faster 0f pi|tl0sijpi|^ - CORE · 2018. 1. 4. · variation (CV) for days to flowering of lentil var. L-4076. Table 22. Esfimates of mean values (X), range, shift in X and coefficient

STUDIES ON THE INDUCTION OF VARIABIL FOR QUANTFTATIVE TRAITS IN LENTIL

(Lens culinaris Medik)

DISSERTATION

'H PARTIAL FULFILMENT OF THE REO' ^̂ ^̂ MENTS 1 *• » , 'T "T" f~

OR THE AWARD OF THE DEGREE OF

f a s t e r 0f pi|tl0sijpi|^ IN

BOTANY

ALKA

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERii.

ALIGARH (INDIA)

2008

rL

\ 8 JAN iflttl,

P

TTTl TnorrnooLTn

Samiullah Khan M.Sc, Ph.D., FISG Reader

MUTATION BREEDING LABORATORY DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH - 202 002 (INDIA) Phone (Res.): +91-571-2709265

(Mob): 9411413437 E-mail: [email protected]

/^•I'i^oft

CE^tfJl'FICA'^

This is to certify that the dissertation entitled "Studies

on the in(duction of variability for quantitative traits in

lentil (Lens culinaris Medik)" submitted by Miss Alka is in

partial fulfilment of the requirements for the award of the

degree of Master of Philosophy in Botany. The research work

embodied in this dissertation is the original piece of work

carried out under my supervision.

0 ^

{Samififfafi l(fian)

Acknowkdaements

<First, I 6cm) in reverence to tfie JifmigAty, the omnipotent, for it is

incCeecffiis Blessing alone wfiicfi provicfecf me enougd zed to complete tfiis wor^

I feel much pleasure to ejq>ress reverence and gratitucCe to my

supervisor <Dr SamiuClah f{fian for peeping a -watchfuC and discerning eye over the

study, providing valuable guidance, and help in overcoming the hurdles throughout

the course of this WOT^

I am highly grateful to Trof (Bahar J?. Siddiqui, Chairman,

(Department of (Botany, Jl.^.V. Jiligarh for providing the necessary facifities,

suggestion and ^nd help to carrying out this wor^

I own an expression ofthan^ to (Dr. Kouser (parveen and<Dr ^Hafiq

J4. Wani research scholars for their cooperation and encouragement.

I feet ^asure to express my appreciation to ^6ina <Perveen and

Sonu goyal, my laS mates for their he[p, suggestion and cooperation throughout

the study.

My gratitude ^ows no hounds when I thin^ of the love,

cooperation andheCp extended 6y my caring and loving friends Youshina, J^al{shi,

Mustaheen, cBusfira, <J(enu, J^asia, Irm, V^onil{a, JConey, ZeSa.

I feet short of vocabutary to express my sincere than^ to my ever Coving and

dearest parents and sisters who tit theftame of teaming in me and whose affection,

sacrifice, devotion and constant encouragement hetpedme to gain this success.

Lastly my tove, regards and Best wishes to all well wishers.

Alkoy

1.1. 1.2. 1.3. 1.4. 1.5. 1.6.

Quality parameter Biosystematics and cytogenetics Botany Origin, area and cultivation Production Induced variability

CONTENTS

Chapter Page No.

1. INTRODUCTION 1-7

2 3 4 4 5 6

2. REVIEW OF LITERATURE 8-27

2.1. Mutagen sensitivity 9 2.2. Mutagenic effectiveness and efficiency 14 2.3. Guidelines for induced mutation techniques 15 2.4. Choice of mutagen 16 2.5. Chlorophyll mutations 19 2.6. Morphological mutations (Macromutations) 20 2.7. Quantitative traits (Micromutations) 23 2.7.1. Effects of mutagen on character association 24 2.7.2. Induced polygenic variability for earliness and other 25

economic traits. 2.7.3. Induce early mutants 26

3. MATERIALS AND METHODS 28-35

3.1. Materials 28 3.1.1. Varieties used 28 3.1.1.1. Variety K-75 28 3.1.1.2. Variety L-4076 28 3.1.2. Mutagens used 29 3.1.2.1. Methymethane sulphonate (MMS) 29 3.1.2.2. Hydrazine hydrate (HZ) 29 3.2. Experimental procedures 29 3.2.1. Preparation of mutagenic solutions 29 3.2.2. Pretreatment 29 3.2.3. Mutagen administration 29 3.3. Ml generation 30 3.3.1. Observations recorded in Mj generation 30

3.3.1.1. 3.3.1.2. 3.3.1.3. 3.3.1.4. 3.3.2. 3.3.3. 3.4. 3.5.

4.1. 4.1.1. 4.1.2. 4.1.3. 4.1.4. 4.2. 4.3. 4.4. 4.5.

6.

Seed germination Seedling height Plant survival Pollen fertility Morphological variants Quantitative traits Cytological studies Statistical analysis

EXPERIMENTAL RESULTS

Biological damage Seed germination Seedling height Pollen fertility Plant survival ANOVA of seed germination and seedling height Meiotic abnormalities induced by mutagens Morphological variations Quantitative traits

DISCUSSION

SUMMARY

REFERENCES

30 30 31 31 31 32 33 33

36-42

36 36 37 37 38 38 38 39 40

43-50

51-52

53-80

List of Tables

Tablel. Percent seed germination in the two varieties of lentil treated with MMS.

Table 2. Percent seed germination in the two varieties of lentil treated with HZ.

Table 3. Seedling height in the two varieties of lentil treated with MMS.

Table 4. Seedling height in the two varieties of lentil treated with HZ.

Table 5. ANOVA for seed germination (for MMS treatments).

Table 6. ANOVA for seed germination (for HZ treatments).

Table 7. ANOVA for seedling height (for MMS treatments).

Table 8. ANOVA for seedling height (for HZ treatments).

Table 9. Effect of mutagens on plant survival and pollen fertility in the two varieties of lentil.

Table 10. Frequency and spectrum of morphological variants induced by mutagens in lentil {Lens culinaris Medik) varieties K-75 and L-4076.

Table 11. Frequency of morphological variants in various mutagens in Mj.

Table 12. Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for plant height (cm) of lentil var. K-75.

Table 13. Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for days to flowering of lentil var. K-75.

Table 14. Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for days to maturity of lentil var. K-75.

Table 15, Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for fertile branches/plant of lentil var. K-75.

Table 16. Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for pods/plant of lentil var. K-75.

Table 17. Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for number of seeds/pod of lentil var. K-75.

Table 18. Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for 100 seed weight (g) of lentil var. K-75.

Table 19. Esfimates of mean values (X), range, shift in X and coefficient of variation (CV) for total plant yield (g) of lentil var. K-75.

Table 20. Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for plant height (cm) of lentil var. L-4076.

Table 21. Esfimates of mean values (X), range, shift in X and coefficient of variation (CV) for days to flowering of lentil var. L-4076.

Table 22. Esfimates of mean values (X), range, shift in X and coefficient of variation (CV) for days to maturity of lenfil var. L-4076.

Table 23. Estimates of mean values (X), range, shift in X and coefficient of variafion (CV) for fertile branches/plant of lentil var. L-4076.

Table 24. Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for pods/plant of lenfil var. L-4076.

Table 25. Estimates of mean values (X), range, shift in X and coefficient of variafion (CV) for number of seeds/pod of lentil var. L-4076.

Table 26. Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for 100 seed weight (g) of lentil var. L-4076.

Table 27. Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for total plant yield (g) of lentil var. L-4076.

List of Figures

Fig.l: Effect of mutagens on seed germination in Mj generation in the two varieties of Lens culinaris Medik.

Fig.l: Effect of mutagens on mean seedling height (cm) in Mi generation in the two varieties QiLens culinaris Medik.

Fig.3: Effect of mutagens on pollen fertility in Mi generation in the two varieties of Lens culinaris Medik.

Fig.4: Effect of mutagens on plant survival at maturity (%) in Mi generation in the two varieties of Lens culinaris Medik.

List of Plates

Plate-I: Morphological variants isolated in Mi generation.

Plate-II: Morphological and foliage variants isolated in Mi generation.

Plate-III: Various meiotic stages in lentil.

Chapter-1

INTRODUCTION

Pulses have formed an integrated part of Indian farming system and

have played an important role in human diet. The country produces a variety

of pulses including chickpea, pigeonpea, urdbean, mungbean, lentil, field pea

and others to the tune of 13-15 million tonnes annually from an area of 22-23

million ha with an average yield of 600-650 kg/ha (Ah and Kumar, 2007).

Lentil or masur is bushy, annual plant that is popular for its lens

shaped seeds. Seeds have a vast range of colours and also have second highest

levels of proteins and fibre after soybeans. The thin lentil plant, which is

named 'Lens culinaris Medik' belongs to the family Fabaceae and gains a

height of 40 to 50 cms at maturity. The tap root system of the plant usually

grows to a depth of around 30 cms that makes it a moderately tolerent to high

temperature and drought.

Lentil have proven to be invaluable in crop rotation, helping to control

weeds, disease and insects, as well as improving soil texture and fertility.

Lentil is a nutritious food legume. It is cultivated for its seed and mostly eaten

as dhal. Lentil has a relatively higher contents of protein, carbohydrates and

calories compared to other legumes and is the most desired crop because of its

high average protein content and fast cooking characteristic in many lentil

producing regions (Muehlbauer etal, 1985). Seeds can be fried and seasoned

for consumption, flour is used to make soups and mixed with cereals to make

bread and cakes, (Williams and Singh, 1988). Even though lentil is considered

to be highly nutritious, it contains antinutritional factors such as, trypsin

inhibitors, hemagglutinins, and oligosaccharides that cause flatulence. These

problems can be greatly reduced by heating and germination (Jambunathan

etal., 1994). Husks, dried leaves, stems, fruit walls and bran (residues), can be

fed to livestock. According to Muehlbauer etal, (1985), when production of

forage crops fall below the level required in the market, lentil residue

commands an equal or a better price than lentil seeds in some Middle Eastern

countries. Green plants make valuable green manure. Seeds are a source of

commercial starch for textile and printing industries (Kay, 1979).

1.1. Quality parameter

Protein concentration of lentils reportedly range from 22-34.6% and

100 g of dried seeds contain 340-346 gm calories, 12% moisture, 0.6 g fat,

65.0 g total carbohydrate, about 4 g fibre, 2.1 g ash, 68 mg Ca, 325 mg P, 7.0

mg Fe, 29 mg Na, 780 mg K, 0.46 mg thiamine, 0.33 mg riboflavin, 1.3 mg

niacin (Adsule etal, 1989; Muehlbauer etal, 1985). Among the cool season

legume crops, lentil is the richest in the important amino acids (lysine,

arginine, leucine and sulphur containing amino acids) (Williams etal, 1994).

The starch content ranges from 35-53% in the seed and 42% in dry matter

while amylase varies from 20.7 to 38.5% of the seed starch (Huisman and

Vanderpoel, 1944; Hulse, 1994). Lentils are a good source of B vitamins,

containing per 100 g: 0.26 mg thiamine, 0.21 mg riboflavin, 1.7 mg nicotinic

acid, 2.23 mg choline, 107 mg folic acid, 130 mg inositol, 1.6 mg

panthothenic acid, 13.2 mg biotin, and 0.49 mg pyridoxine. Dry lentil husks

contain 11.1% protein (1.3 digestible), 0.7% fat, 47.5% carbohydrate, 25.6%

fibre, and 3.1% ash" (Dul<e, 1981). About 90% of lentil protein is found in the

cotyledons with albumins and globulins being the major fractions.

1.2. Biosystematics and cytogenetics

The genus Lens from the tribe vicieae is comparatively small and

comprises five annual species of which only L. culinaris Medik is cultivated.

This name published in 1787 has validity over the earlier common name Lens

esculenta (Gupta and Sharma, 1991). There is existence of two complexes

within the cultivated lentils, the small seeded {Lens culinaris Medik var.

microspermd) and large seeded (Lens culinaris Medik var. macrosperma).

The microsperma are small round seeds, 2-6 mm in diameter, yellow

or orange cotyledons and testa colour ranging from pale yellow to black.

Microsperma types are predominantly grown in India, Nepal, Bangladesh,

Pakistan, Afghanistan, Ethiopea and Egypt.

The macrosperma have large seeds, 6-9 mm in diameter which are

more flattened, yellow cotyledons, and pale-green testa. The macrosperma

types are largely grown in Spain, Italy, Greece and North and Latin America.

Although lentils cultivated in South Asia are exclusively small seeded

belonging to the microsperma type, bold seeded varieties are also grown in

India and their area is concentrated mostly in Madhya Pradesh and Jhansi

division of Uttar Pradesh.

In India, over 20 varieties of lentil have been released for cultivation in

different agro-ecological zones. Most of the varieties are small seeded. In the

recent years, however, a few bold seeded varieties have been developed.

Some of the varieties have resistance/tolerance to rust and wilt.

The chromosome number in the genus Lens is 2n =14. The

chromosome size and small chromosome number makes the lentil a suitable

material for cytogenetic study.

1.3. Botany

Lentil is an annual herb, erect in growth, much branched with slender

stem; leaves are alternate, compound, pirmate, usually ending in a tendril or

bristly; leaflets alternate or opposite, stipules small and linear. Flowers are

small, pale blue or purple, 1-4 flowers in axillary racemes. Pods oblong,

flattened or compressed, smooth containing 1-2 seeds; seeds lens shaped,

greenish-brown or light-red. Germination is hypogeal and this keeps the

developing seedlings blow ground level which reduces the effects of freezing

and other desiccating environmental condition (Muehlbauer etal, 1985).

1.4. Origin, area and cultivation

Lentil probably originated in the near East and rapidly spread to

Egypt, Central and Southern Europe, the Mediterranean basin, Ethiopea,

Afghanistan, India, Pakistan and China and later to the New world including

Latin America (Ladizinsky, 1979; Cubero, 1981; Duke, 1981). It is probably

the oldest of grain legume to be domesticated (Bahl etal., 1993). It is now

cultivated in most subtropical and also in the Northern hemisphere such as

Canada and Pacific North West regions. In India, the introduction of lentil is

linked with the movement of Aryans.

Lentil is widely cultivated globally in regions ranging from temperate

to high-altitude tropical. Cultivated lentils are descended from the wild lentil,

Lens culinaris subsp. orientalis. The wild progenitor is distributed throughout

the Near East, including Turkey, Isreal, Afghanistan and adjacent countries.

Lentils spread very rapidly from their area of early cultivation in

Western Asia to India and Europe. They were later introduced to the New

world by the Spanish and Portuguese to tropical Africa, Central and South

America.

In India, lentil is mainly cultivated in Uttar Pradesh, Madhya Pradesh

and Bihar and to a small extent in West Bengal, Rajhasthan, Haryana, Punjab

and Assam. Lentil is a rabi crop. It is sown during November-December and

harvested during February-March.

L5. Production

Regarding the production of this legume crop, it is considered that

lentil does not contribute much in the world's total production of pulses as

production of other pulses including dry edible beans and field beans is much

higher than this crop. The country that dominate the largest lentil producer's

list is Canada followed by India having a mammoth share in the world

production of around 40 lakh tonns annually. In India, lentil is grown on an

area of about 14 lakh hectares with an annual output of about 8-11 lakh tonns.

This production figure has been almost stable during the last decade.

1.6. Induced variability

Lentils continue to retain some less desirable characteristics such as

bushy and spreading growth habit associated in many cases with excessive

vegetative growth. These characteristics stand the crop well under stress

conditions. Indeed, the presently available varieties of lentils have

incorporated through thousand of years of natural selection, which make them

adapted to conditions, where virtually nothing else will grow. Hence,

considerable proportion of alleles for higher productivity have been lost from

the present population of lentils in view of the overriding role of natural

selection in directing the evolution of the species, even after its domestication.

For this reason, it has been difficult to develop high yielding varieties using

the limited germplasm available. Thus, the creation of lost genetic variability

in lentils is perhaps the most urgent task.

The concept of 'mutation' was first developed by Hugo de Vries

(1901) and its significance was subsequently demonstrated by Mullar (1927)

in Drosophila, Stadler (1928) in barley and maize and Goodspeed (1929) in

Datura and Nicotiana. The discovery of chemical mutagen during the second

world war was another milestone in the history of induced mutations. Though,

there are a few earlier reports, but Auerbach (1943), Oehlkera (1943) and

Rapoport (1946) were the first to demonstrate the mutagens is much greater

than physical mutagens and is continually increasing. The mutagenic activity

of nitroso compound was discovered by Rapoport in Drosophila.

Ethylmethane sulphonate (EMS) has been powerful alkylating agent, used to

induce genetic variability in a number of crop plants. Among the chemical

mutagens, sodium azide (SA) has also been recently demonstrated to be a

potent mutagen (Nilan etal, 1973 and 1976; Kleinhofs etal, 1974 and 1978).

As recombination breeding in lentil is tedious and pollination is

difficult due to its tiny flower size, the approach of mutation breeding is more

advisable in this crop. Induced mutagenesis may be of special use in

broadening the genetic variability of quantitatively inherited characters.

Therefore, planned mutagenesis can create more variability and with efficient

selection procedure, it is possible to get a desirable shift in the mean values of

the characters under consideration. Against this background, the present study

has been carried out to:

(i) study the biological damage caused by mutagens in Mi generation

(ii) study the frequency of morphological variations

(iii) study the spectrum of induced variability and the shift in means for

quantitative traits as compared to the control in M] generation.

Chapter - 2

REVIEW OF LITERATURE

Previous workers have reported significant changes in the desirable

characters in crop plant by using gamma rays as a physical mutagen, which

has been used to develop 64% of the radiation-induced mutant varieties

followed by X-rays (22%). Isolation of mutants of agronomic and economic

significance was a major goal of mutation breeding. Consequently, the high

yielding varieties of rice (RD6, RD15, PNR-102, PNR-381, Kashmir

Bashmati, NIAB-IRJRJ-9) were derived from mutation, while several dwarf

mutants were also induced following chemical and physical mutagenesis.

Similarly in wheat, a light coloured grain mutant 'Sarbati Sonora' was

obtained from 'Sonora 64' whilst a similar light grain colour mutant 'Pusa

Lerma' was derived from another popular line 'Lerma Rojo 64A'. 'Pusa

Lerma' with its high resistance to stem rust and semi hard white grains was

also released for cultivation in peninsular India. Thus mutation breeding acts

as an effective alternate method to conventional breeding.

There is existence of two complexes within the cultivated lentils; the

small-seeded {Lens culinaris Medik var. microsperma) and large-seeded

{Lens culinaris Medik var. macrosperma). Macrosperma and microsperma

genotypes are adapted to short days and are grown during winter in Indian

sub-continent. The spread of the crop into India was accompanied by strong

selection pressure towards increase sensitivity to photoperiod (Malik and

Eriskine, 1994).

The performance of introduced macrosperma genotypes is poor in

India due to photosensitivity. The length of the day in the lentil growing

areas in India decreases to about 11 hours by the time of flowering. Hence,

these short days have limited the utilization of macrosperma lentils as

primary or secondary introduction (Erskine and Hawtin, 1983). The solution

to the successful cultivation and use of macrosperma lentils lies in the

induction of early flowering mutants, which can flower and mature in about

4-5 months. The induced mutagenesis can help in creating genetic variability,

which has been lost due to natural selection.

Inspite of its importance in daily diet, little attention has been paid to

improve the genetic potential of lentil. As the exploitation of exotic variability

through recombination breeding is tedious and pollination is difficult due to

tiny flower, mutation breeding is more advisable for this crop. A few, but

rewarding attempts on experimental mutagenesis with lentils have been made

for the genetic improvement of this crop. The workdone and the experience

gained on induced mutagenesis with lentils have been reviewed.

2.1. Mutagen sensitivity

Sharma (1977) undertook mutagenesis in microsperma and

macrosperma genotypes of lentils with 6, 10, 14, 18, and 22 kR doses of

gamma-rays and 0.005, 0.01, 0.02, 0.03 and 0.04% N-nitroso-N-methyl urea

(NMU). The increasing doses of mutagens caused a progressive increase in

the biological damage measured in terms of reduction in germination, root

and shoot length, plant survival and pollen sterility in Mi generation. The

retardation in root length was more pronounced than that of shoot. The root

system appeared to be relatively more sensitive to mutagens.

Based upon Mi biological parameters, it was confirmed that

macrosperma lentils are more sensitive to mutagens than microsperma (Sinha

and Godward, 1972). It is probably that during evolution an increase in seed

size has been associated with a reduction in seed tolerance to mutagens. A

parallelism was observed between radio-sensitivity and chemo-sensitivity of

the varietal groups and their genotypes. LD50 of varietal groups and their

genotypes for plant survival were found to be 10-14 kR gamma-rays and

0.01-0.02% NMU in microsperma group and 6-10 kR gamma-rays and 0.005-

0.01% NMU in macrosperma group. Hence, these doses of mutagens can be

recommended for mutation studies in different varietal groups of lentils. The

chlorophyll mutation rate increased with an increase in the dose of mutagens

up to a certain level, beyond which it decreased or remained constant (Sharma

and Sharma, 1985). The phenomenon has been attributed to the saturation in

mutational events and vigour of both diplontic and haplontic selection. The

chlorophyll mutation frequency induced with mutagens was 1.5-2 times

higher in the mutagenically sensitive macrosperma group as compared to less

sensitive microsperma group. There was a positive relationship between radio

and chemo-sensitivity of the genotypes and their mutability. The results also

revealed the existence of a parallelism between radio-mutability and chemo-

mutability.

10

Mutagenic sensitivity in Mi generation was determined on the basis of

percentage of germination, chromosomal aberrations, survival, pollen and

ovule sterility and effect on growth and yield parameters.The germination

percentage was reduced in all the mutagenic treatments in comparison to the

control in the var. T-36 but it was promoted in var. the K-333 and K-75 while

var. the K-303 and K-85 showed differing trend in different mutagenic

treatments. Plant height, pollen fertility, maturity, survival percentage,

seeds/fruit in the var. T-36 were adversely affected in all the treatments,

however, simultaneous application of the two mutagens did not show a

synergestic response. Gamma rays and NMU differed with regard to their

effect on branching and number of fruits in the var. T-36 but seed yield and

test weight was promoted by both mutagens (Dixit and Dubey, 1981). In the

varieties K-333, K-75, K-303 and K-85 increase in days to flower, pollen

sterility and test weight was induced while number of branches, pods/plant,

seeds/plant and yield showed a decrease in all the mutagenic treatments.

Survival percentage, plant height and seeds/pod showed differing trends in

different mutagenic treatments (Tripathi and Dubey, 1990a and 1992a). But

when the var. K-85 was treated with three different concentrations each of

EMS (0.05%, 0.125% and 0.25%) and DES (0.25%, 0.50% and 0.75%),

germination and seedling growth was promoted by lower doses of mutagens

while higher doses had pronounced adverse effect. Survival, pollen fertility

and maturity were adversely affected by both the mutagens. Plant height.

branching, pods and seeds as well as yield/plant showed varying response to

different concentrations of mutagens (Vandana and Dubey, 1990a).

Seed treatment with mutagens chemical as well as physical is known to

produce adverse effects on germination, seeding growth and plant growth in

general. Delayed maturity, varying degree of sterility and reduced survival

percentage are the other common features of such treatments (Blixit et al,

1960; Sjodin, 1962; Nerkar, 1970; Sinha and Godward, 1972a, Sharma and

Sharma, 1986 and Sarkar and Sharma, 1989; Reddy et al, 1992; Khan, 2002;

Wani, 2003). However, there were few reports of promoting effect mutagens

of when applied at low doses (Sax, 1963; Singh et a/., 1978; Vandana and

Dubey, 1988 and Kumar and Dubey, 1994). Some of the earlier workers have

found bold seeded types to be more sensitive to mutagens than the small

seeded types (Sinha and Godward, 1972a; Sharma and Sharma, 1986 and

Kalia and Gupta, 1988).

Alterations in the form of chromosomal breakage and later their

reunions induced by physical and chemical mutagens have been of practical

interest. Differential sensitivity and variations in chromosome organization

can be studied through differences in both quality and kind of chromosomal

aberrations (Sinha and Godward, 1972b; Dixit and Dubey, 1983; Malaviya

and Shukla, 1990; Reddy et al, 1992). Mitotic anomalies in root tip cells

were directly correlated to the doses of gamma rays used in the var. T-36.

Chemical mutagen e.g., NMU induced a lower percentage of abnormal cells

in comparision to gamma rays. Combined treatments with gamma rays and

12

NMU showed a direct additive effect of the two mutagens (Dixit, 1985).

Among the three chemical mutagens i.e. EMS, DES and NMU, NMU induced

highest percentage of abnormal cells (Dixit and Dubey, 1984a). There was a

linear relationship between concentrations applied and anomalies induced by

the three chemical mutagens. This supports Sinha and Godward's (1972)

observation regarding linearity between mutagen doses and their effectiveness

in inducing mitotic anomalies in lentil.

In the var. K-333, mitotic anomalies increased with increasing doses of

almost all the mutagens applied (Tripathi, 1995). The main abnormalities

observed were clumping, fragmentation and unorientation of chromosomes,

bridges, laggards and unequal distribution at anaphase; gaint nuclei,

binucleate cells, micronuclei, polyploid cells.

Chromosome breakage and types of aberrations play a role in inducing

sterility which in turn influences the recovery of mutations. The percentage of

cells showing meiotic anomalies in the gamma ray treatments increased with

an increase in the irradiation dose in the var. T-36. However, the combined

treatments of gamma rays and NMU did not show a synergestic influence of

the two mutagens in inducing meiotic abnormalities (Dixit and Dubey,

1983a). Like the mitotic anomalies, NMU induced higher percentage of

abnormal cells than the EMS or DES (Dixit and Dubey, 1986a). In the var. K-

333, meiotic abnormalities increased with increasing dose of mutagens while

highest dose of gamma rays individually induced highest percentage of

anomalies (Tripathi, 1995).

13

2.2. Mutagenic effectiveness and efficiency

Effectiveness means the rate of mutation induction as dependent upon

the mutagenic dose and efficiency refers to the mutation rate in relation to

various biological effects, usually a measure of damage (Nilan et al., 1965).

The usefulness of a mutagen in plant breeding depends not only on its

mutagenic effectiveness, but also on its mutagenic efficiency. Two mutagens

may be equal in effectiveness because they induce the same frequency of

given mutation at a given dose. But, when they differ in their effect on gross

chromosome aberrations, sterility, lethality etc., they may be said to differ in

mutagenic efficiency. The effectiveness and efficiency of NMU and gamma-

rays were compared using microsperma genotypes as test systems (Sharma

and Sharma, 1979a). NMU was about three times more effective than gamma-

rays in all the varieties indicating that the mutation rate per unit dose is about

three times high in NMU than in gamma-rays. The efficiency of NMU was

1.5-2 times higher than that of gamma-rays. NMU can be a useful mutagen at

low concentrations. Both the mutagenic effectiveness and efficiency were

higher at lower dose of NMU in all the varieties. Inspite of a general decline

in mutagenic efficiency and effectiveness with increasing dose, the highest

mutation rates were recorded in the higher doses of gamma-rays as well as

NMU.

Frequency of mutations induced by various mutagenic treatments in

M2 generation appear to have a direct relationship with various mutagen

sensitivity parameters studied in Mi generation (Dixit, 1985 and Tripathi,

14

1995). Thus, the amount of damage produced by mutagens by way of reduced

germination, seedling growth, plant survival and increased mitotic and

meiotic anomalies and pollen and ovule sterility could be correlated with their

mutational efficiency.

The efficiency of mutagens for the var. T-36 in terms of ratio of

chlorophyll mutation frequency and injury or sterility was found to decrease

with the increasing doses of gamma rays. Efficiency of NMU treatment on the

basis of lethality was lower than that based on sterility. The mutagenic

efficiency of the combined treatments was generally lower than the individual

application of either mutagen (Dixit and Dubey, 1986c). In the varieties K-

333 and K-75, mutagenic efficiency of various treatments based on sterility

was lower than that based on injury in either variety. Most of the mutagenic

treatments proved to be more efficient in var. the K-75 than in the var. K-333

(Tripathi and Dubey, 1990b).

2.3. Guidelines for induced mutation techniques

Any proposal to use induced mutations in plant improvement must

consider the likelihood of success when compared with conventional

techniques and the efforts required to obtain the desired genotypes. The

likelihood of success can be considered in relation to the genetic control of

the character to be improved. A successful mutation breeding programme

must have clearly defined and specific objectives with regards to

improvements or mutant types sought. Such objectives could be (a) to

improve one or a few specific characteristics of a variety, (b) to induce

15

morphological marker to establish an identity in a promising line to make it

acceptable for variety registration and (c) to obtain within an adopted

genotype simply inherited mutations useful for breeding by recombination or

inducing further mutations.

2.4. Choice of mutagen

Today the plant breeder has at hand a number of effective physical

mutagens. The choice of the mutagen is not nearly related to its effectiveness

in terms of frequency of desired mutations, but to the kind of material to be

treated and to the availability of a mutagen. The frequency and the spectrum

of mutations differ somewhat depending on the mutagen used and the dose

applied. The physical mutagens, X-rays and gamma rays, are widely used.

They have the advantage of good penetration and precise dosimetry. Whilst,

Ultraviolet Light (UV) has low penetration power and effectively used with

materials such as pollen or in vitro cultured cells in a thin layer. Chemical

mutagens are known to produce a higher rate of gene mutations generally

preferred. However, chemical mutagens present particular problems such as

uncertain penetration to the relevant target cells, poor solubility and finally

the risk of safe handling (Khan, 1990). It would seem advisably to use several

mutagens for a spectrum of mutations as wide as possible.

A few members of alkane sulphonate series have been found to be

mutagenic in a variety of organisms. Freese (1963) and Heslot (1977) gave a

detailed account of EMS and dES. The mutagenic action of EMS was studied

earlier in Drosophila (Fahmy and Fahmy, 1957), bacteriophage (Loveless,

16

1959), Escherichia coli (Strauss, 1964), barley and wheat (Gustafsson, 1960;

Ehrenberg, 1960; Swaminathan et. al, 1962). Gaul (1964) in barley observed

that EMS was capable of producing more number of various morphological

mutants as compared to gamma rays. At molecular level, EMS is known to

react preferentially with guanine and cytosine (Freese, 1963). EMS alongwith

other alkylating agents is reported to induce substitution by two ways;

(i) by substituting a purine for purine or pyrimidine for a pyrimidine

(transition),

(ii) by substituting a purine for a pyrimidine or a pyrimidine for a purine

(trans version).

For causing transversion, EMS alkylates purine at 7* position (N )̂ and

finally leads to its separation from the DNA strand, i.e. depurination. A gap

created because of depurination may be filled up by any of the four bases.

Because of its ability to induce high frequency and wide spectrum of

mutations (Swaminathan et al, 1962; Hussein et. al., 1974; Khan et.al,

1998a; Wani and Khan, 2006), EMS is now being widely accepted as a

powerful mutagen and used commonly in the induction of mutations in

various crop plants.

There is a certain amount of evidence about the mutagenic action of

hydrazine in both, prokaryotes and eukaryotes. It was sometimes classified as

an inactivating agent with weak mutagenic activity (Fishbein etal, 1970) but

studies with bacterial species suggested that it can fairly be a potent mutagen

with little or no toxic effect (Kimball and Hirsch, 1975). A useful review of

17

the earlier work with special emphasis on the chemical basis for mutagenesis

of hydrazine was given by Brown etal. (1966). Hydrazine was reported to

induce a variety of morphological, physiological and colour mutants in

several crop plants such as barley (Shangin Berezovsky et al, 1973), maize

(Chandra Sekhar and Reddy, 1971), potato (Upadhya et al, 191 A), rice (Ratho

and Jachuck, 1971; Reddy and Reddy, 1973; Reddy et al, 1973) tomato (Jain

and Raut, 1966; Jain et al, 1969; Raut, 1969; Yakovleva, 1975) and chickpea

(Parveen, 2006). In general, hydrazine in these studies appeared to be as

successful as the other potent mutagens. However, it appeared to differ in two

ways:

(i) it produced a number of mutations detectable in Mj generation whereas the

other mutagens produced fewer or none.,

(ii) the spectrum of mutational changes (phenotypic classes) for hydrazine

was generally very different from that of other mutagens (Kimball, 1977).

Hydrazine has been reported to react with the pyrimidines in DNA to saturate

the 5,6 double bond, especially of thymine to form N'*-amino-cytosine and to

open up the pyrimidine ring with consequent loss of pyrimidines from DNA

or through intermediate radical reactions including the formation of hydrogen

peroxide depending upon the hydrazine derivatives involved (Kimball, 1977).

There are some unexpected features concerning time of detection and locus

specificity that are not yet explained. The mutations produced by hydrazine

seem to be mainly or entirely single-locus changes (Parveen, 2006).

2.5. Chlorophyll mutations

Mutations for chlorophyll defects have been mostly employed for

assessing the effectiveness of mutagenic treatments in lentils. The scoring of

such mutants is easy and fairly accurate (Singh et al, 1989; Reddy et al,

1993; Vandana et al, 1994; Wani and Khan, 2003).

There is an obvious difficulty in comparing radiations and chemical

mutagens because of the difference in the unit of dose and treatment methods.

However, at any biologically equivalent dose for instance LD50 for plant

survival or sterility the potency of radiations and chemicals with regard to

induction of mutations can be compared. Based upon this consideration, the

mutation rate with NMU was 7.5-2.0 times higher than with gamma-rays in

the microsperma and macrosperma varieties (Sharma and Sharma, 1981). The

general order of the relative frequency of different chlorophyll mutations can

be represented as xantha > viridis > chlorina > albina. The trend was not

altered due to the genotype of material or the mutagen used. The induction of

particular chlorophyll mutation with the same relative proportion in both

types of lentil varieties provides an excellent example of parallelism in

genetic variability in the two types of lentils as was first suggested by Vavilov

(1951). Uhlik (1971 and 1973) reported that viridis was more frequent in

macrosperma varieties followed by xantha-viridis, xantha and albina. Sharma

and Kant (1975) found that the relative proportion of xantha was the highest

in microsperma varieties followed by viridis and chlorina.

19

Mutation frequency showed a dose dependent increase in all the

varieties for various mutagens. Frequency of chlorophyll, seedling

morphology and vital mutations in terms of percentage of segregating M2

families as well as in terms of mutants/1000 M2 plants were higher in gamma

rays combined with NMU or EMS while most of the combined treatments

induced a lower frequency of vital mutations than the individual NMU

treatment (Dixit and Dubey, 1983b, 84b, 86b; Singh etaJ., ]989; Tripathi and

Dubey, 1991, 92b; Vandana etal., 1994).

2.6. Morphological mutations (Macromutations)

The mutagenic treatments induced mutations affecting plant height,

growth habit, branching and stem structure, leaf morphology, inflorescence,

calyx, flower, pod, fertility and seed colour have been reported in lentil (

Sinha et al, 1987; Tyagi and Gupta, 1991; Ashutosh and Dubey, 1992;

Vandana et al, 1994; Ramesh and Dhananjay, 1996; Tyagi and Ramesh,

1998; Solanki and Sharma, 1999; Jeena and Singh, 2000).

There were differences in the mutation spectrum between the two

mutagens (NMU and gamma rays) used (Sharma and Sharma, 1979).

Mutations like "fasciata", "compact", "semi-compact", "condensed

branches", "multiflorate", "ramification" etc., were isolated in NMU

treatments. The "waxless" and "pod" mutations were induced with gamma

rays only. The most important and useful types of mutants in lentils have been

characterized that were simply inherited and usually controlled by recessive

genes (Wilson et al, 1970; Singh, 1978; Sinha et al, 1987; Tyagi and Gupta,

20

1991). These mutants can be used in three general modes for direct release as

improved cultivars, for donor gene sources in standard cross breeding or

hybridization programme.

The isolation and use of induced or spontaneous mutations in crop

plants conferring an altogether new plant type and developmental rythm have

marked the beginning of a major advance in crop productivity (Swaminathan,

1969). Induced dwarf mutants have been used for direct cultivation in crop

plants. Dwarf types are rarely available in the lentil germplasm. Mutants

affecting plant height were classified in two categories viz., dwarf and semi-

dwarf depending upon the extent of height reduction (Sharma and Sharma,

1982a). Semi-dwarf mutant was superior to the parent variety for seed yield

potential. Mutants affecting plant height to different degree were classified as

dwarf, semi-dwarf and bushy dwarf (Dixit and Dubey, 1983; Vandana et ai,

1994).The fasciated stem was hollow from inside except at the nodes (Sharma

and Sharma, 1983). The branches were fused with the main stem at places of

emergence, instead of coming out an angle, which is normal in lentils. This

resulted in flattened stem structure and from the top, the plant appeared like a

cluster of closely fused branches giving a "bunchy top" appearance, carrying

many leaves and flower buds around the nodes of the fasciated stem. The

mutation was under the control of a single recessive gene fa. Induced fasciata

plants were also found to be chimeric in nature (Dixit and Dubey, 1983).

Gelin (1955) used X-rays induced fasciated pea mutant to develop a released

variety 'Stral pea'. The usefulness of fasciated pea mutants in breeding and

21

research has been reviewed (Gottschalk, 1977). Mutants affecting different

degree of compactness had reduced plant height, secondary branches,

pods/plant, pod size, seed size and seed yield/plant as compared to the parent

cultivar (Sharma, 1977; Gupta et al, 1983). However, these had higher

harvest index. Another compact mutant had reduced plant height, leaf size,

but comparable in primary and secondary branches than the parental strain

(Dixit and Dubey, 1986). The leaf variants induced in macrosperma lentils

were large, broad and condensed, narrow, obtuse and small (Sharma and

Sharma, 1981 b). Mutants affecting dimensions of leaves and leaflets such as

large, narrow and small leaf were obtained following simultaneous

application of gamma rays and NMU (Dixit and Dubey, 1986). In case of

large and narrow leaf, seed yield and its component traits were not altered.

Higher frequency of male sterility was induced in lentil (Sharma and Sharma,

1980; Vandana et al., 1994). Male sterile plants, although indistinguishable

from the fertile plants until the onset of maturity, remain green past unusual

maturity and had dark green thick leaves. Semi-male sterile mutants were

similar to the sterile mutants except the development of a few pods (Sharma,

1977). Multiflorate mutation was characterized by four, five or sometimes six

flowers on each peduncle (Dixit and Dubey, 1983). Fused, open and

malformed flower mutants were induced, which did not represent the

characteristic papilionaceous structure (Sharma, 1977). In case of fused and

open flower mutants, the pollen grains were sterile and hence unable to

produced seeds. The induction of mutations for seed coat colour would

22

increase the variability for this character, which is a prerequisite for the

improvement of this crop. Moreover, seed colour in lentil is an important

character having great bearing on the marketability of the produce (Sharma

and Sharma, 1978). The induced seed coat colour alterations did not show any

change in the morphological characteristics such as height, leaf flower and

pod.

2.7. Quantitative traits (Micromutations)

The mutational changes which can be isolated and fixed only through

the adoption of biometrical procedure in a group of plants are called as

micromutations (Gaul, 1961; Swaminathan, 1964). Micromutations can first

be detected in M2 generation, but certainly about the genetic nature of the

selected variants can only be established in later generations (Sindhu and

Slinkard, 1983; Sinha and Chowdhary, 1984; Dixit and Dubey, 1986; Sarkar

and Sharma, 1988; Kalia and Gupta, 1989; Rajput and Sarwar, 1989; Swarup

et ai,1991; Ashutosh and Dubey, 1992; Khan et al, 1999; Solanki, 1999;

Verma et al, 1999; Jeena and Singh, 2000; Solanki and Singh, 2000; Khan

and Wani, 2006; Singh et al, 2006). Micromutations are useful in plant

breeding for two main reasons, (i) they might occur much more frequently

than macromutations. (ii) they often do not affect vitality adversely as do

macromutations, because the minute changes having physiological behaviour

are less drastic.

23

2.7.1. Effects of mutagen on character association

Undesirable linkages involving yield components are known to exist in

crops (Webb et ai, 1968). The correlation between two characters in an

ordinary population is the composite of the effect of selection, gene linkage

and pleiotropy (Sakai and Suzuki, 1964). If the nature of selection practised in

the treated and the control population is the same, any difference in the

correlation coefficients between control and treated population will be due to

the effect of mutagens on gene linkage and altered pleiotropic effect of the

newly mutated genes. Sharma and Sharma (1981 d) examined the utility of

mutagens in altering the association between the traits in lentils. The

association between days to flowering on one side and pods/plant and seed

yield on the other were negative in the control, which increased further

negatively in the mutagenised population. The association of days to

flowering and branches/plant was significantly positive in the untreated

population, which decreased following mutagenesis with gamma rays. The

association of branches/plant with pods/plant and pods/plant with seed

yield/plant were positive, which enhanced further following mutagenesis. Any

character which was either negatively or positively correlated, or not

correlated at all with any other character in the control population, was found

to show alterations in the nature and strength of correlations. These cases of

alterations in the relationships appear to be owing to the effect of mutagens in

breaking or strengthening the linkage of genes. Hence, the mutagenic

treatments could alter the mode of relationships between any two characters.

24

apart from generating variability. Tiie increase in the magnitude of correlation

among the traits may be used to enhance the rate of selection response in a

primary trait (Khan, 1990). The quantum of induced micromutational

variability in lentil was dependent on the mutagens and the genotypes used in

the experiment. It was more distinct for the former than the latter. The

alkylating agents viz., NMU and ethyl methane sulphonate (EMS) were more

effective in inducing polygenic variability for economic traits in lentils than

that of gamma rays (Sharma, 1977 and Ravi et al, 1980). The lower

efficiency of gamma rays in comparison with alkylating agents with respect to

the induction of polygenic variability can be explained by the fact that

ionizing radiations induce a greater proportion of chromosomal aberrations,

whereas the alkylating agents induce changes mostly of point mutation type.

Sarkar and Sharma (1987) observed that NEU (N-nitroso-N-ethyl urea) and

EMS induced more variability for the flowering period, branching, peduncle

number, podding period, podding and seed yield than NaNs (sodium azide).

The lower potential of NaNs for inducing micromutations was due to its

variable non-adjusted high pH (6.5)

2.7.2. Induced polygenic variability for earliness and other economic

traits

The screening of plants with early flowering was undertaken in M2 and

M3 generations (Sharma, 1987). Observations recorded for days to flowering,

plant height, pods/plant and seed yield/plant. Range and mean values for days

to flowering and plant height increased and decreased, respectively, whereas

25

respectively, whereas they increased for pods/plant and seed yield/plant as

compared to the control in M3 generation. The early flowering plants selected

in different treatments of M2 generation were also found to be true breeding in

M3 generation. The induction of earliness was also evident from the shift in

the mean values towards earliness. The induction of earliness was found to be

associated with reduction in plant height. Partitioning of total polygenic

variation into heritable and non-heritable components for days to flowering

revealed substantial induced genetic variation (Sharma, 1987). The quantum

of induced genetic variability was not proportionate to the increase in the dose

of mutagens. Lack of linearity between mutagen dose and induced genetic

variability may be due to the saturation in the micromutational events at the

lower dose or other processes (Brock, 1970). Increase in the heritability and

the genetic advance is mainly due to the increase in the genetic component,

i.e. the induced genetic changes for the quantitative characters. The higher

estimates of genotypic coefficient of variation, heritability and genetic

advance (Khan and Wani, 2005) indicated that the variability may be fixed in

subsequent generations. From the plant breeding point of view, this should

mean a higher response to selection.

2.7.3. Induce early mutants

HPL-4, a tall, very late flowering and maturing with bold seeds was

treated with gamma rays, NMU, EMS and NaNs (Sharma, 1989). M,, M2 and

M3 generations were raised and selection of early flowering mutants and

families was undertaken in M2 and M3 generations. Ten eady flowering

26

mutants were finally established. These were numbered from EMpEMio.

These were induced by different mutagen treatments. The induction of early

flowering mutants was the highest in gamma rays treatments. Of early

flowering mutants, five were induced by gamma rays treatments, two each by

NMU and EMS and one by NaNs. All the mutants flowered and matured

earlier than HPL-4 (Sharma, 1989). These had reduced plant height and

primary branches and no mutant was superior to the control for total branches.

Early flowering mutants were crossed with L-406 and L-639, the cultivated

small seeded {microsperma) genotypes, to combine earliness, cold tolerance,

upright and taller growth habit in macrosperma genotypes (Sharma, 1989).

27

Chapter-3

MATERIALS AND METHODS

3.1 Materials

3.1.1. Varieties used

Two varieties of lentil {Lens culinaris Medik) namely, K-75 and L-4076

were used in the present study. Seeds of both the varieties K-75 and L-4076 were

obtained from Genetic Division of Indian Agriculture Research Institute, New

Delhi. Both the varieties are popular for cultivation in this region. A brief

description of both the varieties is given below:

3.1.1.1. Variety K-75

It is a microsperma variety. This variety matures in 135-140 days, foliage

green, semi-spread medium plant height (40-45 cm), flower violet, pod green in

early stage and brown at maturity, seed bold and mottled gray (2.7 g/100 seeds),

average yield is 10-16 q/ha.

3.1.1.2. Variety L-4076

It is a macrosperma variety. This variety matures in 135-140 days, plants

are dwarf, semi-spreading type, grains are bold (3.0 g/100 seeds) and pinkish in

colour. This variety is resistant to wih disease. It is suitable for growing under

irrigated as well as un-irrigated conditions, average yield is 14-16 q/ha.

28

3.1.2. Mutagens used

3.1.2.1. Methylmethane sulphonate (MMS), CH3OSO2CH3, monoftinctional

alkylating agent, is manufactured by Sissco Research Laboratories Pvt. Ltd.,

Mumbai.

3.1.2.2. Hydrazine hydrate (HZ), NH2-NH2-H2O, a base analogue, is

manufactured by Sigma Chemical Company, USA.

3.2. Experimental procedures

3.2.1. Preparation of mutagenic solutions

All solutions of chemical mutagens were prepared in phosphate buffer of

pH-7. Only freshly prepared solutions used for all the treatments.

3.2.2. Pretreatment

Healthy seeds of uniform size of each variety were used in the present

experiments. The seeds were soaked in distilled water for 9 hours prior to the

treatment with mutagens.

3.2.3. Mutagen Administration

Concentrations used: Four different concentrations viz., 0.01, 0.02, 0.03 and

0.04% of MMS and HZ were used for treating the presoaked seeds.

Treatment time; The treatments were given at temperature of 25±1°C for 6

hours.

Sample size: 273 seeds were used for each treatment.

Controls: For each variety 273 pre-soaked seeds were again soaked in phosphate

buffer for 6 hours to serve as controls.

29

3.3. Ml generation

Three replications of seventy-five seeds each, were sown for every

treatment in each variety in the field. The remaining lot of forty-eight seeds of

each treatment with their respective controls of both the varieties were spread

over moist cotton in petriplates, in order to determine percentage of seed

germination and seedling height i.e. root and shoot length. The petriplates were

kept in the B.O.D. incubator at 25±1°C temperature.

3.3.1. Observation recorded in Mi generation

Following parameters were studied in Mj generafion:

3.3.1.1. Seed germination: After recording germination counts, the percentage

of seed germination was calculated on the basis of total number of seeds sown in

petriplates. Seeds which gave rise to both radical and plumule were considered as

germinated.

^ • ..• /n/^ No. of seeds germinated ,„^ Germmation (%) = x IQO

Total no. of seeds sown

3.3.1.2. Seedling height

On the tenth day, the seedling height was estimated in centimeters by

measuring the root and shoot length of fifteen randomly selected seedlings for

each treatment. Seedling injury, as measured by the reduction in the root and

shoot length, was calculated in terms of percentage of root and shoot injury.

30

3.3.1.3. Plant survival

The surviving plants in different treatments were counted at the time of

maturity and the survival was computed as percentage of the germinated seeds.

3.3.1.4. Pollen fertility

Pollen fertility was estimated from fresh pollen samples. From mature

anthers, some amount of pollen was dusted on a slide containing a drop of 1 %

acetocarmine solution. Pollen grains, which took stain and had a regular outline

were considered as fertile, while empty and unstained ones as sterile.

The following formula was used to calculated the percentage inhibition or

injury or reduction:

Percentage inhibition

or

r, ^ . . Control - Treatment ,„„ Percentage mjury = x 100

Control or

Percentage reduction

3.3.2. Morphological variants

Some induced morphological variants affecting plant form, height and leaf

were isolated in Mi generation. Frequency of each variant was calculated in both

the varieties.

31

3.3.3. Quantitative traits

Observations were also made on 25-30 normal-looking plants in each

treatment and control.

The following eight quantitative traits were studied in Mi generation.

1. Plant height: Plant height was measured at maturity in centimeters from

the base up to the apex of the plant.

2. Days of flowering: Days to flowering were noted as the number of days

taken by the plant from the date of opening of the first flower bud.

3. Days to maturity: Number of days taken by the plant from the date of

sowing up to the date of harvesting of the plant.

4. Number of fertile branches: Number of fertile branches were counted at

maturity as the number for fertile branches which had more than one pods.

5. Number of pods: Number of pods were counted at maturity as the

number of pods borne on the whole plant.

6. Seeds per pod: Twenty best pods were threshed and number of seeds per

pod was counted. The mean was calculated for each plant.

7. 100 seed weight (g): It was the weight of a random sample of hundred

seeds from each plant.

8. Total plant yield: Plant yield was the weight of total number of seeds

harvested per plant and the yield of each plant was recorded in grams.

32

3.4. Cytological studies

For meiotic analysis, young flower buds from each treatment and controls

in both the varieties were fixed in Comoy's fluid (1 part glacial acetic acid : 3

parts chloroform ; 6 parts of ethyl alcohol) for 30 minutes. The material was then

transferred to propionic alcohol saturated with ferric acetate for 24 hours. The

flower buds were washed with and preserved in 70% alcohol. Anthers were

smeared in 1 % acetocarmine solution and pollen mother cells were examined for

their behaviour at various stages of microsporogenesis. Photographs were taken

from temporary preparations.

3.5. Statistical analysis

Assessment of variability

An insight into the magnitude of variability present in a crop species is of

utmost importance, as it provides the basis of effective selection. The variability

present in breeding population was assessed by using simple measures of

variability. Data collected for eight quantitative traits in Mi generation were

subjected to statistical analysis to find out range, mean, standard error, standard

deviation and coefficient of variability.

Range

It is the difference between the lowest and the highest values present in

the observations included in a sample.

33

Mean(X)

The mean is computed by taking sum of the number of values (Xi, X2, ....Xn)

and dividing by the total number of values involved, thus

(X, + X, + X3 X J x = N

or

IX„

N

Where, Xi, X2, X3 Xn = Observations

N = Total number of observations involved.

Standard error (S.E.)

It is the measures of the uncontrolled variation present in a sample. It is

estimated by dividing the estimate of standard deviation by the square root of the

sample and is denoted by S.E., thus

„ P _ S.D. of the sample

Where, S.D. = Standard deviation

N = Number of observations

Standard deviation (S.D.)

The standard deviation is calculated by the following formula for each

parameter of study.

34

cp^p-x.y+CX-x^)^ (x-x„y • V N

Where, X = Mean of observations involved

X], X2 Xn = Observations

N= Total number of observations

Coefficient of variability (C.V.)

It measures the relative magnitude of variation present in observations

relative to magnitude of their arithmetic mean. It is defined as the ratio of

standard deviation to the arithmetic mean expressed as percentage and is a unit

less number. The following formula is applied to compute coefficient of

variability (C.V.).

^ t r /n/\ Standard deviation ,„„ C.V. (%) = = X 100

X

or

^ . 1 0 0 X

Where, S.D. = Standard deviation of sample

X = Arithmetic mean

35

Chapter-4

EXPERIMENTAL RESULTS

Seed germination, plant survival, seedling growth and pollen fertility

are widely used as indices in determining biological effects of various

mutagens. Data on the effects of methylmethane sulphonate (MMS) and

hydrazine hydrate (HZ) on Mi parameters are described below:

4.1. Biological damage

4.1.1. Seed germination

Data recorded on seed germination are presented in Tables 1& 2; Fig.

1. A gradual and dose dependent reduction in seed germination with the

increasing mutagenic treatments was recorded in both the varieties (K-75 and

L-4076) of lentil. Both the varieties differed in the extent of reduction in seed

germination. In the var. K-75, germination was recorded to be 93.75 percent

in the control. The seed germination percentage was 25.00 and 18.75 with

0.04% MMS and 0.04% HZ, respectively. The highest percentage of

germination was recorded in the control of the var. L-4076. In the var. L-

4076, the germination percentage was ranging from 93.75 in 0.01% MMS to

31.25 % with 0.04% MMS, whereas it was 81.25 in 0.01% HZ to 18.75 at

0.04% of HZ treatments. HZ treatments were most effective in inhibiting seed

germination than MMS treatments in both the varieties. Variety K-75 was

found to be more sensitive than the var. L-4076.

36

4.1.2. Seedling height

Data recorded for seedling height of 10 days old seedlings, raised in

petriplates in B.O.D. incubator, are presented in Tables 3 & 4; Fig. 2. Results

showed that all the mutagenic treatments brought about reduction in seedling

height. The decrease concides with increase in the concentration of mutagen

in both the varieties. Seedling injury was more drastic at the highest

concentration of mutagens and it was (74.43 and 90.55 percent) in the var. K-

75, whereas in the var. L-4076, seedling injury was (85.08 and 89.23 percent)

with MMS and HZ treatments, respectively. The reduction in seedling height

was more pronounced in HZ than MMS treatments. Variety L-4076 was

found to be more sensitive than the var. K-75.

4.1.3. Pollen fertility

Although some two percent of pollen sterility was also observed in

control plants of both the varieties, but pollen fertility decreased and gave a

dose dependent relationship in Table 9; Fig. 3. Pollen sterility induced by

MMS was maximum than HZ treatments in both the varieties. The fertility

was lowest (81.48 and 77.50 percent) at 0.04% MMS in the varieties K-75

and L-4076, respectively. At 0.04% HZ treatment, the pollen sterility was

84.27 percent in the var. K-75 and 82.82 percent in the var. L-4076. Variety

L-4076 was found to be more sensitive than the var. K-75 based on the

reduction in pollen fertility.

37

4.1.4. Plant survival

Data on plant survival in Mi generation recorded at maturity in the

field are presented in Table 9; Fig. 4. Percentage of plant survival was

decreased gradually in all mutagenic treatments. However, it was dose

independent. The highest plant survival was observed in the control

population of both the varieties K-75 and L-4076.

4.2. ANOVA of seed germination and seedling height

Both the parental varieties (K-75 and L-4076) differed significantly

(p<0.05) between themselves for both percentage seed germination and

seedling height for MMS treatments (Tables 5 & 7). However, no significant

difference was found between the varieties for HZ treatments (Tables 6 & 8).

Variances among treatments (four doses of mutagen + one control) were also

significantly high.

4.3. Meiotic abnormalities induced by mutagens

Plate-Ill shows the detailes of the cytological effects observed

following treatment with MMS and HZ. In control plants, meiosis was

normal. In the treated plants, the normal meiosis was disturbed as it was

characterized by the presence of sticky chromosomes at metaphase-I,

chromatin bridges at anaphase-I, laggards at telophase-I and irregular

micronuclei at telophase-II. The laggards and chromatin bridges were the

common abnormalities noticed with all the treatments of mutagens. The

number of lagging chromosomes was variable. The chromatin bridges at

38

anaphase-I which might or might not remain persistent up to telophase-I,

were noticed less frequently. Maximum abnormalities were noticed in MMS

than the HZ treatments in both the varieties of lentil. For both the mutagens,

the frequency was positively correlated with the concentration.

4.4. Morphological variations

Inhancement of the frequency and spectrum of mutations in a predictable

manner and thereby achieving desired plant characteristics is an important

goal of mutation research. Different types of morphological variants affecting

different plant parts included changes for plant height (tall and dwarf), growth

habit (bushy and prostrate) and foliage (narrow, broad leaves and

chlorovariants) were isolated in the two varieties (K-75 and L-4076) of lentil

in MI generation (Table 10; Plate I & II). Frequency of morphological

variations was higher in the MMS treatments than those of HZ (Table 11). In

the var. L-4076, frequency was 6.97 percent as compared to 6.33 percent in

the var. K-75 (Table 10). No morphological variants was obtained in the

control (untreated) population. The characteristics of each variant are as

follows:

1. Dwarf: Dwarf variant was only about half the height of control. Its

growth was very slow from early seedling stage to maturity with

significant reduction in number of pods/plant and seed yield. The pod

and seed size was smaller. It was late in flowering and maturity

compared to the control.

39

Table 1: Percent seed germination in the two varieties of lentil treated with MMS.

Variety

K-75

L-4076

Treatment

Control

0.01% MMS

0.02% MMS

0.03% MMS

0.04% MMS Total

Control

0.01% MMS

0.02% MMS

0.03% MMS

0.04% MMS Total

TOTAL

Seed germination

R-I

15

14

12

9

4 54

15

15

13

10

5 58

112

R-II

16

13

13

8

3 53

15

16

14

9

4 58

111

R-III

14

15

11

10

5 55

15

14

12

11

6 58

113

Total

45

42

36

27

12 162

45

45

39

30

15 174

336

Mean

15

14

12

9

4 -

15

15

13

10

5 -

Seed germination

%

93.75

87.50

75.00

56.25

25.00 -

93.75

93.75

81.25

62.50

31.25 -

%age inhibition

-

6.66

20.00

40.00

73.33 -

-

0.00

13.33

33.33

66.66 -

V= 2 varieties; t= 5 treatments; r= 3 replications

Table 2: Percent seed germination in the two varieties of lentil treated with HZ.

Variety

K-75

L-4076

Treatment

Control

0.01% HZ

0.02% HZ

0.03% HZ

0.04% HZ Total

Control

0.01% HZ

0.02% HZ

0.03% HZ

0.04% HZ Total

TOTAL

Seed germination

R-I

15

13

11

8

3 50

15

13

10

7

3 48

98

R-II

14

14

10

7

2 47

15

12

9

8

2 46

93

R-III

16

12

12

9

4 53

15

14

11

6

4 50

103

Total

45

39

33

24

9 150

45

39

30

21

9 144

294

Mean

15

13

11

8

3 -

15

13

10

7

3 -

Seed germination

%

93.75

81.25

68.75

50.00

18.75 -

93.75

81.25

62.50

43.75

18.75 -

~

%age inhibition

-

13.33

26.66

46.66

80.00 -

13.33

33.33

53.33

80.00 -

*

V= 2 varieties; t= 5 treatments; r= 3 replications

Table 3: Seedling height in the two varieties of lentil treated with MMS.

Variety

K-75

L-4076

Treatment

Control

0.01% MMS

0.02% MMS

0.03% MMS

0.04% MMS Total

Control

0.01% MMS

0.02% MMS

0.03% MMS

0.04% MMS Total

TOTAL

Seedling height (cm)

R-I

10.56

13.66

10.60

7.23

4.33 46.38

11.33

10.16

7.26

7.66

5.08 41.49

87.87

R-II

11.66

12.66

11.20

6.51

4.20 46.23

12.02

11.96

6.00

5.24

0.00 35.22

81.45

R-III

11.12

13.01

11.66

7.12

0.00 42.91

10.66

12.48

5.76

5.01

0.00 33.91

76.82

Total

33.34

39.33

33.46

20.86

8.53 135.52

34.01

34.60

19.02

17.91

5.08 110.62

246.14

Mean

11.11

13.11

11.15

6.95

2.84 -

11.33

11.53

6.34

5.97

1.69 -

-

Seedling height

(%age injury)

-

+18.00

+00.36

-37.44

-74.43 -

-

+1.76

-44.04

-47.31

-85.08 -

-

V= 2 varieties; t= 5 treatments; r= 3 replications

Table 4: Seedling height in the two varieties of lentil treated with HZ.

Variety

K-75

L-4076

Treatment

Control

0.01% HZ

0.02% HZ

0.03% HZ

0.04% HZ Total

Control

0.01% HZ

0.02% HZ

0.03% HZ

0.04% HZ Total

TOTAL

Seedling height (cm)

R-I

10.56

9.56

7.85

5.23

3.16 36.36

11.33

10.66

7.02

4.13

3.66 36.80

73.16

R-II

11.66

9.12

8.23

4.66

0.00 33.67

12.02

11.02

6.13

4.02

0.00 33.19

66.86

R-III

11.12

8.16

8.66

4.12

0.00 32.06

10.66

10.12

6.66

0.00

0.00 27.44

59.50

Total

33.34

26.84

24.74

14.01

3.16 102.09

34.01

31.80

19.81

8.15

3.66 97.43

199.52

Mean

11.11

8.94

8.24

4.67

1.05 -

11.33

10.60

6.60

2.71

1.22 -

•

Seedling height (%age injury)

-

-19.53

-25.83

-57.96

-90.55 -

-

-6.44

-41.75

-76.08

-89.23 -

-

V= 2 varieties; t= 5 treatments; r= 3 replications

Table 5: ANOVA for seed germination (for MMS treatments).

Source of

variation

Total Replication Variety(A) Treatment(B) Interaction(AxB) Error

d.f.

30-1=29 3-1=2 2-1=1 5-1=4 1x4=4

18

S.S.

472.80 0.20 4.80

448.80 1.20

17.80

M.S.

-

-

4.80 112.20 0.30 0.98

F

-

-

4.89* 114.48**

0.31

Tabular F

F 0.05

4.41 2.93 2.93

Fo.oi

8.28 4.58 . 4.58

*, * * significant at p <0.05 and < 0.01 respectively.

Table 6: ANOVA for seed germination (for HZ treatments).

Source of variation

Total Replication Variety(A) Treatment(B) Interaction(AxB) Error

d.f

30-1=29 3-1=2 2-1=1 5-1=4 1x4=4

18

S.S.

55.70 5.20 1.40

536.00 1.60

12.80

M.S.

1.40 134.00 0.40 0.71

F

1.97 188.73**

0.56

Tabular F

F 0.05

4.41 2.93 2.93

F 0.01

8.28 4.58 4.58

* * * significant at p <0.05 and < 0.01 respectively.

Table 7: ANOVA for seedling height (for MMS treatments).

Source of variation

Total Replication Variety(A) Treatment(B) Interaction(AxB) Error

d.f.

30-1=29 3-1=2 2-1=1 5-1=4 1x4=4

18

S.S.,

470.58 6.16

20.67 387.86 21.31 34.56

M.S.

20.67 96.96 5.32 1.92

F

10.76** 50.50**

2.77

Tabular F

F 0.05

4.41 2.93 2.93

F 0.01

8.28 4.58 4.58

* * * significant at p <0.05 and < 0.01 respectively.

Table 8: ANOVA for seedling height (for HZ treatments).

Source of variation

Total Replication Variety(A) Treatment(B) Interaction(AxB) Error

d.f.

30-1=29 3-1=2 2-1=1 5-1=4 1x4=4

18

S.S.

467.41 9.34 0.72

422.54 13.27 21.54

M.S.

0.72 105.63 3.31 1.19

F

0.60 88.76**

2.78

Tabular F

Fo.05

4.41 2.93 2.93

Fo.oi

8.28 4.58 4.58

* * * significant at p <0.05 and < 0.01 respectively.

Table 9: Effect of mutagens on plant survival and pollen fertility in the two varieties of lentil.

Treatment Plant survival at matiirity Pollen fertility

% %age reduction

Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ

Control

Variety K-75

87.00

48.00 60.00 65.00 71.00

69.00 72.00 70.00 69.00

Varietv L-4076

89.00

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ

47.00 50.00 59.00 63.00

67.00 69.00 58.00 32.00

98.71

94.17 91.43 89.52 81.48

96.19 93.59 90.42 84.27

97.86

4.54 7.28 9.19 17.23

2.52 5.12 8.29 14.44

92.24 89.60 84.23 77.50

94.16 91.43 87.95 82.82

5.62 8.26 13.63 20.36

3.70 6.43 9.91 15.04

Table 10; Frequency and spectrum of morphological variants induced by mutagens in lentil (Lens cuUnaris Medik) varieties K-75 and L-4076.

Variants

Dwarf

Tall

Prostrate

Bushy

Number observed

K-75

6

7

1

6

in

L-4076

6

8

3

7

Broad leaves

Narrow leaves

Chlorovariants

Total number of morphological variants

Total number of Mi plants

Frequency (%)

8

6

4

38

600

6.33

9

7

5

45

645

6.97

Table 11: Frequency of morphological variants in various mutagens in Mi.

Mutagen

MMS

HZ

Number of M] Plants studied

642

603

Number of variants scored

49

34

Frequency (%)

7.63

5.64

d

100 K-75

L-4076

Control 0.01 0.02 0.03

MMS (%)

0.04

S

I K-75

I L-4076

Control 0.01 0.02 0.03

HZ (%)

0.04

Fig.l: Effect of mutagens on seed germination in Mi generation in the two varieties of Lens culinaris Medik.

14

? 1 2

S 10

5 6

(0 4

5 2

lK-75

lL-4076

1. Control 0.01 0.02 0.03

MMS (%)

0.04

• K-75

• L-4076

Control 0.01 0.02 0.03 0.04

HZ(%)

Fig.2: Effect of mutagens on mean seedling height (cm) in Ml generation in the two varieties of Lens culinaris Medik.

120

100

I so i 60

^ 40 o

OL 20

0 Jl_

R l l

Control 0.01 0.02

MMS C;

0.03 0.04

lK-75

lL-4076

100

95

"T 90 J

1 85

^ 80 i O I Q.

75

nih Control 0.01 0.02

HZ(%)

0.03 0.04

lK-75

lL-4076

Fig.3: Effect of mutagens on pollen fertility in Mi generation in the two varieties of Lens culinaris Medik.

B « E

>

3 W

e a.

100

90

80

70

60

50

40

30

20

10

0

lK-75

lL-4076

Control 0.01 0.02

MMS (%)

0.03 O.M

Fig.4: Effect of mutagens on plant survival at maturity (%) in Mj generation in the two varieties of Lens cuUnaris Medik.

2. Tall: Plant, characterized by longer intemodes and rachis length in the

leaves, was induced by the lowest concentrations of MMS and HZ. The

plant was late maturing with a high degree of pollen sterility and very

low seed set.

3. Prostrate: Plant was spread and occupied more area due to its spread

habit, plant possessed small pods containing shriveled seeds.

4. Bushy: Bushy plant had dense growth with compactly arranged

branches, showed a high degree of secondary branches giving a bushy

appearance to the plant. It was normal in flowering and maturity. It had

large number of pods/plant and pod size compared with the control.

5. Leaf variants: The broad leaves plant, characterized by the increase in

the size of leaflets, was isolated from the lower concentrations of both

the mutagens. Narrow leaves plant, was more frequent at the higher

concentrations of mutagen in both the varieties.

6. Chlorovariants: Chlorophyll variations, chlorina and viridis, were

induced at low frequency at the higher concentrations of mutagens.

They seem to appear in higher number in MMS treatments than HZ.

Such plants died at seedling stage.

4.5. Quantitative traits

Statistical analysis was done to find out mean, standard error, range, shift

in X and coefficient of variation for eight quantitative traits in the two

varieties of lentil. Means for all the quantitative traits shifted in both positive

40

as well as in negative directions. Though increase in coefficient of variation

of the mutagen treated population was of low magnitude, yet it differed from

trait to trait. The highest increase in CV over the control was recorded for

fertile branches per plant, pods per plant and total plant yield in both the

varieties in lentil. Observation regarded on various quantitative traits in two

varieties (K-75 and L-4076) of lentil in Mi generation are presented below:

(i) Plant height: Most of the mutagenic treatments significantly reduced plant

height in the var. K-75 (Table 12). However, plant height was significantly

reduced only at the higher concentrations of mutagens (MMS and HZ) in the

var. L-4076 (Table 20).

(ii) Days to flowering: Two varieties differed in their response to mutagenic

treatments with regard to days to flowering. In the var. K-75, all the

mutagenic treatments increased the days to flowering (Table 13). However,

the increase, except 0.04% MMS and HZ, was nonsignificant. Days to

flowering was slightly reduced by both the mutagens in the var. L-4076

(Table 21).

(iii) Days to maturity: In both the varieties of lentil significant early maturity

was induced by the highest concentrations of mutagens (Tables 14 & 22).

(iv) Number of fertile branches: A significant increase in number of fertile

branches was induced by all, except the highest concentrations, the mutagenic

treatments in both the varieties (Tables 15 & 23). As compared to the var. K-

75, the mean number of fertile branches were recorded higher in the var. L-

41

4076.

(v) Number of pods: Most treatments brought about a significant increase in

number of pods in the var. L-4076 (Table 24). On the other hand, in the var.

K-75, the number of pods showed progressive reduction with the increasing

concentration of MMS and HZ (Table 16).

(vi) Seeds per pod: In both the varieties, no significant difference in number

of seeds per pod was recorded in any of the mutagenic treatments (Tables 17

&25).

(vii) 100 seed weight (g): Data recorded on 100 seed weight are presented in

Tables 18 & 26. As in the number of seeds per pod, most of the mutagenic

treatments showed no significant difference in 100 seed weight in both the

varieties.

(viii) Total plant yield (g): Most of the mutagenic treatments brought about a

significant increase in seed yield in the var. L-4076 (Table 27). However, in

the var. K-75, no significant difference in mean seed yield was noticed in

most of the mutagenic treatments (Table 19)..

42

Table 12: Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for plant height (cm) of lentil var. K-75.

Treatment

Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

MeaniS.E. 54.06±2.32

44.70±1.32 44.40±1.12 47.06±1.07 45.06±2.03

42.80±0.85 43.13dbl.35 40.46±1.29 33.13±2.49

Range 40-67

35-54 35-50 40-55 35-60

36-49 30-53 30-47 19-48

Shift in X -

-9.36 -9.66 -7.00 -9.00

4.66 6.21

-11.26 -10.93 -13.60 -20.93

4.30 5.68

CV (%) 16.61

11.43 9.72 8.81 17.42

7.68 12.12 12.40 28.92

Table 13: Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for days to flowering of lentil var. K-75.

Treatment Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

MeaniS.E. 59.20±0.22

59.26±0.25 59.53±0.19 59.73±0.23 60.13±0.30

59.33±0.18 59.40±0.16 59.53±0.21 60.60±0.27

Range 58-60

58-61 58-61 58-61 58-62

58-60 58-60 58-61 59-61

Shift in X -

+0.06 +0.33 +0.53 +0.93

0.68 0.91

+0.31 +0.20 +0.33 +1.40

0.61 0.81

CV (%) 1.45

1.62 1.24 1.47 1.96

1.21 1.06 1.39 1.73

Table 14: Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for days to maturity of lentil var. K-75.

Treatment Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

MeaniS.E. 69.20±0.19

70.00±0.22 67.26±0.18 67.00±0.22 67.13±0.19

69.40±0.16 70.06±0.20 67.33±0.19 67.86±0.16

Range 68-70

69-71 66-68 66-68 66-68

68-70 69-71 66-68 67-69

Shift in X -

+0.80 -1.94 -2.20 -2.07

0.57 0.76

+0.20 +0.86 -1.87 -1.34

0.52 0.71

CV (%) 1.11

1.20 1.04 1.25 1.10

0.91 1,13 1.06 0.93

Table 15: Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for fertile branches/plant of lentil var. K-75.

Treatment Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

MeaniS.E. 12.00±0.73

18.00±0.96 23.00±1.40 16.60±1.06 10.40±0.76

16.13±0.69 16.46±1.27 15.86±1.17 5.40±0.25

- — ' • - — 1 - . • _ . _ _

Range 8-16

11-25 15-34 10-25 10-18

12-20 10-25 10-25 4-7

Shift in X -

+6.00 +11.00 +4.60 -1.60

2.88 3.83

+4.13 +4.46 +3.86 -6.60

2.54 3.39

CV (%) 23.50

20.77 23.61 24.69 29.42

16.49 29.83 28.75 18.15

Table 16: Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for pods/plant of lentil var. K-75.

Treatment Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

MeaniS.E. 153.66± 11.77

190.66±17.09 201.66±13.25 133.00±16.87 67.33±3.41

169.00±21.71 102.33±22.68 74.33±8.85 64.73±4.19

Range 75-200

75-300 150-300 70-250 50-85

75-300 50-400 45-150 40-90

Shift in X -

+37.00 +48.00 -20.00 -86.33

37.98 50.52

+15.34 -51.33 -79.33 -88.93

44.14 58.71

CV (%) 29.65

34.70 25.43 49.11 19.62

49.69 85.76 46.11 25.05

Table 17: Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for number of seeds/pod of lentil var. K-75.

Treatment Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

Mean+S.E. 1.73+0.12

1.66+0.12 1.66+0.12 1.73+0.12 1.66+0.12

1.73+0.12 1.73+0.12 1.80+0.11 1.73+0.12

Range 1-2

1-2 1-2 1-2 1-2

1-2 1-2 1-2 1-2

Shift in X -

-0.07 -0.07 0.00 -0.07

0.35 0.46

0.00 0.00

+0.07 0.00

0.32 0.43

CV (%)

26.01

28.92 28.92 26.01 28.92

26.01 26.01 26.01 22.77

Table 18: Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for 100 seed weight (g) of lentil var. K-75.

Treatment Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

MeaniS.E. 2.68±0.05

2.67±0.04 2.66±0.05 2.69±0.04 2.64±0.03

2.63±0.04 2.69±0.04 2.67±0.04 2.68±0.04

Range 2.4-2.9

2.4-2.9 2.4-2.9 2.4-2.9 2.4-2.8

2.4-2.9 2.4-2.9 2.4-2.9 2.4-2.9

Shift in X -

-0.01 -0.02 +0.01 -0.04

0.12 0.16

-0.05 +0.01 -0.01 0.00

0.12 0.16

CV (%) 6.72

5.99 6.76 6.32 4.54

5.32 5.95 5.99 6.34

Table 19: Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for total plant yield (g) of lentil var. K-75.

Treatment

Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

MeaniS.E.

4.25±0.33

5.29±0.48 5.36±0.34 4.70+0.23 3.69+0.21

5.15+0.66 3.34+0.61 2.45+0.15 2.21+0.04

Range

2.40-5.90

2.40-8.80 3.80-8.70 3.80-6.40 2.60-4.60

2.30-8.70 2.10-11.60 2.00-3.90 2.00-2.50

Shift in X -

+1.04 +1.11 +0.45 -0.56

2.56 1.25

+0.90 -0.91 -1.80 -2.04

0.28 0.37

CV (%)

29.88

35.54 24.63 19.15 22.22

49.51 70.36 24.08 7.23

Table 20: Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for plant height (cm) of lentil var. L-4076.

Treatment Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

MeaniS.E. 50.86±2.30

49.46±1.47 47.40±3.07 48.33±1.32 42.86±1.59

53.10±1.63 44.60±1.61 36.33±1.15 23.33±0.81

Range 25-60

40-57 26-65 39-59 30-52

43-65 32-53 28-42 18-27

Shift in X -

-1.40 -3.46 -2.53 -8.00

5.82 7.74

+2.24 -6.26

-14.53 -27.53

3.94 5.26

CV (%) 17.52

11.50 25.10 10.53 14.39

11.88 13.99 12.27 13.42

Table 21: Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for days to flowering of lentil var. L-4076.

Treatment Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

MeaniS.E. 62.06±0.20

59.60±0.25 60.66±0.27 61.20±0.24 59.13±0.21

63.06±0.23 64.00±0.22 60.86±0.21 61.80±0.19

Range 61-63

58-61 59-62 60-63 58-60

62-64 63-65 60-62 61-63

Shift in X -

-2.46 -1.40 -0.86 -2.93

17.46 23.23

+1.00 +1.94 -1.20 -0.26

0.61 0.81

CV (%) 1.27

1.64 1.71 1.54 1.40

1.39 1.31 1.36 1.24

Table 22: Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for days to maturity of lentil var. L-4076.

Treatment Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

MeaniS.E. 76.86±0.24

77.93±0.23 75.73±0.20 74.00±0.19 74.93±0.20

77.13±0.21 78.26±0.20 75.06±0.20 75.93±0.20

Range 76-78

77-79 75-77 73-75 74-76

76-77 77-79 74-76 75-77

Shift in X -

+1.07 -1.13 -2.86 -1.93

0.60 0.80

+0.27 +1.40 -1.80 -0.93

0.56 0.77

CV (%) 1.18

1.13 1.04 1.01 1.05

1.07 1.01 1.05 1.04

Table 23: Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for fertile branches/plant of lentil var. L-4076.

Treatment Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

MeaniS.E. 10.06±0.50

20.80±0.91 16.46±1.24 15.66±0.68 8.66±0.57

17.00±0.57 14.00±0.38 11.33±0.56 7.06+0.71

Range 7-14

15-27 8-23 10-20 5-14

12-20 12-16 8-15 4-15

Shift in X -

+10.74 +6.40 +5.60 -2.00

2.32 3.10

+6.94 +3.94 +1.27 -3.00

1.56 2.08

CV (%)

19.28

17.02 29.10 16.79 25.63

12.94 10.43 19.33 38.67

Table 24: Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for pods/plant of lentil var. L-4076.

Treatment

Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

MeaniS.E.

76.33±4.06

438.66±23.37 250±22.57

229.33±18.02 174.66±13.02

373.33±26.23 184.80±23.10 101.86±11.33 48.86±5.74

Range

45-100

200-560 120-450 80-350 100-300

200-500 95-400 40-175 24-100

Shift in X -

+362.33 +173.67 +153.00 +98.33

50.02 66.53

+297.00 +108.47 +25.53 -27.47

47.28 62.89

CV (%) 20.59

20.62 34.94 30.41 28.85

27.19 48.37 43.03 45.47

Table 25: Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for number of seeds/pod of lentil var. L-4076.

Treatment

Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

MeaniS.E. 1.73±0.12

1.80±0.11 1.80±0.11 1.73±0.1l 1.80+0.11

1.80+0.11 1.73+0.12 1.66+0.12 1.73+0.12

Range 1-2

1-2 1-2 1-2 1-2

1-2 1-2 1-2 1-2

Shift in X -

+0.07 +0.07 0.00

+0.07

0.30 0.42

0.00 -0.07 -0.14 -0.07

0.33 0.43

CV (%)

26.01

22.77 22.77 26.01 22.77

22.77 26.01 28.91 26.01

Table 26: Estimates of mean values (X), range, shift in X and coefficient of variation (CV) for 100 seed weight (g) of lentil var. L-4076.

Treatment Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

MeaniS.E. 2.71±0.04

2.68±0.04 2.67±0.04 2.63±0.04 2.68±0.04

2.69±0.04 2.67±0.04 2.68±0.04 2.61±0.04

Range 2.40-2.90

2.40-2.90 2.40-2.90 2.40-2.80 2.40-2.90

2.50-2.90 2.40-2.90 2.40-2.90 2.40-2.90

Shift in X -

-0.03 -0.04 -0.08 -0.03

0.11 0.15

-0.02 -0.04 -0.03 -0.10

0.10 0.14

CV (%) 5.54

6.34 5.99 5.32 5.22

5.95 6.37 6.34 5.75

Table 27: Estimates of mean values (X ), range, shift in X and coefficient of variation (CV) for total plant yield (g) of lentil var. L-4076.

Treatment Control

0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS L.S.D. (p<0.05) (p<0.01)

0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ L.S.D. (p<0.05) (p<0.01)

i

MeaniS.E. 2.43±0.07

12.38±0.62 7.12±0.68 5.91±0.44 4.41±0.31

10.28±0.79 5.27±0.67 3.11±0.20 2.16±0.07

Range 2.00-2.90

10.70-16.70 3.80-12.50 2.30-8.40 2.90-7.20

6.20-14.50 2.40-8.70 2.10-4.20 1.80-2.50

Shift in X -

+9.95 +4.69 +3.48 +1.98

1.34 1.80

+7.85 +2.84 +0.68 -0.27

1.32 1.75

CV (%) 11.52

19.38 37.36 28.59 27.01

29.76 49.72 25.08 12.96

1

Plate-I: Morphological variants isolated in Mj generation.

Fig. 1: Control plant.

Fig.2: Tall variant.

Fig.3: Dwarf variant.

Fig.4: Bushy variant with profused branching.

PLATE!

Plate-II: Morphological and foliage variants isolated in Mi generation.

Fig. 1: Prostrate variant.

Fig.2: Chlorovariant showing yellow leaf colour.

Fig.3: Plant showing broad leaves.

Fig.4: Plant showing narrow leaves.

Fig.5: Seeds of control plant and high yielding variant.

PLATE-II

V

Plate III. Various meiotic stages in lentil.

Fig. 1: Metaphase-I (Control).

Fig.2: Metaphase-I showing stickiness of the chromosomes.

Fig.3: Anaphase-I (Control).

Fig.4: Anaphase-I showing 'anaphase bridge'.

Fig.5: Telophase-I (Control).

Fig. 6 & 7: Telophase-I showing lagging chromosomes.

Fig.8: Telophase-II (Control).

Fig.9: Three micronuclei.

PLATE-III

ft % J. T i

J f̂

^

4 - > • » « • * n i *

#

i

Chapter-5 |

DISCUSSION

The discovery that ionizing radiation and chemical mutagens could cause

genetic changes in an organism and modify linkages offered promise in the

improvement of crop plants. Mutation breeding involves the use of induced

beneficial changes for practical plant breeding purpose both directly as well as

indirectly. Mutation breeding can be used to complement and supplement

existing germplasm resources (Konzak etal, 1975). Mutagenic treatment can

give rise to i} new alleles of known genes and alleles of previously unknown

genes, ii} create useful genetic variability in phenotype, and iii} modify linkages

and retain desirable complexes. Induced mutations are now widely used for

introducing genetic changes, creation of new genetic resources and breakage of

linkages. Although the occurrence of mutations is a random process and the

probability of getting desirable mutation is very low, yet it can be increased if

done under correctly estimated conditions.

The sensitivity of any biological system to a particular mutagenic

treatment depends on various factors such as: (1) properties of biological system,

(2) physical and chemical properties of mutagen, (3) concentration of mutagen,

(4) duration of treatment, (5) temperature during treatment, (6) hydrogen ion

concentration, and (7) pre and post treatment conditions.

43

The sensitivity of lentil varieties to various mutagenic treatments was

assessed by studying the biological damage induced in Mi, in terms of seed

germination, seedling growth, plant survival at maturity, pollen fertility,

frequencies of morphological variations and certain quantitative traits in Mi

generation. In the present study, reduction in seed germination was dose

dependent and linear. Similar observations were made in Vigna radiata (Khan

and Ali, 1987; Khan etal, 1998 a,b; Khan and Wani, 2004, 2005; Parveen,

2006). Inhibition in seed germination after irradiation has been attributed to

chromosome deletion and changes in biochemical and physiological system

(Sparrow and Woodwel, 1962). Decrease in germination was attributed to

inhibitory effect of mutagen (Sahai, 1974). Aman (1968) stated that endogenous

growth regulators play an important role in germination of seed and their exists a

balance between promotors and inhibitors in seed germination. Meherchandani

(1975) also concluded that reduction in germination may be due to disturbances

of balance of promoter and inhibitor of growth regulators, probably in favour of

inhibitory substances. The reduction was slightly more in HZ treatments than

MMS. Inhibition of germination following EMS treatments has been attributed to

the formation of the acids upon hydrolysis, which in turn reduce the pH of the

medium making it toxic (Froese-Gertzen etal, 1963). Delayed and reduced seed

germination caused by various mutagens in the present study may be as a result

of depression in the rate of mitotic proliferations or delay/ or the inhibition of

metabolic activation necessary for seed germination. The denatured DNA after

44

sometime may be repaired resulting in the activation of biological processes

involved in germination and thus delayed germination is observed (Hutterman

etal, 1978).

The growth of seedling were observed after ten days of germination. There

is a definite trend towards the decrease of seedling height with the increasing

concentrations of mutagens in both the varieties. Mutagens induced reduction in

seedling height and grov/th inhibition may be due to destruction or damage to

apical meristem (Patel and Shah, 1974), partial failure of the intemodes to

elongate, decrease in the number of proliferating cells (Van't Hof and Sparrow,

1965) and chromosomes structural damage in meristematic cells (Gray and

Scholes, 1951). Reduction in seedling growth might have arisen due to mitotic

delay or arrest promoted by chromosomal abnormalities which in turn is

proportional with the increase in concentration of mutagenic treatment.

Reduction in seedling growth was explained due to auxin destruction and

changes in ascorbic acid content (Usaf and Nair, 1974). The extent of decrease in

seedling height was not uniform in the two varieties of lentil studied. It may be

due to the uneven damage to the meristematic cells as a consequence of the

genetic injury. Reduced growth followed by mutagen treatments has also been

reported by Rehman etal. (2000) in urd bean, and Sinha and Singh (1987);

Sharma and Sharma (1983) and Wani and Khan (2003) in lentil.

Sterile pollen observed in both the control as well as in the mutagenic

population of both the varieties of lentil used in the present study. However, the

45

percentage sterility increased considerably in mutagen treatments. A depression

in pollen fertility was also reported in Vigna radiata (Khan and Hashim, 1978;

Ganguli and Bhaduri, 1980; Khan and Siddiqui, 1992; Wani, 2007) and Lens

culinaris (Sinha and Godward, 1972; Reddy and Annadurai, 1991; Wani and

Khan, 2003). The pollen sterility found in the mutagenic treatments could be

attributed to genetic and cytogenetical changes. Since in lentil the pollen sterility

is being used as a parameter for estimating translocation frequencies (Sinha,

1967), therefore, it is likely that increase in the cytological abnormalities

including the frequency of quadrivalents are responsible for increase sterility in

lentil. These results are in confirmity with earlier reports in Pisum (Kaloo and

Das, 1971) where it has been noted that increase in pollen sterility was associated

with the increase in frequency of interchanges in Vigna mungo (Kumar and

Gupta, 1978; Rehman, 2000), the pollen sterility was associated with asynapsis

and/ or desynapsis. In chickpea (Mehrajuddin, 2001), meiotic irregularities were

shown to be responsible for high pollen sterility. Maximum reduction in fertility

was observed in MMS treatments than HZ. A high frequency of pollen sterility in

EMS treated barley was attributed by Sato and Gaul (1967) to gene mutations.

The chromosomal aberration frequency following treatments with MMS and HZ

was found to be dose dependent. Earlier, dose dependent increase in meiotic

abnormalities were recorded in mung bean (Ignacimuthu and Babu, 1989; Azad,

1998), urdbean (Rehman, 2000), lentil (Sinha and Godward, 1972; Agrawal,

2007) and in faba bean (Fatma, 2007). Considerable number of abnormalities in

46

MMS and HZ treated population also revealed that they are capable of inducing

mutation for cytological characters. The sticky chromosomes following

mutagenic treatments had been reported in pea (Skorupska, 1975). Stickiness is

attributed to the alteration in chromosomal protein resulting in the change of

surface nucleoprotein configuration. The occurrence of single and double

chromatin bridges involving several chromosomes/chromatids breakage followed

by exchange is in agreement with similar observation in Lathyrus sativus and

Vicia sp. (Shaikh and Godward, 1972). Besides sticky chromosomes and bridges,

laggards were also noticed in the present study. The delayed terminalization and

sticky ends may lead to the formation of a laggard and chromatin bridges.

Battacharya (1974) attributed the formation of laggards to chromosomes spindle

interaction. The formation of chromatin bridges following mutagenic treatment

had been noticed by various workers in different crops (Rees and Thomson,

1955; Ghatnekar, 1964; Shaikh and Godward, 1972; Azad, 1998; Rehman, 2000;

Khan, 2002).

Plant survival at maturity, in both the varieties, decreased over the

control and it was dose dependent. These results are contrary to the earlier

findings of Raghuvanshi and Singh (1979) in chilli, Anwar and Reddy (1981) in

rice. Khan and Ali (1987) in mungbean and Mehrajuddin (2001) in chickpea,

who reported positive relationship between the dose of the mutagen and final

plant survival. The mutagens are capable of creating chromosomal damage

47

leading to mitotic arrest and have lethal effects on different stages of plant

growth.

Morphological variations affecting different plant parts were isolated on

screening of Mi population. Some of the morphological variations viz., mutation

affecting foliage and plant height appeared more frequently than growth habit.

Similar results have been reported earlier in lentil (Tripathi and Dubey, 1992;

Vandana etal., 1994; Ramesh and Dhananjay, 1996; Tyagi and Ramesh, 1998;

Solanki and Sharma, 1999; Khan etal., 2006), mungbean (Khan and Siddiqui,

1996), black gram (Rehman etal., 2001), Vicia faba (Sjodin, 1971) and chickpea

(Atta etal., 2003; Khan etal, 2004a, 2004b). MMS induced higher frequency of

morphological variations than HZ. The superiority of alkylating agents to induce

the highest frequency of morphological mutations has been demonstrated by

several workers in different crops (Vandana and Dubey, 1990; Khan and

Siddiqui, 1993; Wani and Khan, 2003; Wani and Khan, 2005). Dwarf plants

showed reduction not only in the number of intemodes but also in the intemode

length, resulting in significant decrease in plant height. Such plants also showed

marked reduction in yield and yield component. Though these plants may not be

useful for direct commercial cultivation because of reduced yield, it may be used

in hybridization programmes to transfer some of its useful traits to high yielding

cultivars of lentil, if breed true in next generations. Though it is not easy to

eliminate the negative traits of the pleiotropic spectrum from the positive ones,

the pleiotropic pattern of a mutant gene can be altered to some extent by

48

transferring it into specific genotypic background (Sidovora, 1981). Variety L-

4076 gave higher frequency of morphological variants than the var.K-75. This

reflects to differences in their mutagenic sensitivity.

A number of plants with variation in the pigmentation of leaves were

observed in treated population. Millar etal. (1984) attributed that the variants

with chlorophyll pigments resulted due to deformation or degeneration of stroma,

lamella which in turn arised from changes in the nature of gene controlling

chlorophyll development. In the present study, MMS produced higher number of

chlorophyll variants than HZ treatments, confirming that alkylating agent are

highly potent mutagens in producing chlorophyll chimeras. Reddy and Gupta

(1989) suggested that high frequency of chlorophyll mutation in EMS treatments

is perhaps due to preferential action of EMS on genes for chlorophyll

development located near the centromere. Freese (1963) suggested that EMS is

more specific to guanine and cytosine and there is a alkylation of the chloroplast

DNA.

Two lentil varieties differed in response to the mutagenic treatments

regarding quantitative traits viz., plant height, days to flowering, days to

maturity, number of fertile branches, number of pods, seeds per pod, 100 seed

weight (g) and total plant yield (g). Generally, reduced plant height, days to

flowering, days to maturity, seeds per pods and 100 seed weight (g) were

recorded in both the varieties. Similar finding have been reported earlier in

mutagenic treatments in various crops (Nerkar, 1970; Sinha and Godward, 1972;

49

Dixit and Dubey, 1981, 1986, 1987; Dixit 1985; Sharma and Sharma, 1979,

1980, 1986; Sinha and Chaudhary, 1987; Kalia and Gupta, 1989; Khan

etal.,1994; Khan and Wani, 2004; Wani and Khan, 2007). Reduction in mean

number of days to flowering in some of the mutagenic treatments indicates the

possibility of isolating early maturing type in later generations. Kaul (1980)

suggested that mutation of two dominant gene to their recessive forms makes for

an early flowering in peas. The shift in X and the nature of induce variability

and their relationship with increased concentrations of mutagens indicate

differential mutagenic sensitivity between the two lentil varieties. Borojevic

(1970) reported that the genetic differences even though very small (as single

gene difference) can induce significant changes in the mutagen sensitivity which

influence various plant characters in Mi generation. Present study confirms the

reports of some of the earlier workers that bold seeded types are more sensitive

to mutagens than the small seeded types (Sinha and Godward, 1972; Sharma and

Sharma, 1986; Kalia and Gupta, 1988).

50

Chapter-6

SUMMARY

The objective of the present study was to explore the possibihty of inducing

variabiUty for quantitative traits namely, plant height (cm), days to flowering,

days to maturity, number of fertile branches, number of pods, seeds per pod, 100-

seeds weight (g) and total plant yield (g) by using two chemical mutagens viz.,

methyl methane sulphonate (MMS) - an alkylating agent and hydrazine hydrate

(HZ)- a base analogue, in the two varieties (K-75 and L-4076) of lentil (Lens

culinaris). The other aspects of this study were:

(i) to study biological damage in Mi generation, and

(ii) to estimate frequency of morphological variations and its spectrum

A gradual and dose dependent reduction in Mj biological parameters, except

plant survival at maturity, was recorded in both the varieties of lentil. When a

critical assessment was made for the average of biological damage done by the

mutagenic treatments in the two varieties, greater biological damage was

observed in the var. K-75 than the var. L-4076. Based on inhibition in seed

germination and seedling injury, HZ was found to be more effective as compared

to MMS, whereas pollen sterility induced by MMS was maximum than HZ

treatments in both the varieties. Both the chemical mutagens induced

chromosomal aberrations. The maximum abnormalities was noticed with MMS

than the HZ treatments in both the varieties of lentil.

51

Morphological variations affecting foliage, plant height and growth habit

were isolated on screening of M] population. If breed true in next generations,

few variants may be used in hybridization programmes to transfer some of their

useful traits to high yielding cultivars of lentil.

Two lentil varieties differ in response to the mutagenic treatments

regarding quantitative traits. Mean values for the quantitative traits shifted in

both positive as well as in the negative direction of the control (untreated) means.

Reduction in mean number of days to flowering in some of the mutagenic

treatments indicates the possibility of isolating early maturing type in later

generations.

52

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