Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
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
9J^<* "0
rvt. /\
J*
QkmEmMk §3{m (d^
(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.
,^iC^ (Sammdafi %fian)
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 ^eping 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 thin^of the Cove, cooperation
and heCp extended 6y my caring and loving friends 9^oushim, iMustadeen, (Bushra,
(Renu, Jiasia, Irm, [Manilla, Jfotiey, Zeha.
"^ocaSulary 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.
Table 17: Estimates of mean values (X), shift in X and coefficient of variation (CV)
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.
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.
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.
Table 23: Estimates of mean values (X), shift in X and coefficient of variation (CV)
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.
Table 26: Estimates of mean values (X), shift in X and coefficient of variation (CV)
for days to flowering of Vicia faba var.05/233HBP.
Table 27: Estimates of mean values (X), shift in X and coefficient of variation (CV)
for seed for days to maturity of Vicia faba var. 05/233 HBP.
Table 28: Estimates of mean values (X), shift in X and coefficient of variation (CV)
for number of fertile branches / plant of Vicia faba var.05/233 HBP.
Table 29: Estimates of mean values (X), shift in X and coefficient of variation (CV)
for number of pod/plant (grain) of Vicia faba var.05/233 HBP.
Table 30: Estimates of mean values (X), shift in X and coefficient of variation (CV)
for pod length (cm), of Vicia faba var.05/233 HBP.
Table 31: Estimates of mean values (X), shift in X and coefficient of variation (CV)
for number of seeds/ pod of Vicia faba var.05/233 HBP.
Table 32: Estimates of mean values (X), shift in X and coefficient of variation (CV)
for 100 seed weight (g) of Vicia faba var.05/ 233 HBP.
Table 33: Estimates of mean values (X), shift in X and coefficient of variation (CV)
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
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
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
Control 0.02%EMS 0.04%EMS 0.06%EMS 0.08%EMS
Control 0.02%EMS 0.04%EMS 0.06%EMS 0.08%EMS
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
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
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.
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
* * * Significant at p <0.05 and < 0.01 respectively.
Table 4: Seed germination in two varieties of Viciafaba treated with HZ.
Variety
05/249 local
05/233HBP
Treatment
Control 0.02%EMS 0.04%EMS 0.06%EMS 0.08%EMS
Control 0.02%EMS 0.04%EMS 0.06%EMS 0.08%EMS
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
-
-
V = 2 varieties; t = 5treatments; r =3 replications
Table 7: ANOVA for seed germination (for HZ 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.
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
Control 0.02%EMS 0.04%EMS 0.06%EMS 0.08%EMS
Control 0.02%EMS 0.04%EMS 0.06%EMS 0.08%EMS
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
-
-
V = 2 varieties; t = 5treatments; r =3 replications
Table 11: ANOVA for seedling height (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.
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
Control 0.02%EMS+DMSO 0.04%EMS+DMSO 0.06%EMS+DMSO 0.08%EMS+DMSO
Control 0.02%EMS+DMSO 0.04%EMS+DMSO 0.06%EMS+DMSO 0.08%EMS+DMSO
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
-
-
V = 2 varieties; t = 5treatments; r =3 replications
Table 12: ANOVA for seedling height (for EMS+DMSO 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.
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
* * * Significant at p <0.05 and < 0.01 respectively.
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
-
-
V = 2 varieties; t = 5treatments; r =3 replications
Table 13 : ANOVA for seedling height (for HZ 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.
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
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<0.05)
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
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<0.05)
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
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<0.05)
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
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<0.05)
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
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<0.05)
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
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<0.05)
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
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<0.05)
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
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<0.05)
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
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<0.05)
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
Control 0.02%EMS 0.04%EMS 0.06%EMS 0.08%EMS
LSD (p<0.01) (p<0.01)
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)
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
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<0.05)
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
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<0.05)
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
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<0.05)
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
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<0.05)
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
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<0.05)
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
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<0.05)
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
References
Ailard, R.W. (1960). Principles of Breeding, New York, London, John wiley
and sons Inc.
Auerbach, C. (1967). The chemical production of mutation, science. 158: 1141-
1147.
Ananthaswamy. H.N.; U.K. Vakil and A. Srinivasan (1971). Biochemical and
physiological changes in gamma irradiated wheat during germination.
Rad.Bot.JLi: 1-12.
Ahmad, Q.N.; E.J. Britten; D.E. Byth (1977). Inversion bridges and meiotic
behavior in species hybrids of soybeans. J. Hered. 68: 360-364.
Abdalla, M.M.F. and H.A.S. Hussein (1977). Effects of single and combined
treatments of gamma- rays and EMS on M2 quantitative variation in Vicia
faba L. Zeitschrift Pflanzenzueehtung 78: 57-64.
Abdel - Hafez, A. A.G.I, and G.Robbelen (1979). Difference in partial
resistance of barley to powdery mildew {Erysiphe graminis De.
F.sp.hordii Marchal). after chemomutagenesis .III. Agronomic
performance of the mutants, Z .? flanzenzucht 86: 99-109.
Aziz, A. Abdel-Hafez, G.T. and G. Robbelen (1980). Difference in partial
resistance of barley to powdery mildew {Erysiphe graminis) Def sp.
hordei Marchal) after chemomutagenesis. H. Reaction of mutants to
pathotypes, Euphytica 29: 755-768.
46
Abdel- Hafez, A.A.G.I. and G. Robbelen (1981). Differences in partial
resistance of barley to powdery mildew (Erysiphe graminis DC. F. Sp.
hordei Marchai) after chemomutagenesis. I. Screening of mutant under
field conditions, Z. Pflanzenzucht 81 "• 321-339.
Awan, M.A; A. Magbool and A.A. Cheema (1982). Evaluation of short stature
mutants of Basmati 370 for yield and grain quality characteristics. Pak.
J.sci.Ind.Res.25(3V.67-71.
Awan, M.A. and A.A. Cheema (1988). New mutant genes for early maturity
and dwarfism in Basmati rice {Oryza sativa L.) SABRO J. 20(5) 56-61.
Bhaduri, P.N.; A.T. Natarajan (1953). Studies on the effect of nitrogen
mustard on chromosomes in somatic and gametic plant tissue .Ind. J.
Genet. 16: 8-23.
Blixit, S. (1960). Quantative studies of induced mutations in peas. IV.
Segregation after mutation. Agri. Hortique Genetica 18: 219-227.
Bianchi, A.; G. Mariani and P. Uberti (1961). Mutations induced in endosperm
and seedling of maize following X-irradiation and Die-poxbutane
treatment of mature pollen. Proc. Effects of Ionizing Radiation on seeds
(Symp. Karlsruhe, 1960), IAEA, Vienna.
Bhatia, C.R. (1967). Increased mutagenic effect of ethylmethane sulphonate.
Mutation Research 4: 375-376.
47
Borojevic, K. (1970). Factors influencing the mutant spectrum and tiie quality of
mutants. In: Mannual of mutation breeding. IAEA. Tech.Rep Series.
No.l 19. Vienna, pp.125.
Bond, D.A.; D,A. Lawes; G.C. Hawtin, M.C, Saxena, and J.S. Stephens
(1985). Faba bean {Viciafaba L.). pp. 199-265. In: R.J. Summerfield and
E.H. Roberts (eds.), Grain Legumes Crops .William Collins sones Co.
Ltd. 8 Grafton street, London WIX SLA, UK.
Bond, D.A. (1987). Recent developments in breeding field beans {Viciafaba L.).
Review of Plant Breeding. 99: 1-26.
Bakheit, B.R. and E.E. Mahady (1988). Variation correlations and path
coefficient analysis for some characters in collections of faba bean (Vicia
faba L.) FABIS Newsletter 20: 9-14.
Bhat, T.A.; A.H. Khan; S. Parveen and F.A. Ganai (2005). Clastogenic effect
of EMS and MMS in Viciafaba L. J. cytol. Genet. 6{NS}: 117-122.
Cubero, J.I. (1974). Evolutionary trends in Viciafaba. Theoretical and Applied
Genetics 43: 59-65.
Chrispeeds, M.J. and J.E. Varner (1976). Gibberllic acid induced synthesis
and release of A- analysis and ribonuclease by isolated barley aleurone
layers, Plant Physiology 42: 346-406.
Chapman, C.P. (1981). Genetic variation within Vicia faba L. FABIS.
ICARDA, June. ICARDA, Aleppo, Syria 12 pp.
48
Chapman, C.P. (1986). Third conspectus of genetic variation within Viciafaha
L. FABIS, ICARDA, Aleppo, Syria 54 pp.
Chavan, J.K., L.S. Kute and S.S. Kadam (1989). In: CRC Hand book of world
Legumes.pp.223-245. D.D. Salunkhe and S.S.kadam (eds.), Boca Raton.
Florida, USA: CRC Press.
Caetano-Pereira, CM. and M.S. Pagliarini (1997). Cytomixis in maize
microsporocytes. Cytologia 65: 351-355.
Dixit, P. and Dubey, D.K. (1981). Studies on the effect of separate and
simultaneous application of gamma rays and NMU on lentil {lens
culinaris Medic). 1. Germination, growth, fertility and yield.Bot. Progress
4: 10-15.
Duke, J.A. (1981). Hand book of legumes of world economic important. Plenum
Press, New York. pp. 199-265.
Donini, B.; T. Kawai and A. Micke (1984). Spectrum of mutant characters
utilized in developing improved cultivars, in: Selection in Mutation
Breeding Vienna, International Atomic Energy Agency, pp. 7-31.
Daskalov, S. (1986). Mutation breeding in pepper, Mutat. Breed. Rev. 4:1-26.
Ehrenberg, L.; and A. Gustafsson (1957). On the mutagenic action of ethylene
oxide and diepoxybutane in barley. Hereditas 43: 595-602.
Ehrenberg, L. (1960). Chemical mutagenesis: Biochemical and chemical points
of view on mechanism of action. Erwin - Baur- Gedachtnis-vorlesungen I.
Abh. Dt. Akad. Wiss. Berl. (Med.), i : 124-136
49
Enken, V.B. (1967). Manifestation of Vavilov's law of homologous series in
hereditary variability in experimental mutagenesis, in: Induced Mutation
and their utilization, Berlin, Akademic verlag, pp. 123-129.
El-Shouny, K.A. and A.A. EI- Hosary (1983). Effect of some chemical and
physical mutagens on Vicia faba L. 3. Effect of EA, DES, gamma rays
and some combination of them and frequency and spectrum of
morphological and physiological mutations induced in M2 and M3 Pages
73-83 in proceedings of the first Conference of Agronomy, Cairo (Egypr).
Vol.2. Egyptian Society of Crop science, Cairo, Egypt.
Freisleben, R. and A. Lein (1942). Uber die Auffindung einer
mehtauresistenten MU tante nach roentgen bestrahlung einer anfalligen
Linie Von Sommergeste, Naturewissenschaften. 30: 608.
Freisleben, R. and A.Lein (1943a). Vorabeiten Zur Zuchterischen Auswertung
rontgeninduzierter Mutation I, Z. Pflanzenzucht 25: 235-254.
Freisleben, R. and A. Lein (1943b). Vorabeiten Zur Zuchterischen Auswertung
rontgeninduzierter Mutation 11, Z. Pflanzenzucht 25: 255-283.
Filippetti, A. and C. De Pace (1983). Improvement of grain yield in Vicia faba
L. by using experimental mutagenesis. I. Frequency and types of
mutations induced by gamma radiation. Genetica Agraria 37: 53-68.
Filippetti, A. and C.F. Marzano (1984). New interesting mutants in Vicia faba
L. after seed treatment with gamma rays and ethyl- methane- sulphonate.
FABIS Newsletter No. 9: 22-25.
50
Filippetti, A. (1986). Inheritance of determinate growth habit induced in Vicia
faba major by ethyl methane sulphonate (EMS). FABIS Newsletter jo:
12-14.
Fillipetti, A. and C. De Pace (1983). Improvement of grain yield in Vicia faba
L. by using experimental mutagenesis .1. Frequency and types of
mutations induced by gamma- radiation. Genetica Agraria XXXVII: 53-
68.
Fillipetti, A. and C. De Pace (1986). Improvent of seed yield in Vicia faba L. by
using experimental mutagenesis. II. Comparison of gamma radiation and
ethyl methane sulphonate (EMS) in production of morphological mutant.
Euphytica 35: 49-59.
Fatma, S. (2007). Studies induced variability in faba bean. M.sc. dissertation,
Aligarh Muslim University, Aligarh
Gager, e.s. (1908). Effects of the rays of radium on plant, Mem. N.Y.Bot.Gard.
4: 278.
Gustafsson, A. (1947). Mutation in agricultural plants. Hereditas 31: I-IOO.
Gregory, W.C. (1956). Induction of useful mutations in the peanut, Brookhaven
symp. Bio I. 9:177-190.
Gichner, T. and J. Veleminsky (1967). The mutagenic activity of 1-alkyI-l-
nitrosoureas and I- alkyl-nitro-I-nitroso guanidines, Mutat. Res. 4: 207-
212.
Goud, J.V. (1972). Mutation studies in sorghum. Genetica polonica 22: 30-40.
51
George, K.P. and G.C Nayar (1973). Early-dwarf mutant in seed induced by
gamma- rays, curro sci. 42: 137-138.
Gopai, J. (1977). Cytogenetical studies in Trigonella M.sc. thesis, submitted to
Punjab Agricultural university.
Gottasehalk, W. and M.L.H. Kaul (1980a). Open problems in phyploidy
research. The Nucleus 21.: 99-112.
Gottasehalk, W. and M.L.H. Kaul (1980b). Asynapsis and desynapsis in
flowering plants: II. Desynapsis. The Nucleus 23: 97-120.
Gottschalk, W. and G.Wolff (1983). Induced Mutations in plant Breeding.
Monographs on Theoretical and Applied Genetics No.7, Berlin, Springer-
Verlag.
Gaulden, M.E. (1987). Hypothesis: some mutagens directly alter specific
chromosomal proteins (DNA topoisomerase II and peripheral proteins) to
produce chromosomes stickiness, which causes chromosomes aberration.
Mutagenesis 2: 357- 365,
Hoffmann, W. (1959). Neuere Moglichkeiten Mutationszuchhtung Z.
Pflanzenzucht 41: 371-394.
Herskowtz, I.H. (1962). Genetics, Boston, Toronto, Little, Brown and company.
Hanna , W.W. (1982). Mutation breeding in pearl millet and sorghum, Mutat.
Breed. Rev. i : 1-13.
Hussein, L.A. and M.Saleh (1985). Antinutritional factors in faba beans.
pp.257-269. In: M.C. Saxena and S.verma (eds.), proceedings of the
52
International workshop on faba bean, Kabuli chickpeas and Lentils in the
1980s. ICARDA, 16-20 May, 1983. Aleppo.Syria.
Heath, M.C; C.J. Pilbeam; B.A. Mckenszier; and P.D. Hebblethwaite (1994).
Plant architecture, competitive ability and crop productivity in food
legumes with particular emphasis on pea (pisum sativum L.) and faba bean
(Vicaifaba L.).pp. 771-790. In: F.J.Muehlbauer and W.J. Kaiser (eds.).
Expanding the production and use of cool season legumes, Kluwer
Academic publishers, Dordrecht, The Netherlands.
Hulse, J.H. (1994). Nature, Composition and utilization of food legumes.77-97.
In: F.J.Muehlbauer and W.J.Kaiser (eds.). Expanding the production and
use of cool season Food Legumes. Kluwer Acedemic Publishers,
Dordrecht, The Netherlands.
IAEA (1961). Effect of ionizing Radiations on seeds. (Proceedings of
symposium) Vienna, Intenational Atomic Energy Agency.
IAEA (1965). The use of Induced Mutations in Plant Breeding. (Report of the
FAO/ IAEA technical Meeting. Rome, 1964), Oxford, Pergamon press.
IAEA (1971). Rice Breeding with Induced Mutation III., Vienna, International
Atomic Energy Agency.
IAEA (1977). Induced Mutations against plant Diseases, Vienna, International
Atomic Energy Agency.
IAEA(1983). Induced Mutation for Disease Resistance in crop plants II, Vienna,
International Atomic Energy Agency.
53
IAEA (1884a). Conclusion and recommendations, in: Selection in Mutation
Breeding, Vienna, International Atomic energy Agency, 157-169.
IAEA (1984b). Cereal Grain protein Improvement, Vienna, International Atomic
Energy Agency.
IAEA (1984c). Semidwarf cereal Mutants and their use in cross Breeding II,
Vienna, international, Atomic Energy Agency ,TEC-DOC- 307.
Joergenson, J.H. (1975). Identification of powdery mildew resistant barley
mutants and their allelic relationship. Barley Genetics III, 446-455.
Jaranowski, J. and A. Micke (1985). Mutation breeding in peas. Mutation
Breeding. Review No.2, Vienna, International Atomic Energy Agency.
Jambunathan, M.M.; R.H.L. Blain; K.H. Dhindra; L.A. Hussein, K. Kogure,
L.Li-Tuan, and Youssef (1994). Diversifying use of cool season food
legumes thorugh processing pp.98-112. In: F.J. Muehlbauer and W.J.
Kaiser (eds.), Expanding the production and use of cool season Food
Legumes. Kluwer Academic publishers, Dordrecht, The Netherlands.
Joshi, P. and R.C. Verma (2004). Radiation induced pod and seed mutants in
faba bean {Viciafaba L.). Indian J. Gent. 64 (2): 155-156.
Keil, D.L. (1965). DMSO shows great promise as carrier of agricultural
toxicants. Agri, chem. 20: 23-24.
Kawai, T. (1969). Relative effectiveness of physical and chemical mutagens
Induced Mutations in plant IAEA Vienna, 137-151.
54
Kulkarni, L.G. (1969). Induction of useful mutations in castor. Radiation and
Radionimetic substances in Mutation Breedin^i"-Proceeding ' C^a
symposium Bombay, pp. 293-299.
Kay, D. (1979). Crop and product digest No. 3- Food legWe^. London: Tropical/
Product Institute. UK. pp. 26-47. --^-.T - -
Kurobane, I.H.; H. Yamaguchi; C. Sander and R.A. Nilan (1979). The effects
of ganmia irradiation on the production and secretion of enzymes and
enzymatic activities in barley.Env.Exp.Bot. 19: 75-84.
Konzak, C.F. (1984). Role of induced mutation, in: crop Breeding, a
contemporary Basis (Eds vose, P.B. and Blixt , S.G.). Oxford, Pergamon
press, pp. 216-292.
Kaui, M.L.H. and T.G.K. Murthy (1985). Mutant genes affecting higher plant
meiosis. Theo. Appl.Genet. 70: 449-466.
Khan, S. (1990). Studies on chemical mutagenesis in mungbean [Vigna radiate
(L.) Wilczek]. Ph.D. thesis, Aligarh Muslim university, Aligarh
Kumar, S., Nandana and Dubey, D.K. (1993). Studies on the effect of gamma
rays diethyl sulphate (DES) on germination, growth, fertility and yield in
faba bean. FABIS Newsletter B^: 15-18.
Kumar, S. and D.K. Dubey (1996). Influence of separate and simultaneous
application of gamma rays, DES and EMS on meiosis in khesari {Lathyrus
sativus L.). J. Genet Breed (In press).
55
Khan, S. and B.A. Siddiqui (1996). Mutation genetic studies in Mungbean 11.
Frequency spectrum of morphological mutants. Thai J. Agri. Sci. 29
(April): 173-182.
Khan, S.; Rehman, M.; M.Bhat and B.A. Siddiqui (2000). MMS induced
biological damage and polygenic variability in green gram [ Vigna radiate
(L.) Wilczek]. 23(2): 126-129.
Khan, S. and M.R. wani (2004). Studies on the effect of EMS and MMS on
biological damage and quantitative characters of mungbean. VEGETOS
17: 15-20
Khan, S. and Wani, M.R. (2006). MMS and SA induced genetic variability for
quantitative traits.in mungbean, Indian J. Pulses Res. 19 (1): 50-52.
Love lock, J.E. and M.W.H. Bishop (1959). Preventation of freezing damage to
living cells by dimethyl sulphoxide. Nature 183: 1394-1395.
Ladizinsky, G. (1975). On the origin of the broad bean Vicia faba L. Isreal J.
Botany 24: 80-88.
Lawley, P.D.; D.T. Orr and M. Tarman (1975). Isolation and identification of
products from alkylation of nucleic acids: Ethyl and isopropyl purines.
Biochem. J. 145: 73-84.
Lawes, D.A. (1980). Recent developments in understanding, improvement and
use of Vicia faba. pp. 625-636. In: R.J. Summerfield and A.H. Bunting
(eds.), Advances in legumes science. Proceedings of the International
56
legume conference, Kew, 31 July-4 August 1978, Royal Botanic Garden,
Kew, the Missouri Botanical Garden, And the university of Reading , UK.
Loidl, J. (1989). Effects of elevated temperature on meiotic chromosomes
synapsis in Allium ursinum . Chromosoma 97: 449-458.
MuIIer, H.J. (1927). Artificial transmutation of the gene, science 66: 84-87.
Moos ,W.S. and S.E. kim (1966). Radioprotective effect of topically applied
dimethyl sulphoxide in mice. Experientia 22 : 814.
Mabuchi,T. and T.J. Arnason (1969). EMS induced mutations in maize .Maize
Genet. Coop. Newsl., 43: 166-167.
Meherchandani, M. (1975). Effect of gamma radiation of dominant seeds of
Avena sativa L. Rad Bot. \5_: 439-445.
Micke, A. (1979). Use of mutation induction to alter the ontogenic pattern of
crop plants, in: crop improvement by induced Mutation, Gamma Field
Symposia No. 18, Ohmiya, Japan, Institue of Radiation Breeding, pp. 1-
23.
Micke, A.; B. Donini and M. Maluszynski (1980). Induced mutation for crop
improvement. Mutation Breeding Review No. 7. IAEA, Vienna.
Micke, A. (1983). International research Programmes for the genetic
improvement of grain proteins, in: seed proteins: Boichemistry, Genetics,
Nutritive value (Eds Gottschalk,W. and MuUer , H.p). The Hauge, Boston,
London, Martinus Nijhoff / Dr. W. Junk Publishers, pp. 25-44.
Micke, A. (1984). Mutation breeding of grain legumes, plant soil 82: 337-358.
57
Muller, H.P. (1984). Breeding for enhanced protein, in: crop Breeding, a
contemporary Basis, Oxford, Pergamon Press, pp. 382-399.
Maluzynski, M.A. Micke and B. Donini (1986). Genes for semidwarf in rice
induced by mutagenesis, in: Rice Genetics (Proc. International Rice
Genetics Symposium, Los Banos.
Micke, A; B. Donini and M. Maluszynski (1993). Les mutaions induties en
amelioration des plantes, Mutat. Breed. Rev. 9: 1-44.
Malusznski, M. L.; H. Vanzanten; B, Brunner; F.J. Ahloowalia; Zapata and
E.Week (1995). (Mutation techniques in plant Breeding, in: Induced
Mutations and M (llcellar Techniques for crop Improvement,
International 'Atomic Energy Agency, pp. 489-504.
Natarajan, A.T. and G. Shiva Shanker (1965). Studies on modification of
mutation responses of barley seeds of ethylmethane sulphonate. Z.
Vererburgstehre. 43: 69-76.
Nilan, R.A.; C.F. konzak; J. Wagner and R.R. Legault (1965). Effectiveness
and efficiency of radiations for induced genetic and cytogenetic changes,
in: the use of Induced Mutations in plant Breeding, (Report of the
FAO/IAEA Technical Meeting, Rome 1964), Oxford , Pergamon Press,
pp. 71-89.
Natarajan, A.T. and M.S. Ramanna (1966). Modification, AT. and M.S.
Ramnna .Modification of relative mutagenic efficiency barley of
mesyloxyesters by different. Nature 211: 1099-1100.
58
Narayan, K.R. and C.A. Konzak (1969). Influence of chemical post treatment
on mutagenic efficiency of alkylating agents Induced Mutation in plant,
IAEA Vienna, 281-304.
Nerker, Y.S. (1970). Study on the induction of mutation in Lathyrus sativus with
special reference to the elimination of neurotoxic principle. Ph.D.Thesis,
lARI, New Delhi.
Nayar, G.G. (1974). Yield potential of a radiation induced early-dwarf mutant in
linseed in: use of radiations and radioisotopes in studies of plant
productivity, proceedings of a symposium, Pantnagar, India, pp. 109-107.
Natarajan, A.T.; S.M. Sikka and M.S. Swaminathan (1985). Polyploidy,
radiosensitivity and mutatation frequency, in wheat .Proc. 2 UN Int.
conf. peaceful uses Atom Energy. UN, New York. 27: 321-331.
Nanda, H.C.; M. Yasin; C.B. Singh, and S.K. Rao (1988). Effect of water
stress on dry matter production, harvest index, seed yield and its
components in faba bean (Viciafaba L.) FABIS Newsletter 2i: 26-30.
Oehlkers, F. (1943). Die Auslosung Von choromosomes mutation en in der
Meiosis durch Ein Wirkung Von Chemkalien.Z. induct. Abstamm-U.
Vererlehre. 81: 313-341.
Osterman-Golkar, S., K. Ehrenberg and C.A.,Wachtmaster (1970). Reaction
kinetics and biological action in barley of monofunctional methane
sulfonic esters. Radiat. Bot. 10: 303-327.
59
Parveen, K. (2004). Studies on the induction of polygenic variability in chick
pea (cicer arietinum L.). M.Phil dissertation, Aligarh. Muslim. University,
Aligarh
Prashant, J. and R.C. Verma (2005). Ethyl methane sulphonate (EMS) induced
(Partial). A synaptic mutant in faba bean (Viciafaba L.). Cytologia 70(2):
143-147.
Perveen, R. (2006). Cytomorphological studies in two economically important
mutants of Viciafaba L. M.sc. dissertation, Aligarh Muslim University,
Aligarh
Ramiah, K. and M.B.V.N. Rao (1953). Rice breeding and genetics. Indian C.
Agri. 40: 335-355.
Rao, R.N. and A.T.; Natarajan (1965). Mutagenecity of some alkylalkane
sulphonates in barley. Mutat. Res. 2: 132-148.
Ramanna, M.S. and A.T. Natarajan (1966). Chromosomes breakage induced
by alkylalkana sulphonate under different physical treatment conditions
Chromosomes (Berlin). 18: 44-59.
Rao, R. N. and A.T. Natarajan (1967). Somatic association in reaction to
chemically induced chromosomes aberration in Vicia faba Genetics 57:
821-835.
Raut, R.N.; H.K. Jain; and R.S. Panwar (1971). Radiation induced photo-
insensitve mutants in cotton, curre. sci. 40: 383-384.
60
Rao, C.H.; J.L. Tickoo; H. Ram and H.k. Jain (1975). Improvement of pulse
crops through induced mutations: Reconstruction of plant type, in:
Breeding for seed protein Improvement using Nuclear Techniques,
Vienna, International Atomic Energy Agency, pp. 125-131.
Rao, S.A. and Jana (1976). Leaf mutations induced in black gram by X rays and
EMS. Enviromental and Experimental Botany 16: 151-154.
Robbelon, G.; A.F. Abdel -Hafen; and M. Reinhold (1977). Use of mutants to
study host/Pathogen relations, in: Induced Mutation against plant
Diseases, Vienna, International Atomic Energy Agency, pp. 359-374.
Reddi, T.A. (1979). Effect of dimethyl sulphoxide on mutagenic action of
diethyl sulphate on rice. Curr. Sci. 48: 540-541.
Reddy, T.P. and G.M. Reddy (1979). Presoaking and the inducing of
chlorophyll mutations with diethylsulphate on oryza sativa L., Oryza. 7:
77-84.
Ricciardi, L., C. de Pace and A. Filippetti (1982). Intergenotypic competition
in Viciafaba. Preliminary results. Genetica Agraria XXXVII, i ^ : 84-104.
Robbelen, G. (1990). Mutation breeding for quality improvement -A case study
for oil - seed crops. Mutat. Breed. Rev. 6:1-44.
Rutger, J.N. (1992). Impact of mutation breeding in rice -A review, Mutat.
Breed. Rev. 8: 1-24. 17'0}
Stadler, L.J. (1928a). Mutation in barley induced by X-rays and radium, science
68: 186-187.
61
Stadler, L.J. (1928b). Genetic effects of X- rays on maize, proc.Natt Acad. Sci.
14: 69-75.
Swain, C.G. and C.B. Scott (1953). Quantitative correlation of reactive rates:
Comparison of hydroxide ion with other nucleophilic regions towards
alkyl halides, ester, epoxides and acyl halides. J. Am. chem. soc. 75: 141-
147.
Sarvella, P. (1958). Cytomixis and the loss of chromosomes in meiotic and
somatic cells of Gossypium. Cytologia 23: 14-24.
Scholz, F.; and ch. Lehmann (1958). Die Gaterslebener Mutation der Saatgerste
in Bazieehung zur Formen - mannigfaltigkeit derArt Hordeum Vulgare
L.S.I.1., kulturpflanze. 6: 123-166
Sjodin, J. (1962). Some observations in Xi and X2 of Vicia faba L. after
treatments with different mutagens. Hereditas 48: 565 - 586.
Swaminathan, M.S.; V.L. chopra and S. bhaskaran (1962). Chromosomes
aberration and the frequency and the spectrum of mutations Induced by
ethylmethane sulphonate in barley and wheat. Ind .J. Genet. 22: 192-207.
Sax, K. (1963). The stimulation of plant growth by ionizing radiations. Radiation
Botany 3: 179-186.
Sjodin, J. (1970). Induced asynaptic mutant in Vicia faba L. Hereditas 66: 215-
232.
Sjodin, J. (1971). Induced morphological variations in Vicia faba L Hereditas.
67: 155-180.
62
Sudhakaran, I.V. (1972). Influence of gamma rays on the cell division in the
seed roots of irradiated dry seeds of Vinea rosea L.Cytologia 37: 445-
456.
Swaminathan, M.S. (1972). Mutational reconstruction of crop ideotypes, in:
induced mutation and plant improvement, Vienna, International Atomic
Energy Agency, pp. 155-171.
Saree Ramulu K. (1972). A comparison of mutagenic effectiveness and
efficiency of NMU and NMG in sorghum. Theor. Appl. Genet. 4: 101-
106.
Sinha, S.S.N, and M.B.E. Godward, (1972). Radiation studies in lens culinaris.
Indian Journal of Genetics and plant Breeding 32: 331-339.
Sigurbjornson, B.; and A. Micke (1974). Philosophy and Accomplishments of
Mutation Breeding. In: Polyploidy and induced Mutation in plant
Breeding, Vienna, International Atomic Energy Agency, 303-343.
Singh, R.B.; B.D. Singh; R.M. Singh and Vijay Laxmi (1978). Seedling
injury, pollen sterility and morphological mutations induced by gamma
rays and EMS in pearl millet. Indian Journal of Genetic and Plant
Breeding 38 (3): 380-389.
Shaikh, M.A.Q. ,Z.U. Ahmed ; M.A. Majid; A.D. Bhuiya; A.K. KauJ and
M.M. Miz (1980). Development of high yielding chickpea mutant.
Mutation Breeding News letter No. 16:1-3, IAEA, Vienna.
63
Singh, R.R. and S.S. Raghuvanshi (1980). Effect of DES in combination with
DMSO on 2x and 4x Trigonella foenum greacum L., Ind. J. Hort. 34
310-313.
Sokou, J.P. (1982). Callose formation responsible for powdery mildew
resistance in barley with genes in the ml-o- locus, phytopathol .Z., 104:
90-95.
Singh, V.P. (1984). Natural out -crossing in faba beans under north Indian
conditions. FABIS Newsletter. No. 9: 27-28.
Sato, M. and H.Gaul (1987). Effect of EMS on fertility of barley, Radiation
Bot.. 7: 7-15.
Smart, J. (1990). Grain Legumes: Evolution and genetic resources. Cambridge
university press, Cambrodge, UK. 200p.s
Spiegel-Roy, P. (1990). Economic and agricultural impact of mutation breeding
in fruit trees, Mutat. Breed. Rev. 6: 1-44.
Scarascia - Mugnozza, G.T. et ai, (1993). Mutation breeding for durum wheat
{Triticum turgidum sp. durum Desf). Improvement in Itlay, Mutat. Breed.
Rev. 10: 1-28.
Sinha, A.K. and Gandhi (1994). Mitotic abnormalities caused by gamma rays
in some Vicia species. 11 (3-4): 183-184.
Tomar, Y.S.; V.P. Singh and S. Lai (1986). Promising new grain legumes
(Faba bean and Rice bean) for irrigated areas. Indian Farming. 36(7): 11-
12.
64
Trivedi, S.C. and D.K. Dubey (1998). Effect of gamma rays on seed
germination and seedling growth in Triticale var. Rahum. Abstract in
proceedings of the 69* Indian science congress part III- 24.
Usaf, K.K. and P.M. Nair (1974). Effect of gamma irradiation on the lAA
synthesizing systems and its significance in sprout inhibition of potatoes.
Rad.Bot.,i4:251-256.
Venkateshwarlu, S.; R.M. Singh and B.D. Singh (1978). Radiosensitivity and
frequency of chlorophyll mutations in pigeon pea. Indian Journal of
Genetics and Plant Breeding. 38: 90-94.
Vogel, E. and A.T. Natarajan (1982). The reaction between reaction kinetics
and mutagenic action of monoflinctional alkylating agents in higher
eukaryotic systems: Inter species comparisons. In chemical Mutagens:
Principles and method if their Detection (eds De Serres, F.J. and
Hollander, A.), Plenum Press, New York. 7: 295-336.
Vandana and D.K. Dubey (1988). Effect of ethyl methane sulphonate (EMS)
and diethyl sulphate (DES) on germination, growth fertility and yield of
Viciafaba L. FABIS NEWSletter 20: 25-29.
Vandana (1990). Studies on mutagenesis and polygenic variability induced by
EMS and DES in faba bean {Vicia faba L.) P.hD. Thesis.Kanpur
university, Kanpur.
65
Vandana, D.K. Dubey (1991). Frequency of chlorophyll, sterile and vital
mutations induced in faba bean {Vicia faba L.). J- Indian bot. soc, 79:
419-420.
Vandana (1991). Studies on mutations induced by EMS and DES in faba bean: I
chlorophyll and sterile mutations. FABIS Newsletter 28-29: 11-14.
Vandana and D.K. Dubey (1992). Mitotic anomalies induced by EMS and DES
in Vicia faba. L. J. Indian bot. Soc. 1\: 179-180.
Vandana (1992a). Studies on mutations induced by EMS and DES in faba
bean.III. vital mutations affecting maturity period and reproductive parts.
FABIB Newsletter 30: 7-
Vandana (1992b). Studies on mutations induced by EMS and DES in faba
bean. 11. Vital mutations affecting vegetative organs, FABIS Newsletter
30:3-6.
Vandana, D.K. Dubey (1993). Path analysis in faba bean. 32: 23-24.
Vandana, (1993). Mutagens sensitivity effects by ethyl methane sulphonate
(EMS) and diethyl sulphate (DES) in faba bean. FABIS Newsletter.
Vandan, K. Sushi! and D.K. Dubey (1996). Meiotic anomalies induced by
EMS and DES in faba bean {Vicia faba L.). J. Indian bot. Soc. 75: 237-
240.
Vandana, S. Kumar and D.K. Dubey (1996). Meiotic anomalies induced by
EMS and DES in faba bean.(Vicia faba L.). J. Indian bot. Soc. 75: 237-
240.
66
Wang, L.; Q. Fan; J. Shi; and Z. Wang (1986). Studies on improving
efficiency for inducing mutation of wheat hybrid by irradiation, in: Proc.
International symposium on Plant Breeding by inducing Mutation and In-
vitro biotechniques. pp. 39-44.
Wani, M.R. and S. Khan (2006). Estimates of genetic variability in mutated
populations and the scope of selection yield attributes in Vigna radiata
(L). wilezek. Egyptain Journal of Biology. 8L1-6.
Wani, M.R. and S. Khan (2007). Genetic variation and scope of selection for
high yielding mutants in mungbean. J.phytol.Res. 20(2): 309- 311.
Yen, C; J.L. Yang and G.L. Sun (1993). Intermeiocyte connection and
cytomixis in intergeneric hybrid Roegeneria ciliares (Trin) Nevski with
Psathyrosatachys luashancia Keng. Cytologia. 58: 187-193.
Yasin, M. (1996). Induced leaf variationsin faba bean. FABIS Newsletter 38/39
: 18-20.
Yasin, M.; G.B.Singh; V.K. Gour and H.S. Yadava (2006). Induced mutation
in faba bean {Vicia faba L. Var. minor).Indian J. Applied & Pure Bio.
Vol. 21 (2): 325-332.
Zickler, D. and N. Klecker (1999). Meiotic chromosomes: Intergrating
structure and function. Annu. Rev. Genet. 33: 603-754.
67