faster ^ijtJLasopijg · to my father, Mr Mohd Qadeer who is extra-curious and interested in my...

STUDIES ON THE EFFECTS OF EMS ALONE AND IN COMBINATION WITH DIMETHYL SULFOXIDE IN THE

INDUCTION OF VARIABILITY IN Vicia faba L.

DISSERTATION

SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREM^ FOR THE AWARD OF THE DEGREE OF

f a s t e r ai ijtJLasopijg IN

BOTANY

RUBINA PERVEEN

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2008

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(M immm l%Ami^ IMi

Ik

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-maif: [email protected]

l t ' 7 i « o g

cEmiTicji^

This is to certify that the dissertation entitled "Studies on

the effects of EMS alone and in combination with

dimethyl sulfoxide in the induction of variability in Vicia

faba L." submitted by Miss Rubina Perveen 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 guidance and supervision.

,îC^ (Sammdafi %fian)

mailto:[email protected]

Ac^nowCecCgements

Tirst, I Bow in reverence to the ACmighty, the omnipotent, for it is indeed

his 6(essing atone which provided me enough zeaCto complete this wor^

I feeCmuch pleasure to express reverence and gratitude to my supervisor

(Dr. Samiuttah l{hanfor êping a watchfuCand discerning eye over the project wor^

providing vaCuaSCe guidance, and help in overcoming the hurdles throughout the course

of this wor^

I am highCy gratefid to (prof (Bahar .JA. Siddiqui Chairman, <T>epartment

of (Botany, Ji.MV. JlCigarh for providing the necessary faciCities, suggestion and ^nd

guidance to carrying out this project wor^

I own an expression ofthan^ to ^Dr 'Kpuser (parveen and (Dr. (Rafigji.

"Wani research scholars for their cooperation and encouragement.

IfeeC(Pleasure to ej^press my appreciation to j^Qia andSonu goyaC, my

Ca6 mates for their he^, suggestion and cooperation throughout the study.

My gratitude ^jiows no hounds when I thinôf the Cove, cooperation

and heCp extended 6y my caring and loving friends 9ôushim, iMustadeen, (Bushra,

(Renu, Jiasia, Irm, [Manilla, Jfotiey, Zeha.

"ôcaSulary faiCs to express than^ to myJLmma my sisters ^Hida and

!Kahid, my Brothers, TasCeem, Waseem, Jfammza, andSher-uz- Zama, whose Cove and

affection has aCways Been a constant source of inspiration for me. My heartCy thanks

to my father, Mr Mohd Qadeer who is extra-curious and interested in my emergence

with high potentiaC and efficiency.

CastCy my Cove, regards and Best wishes to aCC wed wishers (Jlmin)

(RiiSina (Perveen

CONTENTS

Contents Pages

Chapters-1

1. 1.1. 1.2. 1.3. 1.4. 1.5.

INTRODUCTION Botany Origin Uses Quality parameters Induced variability

Chapter-2

2. 2.1. 2.2. 2.3. 2.4. 2.4.1. 2.4.2.

2.5.

Chapter-3

3.

3.1. 3.1.1. 3.1.2. 3.2. 3.2.1. 3.2.2. 3.3 3.3.1 3.3.1.1. 3.3.1.2. 'J^JtX a*?*

3.3.1.4. 3.3.2. •J*Jm-3m

3.4.

REVIEW OF LITERATURE Mutation spectrum Achievements Chemical mutagens Alkylating agents Studies with higher plants Modification in the effect of alkylating Agents in combination with DMSO. Induced mutation in Viciafaba

MATERIALS AND METHODS

Material Varieties used Mutagens used Experimental procedure Pretreatment Mutagens administration Ml generation Observation recorded in Mi generation Seed germination Seedling height Plant survival Pollen fertility Morphological variants Quantitative traits Cytological studies

1-7 2 3 3 4 6

8-24 13 13 16 18 18

20 20

25-31

25 25 25 25 25 26 26 26 26 27 27 27 28 28 29

3.5. Statistical analysis 30 3.5.1. Assessment of variability 30 3.5.1.1. Mean 30 3.5.1.2. Standard error (S.E.) 30 3.5.1.3. Standard deviation (S.D.) 31 3.5.1.4. Coefficient of variability (C.V.) 31

Chapter-4

4. EXPERIMENTAL RESULTS 32-37

32 33 34 34 34 35 36 37

4.1. 4.2. 4.3. 4.4. 4.5. 4.6. 4.7. 4.8.

Chapter-5

Seed germination Pollen fertility Seedling height (cm) Plant survival ANOVA of seed germinations and seedling height Morphological variations Cytological abnormalities Quantitative traits

DISCUSSION

Chapter-6

SUMMARY

REFERENCES

38-43

44-45

46-67

List of Tables

Table 1: Effect of mutagens on seed germination, plant survival, pollen fertility and

seedling height in two varieties of Viciafaba L.

Table 2: Seed germination in two varieties of Viciafaba treated with EMS.

Table 3: Seed germination in two varieties of Viciafaba treated with EMS+DMSO.

Table 4: Seed germination in two varieties of Viciafaba treated with HZ.

Table 5: ANOVA for seed germination (for EMS treatment).

Table 6: ANOVA for seed germination (for EMS+DMSO treatment).

Table 7: ANOVA for seed germination (for HZ treatment).

Table 8: Seedling height in two varieties of Viciafaba treated with EMS.

Table 9: Seedling height in two varieties of Viciafaba treated with EMS+DMSO.

Table 10: Seedling height in two varieties of Viciafaba treated with HZ.

Table 11: ANOVA for seed germination (for EMS treatment).

Table 12: ANOVA for seedling height (for EMS+DMSO treatment).

Table 13: ANOVA for seedling height (for HZ treatment).

Table 14: Frequency and spectrum of morphological variants induced by mutagens

in faba bean {Viciafaba L.) varieties.

Table 15: Frequency of morphological variants in various mutagens in Mi generation.

Table 16: Estimates of mean values (X), shift in X and coefficient of variation (CV)

for plant height (cm) of Viciafaba var.05 /2491ocaI.


for days to flowering of Viciafaba var.05/2491ocal.

Table 18: Estimates of mean values (X), shift in X mean and coefficient of variation

(CV) for days to maturity of Viciafaba var.05/2491ocal.


for number of fertile branches/plant of Viciafaba var.05/2491ocal.

Table 20: Estimates of mean values (X), shift in X and cofficient of variation (CV) for

pods/plant (grain) of Viciafaba var.05/2491ocal.

Table21: Etimates of mean values (X), shift in X and coefficient of variation (CV) for

pod length (cm) Viciafaba var.05/2491ocal.

Table 22: Estimates of mean value (X), shift in X and coefficient of variation (CV) for

number of seed /pod of Viciafaba var. 05/249 local.


for 100 seed weight (g) of Viciafaba var. 05/249 local.

Table 24: Estimates of maen values (X), shift in X and coefficient of variation (CV)

for Yield/plant (g) of Vicia faba var. 05/249 local.

Table 25; Estimates of mean values (X), shift in X and coefficient of variation (CV)

for plant height (cm) of Vicia faba var.05/233 HBP.


for days to flowering of Vicia faba var.05/233HBP.


for seed for days to maturity of Vicia faba var. 05/233 HBP.


for number of fertile branches / plant of Vicia faba var.05/233 HBP.


for number of pod/plant (grain) of Vicia faba var.05/233 HBP.


for pod length (cm), of Vicia faba var.05/233 HBP.


for number of seeds/ pod of Vicia faba var.05/233 HBP.


for 100 seed weight (g) of Vicia faba var.05/ 233 HBP.


for yield/ plant (g) of Vicia faba var.05/233 HBP.

List of Figures

Fig. 1. Effect of mutagens on seed germination (%) in M| generation in the two varieties of Viciafaba.

Fig. 2. Effect of mutagens on seedling height (cm) in Mj generation in the two varieties of Viciafaba.

Fig. 3. Effect of mutagens on pollen fertility (%) in M] generation in the

two Varieties of Viciafaba.

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

List of Plates

Plate - 1 : Leaf variants isolated in Mi generation

Plate -II: Leaf and chlorovariants isolated in Mi generation.

Plate - III: Morphological variants isolated in Mi generation

Plate IV. Mitosis & meiosis in untreated and the mutagens treated faba bean.

ililiili

chapter 1

INTRODUCTION

Grain legumes, commonly known as pulses, play an important role in

world agriculture by virtue of their high protein content and capacity for fixing

atmospheric nitrogen. The amino acid composition of pulse protein is such that

cereals and pulses complement each other in order to provide a mixed and

balanced diet of high biological value to the human population. They are also

rich in carbohydrates and vitamins. They are very important for large portion of

the population in the developing countries who can hardly afford to consume

animal protein in adequate amounts because of the cost factor. The major

segment of population in India, being vegetarian, depends largely, for a large part

of their dietary protein on pulses. The production and productivity have remained

stagnant all through the last four decades and the growth in pulses production

could not keep pace with the demands of the increasing population.

The pulse crops can improve soil fertility by fixing atmospheric nitrogen

and increase organic matter of the soil by adding leaves and other plant parts. In

order to achieve higher production of pulse crops, a major emphasis has been

given on the development of high yielding genotypes in all major pulse crops

like chickpea, mungbean, urdbean, lentil and cowpea. However, one of the

strategies of pulse improvement programme is to achieve a higher level of pulse

production by the exploitation of unconventional pulse crop like faba bean.

ricebean, and Phaseolus vugaris.

1.1. Botany

Faba bean, also known as bakla in India, is an annual herb with coarse

upright stems, unbranched tall, with 1 or more hollow stems from the base (Bond

et al, 1985; Duke, 1981; Health, et al, 1994). The leaves are alternate, pinnate

and consist of 2-6 leaflets and unlike most other members of the genus, it is

without tendrils or with rudimentary tendrils (Kay, 1979; Bond et al., 1985).

Flowers are large, white or purple, borne on short pedicels in cluster of 1 -7 on

each auxiliary raceme; 1-5 pods develop from each flower cluster; stamen 10,

anther generally bilobed, intrude usually open longimdinally; monocarpellary,

unilocular ovary, marginal placentation. In most cases, the ovary has small,

ovules arranged in two alternating rows along the ventral structure, ovules

generally anatropous, style simple, ovary superior or half inferior; legume (pod)

dry dehiscent type of fruit, develop from monocarpellary ovary breaking through

both margins; non endospermic with two cotyledons, more or less round or oval.

Tap root with profusely branched secondary roots. Based on seed size, two

subspecies were recognized, paucijuga and faba. The latter was subdivided into

var. minor with small rounded seeds, var. equine with medium sized seeds and

var. major with large broad flat seeds (Kay, 1979; Bond et al, 1985). Cubero

(1974) suggested four subspecies, namely.- minor, equine, major, and paucijuga .

Faba bean belongs to the family Leguminasae (Fabaceae) and to the Genus Vicia

(Bond era/., 1985; Smart, 1990).

1.2. Origin

Faba bean assigned to the Central Asian, Mediterranean, and South

American centre of Diversity. Cubero (1974) postulated a Near Eastern centre of

origin, with four radii (1) to Europe (2) along the North African coast to spain,

(3) along the Nile to Ethiopia, and (4) from Mesopotamia to India. Secondary

centre of diversity are postulated in Afghanistan and Ethiopia. However,

Ladizinsky (1975) reported the origin to be Central Asia. The wild progenitor

and the exact origin of faba bean remain unknown. Several wild species

(V.narbonensis L. and V.galilaea Plitmann Zohary) are taxonomically closely

related to the cuhivated crop, but they contain 2n =14 chromosomes, whereas

cultivated faba bean has 2n = 12 chromosomes. Numerous attempts to cross the

wild species to cuhivated faba bean have failed (Bond et al., 1985).

1.3. Uses

Cuhivated faba bean is used as human food in developing countries. It

can be used as a vegetable either green or dried, fresh or canned. It is a common

breakfast food in the Middle East Mediterranean region, China and Ethiopia

(Bond et al., 1985). The most popular dishes of faba bean are Medamis (stewed

beans), Falafel (deep fried cotyledons paste with some vegetables and spices),

Bissara (cotyledons paste poured onto plates) and Nebet soup (boiled germinated

beans) (Jambunathan et al., 1994). Feeding value of faba bean is high, and is

considered in some areas to be superior to field peas or other legumes. It is one

of the most important winter crops for human consumption in the Middle East.

Faba bean has been considered as a meat extender or substitute and as a skim-

milk substitute. Sometimes grown for green manure, but more generally for stock

feed. Large seeded cultivars are used as vegetable. Roasted seeds are eaten like

peanuts in India (Duke, 1981). The straw can be used for brick making and as a

fuel in parts of Sudan and Ethiopia. In India, its cultivation is contained as a

minor crop in Himalayan hills, Bihar, Eastern U.P. and around cities and town

where its green pods are sold as vegetable and they fetch a good premium.

1.4. Quality parameter

Wide variation of protein content (20 - 41%) has been reported

(Chaven et al, 1989). Protein concentration is influenced by both genetic and

environmental factors and it has been reported that inheritance of this trait is

additive with some partial dominance (Bond et al, 1985). Amino acid content as

mg/g of nitrogen varies from 36-69 mg for methionine, 44- 94 mg for cystine and

333-400 mg for lysine (Chevan et al, 1989). Legumin is the predominant

globulin and has a larger proportion of arginine, threonine and tryptophan

(Hulse,1994). Faba bean contains small amount of several possible

antinutritional factors, however, their effect are less cute, and protease inhibitors

are much at lower concentrations compared to soybean (Lawes, 1980; Bond et

al., 1985). Inhalation of pollen or ingestion of the seeds may incite the condition

known as favism, a several hemolytic anemia, perhaps causing collapse (Smart,

1990). The main factors responsible for favism, which can occur in susceptible

people, are believed to be glucoside vicine and convicine and their hydrolytic

derivative divicine and isouramil, respectively. These anti-nutritional factors

render the red blood cells gucose-6-phosphate dehydrogenase deficient patients

vulnerable to oxidation and destruction (Bond et al, 1985; Hussein and Saleh.

1985 and Smart, 1990) which are uncommon in cooked beans (Lawes, 1980).

The whole dried seeds contain(per 100 g) 344 calories, 10.1% moisture, 1.3g fat,

59.4g total carbohydrates, 6.8g fiber, 3.0 g ash, 104 mg Ca, 301 mg P, 6.7 mg

Fe,8 mg Na, 1123 mg K, 130 jxg P-carotene equivalent,0.38 mg thiamine,0.24mg

riboflavin, 2.1 mg niacin, and 162 mg tryptophan. Flour contain: 340 calories,

12.4 % moisture, 25.5 g protein, 1.5 g fat, 58.8 g total carbohydrates, 1.5 g

fiber, 1.8 g ash, 66mg Ca, 354 mg P,6.3 mg Fe,0.42 mg thiamine, 0.28 mg

riboflavin, and 2.7 mg niacin. The amino acid content except for methionine is

reasonable well balanced (Bond et al, 1985).

Faba bean, is an often cross pollinated crop with a natural out crossing

percentage ranging from zero to 45 percent (Singh, 1984). The lack of adequate

pollination and reduced seed setting can be major constraints to yield. Flower

drop and seed abortion and pests such as Botrytis fabae, Ascochyta fabae,

Uromyces fabae, Orbanche crenata, and Aphis fabae are also major constraints

to yield. Faba bean is grown as a minor vegetable crop in India. Inspite of its

substantial production potential, no attention has been paid to its improvement

and to increasing the production of local strains in different parts of the country.

1.5. Induced variability

The possibility offered by mutagenic agents to induce new genetic

variation is, therefore, of extreme interest. It might in many cases be the only

answer to problems posed upon the practical breeder. A mutation event is

induced very important even when it has a small effect for a specific

morphological or physiological character, because it changes the balance

established by natural selection in co adapted blocks of genes and it, therefore,

offers new situation for natural and artificial selection.

Mutagenesis is a tool to increase variability in species in which

natural variation is not large or, as often happens, where phenotypes desired by

plant breeder are not available because they have disappeared due to their poor

competitive ability in natural condition (Ricciardi et al, 1982).

Dimetlily sulfoxide (DMSO)

DMSO (dimethyl sulfoxide), a by product of the wood industry, has

been in use as a commercial solvent since 1953. The introduction of DMSO in

clinical medicine as a carrier for drugs was accompanied by reports emphasizing

its quick penetrartion through biological membrane. DMSO is also reported to

show protective effect against freezing damage in R.B.C.'S (Love lock and

Bishop, 1959) and biological damage caused by X- irradiation in mice (Moos

and Kim, 1966). Several workers have also reported that DMSO acts as a useful

carrier for chemical mutagens in plants (Bhatia, 1967; Reddi, 1979; Singh and

Raghuvanshi, 1980).

There has been a number of attempt to assess mutagens induced genetic

variability in Viciafaba L. (El-Shouny and El- Hosary 1983;Filippetti and

Marzano, 1984; Vandana, 1992; Kumar e? a/, 1993; Yasin, 1996s).

In the present study, a breeding programme for Viciafaba L. varieties

05/249 local, 05/233 HBP using ethylmethane sulfonate (EMS) alone and in

combination with dimethyl sulfoxide (DMSO) and hydrazine hydrate (HZ) has

been under taken to induce genetic variability in the crop.

The objective of the study were:

1. to study the effect of chemical mutagens on such biological parameters as

seed germination , seedling height, plant survival, cytological

abnormalities and pollen fertility in Mi generation

2. to test effectiveness of chemical mutagens for the induction of quantitative

variability

3. to study the frequency and spectrum of morphological variations.

Chapter-2

REVIEW OF LITERATURE

Mutations have served as a vehicle of progress in evolution as well as

improvement of living organisms in terms of their economic utility (breeding).

Variability at the level of gene (DNA) can be created through mutations. Grossly

speaking, mutations are grouped into two major categories on the basis of their

phenotypic manifestation:

(i) Macromutations - with large change in the characters which can be detected

even without instrumental help at the level of individual organism (plant), and

(ii) Micromutations - with minor changes in the properties which are practically

unidentifiable in an individual plant but can be measured at the level of

population using various statistical parameters, such as, character mean,

variance, etc.

Macromutations, whether resulting from single-gene changes or

chromosomal aberrations, behave as monogenic traits and follow the Mendelian

pattern of inheritance. On the other hand, micromutations are governed by the

principles of quantitative genetics. Even since the early part of the history of

induced mutagenesis, it has been a well known fact that even "monogenic"

macromutations are invariably associated with multiple pleiotropic effects. Some

of which (e.g. chlorophyll deficiency, sterility, reduced productivity,etc.) make

them unsuitable for plant breeding. In contrast, micromutations, being subtle

changes in a large number of loci associated with determination of plant

morphology or physiology, have negligible "side effect". It has been generally

believed that such mutations for any economic trait could be accumulated in a

single genotype to great advantage.

In spite of these expectations, micromutations for polygenic traits have

not been of much consequence in plant breeding, whereas hundreds of plant

varieties have been evolved using macromutations directly or indirectly.

Compiled information on this aspect can be found in the issues of the Mutation

Breeding Newsletter published by the International Atomic Energy Agency

(IAEA), Vienna.

It is well known that a crop plant can be improved in productivity,

resistance to pest and adaptation to environment when genetic variability for the

specific trait is available in the considered population or species. The process of

breeding crop plants has been successful for a long time, because genetic

variation already present in the population had been used, and subsequently

further genetic variation was made available by crossing plants from different

populations, varieties, species and genera. In some cases, however, for instance

in bread wheat, the progress obtained for productivity has exploited the

variability present in nature to such large extent that only further progress from

the classical methods of breeding become more and more difficult (Natarajan et

al., 1985).

The possibility offered by mutagenic agents to induce new genetic

variation is, therefore, of extreme interest. It might in many cases be the only

answer to problems posed upon the practical breeder. A mutation event is indeed

very important even when it has a small effect for a specific morphological or

physiological character, because it changes the balance established by natural

selection in co- adapted blocks of genes and it, therefore, offers new situations

for natural or artificial selections.

Exposure of a biological material to a mutagen in order to induce mutation

is known as mutagenesis. When mutations are induced for crop improvement, the

entire operation of induction and isolation of mutants is termed as mutation

breeding. Various considerations like part of plant to be treated, mutagens, dose

of mutagens, methods of treatment, modifying factors, and methods of pre-and

post- treatments constitute what is precisely known as mutagenesis technique.

First observations about artificial induction of genetic changes date back

to the beginning of the 20 century (Gager, 1908), but proper proof of Mendelian

inheritance of such induce changes came only in the late twenties by Muller,

Stadler and others using X-rays as mutagens (Muller, 1927; Stadler, 1928a, b)

Although Muller, being an entomologist, assumed that induced mutations could

play an important role in further genetic improvement of plants, Stadler as a plant

breeder became rather special about such prospects when he noticed so many

useless and even deleterious mutations in maize and barley. Stadler's specticism

has influenced almost two generations of plant breeders, especially in North

10

America, and has led to a widely spread preconceived notion that mutation

induction will be of high interest to genetictists, but is a rather wasteful

undertaking for plant breeders. Stadler's view, primarily, was based upon this

experiments with maize, where a lot of genetic diversity exists (as in most cross-

pollinated crop), from which improved varieties could still easily be developed

simply through selection or a combination of cross breeding and selection.

Among the first researchers who used mutagenesis strictly for plant

breeding were Freisleben and Lein in Halle (Germany). They succeeded in

obtaining mildew resistance in barley (Freisleben and Lein, 1942) and developed

a practical mutation breeding procedure (Freisleben and Lein, 1943 a, b), but due

to World War II this work was not followed up properly (Hoffmann, 1959). In

the meantime, primarily in Sweden, plant geneticitst such as Nilsson Ehle.

Gustafsson, Hagberg, Gelin and Nybon continued to experiment mainly with X-

rays and carried out rather systematic studies as to optimal doses, treatment

conditions, mutation frequency and muatation spectra. They also compared X-

ray effects with those of certain chemicals which became known as mutagens,

such as (EI) ethylenimine (Micke et al, 1980). Although most of this work was

of a flindamental nature, there were by-products which turned out to be of

interest to breeder- easily recognizable mutants of barley, wheat, oats with early

or later heading, short straw or different spike architecture but also mutants of

pea, soybean, flax, mustard and rape (Gustafsson, 1947).

11

For more than 10 years, major research efforts went into the search for

radiation treatment conditions or additional treatments (before or after

inadiation) that could modify the random mutation induction into something

more specific, more directed, more economically useful (Nilan et al,

1965).Water, oxygen and time were the main factors discovered to be of

influence, but their deliberate control only brought about quantitative differences.

which could also be obtained from different doses, and did not really lead to any

useful methodological improvement (IAEA, 1961, 1965). Later on, developing

countries began to play an increasing role in mutation breeding work particularly

in Asia. New varieties of rice soon appeared on the market which derived some

valuable characteristics from mutation induction (IAEA, 1971; Sigurbjomsson

and Micke, 1974; Wang, 1986). In the beginning, mutation breeding was based

primarily upon X-rays but now mainly gamma rays and to a smaller extent fast or

thermal neutrons also started to be used.

In 1969, the joint FAO/IAEA Division started to organize course for plant

breeders on the induction and use of mutation, and in the same year published the

first edition of the Manual and Mutation Breeding. It may, therefore, be justified

to consider 1969 as the year that marked the establishment of mutation breeding

as a practical tool available to plant breeder in their endeavours to develop more

productive cultivars with better resistance to stresses, pathogens and pests, and

with improved quality characteristics for plant products used as food, feed or

industrial raw material.

12

2.1. Mutation spectrum

Known mutant collections, contain only selected mutants, mostly of easily

recongnizable type and. therefore, not fully representative of the potential

spectrum of induced mutations. A specific advantage of mutation induction,

however, is the possibility of obtaining unselected genetic variation, whereas all

other available germplasm has already passed screens of selection by nature or

man. The question whether induced mutations duplicate the genetics variation

produced by nature (AUard, 1960; Herskowitz, 1962) is rather theoretical since

both natural or man-made germplasm do not represent all the possible

spontaneous mutations or recombinants. When new breeding objective come up-

and this will be more often in the ftiture-it will be a matter of lucky chance if the

desired variant exists among stocks in germplasm collections or inhabitats of

high diversity. Spontaneous mutation rates, on the other hand, will not give much

new variation to breeders. There is sufficient evidence that induced mutations fit

Vavilov's law of homologous genetic variation (Scholz, and Lehmann, 1958;

Enken, 1967). It has logically been concluded that limitations of mutation

breeding are not in mutagenesis as such but rather in identification and selection

of desired variants (Gregory, 1956; IAEA, 1984a).

2.2. Achievements

An early record of an induced valuable mutant has been shown by Ramiah

and Rao (1953).They reported about 36 X-ray induced mutations affecting

different characters in rice. Of these one mutant proved useful from the economic

13

point of view. It had a slight shorter stature with a large number of tillers than the

original parent material and proved valuable in that it performed well in rich soil

where problem of lodging was serious. Looking at the progress of mutation

breeding, it seems that as far as cereals are concerned major emphasis has been

on obtaining mutant for improved disease resistance and improved grain quality

(protein), but main results were in improving lodging resistance (short or /and

stiffculm) (IAEA, 1984c; Maluszynski et al, 1986) and altering crop duration i.e.

photoperiod sensitivity (Awan et al, 1982; Gottschalk and wolff, 1983; Donini et

a/., 1984; Konza, 1984). Results in terms of improved grain protein were not

discouraging, but remained below the rather exaggerated expectations (Micke,

1983; IAEA, 1984b; Muller, 1984; Awan and Cheema, 1988). This on one hand,

is certainly due to the low heritability of quantitative endosperms characters and,

the inefficient selection. With regard to disease resistance, applied selection

procedures generally have been inadequate to a large extent because objectives

were poorly defined due to insufficient understanding of epidemiological

principles and host /parasite interaction. Neverthless, some results have been

rather spectacular (IAEA, 1977; 1983, Konza, 1984). On the other hand, it is

worth noting that more than 40 years after its discovery one has begun to

understand the nature of mutations in the famous ml-o locus of barley and the

reasons for the universal, non specific resistance rendered by a series of recessive

alleles in that locus (Jorgensen 1975,Sokou 1982). It is also the barley powdery-

mildew complex where first clear experimental proof was obtained as to the

14

possibility of improving quantitative resistance by monogenic mutations

(Robbelen, et al, 1977;Abdle-Hafez and Robbelen, 1979,1981, Aziz et ai,

1980).

Since most mutation breeding work was performed with annual and self-

pollinating cereals, most experiences relate to them. The problems in other

groups of crop plants, however, are quite different. For example, in grain

legumes, where breeding advances lag far behind the cereals, we have still a

relatively poor adaptation of the plant architecture to modem farming systems.

The plant architecture of course, being the uhimate result of numerous

physiological reactions and interactions, is therefore not likely to be inherited as

simply as the culm length in cereals (Micke, 1979,1984). on the other hand,

reports confirm that even with single monogenic mutation a remarkable r

reconstruction of plant architecture is achievable in grain legumes and in other

dicotyledonous plants, e.g. in chickpea (Shaikh et al., 1980), pigeon pea and

mungbean (Rao et al., 1975; Khan and Siddiqui,1996), pea (Jaranowski and

Micke, 1985), castor bean (Kulkami 1969), cotton (Raut et al., 1971;

swaminathan, 1972), linseed (George and Nayar, 1973; Nayar, 1974). Fast

development of computer technology enabled FAO/IAEA to organize the data

base in 1987. The information contained in this data base is based on data on

mutant cultivars published in various issues of Mutation Breeding Newsletter.

According to the latest information available there are 1239 accessions in the

FAO/IAEA Mutant Varieties Database (Maluszynski et al., 1995). These crop

15

varieties were developed either directly after mutagenic treatment or through

crosses involving mutant varieties or mutant lines. The cumulative number of

officially released mutant cultivars indicates that more than 50 percent of these

varieties were released during the period between 1980 -1995. Maximum

numbers of crop varieties 304 have been released in China followed by India

(243), the former USSR and the Russia Federation (209), the Netherlands (176),

Japan (115) and USA (93). Mutant cultivars of cereal dominate (828) followed

by legumes, oil crops, and industrial crops. In cereals mutation techniques were

most successfully applied for improving rice (322 mutant cultivars ) and barley

(240) followed by wheat, maize, durum wheat and other cereals such as oat,

millet, pearl etc. Application of mutation techniques for improving a particular

crop or group of crops has been the subject of review papers published by the

International Atomic Energy in Mutation Breeding Reviews (Hanna, 1982:

Jaranowski and Micke, 1985; Daskalov, 1986; Spiegel-Roy, 1990; Robbelen,

1990;Rutger, 1992; Micke era/., 1993, ScarasciaMugnozzae/a/., 1993).

2.3. Chemical mutagens

Seeds and buds may be treated either in the dormant state or in the

actively metabolizing stage. In the literature several methods for treating growing

plants and pollen have been described.

a) Soaking in the mutagenic solution of appropriate concentration for seeds,

buds and dormant cuttings.

b) A shallow cut is made in the plant stem and the mutagens applied through

a wad or wick of cotton saturated in the chemical agent. This method can

be used either for intact plant (Oehlker, 1943) or the developing intact

inflorescence (Bianchi et al, 1961).

c) A suitable amount of the mutagen may be injected in or near the organ to

be treated.

d) Although the roots are very sensitive, the mutagens in low concentration

may be applied to the growth medium and allowed to enter the plant

through the roots. The simple method offers the advantages of studying.

I. Choronic mutagen exposure, and

II. Sensitivity of different stages of growth and development to

chemical mutagens.

e) Pollen in monolayer may be exposed to the vapour of the mutagen in a

closed humid chamber (Mabuchi and Amason, 1969).

Although chemical mutagens have been rather disappointing as compared

with ionizing radiation in asexually propagated crops, it is well to compare

the advantages and disadvantages of the two methods.

a) At least in sexually propagated crops, chemical mutagenesis has yielded

very high chlorophyll mutation frequencies and in most instances it was

more efficient than ionizing radiation with regards to mutation quantity.

b) Chemical mutagenesis is very economical due to following reason: I) a

small amount of a suitable chemical mutagens II) normal laboratory glass

17

ware, and III) the use of a fume hood. On the other hand, when working

with ionizing radiation one must have access to an X-ray machine or a

more expensive gamma-rays source and must ascertain proper dosimetr\

of these machines. In fact, a 100 ml bottle of EMS or NMU can go along

way in a plant breeders, laboratory,

c) Since most chemicals are also carcinogenic agents, extreme caution must

be exercised in their use.

2.4. Alkylating agents

2.4.1. Studies witti higher plants

Alkylating agents (AA) are potent mutagens can be classified broadly into

monoflinctional and bi- or polyfunctional ones, depending upon the number of

alkyl groups present in the compound. The first chemical tested at the Indian

Agriculture Research Institute was nitrogen mustard, a bi-functional alkylating

agents (Bhaduri et al., 1953). However, systematic studies on different crop plant

using AAs were initiated by Swaminathan and his student in the late fifties

(Swaminathan et al, 1962).

From the pioneering studies of Ehrenberg and coworkers in Sweden

(Ehrenberg et al, 1960 and Ehrenberg et al, 1957). It was clear that AAs are

particularly suited for mutagenicity studies in plants. Thus, indepth studies

employing different AAs were started to correlate various biological effects, such

as killing, induction of chromosomal aberrations and mutations with their

chemical reaction patterns (Rao et al, 1965; Ramanna et al, 1966 and Natarajan

18

et al, 1966). The reactivity of AAs towards nucleophiles can be defined in terms

of reaction mechanism and the dependence of reaction rates on nucleophiiic

strength of receptor atoms (Swain et al, 1953; Osterman et al, 1970). An useful

expression of the reactivity of AAs is Swan-Scott substrate constant s, which is

the measure sensitivity of AAs to the strength of nucleophiiic substitution

reactions have been invoked. The reaction types are generally referred to as

unimolecular (SNl) and bimolecular (SN2). (Vogel and Natarajan, 1982). The

ability of various alkylalkane sulfonates (such as, methyl methanesulfonate

(MMS), ethyl methanesulfonate (EMS), isopropyl methane sulphonate (IPMS) to

alkylate various sites in DNA was found to vary in accordance with the

expectations based on s values (Swain et al, 1953).The most common adducts in

DNA alkylated in neutral solution was 7- alkylguanine (Lawley et al, 1975).

However, the proportional extent of reaction at the N-7 position varied according

to the s values, the high s value of the AA was correlated with high N-7

alkylation. Conversely, the alkylation of 0-6 alkyl guanine was higher for AAs

with low s values. The biological effects of different AAs were found to be

correlated with the s values of the AA employed. For example, in barley, AA

with high value s value (MMS) was found to be more cytotoxic and less

mutagenic in comparison to an AA with low s value (propyl

methanesulfonate,PMS) (Osterman et al, 1970). (Roa et al, 1965; Ramanna et

al, 1966; Natarajan et al, 1966). Studied extensively the frequencies of

chromosomes aberrations induced by different alkyl alkanesulfonates in both

19

mitotic and meiotic cells of barley. They found that AAs with low s values

(EMS, butyl methanesulfonate -BMS and PMS) were poor inducers of

chromosomal aberrations in comparison to those with high s values (MMS and

methyl ethanesulfonate (MES)).(Rao et al, 1965).Higher chromosomes breaking

ability of MMS in comparison to EMS was also found in studies employing root

tip cells of Viciafaba {Rao et al, 1967).

2.4.2. Modification in the effect of alkylating agents in combination with

DMSO

In chemical mutagenesis, secondary steps, other than the alkylation of DNA

perse, are more important in the final realization of the induced mutations. This

is all the more true for higher plants where multicellular systems are treated

invariably. An induce alterations at the DNA level has to pass through several

cellular sieves in competitions with unaffected cells (Keils, 1965; Auerbach,

1967). The fate of alkylated DNA thus depends upon the cellular processers that

follow. Mutation yield and efficiency of mutagenic treatments can be

considerably enhanced by manipulating the secondary factors (Kawai, 1969;

Narayan et al, 1969,). Several workers have reported that DMSO acts as an

useful carrier for chemical mutagens in plant (Bhatia, 1967; Gopal, 1977; Reddy

andReddy, 1979; Singh and Raghuvanshi, 1980).

2.5. Induced mutation in Viciafaba

Seeds of field bean or faba bean (Viciafaba L. > are an important source

of protein in the diet of many people in countries like China, Syria, Egypt,

20

Ethiopia, Sudan and Morocco. On the basis of area and production, faba bean

ranks fourth among pulse crops of the world, the first three being dry pea, dry

bean and chick pea (Bond, 1987). In India it is cultivated throughout northern

states for the sake of its broad and succulent seed pods which are used as

vegetable. Its dried seeds are also used as pulse in hilly areas of U.P., H.P.

and Jammu and Kashmir. Experiments at H.A.U. Hissar have revealed that

faba bean out yielded chick pea, field pea and lentil (Tomar et al, 1986).

Thus, a possibility exists to popularize faba bean as a new pulse crop in our

country. However, the genotypes in our country have a low yield potential.

In Vicia faba L. there is a lack of variability for most agronomic traits.

Germplasm collections from diverse sources have been made to bring all the

variations at one place. The number and size of collections have increased

substantially during last 10 years. The largest collecfion is now held by

ICARDA at Aleppo, Syria. Attempts on creating new genetic variability

through mutation breeding have received only limited attention in this crop.

Mutagen sensitivity in Mi generation was worked out on several growth

and yield parameters. Germinadon, seedling growth, pollen fertility, time

taken to maturity and survival were adversely affected by the mutagens. Plant

height, branching, number of leaves, pods and seeds as well as yield/plant

showed varying response to different concentrations of mutagens. However,

DES at all doses and EMS only at the highest dose of 0.75% had an adverse

effect on these traits. Whereas, the lower doses of EMS had either no effect or

21

a slight promoting effect (Vandana and Dubey, 1988). In another study. lOio-

of gamma rays and 0.75% DES were applied individually or in combined

application, it was found that gamma rays induced more sever effects than

DES (Kumar e/fl/., 1993).

Chromosomal aberrations formed another criterion to judge the mutagen

sensitivity. Vandana and Dubey (1992), Vandana (1993), Sinha and Gandhi

(1994), Vandana and Dubey (1996), Bhat et al. (2005), and Prashant and

Verma (2005) reported that the main types of anomalies in root tip cells were

fragmentation, clumping and stickiness of chromosomes, star metaphase,

giant nuclei, saucepan arrangement of chromosomes, binucleate cells.

micronuclei, bridges and laggards while meiotic abnormalities included

multivalent associations such as rings or chain of bivalents, fragmentation of

nucleolus, precaucious separation of bivalents at Metaphase -1, single, double

and multiple bridges and unequal distribution of chromosomes at the two

poles at Anaphase I/II etc. The percentage of mitotic or meiotic anomalies at

various stages was directly correlated to the dose of mutagen used. DES

dosage inducing a higher percentage of abnormal cells than EMS (Vandana,

1993; Vandana and Dubey,1992 and Vandana et al, 1996; Perveen, 2006).

Similar anomalies have been reported by Singh Joshi (1967) and by Sjodin

(1971) who investigated nearly 200 induced translocations in the species.

The frequency and spectrum of mutations in M2 generation were

partitioned into those for chlorophyll, sterile and vital categories of mutations.

22

Among chlorophyll mutations, xantha, viridis, viridoxantha and straita t)pes

were observed while sterile mutants were flowerless, cleistogamous, fruitless

and under developed seed mutants (Vandana,1991; Fatima, 2007). Vital

mutations were classified on the basis of the characters involved into mutants

for cotyledonary leaf, plant height, branching, leaf, bristle, plant surface.

colour and texture, floral characters, maturity period and pod and seed

characters (Vandana, 1992a,b). The frequency and spectrum of chlorophyll

and leaf mutation of gamma rays, EMS and nitrous oxide (N2O) seed

treatment in two varieties of faba bean were studied by Yasin (1996). The

frequency of chlorina type mutations was higher than that of xantah. EMS

treatment was found to be most effective than the gamma rays treatment.

Mutation frequency in terms of percentage of families segregating as well

as mutants/1000 M2 plants increased with an increase in concentration of

chemical mutagen. DES induced higher frequency of mutations than EMS.

Frequencies of vital mutations were always higher than the chlorophyll and

sterile mutations (Vandana and Dubey, 1991). In the study involving gamma

rays and DES applied individually as well as in combined treatment,

individual DES treatment induced highest percentage of families segregating,

while combined application of gamma rays and DES induced highest

percentage number of mutant/1000 M2 plants (Kumar and Dubey, 1996).

Few studies have been undertaken to compare mutagenic agents for their

ability to induce genetic variability in quantitative characters (Sojodin, 1971;

23

Abdalla and Hussein, 1977; Filippetti and De pace, 1983,1986; Chapman.

1981, 1986; Joshi and verma,2004; Khan et ai, 2006; Yasin et ai, 2006).

Enhanced variability for polygenic traits was also induced by various

mutagenic treatments which was reflected by shift in mean values and

increased inter and intra family variability for these traits in M2 populations.

Coefficients of interfamily variability were much higher than those for

intrafamily variability indicating better scope of selection between the

families than within the families (Vandana and Dubey, 1990b; Vandana,

1990). A study of root of AV-8 mutant revealed a heavier nodulation in

comparison to the control (Vandana and Dubey 1993). Highest phenotypic.

genotypic and environmental coefficient of variability was recorded for seed

yield which was closely followed by those for number of pods. Days to

flower and test weight had rather small coefficient of variability (Vandana.

1992a). High heritability values for seed yield and traits like test weight,

seeds/plant, seeds/pod and pods/plant have been reported in faba bean by

Bakheit and Mahday (1988) and Nanda et al., (1988). On the other hand,

Bond (1987) has observed that as in most legumes, yield in faba bean has low

heritability because of the major effects of the environmental factors.

24

Chapter - 3

MATERIALS AND METHODS

3.1. Materials

3.1.1. Varieties used

Two varieties of faba bean {Vicia faba L.) namely, 05/249 local and

05/233 HBP were used in the present study. Seeds of both the varieties were

obtained from Genetic Section of the Indian Agricultural Research Institute of

New Delhi.

3.1.2. Mutagens used

Ethylmethane sulphonate (EMS)-CH03 SO2C2H5)-, an alkylating

agents, manufactured by Sisco Research Laboratories Pvt.Ltd.,Mumbai.

EMS was used alone and in combination with dimethyl

sulfoxide(DMSO)-CH3SO.CH3)-manufactured by Ranbaxy Laboratories

Pvt.,Ltd., S.A.S Nagar, Punjab.

Hydrazine hydrate (HZ)-NH2 -NH2 -H2O, a base analogue, is manufactured by

Qualigens Fine Chemical, Mumbai,,

3.2. Experimentai procedure

3.2.1. Pretreatment

Healthy seeds of uniform size of each variety were used. Seeds were

soaked in distilled water for 9 hours prior to the treatment with mutagens.

25

3.2.2. Mutagens administration

Concentrations used; Four different concentrations viz., 0.02, 0.04, 0.06, and

0.08% of EMS, EMS+2%DMS0 and HZ were used for treating the presoaked

seeds.(2% DMSO was prepared by dissolving 2mi of DMSO in 100 ml of

distilled water).

Treatment time: The treatments were given at temperature of 22 ±1°C for 6

hours.

Sample size: 255 Seeds were used for each treatment and control.

Controls: For each variety, 255 pre-soaked seeds were again soaked in

phosphate buffer for 6 hours to serve as controls.

3.3. Ml generation

Three replications of seventy seeds each were sown for ever>

treatment and control in each variety in the field. The remaining lot of forty five

seeds of each treatment with their respective controls of both the varieties was

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 B.O.D. incubator at 22 ±1°C temperature.

3.3.1. Observations recorded in Mj generation

Following parameters were studied in Mj generation

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

26

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

germinated.

„ . . ,„,. No. of seed germinated ,„„ Germmation (%) = x 100

Total no. of seed sown

3.3.1.2. Seedling height

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

measuring the root and shoot lengths from each treatment and control. Seedling

injury was calculated in terms of reduction in seedling height with respect to

control.

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 in

the field.

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 regular outline

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

The following formula was used to calculate the percentage inhibition or

injury or reduction:

27

Percentage inhibition Or

. . Control - Treated ,^^ Percentage injury = x 100

Control Or

Percentage reduction

3.3.2. Morphological variants

Some induced morphological variants affecting plant from, plant height

and leaf were isolated in M] generation. The frequency of morphological variants

was calculated by the following formula:

„ ..,, Number of variants ,^^ Frequency (%) = xlOO

Total number of M,

3.3.3. Quantitative traits

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

treatment with their controls.

The following nine 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 to flowering: Days to flowering were noted as the number of days

taken by the plant from the date of sowing to 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 upto the date of harvesting of the plant.

28

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

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

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. Pod length: The pods were measured in centimeters and the mean for

each selected plant was calculated for pod length.

8. 100-seed weight (g): It was the weight of random sample of hundred

seeds from each plant.

9. 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 gram.

3.4. Cytological studies

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

control in both the varieties were fixed in Camoy's fluid (1 part glacial acetic

acid: 3 parts chloroform : 6 parts of ethyl alcohol)for 30 minutes. The material

was 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% propiono aceto carmine solution and pollen mother cells were

examined for their be behaviour at various stages of microsporogenesis.

.Photographs v/ere taken from temporary preparation.

29

3.4. Statistical analysis

3.4.1. 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 nine quantitative traits in M] generation were subjected to

statistical analysis to find out ranged, mean, standard error, standard deviation

and coefficient of variation.

3.4.1. Mean(X)

The mean was computed by taking the sum of the number of values and (Xi,

X2, .. ..Xn) dividing by the total number of values involved, thus

(X, + X, + X3 X J X

N

Or

IX „

N

Where, Xj, X2, X3, Xn = Observations

N= Total number of observations involve

3.5.1.2, Standard error (S.E.)

It is the measure of the uncontrolled variation present in a

sample. It is estimated by dividing the standard deviation by the square root of

the number of observations in the sample and is denoted by S.E.

„ ^ _ S.D. of the sample VN

30

Where, S.D. = Standard deviation

N =Number of observations

3.5.1.3. Standard deviation (S.D.)

The Standard deviation was calculated by the following formula for

each parameter of study.

S.D.= (X-X,)^+(X-X,)^ ( X - X J

N

Where, (X) = Mean of the observations involved

X] X 2 Xn = observation

N= Total number of observations

3.5.1.4. 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 was applied to compute coefficient of

variability (C.V).

C V (%) = ^ ^ ^ ^ ^ ^ 1 " ^ ^ ^ ^ ^ " " xiOQ X

Or S.D X

xlOO

Where, S.D. = Standard deviafion of sample

(X) = Arithmetic mean

31

Chapter 4

EXPERIMENTAL RESULTS

Mutagenic sensitivity is known to be influenced by a variety of factors of

which the type of mutagen used and dose applied, pre and post treatment

conditions and genotype of the material are important. Different parameters such

as percent of seed germination, seedling growth depression, pollen fertility and

certain quantitative characters in Mi generation were used to study mutagenic

sensitivity.

The Ml generation arisen directly from the chemically treated seeds.

Hence, maximum mutagenic damage can be anticipated in Mi in terms of

morpho-physiological changes. Thus, two types of experimental studies were

conducted with treated seeds. First, Laboratory petriplate experiment to evaluate

seed germination and seedling height in mutagenized population as well as in

control (untreated) population, and second, field experiment to study pollen

fertility, certain quantitative characters and to collect Mi seed.

4.1. Seed germination

Data recorded on seed germination are presented in Tables 1-4. The

treated seeds germinated 3 to 7 days after germination of control seeds. The

maximum seed germination was observed to be 100% in control population of

both the varieties (Table 1). In the treated population of the var. 05/249 local, it

ranged 93.22 - 46.67 % in EMS, 93.33 - 40.00 % in EMS in combination with

32

DMSO and 88.89 - 51.11% in HZ treatments. It showed that the percentage of

seed germination gradually decrease with increasing concentrations of mutagens.

Seed germination was drastically affected in the combination treatments than the

mutagens used singly. Comparatively the seed germination was highest in EMS

treated population followed by HZ and EMS in combination with DMSO.

Consequently, the degree of inhibition in seed germination was recorded higher

in the combination treatments. The other var.05/233HBP behaved more or less

similarly. Variety 05/249 local was found to be more sensitive.

4.2. Pollen fertility

Pollen character is one of the important stable and genetically

controlled characters, which may be considered for preparing index to asses the

effect of any internal change in plants.

Although some 2% pollen sterility was also observed in control plants of

the two varieties, but it increased with increase in the concentrations of

mutagens used singly or in combination with DMSO revealing a linear

dependence of fertility on dose (Table 1). Pollen fertility ranged from 97.77 -

90.86% in EMS, 96.98 - 88.75% in EMS used in combination with DMSO and

98.26 - 94.22% in HZ treatments. The highest percentage of reduction in pollen

fertility was recorded in EMS+DMSO followed by EMS and HZ treatments. The

degree of sterility of pollen grains was appreciably more in the var. 05/249 local

than the var. 05/233HBP.

33

4.3. Seedling height (cm).

Data on the height of the ten days old seedlings indicated a gradual

decline in seedling height with increasing concentration of EMS alone and in

combination with DMSO and HZ in both the varieties used in the present study

(Tables 1, 8-10). Total seedling length (root + shoot) in the var.05/233HBP

ranged 45.01-23.33 cm in EMS, 42.30-27.55 cm in combination treatments and

41.92- 29.91 cm in HZ treatments (Tables 8-10). It was 47.01 cm in the control

of var. 05/233 HBP. Results show that EMS in combination with DMSO was

most effective in reducing seedling height. Percentage injury in seedling height

was noticed in increasing order with increasing various mutagenic treatments

(Table 1). Reduction in seedling height was more in the Var. 05/233 HBP as

compare to the var.05/249 local.

4.4. Plant survival

Data on plant survival in Mi generation recorded at maturity are given in

Table 1. Percentage of plant survival was noted to decrease gradually in all

mutagenic treatments. However, it was dose independent. The highest plant

survival was observed in control of both the varieties. Both the varieties

responded more or less in the same manner.

4.5. ANOVA of seed germination and seedling height

Variances among treatments (4 concentrations of EMS alone and in

combination with DMSO and HZ + one control) were significantly high for both

percentage seed germination (Tables 5-7) and seedling height (Tables 11-13).

34

Table 1: Effect of mutagens on seed germination, plant survival, pollen fertility and seedling height in two varieties in Viciafaba L.

Treatment

Control 0.02%EMS 0.04%EMS 0.06%EMS 0.08%EMS

0.02%EMS+DMSO 0.04%EMS+DMSO 0.06%EMS+DMSO 0.08%EMS+DMSO

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ


0.02%EMS+DMSO 0.04%EMS+DMSO 0.06%EMS+DMSO 0.08%EMS+DMSO

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

Seed germination

Actual (%)

100.00 93.33 80.00 66.67 46.67

93.33 80.00 60.00 40.00

88.89 73.33 66.67 51.11

100.00 93.33 86.67 73.33 60.00

93.33 93.33 80.00 60.00

86.67 82.22 66.67 57.78

%age inhibition

- 6~67 -20.00 -33.33 -53.33

- 6.67 -20.00 -40.00 -60.00

-11.11 -26.67 -33.33 -48.89

-6767 -13.33 -26.67 -40.00

-6.67 -6.67

-20.00 -40.00

-13.33 -17.78 -33.33 -42.22

Plant survival at maturity (%)

Van 05/249 local

79.00 65.00 67.00 59.00 59.00

67.00 68.00 69.00 72.00

65.00 66.00 52.00 54.00

Var. 05/249 HBP

72.00 58.00 59.00 52.00 48.00

67.00 68.00 70.00 68.00

58.00 59.00 52.00 45.00

Pollen fertility

Actual (%)

99.18 97.77 96.39 94.99 90.86

96.98 95.98 92.67 88.75

98.26 97.27 95.91 94.22

98.76 97.80 96.44 94.42 91.31

96.89 95.65 94.40 92.64

98.23 96.44 95.65 94.11

%age inhibition

-l742 -2.81 -5.03 -8.39

-2.22 -4.03 -6.56

-10.52

-0.96 -1.93 -3.30 -5.00

-0797 -2.32 -4.40 -7.54

-1.89 -3.15 -4.42 •6.23

-0.54 -2.32 -3.15 -5.03

Seedling height f% a£

• • X mjury)

1

- 7.20 -15.14 -17.62 -41.56

- 5.15 -17.62 -19.83 -28.22

+ 1.44 -17.62 -30.36 -40.02

-~4.25 -12.89 -31.29 -50.37

-11.13 -20.19 -32.99 -41.39

-10.83 -12.89 -23.69 -36,37

t 35

% 25

O

105/249 local

105/233 HBP

Control 0.02 0.04 0.06 0.08

BMS (%)

^ 40 e 35 g 30 « 25 | .

• 05^9 local

• 05/233 HBP

Control 0.02 0.04 0.06 0.08

BWS-K3MSO(%)

C. 35

I 30 « 25

I 20

• OS/249 local

• 05/233 HBP

Control 0.02 0.04 0.06 0.08

HZ (%)

Fig -1 . Effect of mutagens on seed germination in Mi generation in the two varieties of Viciafaba.

105/249 local

105/233 HBP

Control 0.02 0.04 0.06 0.08

BMS(%)

• 05/249 local

• 05/233 HBP

& 10

Control 0.02 0.04 0.06 0.08

BMS-i-DMSOC/

• OS/248 local

• 05/233 HBP

Control 0.02 004 O06 0.08

HZ(%)

Fig. 2- Effect of mutagens on seedling height (cm) in Ml generation in the two varieties of Viciafaba,

£ 92

• 05/249 local

• 05/233 HBP

Control 0.02 0.04 0.06 0.08

EMS (%)

5? 96

105/249 local

105/233 HBP

£ 90 i

I 86

Control 0.02 0.04 0.06 0.08

BNS+DMSO(%)

100 99

97 3 96 I" f .S 95 I 94 I 93

92 91

• 05/249 local

• 05/233 HBP

Control 0.02 0.04 0.06 0.08

HZ (%)

Fig. 3- Effect of mutagens on pollen fertility in Ml generation in the two varieties of Viciafaba.

105/249 local

105/233 HBP

Control 0.02 0.04 0.06 0.08

BMS (%)

C 72

£ 68 S 66

105/249 local

106/233 HBP

60 + Control 0.02 0.04 0.06 0.08

BMS+DMSO (%)

105/249 local

105/233 HBP

Control 0.02 0.04

HZ(%)

0.06 0.08

Fig.4 - Effect of mutagens on plant survival at maturity in Mj generation in two varieties of Viciafaba.

Variety x treatment (AxB) interaction was significant (p<0.01) for seed

germination for EMS used in combination with DMSO (Table 6), and for

seedling height for EMS in combination with DMSO and HZ treatments (Tables

12&13).

4.6. Morphological variations

Frequencies of various morphological variations affecting vegetative parts

of the plants isolated in Mi in various mutagenic treatments are given in Table

14, Plate I -III. Variety 05/233 HBP was more sensitive than the var. 05/249

local, given a higher percentage of morphological variations. Several leaf

variation were recorded at higher frequencies in 05/233HBP than in the

var.05/249 local (Table 14). EMS+ DMSO treatments resulted in the highest

frequency (8.79%), followed by EMS (8.58%) and HZ (7.52%) treatments (Table

15).

Height variants

Plant height in control ranged from 62 to 70 cm. Treated population

showing variation from the normal plant height are grouped into the following

categories: dwarf variants - 32 to 38 cm; tall variants- 86 to 94 cm.

Branching variants

In the control plant, 2 to 3 branches arise from the base of plant and no

secondary branching occur above this level. Among the treated Mj population,

unbranched variants showing no branching at all as well as those showing an

increased number of basal branches leading to a bushy appearance of the plant

35

Table 2: Seed germination in two varieties of Viciafaba treated with EMS.

Variety

05/249 Local

05/233HBP

Treatment



Total

Total

Seed germination

R-I

15 14 12 10 6

57

15 14 13 11 8

61

118

R-II

15 13 13 9 7

57

15 15 12 12 9

63

120

R-III

15 15 11 11 8

60

15 13 14 10 10

62

122

Total

45 42 36 30 21

174

45 42 39 33 27

186

360

Mean

15 14 12 10 7

-

15 14 13 11 9

-

-

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

Table 5: ANOVA for seed germination (for EMS treatment)

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.

214.00 0.80 4.80 189.00 4.20 15.20

M.S.

4.80 47.25 1.05 0.84

F

5.71* 56.25** 1.25

Tabular F

F 0.05

4.41 2.93 2.93

Fo.oi

8.28 4.58 4.58

„ 1

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

Table 3: Seed germination in two varieties of Viciafaba treated with EMS+DMSO.

Variety

05/249 Local

05/233HBP

T(

Treatment

Control 0.02%EMS+DMSO 0.04%EMS+DMSO 0.06%EMS+DMSO 0.08%EMS+DMSO


Total

3tal

Seed germination

R-I

15 14 11 8 6

54

15 13 14 12 8

62

116

R-II

15 13 12 9 7

56

15 15 13 13 10

66

122

R-III

15 15 13 10 5

58

15 14 15 11 9

64

122

Total

45 42 36 27 18

168

45 42 42 36 27

192

360

Mean

15 14 12 9 6

-

15 14 14 12 9

-

-

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

Table 6: ANOVA for seed germination (for EMS+DMSO treatment).

Source of

variation


d.f

30-1=29 3-1=2 2-1=1 5-1=4 1x4=4 18

S.S.

291.00 26.90 19.20

219.00 13.80

M.S.

19.20 54.75 3.45 0.67

F

28.65** 81.72** 5.15**

Tabular F

Fo.05

4.41 2.93 2.93

Fo.oi

8.28 4.58 4.58


Table 4: Seed germination in two varieties of Viciafaba treated with HZ.

Variety

05/249 local

05/233HBP

Treatment



Total

Total

Seed germination

R-I

15 13 10 10 8

56

15 13 12 10 7

57

113

R-II

15 13 12 9 7

56

15 14 12 11 9

61

117

R-III

15 14 11 11 8

59

15 12 13 9

10

59

m

Total

45 40 33 30 23

171

45 39 37 30 26

111

348

Mean

15.00 13.33 11.00 10.00 7.67

-

15.00 13.00 12.33 10.00 8.67

-

-


Table 7: ANOVA for seed germination (for HZ treatment).

Source of

variation


d.f.

30-1=29 3-1=2 2-1=1 5-1=4 1x4=4 18

S.S.

189.20 1.40 1.20 170.20 3.13 13.27

M.S.

1.20 42.55 0.78 0.74

F

1.62 57.50**

1.05

Tabular F

Fo.05

4.41 2.93 2.93

Fo.oi

8.82 4.58 4.58

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

Table 8: Seedling height in two varieties of Viciafaba treated with EMS.

Variety

05/249 localch

05/233HBP

Treatment



Total

Total

Seed height (cm)

R-I

14.85 13.16 13.01 11.50 9.03

61.28

16.77 15.78 14.17 11.23 8.23

66.18

127.46

R-II

15.01 14.56 12.23 12.12 8.24

62.16

15.01 14.00 13.78 10.84 7.85

61.48

123.64

R-III

14.02 13.00 12.87 12.53 8.37

60.79

15.23 15.23 13.00 10.23 7.25

60.94

121.73

Total

43.88 40.72 38.11 36.15 25.64

184.50

47.01 45.01 40.95 32.30 23.33

188.60

373.10

Mean

14.63 13.57 12.70 12.05 8.55

-

15.67 15.00 13.65 10.77 7.77

-

-


Table 11: ANOVA for seedling height (for EMS treatment).

Source of variation


d.f

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

18

S.S.

208.04 1.69 7.27

185.03 7.78 6.27

M.S.

7.25 46.33 1.95 0.35

F

2.70 17.22** 0.72

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 9: Seedling height in two varieties of Viciafaba treated with EMS+DMSO.

Variety

05/249 local

05/233HBP

Treatment



Total

Total

Seedling height (cm)

R-I

14.85 13.26 11.50 12.87 11.23

63.71

16.77 14.28 13.24 10.27 9.28

63.84

127.55

R-II

15.01 14.28 12.12 11.28 10.27

62.96

15.01 14.00 12.27 10.12 9.27

60.67

123.63

R-III

14.02 14.08 12.53 11.03 10.00

61.66

15.23 14.02 12.01 11.11 9.00

61.37

123.03

Total

43.88 41.62 36.15 35.18 31.50

188.33

47.01 42.30 37.52 31.50 27.55

185.88

374.21

Mean

14.63 13.87 12.05 11.73 10.50

-

15.67 14.10 12.51 10.50 9.18

-

-


Table 12: ANOVA for seedling height (for EMS+DMSO treatment).

Source of

variation


d.f.

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

18

S.S.

89.26 0.25 1.57

70.22 9.93

M.S.

1.57 17.56 2.48

F

3.83 42.83** 6.05**

Tabular F

F 0.05

4.41 2.93 2.93

Fo.oi

8.23 4.58 4.58


Table 10: Seedling height in two varieties of Viciafaba treated with HZ.

Variety

05/249 local

05/233HBP

Total

Treatment

Control 0.04%HZ 0.02%HZ 0.06%HZ 0.08%HZ

Control 0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

Total

Seedling height (cm)

R-I

14.85 15.01 11.50 10.27 8.27

59.90

16.77 14.27 14.17 12.87 9.78

67.86

127.76

R-II

15.01 15.23 12.12 10.28 9.28

61.92

15.01 13.88 13.78 11.78 10.25

64.70

126.62

R-III

14.02 14.27 12.53 10.01 8.77

59.60

15.23 13.77 13.00 11.22 9.88

63.10

122.70

Total

43.88 44.51 36.15 30.56 26.32

181.42

47.01 41.92 40.95 35.87 29.91

195.66

377.08

Mean

14.63 14.84 12.05 10.19 8.77

-

15.67 13.97 13.65 11.96 9.97

-

-


Table 13 : ANOVA for seedling height (for HZ treatment).

Source of variation


d.f

•

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

18

S.S.

172.66 0.70 5.51

152.91 8.54 4.99

M.S.

5.51 38.23 2.14 0.28

F

19.68** 136.53**

7.64**

Tabular F

Fo.o5

4.41 2.93 2.93

Fo.oi

8.28 4.58 4.58

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

Table 14: Frequency and spectrum of morphological variants induced by mutagens in faba bean {Viciafaba L.) varieties.

Variants

Dwarf

Tall

Bushy

Leaf variation

Shape

Texture

Size

Arrangement

Foliage colour

Total number of morphological variants

Total number of Ml plants

Frequency (%)

05/249 local

12

23

8

35

10

26

7

11

132

1598

Number observed in

Frequency (%)

0.75

1.44

0.50

2.19

0.63

1.63

0.44

0.69

8.26

05/233 HBP

9

20

10

25

16

22

8

14

124

1474

Frequency (%)

0.61

1.36

0.68

1.70

1.08

1.49

0.54

0.95

8.41

Table 15: Frequency of morphological variants in various mutagens in Mi generation.

Mutagen

EMS

EMS+DMSO

HZ

Number of M) Plants studied

979

1149

944

Number of variants scored

84

101

71

Frequency (%)

8.58

8.79

7.52

were recovered. The bushy variants are further classified as bushy dwarf and

busy tall depending upon the plant height.

Leaf variants

Many leaf variations were recorded which could be useful in crop

improvement programmes. The leaf in faba bean is compound with the number

of leaflets varying from 2 to 5. Leaf variants were grouped into five categories:

(i) Shape: Mutagens treated plants had narrow or rounded leaflets compared

with the intermediate leaflets of the controls

(ii) Size: Both smaller and larger leaflets than normal were observed in variants

(iii) Texture: Leaflets had rough, thick and leathery surfaces rather than the

smooth surface of the parental varieties

(iv) Arrangement: Variants showed more diverse leaf attachment to the base

than the parental varieties. These changes included droping leaves with short

intemode and leaflet leading to a change in leaf arrangement and canopy

(v) Foliage colour: Mutation causing alteration in the colour of leaves were

included in this category. Compared with the control some plants were lighter

yellow, lacking proper chlorophyll content and a few plants were darken green

apparently rich in chlorophyll content.

4.7. Cytological abnormalities

Various anomalies scored at different stages in root tip cells and pollen

mother cells are given in plate-IV: Figs. 1-12. The proportions of chromosomal

aberrations increased with the increase in the dose of mutagens. Chromosomal

36

abnormalities were directly correlated to the dose of mutagens used; EMS in

combination with DMSO induced a higher percentage of abnormal cells in

comparison with EMS alone and HZ treatments. Various chromosomal

abnormalities of noticed at various stages involved bridges, stickiness of

chromosomes, fragmentation of chromosomes cytomixis and micronuclei.

4.8. Quantitative traits

Data on the effect of various treatments of EMS alone and in combination

with DMSO and HZ in two varieties are given in Tables 16-33. Statistical

analysis was done to find out mean, standard error, shift in mean and coefficient

of variation for nine quantitative traits namely plant height (cm), days to

flowering, days to maturity, number of fertile branches, number of pods, pod

length(cm), number of seeds per pod, 100 seed weight (g) and yield per plant(g).

In the present study, means for quantitative traits shifted in both positive

as well as in negative direction, being more in the positive side for the traits like

days to maturity, pods per plant, 100 seed weight and total plant yield. However,

the shift in mean values, except in few mutagenic treatments, was insignificant.

Coefficient of variation (CV) of the mutagens treated population

differed from trait to trait. The highest increase in CV over the control was

recorded for plant height, fertile branches per plant, pods per plant and yield per

plant.

37

Table 16: Estimates of mean values (X), shift in (X), and coefficient of variation (CV) for plant height (cm) of Viciafaba var.05 /2491ocal.

Treatment

Control 0.02%EMS 0.04%EMS 0.06%EMS 0.08%EMS LSD

(p<0.01) (P<0.05)

0.02%EMS+DMSO 0.04%EMS+DMSO 0.06%EMS+DMSO 0.08%EMS+DMS0 LSD

(p<0.01) (p<0.01)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ LSD

(p<0.01) (p<0.05)

Mean ± S.E.

71.33±3.25 49.53±5.18 63.47±3.02 63.93±2.55 56.60±2.41

66.33±3.43 30.13±3.20 74.33±4.72 67.40±4.50

55.80±6.56 49.86±6.88 48.27±6.58 44.80±3.73

Shift in X

-21.80 - 7.86 - 7.40 -14.73

12.88 9.68

- 5.00 -41.20 + 3.00 - 3.33

14.58 10.96

-15.53 -21.47 -23.06 -26.53

21.22 15.96

C V (%)

17.66 40.54 18.48 15.48 16.53

20.05 41.02 23.77 25.86

45.46 53.40 52.83 32.27

Table 17: Estimates of mean values (X), shift in X and coefficient of variation (CV) for days to flowering of Viciafaba var.05/2491ocal.

Treatment

Control 0.02%EMS 0.04%EMS 0.06%EMS 0.08%EMS LSD

(p<0.01) (p<0.05)

0.02%EMS+DMSO 0.04%EMS+DMSO 0.06%EMS+DMSO 0.08%EMS+DMSO LSD

(p<0.01) (p<0.05)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ LSD

(p<0.01) (p<0.05)

Mean ± S.E.

49.67±0.18 50.93±0.23 51.06±0.21 52.00±0.22 52.87±0.27

51.13±0.32 51.26±0.22 50.60±0.19 51.07±0.21

50.00±0.22 49.07±0.21 49.13±0.22 48.87±0.17

Shift in X

+1.26 +1.39 +2.33 +3.20

0.82 0.62

+1.46 +1.59 +0.93 +1.40

0.87 2.54

+0.33 -0.60 -0.54 -0.80

0.78 0.59

CV. (%)

1.45 1.73 1.56 1.62 2.00

1.72 1.72 1.45 1.56

1.69 1.63 1.69 1.70

Table 18: Estimates of mean values (X), shift in X mean and coefficient of variation (CV) for days to maturity of Viciafaba var.05/2491ocal.

Treatment


LSD (p<0.01) (p<0.05)

0.02%EMS+DMSO 0.04%EMS+DMSO 0.06%EMS+DMSO 0.08%EMS+DMS0

LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

LSD (p<0.01) (p<0.05)

MeaniS.E.

109.33±1.08 109.00±1.00 110.00±1.19 111.33±1.14 110.67±1.08

111.07±0.22 111.00±0.28 1I1.27±0.23 111.47±0.19

110.00±0.53 109.67±0.50 110.20±0.52 110.67±0.48

Shift in X

-0.33~ +0.67 +2.00 +1.34

4.15 3.12

+1.74 +1.64 +1.94 +2.14

1.97 1.48

+0.67 +0.34 +0.87 +1.34

2.50 1.88

CV. (%)

3.81 3.55 4.21 3.97 3.99

0.80 0.83 0.79 0.67

2.07 1.77 1.81 1.69

Table 19: Estimates of mean values (X), shift in X and coefficient of variation (CV) for number of fertile branches/plant of Viciafaba var.05/2491ocal.

Treatment


LSD (p<0.01) (p<0.05)


LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ OMVoUZ 0.08%HZ

LSD (p<0.01) (p<0.05)

MeaniS.E.

9.40±3.35 10.27±3.02 7.40±0.99 7.20±1.19 9.60±0.87

7.13±1.12 7.07±1.11 6.53±1.17 4.20±0.43

3.87±0.75 4.80±0.88 4.86±0.79 10.87±1.76

Shift in X

+0.87 -2.00 -2.20 +0.20

8.11 6.09

-2.27 -2.33 -2.87 -5.20

6.52 4.90

-5.53 -4.60 -4.54 +1.47

6.73 5.06

CV. (%)

124.47 113.98 51.53 63.90 35.17

60.84 61.32 69.14 39.43

2.80 3.29 2.94 6.58

Table 20: Estimates of mean values (X), shift in X and cofficient of variation (CV) for pods/plant (grain) of Viciafaba var.05/2491ocal.

Treatment


LSD (p<0.01) (p<0.05)


LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

LSD (p<0.01) (p<0.05)

Mean ± S.E.

17.87±2.77 20.33±3.56 24.33±3.54 34.73±9.04 43.00± 10.68

8.67±3.69 24.93±4.77 28.40±5.33 36.6. ±8.28

52.20±6.92 28.13±3.35 25.73±5.14 14.60±3.77

Shift in X

+2.46 + 6.46 +16.86 +25.13

25.43 19.12

-9.20 +7.06 +10.53 +18.73

19.89 15.02

+34.33 +10.26 +7.86 -3.27

16.67 12.55

CV. (%)

59.99 67.78 56.39

100.74 96.14

164.82 74.08 72.67 87.65

51.26 46.21 77.26 61.57

Table21: Etimates of mean values (X), shift in X and coefficient of variation (CV) for pod length (cm) Viciafaba var.05/2491ocal.

Treatment


LSD (p<0.01) (p<0.05)


LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

LSD (p<0.01) (p<0.05)

Mean ± S.E.

5.32±0.16 5.11±0.17 4.70±0.24 4.72±0.23 4.60±0.18

5.13±0.13 5.13±0.15 4.80±0.13 4.74±0.20

5.05±0.19 4.40±0.21 5.11±0.11 4.98±0.12

.

Shift in X

-0.21" -0.62 -0.60 -0.72

0.59 0.57

-0.19 -0.19 -0.52 -0.58

0.61 0.46

-0.27 -0.92 -0.21 -0.34

0.63 0.47

CV. (%)

12.04 12.91 20.14 18.98 15.28

9.90 12.00 10.88 21.09

14.99 18.88 8.80 9.42

Table 22: Estimates of mean value (X), shift in X and coefficient of variation (CV) for number of seed /pod of Viciafaba var. 05/249 local.

Treatment


LSD (p<0.01) (p<0.05)


LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

LSD (p<0.01) (p<0.05)

MeaniS.E.

3.13±0.13 2.33±0.21 2.60±0.19 2.53±0.16 2.20±0.22

2.53±0.25 2.86±0.27 2.47±0.22 2.40±0.21

2.80±0.17 3.06±0.15 2.53±0.13 2.73±0.18

Shift in X

-0.80 -0.53 -0.60 -0.93

0.70 0.53

-0.61 -0.27 -0.66 -0.73

0.84 0.63

-0.33 -0.07 -0.60 -0.40

0.58 0.44

CV. (%)

16.48 34.99 28.33 25.26 39.17

39.09 36.98 33.80 34.50

24.15 19.36 20.38 25.75

Table 23: Estimates of mean values (X), shift in X and coefficient of variation (CV) for 100 seed weight of Viciafaba var.05/249 local.

Treatment


LSD (p<0.01) (p<0.05)


LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

LSD (p<0.01) (p<0.05)

Mean ± S.E.

33.31±0.24 33.34±0.24 33.65±0.22 33.51±0.25 33.71±0.22

34.26±0.21 34.12±0.26 33.77±0.17 34.08±0.17

32.96±0.19 33.47±0.25 33.36±0.24 33.69±0.26

Shift in X

+0.03 +0.34 +0.20 +0.40

0.88 0.66

-0.95 -0.18 +0.46 +0.77

0.80 0.60

-0.35 +0.16 +0.05 +0.38

0.92 0.68

CV. (%)

2.79 2.79 2.53 2.56

2.33 2.95 1.89 1.89

2.26 2.85 2.78 3.30

Table 24: Estimates of mean values (X), shift in X and coefficient of variation (CV) for yield/plant of Viciafaba var.05/2491ocal.

Treatment


LSD (p<0.01) (p<0.05)


LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

LSD (p<0.01) (p<0.05)

Mean ± S.E.

15.60±2.30 15.18±2.45 20.98±2.60 29.48±7.62 37.56±8.33

24.51±2.75 38.53±4.41 53.99±4.89 61.05±7.76

48.21±6.45 25.71±3.16 22.73±5.87 12.61±2.22

Shift in X

-0.42 +5.32 +13.88 +21.96

19.46 14.62

+ 0.91 +22.93 +38.39 +45.45

17.98 13.52

+32.61 +10.11 +7.13 +2.99

15.64 11.76

CV. (%)

57.08 62.63 47.89 89.44 85.87

43.56 41.63 35.09 49.21

51.75 47.72 84.71 67.91

Table 25: Estimates of mean values (X), shift in X and coefficient of variation (CV) for plant height (cm) of Viciafaba var.05/233 HBP.

Treatment


LSD (p<0.01) (p<0.05)


LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ OM%UZ 0.08%HZ

LSD (p<0.01) (p<0.05)

Mean ±S.E.

70.46±3.95 62.93±5.70 48.20±8.67 42.20±5.08 36.20±3.13

71.06±5.20 72.53±3.89 67.86±5.59 55.00±2.85

69.40±4.39 68.47±2.01 64.40±5.13 56.46±3.90

Shift in X

-7.53" -22.26 -28.26 -34.26

21.20 15.94

+0.60 +2.07 -2.60 -15.46

97.14 73.04

-1.06 -1.99 -6.06 -14.00

15.91 11.96

CV. (%)

21.71 35.07 69.58 46.67 33.46

28.32 20.74 31.90 20.05

24.48 24.61 30.82 26.74

Table 26: Estimates of mean values (X), shift in X and coefficient of variation (CV) for days to flowering of Viciafaba var.05/233HBP.

Treatment


LSD (p<0.01) (p<0.05)


LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

LSD (p<0.01) (p<0.05)

Mean ± S.E.

45.80±0.20 45.00±0.22 45.73±0.23 45.00±0.22 44.80±0.22

43.80±0.22 44.00±0.22 44.93±0.23 45.07±0.18

44.93±0.23 44.80±0.20 45.27±0.23 49.87±0.22

Shift in X

-0.80" -0.07 -0.80 -1.00

0.82 0.62

-2.00 -1.80 -0.87 -0.73

0.80 0.60

-0.87 -1.00 -0.53 +4.07

0.81 0.61

CV. (%)

1.68 1.87 1.92 1.87 1.92

1.96 1.92 1.96 1.55

1.85 1.72 1.95 1.67

Table 27: Estimates of mean values (X), shift in X and coefficient of variation (CV) for seed for days to maturity of Viciafaba var. 05/233 HBP

Treatment


LSD (p<0.01) (p<0.01)


LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

LSD (p<0.01) (p<0.05)

Mean ± S.E.

102.27±0.23 104.07±0.23 103.87±0.19 103.80±0.20 103.03±0.16

101.00±0.19 101.20±0.28 102.13±0.24 102.33±0.23

102.73±0.21 102.8. ±0.17 102.93±0.21 103.20±0.22

Shift in X

+1.80 +1.60 +1.53 +0.76

0.77 0.58

-1.27 -1.07 -0.14 -0.06

0.88 0.66

+0.46 +0.53 +0.66 +0.93

0.78 0.16

CV. (%)

0.86 0.85 0.72 0.75 0.62

0.75 1.07 0.90 0.88

0.78 0.66 0.78 0.83

Table 28: Estimates of mean values (X), shift in X and coefficient of variation (CV) for number of fertile branches / plant of Viciafaba var.05/233 HBP.

Treatment


LSD (p<0.01) (p<0.05)


LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

LSD (p<0.01) (p<0.05)

Mean ± S.E.

10.47±1.58 12.26±2.65 7.40±1.15 8.20±1.36 14.27±2.79

10.73±1.50 8.73±1.23 9.60±1.08 7.80±1.42

5.20±0.95 6.60±0.86 6.27±0.75 7.33±0.87

Shift in X

+1.79 -3.07 -2.27 +3.80

5.72 7.61

+0.26 -1.74 -0.87 -2.67

3.88 5.17

-5.27 -3.87 -4.20 -3.14

2.95 3.93

CV. (%)

58.44 83.61 60.61 64.39 75.79

54.26 54.47 43.80 70.41

95.27 86.12 46.12 45.71

Table 29: Estimates of mean values (X), shift in X and coefficient of variation (CV) for number of pod/plant (grain) of Viciafaba var.05/233 HBP.

Treatment


LSD (p<0.01) (p<0.05)


LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

LSD (p<0.01) (p<0.05)

Mean ± S.E.

18.67±3.65 25.40±4.37 24.80±3.62 27.67±5.47 32.73±3.29

29.40±6.53 33.53±8.67 27.53±7.11 26.80±6.92

19.13±4.82 23.80±4.67 25.57±4.43 40.73±8.23

Shift in X

+6.73 +6.13 +9.00 +14.06

11.76 15.64

+10.73 +14.86 +8.86 +8.13

19.84 14.92

+0.46 +5.13 +7.06 +22.06

20.30 15.26

CV. (%)

75.79 66.73 56.45 76.64 38.98

86.08 68.98 72.05 66.45

95.51 75.89 66.67 78.17

Table 30: Estimates of mean values (X), shift in X and coefficient of variation (CV) for pod length (cm), of Viciafaba var.05/233 HBP.

Treatment


LSD (p<0.01) (p<0.05)


LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

LSD (p<O.Ol) (p<0.05)

Mean ± S.E.

5.23±1.35 5.14±1.32 4.68±0.24 4.76±0.24 4.47±0.20

5.04±0.21 5.06±0.21 4.93±0.20 4.63±0.22

5.05±0.20 9.39±0.24 5.12±0.15 4.80±0.14

Shift in X

-0.09 -0.55 -0.47 -0.76

1.18 0.86

-0.19 -0.17 -0.30 -0.60

0.78 0.59

-0.18 +4.15 -0.11 -0.43

0.72 0.54

CV. (%)

14.86 12.92 19.88 19.10 17.71

16.13 16.03 15.54 18.18

15.18 10.12 11.38 11.05

Table 31: Estimates of mean values (X), shift in X and coefficient of variation (CV) for number of seeds/ pod of Viciafaha var.05/233 HBP.

Treatment


LSD (p<0.01) (p<0.05)


LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

LSD (p<0.01) (p<0.05)

Mean ± S.E.

3.13±0.01 2.53±0.19 2.47±0.17 2.00±0.24 2.07±0.2I

2.33±0.25 2.93±0.25 2.47±0.19 2.40±0.19

3.07±0.21 2.67±0.19 2.93±0.15 2.60±0.16

Shift in X

-0.60~ -0.66 -1.13 -1.06

0.72 0.54

-0.80 -0.20 -0.66 -0.73

0.78 0.59

-0.06 -0.46 -0.20 -0.53

0.64 0.48

CV. (%)

16.48 29.33 25.94 46.29 38.65

41.82 32.76 30.13 30.70

26.05 27.14 20.24 24.33

Table 3X: Estimates of mean values (X), shift in X and coefficient of variation (CV) for 100 seed weight (g) of Viciafaba var.05/ 233 HBP.

Treatment


LSD (p<0.01) (p<0.05)


LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

LSD (p<0.01) (p<0.05)

MeaniS.E.

34.21±0.26 34.38±0.21 34.25±0.30 34.09±0.27 34.53±0.22

34.11±0.25 34.20±0.23 34.21±0.24 34.28±0.26

34.50±0.27 34.74±0.25 34.55±0.26 34.69±0.26

Shift in X

+0.17 +0.04 -0.21 +0.32

0.96 0.72

-0.10 -O.OI 0.00 +0.07

0.94 0.70

+0.29 +0.53 +0.34 +0.48

0.92 0.70

CV. (%)

2.93 2.33 3.37 3.08 2.47

2.83 2.63 2.73 2.96

3.05 2.78 2.70 2.20

Table 33: Estimates of mean values (X), shift in X and coefficient of variation (CV) for yield/ plant (g) of Viciafaba var.05/233 HBP.

Treatment


LSD (p<0.01) (p<0.05)


LSD (p<0.01) (p<0.05)

0.02%HZ 0.04%HZ 0.06%HZ 0.08%HZ

LSD (p<0.01) (p<0.05)

—

MeaniS.E.

17.08±3.3 22.68±5.86 23.13±3.36 28.58±6.93 27.95±3.06

27.22±6.14 30.03±5.64 25.19±4.89 25.37±4.18

17.05±4.63 21.40±4.51 23.47±4.13 27.77±7.49

Shift in X

+5.60 +6.05 +11.50 +10.87

16.57 12.46

+10.14 +12.95 + 8.11 +. 8.29

18.58 13.96

-0.03 +4.32 +6.39 +10.69

18.88 14.18

CV. (%)

75.63 71.17 56.16 93.78 42.40

87.37 72.69 75.10 63.71

105.17 81.61 68.22 104.40

Plate - 1 : Leaf variants isolated in Mi generation.

Fig. 1: Leaf control plant showing four leaflets.

Fig.2: Narrow leaflets.

Fig.3: Two leaflets with round margins.

Fig.4: Multileaflets

Plate -1

Plate -II: Leaf and chlorovariants isolated in Mi generation.

Fig. 1: Leaflets with rough, thick and leathery surface.

Fig.2: Albina variant showing white patches on the surface of the leaflets.

Fig.3&4: Leaflets showing dissected margins.

Fig.5&6: Chlorovariants showing yellow leaflets.

Plate - II

Plate - III: Morphological variants isolated in Mi generation.

Fig. 1: Control plant.

Fig.2: Plant

Fig.3: Dwarf variant.

Fig.4: Tall variant.

Fig.5: Bushy variant.

Plate - III

Plate IV. Mitosis & meiosis in untreated and the mutagens treated faba bean.

*

Fig.l: Prophase of mitosis (Control).

Fig.2: Metaphase of mitosis (Control).

Fig.3: Anaphase showing chromatin bridge.

Fig.4: Anaphase showing sticky chromosomes.

Fig.5: Metaphase showing fragmentation of chromosomes.

Fig.6: Metaphase-1 showing sticky chromosomes.

Fig.7: Anaphase-I (control).

Fig. 8: Anaphase - 1 showing chromatin bridge.

Fig.9: Telophase -I (control).

Fig. 10: Cytomixis and disturb polarity at Telophase -II

Fig. 11: Micronuclei at Telophase-II

Fig. 12: Disturbed Telophase-II showing two nuclei at one pole and unseperated nuclei at second pole.

Plate - IV

% ^

(4J

**

# *

f* i;-

8

H

SI

f* '.

%

.4W

12 «i

Chapter 5

DISCUSSION

Chemical mutagens are known to produce adverse effects on germination,

seedling growth and plant growth in Mj generation. Delayed maturity, varying

degrees of sterility, and reduced survival are other features recorded in M,

generation after mutagen treatments (Blixit, 1960; Sjodin, 1962; Nerkar, 1970;

Goud, 1972; Sinha and Godward, 1972; Dixit and Dubey, 1981; Parveen, 2004;

Fatma, 2007). The above mentioned attributes are generally taken as an index of

the efficiency of various treatments in inducing mutations. Both the chemical

mutagens applied during the present study produced adverse effects on

germination, seedling height, pollen fertility, and plant survival at maturity.

Concentrations of EMS used in combination with DMSO were found to be more

effective than those of EMS and HZ used in alone in most cases.

Reduction in seed germination in mutagenic treatment has been explained

due to delay or inhibition in physiological and biological processes necessary for

seed germination which include enzymatic activity (Kurbone et al, 1979),

hormonal imbalance (Chrispeeds and Varner, 1976) and inhibition of mitotic

process (Ananathaswamy et al, 1971). Reduction in seedling height may be due

to inhibition of energy supply caused by mutagens and as a resuh of inhibition of

mitosis which is primary requirement for seedling growth. Usaf and Nair (1974)

inferred that gamma irradiation interfered with the synthesis of enzymes involved

38

in the formation of auxins and thus reduced the seed germination in potatoes. The

reduction in seedling survival is attributed to cytogenetics damage and

physiological disturbances (Sato and Gaul, 1987). The biological damage was

higher at higher doses of mutagens. The greater sensitivity at higher mutagenic

level has been attributed to various factors such as changes in the metabolic

activity of the cells (Natarajan and Shivashankar, 1965), inhibitory effects of

mutagen (Sree Ramulu, 1972) and to disturbances of balance between promoters

and inhibitors growth regulators (Mcherchandani, 1975). The mutagens may also

cause disturbances in genetical and physiological activities leading to the death

of the cells.

The dose dependent pollen sterility with the increase in mutagenic

concentrations was observed in the present study. Similar results were also

reported by Vandana and Dubey (1988), Fatma (2007) in Viciafaba and Khan et

al, (2000) in Vigna radiata. The high sterility observed in the treated population

may be attributed to vast array of meiotic aberrations that were induced by

chemical mutagens leading to aberrant pollen grains. The reason of pollen

sterility caused by these chemical mutagens may be attributed to a gene mutation

or more probably invisible deficiencies. The lower concentrations of mutagens

showed less pollen sterility compared to the higher concentrations. It may be

concluded the such mutagenic treatments could be used favourably for increasing

mutation rate and obtaining a desirable spectrum of mutations in faba bean.

39

Chromosomal aberration produce by chemical mutagens are of practical

interests. Differences in both quality and kind of chromosomal aberrations

provide excellent data for the study of differential sensitivity. Sticky

chromosomes, observed at metaphase and anaphase of mitosis and metaphase- I

of meiosis, might have caused due to failure of the spindle mechanism.

According SudhaKaran (1972) the stickiness and clumping of chromosomes are

the out come of physiological effects. The alteration in the surface property of

chromosomes results in their stickiness. However, the primary cause and

biochemical basis of chromosomes stickiness are still unknown. Gaulden (1987)

postulated that sticky chromosomes may result from the defective functioning of

one or two types of specific non- histones proteins involved in chromosomes

organization, which are needed for chromatid separation and segregation. The

altered fiinctioning of these proteins leading to sticikiness is caused by mutations

in the structural genes coding for them (hereditary stickiness) or by the action of

mutagens on the proteins (induced stickiness). Chromatin bridges and micro

nuclei were described for the first time in interspecific hybrids of Glycine max x

Glycine soja by Ahmad et al, (1977) who found that the extent of abnormalities

was influenced by environmental conditions. In general, cytomixis has been

detected at a higher frequency in genetically imbalanced species such as hybrids,

as well as in apomitic, haploid and polyploidy species (Yen et al, 1993). Among

the factors proposed to cause cytomixis are the influence of genes, fixation

effects, pathological conditions, herbicides and temperature ( Cactano- percira

40

and Pagliarini, 1997). Cytomixis may have serious genetic consequences by

causing deviations in chromosome number and may represent and additional

mechanism for the origin of aneuploidy and polypoidy (Sarevlla 1958).

From the present study and the work presented by earlier workers on this

aspect especially Sjodin (1970), Gottschalk and Kaul (1980 a, b) Kaul and

Murthy (1985), Loidl (1989), Zickler and Kleckner (1999) and may other, it is

reaffirmed that meiosis is a complex, coordinate activity involving several genes

and that mutation in any one of these leads irregularities.

Enhancement of the frequency and spectrum of mutation in a

predictable manner and consequent achievements of desirable's plant

characteristics is an important goal of mutation research. Although high seed

yield is the ultimate goal for legumes breeders, yield is a composite character

and, therefore, can be manipulated through the various components

characteristics. Thus, manipulation of plant structural component to induce

desirable alternations in the yield components provides valuable material for the

breeders. A wide of range morphological variations was induced in the present

study, several of which are useful from a breeder's point of view. The differences

in the frequencies of leaf mutations may be due to the number of gene with

pleiotrophic effects as has been reported by Sjodin (1971), Rao and Jana (1976),

Filippetti and De pace (1986) and Fatma (2007) also succeeded in inducing the

leaf mutations in faba bean similar to the present finding. Study of genetic aspect

of such variations would be useful in understanding the systematic development

41

of this crop and also in the formulation of various plant types. Bushy plants

characterized by increased branching have better yield potential because of their

greater number of nodes and consequently increase number of fruits and seeds.

Small leaflet coupled with compact arrangement could be utilized to develop

dwarf plant types which could be grown at higher plant density.

Mean and coefficient of variation for nine quantitative traits of faba

bean provided ample evidence that mutagenic treatments could alter mean values

and create additional genetic variability for polygenic traits. Khan (1990) and

Wani and Khan (2006) reported variable response of quantitative characters to

various mutagenic treatments in Vigna radiata. The extent of variation in mean

values and CV was not same in two varieties showing the varietals differences.

Variety 05/249 local was found to be more sensitive than 05/233HBP. The

sensitivity of an organism depends upon the mutagen employed, genetic makeup

and physiological factors such as pH, oxygen and temperature. Genetic

differences even though very small can induce significant changes in the

mutagen sensitivity which influenced various plant characters in Mi generation

(Borojevic, 1970). Growth and yield parameters were affected by EMS alone and

in combination with DMSO and HZ treatments in various ways. Higher

concentrations of mutagens produced adverse affects on all the traits on the other

hand, lower concentrations of mutagens had no significant adverse effects on

them. Growth promoting effects of mutagens when applied at low doses have

earlier been recorded in a number of crops (Sax, 1963; Singh et ai, 1978,

42

Venkteshwarlu et al, 1978; Trivedi and Dubey, 1998; Khan and Wani 2004;

Khan and Wani, 2006; Wani and Khan, 2007).

43

Chapter-6

SUMMARY

The present study was carried out using ethyimethane suiphonate (EMS)

alone or in combination with dimethyl sulfoxide (DMSO) and hydrazine hydrate

(HZ) on faba bean {Vicia faba L.). The main objective of this study was to

explore the possibility of inducing variability for quantitative traits the in two

varieties viz., 05/249 local and 05/233 HBP of faba bean. Various other aspects

of this study were; (i) to study biological damage in Mi generation, and (ii) to

determine the frequency of morphological variations.

Ml generation was studied for such parameters as percentage of seed

germination, seedling height, pollen fertility and plant survival at maturity. A

depression in seed germination, seedling height and pollen fertility was noted

with increasing concentrations of mutagens. Such parameters were drastically

affected in the combination treatments than the mutagens used singly in both the

varieties. Chromosomes abnormalities, recorded in the present study, involved

stickiness, chromatin bridges, fragments and micronuclei. Such anomalies were

found to be directly correlated to the concentration of mutagens, EMS in

combination with DMSO induced a higher number of abnormal cells than EMS

and HZ used singly.

44

A wide spectrum of morphological variants was obtained in Mi

generation. EMS+DMSO treatments resulted in the highest frequency of

morphological variants, followed by EMS and HZ treatments.

Induced quantitative variability was studied for certain quantitative traits.

Mean values for all the nine quantitative traits shifted in both positive as well as

in negative direction. However, the shift in mean, except in few mutagenic

treatments was insignificant. The highest increase the coefficient of variation

over the control was recorded for plant height, fertile branches per plant, pods

per plant and yield per plant.

Lower concentrations of mutagens and EMS in combination with DMSO

were found to be effective in inducing variability in the two varieties of Vicia

faba.

45

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