MUTATION BREEDING FOR DISEASE RESISTANCE …eprints.icrisat.ac.in/13271/1/RP 3323.pdfMUTATION BREEDING FOR DISEASE RESISTANCE USING IN:VITRO CULTURE TECHNIQUES RF~'~JHT ~i Ard Ar)viwRY

MUTATION BREEDING FOR DISEASE RESISTANCE

USING IN:VITRO CULTURE TECHNIQUES

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A TECHNICAL DOCUMENT ISSUED HY T H E

INTERNATIONAL A T W l C ENERGY AGENCY VIENNA 1885

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MUTATION BREEDING FOR DISEASE RESISTANCE

USING IN-VITRO CULTURE TECHNIQUES

REPORT OF AN ADVISORY GROUP MEETING ON MUTATION BREEDING FOR DISEASE RESISTANCE

USING IN-VITRO CULTURE TECHNIQUES ORGANIZED BY THE

JOINT FAOIIAEA DIVISION OF ISOTOPE AND RADIATION APPLICATIONS OF ATOMIC ENERQY FOR FOOD AND AQRICULT URAL DEVELOPMENT

HELD IN VIENNA, 8-12 OCTOBER 1984

A TECHNICAL DOCUMENT ISSUED BY THE

@ INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1985

MUTATION BREEDING FOR DISEASE RESISTANCE UStNG IN-VITRO CULTURE TECHNIQUES

IAEA. VIENNA. 198s IAEA-TECMW=-342

PrinW by the t AEA in AusWia July 1-

I n t r o d u c t i o n ...................................................... 5

C o n c l u s i o n s a n d r r c o n u n e n d a t l o n ~ ..................................... 7

A . G e n e r a l c o n s i d e r a t I o n s o n r h c u s r of i n - v I t r o t u l t u r r i n b r e e d i n g f o r d i s e a s e r e s l s t a n r r ................................ 9

8. I n - v i r r o m u r a g r n r r i s ........................................... 13

C . I n - v l t rti c~c.lr-ct i o n o f d t s e n s ~ . r r s l n t a n r p l a n t n . . . . . . . . . . . . . . . . . I S

D. S u c r c s s e s w i t h r I s s u e c u l t u r c e f f o r r s f u r Imprrrvvd d l s ~ + r s r r r s i s r a ~ l t p l i tn t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , 19

E. Crup p , ~ t h o p r n s y s t e m s w h e r e ~ n - v i t r r , t e ~ . l ~ ~ ~ r i q u r ~ m a y bra a p p r o p r l n t c . t o t r s r ............................................. 19

F . Sprc ~ l i c c.xamp1c.s a s t o u h r r r and u h r n t t r u s e i n - v l t r o ~ e c h n l q u r s f o r i m p r o v ~ n g d i s r a s e r e ~ l ~ t n n c e . . . . . . . . . . . . . . . . . . . . 2 1

C. The u s e of i n - v i t ro t r c h n i q u c - a t o r s t u d i r t i o f h n r t - p a r a s i t r * i n t r r a c t i o n s ................................................... 2 5

Lisr of p a r t i c i p a n t s a n d o b s e r v e r s . . . . . . . . . . . . . . . . . . . . . . . . . . . m . . n . . . 41

EDITORIAL NOTE

In pnporing rlrls matrrral for rht press. rra ff of rht Internarional Atomic Energy Agency haw mounted arrd paglnatrd rhc origitral mnurcriprs and glwn some attention ro pnsenration.

The ~.ie\vs e.rpresscddo nor necrmrily rej7ecr thare of the governments of rhe Member Slates or organizatioru under whose auspices the m u c r i p m wee produced.

Thc use in this book of prticulor dtsignorioru of counrm or rerritories does nor lmply any iudgemtnr by rht publisher, the IAEA, as to tire legal status of such countries or ~errirories, of their authorities and lturitutions w of rhc dellmirarion of their boundaries.

?Re menrion of specific compPnies or of their products w brand names does nor imply any endorsement or recommen&rion on rhc pwr of the IAEA.

In 1982. the Jornt FW/IAE(I Division arranged an empert contultrtion to discurt the problenr arasrng in u t a t on bread~ng by the need to f lnd relativvly rare mutants wrth desired tra~t;) or characteristics in larqe populat~onr (Smlectron in htatron Bredinq, lAEA 19@4) It w s pointed out thal choosing a high standard uarlety a d apply lng an effective rautagen treatmnt Is not enough Clear defrnrtion of breeding objectiues and the establithment of appropriate relection criterla are crucial prerequirltes for the ruccerr of a nutatlun breed~ng program Since mutation lnduct~on cannot be directed ir~ any way towrrds part~cular trarts (not even to thrnk about changing thrt trrlt into a desired d~rect~on), mutant selection play8 a crucral role

htant selection generally is phenotypic selection, which ~aaant lt~at the mutated gene has to have a fairly strong oxprOs8lon with little dlsturbanc:~ by environnontal interaction for a reatonable chance of d i n g detect.~d. Unfortunately, many genes do not have such a strong phenotypic expresnion bucrute they share rerponsibility for a trait or characteristic with noveral or nwny other goner. Nevorthelers also these genes have a gene product and thus M oignal thrt could be identified, if one would know what to look for. Selection guts of course m r e complicated by the fact that many genet are "silent" through long periods of the plant's life and are only activated dt curtain stager o f plant development, in certain organs, at certain times of the daily mtrbolic cycle etc

The phenotype looked at is alwny~ the result of genotypulenuironmunt interactions. The plant breeder is used to deal with environmental problurnu by using established experimental designs, which by standardization, replication, randomization etc, silence t k enuironmentul noise and thun let the relevant genelenvironlrrpot interaction become more clearly recoqnirablo. Going one step further, the breeder may oven provoke a particular genotypolenvironment interaction, e.g, by varying planting dates, by differant fertilizer levels or by inaculrtion with pnthogens. Such practtces are c-n, but considering thu persisting difficulties in plant selection. one wonderr whether there are not W r e possibilities to manipulate the enviromnt so that gene expression can be more dirtincly recognized.

Earlier selection, e.9. using seedlings, could true breeder's tima a d allow the rcreening of larger populations, which would Increase the probability of success. Unfortctnately. many useful plant characters are not erpressed in the seedling stage. When adult plants are required for selection, this pose8 limitations on the number of individuals that can be handled and considerably extends the time required for decision making. The technology of i_n-uJ>ro culture stimulated hopes for new dimensions in plant breeding as far a~ population sizes are concerned, and certainly if one considers single cell cultures, there is virtually no limit as to the nunkr of individuals that can be hndled. On the other hand, the problem with gone expression, pointed'out for seedlings, is worse for lingle cells or protoplasts.

Of course, in-uitro cultured plant calls are w n a b l o to vdriour kids of genetic unipulation, thus producing not only gemtic variability, but eventually genotypes hitherto not eristing in nature. Marover, the phenotype is ~ b j e c t of selection and if w want to n k e use in plant breeding of the inonre population sizes mrugeable through in-vitro culture, w uill have to use single cell selection. for that w might h u e to learn the unipulation of gene exprenclion at th. cell level. thore is a wide field open for research. Once we kncw hou to

t u r n on and o f f p r r t r c u l a r gornea, once uc h u e learned how t o make g e n s exprerr rng mare d r s t r n c t l y . once w &re able t o mcoqnlze genes throuqh r igrut la rat t ler than through the* phenotype o f d i fCermt iaLPd p l a n t s i n th f t v l d . w can d v s ~ q r ~ u l f l r tent r n - v r l r o r e l e c t i o n procobumv c-rabla t o those applred i n mtcrub~alugy

Tho Wv lsu ry Group Pkset~ng u d s no t convened t o deal w i t h such b r w d issues I t uns fo ru r red on d~acwugq rnsrstance f o r two reason8 F i r s t , d i s ~ a s e e r r e r u n g the b ~ q q e r t problvmr I n t r v p product ion Although rea ia tan t v a r i e t i e s appear l o be LIIU nu)$t elegant and ac(eptable wry o f so lv inq these problemr, b r e e d a r ~ drd p a t h o l n y ~ s t s screm t o t o t t e r betwc.cn f r u s t r a t i o n and desperatlon, on ly consoled by the a$.lurarlte o f purmanunt umployment Secondly, there i s optimrrm t h a t i _ n - v i t ~ cu l tu re tarhnolcyy t o u l d be p a r t i c u l n r l y use fu l f o r r o l u t n q problems oF t t . * I r t anr n t o ~ t h o q e n r

1t1n proapor t r dnd I ~ m r t * t ~ o n s of us ing !rr~lt_ro c u l t u r e f o r mu t r t ron brscrd~ry hdvu boon d l s t u s ~ e d already once i n October 1983 a t a mcr t ing hvre i n V~erlnd drwf thcr rur r~mn~urdat lor15 ar u nuw f o l l w d by d qroup o f 20 rclscla~ch r r ~ a t i t ~ ~ t e r Ln I? r o u r ~ t r l u s am w e l l as by the TW/lRFA A g r i c u l t u r a l B l < ~ t e t t ~ r u ~ l o y y Un i t o f t t ~ a S e i k r s d r ~ r f Idhordtory rxper~enre ' l and f u t u r e trends w ~ l l be reviewed d u r i r q en i n t e r n a t ~ o n a l ~ymposium t o be he ld I n Vienna, 19 - 2 3 August lP85

Thp suhjor t ma t t r r o f d1soa.10 r l r r ia tance i s Ilkewrao not new t o the f /K)/IAEW proqramo f o l l c u ~ r q d Pdnel o f experts i n 1970. the J o r r ~ t I O / l R l 6 U ~ v i a i o r ~ u r y a n ~ / o d a c o ordlncrted requarch prcr)ran~me armed at the devcllopmer~t o f improved tethnrqrres f u r i r d u r t l o n , *election and u t i l ~ t d t r o n o f mtJtdtlunu fo r but tar d l sear@ r o r i ~ t u r i r e Thrs work, f rnancrd l l y upp ported by the S ~ e d r ~ h Goverr~murrt thrrbugh Sf06 ~ r ~ v c ~ l \ t 11 olnnru than 10 l n ~ t ~ t u t t o n s I n 2 1 Cour~lr IPY I n r t s f ~ r s t phdso from 1971 1919. ~t focuted p r l m r i l y upon c e r e a l ~ t a n d t h l > i r dlaea%es, but from l 9 l 8 on !nore and mow emphdr13 has been gruun to g r d l n logumes Rnsulta o f t h ~ a work havp been very much up t o expectdt#onn wr th regard to tcsroal disuavua cat13od by a numbor o f fun91 dnd bacterra, but llut so much regarding v l r u l dlyoasas w h r t t ~ drc p a r t l t u l n r l y important I n 1egu4inou.i p lan t apaclus It I r d r ~ t ~ r ~ p a t e d Chat LC-vrt_ro r u l l t ~ r v technlquer w ~ l l be p d r t r c u l a r l y u $ a f u l I n s a l c e t i r g for re t l a tdnce t o f a c u l t a t ~ v e paras i te%, nanywf w h ~ r h seem t o b0 nutr-otropha dnd entur the host p lan t v t a toxrn3 Wheth~r c u l t u r e can he lp i n naklnq advances I n broedur f o r vr rus r e s r . r t a r ~ r r remains t o be leen So f a r we mcogn i re the p o s s r b r l r t ~ e s f u r v i rus - f ree c lona l propagdlron.

Conr.1~8 runs dnd rccomner~ddtrons from th rs qroup w i l l be use fu l fu r I M . IfiCf~, t h e r r Mumbur States crnd numorous s r l e n t i s t s In making a d e c i s i u r ~ ~ t n ~ u t p r i o r i t t e s i n re8earch e f f o r t s arrd funding

Breod~ng f o r d israse m s ~ $ Z a n t u i $ a u j o r arpor t o f p lant brr1drru3, which u y taka at l e v s t 2M OF a p lan t bmeders$ trae, v f r o r t cmd burlqt*t N o v e r t h I e r r , mmerous r e s l ~ t w n t e p m b l o r r r e u l n unsolvwd and praswnt mnjor c u n s l r a ~ n l ~ t o the production of road, F n d , flbr and r n d u r t r i r l t r ~ n m d r ~ i o r

Ihe a p p l r c a t ~ o n of novel btotactrtwlcqy a rd qanwt~c a r y l n s c r l r q w i l l omtund the purrrbi l111e.r o f c o n v ~ n t ~ o r ~ a l p l m t breeding I)lerwfure r nrcul irq u l u m p r l s I n p lant p ru luc l ion . p lant b raed~ng a r d ~ r t - t ~ t r o i u l t u r v tc l thn~l l rqy wnq cor~vwnud by the J o ~ n t I W/IhFh D I v i r l o n An Viunnr %a exparta w r w arkcd t o d~scu.tr ard qtvc rdv l9e on p r o s p c t s o f blotcchnoloqy, Qspor l r l l y p lant !r! v l t r u t u l t u r u ~ . t t ~ t u r r t r ~ b u t e towards rmproved t h c e s 11T success i n m u l r l l u n brr*crd~nq l u r d~%t.ane r v ~ l a t r n r e

The p lant b r u d o r , i n searchtry l u r r c * ~ t % I & ~ ~ c u I t ) rr p , w l ~ < u l e r pathwpn, I l k o f o r any o lhur d ~ r r r a b l e ctwvactsr, neadr qonrt lc urr r In t lon t o t t l t j ~ n w i t h I n addr l l on he rrvedr an dppropr l a tn w raonrnq mptt~od Lcr dwlw 1 thu dvr t rud I haracter Sr ~ e n c o ha3 developed ao ran t t h t i t 1 s n a ~ t rlno t o arrply Ittu emtstlng kn.owlrdqs> o f brot rchnol rqy t o ptact'icnl prcrhlumn I n aqr i c t r l l u ru , a l so I n duvcrlnp~nq ct~unl r ra, I n the near fu tu rv l h ~ s my bv ( rue a1.611 for r~uvu l te rhnrqu~s o f qenot I( erglnoer 13 lhc, un~~fu l r !en \ a d f c ~ d s i b ~ l l t y o f t t ~ u dpp l r ta t ron o f i n - v i l r o t u r h n l q u s ~ fur l h r r u purpusvn u*r awe w t l h ctcrpu ard p&thoyen.r but d l s r ~ daprr~ds or1 tt10 at r wrugt h o f plartt brursdlnq ward p l a r ~ t pathulo l~y m d the f a c l l ~ t r e r a v a ~ l n h l v i n a p a r l r t u l a r cour~ l ry The ~ m b o r a uF the lk lv i rory Group at tempted t o d r? tus r the varri lur arpor ta arld to rvar h snurd ront lus iunr

A . iu ~lMl a)llSIDf#nl loYs Q1 1- USE 01. IWVI IRO CULTURL IN ERLLUfllK; F00 DZSL(\SI RtSLSl(YlltE.

A 1 problem d e f l n l t l o n L

Before s t a r t i n q bmodinq Cqr res is tance and consider ing the l n ~ l u r r u r ~ o f i n - v i t m c u l t u r e techn~ques, tH divoase o r p e l t pmblens have t u be Julincld - -- pmcrse ly , t v h ~ n g i n t o account - the crop, ( ~ t s c u l t l v a t ~ o n and us*, qonotlcs, typo of pruprgsrtion) - the pathogen ( i t s 11Fe c y c k , e v o l u t i o m r y c a m c i l y wtr ) - tha losses caused by t h ~ p&thogen or pvst - tho cost o f a l te rna t rve w t s o f co r~ t rw l , conniderrnq r l r o tho e t a ~ l o g ~ c ~ l

1.p.c t - the typc end l e v e l of reslstsrnco ruqui rvd - the e r t e n t o f r r d umporroncur walh cunvent~unal b r w d t n q f u r r e a r a t a n r ~ - the a v d r l a b ~ l ~ l y of e s l s b l i s l ~ u d in -y l t_ ro c u l t u r u m l l ~ u d s a rd o f s u ~ t u b l u

i n - v l t r o screening procodurur - - the r v u i ldb111I.y o f tho nocvssary laboratory lac 111lru.l dnd a u p p l ~ r n

( inc lud in3 r u l ~ a b l e w t e r and pwr supply) - the avar l a b i l l t y o f auif I< ient and q u ~ 1 r f r d purst ) r t~~al - the ~ o s t s of i n v r t r ? Luttmlque d p p l l c d t ~ u n i n r o l d t on 11) . rvd~ l d t ~ l s ,

resources the p v s a i b ~ l r l y u f ualnq n w t * r i a l r o ~ u l t t r q frvm thu a p p l ~ t r l ~ r ~ r ~ u l l r l v i t r o t e ~ h n l q u ~ l i n rugular p l d r ~ t trrcudlrw) pruyrammur Fur t u l t r v r r d o u v l ~ ~ ~ n ~ ~ u r ~ t

l h u p u l e r ~ t ~ d l efonomlc r n ~ p r r l o f r surck~r r fu l I n wr t ru drsuavv rw lc~wld r l~v brc~rdr rrg p r w y mme tickpunds not only upln lhu Inagrll l ~ ~ d r u f l hu r r r lp l < t n . ~ e a I be avoldbd, but a l ~ u on Ihe r a ~ ~ r d l l y w ~ t t l w h ~ r 11 suciu'cs (MI bn c~hldrr~ocj

In-v_rtry t t r rhn iquo~ p r o p ~ r l y appl led i ou I d bu a rnkaans l u ?pt*ud up c i ~ r ~ v c ~ r ~ t l u r ~ n l p lan t breeding aa oxamplif tad by the use o f anther t u l t u r a der l vnd hnplnids i n breeding p r g r r m r of r l c o end barley

Regard~rq the cost /benef i t r a t i o o f the d p p l l c r t l u n u f i n - v j l r o t u l l u r a Lechnlqucs, the p o r r l b l e mu l t lp lp us@ o f I n u l t r o f r r 1 L 1 t i u ~ (0 q r l ~ n r r l m u l t ~ p l ~ c a t ~ u n ) must be kept i n mind, e rpac ia l l y i n v o g a t r t ~ v a l y proprqatsd crops, s lnro 11 can makc the investment i n t o establ i rhmur~l o f rn y j t r o cu l tu re f e c ~ l i t ~ e s more vcur~umicdl

Gpnetlc v u l n e r d b l l l t y has been i d e n t ~ f t e d i n $everel cropn duv t o widerprudd use o f a c e r t a i n gerrnplasm The r l s k u f crop losses, however, i s p a r t t c u l a r l y great (and the erponses f o r f u n g ~ c i d e s p a r t j t u l a r l y h ~ g h ) irt vvqcltdttuc~ly propagatrd crop$, whrch general ly consis t u f uniform clones and i n d d r l l un of ter l have t o F u l f l l p a r t ~ c u l a r l y h ~ g h consumer ruquiremur~ts Hero, ~ c + - y ~ t r o c u l t u r u of fers a chance qdd i t tona l t8 o r i n combination wrth c lass ica l mutation b r a ~ d i r y t o create genetic d l v e r s l t y wl thout disrupting the h e i c genotypa. and i t f a c i l i t a t e s se lec t ion o f mutant clones posvesving spec i f i c improvvinunts The $ i t u a t ~ o n o f bananas i n the Caribbean eree p r o v r d ~ s an ~ n s t r u ~ t l v e orample f o r the approach needed ( re f chapter F 1 2 )

6 good p l a n t breeding in f ras t ruc tu re would be e very p v s i t l v e asliet, but i t i s no t necessar i ly a p re requ is i te f o r s t a r t i n g h-ero cu l tu re wrk. $om in -v i t ro techniques app l i ca t ions are so simple t h e t they can be roconmerded, -- without hes i ta t ion , even t o lesr4eveloped countr ies. Ln-*r:o select ion f u r disease res is tance nust i n m y case be folloued-up by good f i e l d expar~ment$ along conventional l i n e r o f p lan t breadinq.

4 . 2 Genetic war iat ion i n th host smc ies Without qenetic v a r i a t i o n there i s no chance f o r devolonins improved

v a r i e t i e s . 1; w y u s e s r u f f i c i o n t genet ic v a r i a t i o n f o r the ~ e s i k l character i s ava i lab le i n c u l t i v a r o r o ther qern plasm c o l l e c t i o n r . I n other case9

scouting i n centres o f h i q h g e n r t f c d i u e r s i t y w w l d k m4.d t o evmtuml l y find tho de r i rad character Not I n f r ~ q u e n t l y , tha desi red v a r i a t i o n i s a i s t i n q in the c u l t i v a t e d rpoc ies, but prorent I n more o r less r e l a t e d species To c w t e ~ n t e r s p e c l f i c hybr ids, s t e r i l i t y b a r r i e r s o f ten h u e t o k overcaw ud In t h i s context rn-v&o techniques uere found t o k usefu l o r even indi,ponsable (e g f o r embryo rescue)

I n several cases in tens iue g e m plasm studles have led t o tho conclusion tha t su i tab le genet ic va r ian ts do n o t e x i s t md therefore v a r i a t i o n muct be inducvd ( e g f o r res i r tance mgainst bean golden l o s a i c v i rus o f P h r e o l u s k r n s o r against s w l l e n shoot v i r u s o f cocoa) The c-n w y t o c n a t e g e m t i c va r i r r t i on i s w t a t i o n i n d u t t l o n by r e d i r t i o n o r chem$cal nu twens ( r e f chapter B ) , bu t apparently a l so t_n -vKo c u l t u r e as such leads, under c e r t a ~ n circumstances, t o cons~derable genet ic v a r i a t i o n ("sonuclonal va r ia t i on " ) Novel blutechnology has extended the means o f inducing genet ic v a r i 4 t i o n by us ing t l ssue , c e l l mnd protopl r rs t cu l tu res , and i s a l so p rov id ing new ways o f nutant 8elect1on, i nc lud ing screening f o r disease res is tance Therefore, i t i s u o r t h enaminlmy whl rh advantarjr's are o f fe red by !_k-vgrg cu l tu res t o so lve dipease r ~ r l r t a n c e problems

The major types u f p lan t !n-v_itco c u l t u r e are - mer i r ten c u l t u r o - anther c u l t u r e

embryo c u l t u r e - ca l l us c u l t u r e - c e l l cu l tu re , and

- protoplast c u l t u r e

rach o f tho le t u l l u r a l typos has specific ddvzlntdqes and d iudv r r~ tdqh ls fo r lnduc- t ior~ o f udr tat tori. for select 10r1 and f o r subscrqu~nt regenerat ion and propdgr t ion l h e use o f a p d r t l c u l d r t y p p o f c u l t u r e I n brcwding f o r d ~ s ~ w s e res is tance wi 11 a l s o depend upon the pdt hoqen and tho host/pnthoqm i n t e r a c t i o n

I-or rea is tn r~co s ~ l e c t IUD purpu.t(*s, the ip-v_itr_u techniques pliBser~t rln r t t r a r t tvc advantdqr. I n tha t lurge p<~pul i r t lons ran be srreened e d d l y The populat ion cllfos r e q u ~ r e d f o r s o l e c l l o n d ~ p u n d upon the ewpected $bcceu$, which may b~ i n the order o f 1 I n lo4 t o lo6 ~ n d ~ u t d u a l s Such pupulat ions would requtre, for example, i n c ~ r e ~ l b r e ~ d i n g approxlnvltely 1 tw nurseFy Uslng i_n-uitro c u l t u r e . only a few square meters o f laboratory qpace would be requi rud I h e i r r yl t_ro cu l tu red n a t e r ~ a l tan a130 be subjected t o uarlous 3tre8s0s. such as h ~ q h trmperature o r s a l i n i t y , so tha t s~mul l r l r~eous o r consecutive se lec t ion for m u l t i p l e ohlect1ve3 i s posqlb le us ing the sdme establ ished t u l t u r e r Af ter w s r r n g through the selection screens, hcw>ver, the ! r+ymo w t e r i a l f i r r t ha$ t o be regenerated t o normdl p lan ts , then propagated, und f i n a l l y subjected t o usual procedures o f repeated t e s t i n g and eva lua t ion i n the f l e I d

I t has been found tha t p lan t genotypes vary i n t h e i r s u i t a b i l i t y f o r c u l t u r e and regeneration, and th i , " c u l t u r a b i l i t y " soems t o be i n h e r i t e d . Therefore, i n passing p lan t populat ions through iyyi* c u l t u r e there i s a r i a k o f reducing v a r i a t i o n o f g e m plasm by inadver tent se lec t ion f o r c u l t u r a b i l i t y . I t w l d , o f course, a lao be a disadvantage t o r e l y i n biotechnology only on p l a n t v a r i e t i e s tha t comply w i t h the ra the r unusual condi t ions o f c u l t u r e . I n t r a i n i n g and technology t r a n s f e r a t t e n t i o n should be c a l l e d to t h i s problem.

A.3 Lypps o f c m p p l a n e Considerinq d i f f e r e n t types of c rop p lan ts , w i t h regard to t he use o f

iwvitm c u l t u r e i n breeding f o r r e r i r t a n c e , the re are f i r s t tho perenn ia l t rpo -- crops, b c h a r a c t e r i s t i c of these crops i s t h a t t h e i r l i f e t ime i s a m u l t i p l e o f

t he i r 0.mrat ion tima and t h a r q h m qomt i c iwrouwunt by c l a r s ~ c a l awns i s ntkr cdmrscu r . Wj o f the@ a m clonal ly propy)atd i n order to maintain thir der i rod heteroayqwr gproSypss. rubber. cocoa. c i t rus . apple, tvnrru and black popper, t o aenllon a few A 1 p lantat ion cmpn they are c h r a c t e r i z d by genetic u n i f o l r ~ t y over large Faas The cu l t iua t lon o f u n y thourands o f hectrrcrr o f gerurtically unlfor* clones. hawuer, pmsenta a sertour r i sk of vulnerabi l i ty , r f an epideaic wcurred *dt.r_y cul ture u l t i p l r l * t ,on as L B u s d c m r c i a l l y rn o i l palns edds t o the problem Durable resistunce i s therefor. a necessity ~f one w n t s t o get way from the covts and h r ~ a r d r u f cheaical protection. Resistan- screens durrng tha Lp-vttro propagation phase a n indispensable, but genetic d~ve r r r f kca t i on br also mandatory

lkonq the vegetatively prg(yqated crop: are also annunl o r b iennial ones such as potatoes, sugar c a n e and cassava, which urn lihawiae highly hulurorygous and for which c l a s s ~ c a l cross breading hus Ll~o drsadvantage of brerklny up many useful gene c o n b r ~ t i o n r . In-uJt9 inductlun o f genetic vwrlatron, f o l I d by i n - v i t r o screening a d @ih diserrse-frua mu l t i p l~ca t run w i l l be r n a t t rac t ive -- -- methodolog~cal pack*~. A special plea 11 made here for cassava and two o f ~tm dlseasos thvt are vory iaportant I n Africa, namely cusaava muaaic virus ard cassava barterral b l ~ g h t

b o t h e r category ere the annual o r biennial seed-propiayatud crops. 7here can be dif ferent iated in to sel f -pol l inators and crorv- pollinator^, and lhe l a t t e r my ba mnoecioua or dioecioun. The d in t inc t ion botwvvn thvue qroupa krs implications for the strategy of genetic nunipulatlon Tho production o f haploids from m t h c r cultures has already bemn used successfully fo r a few sel f -pol l inat ing specie$ (nawly cerealr). but the method o f f e r r groat opportunities f o r tho future also f o r crorr pol l inators. D ip lo id i r r t l on o f haploids lead$ immediately t o honocygous forms wlth genetically Pimud t r a i t 8 including resi$tanues. This wwld be true fo r "qual i tat ive" and "quantitative" re$istance. fo r immunity, hyperrensi t iv i ty or p r r t i a l resistance, no mottur whether controlled by ona o r several gener.

The development o f tmchnology for rn-v_(tr_o drrease reristance breeding i s st111 rn en rnfant stage f o r most seed propagatud species Ef for ts seen partrcuiar ly urgent fo r leguminous cropr, l i k e Phaseolu? bean, cowpea (we u_ngu~culata), and groundnut ( A r e i t hma), because o f their importance an food crops, the d r f f r cu l t y encountered I n conventionbl di#vase resistance breedrng, and the great number of d~seares. especially virus disease#, 8overal o f wh~ch are s t r l l 111 dercrrbed

6 . 4 Typos of pathogens I n p r i nc~p le , m y kind o f plant pathogen can a180 be used fo r km

screening, whethdr fungi, bacteria, nycoplasms, viruses, nelutoder and possibly even insects. However, there are pract ical differences wi th fungi there are already a number o f posi t ive erperiences, bacteria cause r o w technical problems, mycoplasmr have hardly been studied, viruses a d nomatoden seem t o o f fe r good poss ib i l i t ies , but information on insect .1--r2 screens i s scanty.

hang fungi, tho toxigenic ones, usually o f the necrotrophic type, provide par t icu lar ly good prorprcts (e.g. Hglminthosooriun i n sugar cans) (ref . ' chapter E.2). l k n g biotrophic fungi, opportunitres are indicated by resul ts obtained wi th Peronosoora rpp., Phvtwhthora spp. and PlaHodioPhora braraicaq. Care inuct be taken t o use the proper prt o f the l i f e cycle o f tho fungus i n a resistance screen. Two groups of fungi donrnd special attention: (1) Soil-borne fungi, oepecially i n c lonal ly propqated plantat ion crops wi th

high genetic uu l rurab i l i t y . ( 2 ) Loaf-flecking toxigenic fungi that are par t icu lar ly releuant t o crops g ram

i n lowinput agr icul tum.

Bac te r ia as a group have been neglectad because o f spefCic M h n l u l d i f f i c u l t ~ o s Since bac te r ia a r e p o t e n t i a l t o r i n producers, Uwtr t o r l g o n i c properticvs cihould be exp lo i ted as j n P s ~ u d ~ ~ ~ y r i n e y pmthovar phar*p l tco la on Pha?.o_lu~ bean*.

Viruses as a qroup need t o b~ studied u rgon t l y w i t h a v i e u t o deuelop appropr i r te ¶croons, w i t h specia l m p h ~ i r on t r e e v i rus01 and viruses o f lvquminous p lan ts ( r e f . G . 2 )

lonutcdar and jnsects producing phys io log ica l drrgo need f u r t h e r t n u n a l l q a t ~ o n 4 t h rerpect t o ip-ur_o sc rems Soae p lan ts produce undesi rab le ~ n a a c t attractant^, and i t n i g h t be possib le t o screen them wt I f insect r e p e l l r n t s are i d e n t i f i e d , i t m y be possib le t o se lec t fur them

h n y harmful b ~ o l o g r c a l aqents (including fungl , bacteria, v i r u r r s , nematodes and insects) a re ab le t o undergo genot ic d i f f e r . n t i r t i o n and, as a consequen( a, pathogenit s p e c i a l i z a t i o n f o r particular host genotypes through phynio log ic races ( i n a p a r t i c u l a r con te r t a l s o c a l l e d biotypos. pathotype*, pathovurs, I so la tes o r s t r a i n s ) I n p lan t breeding fo r d~sease re%lstance the appearance and spread o f new p h y s ~ o l o g ~ c races i s causing a r a p i d t u m v e r o f c u l t i v a r s but nuverl t ieless IB accepted as a " f a c t o f l i f e " The e f f e c t ~ v e rountermeatur@ i i s the auoidancr o f genet ic un i fo rm i t y w i t h i n and mwj crop e u l t i v a r y Fundamental research on res ls tnnce phenomena nay p r o f i t a b l y use i n - y i t r o ~ u l t u r ~ s t o f l n d means f o r re r i s tanca which would erc lude the pathogen's p o s s i b ~ l t t y for response I n terns o f phys io loq lc s p ~ r i a l i r a t i o n

R 5 lypesoC res!s&it$c_o lhe type o f reslstar8re t o be r e c o q n ~ l e d d e t e m ~ n e s the type of

~ u l t u r e Aes~stances which depend on c e l l u l a r physiology. such 4s r e s t r r c t i o n o f v l rua r e p l ~ c u t ~ o n , can be ~ d e n t ~ f l e d I n p ro top las t and c e l l cuwures This type o f res ls tance r g a l n s t v i ruses 1s l ~ k e l y t o be axpressad a lso Jn regenerated p lant3 liawcwer, nu ~ n f o r m a t ~ o n i s a v a i l a b l e as t o the d u r u b ~ l i t * o f t h ~ s c o l l u l a r type uf res is tance, nor o f i t s c o m p a t i b ~ l l t y u ~ t h p lan t v iqour as r ~ q u l r e d I n farmora' f l v l d r ,

I n another type o f res ls tance, i n t e r n a l p lan t s t ructures are Involued (e q r e s t r i c t i o n o f movoment o f v i r u s from c e l l t o c e l l ) Such t y p s db res is tance probubly c a n only be observed i n t issues ( c a l l u s ) o r d i f f e r e n t ~ a t a d organisms (ombryo, p l a n t l e t )

Other types o f res is tance c e r t a i n l y requ i re normally developed and d i f f e r e n t i a t e d p lan ts o r p l a n t p a r t s . For example, t o judge re r i c tance against powdery mildew an i n t a c t epidermis i r needed To t h i s category a l s o belong mechanical b a r r i e r s such as ha i r i ness i n res is tance t o insects . Such res i r tances may be t es ted us ing detached p l a n t pa r t s k*, as i s done m u t i n e l y f o r ce rea l powdery mildew.

Detect ion o f au jo r d i f f e rences between suscept ib le and r e s i s t a n t i s r e l a t i v e l y easy. More fundamental r ~ s e a r c h i s needed. however, t o de tec t s w l l e r d i f f e rences . Tor t h i s purpose, r e f i n e d t e s t i n g techniques (perhaps the use o f chemical markers) combined w i t h appropr ia te s t a t i r t i c a l analyses a m requi red. kyitn, c u l t u r e o f f e r s the p o s s i b i l i t y o f qu ick l y m u l t i p l y i n g any selected p l a n t by maristern c u l t u r e . I n c e r t a i n case8 anther c u l t u n s could be used t o d v a n c e development o f b z y g o u s l i n e r .

Epidemiological r ~ l y s i s should u s e f u l l y be undertaken p r i o r t o the breding p r o g r u w t o determine the requi red l e v e l and tho w e t a f P ~ c t i v a t y p a o f res is tance. D i f f e r e n t mechanisms o f res i s tance f o r OM and t h e same host/pathogen syston may poss ib ly be detected w i t h the h e l p o f i n - v i t m

t.chniqwr. T h s e could thon b) ccdinad to provide mom camplea. and therefore likely ro*r durable n s i ~ t m c e . ~

<

6 . 6 R_vailable n#w-5 For the establishwnt of &-v* laboratories, prrtrcularly in developing

countries. reueral arwcto hvalto k taken into consideration

I_"witro laboratories can $erv~ ultrple purposes - incmase of qewtic v.rIation and !_n-yJWtr rcreenirq in suppurt of 0n onping breading pragrama

- diswrrfree wlt~plication of cultivars for cornorcis1 use or nwly selected genotypes conseruing their desirable heterorygosily - convanlent mnd mre etanamical wintenance of g e m plarm colleclion#. espwi4lly of tmes, clvclntwlly coablned with cryoprerervation - drrease-free international exchnqe of germ plasm (as already pract ~ c d by the International Potato Centre ( C I P ) in Peru)

In viw of the limitad rerourcer and rcarcity of qualifiud por~onnal in most developing countries it Is suggested that f~rst national or regional j w y & , p Iaborutorles are established Such cantre* rhould thon oPPer training ard services for plant breeders, but also fur associatud phytoprtholoqistr, vrrologists, nenurtoloqistr and entanologlrts Dvsrrable would also b trhinlng of scientists having responsibl~ position3 in devuloplng countrrea to pave the way for appropriate use of nw technologies that can be r>P benefit for thore countries

&n important question is also whethr farllrties are availrbla for confimlng i_n-c\r_o sele~tod resistance in whole plants, for tcbrling tho [email protected] Irnes, clones and populations using common greenhouse or Piuld procadurea and for relecting the sama origrnal populations under natural conditions This I r a serious matter since the expro~sion of reristanru In culture Is o f t ~ n abnormal It is vital that awresistmce b r e a d i ~ r p g y ~ ~ ~ - l n ~ ~ _ I . y l - q , ~ ~ , n ~ ~ v ~ ~ ~ ~ _cu!t,ur?. i_r -- - -. .- - . . -. -. - . - - - -- - - !uantdkinf!ctpl.ntt!stiml u s l q aptrb!ished-pro_r_epurqs

Resistance of agricultural crops can ba improved by s~lection, hybridiration, induced mutations or &*-r_o manipulation and various tombrned approaches

The first and second approach can only be suc.cessfu1 when the rclaietance to a certain disease is at hand in the parental genotypes. On the uthmr hand, the selected type or the recombinant may not be resistant enough or resistant only for a short tima or may posnra 1 0 ~ ) other characters that limit the practical utilization and therefore rquires additional crosses or mutation induction

Cartain types of in-vitro culture create genetic variation which may contain ncu resistance traits usePul for the creation of now cultivars. The genetic changes observed, harover, are often very compler. In most cases, such roartic mutants require genetic reconstruction by hybridization from which segregmts with desired resistcmnce and 8gronoaically acceptable pertonrunce have to b. relactad. Unfortunately such recombination is not fearible in movt vegetatively propmgatad plants and this limits the usefulnems of b u o culture derived variation. Experimental ~t.genesis, on the other hard, otters the possibility (using appropriat. doses) to induce chanqer for desired traits while limiting the disruption of the genetic con~titution of the valuable original material, Experimental utagenesir combined with in-vitro culture offers corbin dwantager. For example, dainant mut8tions are a rOlatlwely riire event, but

in-u!t? urtrg.~sis in lrrqe populations will give .n i n c m r d probabibity of obtaining such wtants Thir would be particularly nlauant for irprowing d~nease resistance of cross pollinat inq crop plants.

0 1 [email protected]+tmen trttatcwnt The trqet of an in-uJf utujenic treatront m y differ in ploidy level,

k ~ n g haploid, diploid ur polyploid (OU- or aneuplotd).

li 1 1 Tho hapjotd slttem (anthers containing ricrospono or pollen grains, protoplarts, nrrricrtom tioue, etc ) allwc phenotypic expmssion of all ~Lationo directly in the Nl gonerallon Solaction could be applied to haploid cellr or haploid plontr reqenerrted from them But there would #till be a big advantage Prom haploid nutagenorit, even If telection for utantr is carrid out later, fullau~y q e n m dupl~cation of the wtagen treated hplord individruls The reason is ttut the fl2 would only contain honozygous mutants and orrq~nal typer. thus the selectrnn of nutmts (e g by chemical analysis) uwld be free froa the problomo rroatd by the presence of hetarozygotes On the other hand, all mutant8 would be fixed without any further regrogat~on and recolbinati~ of inultiple ncctations I'hir system 1s applicable for species, *here haploids can easily be obtained (at prssent e g rice, barley, t o ~ t o , pepper).

0 I 2 Tha.dipltiP_ or polyelold ryatem~ (usually budm, embryos. shoot tips, but potentially also protoplasta, cells, tissues, call1 etc ) require, in the case of huwrygous material of suud propagated speclea, for select~on o f recessive nwtations a serual cycle and acreonirq in subsequent generations Therefore, in-vitro ncctrgennris uffers little advantage Screening for race8slve mutations, howver, arey be performed already In the fll generat~on ~f the oripinal mater~al is hetaruzygoua 7hir 1s pcwrtrcularly the case In nost veg~tatlvely propagated crops liko bandna, cassaur, yam, potato, sweet potato. as well as many fruit crops snd ornamentals Hare in-y&r_o mutagonesis offers many advmtages ranglng from mutagen treatment of large cell numbers, possible teat tube ((alection. noc~chinurric regeneration for p - u r n selection to quick clonal M a s propagation of promising mutdrrts for field eualuation end subsequent distribukion to farmers lhe tw-qet of an !w-ct_yo muturjerric treatment my be a eingle cell or a multicollutar ontlty In lhe lattor case one must be aware of po8s~ble.chimrrism and may have to dissolve the chimer~c structure of rogenerant Nl plants S w t r c ombryogonesis generally has single cell origin so that fl1 plants regenerated in this m y are supposed to be homhistonts

0 1 . 3 Tho.atoeLa~-n_ L n - a o culture methods may be necessity h e n the trait neoded is

cytoplaomically inher~ted. Some resistances to pathotoxino ware rhown to be based upon mitochondrial characteristics. Nitochondrial DNA will likely respond to the same nmtagenic treatments as chromsoto.1 DNA, but tho selection of mutated mitochondria by application of tomins will require in-vitro techniques. Research thould be encouraged on the role of mitochondrial plasmids, their interaction with nuclear DNA and on practical aspects of cytoplasmic mutagenesis. (Ref. Report of FM)/IAEA Consultants heting on the Induction of Plrtation in E~tranucloar Hereditary Cell Elements, STI/PUB/591, IAEI, Vienna 1981).

8.2 Choice of u t a g ~ n s Basically, both physical or chemical mutagens can be used, the choice

depending usually upon availability. The spectrum of u t ~ t i o n m y differ, but nothing specifically is knwn in thin respect concerning disease resist.nce mutation$. Using several different mutagens will ascertain a wide spectrum of mutations. Both categories o f mutagens offer dvantaqoa and dispdvant.ges.

9 .2 .1 Physical u t W e n a Ionizing radiations like X-rays, g e ~ rays, faat neutrons, but also UV.

f!&wwe!: Good dosirtry and rwasonrble rJproduclbillty. High urd uniform panetration of ulticellulr systPrr particulrly by pur rays

O~advantrePs. R d i a t ~ o n swrce w i n d . i

Unlike s d r , culture1 flasks d m o t easily k #hi& lonq distances to the sltr of th. 80UrCO. Siultanewr induction of chrororoul a d gene u t e t ~ o n s UV can only be urod for single cell layern

0 . 2 . 2 ama1c.l utrpon, lbst c m n l y used are ethyl-nitroso urea ([NU), methyl--nitroso-urea (PIYH),

ethyl-wthane-sulphonato (m), di-ethy ltulphate (DCS). ethy lune~mine (11). sodium azide (SA). e t h i d i ~ broaide ( L I ) ) .

- ~ V M ~ M U : Po~nt nutations m y predoninato for some of the chemiral nutagens High mut,ation rates can ba obtained from certain mutagens . D~sadvantmer: Dtfficultias in effective dose assosswr~t and repruducibility Nun-uniform penetration of ~nulticellular sywtums. Problems with breakdwon conpounds of the mutagon8 nppllad, as they cannot eaaily be removed Several nutagonic chemicals are cancorqenlc.

Compared to seed or dormant bud inutagonosrr, the celln of in-vltro cult~vated targets generally lack synchwn~tut~on us to the cull cycle #t.qe, which lim~tr the raproduc~bll~ty of erparlmvnta bans Tor synchronir~tiur~ may be found )bny relevant aspects of mutat~on brrod~ng arc* doall wlth in the "tlanual on Wtation Breed~ng" end othur books publtnhad by IALA (sae b~bl~oqraphy) Howver, l~ttlo is k n w n so far as to tho optimal doaes, Limo of treatment, physiological condktions of ob]erte For ipv_!-tr~ mutrgenurir Research in t h ~ s respect i s needed a d di.rseninut~on of rusulta would svon err appropriate task for the Jolnt FIK)/IAEA D~viaion

C I&-VITA0 SELtCT1CN OF DISEASE. RCSISTW PLWIWTS

In-vitro selection my be appliod ~rrespvctlve of tho source of gvnvtic - variation. (e.9, "somaclonal variation" or induced mutations). Also the distinction betwoen monogenic or polygenic inheritance is rathor irrelevant, since at the selection staqr only the phenotype dif ferenca betwaen susceptible and resistant is important. (The genetic classification i n a nrttor of later concern, more critical is the verificrtion of &-v_l_fi aalected ruai~tance under field conditions). Howvor, ip-fiui culturu conditions nuwt bu suitable to screen for resistance to the various plant pathogens. It would seem most efficient if such screening could be performed imnodiately after the generwtion of genetic variation, e.g. protoplasts subjected to nutclqons could subnequently be plated on toxin-containing udia. Suruivors reristant to the torin thould be regenerated and tasted for resirtance to thp pathogen as such.

It is one of the u i n attractions of i & - y y ~ techniques that thoy can provide r scale for selection tlut uould k difficult, if not inposaible, to achieve in o t k r ways. For exwple, a potato breeder m y screen In th. graenhouse or the field perhps 50,000 to 100,000 seedlings per year for disease resistance. On tho other hand, 20 million protoplasts obtained from only I g of

leaf issue, could e&si l y be cu l t u r d i n a laboratory. I f a Iven b o ~ f i t i . l u t a t i o n such as d18ea.e n s i s L u K e M a Orobrbi l i ty of 104, ttmn peehps o m such w t r n l w u l d occur among the 100,000 aoodlingr. Mwmvar, 200 utmt plants might bo obtainud frw protoplast cul ture screening

lhus. #erious considerallon o f th potent ia l o f &-v& aalect im, both wrlh respect t o v r r r w s pathogen types ond t o various host plant materials i s ( Iear lv wrrruntud

C I Ihe use of ~+r_iou_r-l lssuo_ulturs systems fo r selecting disease resistant plant*

Seuaral systems of d i f fo rent conplemity can be subjected t o screening. Srnglu c ~ l l r without w l l s (pro top l r r ts ) or wi th w l l s (ce l ls ) , u l t i c e l l u l a r nuspun8 ion culturea, callus culturea, cultured anthers or nicrospores, rogerturst~d p l m t l e t r and even in tac t p l&nt1 may be useable i n such selecticn prorodurus

C 1 1 Protoplart c u l l u r a ~ Protoplaats cun bo adequate f o r screening for tomin resistance I f

relistunce i s not the resul t o f c o l l w l l ~ m p e m a b l l r t y ht rea l ly o f rnaensit ivrty to tho tom~n ( ~ n a b r l l t y fo r certarn reactions or detox i f l ca t lon by cartain r u r ~ t i o n r ) , thun such screens can be qurte e f fec t ive h c h r n f o r ~ t ~ o n r r needed, houovor, ort the brochemical and physiolagical ef fects o f tomins, on the requirad pu r i f i ca t i on o f the t o ~ i c materrals and particularly on the importar~ccr of sut h tom rn l i n ho~t"pcrthogen interactions

Ir lternction of protoplasts w i th intdct pathogens m y d i f f e r From that o f inC@ct plants Whereas there are i nd~ ra t l ons that protoplasts re t l s tan t t o virus infect ion y ie ld plants that are resistant too, pu r t i cu l r r l y v ~ r u r e o f ten l n f e ~ t protoplasts which are dorived from rasistance plants r

On the uthar hand, protoplasts may not be affected subntant ia ly by vlruses, and dntectibn of i n f e c l ~ o n n a y only be posaible by destrurtrve prQCedures such as staining w ~ t h flourascent antibodies not allawing any selection wYh subsoquont rognorat ron. Barterra1 and fungal infectrons, where pathotomins e r the r - hort-sperr f ic or ho,t non-specific) are not ~nvolved, m y not & *able us protoplrrst wcreons Often protoplasts from resrstant and susceptible plants are k i l l e d at apually raprd rates For selection o f reslrrtance agarnrt b a c t e r ~ a l diseases, parhap5 rrt some tarses changes i n bacter ia l attachment t o ce l ls could be cibsurvod and used Uesistance aqainst cer t r rn bacteria, where the bacterium doesn't caute raprd deuth of protoplasts, m y be screened for by d i rec t rnrrlng of vrrble plant and bacter ia l ce l ls

Recently i t has been suggested that protoplasts may respond to fungus-derived e l i c i t o r 8 by producing phytoalexins and tbt these responsea are under genetic cont ro l . Thus screening for resistance a t the protoplast level i n the near future may involve the use o f pathogen components (which could m r e easily be standardized) When molecular aspects o f host-pathogen interactions become clearer, more e f fec t ive screens wi th protoplasts can pmbably be deviaed.

C 1.2 3 1 1 suspension cultures Ce l l suswnsion cultures huve been usad fo r studies o f . ~ ~ t & o l i c reswnses

to pathogenr and as systems t o give c o l l l ines that are resistant t o v r r i & r toxin analogs o f amino acids. HaRuer, fw rigorous studies have been made using these as mans to screen fo r disease resistant plants. Considerable research is meded e.g. on the mans that host c e l l s use t o rocoqnize pathogens, ud v& versa. Such information could provide r basis f o r devising screening procoduns. -

C.1.3 Flluv cultuns C.llur cultuns b v o h n u s d already to elect for resistant plantn

Tonic c n d e filtrates of fmqal broth killod many cell* but s o w @urvlvora rhoued enhmncad resirknce rh.n inkrct,pl.ntr derlvd froa the culturea were Inoculated w i t h tho fwgus R ~ c t i o n of eCllus tissuos to inoculation with living fungi h r s also been ex~glifiod U k n to&cco Ioave~ w r o inoculated with virur and callun derived from tho laavos wm selected for rerittance, the resintant material -.red gmen, tho susceptible yella, m r r studien with viruses producing good visible msponses such as there a m destrable

fn-v&yo_ rcroenin~ for roslstmce to hcteria c&n b4 difficult IC no tomin production ir involvd in pathogenosir. Often bctoria colonize the tir~urs a d v d i a very quickly; rtructural repuirenrntr for recoqnixiq diffbwncen In pathogenesis u y not be fulfilled in rallu8 culturer

F i ~ l l y , expression of reriatance in c@llu8 tissue m y bf, qurntit~tiue and in that care difficult to identify by anresriq a aliqhtly more or less viqorous growth of pathogens. R o n resurch vould k, noodod as to the erpression of levelr of rbsietance aqainst variour prthogepr in callus culturor.

C 1.4 Embryo cultures Embryos in culture might be u~eful in screen8 by virtue of their organized

structure For any selected embryo, there 19 also a greater lihol~hocd OF regenerating Intact wholo plants. By somtic embryogenesia, larqb numbara of embryos for rcreonlng could bo gen~rclted. It seems fe&siblr for enample, to have embryo* of Brarsica or Hio=!ri plated on tomin containing mdia or be challenged by the fungus ~tself Susceptibility to loore snut could be detected using Ln-vitro inoculation of young embryos.

C 1.5 hnther cultyra! bnther and microspore culturen seam particularly attractive for induced

nutant screening D o m i ~ n t as wall a8 rscossive traits Por rasistance might bo rhoun ~mmediat~ly and doubling genome8 will then glue howryqoua breedlny llnas One atill needs, howover, reliable nothods Par obtaining embryogenic microsporer of many crop plants

C.1.6 Whole plant in-vitro cultu"rs_! &-w screening methods can also bo applied to intact Ln-u-it~o_ cultured

plants using the opportuntty of rtmndardiratron, 04 total enuironnsntal conlrol and of envirornontal manipulation for accentuating disease roaction~. In particularly dtfftcult caser, like longqeneration-cycle host plant8 arrd long latent-infoctiowperiods. int~ct plant ipv-ts culture might opon ways to early selection and thus represent a real qroat advantage. By enamining changes in propertie8 of diseased versun nor*-diseased susceptible plants, "early rcrooning" cwld porribly be furthar improved. Perhaps changer in conductivity could indicate the state of plant infection, and thus dlfferrnceu in susceptibility . C.2 General r e c ~ n d a t i o n r on in-vitro selection for dise5y

C.2.1 Fungal toxins at pnsont are the oasiest applicable screens for resistance. Therefore, &em porslble, determine if a toxin ir inuolvd in pathogemsir and thon use it in screoninp. Uhothor tonin nelection lads to plants with satisfactory resistance rouins to be seen.

C.2.2 CImhtl study of host, pathogon and enviromntal p.rwters in p~thogonesis should be d e prior to attorpts of designing a rcreon. Uesirtance m y ba needed at prticular stqem of host's life cycle. It may or m y not bo exprossad at othor stqos. Biotrophr and nrcmtmphs uill behave quite diffonntly in culture.

HOST P L M

Helminthosporiua mydis Zea nays toxins

Phoma lingan Brass ica napus toxin, germinating conidia c

Phytophthora infestms Solanmi tuberosum culture filtrate, infection a

Altenuria solmi S o l w n tubero3ur toxin, infection a

Pseudor~nar tabaci Nicotiana tabacum toxin c

Sc lerorpora racchari Saccharun oPficinarum infection a Mint st al. 1977

Helminthosporiun sacchari Saccharum officinarum infection

Fusariur oryrporun Lycopersicon esculentw culture filtrate

Fusariun oxysporum Solanun tuberosun culture filtrate

Heinz st al. 1977

Scala et a1. 1984

Behnke 1 9 M

P8eudomo~s syringae Nicotiana tabacum tor in a Thnutorq et rl. 1983

Altsrruria alterrut. Nicotiana tabcum tor in a Thnutong et 81. 1983

Fusariua oxyrporua W i c a g o sativa culture filtrate a

H.lrinthosporium rativun Triticun aestivum culture filtrate, toxins a

Hartmn st a1. 1914

Dutrecq 1981

- , - a = Cell lines

b = Regenerated plants

c = Pmgeny of regenerated plants

C 2 3 l t tampts t o determin c o m l a t i o n r hewn pruportiea of tho tiaaue cu l t um a y a t n and m a i r k n e e under ti& conditions should be d e . Studies on w n a t o e n W e d i r u a e ms is tmce expr)aaion An tAsaue c u l t u n should ba conductad. It mould k ureful t o camfu l ly ex@ine m y tLssue cu l tun /pr thoqm ryrtvna t o obtain the k r t c r t t e r ra for d i # w s r raslstanca dlftermncea.

C 2.4 Too l i t t l e in fonwt ion i a currently available on &-dk% cul ture screening f o r nentoda, v i r o i d or my cop la)^ r a s l a t ~ c e m . Moat uork w i th nematodes appvars t o have been done using in tac t p lant#, k c w a e thore a r i s t ap.c i f lc atructural r.quil..lonts For infection, but also root t i p culturea h u e been u o d .

C . 2 . 5 I t would w a r -st u ra fv l t o choose a selection ayaten tht would l e d i n tho shorteat and m a t d i rec t way to in tac t p lantr . Regeneration from i n - y i \ s cultures a t i l l presents problma f o r nrny lwrtmt plant apacies and therefore 1s a w j o r handicap o f &+wJtrtr selection f o r disease resiatanca.

C . 2 . 6 Then i r much noad For information on the genetics o f host-pathogen interactiona and on the components o f the interart tons. With t h l r information, screen8 fo r the presence or abaence o f spacif l c nolecules Involvad and fo r the omrat ion of part icular genes w i l l f a c l l i t a t b acreeninq a t the singlo c e l l Ieuol, the level that w i l l allow t o handle a marinurn nunber uf individuals w i th tho mini~rmm of e f f o r t .

F i r s t , I n the early seventies C.rlson suggested that t issue cul ture methodm could give disoste resirtance and a fw years la ter th is wrr proven by obtaining l r ~ i z e l ines r e l ~ ~ t ~ l r ~ t to U a l m i n t h o r o o r i ~ wydb Screening fo r ru t i t tance was done a t the cal lus level, then plants w r a regenerated and ruhjuctud t o genetic analysis and evaluation. Good examples o f tho potent ia l of j-py&rrrq diseasv resistance breeding are the rocesriue roristance ngainrt h r l o y yollow manaic v i rus found i n sel f -pol l inat ing h r l e y and tho dominant resistance t o &?% 11- i n crosa-poll inating @rareica r u w n . I n the meantime, a number o f other iramples have been published. S m are prorentad i n the table on page 18

E . CROP-PATI1OCEN SYSTEMS WERL TLCHNlQUES MAY BC APPROPPIAlL TO USE

The choice o f the hOst/paraaite systems Where *&tAt~ techniques could be used as an aid t o disease rasirtance breeding hue t o be made taking i n t o consideration both the crop and the pathogen as mentioned before. The investigator should have a thorough knouladge o f the interact ion botwen the host, the pathogen and the e n v i r o ~ ~ n t . $ 0 ~ ) w r e genera1 guldelinas given hero are rupplerented by specif ic eramples i n Chptor F . 1.

E . l The crops Crops which are normally propagated vegetatively, or which huvo a long

generation tim, are obvious p r i m candidates f o r tho use OF _in-vitro culture tachniques. There are aeven l reasons f o r t h i s : c o r o r c i a l cu l t i vara o f t h a o crops generally are expected t o have very har4~onoous phenotypes. On the other had, t he i r leve l o f hetorozygoaity i s generally very high. I t i s therefore d i f f i c u l t t o introduce a l i en roaiatance gone o r even to uncover a recotsive resistance gem possibly present I n heterozygous clone, without disrupt ing the coaplex valuabla type o f an exist ing cu l t i var .

Momover, i n seven1 lnst.nces, crosses both u i t h i n ud ktween species aro d i f f i c u l t due to a non- f la r r ing Mit, incompatibi l i ty barrierm, and d i f f e ren t

ploidy level*. Reconstruction o f a eu l t i var a f te r a cross i s e r t n w l y difficult Th. ti- requlrod f o r reselection using trditlavl tools k quite long and th. developrnt o f a resistant variety may not k fas t .nough to cops with the urgency of a d iswse problem and th. evolutiorury capacity o f a prthogen. For those reasons, disease resistance breading i n t h i s group o f plants i s wel l d v i s e d t o look f o r tpontanows or induced u t . t l ons . But even f o r tha l a t t e r , t he i r frequency requires large scale screening by e f foc t iv r techniques. Ln-kttr_a culture, pronine auch posslbi l i l i e s

The ubstacler to t rad i t iona l selection for disease resistance increase wi th the langth of lhe plant generation time and are alaast u n s u m n t a b l e I n cases where uoqatative propmgatron i s oblrgate I t i s rather unfortunate that &-fits culture techntques fo r these groups o f plants, w i th a fw notable erceptionc (as w shal l sre l a te r ) . are f a r from being established. I n case o f urgency, howuer, the mthodoloqy m y only be a matter of tim, provided thmt genrrel f e a r i b i l i t y c r i t e r i a have been f u l f i l l e d To increase genetic va r i ab i l i t y w y be a crucial point i n cases where veqetative propmqation h a s r r s t r i c ted o r eliminated previously exrrtent genetic va r i ab i l i t y Where cros8 brsodipg i s poesible, the introduchon of a1 ien res istanco genes v ia intra- and interspeci f i c crorsas my & fac i l i t a ted by &uur_a fe r t i l i za t i on , d r y 0 rescue, the use o f t r r e d i a t d protoplaatr as donors rn fusron or by donating DNA from &-* g e r m i ~ t ~ n q pol len

I n the case of lonq-qeneretion-time seed propagated plants tho breeder lay also be lacking genetic va r i ab i l i t y , which could be augmented by mtageneais and nelectad using L_n-c t~ cul ture. But the breeder w i l l face segregation, and therufore h s to screen large populations f o r the most desired rooombinant. Anther cultures, aa fa r as available, could provide a rea l help for quickly developing improved cu l t i vv rs or populations once the desired r e c w i n e n t s have been i den t i f i ed . I n the case of short-qeneration-tine seed propaptad crops, the use of fn-vftt cultures may be of only subsidiary value, a t leas6 when suitable sources of disease resistmco genes are auailable. Direct in-v i t* selection f o r disease re~ i s tance does not seem generally recommendable i n t h i n .tare, even i f better selection techniques are becoming avai lable. But in-v i t ro Systems could of course be helpful i f used for the increase o f genetic var iab i l i ky i n . self-pollinmted crops, f o r ov~rcominq incompatibi l i ty barr iers theugh one o f the already mentioned techniques and as an a id to establish homozygosity v ia haploids.

lwo general conridarattons m y be added which rhould be taken i n to account rn decidinq whether a disease resistance breeding programme involvinq techniques &hould be started P r i o r i t y should be given t o plants f o r which re l iab le in -v i t ro techntquos are already available. Uhen th i s i s not yet the case, preference should bo given t o species which &long t o groups &re a t least good tisuue cul turu nodal systems have been developed. For example, d icots a t the present state o f the a r t are easier t o handle than monocots and gymspprms. Secondly, i t should ba obvious that progress i n brooding f o r disease resistance using ir~-- cultures w t l l be more l i k e l y if round breeding experiences as w l l as a good knowledge of tho etiology o f the disease i n question en is t .

E.2 The pathosen To judge whether i t would be appropriate t o ol&rk on the use o f i n - v i t m

procedures i n breeding fo r resistance against a par t icu lar pathogen, tho f o l l w i n g questions should be considered:

(a) Is the l i f e cycle of the pathogen, i t s means o f in fec t ion and the u u s e o f disease suff ic ient ly understood? Which i s the u r s t infectious spom stage7 Mat i s the survival mchanism? Am them a l t r m t i u e harts? I s a par t icu lar environment mquired f o r infect ion7 W s the pathogen produce a toxin? I s tomin pruduction a #.na o f i n f w t i o n o r tho m a u l t o f the established infect ion? I n which organs and s t . r J I o f host p lant devrlopllpnt

is tha disuse enresHd7 bra t h e n seueral o q m s or stoqes uh.n the disusb is doteetable?

It will be crucial to have this informtion for devising &y&rtr relbction procdums and themtom it is very Important to hrve a plant pathologirt'r co-oporation.

(b) Ia it likely possible to dwise an ipw rcmenlng pracedurr7 The prodiction uill k difficult kcausr th* erlrtin) evidence and precedentr are limited The followiq guidelirwr, hanvor, may ba useful - If the pathogen is a mcrctmphic fungus or bacterium, It Is porc~ble thrt a toxin (or tonins) is involved in pathogenesis Them is masonably gpod evidence that scrwning h~-~-i&? using c w d e toxin preparations or culturn filtrates is feasible If the p.thogun is not producing A torln, it will probably bonecbrrary to screen by uringuholeorganlsns IImajor practical difficulty In thls procedure would be eacereive growth of the pathogen on tha culture medium as ruch.

- If the pathogen is a biotrophic fungus or a bactorlun, or a nemrtode, It will probably k mcessary to ocreen by utinq the thole orqmirmo, Therr are strong indications from publirhod work that the erpresmlon OF resistance to a11 there groups of pathogens in culture is ~ b f ~ variable and depends upon the ruture of the culture enuiromnt Dual cultures would bu valuable for maintaining pathogen collections and as sources of aseptic inoculum.

- The type of resistance searched for 111111 have a stronq bearlng on the probability of ruccers. For eruple, structural barriers are lens lilrbly to be erpresrod in cell or tissue culturs than phyrioloqic~l machmisns, and partla1 resistance is likely to be m r e difficult to identify i~-u_trq than complete resistance or hypersensltlvity

- If the pathogen is a virus or mycoplasmr it will most likely be necessary to use the disease cauring agent as ruch. mans for detection of rerintance/~usceptibility must be developed by stronq renoarch eflortr.

F. S P E C I f I C E X W P L E S AS TO WERE AND WHEN TO USF IN-VITA0 TLCHNIQUES F M InPRovIruc OISE~SE R E S I S T ~ E

F.1. Cropn that are usually v.qetatively proprqrted

F . I .1 Sugarcane The suaarcane breedinq system is c o m n l y bared upon hybridization followed

by vogetativ; propmgation through stem cutting; and subaoqu&t smlection of clones meting agronomic and industrial requirenents. Haurver, in sono breeding stations nutation induction as well as in-vitro culture have already been used succesrfully for new variety developont.

Disease problems are many ud loasor are high due to snut, mat, Fusariua sp., Helninthosporiun sp., ratoon stunt (virus), Fiji disearea (virus) etc. Bmeding for resistance is recognized as the m a t desirablr mans of controlling crop losses, but traditional breeding methods (incl. distant hybridization and clonal selection) are hampered by sp.ce, time, labour and other requirements.

Present role of in-vitro tachniauev Plant tugenerrtion (via organogeneris and rolutic erbryoganeria) is w l l

establishad fm diffemnt anplants including cell and protoplast cultures. Mcuevrr, ddifferentiatd cell cultures usually possess high gemtic inatability

(osp.cially chromsawl irmlrritles). Scutic variation in rcw).mmkd plants appears to b. a useful source of gonotic variability. Indued wtations -9 suppleniant this gemtic varlabil ity .

In breeding for disease rosist~ce, i w i t r o cultums c u m n t l y could sorue (a) as a quick tool for selection against f u w l pathogens producinq

torinr, e.9. Hol~inthowwrlua. (b) as a source of genetic variability (c) for rapid propagation of solocted type* by using mriatem culture.

lknong limitations of tho application of im-vitro techniques for disease resistance brobding of sugarcane are tho non-controllablo gonetic variabi lity and abnormal wt.bollsa of cultured cells rhich right bias @* solectim procodura s

Prowppcts for tho tutu9 gn-vitro technology has boon usod since 1960 in ruqarcane and w s t bo

coneiderrid as a rogular component of sugarcane brooding. Research on f w t i c and inducod qenotlc variability houover is still needed. Protoplast technology should be dovelopod for use in solving inportant breeding problems. In-vitro technolopy will certainly be uroful for g e m plasm preservation (cryopreservation) and for interruttonal g e m plasm distribution.

F 1 2 & a m C-rcial b ~ m s and plantains are vegetatively propagated

parthenocarplc trlploldr . 6onatic variability is very r u r w in cultivated types In comarc~rl banana plantation$ urnly tw groups of cloms are found i e. Gros niche1 and Cavendrsh

Di804le ~roblenr Panau disease causod by Fu~araum Oaysporum f.sp, cubenee i b r serious

problem in Cross niche1 clones. The nrjor limltatlons of the h v u d i s h clones on tho other hand are thoir susceptibility to the Yellor Slgatoka or &man8 loaf spot diseaso caused by Mycosph.er01la muslcola and to the black 1-f streak or Black Sigatoka di seaso caused by Pjycos~haerella f i j iensls var. d r h m i s Other serious problums are bunchy top vrrus, M k o drsease (Pseudonorum & a c ~ u ~ ) and netnbtodbt (Rdopholus simrlls)

Breeding for disease r o s i a t ~ c ~ Corm plram for disease resistance is aurilable among diploid wild species

but their vary inferior bunch cbracterirtics have handicapped the synthesis of relistant clones with good coaercial characteristic. Tho contemporary breeding rtratrqy is based on the devolopcusnt of acceptable banana diploids f m m crosser with diseaae nsistant wild species which then could be cmsrod with tetraploids for obtaining synthetic triploids. Rutation breeding is constdored a tool to improve currently cultivated clones without disrupting their conercially ualuablo genetic structure.

Rolo of in-vitro techniauas The established steps of banana in-uitm culture a n : (1) banana rhoot tip culture ( 2 ) stimulation of multi-shoot formation from dvmtitiouc buds ( 3 ) shot rooting and plantlet developclont.

Proswcts for the future In-vitro cultures must bo considerod as a Putum regular component o f &rum

bnoding. This technology will certainly bocow uroful also for g e m plasm rtomge a d for international g e m plasm distribution. In-vitro cultuns as a

u r n s of clonal propr)rtion u l l l have uide am l i ca t l on f o r both banana8 and plantatns. Thr pmduction of r(genorating ca l lu r and .orphogemtlcally cocrprtent c e l l susp.nslons w i l l ba th. u ) o r mqulrownt for s u c c e r ~ h l appllcatlon o f

technology. for obtainlnq now gemt ic ua r i . b i l i t y and f o r u k i n g use of in -v i tm selection procdurrr. -- F . 2 . Crop8 that are ~ ~ s u a l l ~ v ~ t a t i v e l v pmmaated kt producq 11110 seeds

F.2.1 Clrsava This i a o w of thr nost iaportant t ropical t u k r crops used a8 a otrple

food i n u n y developing countrim* but also am an export conrodity f o r a n i w l f e d I t i s vegetatively prop.gated by stem cut t ing#. The m i n swrceo o f genetic va r i ab i l i t y arm cu l t i va r r umd i n cmcoes. Camtic variability appr.rv t o k f a i r l y brvwd for various #rononlcally u r r r u l character ist ic#, but l i n l t r d f o r disease m r i ~ t a n c e . The u i n disease problws are caused by vlwm (Caraava mosaic v i rus) and bacteria (bacter ia l b l i g h t ) .

B m d i f o r dlmuae r s i s t u K ~ Szrces o f virus ZsiBtance are l i m i t & Selection of vlrus symptom free

plants and the use of thennotherapy are pract ical waym of providing healthy plant ing material, but t h i r i s only a short term solution. Shoot t i p c u l t u n can be used f o r production o f v i rus free luter rial. Scnanlng o f germ plasm fo r resistance o r tolerance t o bacter ia l and virus direamem i s u rqmt l y recon rndd .

Fmsent ro le o f in -v i tn , tech iauet Shoot t i p cul ture and i w v i t r o nnr l t ip l icat ion o f cut t ing# are established

procedures i n certain clones. High genetic s tab i l i t y i r associated wi th these in -v i t ro systems Hawever, no established nethod fo r plant regeneration from -. -- c e l l and c a l l w cul ture i s available.

Prospects f o r the future Virological research should be rtrengthened, par t icu lar ly the development o f

indexing techniques to ident i fy virus free plant# i n the f i e l d and t o check re- infect ion of clean stocks. In -v i t ro technolcqy w i l l certainly be urefu l f u r gem plasm preservation and f o r international gem plasm d is t r ibu t ion . Resrarch on using shoot t i p cul ture technology f o r induction of mutations and f o r + v A t ~ selection i s advisable. Developcwnt o f procedures of plant regeneration from single ce l ls i s urgent. This w i l l o f f e r the scow for ii~--v_LCo selection against bacter ia l , fungal and virus diseaser.

F . 3 Seed propaqatod crops wi th a very long qeneration tima

F.3 .1 * Cocoa i s among the most important export crops o f West Afr ica and, t o a

lesser ertent, o f &ny South her ican, Caribbean and South East Asian countrleo Swollen shoot v i rus i s I n Wort Africa perhaps the l o s t important disase o f cocoa and the crop losses caused by t h i s disease are d ramt i c . Other inportant diseases include black pad (Phytophthora spp.) and witch.# ' broom (Cr in ipg l l i s perniciosa).

Broedim svste.8 a n u a l po l l i na t i on for the production o f seed i# a c ~ l l o n practice.

Seedlings of evaluated hybrids are suppliod t o f a m r s frm r e d gardens. However, the period between plant ing and f ru i t i ng rangem from 3 year* t o r improved hybrids t o about 10 years f o r othbr stocks. Vegetative propagation has been developed, but so far ha5 been used mainly f o r ewprr imntal purpomes. Tha gonetic base of the cul t ivated cocoas u n f o r t u ~ t e l y i s Mrrar, and p rogn ts i n

the b reod~m] progr- has r e l l d on the i n t r o d u c t i o n o f g e m p l a m fr# the fo res ts of l b u l h h o r i r a

B r _ e f @ ~ ! Cot_Airoa-?-ee cec_l_C&t$sl B r e r d ~ n g for s w l l a n shoot and black pod res is tance h . s been qo lng on f o r

u n y yearr . For s w l l e n shoot resistance. a screening technique u s r q ~ (aed l ings has boon deve1opc.d ohrch I $ basud on tho ~ n o c u l a t i o n o f seeds us ing p u r i f i e d v r rus o r v r w s ~ n f e c t o d vectors Appropriate m t h o d s f o r i n i t i a l scrueninq have a lso been daveloped f o r fungal and netnutode dlseases These screeninq rothods #re, h w e ~ e r , time connluming Recently the ea r l y de tec t ion o f r w l l e n shoot v i r u s by €1151) h r haen developed, and i t s i n c l u s ~ o n i n the breeding progr-s w i l l be a u l e f u l asset

Prepp_nt r o l e OJ IW-UI t r o t e c h n o j q y In -v i * technology has, so f u r , not been en tens iwe~y used I n p r a c t i c e b u t

sono t l ssue c u l t u r e methods are ava i lab le , espec ia l l y shoot t i p c u l t u r e I n y G r o c u l t u r e has, so far . no t been used f o r d ~ s e a s e res is tance breading attempt^ are nw b e l r q made t o Induce w t a t l o n s by ~ r r d i r t l o n o f v e q e p t i v e mater ia ls and pol1en;so as t o p r o d u ~ r genetic v a r i a b ~ l i t y f o r disease react ion, and there are plans t o combine mutat ion induction w i t h &_n-u&tro c u l t u r e

Proopects t o r t h e f u t u r e Shoot t i p c u l t u r e t o produce v l r u a f r e e p lan ts should ba an e r s a n t l a l

rupplomont t o t r u d ~ t ~ o r m l breedlnq, t o be able t o r e t a r n precious germplasm i n the f i e l d l o r bruudlncj pclrpuser, t19sue c u l t u r e technology m y a l s o he lp i n rpvodlnq up t r a d ~ t l o n ~ l b rned~ng prvgramne3, eventual ly p e r n i t t l q e a r l y and gurck rcreeninq f o r d~secrsc? res ls tan tc

When an i n - u i t r c ) ryatem f o r clona.1 p r o q a t ~ o n I S established / t could be used f o r nnrtetron b r u e d ~ r q by ~ r r r d ~ a t ~ n q t h e ~ n l t ~ a l enplants (ahoot t i p s ) and sub~equant ly i s o l a t lncg nun ch~mor IC m t e r l a l t o be screened f o r disease r e s t s tant ntutcrnts

H recen t l y In~tiated,proqramcne o f lnduclnq mutations i n c o c d t o in t roduce genet IC u a r r r b ~ 1 l t y I n t o b r ~ e d i n q materra ls should be v igorously y r r u a d Such u a r i a b i l l t y 1s dealred not only f o r d i seu le re$ ls tancs bu t a l s o f # r o the r u s e f u l charactors l l l c h ,as drought resrstanca, dwarf lng o f p lan ts , and for overcoming g a n u t ~ c i n a o m p a t ~ b i l r t y

F . 4 ~_a f_paqa tPd-c ropr w i t h a s h o ~ ~ e n e r a t i o n t ima

F . 4 . 1 $0-n Soybe4n i s considered her^ as an example o f the la rge group o f important

crop p l a n t s belonging t o the fami ly o f &umlnr.o e. I t i s one o f the major p r o t e i n f o a l md feed crops i n the wor ld . I t s importance as vegetable o i l and

i n d u s t r i a l crop i s a l so w e l l recognized. It i s an autoganwws species. Many

developing countr ies such as Indonesia, I n d i a and Thai land use soybean as a major p r o t e i n food source, but product ion i s usua l l y unsa t i s fac to ry .

Diseare p r o b l e ~ -- Soybean r u s t caused by Phakopsor- pachy rh iz i i s a ser ious disease l i m i t i n g

soybean product ion i n the t r o p i c s . r h i s disease w y becoma a p o t e n t i a l t h r s a t f o r temperate c l ima te soybean growing ragions as w l l . A number of v i r u s diasaser such as soybean mosaic. ye l low mosaic and b lack eye cowpoa mosaic a r e a l so serious problems.

Bwodins t o r d i s ~ l e n s i s t w w e Thr en t i r e vor ld soybean gwm planr co l l r c t i on h e been screened f a r soybean

rust and uirus resistances W u e r , for soybean rust only a l l r i t o d I r v a l o f r ~ s i s t a n ~ e il available U l l d mrenn ia l Cilih sp.cien sew t o include typru possessing better loybean rust I r r iutance, but aost a m polyploids and cannot ~ s i l y be crested due t o hybrrd breakdown Resimtances t o a t l u s t r o m of the v i rus diseases have been identr f led and ere used i n conventional broodirq

Prerent ro le o f in -u i t ro techniwer So far a w i l d perennral, GlycLz w%acc_nn, and one particular uariety o f C

R& (cul t ivated soybean) havr boon ~uccessfu l ly rugeneratd through orq.nogenasls and enbryogenesi~ ualng unique s d i a and spocialitPd prorodur@s I n G. canscens, the r~gonera t ion 0ccurr.d a f t e r two years I n cul ture M d i t i u n a l research l a needed to understund the f l ne r d e t a ~ l s involved i n problens of i n - v i t n , cul ture of soybean and other lequninwr crop plants --

Embryo cul ture has h e n successfully used to rarcuo hybrids betwen (; p~ and 5 . canscens. Thin method a8 w l l as &her In-ul t ra culture technique# w i l l hopefully pave the wy for i n te rspec i f~c hybr id i ta t ion t o transfer soybean rus t resistance f m n other species

future proapocts cul ture nsthods uo fa r are not well eatab l l~had for qrain legumes

I t can be expected that current l a q e e f fo r ts w i l l soon lard t o advances i n v i w o f the unique ro le legua~s play i n symb~otic f i xa t ion o f atnoapher i~ nltrogen Without such advances I n i n - v i t q technology, there are l i t t l e prospects f o r usrng e ~ t ~ culturos i n breed~ng of grain legume^ fo r di80aae rusintuncu

F 4 2 P_icc~ Rice 1s cons~derod as an example of a cereal crop i n whlrh good

convent~onal breading procoduros are well ert~ablishad, and r i ce breeding has led to major advances I n productivity as we l l as diaevae resistance Nevurthulons. rrce breeders are eager to make U ~ U of j_n-&r_o culture procudures, especially anther cultures, fo r the purpose of rapidly f i r i n g recombinants ard obtaining homocygosity However, aurh l~uthods +re not yet uppl~cable to a l l r i ce typun ard to other inportant cereals Cfforts should therefore be wxerled t o develop e f f i c rent haploid produrt~on i n reca lc i t ran t genotypes o f r l r e ( l r d i ca ) and other cereals

Future prospsc& The use of & w i t y o cultures i n r i c e brooding fo r improving disease

reriatance may not be l imi ted to the support of classical croc8 breadlnq via anther cul ture. There are m y disease and insect p ~ s t problams I n r i c e and the poss ib i l i t y f o r screening huge populations under 1z-vitr-9 condition# m y provide a chance fo r new genetic rewurce$. The same would apply fo r other cereLals.

C. THE USE OF I N - V I T R O TEWWIQUES FOR STUDIES OF HOST - PARASITE IWTERIICIIO(Y8

In-ui t ro cul ture techniques m y have an l r p o r k n t ro le t o play i n . elucidating host - p a r a ~ i t e interactions. The re ru l t s o f such studies could be of d i m c t value i n tho development o f practical procoduros fo r tha use o f in-v i t ro techniques i n disease resistance breeding, but I n many Instancar would - - l e d t o fundamental knowledge useful i n dvanclng current concepts of plant protection.

G. l Research wi th p.thogonic f um i , bacteria and nrwtodes Souoral topics o f research m y be i dent i f ied i n l h i c h cultures, by

providing a control lad r r t po r iwn t r l system, c w l d be an relevant aid:

Nutur l r e c q n l t i o n of host and parasite. Resisknce reactions (phytorlenins, hyparaensit lvity) vs. t o l w m c e Trnour phyrLoloqy. T h e aode o f act ion ( Interact ion) o f pathotorins and enzymes Systemic infections. Nutrient ud signal enchange batwen hort and parasite. Cross protect ion. Seruai and asexual sporulation E v o l u t i o ~ r y capacity of pathogenr.

Three o f there topics havr par t icu lar relevance t o ths developlont o f methods fo r j r p v i t r g screening studies about recognit ion, about r e ~ i s t ~ c e reactions and about the node of a t t i o n o f pathotox~ns lbrt useful w u l d also be progress i n the ip-sttrg cul ture of obligate parasitas This could be advantageous wi th regard t o providing i d e n t ~ f ~ o d and stunlard~zud i nocu lw and w u l d a l l w nvtntenance as w l l as transport o f col lect ions o f such pathogens

Despite the potent ia l importance, r t u d ~ e r of the interact ion b e w e n cultured plant matorla1 and pathogenic fungi, bac te r~a and n e ~ t 0 d ~ l have so far h e n rather l im i ted I n the care o f bacteria, nost at tent ion has been focussed on the crown g a l l syndrome (%cqm~ter~um tumefaciens) I n the case o f fungi ard nematodes, tho dual culture of obl igate or sem~obl~gate parasites as w l l as the i r hosts and the d o of act ion o f pathotorinn have received m s t at tent ion I h r wel l defined &e l system of tobacco and Ph~ toph thoz parasi t ica nlcotianae i s available f o r resistance studies, but only few researchers have taken advantage o f the p o s a ~ b ~ l t t i e s Too l i t t l e at tent ion has been devoted t o studying the ef fects of c e l l frae component8 o f pathogens on the phys~ology o f host ce l ls and tissues, wh~ch rhould rllow to d ~ s s ~ c t complex intmractlons

rhore are three probable reasons fo r t h i s lack o f e f f o r t s F i r s t l y . p l r n t pathologists seem t o have been u r w ~ l l i n g to explore the posa ib~ l t k i es offered by iryvi>r-q t*chniques, perhaps par t ly through prejudice and part ly through ignorance o f vhst i s available Second, by an irony, the lack o f understanding of the genetical and biochemical ba51s of many aspects o f the ~n t * rac t i on between plants and the i r pathogens may have prevented recoqn~t ion of both the ~4rengthr and the weaknesses of i n - v i t g rystem$ and, therefore, the correct i d e n t ~ f l c a t i o n of those ctrcumstanc.s under uhich they could be of value Finalty, i n - e o culture technology i t s e l f . despite i t s masalve research e f f o r t . st111 has many unsolved problem9 Those most relevant t o atud~es o f host - parapita interact ion (and. ~nc identa l ly , t o the use of in-%[rz culture i n plant breeding) are (1) genetic i no tab i l i t y . (2 ) abnormal and stress metabol~sm I n culturu, and ( 3 ) the control of d ~ f f e r a n t r a t ~ o n and regeneration

G . 2 . Research wi th viruses Plant virolcqy provides a wrked contrast t o the above. The develorment of

martstem culture tPchniques during the f i f t i e s lad t o the perfection of Athods for the production o f virus free clones o f a wide range o f vegetatively pruwqatad crop and ornamental species. The techniques are s t i l l o f great irnportanca. Cater, I n the s ixt ias, the deve lomn t o f methods f o r cu l tu r ing and infect ing plant protoplants r~vo lu t i on i sod many aspects o f plant virology, by opening up now avenues of research not possible w i th i n tac t p lants. The f o l l w i n g topic# are currently under act ive invest igat ion using protoplast syrtans: - The infect ion processes. - Replication a d the enpression o f the v i r a l genome

- Genetic var iat ion - Cross protection and other v i rus interact ions. - Resistance mchanisns - DN&viruser as tools for genetic modification o f plants

The f i r s t f i v e topics h u e ralevame fo r tho developnmt o f &--yLtLp scmwinq procedures but e l m for c lassical d l s w r e ns l r t ance breading. The las t topic u y b e c a m l e u m t fpr tho dovelopunt o f ruu &- plant h e d i n g prrcdurclr

The momentum of the reaearch on u i r u s e ~ i s considerable. and there I s every n r a o n t o suppose thet i t w i l l continua It should ba strongly encoura+pd. Important challenges e n l l t fo r the future, especialty tho solution of tho problon o f metabolic stress imposed by the a r t r f i c i a l enulrorwnt of pmtop lar t cultura, the understanding o f how the events i n i s o 1 ~ t . d protoplasts are related t o those I n Intact tissues. and the uncovering of the i n te rn l r t i onsh tp htwaen c e l l growth and virus mul t ip l i ca t ion

C 3 R e c m n d a t i o n Studles o f the interaction betwen plant pathogens and the i r hosts using

in -v i t ro technique, should ba strongly encourrqrd and supportad because t h y w i l l -- t o m the b o a ~ ~ fo r the dovelopwant o f n w , re l iab le and efficient kt& sLreenlng pmceduros But such research w i l l also praluce f u d u w n t a l knwl.dp@ about plant diseaser, contribute to the understanding o f resistance nochanlsas a d thus help An dsslqning mom e f f e c t ~ v e und'durrble measures fo r cor l t ro l l ing crop losses

WYrWWS. Induced Wutations for Dirsasa Resistmnc~ i n Crop Plmtn, XAEA, Vienna (1974)

IWONYWS., Irduced rCltat~ons rga in r t Plant Disearas. IAEA, Vionnr (1YII)

oNYKX)S, lnduted Rutations - A Tool i n Plant Reasarch. I A E A . Vienna (1981)

WcmYrouS. Induced h t a t i o n s i n Vogutdtively Propqatad P lm ta 11, ILEA, Vienna (1982)

W Y W S , Induced b t r t i o n m for Disorse Resistance In Crop Plants 11, IflEL. Vienna (1983)

WOIYYWS. lho potentla1 f o r u s ~ n g i n - v l t m techniques fu r gemplasm co l lsc t lon K P C . IBPGR/83/108. Rome (1984).

WYPWLIS, l n t e r n d t ~ o r u l Symposium on h n e t ~ c Manipulation i n Crops r h ~ r d l n t e r n r t t o r l ~ l Sympoaiun on Haplo~dy. F i r s t Internat ional Sy*pocIum on S o m t ~ c Cel l Gpnet~c- i n Crops, 22 - 26 October 1984, Bs i j i r q , China

CONSlANflN, PI J . HLNKE. R R . . HUGHES, K W and W t R , B V . . Propaqation o f Higher Plants through l issuo Culture Laorginq fochnologies a d Slratogien. fnulronmental and Frper~montal Botany 2_1 (19B1) 209-452.

DALY, J PI. artd DtVERALL (Eds.), roxlns and Plant Prthogone~tr . Acadumic Press o f A u ~ t r a l i u , Sydney (1983).

DUDlTS, D., FARKAS, C.L. 4 rVIL1GA. P., Ce l l Genetic8 i n Higher Plarrts Akademiai Kiado, Budapest (1976).

DURBIN. R.D. (Ld.) , Toxins i n Plant Disease. Aculenic Press, Now York (1981)

EVWS, D.A., SHARP. U . R . . IV+IIRATO, P . V . and Y W L , Y . , (Eds.), Handbook uf Plant Ce l l Cul ture Vol.1 Techniques f o r Propagation a d Brooding. tbcmi l lan Publishing Co. N w York (1983).

FRIEND, J. AND THRELFALL, D.R., Biochemical Anpectr o f Plbnt-Paraslto R~la t ionsh ips . Academic Press, London (1976).

FUJMRA, A,, Plant Tissue Culture. (Proc. o f 5th Internat ional Congress Plant Tissue ud Ce l l Culture). Jap.Aasoc. f o r Plant Tissue Culture, Tokyo (1982).

GAUTHERET, R.J., La Culture dcs Tisnes Vigbtaux, lechniques a t ROalisations Masroo Cie., Paris (1959).

WTTSCHIILK, W. and WOLFF, G.. Induced mta t l ons I n Plant Breeding. Sprloger, Bs r l i n (1983).

HELGESON. J.P. and DEVERALL, B . J,, Use o f T iasw Culture and Protoplasts i n Plant Pathology. Academic Press o f k r s t m l i a , Sydney (1983).

HOLL(KWOtR, A , , LASUIN. A X , ROGERS. P . DW;LEY. S , MWXi#, R , K X A V . L urd IYSGINC. J , h r i c B io logy of Y.w Oevo lopun tc in B~u techno loqy . P l e n u Press, N w York (1983)

HUGHES, K.W., HENKE. R . , COUSTPMTN, R . (Eds. ) . P r o p q a t i o n o f Higher P l a n t s through Tirwue C u l t u r e . (Proc. o f a Symposium K n o r v i l l ~ 1978). Techn, I n f u r r Centre US Department of Eneqy (1979).

I f f iRW, D.S. and HEL.(;ESDN, J .P . , T issue Cu l tu re kthods f o r P lan t Pa tho log is t s . t l l a ~ l r u u l l Sc ient .Pub1 Onford (1980).

K0SW.X. 7 , MREDITM, C P. and HOILAENDER, A . Canetic Eoginaer ing of P lan ts Plenum Pross, Now York (1983).

LLWONTI, J.F. and MNLAOSO, W.R. (Eds. ) . h l e c u l a r and C e l l u l a r PIPchani,ms o f IkrLaqenuris Plenum Press, Nw York (1982).

POHIER. A , b l e c u l a r Go lw t l c r o f t he Bacler ia-Plant 1nt.eract ion Springer-Vur lag, B e r l i n (1983)

RACHIL, K.0 and LYMAN. J u d l t h R . , Gvnetic Engineering f o r Crop Improvement, Working Papers: Rocke fe l l e r Fourdat ion, N.Y.. USA (1981).

RIY), P O , HLBLE, R R and CHADHA, n S , P lan t T ~ s s u e Cu l tu re G I o o t ~ c b n i p u l a t l o n and Somatic H y b r ~ d ~ t a t i o n o f P lan t C e l l s Bhabha A tOn~c Research Cantre. Bombay (1980)

RLINtWI, J and BAJA3, Y P S (Eds ) , P lan t C e l l , Tissue end Organ Cu l tu re Springer-Verluq, B e r l ~ n (1917)

!;&LA, F , PARISI, B , CFLLA, R and CItFRRI, 0 (Ede ) . P lan t c e l l Culture:, Rssul ts and Proupac t i v ~ s F lsev ler /Nor Lh 1401 lwd, Amsterdam (1980).

SEN, S K mnd G1119, K L (Eds.) , P l a n t C e l l Cu l tu re i n Crop Improveme?t Plonum Prurs, New Yurk (1983)

SHARP, U.H., EVANS. 0 , AMIRA10, P . and YMADA, Y. (Eds. ) , H a n d m k o f P lan t C e l l C u l t u m Vol 2 Crup Species. &cmi l l an Pub l i sh ing Co , N w Yctrk (1983).

SIinRP, W R . . LARSCN. P.O. , PADDOCK, F F. and RfWAVAN (Edrl .) . P lan t C e l l and Tissue Cu l tu re . Ohio S ta te U n i v e r s i t y Press, Columbia, Ohio (1979).

STREL1. H E . (Ld ) . P lan t Tissue and C e l l Cu l tu re . B l a c k w l l S c i e n t i f ~ c Publ . Oxford ( l Y t 3 )

THOWPL. T.A., frontiers o f P l a n t T issue Cu l tu re The I n t e r n a t i o n a l Aesoc iat ion o f P l s n t l r s r u e Cu l tu re , Ca lq r ry (1978).

VASIL, I. K . , SCOWCROf 1, W. R , and FREY, K. J . , P l a n t Improvement and Somatic C e l l Genetics. kcadurnic Press. NW York (1902).

BINDING, H., Se lek t i on i n K a l l u r k u l t u r e n l i t haplo iden Z e l l e n . 2 . Pf lanzenz. 67 (1972) 33-38.

BINDING, t i . , BINDING, K . and STRAUB, J., So lek t i on i n Ceuebekulturen m i t haplo iden Z e l l e n . Natunr i rsenrch. (1970) 138-139.

BPttl'EL. R.I .8 . and 1W;PWI. 0.S.k Tlosaa c u l t u n and the production o f novel d ~ s u r c r r r l s t u l t crop plants. B io l .Rev i.( (1979) 329-345

BaOTJES, C , Signlf icmce o f &-* duen t i t i ous bud techniqurs f o r wt.tlon b n d i n g of vegetativrly proprg.tmd craps. I n : Indued Ib Ia t ions i n Vegetat~vely Prop.9. t .d Plants It. I M 4 . YIMWU (1901) 1-10.

C&ILLOOX. M. , Plant t issue cul tuto: Rapid promqmtion, inducod ur ta t ionr and the potent ia l r o l e of protoplast technique#. In : Crop Breading, a Contmporary Bar i r (VOSE. P 8. end B L I X T . S . G Ed*.). Perpuon Press. Onford (1904) 311-346.

CIIRLSOW. P.S.. Somatic c e l l qomtica as a too l f o r plant breeding. I n : Induced Rutations I n Vegetatively Pmp.9.t.d Plantr. IN&, VLrnna (1973) Ibl-177.

CARLSON, P.S. and WLACCO, J . C . , Plant c e l l cul tures, genetic aspoct~ o f crop improvement. Science (1975) 622-625.

CHALEFF, R.S . Iso la t ion o f agronomically useful nwtants Iron plant c e l l cultures Science 2 1 9 (1983) 676-682

CHALEFF, R.S. and CARLSON. P.S.. Sorvtic c e l l gerwtics o f higher p la r~ t s . Arm Rev.Cenet 0 (1974) 267-278.

DARVILI., A . C . arld ALBERSHLIR, P., Phyloalenins and the i r elicitors - fi dofenas against microbial infect ion i n plants. Ann.Rev. of Plant Physiology 35 (1984) 243-275.

DIXON. R . A . , Plant tissue culture methods i n tho study of phytoaleiin inductlon. I n : Tissue Culture Rothods for Plant Pathologists. (INCRIYI, 0.5. and HELCESON, J.P. Eds.), B lackw l l Sc ient i f i c Publ., Oxford (1980) 185-196.

DUTRECQ, A , , I c -v i t ro selection of plants res i l t an t or tolerant to pathogenic fungi. I n : Induced Ilutationr against Plant Diaea~es, IAEA, Vienna (1977) 471-478

EARLE, E D. , Phytotorin studies wi th plant ce l ls and protoplast. I n : Front ierr o f Plant Tiasue Culture (THORP€, T.A. Ed.) (Proc. of a Conqrers Alberta, Canada). I n t e r ~ t i o n r l Association f o r Plant Tiasue Culture (1978) 363-372.

EARLE, E .D., The use o f phytotor ins f o r in -v i t ro selection of ditoare,-resistdnt plants. I n : Ce l l Titsue Cuture Techniques for Cereal Crop Inprovoment (Proc, o f a Workshop). Science Press ki j ing; Cordon L Broach N w York (1983).

EARLE, E.D and GRKEN, V.E., The ro le o f protoplastr ard c e l l cultures I n plant disease research. I n : Plant Disease Control: Resistance and S u ~ c e p t i b i l i t y (STAPLES, R.C and TOENNIESSEIY, G.H. Eds. ), John Wiley and Song, New York (1981) 285-297.

EVANS, D. A , , FLICK, C.E., JENSEN, R.A., Disease roaistance: Incorporation in to serually incompatible s w t i c hybrids o f the gsnur Nicotlary, Bciancs (1981) 907-909.

do FhZIO, G.n. , Plant t issue culture - proswctr of i t 8 pract ical appl icat ion i n plant pathology. S u m Phytopathologica 2 (1976) 145-153.

FOXE, H. J. and PRAKASH, J., Us0 o f protoplarts i n regeneration and virus resistance studies. I n : Protoplast Symporiw, &st. 168, Birkhiiurar Basrl (1983).

GREEN. C.E., Prospects f o r crop iaprounont i n tb f i e l d o f c e l l culturcr. Hortsci. 12 (1917) 131-134.

da GR001, 8 . and PUITE. K . 3 . . Corntic eng imer in~ : rothods fo r plant broadlng; a review and proswcts. I n : I nducd Var iab i l i t y i n Plent Omding. Centn f o r hqric .Publ. and Docwcmtation Y.qeningen, h th . r l . nds (1981) 34-41,

GROUT, 0.U.W. and UEAWRHE~. M.A., A strategy Tor tho production u f d isease-re~isturt mutants. I n : Tissua Culture tbthods fo r Plant P a t h o l q i r t s (~ffiunm, 0 8 , and HELCESOW, J.P. U s . ) (1980) 249-254.

M CVUUMI, E.V., OEL ROGARIO. A.G. and PMXALIYK;IY3. P.C., Production o f mutants by i r r d i a t i o n o f in-wits c u l t u r d tissues o f coconut and banma &nd the i r mass propaqmtion by tho tissue cul ture technique. I n : Inducod Rutations i n Uegetatiuely Propagted Plants 11, I M A , Viennr (1982) 113-130.

HADRO. U . . fWlagenesir and in -v i t ro selection. I n : Plant Tisrue Culture, k thods and Application i n Agriculture, (THORP€, T.A. Ed.), Academic Press, NemVork (1981) 155-180.

HAM. C . T . , Gonetic engineering and tisnue culture i n crop protection. I n : 10th Intercut ional Congresrr of Plant Protection (Proc. o f Conference Brighton England) Vol. 2 (1903) 810-019.

HARRISON, B . D . and H I Y O , M.A., The ure o f protoplasts i n plant virus research. I n . Use o f Tissue Culture and Protoplartr i n Plant Pathology, k rdPaic Press o f Australia. Sydney (1983) 69-137

HLINZ, 0 J., Suqarcano iaprovel l~nt through induced u t a t i o n s using vegetatiue propaguler and c e l l cul ture technique$ I n : Inducod Mutations i n Vagetatively Propagatad Plants, I R A , Vienna (1973) 53-59

HEINZ, 0 J., KWISHNWJRTHI, M , NICKELL. L C and WRLTZKI, A , Cel l . t i r r u e and oqan culture rn sugarcane rmprovemant I n . Applrod and ~undwn te ] . Aspects o f Plant Cell, Tissue and Orqan Culture (REINERT, J and BAJAJ, Y . P 8 Eds '), Sprinqer-Verlaq, Ber l ln (1977) 3-17

HELGESON, J,P , Studies of host-pathogen interactions in -u i tm. I n : Use o f Tlraue Culture and Protuplastr i n Plant Psthology Academic Press of Australia, Sydney (1983) 9-38.

INCUIYI, D.S,, Growth o f biotrophic parasites i n t i s rue cul ture. I n : Enxyclopeedia o f Plant Physiology, N w Series, Vo1.4 (HEITEFUSS, R. and WILLIIYs, P.H. Eda.), Springer-Verlaq, Ber l in (1976) 743-759.

INGRM, D.S., The ertabl ishmnt o f dual culturns o f dany n i l d w fungi and tha i r horts. I n : Tiasue Culture Bathods f o r Plant Patb log is ts (INGUM, D.S. and HELCESOIY, J.P. Eds.). Blackus11 Scient.Pub1. Oxford (1980) 139-144.

INGRM, D.S., Tissue cul ture methods i n plant pathology. I n : Tissue Culture mthodr f o r Plant Pathologists (INGRM, D.S. and HELCES(MI, J.P. Edr.), Blackus11 Scient .Publ. Oxford (1980).

IWORW, D.S.. Prospects and ch l lenqes f o r the future. I n : Use o f T iasw Culture and Prwtoplasta i n Plant Pathology. Acdsr ic Press o f h r t r a l i a , Sydney (1983) 163-188.

JACATHESIOI. D . . I m p r o v ~ l ~ n t o f sugarcane through induced mutations. In: Induced h t a t i o n s i n Vaqetatively Propaqated P l w t s XI, IWA, Vienna (1982) 139-153.

KO&, A J . , Inducad and rpnturmwr wta taonr i n p l m t c e l l c u l t u n r ud tkir p o t ~ l t i a l use f o r plant brradlrq* I n . Induced V a r h b i l l t y In Plant Breedlrq. Cen tn f o r 4 r i c Publ. and Oocuaentatkon. Ulpminqen. Wetherlands (1981) 45-50.

3 KLEIWMOfS, L. and BEKWI, R . , Pro(prcts f o r p lant g e m m d i f i c a t l m by n o n c o n u r n t i o ~ l wthods. (hn ReW.G.net M (1977) 79-101

LAINIIN, P.J. md SOOYUKWT, Y.R . SDvclonal var iat ion - A novel source o f ua r i ab t l i t y from c a l l cultures for plant iapmvemnt Thror.App1.G.net. @ (1901) 197-214

W A L D , n v. and IffiRw. D.S., lha u8e o f t i s rua cul tura i n o i l seed rape breeding. I n : Aspscts of bpp l~od Biology 2 (1984) 37-48.

MLIM. P . I so la t i on and charac ter l ra t~on of mutdntr i n plant c e l l cultutw. Ann.Rcv Plant Phys~o l . (1984) 519-542

BfLCHERS, C . Haploid6 fo r breeding by mutation and recumbrnation. I n , Polyploidy and Induced Rutatlonr i n Plant Brooding, IAEA, Vienna (1974) 221-231,

N I CHERS, G . K o m b ~ ~ t i o n ~ w t i s c h e r und konvontioneller Genetik fu r d ie Pflanzenzuchtung. Naturwissensch 6_f ( 1 9 7 / ) 184-194.

MCNENDFZ. T , A p p l i ~ a t i o n o f mutation mathuds to bunma breeding I n Inducad Mutations i n Vegetatively Propsgated Plants, IAEA. Vienna (1973) 7590

MILLER, S.A, and MAMYELL, D.P.. Evaluation o f d i s e r ~ e rarlstance. I n : Handbook o f Plant Ce l l Culture, Val .1: Techniques for Propagation and Breeding (EVANS, 0 . 6 . . SHARP. W . R . . WIRATO, P.V. and YlYVlOA, Y . Edn.), h c m i l l a n Publ.Co. N w York (1980) 853-879.

fWIOYOSH1, I . , Protoplasts i n virology. In: (FOWKE, L.C, and CMYSTABEL, F . C . Edr.), Plant Protoplasts, CRC Press, I n press.

IWRAKISt41, H., LESNEY, U. and CARLSON, P . , Protoplastr and plant viruses, Advancer tn Ca l l Culture 1 (1984) 1-55.

NABORS, M . W . , Using spontaneously occurring and induced mutations to obtaln agr icu l tu ra l ly u rv fu l p lants. Biosci . ?_d (1976) 761-768,

NELSON, O.E., The app l icab i l i t y o f p lant c e l l and tissue cul ture techniques t o pldnt improvement. I n : holecular Genetic Rodif i ca t ion o f Eucaryoter (RUBENSTEIN. I., PHILLIPS, R , and GREEN, C.E . Eds.), Academic Press, Maw York (1977).

NELSON. R . R . , Strategy o f breeding for disease resistance I n : Crop Braeding, Contemporary Basra (VOSE. P.6. and BLIXT, S C. Ed*.), P e r g w n Prsrs, Oxford (1984) 32-50,

NICKELL, L.G., Crop inprovewnt i n sugarcane: studies using h v i t r o mthodr. Crop Sci. ,Q (1977) 717-719.

ORTON, T.J., Genetic var iat ion i n s o w t i c t issues: method or madness? I n : Advancer i n Plant Pathology Vol. 2 (IUGRIY), D . S . and UILLIMS, P.H. Eds.), Acdmic Press, London (1984) 153-189.

PATIL, 8.8. . Toxins produced by phytop.thogenic brc tor ia . In : Annual Reviw of Phytoplthology (1974) 259-279.

QUALSLT, C.O., hgricultural applications: Inteqrating convent Ionrl ud mlecul&r genetics. In: (krutlc Engimrriq of Crop Plants. Calif . M r . 311 (1912) 29-30.

QUAK, F . , Maristen culture and virus-free plants In. Plant Cell, Tissue and Organ Culture (YEIMERT, R.J. and BEJAJ, Y.P.8, Ms.). Springer Verlag, llerlin (1977) 590-615.

SCHIFILR, R P and LIVIWGSTMY, R S . Host-telective toxlns and their role in plant d1seas.r. Sc~once 22J (1994) 17-21.

SCOWCROFT, Y.R , $omtic cell genetics and plant improvsnont Adv.fqron. 19 (1977) 39-81

SmCROFl, Y.R., LhRKIN, P.J. and BREITELL, R.I.S., Genetic variation fron tissue culture. In: Ume of Tissue Culture and Protoplasts in Plant Patholog9 (HELCESQN, J.P. and DFVCRALL, 0. J. Eds. ). Academic Press of Australia. Sydney (1983) 139-162.

SELA, I., Plmt-virus interactions related to resistance and localization of viral infoctiun~. Advancer in Virus Research 26 (1981) 201-237.

SEWEIRA. L , Mechanirms of induced resistance in plants. Ann.Rew. Plicrobiol. L7 (1903) 51-79.

SHEPARD, J.F , Protopla?ts as sources of disease resistance in pllnts Annual Review of Phytopatholoqy )_P (1981) 145-166

SIEGEMJND, r , Selektiorl won Resrstenzmutanten in pf lanzl ichvn Zel lkulturen, elne Obersicht Biol ZenCralbl @ (1981) 155-165

S I M S , Pl.0.. Modification of host-parasite interactions through 8rtifici.l rautagenesiv. Ann.Rou. Phytopathol. 1: (1979) 75.-96.

SPILCCL-ROY, P and KOCHBA, J , htation breeding in citrus In Induced Rutations in Vegetatively Propaqdted Plants, IAEA, Vienna (1973) 91-103

SPIECEL-ROY, P. and KOCHBA. J . , Rutant selection in cltrus callus. In: Muances in Riochemical Engineering. Springer Verlag (1980) 40-44.

TAKEBE. I., The use of protoplasts in plant virology. Ann.Rev. of Phytopathology (1975) 105-125,

THOrVIS, E . , KING, P.J. and POTRYKUS, I., Improvement of crop plants via single cells an astess~wnt. Z.Pflanren2. 82 (1979) 1-30.

UALTOIY, J.D. and EARLE, E.D., Fungal phytotoxins - Isolation and bimrsay. In: Cell Culture and S o ~ t i c Cell Genetics of Plants, Vol.1. (VRSIL, I.K. Ed.). Rcademic Presr, N w York (1985) 598-607.

WENZEL, C . , Protoplast techniques incorporated into applied breeding programer. In: Advances in Protoplast Research (FERENCZY, L. and FARKAS, G.L. Eds.), P e q m n Pmss, London (1980) 327-340.

WENZEL. G . . SCHIEDER. O., PRZEWOZNY, T., SOPORY, S.K. and IFLMERS, G., CMIplrison of single cell culture derived Solmum tubemsum plants and a mdel far their application in breeding programas. lhor.App1. Genet. 55 (1979) 49-55,

ULWOUI, J . R . . (klwtion and c h n c t * r l s t l c s o f b i o c h w i c a l mutants o f c u l t u r e d p l a n t c e l l # . In: Tis8ue Cultury and P l m t Science (STREET, W.E. Ed.), ( k . d w l c Press, London (1974) 207-299.

WIDHOW. J . R . , The s e l e c t i o n o f * J r i c u l t u r a l l y d e s i r a b l e t r a i t s w i t h e u l t u n d p l m t c e l l s . I n P r o p q e t i o n o) W i g k r P lan t8 t h r w g h T issue C u l t u r e T e c h Inf . Cent re US Oepr tmen t @f E m q y , k s h i n g t o n (1978) 109-199

U)o, I .R.. BOULTOU. R . I . ud MIICE. A. 3 . . Ampl icat ion o f p r o t o p l a s t s in p l a n t v i r u s research I n . P lan t C e l l Cu l tu res . Resul ts and Perspect iver (CIFERRI, 0

and PARISI, 0 . Eds. ). L l r e u i e r , CY8terd.n (1979) 405-410

111. Specia l re ferences

O'IIPUITO. F . , BENNICI, A. , CIOUINI, P.G., BARONCELLI, S. and LUPI. M.C., Nuc lear f ragmentat ion fo l l owed by m i t o s i s as mechanism f o r v i d e chrormsow n u m h r v a r i a t i o n i n t i s s u e c u l t u r e s : I t a i m p l i c a t i o n s f o r p l a n t r q e n e r r t i o n . In: P lan t C e l l Cu l tu res : Resul ts and Perspect ives <SOLA, F., PRRISI, 8 . , CFLLn. R. and CIFERRI, 0 Eds. ) . E l sev ia r /No r th i b l l a n d , knstordam (1980) 62-72.

CARLSON, P.S. , h t h i o n i n r su l f o r im ine - res i s tan t mutants o f tobacco. Science KO (1973) 1366-1368.

DIIVIS, J.R , PAVEK, J . J . and WRSINI, 0 . 1 . A s e n r i t i u e method f u r q u & n t i f y l n q V o r t i c i l l i u a d a h l i a 0 c o l o n i z a t i o n i n p l a n t t i s s u e and eva lua t i ng r e r i s l m c e u o n g po ta to genotypes Phytopathology 73 ( 1983) 1009-1014.

OEATON, W.R., KEYES, G .J and COLLINS, G,B. , Erprossod rea l s tance t o b lack shank among tobacco c d l l c u l t u r e s . Theor.App1. Genet. g (1992) 64-70.

OIX. P . J . , C h i l l i n g r o s i r t a n c e i 3 n o t t r ansmi t t ed s e ~ u a l l y I n p l a n t s regenerated from N i c o t i a ~ y l v e r t r i s c e l l l i n e r . 2 . Pf lanzenphys io l . 84 (1977) 233-226.

EBEL, J . , AYERS, 6.R. and IILBERSHEIM, P. , Host w thogon i n t e r a c t i o n X I I . Response o f suspension c u l t u n d ~ o y b e a n c e l l s t o t h e e l i c i t o r i s o l a t e d from P h ~ t o p h t h o r a ~ 0 9 . s m ~ var . ,el.@, a fungal pathogen o f noybernr. P l a n t Phys io l . (1976) 775-779.

ERSEK, T . and SZIRAKI, I , Product ion o f serqu i terpene phy toa le r i ns In t i s s u e c u l t u r e c a l l u s o f po ta to tubers . Phytopath. 2 . 97 (1980) 364-368.

GABORJANYI. R . , ERSEK, T . and KIRALY, 2 . . L e c t i n l i k e m a t e r i a l responstb le f o r s p e c i f i c a t t a c t m n t o f P s e u d m n r s s l ~ c t n o a t o r e s i s t a n t soybean l e a f c e l l # . P ro top les t Symposium, Abst . 172, B i r k M u s e r , B a r e l (1983)

HABERLIICH, G.T., BUDDE, A . O . , SEpUEIRO, L . and HELEESOU, J .P . , E a d i f i c a t i o n o f disease res i s tance o f tobacco c a l l u s t i a r u e s by c y t o k i n i n s . P l m t P h y t l o l . a (1978) 522-525.

HEINZ, O.J. and PEE, G.Y.P., Horpholog ic , cy t cgene t i c a d enzymatic v a r i a t i o n in Saccharm species h y b r i d clones de r i ved Prom c a l l u s t i s s u e . h . J . Bot . 3 (1971) 257-262.

HELGESOW, J.P., HABERLIICH, G.T. and UPPER, C.D., O d o r i ~ n t gene c o n f e r r i n g d isease r e s i s t a n c e o f tobacco p l a n t s i s erpreseod i n t i s s u e c u l t u r e . Phytopathology 66 (1976) 91-96.

HELGEWft, J.P., Y E W , J .D . , HABERLm, G.T. ud MMLL, D.P., d t i s s u e c u l t u r e

syster f o r studying dioaase m s l s t u u a : tho Black Sbn& D i s u w i n tobacco ca l lus cul tums. Phytopatholapy 62 (1972) 1439-1443.

WFMMN, F., WUW, E . and YWZEL, G., h t t m r cu l t um as a b m d i n g too l i n rape 11: Progeny aculyser o f androgenic l i ~ s and induced u h t s tra h p l o i d cultures. Thoor. UI OgPl.OII1. 61 (3982) 225-232.

HOLI.IDAY, M.J. and KLARMIY, W.L.. Expresrion of d i s o ~ s v reaction typls i n soybou, cal lus Proa tusistant and susceptible p l u i t s . Phytopatholoqy (1970) 576-571.

INGRM. D S., LW, C.S., WCOONhLD, M . V . and YEYBWUE, D.H., Secondary embryoqenesis i n Brassica: A tw l f o r research and crop illproveaent. I n : Tha Cvnetic Pbnipulation o f Plants and i t s Application t o hgr icul turo (1984) 219-242.

XNGRW. D S and ROBER7SON. N , In terac t ion betwen Phytophthora infestans and t~snue rul tures of Solanum tukrosum J gen n ic rob io l 40 (1965) 431-437.

KMINSKYJ, S.G.W. and DIIY, A.W., Chamical induction o f in fec t ion strucf i reo i n rus t fungi. 1. Sugars and conplvr media. Exparim. tlycology &I (1994) 63-72.

NEWSHOLfii, O.R., WDONALU, tl V . , LOH, C.S. and INGRAFI, D.S., Secondary embryogeneair and the production of novel diaeaao r e s i a b n t Brassicas. I n : 1984 B r i t i ah Crop Production Conference - Pests and Diseaaer p.193-198.

SKRISTMJ, R.D., Rvsistance responses t o c+ lim of plants regenerntad f r u r salected c e l l and embryogenic culturen of h p l o ~ d Brrcsica m e . : Theor Appl. G n a t . ql (1982) 193-200.

SIICRISIAN, R D , Zellkulturen fu r d ie Erzvugung rer iotenter Pflangen Grenzen und Roqlichkuiten Vortrage Pflanzoncuchtung 3 (1983) 97-103, G.F P . iBonn

SACRISlhN, n D nnd HOFIIIANN, F , Direct infect ion of embryoqeni~ t issue cultures o f hap lo~d & r a ~ & j f l WIJJ with rest ing spores o f Platnodiophora bpss icae. Theor Appl Genet 54 (1979) 129-132

SCALA, A , B t l l I N I . P , PCLLECRINI, C , BOGANI, P , COILINA, F , / I P U l X I , tl , TOCNONI. F and BUIIITll. tl . Preliminary data on l o - v i t r o selection systems for pathoqen reg1 stance i n t o w t o (&coperalcon esculentum) (abstract). I n A l t i del W x V I Cmvegno de l la Sociatn i t a l l a m d l gunetica agraria Bordighora 1982 1st Spv r i l ~ r r t a l e per l a C~rca l i co l t u ra . Rorv (1982) p.116

VhRDI, h . GPICGLL-ROY. P and MLUN. E , Ci t rus c e l l culture iso la t ion of p r o t o p l a ~ t ~ , p la t ing denr l t iee, e f fec t o f mutagens and regeneration of eAbryos Plant Science Letter8 c(1975) 231-236

WARREN. R.S. and ROUTCEY, D G.. The use of t i s w e cul ture i n the study o f single gene rosiatance o f t o u t o t o Phytophthora infestans. J . h . Soc.Hort. Sci. 95 (1970) 266-269.

BEHNKE, f i . , S v l ~ c t i o n o f potato ca l lus f o r resistanca t o cul ture f i l t r a t e s o f Phytophthora infestans and regeneration o f resistant plants. Theor.Appl. Gonet. 5_1 (1979) 69-71.

BEHNKE, fi., General resistance t o l a t e b l i gh t o f Solanua t u b s m s u plants rsqensrated from callus resistant t o cul ture f i l t ra tms o f Phythophthora infvstans. Theor. Appl. Genet. 56 (1980) 151-152.

BEWYE. n.. Se lw t ton o f d l h p l o M potato ca l lu r f o r revir t .nce to t h cul ture f r l t r a t e o f f u a a r i u o r y s w w . Z Pf lanzenz 85 (1980) 244-259

BTRVILLE, R., Chracter iza t ion of r i toelmndria P r o a u t a n t u n r l r t r n t t o M l a i n t h o r p o r r u a race 1. I n : P l u t t n i tochonlr ia (WCCT, C and LW. C. Edr.) E i sev ie r /h r th Holland B toud i c& l P n r a (1978) 427-434.

BRtnTL. R . 1.8.. 0300(rRD, B.V.0. ud IWCRW, D.6.. S ~ l e c t i o n o f Tn8-cytoplasm u i z e t issue culture* resistant la Drechclera uma~ 1-tor in . myd ica &4 (1979) 203-2 1 3 .

BRE7TEL. R.I.S., IWGRM, O.S. and T W M S E . , Selection o f u i z ~ tissue cul turer reslutant t o O n c h ~ l e r a ( ~ m i n t h o 8 p o r i u n ) ~ d h T-torin. I n : f i r r u e Culturn Methods for Plant Pathologists ( W R M , D.S. and HELCESON, J . P . Eda.), B l a c k w l l Scient.Pub1. Orford (1990).

BRCTTEL, R I S , lHO((AS. E. and INGRWl. D.S , Reversion o f T P X ~ S la-sterile cytoplasm m i t o i n culture t o give f e r t i l e . T-tonin resiglant p lant# , Theor Appl Genet 58 (1980) 55-58

BRONSON. C R . and SCHEFFER. R.P., Heat a d aqring induced t o l o r m e o f r o q h and oat tissues to host-selective t o r i n r . Phytop~thology 6_! (19'17) 1232-1238.

COSSINI. R , CORNU, A , BLRVILLE, A , VUILLAWK, E and PANOUILLE. A , , IIGrLdlt6 e t chrac ter is t iques des sources do r b ~ i s t m c e A HelminthorppJuu u-2 race 1, obtenuea par aut&qani)se chez der u i r cytoplasm &lo r t b r i l e Texag. Ann. fM6l ior . Plantes 2.T (1977) 753-766

OIXON. L.K., L t A V € R , C . J . , BRETTEL, R . I . S . and CENCENBKH, B . C . , Mltochondrial s e n s i t i v ~ t y to Drechslera u y d i s T-torin and the syntheris of a variant mitochondria1 polypeptide i n plants derived from ~ I z e tisaue culture w i th lerav male-steri le cytoplasm. Theor.App1. GPn. 63 (1982) 75-80.

DUTRECQ. A , , Studies of the e f fec t o f toxic preparations o f Hglninthosoorium satiuum P.B. and 0 , on barley and wheat and perspectives of Lrv- re lect lon o f these cereals. hgronomie 1 (1981) 167-176.

DUTRECQ, A . , SWMREVNS, G , and SEWL, J . , Using resiatmnce to tho tox in of Helminthojporiur rativum as a uana of selecting cereals: t o r i c l t y tests and toxin preparation. Ann.App1. B io l . Q (1978) 370-371.

EARLE, E.D., Cytoplasm-specific ef fects of & l m t n t h o s p o r ~ nyvdir race T-torin on corn protoplasts and mitochondria. I n : Var iab i l i t y i n Plants Rageneratad from Tissue Culture (EARLE, E D . and OERARLY, V . Eds.), P ruge r Sc ient i f i c , Ww Vork (1982).

EIRLE, E 0 , and GRKEN, V . E . , Fused soybean and T cytoplasm corn protopiarts are resistant t o Ho l r i n tho rpo r i q race 1-tor in. Plant Physiol. 63 (ruppl.) (1979) p. 136.

EARLE, E.O., CRCICEN, V.E., VOOER, O.C. and GEI*IILL. K.P., cytoplasm-rp~c'ific effects o f I h l a i n tho rpo r im race T-torin on survival o f corn wsophy l l protoplasta . Plant Physiol . 6_1 (1979) 22-24.

GEWCENBRCH, B.G. and GREEN, C.E., Selection o f T-cytoplarr u i z o cal lus culture# reaistant t o HPlninthoswriucl uY~I, race T pathotorin. Crop Sci. 11 (1975) 64-49.

GENCENB(ICH, B.G., GREEN, E.C. and W V W , C.M., Inheritance o f selected

prthotoxin rerirtance in maize plurtr rrgmniod tram cell cultunr. Proe.*rt. Ikd.Sci US4 74 (1977) 5113-5117.

'HARRVUI, C.L., Selection of alfalfa ( M i - -a) cell liner and -noration of plants realst.nt to the toxin(r) produced by Furaricu o ~ v r m r i u f.rp. d i c y ~ i n i r . Plant Science Lettors 3) (1984) 193-194.

Wl'ERN, U., SIROBEL, G. and SHEPARD, J., Reaction to phytotorinr In a potato population derivud f r w wsophyll prutoplasts. Proc.Net Rcd.Sci. USA (1978) 4935-4939

OSWALD, 1 . t i . , SRI'IH. R.E. and PHILLIPS. D.V., Phytotoricity and detoxification of Metribuzin in dark grown ruspllnsion cultures of uoybean. Perticide Biochur. Physiol. g (1977) 73-83.

PAYNL, G , KOWO, Y. and DALY, J.M.. A comparison of purified host specific torin from &lmintho~poriu_r I?&"i,, Race 1, and its acelato derivativm on omidrtion by mitochondr.ir from susceptible and resistant plants. Plant Physiol. Ge(1980) 785-791.

POLMM), J.C. and POLACCO. M.L . Inducing and selecting valuable utation in plant cull culture: R tobacco mutant resisLant to carboxin. Ann. N w York Acad.Sci. 282 (1977) 385-400.

RANCILLAC, M , KAUR-SMNLY, R., SIASKWICZ, B and GALS1011, A W , Effects of cyclohex~mide and kinetin pretrebtments on responses of susceptible dnd resishnt

leaf protoplasts to the pytotomin ulctorln Plant Cell Phyllol Q (1976) 987-905

SCALA. A . BETTlW1. P , W N I , P . PkLLEGRINI, M G. and BUIAT11. fl , 1-vltro analysis of several pathogenic factorr In tomato - Fusar~um oxys~uru system VI Congreua ol' the Iled~terranean Pt~ytopatholog~cal Union 1-6 Oct~ber 1905, C a ~ r o (Egypt)

1WNUTONC. P , FURUSWfi, I and Y W H O T O , M , Resistant tobacco pbnts from protoplust-derived culluses selected for t h e ~ r resistance to Pseudoloner drd Slternariato&inu Theor Appl Genet $5 (1983)209-215

WILTON, J.D.. EdRLE, E.D., YODER. O.C, and SPMSWICK. R . M . . Reduction of steady stat.@ AlP levels i r ~ susceptible maize ~ s o p h y l l protoplasts by t&lminthoswrium ma- race 1-torin. Plant Phyaiol. $3 (1979) 806-810.

ZlLKAH, S, and GRESSEL, J . C . , Cell cultures vs. whola plants for measuring phytotoxlcity. I. The uatablist~mnt and growth of callus and suspcmsion culturea, definition of factors affecting tonicity on calli. Plant Cell Physiol. 18 (1977) 641-655, - ZILKRH, S., BOCION, B.F. and GRESSEL, J.. Cell cultures vs. whole plants for measuring phytoton icity . 11. Correlations between phytotoxicity in seedlings and calli. Plant Cell Physiol. (1977) 657-670.

ZILKRH, S. and GRLSSEL. J., Cell cultures vs. whole plants for w s u r i r q phytotoxicity. 111. Correlations between phytotoxicities In cell surpension cultures, calli and seedlings. Plant Call Phyaiol. & (1977) 815-820.

ZILKAH. S. and GRESSEL, J., Cell cultures us. whole plants for measuring phytoxicity . IV . Correlations in phytotoxicity bptuoen white ud green calli of R- obtuaifolius. Nicotiana tabaccum and Lycop~rricon e~culentua. Plmt -11 Phyriol. (in pnss).

BEIER. H.. SILEV. D J . RUSSEL. a.L and BRUEWIWC, C , Survey o f r u r c o p t l b l l l t y t o -4 t b s a i c V i rus mng p r o t o p l a s t s and I n t a c t p l a n t s from &M ~ I ~ n s h l i n c r . Phytopathology 6_1: (1977) 917-921.

CIISSELLS. A.C and COCWER. F.R . . ?RV i n o c u l a t i o n o f tobacco p r o t o p l a a t r in the presence o f p r o t o p l a a t f u s i o n a q m t s . 2 . Natur forach. )5C (1900) 1057-1061

DERAW, Y.. BICCS. S. and SELA. 1.. M u l t i p l i c r t i o n o f tobacco mosaic v i r u v i n tobacco l e a f spots d i s k s in i n h i b i t e d by ( 2 ' - 5 ' ) o l i q a ~ o n y l a t o . Sciunce (1982) 1415-1416.

K I K K M , H . . NIK;(ITA, 1'. . MTSUI, C , a d TAKEBC, I . , I n f e c t i o n o f p r o t o p l a a t s f rom tobacco suspension c u l t u r e s by t o b c c o moaaic v i r u r . J.(;en. V i r o l . 63 (1982) 451-456.

NURAKISHI, H H and CARLSON. P S. , I n - v i t r o r ~ l e c t l o n o f t4'4ot.j.n. ~ y l v p s t r i ~ v a r i a n t s w i t h l i m i t e d res i s tance t o THW. Plan t C e l l Reports ! (1982) 94-.97.

WnWn, fl , SAKAI, F and TAKEBt, I , A messenger RNA f o r tobacco mosaic v i r u n coat p r o t e i n In i n f e c t e d tobacco laorophy l l p r o t o p l a r t s Phytopnth 2 . 101 (1983) 146-158.

ORCHANSKY. P , RUBINSTEIN, R . and SELA. I., Human i n t e r f e r o n s p ro tec t p lan t8 from v i r u s i n f e c t i o n . Proc.Nat1. Acad.Sci. USA ?q (1982) 2278-2280 .

OTSUKI, Y , 'TAKEBE, I . , Double i n f e c t i o n o f i s o l a t e d tobacco l e a f p r o t o p l a s t s by two s t r a i n s o f tobacco nosa ic v i r u s . I n : (TORIYIYIA, K , DALY, I.N., UUlTANI, I., OKU, H. and OUCHI, S Eds. ) , B iochemis t ry and Cytology o f P lan t Pa ras i t e I r r t e r a c t i u n . Kudanrha, Tokyo (1916) 2 13-972.

TAKEBE, I., Protoplast:, i n the study o f p l a n t v i r u s r e p l i c a t i o n . I n : (FRAENKEL-CONRAT, H, and WAGNER, R.H. E d s . ) . Comprohonsiue V i ro logy. Vo l . 11, Plenum Press, London, Now York (1977) 237-203.

WENZEL, G . and UHRIG, H . , Breeding f o r nematode and v i r u s r e s i r t a n c e i n p o t a t o v i a anther c u l t u r e . Theor.App1 Genet. (1981) 333-340.

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