only dealing with mutations at the chrom osom al le v e l rather than with those at the DNA le v e l (base a lterations), no m atter which mutagen we use. When any mutation induced by radiation or EMS has been analysed in any detail, it has turned out to he som e type o f chrom osom e rearrangem ent.

With rega rd to the chances o f success fo r a plant b reeder using mutation techniques, it was concluded that it is never re a lly possib le to p red ic t whether any plant breeding p ro jec t - no m atter i f it is based on sim ple selection , on hybridization , on polyp lo id ization , o r on induced mutations - w ill lead to a new va rie ty . E ve ry technique has its particu lar chances and its lim ita tions, and the plant b reeder needs to know not on ly the techniques as such, but a lso which technique is m ost appropriate fo r a particu lar plant type and the particu lar breed ing ob jectives.

The assem b ly also attempted to look into the future and made various recom m endations with rega rd to research areas where m ore ac tiv ity should be developed. Among them, high p r io r ity was g iven to mutation research on asexually propagated plants, which com prise a g rea t number o f important crops in La tin A m er ica and other trop ica l a reas. Quite often the genetica l va r ia b ility fo r the im provem ent o f these crops is lacking o r v e ry lim ited . Induced mutations could be a basic means to ach ieve p rog ress . It was also fe lt that c ross-po llin a tin g plants have unfortunately been somewhat neglected by mutation b reeders and that suitable steps should be taken by national and international institutions to stim ulate research in this fie ld .

The suggestions put fo rw ard went even a further step beyond the present considerations; one o f the eminent plant breeding experts expressed his v iew that future plant breeding would tend to go much m ore deeply into the basic ph ysio log ica l p rocesses of plant l i fe , such as C O 2 assim ilation , and to in crease th e ir e ffic ien cy . Even the recen t rem arkab le successes o f

SUMMARY OF GENERAL DISCUSSION 525

plant breeding, which have resu lted in the so -ca lled green revolu tion , w ere based m ostly on changes in the re la t iv e proportion o f useful to u seless plant products, and not on an inherent ab ility of plants to produce m ore substance. Consequently, ass im ila tion m odel studies, including mutation techniques, should be in itiated to further the use o f induced mutations as an e ffec tive too l fo r the production o f even better yie ld ing types o f crop plants fo r feeding mankind in the future.

526

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LIST OF M U TANT VARIETIES 535

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536 APPENDIX

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LIST OF M U TANT VARIETIES 537

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538 APPENDIX

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LIST OF M U T A N T VARIETIES 539

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544 APPENDIX

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Ag

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unda

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A R G E N T IN A

Anitua, J.

Antonelli, E.F.

Arriaga, H.

Baracco, N.

Barbagelata, P.

Boggiato, A.J.

Bokde, S.

Bruni, О.

Burgnoni, L.

Calvar, D.

Cálvelo, A.

Campagnac, N.

Chabrillon, A.

Claver, F.

Correnti, J.

LIST OF PARTICIPANTS

Northrup, King and Co.,Buenos Aires

Centro de Investigaciones en Ciencias Agronómicas (CICA),

Instituto Nacional de Technología Agropecuaria (INTA), Castelar, Prov. de Buenos Aires

Facultad de Agronomía,La Plata, Prov. de Buenos Aires

Compañía Continental,Murphy, Prov. de Santa Fe

Estación Experimental Agropecuaria (EEA),Instituto Nacional de Technología Agropecuaria (INTA), Paraná, Prov, de Entre Ríos

Facultad de Agronomía,Universidad de Tucumán,San Miguel, Prov. de Tucumán

EEA, INTA,Pergamino, Prov. de Buenos Aires


Centro Argentino de Ingenieros Agrónomos,Buenos Aires

ÉEA,INTA,General Roca, Prov. ú é Río Negro

Bolsa de Cereales,Buenos Aires

EEA, INTA,Roque Sáenz Pefia, Prov. del Chaco

EEA, INTA,Paraná, Prov. de Entre Ríos

Facultad de Agronomía,La Plata, Prov. de Buenos Aires

CICA, INTA,Castelar, Prov. de Buenos Aires

545

546 LIST OF PAR TIC IPAN TS

Crnko, J.

Damilano, A.

Dihaice, J.A.

Di Pardo, R.

Erejomovich, J.

Ezcurra, O.

Favret, E. A.

Fogante, R.

Frecha, J.H.

García, U.

Gardenal, L.

Gear, J.

Giandana, E.

Gnoatto, I.

Godeck, W.

Godoy, E.

Gutiérrez, M.

Hunziker, J.

Induni, C.

EEA, INTA,Prov. de Mendoza


Diharce y Cfa,Buenos Aires

Criadero y Semillero,Morgan, Buenos Aires

EEA, INTA,Paraná, Prov. de Entre Ríos

Consejo Federal de Inversiones, Buenos Aires

CICA, INTA,Castelar, Prov, de Buenos Aires



Director Nacional del INTA,Buenos Aíres

EEA, INTA,Roque Sáenz Pefla, Prov. del Chaco

Criadero el Boyero,Rojas, Prov. de Buenos Aires

EEA, INTA,Manfredi, Prov. de Córdoba

EEA, INTA,Corrientes, Prov. de Corrientes


EEA, INTA,Marcos Juárez, Prov. de Córdoba

EEA, INTA,Roque Sáenz Peña, Prov. del Chaco

Laboratorio de Genética, Universidad de Buenos Aires,Buenos Aires

EEA, INTA,Balcarce, Prov. de Buenos Aires

LIST OF PARTICIPAN TS 547

Jetter, W.

Jimenez, H.

Kirschbaum, W.

Klein, F.A.

Klein, O.

Koenig, G.

KrulU C.

Lifschitz, E.

Lifschitz, F.M.

Llórente, С.

M alvarez, Elsa

Manghers, L.

Mariotti, J. A.

Marteau, V, G.

Martin, G.О.

Mazar Barnett,

Merzari, A. H.

EEA,INTA,Corrientes, Prov. de Corrientes

EEA,INTA,Paraná, Prov. de Entre Rfos

Facultad de Ciencias Agrarias, Universidad Católica,Buenos Aires

Criadero y Semillero Klein,Plá, Prcv. de Buenos Aires

Criadero y Semillero Klein,Plá, Prov. de Buenos Aires

Consejo Federal de Inversiones,Buenos Aires

De Kalb Argentina SA1C,Buenos Aires


CICA, ШТА.,Castelai, Prov. de Buenos Aires

Asgrow Llórente SAIC,Buenos Aires



Facultad de Agronomía y Zootecnia, Universidad de Tucumán,San Miguel, Prov. de Tucumán

Ingenio Nuñorco,Monteros, Prov. de Tucumán

Facultad de Agronomía y Zootecnia, Universidad de Tucumán,San Miguel, Prov. de Tucumán

Beatriz Comisión Nacional de Energía Atómica,Buenos Aires

Facultad de Agronomía y Veterinaria, ■Universidad de Buenos Aires,Buenos Aires

Miro De Rosbaco, Aurora EEA, INTA,Paraná, Prov. de Entre Ríos

548 U S T OF PAR TIC IPAN TS

Muhlenberg, C.E,

Mujica, F.L.

Olsen, H.A.

Pacagnini, H.

Pahlen, A. von der

Parodi, R.

Parodi, Rosa

Pavoni, J. C.

Pérez, J.A.

Piterbarg, B.

Ponce, M.G,

Popovich, M.

Quinteros, I.R.

Universidad Católica de Santa Fe, Esperanza, Prov, de Santa Fe


Criadero Vilela,Bahía Blanca, Prov. de Buenos Aires

Compañía Continental,Murphy, Prov. de Santa Fe


EEA, INTA,Manfredi, Prov. de Córdoba

Venado Tuerto,Prov. de Buenos Aires

EEA,INTA ,Marcos Juárez, Prov. de Córdoba

Universidad de La Pampa,Santa Rosa, Piov. de La Pampa


Mendoza 327, Prov. de Córdoba


Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata,La Plata, Prov. de Buenos Aires

Re, R.R.

Remussi, C.

Roby, F.

Rodríguez, D.S.

Rodríguez, N.

Rodríguez Amieva, P.

Facultad de Agronomía,Universidad Nacional de La Plata,La Plata, Prov. de Buenos Aires

Facultad de Agronomía y Veterinaria, Universidad de Buenos Aires,Buenos Aires

EEA, INTA,Luján de Cuyo, Prov. de Mendoza

EEA, INTA,Bella Vista, Prov, de Corrientes

EEA, INTA,Corrientes, Prov. de Corrientes



Rojo, N.

Rosbaco, U.

Rosenvaig, J.

Ryan, G.S.

Sancho, R,

Sarasola, J.

Saumell, H.

Saura, F.

Schnack, В.

Senano, H ,,

Silvero Sanz, O.I.

Solari, Rut M.

Terraciano, L.C. de

Tirant!, I.

Troncoso, J.M.

Vallega, J.

Vrdoljak, J.

Zubrzycki, H.M.


EEA, INTA,Paraná, Prov. de Entre Rios

Venado Tuerto,Prov. de Buenos Aires


EEA, INTA,Campana, Prov. de Buenos Aires




Facultad de Agronomía,Universidad de La Plata,La Plata, Prov. de Buenos Aires


EEA, INTA,Roque Sáenz Peña, Prov. del Chaco



Departamento de Agronomía, Universidad Nacional del Sur,Bahía Blanca, Prov. de Buenos Aires

EEA,INTA,Paraná, Prov. de Entre Ríos

Consejero Agrícola,Embajada Argentina en Roma,Roma, Italy

EEA, INTA,Roque Sáenz Pefia, Prov. del Chaco



Brock, R.D. Commonwealth Scientific and IndustrialResearch Organization (CSIRO),

Canberra

AUSTRALIA

AU S TR IA

Hfinsel, H. Institute for Crop Husbandry and Plant Breeding,Hochschule für Bodenkultur, Vienna; Probstdorfer Saatzucht,Probstdorf, N.O.

B R A Z IL

Department of Genetics, ESALQ, University of sao Paulo,Piracicaba

Department of Genetics, ESALQ, University of SSo Paulo,Piracicaba

Universidad de Campinas,Campinas, S.P.

Facultad de Agronomía Eliseu Maciel, Universidad Federal de Pelotas, Pelotas, Rio Grande do Sul

Peixoto Gomes, E. Estación Experimental de Passo Fundo,IPEAS, Ministerio de Agricultura,Rio Grande do Sul

Ando, A.

Blumenschein, A.

Brieger, F.G.

Osorio, E.A,

CANAD A

Kamra, O.P.

CHILE

B ra v o , A .

Mora González, S.

Laboratory of Radiation Biology, Dalhousie University,Halifax, N. S.

F a c u lta d de Agronomía, Universidad Católica, Santiago

Instituto Producción Vegetal, Universidad Austral de Chile, Valdivia

Ramírez Araya, I. Instituto de Investigaciones Agropecuarias, Santiago


CO LO M BIA

Gómez Cuervo, P.L. Departamento de Biología, Universidad del Valle,Cali

COSTA R ICA

Moh, C.C. Inter-American Institute of Agricultural Sciences, Turrialba

DENM ARK

Doll, H. Agricultural Research Department, Danish AEC Research Establishment Risô, Roskilde

D OM INICAN R E PU B L IC

Diaz Caraballo, F. Proyecto FAO-SA, Santiago de los Caballeros

ECUADOR

Bueno Cifuentes, A. Ministerio de Agricultura y Ganadería, Guayaquil

G ERM ANY, F E D E R A L R E PU B L IC OF

Gaul, H.

Gottschalk, W.

Abteilung fur Pflanzengenetik, Gesellschaft fiir Strahlenforschung, K61n - Vogelsang

Institut fiir Genetik,Universitât Bonn,Bonn

G U A T E M A LA

Fuentes Orozco, A. Ministerio de Agricultura, Guatemala

IND IA

Swaminathan, M.S. Indian Agricultural Research Institute, New Delhi

ISR AE L

Ashri, A. Faculty of Agriculture, The Hebrew University, Rehovot

552 U S T OF PAR TIC IPAN TS

IT A L Y

Scarascia-Mugnozza, G.T. Istituto di Agronomie, Università di Bari,Bari

JAM AIC A

Panton, C.A.

M EXICO

De Alba, G.

Trujillo Figueroa, R.

Department of Botany, University of the West Indies, Mona, Kingston

Instituto Technológico y de Estudios Superiores de Monterrey,

Departamento de Agronomía,Monterrey

Rama de Genética, Colegio de Postgraduados, Escuela Nacional de Agricultura,Chapingo

NETH ERLAN D S

Broertjes, C. Institute for Atomic Sciences in Agriculture, Wageningen

PA N A M A

Lasso Guevara, R. Instituto Nacional de Agricultura, Ministerio de Agricultura y Ganadería, Divisa

PERU

Delgado De La Flor B., L.

Grobman, A.

PU E R TO RICO

Abrams, R.

Universidad Técnica del Altiplano, Puno

Northrup, King and Co.,Lima

Agronomy Department,College of Agriculture, University of Puerto Rico,Mayaguez Campus,Río Piedras

Vélez Fortuño, J. Department of Genetics, Agricultural Experiment Station, Rio Piedras


SWEDEN

Gustafsson, Â. Institute of Genetics, Lund University,Lund

Hagberg, A. Swedish Seed Association, SvalSf

UNION OF SO VIET SO C IALIST REPU BLICS

Privalov, G.F. Institute of Cytology and Genetics,Siberian Branch, USSR Academy of Sciences, Novosibirsk

U N ITE D STATES OF AM E R IC A

Konzak, C.F.

Loomis, R. S.

Nilan, R.A.

Smith, H.H.

Department of Agronomy and Program in Genetics, Washington State University,Pullman, Wash.

Department of Ecology,University of California,Davis, Calif.

Department of Agronomy and Program in Genetics, Washington State University,Pullman, Wash.

Department of Biology,Brookhaven National Laboratory,Upton, N.Y.

URUGUAY

Rodriguez Zapata, M. ПСА, OEA, Montevideo

V E N E Z U E LA

Gonzalez Rosquel, V. Centro de Investigaciones Agronómicas, Ministerio de Agricultura,Maracay, Estado de Aragua

YU G O SLAV IA

Borojevic, Katarina Department of Genetics, Faculty of Agriculture, Novi Sad


IN T E R N A T IO N A L O RG ANIZATIO NS

Micke, A. (Scientific Secretary) Joint FAO/IAEA Division of Atomic Energyin Food and Agriculture, IAEA,

Vienna, Austria

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Vienna, Austria

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