STUDIES ON THE INDUCTION OF POLYGENIC
VARIABILITY IN CHICKPEA {Cicerarietinum L.)
DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF
iEaHter 0f Pi|tloHO|ji|g IN
BOTANY
KOUSER PARVEEN
DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
2004
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DS3437
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S A M I U L L A H KHAN M Sc , Ph D , FISG Lecturer
Department of Botany ALIGARH MUSLIM UNIVERSITY ALIGARH - 202 002 (India) Phone (Res) 0571-2709265
Date ^ 3 "^ * -2_oolj
CERTIFICATE
This is to certify that the dissertation entitled "Studies on the
induction of polygenic variability in chickpea {Cicer arietinum L.)"
submitted by Miss Kouser Parveen 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.
(Samidllah Khan)
Acknowledgements
I thank Almighty Allah for guiding and providing all the
channels.
I feel myself enough privileged to have completed this work
under the able guidance of Dr. Samiullah Khan, Lecturer,
Department of Botany, Aligarh Muslim University, Aligarh. I owe a
great deal of gratitude to him for solving problems, critical
suggestions and consistent encouragement throughout the
preparation of this dissertation.
I express my deep sense of thankfulness to Prof. Mohammad
Ishrat Husain Khan, Chairman, Department of Botany, Aligarh
Muslim University, Aligarh for providing me all the necessary
laboratory and field facilities.
My heartful thanks are due to all the teachers of the 'Faculty
of Cytogenetics and Plant Breeding' of the department especially to
Prof. Bahar A. Siddiqui for their valuable suggestions.
It is an opportunity to express my sincere thanks to Dr. S.K.
Chaturvedi, Senior Scientist, Indian Institute of Pulses Research,
Kanpur for providing me the seeds of chickpea varieties.
For giving me strength and support I would like to thank all my
friends particularly Nikhat, Aafia, Surraya, Shabina, Salma, Sheema
and research scholars of the department, Mohd. Rafiq Wani, Parvez
Maqbool Lone and Mohd. Shaikhul Ashraf.
I feel immense pleasure in thanking my uncle Dr. Sanaullah
Mir for his valuable advices and fatherly concern.
I am also very thankful to Mr. Kafeel A. Khan for the excellent
typing.
Last but not the least I would like to thank my parents, all
brothers and sisters for their constant encouragement, inspiration
and for always being there by my side whenever I needed them.
^ / y / ^ V ) ^ ^ ^
(Kouser Parveen)
Chapter Contents Page No.
1. INTRODUCTION 1-7 1.1. Area, production and productivity of chickpea 3 1.2. Chromosome number 4 1.3. Botany 4 1.4. Induction of genetic variability 5
2. REVIEW OF LITERATURE 8-29 2.1. Origin and distribution 8 2.2. Origin and cytological relationship 9 2.3. Some concepts in induced mutagenesis 11 2.3.1. Dose effect / LD-50 14 2.3.2. Mutagenic sensitivity 16 2.3.3. Biological damage 18 2.3.4. Induction of cytological abnormalities 20 2.4. Mutagenic effectiveness and efficiency 22 2.5. Chlorophyll mutations 24 2.6. Morphological mutations 26 2.7. Induction of polygenic variability 27 2.8. Desirable mutants 29
3. MATERIALS AND METHODS 30-36 3.1. Materials 30 3.1.1. Varieties used 30 3.1.1.1. Variety Pusa-212 30 3.1.1.2. Variety BG-256 30 3.1.2. Mutagens used 30 3.2. Experimental procedures 31 3.2.1. Preparation of mutagenic solutions 31 3.2.2. Pretreatment 31 3.2.3. Mutagen administration 31 3.3. Ml generation 32 3.3.1. Observations recorded in Ml generation 32 3.3.1.1. Seed germination 32 3.3.1.2. Seedling height 32 3.3.1.3. Plant survival 33 3.3.1.4. Pollen fertility 33 3.3.2. Morphological variants 33 3.4. Statistical analysis 34 3.4.1. Assessment of variability 34 3.4.1.1. Range 35 3.4.1.2. Mean (X) 35
3.4.1.3. Standard error (S.E.) 35 3.4.1.4. Standard deviation (S.D.) 36 3.4.1.5. Coefficient of variability (C.V.) 36
4. EXPERIMENTAL RESULTS 37-42 4.1. Seed germination 37 4.2. Seedling height 38 4.3. Pollen fertility 38 4.4. Plant survival 39 4.5. Morphological variations 39 4.6. Effects of mutagens on quantitative characters in M] 41
5. DISCUSSION 43-48
6. SUMMARY 49-50
REFERENCES 51-79
List of Tables
Table 1. Essential amino acid composition of chickpea (g/100 gm protein).
Table 2. Nutritional composition of chickpea (100 g of dry edible parts).
Table 3. Area, production and productivity of chickpea in India.
Table 4. Effect of mutagen on seed germination, plant survival and pollen fertility in two varieties of chickpea {Cicer arietinum L.).
Table 5. Effect of mutagens on seedling height in chickpea {Cicer arietinum L.) var. Pusa-212.
Table 6. Effect of mutagens on seedling height in chickpea {Cicer arietinum L.) var. BG-256.
Table 7. Frequency and spectrum of morphological variants induced by mutagens in chickpea {Cicer arietinum L.) varieties Pusa-212 and BG-256 in Mi.
Table 8. Frequency of morphological variants in various mutagens in Ml.
Table 9. Range, mean, shift in X, standard deviation (SD) and coefficient of variation (CV) for plant height (cm) in two varieties of chickpea {Cicer arietinum L.).
Table 10. Range, mean, shift inX, standard deviation (SD) and coefficient of variation (CV) for days to flowering in two varieties of chickpea {Cicer arietinum L.).
Table 11. Range, mean, shift inX, standard deviation (SD) and coefficient of variation (CV) for days to maturity in two varieties of chickpea {Cicer arietinum L.).
Table 12. Range, mean, shift in X, standard deviation (SD) and coefficient of variation (CV) for fertile branches per plant in two varieties of chickpea {Cicer arietinum L.).
Table 13. Range, mean, shift in X standard deviation (SD) and coefficient of variation (CV) for pods per plant in two varieties of chickpea (Cicer arietinum L.).
Table 14. Range, mean, shift in X, standard deviation (SD) and coefficient of variation (CV) for seeds per plant in two varieties of chickpea {Cicer arietinum L.).
Table 15. Range, mean, shift in X, standard deviation (SD) and coefficient of variation (CV) for 100-seeds weight in two varieties of chickpea {Cicer arietinum L.).
Table 16. Range, mean, shift in X, standard deviation (SD) and coefficient of variation (CV) for yield per plant in two varieties of chickpea {Cicer arietinum L.).
List of Figures
Fig. 1. Effect of mutagens on seed germination in Mi generation of chickpea (Cicer arietinum L.)-
Fig. 2. Effect of mutagens on seedling lieight in Mi generation of chickpea {Cicer arietinum L.).
Fig. 3. Effect of mutagens on pollen fertility in Mi generation of chickpea {Cicer arietinum L.).
Fig. 4. Effect of mutagens on plant survival at maturity in Mi generation of chickpea {Cicer arietinum L.).
Chapter 1
INTRODUCTION
Our country can take a rightful pride in attaining self sufficiency
in the production of food grains but it has miserably failed in case of
pulses, for which we are even today dependent on import to a large
extent. For vegetarians, pulses constitute a major source of protein.
The food production has crossed 200 million tonn mark, yet crop
imbalances are still there. This is because certain crops like pulses and
coarse grains have not experienced the impact of green revolution.
Pulses form an integral part of vegetarian diet of Indian
subcontinent. India is the major pulse growing country of the world
accounting 1/3 of the total world production of pulses (57.63 metric
tones). India contributes about 14.42 metric tones from an area of
22.24 million hectares (Lai, 1997; FAO, 1998). Pulse crops, also called
as grain legumes, have also been valued as food, fodder and feed and
have remained as a mainstay of Indian agriculture for centuries. Pulse
crops play an important role in the agricultural economy of India by
virtue of their ability to fix atmospheric nitrogen in symbiotic
association with rhizobium.
The second unique feature of pulse crops is their deep
penetrating root system which enables them to utilize the limited
available moisture of soil more efficiently than any other crops
including cereals and also contribute substantially to the loosening of
the soil.
Chickpea (Cicer arietinum L.), also called as Bengal gram, is an
important source of human food and animal feed and plays a key role
in the maintenance of soil fertility in the wheat based systems of the
dry rainfed areas of Indian subcontinent, West Asia and North African
regions. Nutritionally, the importance of chickpea in human food is
reflected by its high seed protein content (Table 1 and 2), a better
protein digestibility than several other pulses. The amino-acid
composition of chickpea protein complements that of cereals and
therefore chickpea and cereals make an integral part of several
traditional foods in India.
Table 1: Essential amino acids composition of chickpea (g/lOOg protein)
Amino acids Chickpea
Lysine 6.3
Threonine 3.4
Valine 5.5
Leucine 8.2
Isoleucine 6.0
Methionine 1.2
Tryptophan 0.8
Phenylalanine 4.9
Arginine 6.9
Histidine 2.3
Source: Plant Breeding Advances and in vitro Culture (eds.) Bahar A. Siddique and Samiullah Khan
Table 2: Nutritional composition of chickpea (100 g of dry edible parts)
Crop Energy Protein Oil Total Fiber Ash Ca P Fe k.cal. (g) (g) carbo- (g) (g) (mg) (mg) (mg)
hydrate (g)
Chickpea 396 19.4 5.5 70.5 7.4 3.4 280 301 12.3
Chickpea has two principal cultivated types namely desi or
brown chickpea and kabuli or white chickpea. Desi type is generally
grown in Indian subcontinent and has seeds normally smaller in size
whereas seeds of kabuli type are bold and is preferably grown in
Afghanistan, Iran and other Mediterranean countries.
1.1. Area, production and productivity of chickpea
India occupies the maximum area in the world followed by
Pakistan in chickpea production. It is grown as cold weather rabi crop
either as monoculture or mixed with wheat, barley and rice. Being a
legume crop it is much esteemed as a rotation crop with cereals like
bajra, jawar, wheat, barley and rice. The best time for sowing of
chickpea is mid October with onset of cool weather when the daily
average temperature is around 25°C. Among different pulse crops,
chickpea occupies a premier position in the country both in area and
production. The country's chickpea production during 2001-2002 was
5.27 million tones (Table-3) against a target of 6.5 million tones. The
area under chickpea fell sharply in major chickpea growing regions
contributed to the short fall in chickpea production.
Table 3: Area, production and productivity of chickpea in India.
Year
1998-99
1999-2000
2000-2001
2001-2002
Area (million hectares)
7.22
8.69
6.82
6.09
Production (million tones)
6.01
6.96
3.87
5.27
Productivity (kg/hectare)
832
802
806
865
Source; The Hindu Survey of Indian agriculture
1.2. Chromosome number
The somatic chromosome number of chickpea and in several
other species of Cicer is known to be 2n = 16 (Lidizinsky and Adier,
1976; Singh et ai, 1984; Gupta and Sharma, 1991). However there are
also reports of 2n = 14 (Vander Maesen, 1972) but such plants with 2n
= 14 are rare and may not be able to maintain themselves in nature.
1.3. Botany
Chickpea belongs to family Fabaceae. The plant has tap root,
provided with nodules. Stem is erect or suberect and covered with
glandular hair. Leaves are imperipinnately compound, oval with 9-15
pairs of leaflets. Flowers are bisexual with papilionacious corolla,
usually borne singly. The stamens are ten (9+1) in number. The ovary
is superior with a terminal slightly bent style and blunt stigma. Fruit is
inflated pod with 1-2 seeds. Seeds are wrinkled or smooth. Germination
is hypogeal, cotyledons are thick and yellowish.
1.4. Induction of genetic variability
Macro-mutations, whether resulting from single gene changes or
chromosomal aberrations, behave as monogenic traits and follow the
Mendelian pattern of inheritance. On the other hand, micro-mutations
are governed by the principles of quantitative genetics. It has been
found that monogenic macro-mutations are invariably associated with
multiple pleiotropic effects, some of which (e.g. chlorophyll
deficiency, sterility and reduced productivity etc.) make them
unsuitable for plant breeding. In contrast, micro-mutations bring subtle
changes in a large number of loci associated with determination of
plant morphology or physiology have negligible side effects. It has
been generally believed that such mutations for any economic trait
could be accumulated in a single genotype to a great advantage.
It is well known that a crop plant can be improved in
productivity, resistant to pests and adaptation to environment when
genetic variability for the specific trait is available in the considered
population or the species. The process of breeding the crop plants has
been successful for a long time, because genetic variation already
present in the populations had been used, and subsequently further
genetic variation was made available by crossing plants from different
populations, varieties, species and genera. In some cases, the
variability present in the population has been exploited to such a large
extent that only further process from the classical methods of breeding
become more and more difficult. 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 considered 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 or artificial selection.
Despite the release of so many cultivars, chickpea production has
not increased to any noticeable extent. The situation is very
disappointing considering ever increasing human population, leading to
a sharp reduction in the per capita availability of proteins particularly
in developing countries. Therefore, there is an urgent need to
strengthen efforts aiming for further improvement in the quality and
quantity of chickpea.
The conventional approach of plant breeding (like selection and
hybridization) have exploited the available genetic variability in
chickpea. The use of the above approaches for a longer period in this
crop, might have concentrated genetic variability in to a narrow genetic
base. Therefore, further improvement in this crop requires induction of
genetic variability. For this purpose, induced mutations may be helpful
in the breeding programmes. Mutations provide an opportunity to
create unknown alleles, so that the plant breeder does not remain
handicapped due to limited allelic variations at one or more gene loci
of interest. The significance of induced mutations is well documented
from numerous reports. More than 1700 varieties developed through
mutation breeding have been released for cultivation (IAEA, 1996).
7
The natural variability for yield and yield components is very
narrow in chickpea - normally a self pollinated crop. In the recent
years there has been a number of attempts to assess radiation induced
genetic variability in quantitative characters of chickpea (Nerker and
Mote, 1978b; Kharkwal, 1979; Bhatnagar et ai, 1979; Farooq and
Nizam, 1979b; Kalia et al., 1981; Singh, 1988b; Khan and Siddiqui,
1992; Atta et al., 2003). However, little information exists concerning
the influence of chemical mutagens on quantitative characters in
pulses, particularly in chickpea.
In the present study, an attempt has been made to evaluate
quantitative characters in Mi generation following mutagenesis with
hydrazine hydrate (HZ), methylmethane sulphonate (MMS) and sodium
azide (SA) in two varieties of chickpea viz. Pusa-212 and BG-256.
The objectives of the study were:
1. to study the effect of chemical mutagens on such biological
parameters as seed germination, survival, seedling height and
pollen fertility in Mi generation
2. to test effectiveness of chemical mutagens for the induction of
quantitative variability in chickpea
3. to study the frequency and spectrum of morphological variations,
if any
4. to study the differential response of two varieties (Pusa-212 and
BG-256) to various chemical mutagens.
Chapter 2
REVIEW OF LITERATURE
2.1. Origin and distribution
Chickpea (Cicer arietinum L.) is the most important pulse crop
of India. It is considered to be one of the oldest pulses known and
cultivated from ancient times both in Asia and Europe. Vavilov (1926)
postulated two centres of diversity of chickpea - one in the south-west
Asia and Mediterranean and second in Ethiopia. He noticed that like
other grain legumes, large seeded cultivars around the Mediterranean
bases where as small seeded cultivars predominated eastwards. There
are linguistic indications that the large seeded chickpea reached India
through Afghanistan. Cicer had been regarded as a comparatively
young and incompletely differentiated group in which the process of
individualization of types still continue and that due to geographical
isolation, races of one species might differ more sharply among
themselves than from the neighbouring closely related species De
Candolle (1986) considered that the original place of chickpea is in the
broad area in between Greece and Himalayas. It is supposed that the
chickpea has been cultivated in Egypt from very earliest times of the
Christian era. But it is most likely that it was introduced into Egypt
from Greece and Italy. Its introduction into India is of a more early
date for there is a Sanskrit name and several other names in the modern
Indian languages. It is considered to have originated in the track lying
9
between Caucasus and the Himalayas, where from it has spread into
southern Europe, Persia, Egypt and India.
Its cultivation as an important field crop is extensive only in
India, where it extends from the north western frontier provinces down
to the forth east south of the peninsula and likewise from Gujarat in the
west to the eastern limits of Bengal. Outside India, chickpea is grown
in many countries like Italy, Greece, Romania, Russia, Egypt, north
and east Africa, Iran and Turkey, in central and south America and in
parts of Australia.
2.2. Origin and cytological relationship
The middle east is considered as the homeland of several annual
wild chickpeas. Three species viz. C. judaicun BOISS, C. pinnatifolum
JAUB et SPACH and C. bijojum RECH known from that area have the
same chromosome no. (2n=16) as that of the cultivated species (Vander
Maesen, 1972). However, morphologically and according to seed
protein profile, these species are apparently unrelated to the cultivated
chickpea. (Ladizinsky and Adier, 1975). Later on two new species have
been found in south east Turkey viz., C. chinospermun Davis (Davis,
1969) and C. reticulatum Ladiz (Ladizinsky 1975). Morphologically
and by their seed protein profile these two species are close to the
cultivated species could be, therefore, suspected as their progenitor(s).
The two wild species viz. C. echinospermum and C. reticulatum
share the same morphological features as the cultivated chickpea and
the differences between them are small. The cytological differences
10
between them are small. The cytological observations of these two
species revealed the chromosome no. (2n=16) with eight bivalents
being formed regularly at meiosis (Ladizinsky and Adier, 1976). With
the help of inter specific crosses cytogenetic relationships were studied
between these two wild species and the cultivated chickpea (C.
arietinum), which brought about a new information regarding the origin
of cultivated chickpea (Ladizinsky and Adier, 1976). The inter specific
crosses between the two wild chickpea C. reticulatum and C.
echinospermum indicated that these are two different biological
species. These two wild species differed from each other by a major
reciprocal translocation and their hybrid was also highly sterile.
However, the presence of regular meiosis and normal fertility of the
hybrid between C. reticulatum and C. arietinum indicated a remarkable
similarity between the genomes of the two species. Consequently their
hybrid behaved like any other inter specific hybrid of C. arietinum.
Therefore, C. reticulatum could be considered the wild progenitor of
the cultivated chickpea C. arietinum (Ladizinsky and Adier, 1976).
Genetic relationships in the genus Cicer were also revealed by
polyacrylamide gel electrophoresis of seed storage proteins (Ahmad
and Slinkard, 1992). C. reticulatum was found to be the closest relative
of C. arietinum followed by C. echinospermum and other species
whereas C. cuneatum was the farthest relative. Thus the suggestions
that the C. reticulatum is the wild progenitor of the cultivated chickpea
was therefore further supported by Kabir and Singh (1991) and Ohri
11
and Pal (1991) also supported the similar view about the origin of
chickpea.
2.3. Some concepts in induced mutagenesis
The discovery of mutagenic role of ionizing radiations (Muller,
1928) and some chemicals (Auerbach and Robson, 1942) initiated the
furry of activities in the field of mutation breeding. Induced mutations
are considered as an alternative to naturally occurring genetic
variations that serve as the source of germplasm for crop improvement
programmes and also as an alternative to hybridization and
recombination in plant breeding. Mutagens have remarkable potential
of improving plants with regard to their quantitative and qualitative
characters and where appropriate selection has been applied,
improvement in yield (Brock, 1965; Gregory, 1968), adaptability
(Gustaffson, 1965), maturity time (Brock 1970), disease resistance
(Yamasaki and Kawai, 1968; Yamaguchi and Yamashitra, 1979) and
numerous other traits (Sigurbijornson and Micke, 1969) have been
reported. The extent to which induced mutations provide a useful
alternative to the natural variations as a source of germplasm for the
improvement of such traits is largely determined by the importance of
linked groups of genes and the degree to which natural selection has
built up linked gene complexes of adaptive significance in the naturally
occurring population (Brock, 1971).
The generation of genetic variability through induced
mutagenesis provides a base for strengthening crop improvement
12
programmes. Various classes of physical and chemical mutagens differ
in their efficiency in inducing mutations and spectrum of mutations
induced. Ever since the discovery that the mutations could be produced
artificially, one of the aims of the studies on mutations has been to find
the treatment combinations of the mutagens that could induce higher
magnitude of useful mutations. Combined mutagen treatments increase
the higher mutation frequency and alter the mutation spectrum than the
individual treatment was reported by Sharma, 1970. Some of the
monofunctional alkylating agents, EMS in particular, have been shown
to be very efficient in induction of mutations than radiations. As
certain genes are mutated by radiations and not by EMS (Favert, 1960)
and mutation spectrum induced by radiations and chemical mutagens is
different (Heiner et al. 1960; Ehrenberg et ai, 1961), it was thought of
interest to find the mutation frequencies when the physical and
chemical mutagens were used in combination by many workers
(Sharma, 1970; Khalatkar and Bhatia, 1975; Gupta and Yashvir, 1975;
Jayabalan and Rao, 1987a; Suganthi and Reddy, 1992).
Alkylating agents are by far the most extensive and important
groups of mutagen. These compounds bear one or more reactive alkyl
groups capable of being transferred to other molecules at positions
where the electron density is sufficiently high. They cause alkylation
of phosphate groups of DNA as well as the bases, the most frequent
event being the formation of 7-alkyl guanine. In practice, however,
only a few of the mutagens belonging to the group of alkylating agents
such as EMS, MMS and diethyl sulphate (dES), ethylamine (EI) and N-
13
nitroso-N-methyl urea (NMU) have been used more frequently. Of
these, nitroso compounds have been reported to be most effective
(Rapoport, 1962, 1963; Swaminathan, 1966, IAEA, 1970).
Mutagens affect the metabolism of individuals and influence the
activity of synthesis of enzyme and growth regulators. Such harmful
affect of mutagens lead to various forms of physiological expressions
of damage such as retarded plant growth, sterility and death. Mutagen
induced biochemical and physiological changes during seed
germination have been reported in rice (Inoue et al., 1975) and cowpea
(Khanna, 1988, 1991). Radiations have been found to produce genetic
changes such as mutations, chromosomal rearrangements and
disturbances in the cell division (Khanna and Maherchandani, 1981;
Khanna, 1986; Singh and Khanna, 1988). Low doses of radiations have
been found to have stimulatory effect in different crops (Sparrow,
1966; Khanna, 1988).
For combined treatments of gamma radiations and chemical
mutagens on seeds, the mutagenic effects were reported to be
synergistic when radiation was given first followed by chemical
treatment (Nilan et al., 1962; Sharma, 1970). When treatments were
given in reverse order, the mutagenic effects were not synergistic
(Sharma, 1970). Mohan Rao (1972) obtained a synergistic effect for Mi
seedling injury and mitotic anaphase fragment frequency, whereas the
effect was only additive for mitotic bridges and M2 chlorophyll
mutations. Favert (1963) and Doll and Standfer (1969) however could
14
not obtain any synergistic interaction between radiations and chemical
mutagen treatments. In chickpea, intentional exposure of seeds to
various mutagens has produced many new and desirable characteristics
(Pundir and Vander Meson, 1977; Kalia et al, 1981; Haq et ai, 1988;
Hassan and Khan, 1991).
Our knowledge on fundamental aspects of mutational processes
and mechanism of action of various physical and chemical mutagens
and their combinations have been fairly widened with the reports of
Blixt and Gottschalk (1975), Gottschalk (1978a, 1978b), Gottschalk
and Wolf (1983), Sharma (1985) and Khan (1986). Though there are
several unanswered questions regarding the classification and
mechanism of action of mutagens, yet a more comprehensive account
of them was given by Kaul (1989).
2.3.1. Dose effect / LD-50
The dose required for high mutation efficiency of a physical or
chemical mutagen depends on properties of mutagenic agents and of
biological system in question. In general, the dose effect of physical
and chemical mutagenic treatment comprises several parameters, of
which the most important are dose rate, concentration, duration of
treatments, temperature and pH during treatments.
Lethal dose (LD-50) gives an idea about the appropriate dose of
mutagen in an experiment on the induced mutagenesis. In chickpea
(Singh, 1988a) reported LD-50 value for gamma rays at 460 Gy (var. G
130) and 483 Gy (var. H208) and for EMS at 0.25% (var. G130) and
15
0.2% (var. H208). In both the varieties 0.4% EMS treatment was most
lethal. Kharakwal (1981a) reported higher lethality in 0.2% EMS in
comparison to 400 Gy and 500 Gy gamma rays. Higher LD-50 values
for gamma rays in chickpea in comparison to other pulse crops such as
30 Gy in black gram (Khan, 1988a), 200 Gy in lentil (Singh, 1983) and
100 Gy in pea (Singh, 1988b) indicate its greater resistance to the
mutagen. Further, differences have been observed for LD-50 values in
different chickpea varieties, which is attributed to their differential
radiosensitivity. A decline in the survival of a mutated population has
been associated with the increase in the dose of mutagen (Farooq and
Nizam, 1979a; Singh, 1988b), which may have resulted from cytogenic
damage and/or physiological disturbances as also reported earlier by
Sato and Gaul (1967).
Both gamma rays and EMS have shown to have a dose related
reduction in seed germination and pollen fertility (Nerker, 1970a; Rao
and Laxmi, 1980; Khanna and Maherchandani, 1981; Gautam et ai,
1992). Dose linked effectiveness of EMS and gamma rays was noted in
chickpea in terms of germination, reduction in pollen fertility,
chlorophyll mutations and seedling height (Kalia et ai, 1981;
Kharkwal, 1981a; Khanna, 1991; Gumber et ai, 1965).
Similar effects were also reported in peas (Salim et ai, 1974),
pearlmillet (Singh et ai, 1978), Vigna radiata (Singh and Chaturvedi,
1980), Lens culinaris (Sharma and Sharma, 1981b), Arachis hypogea
16
(Vankatachalam and Jayabalan, 1995) and Nigella sativa (Mitra and
Bhowmik, 1999).
With a view to enhance the mutation rate and also to alter the
spectrum of mutations, many variations in the treatment methodology
have been used by different workers. Treatment with chemical
mutagens have been given to dry as well as to soaked seeds. Seedlings
at different developmental stages, different phases of cell cycle at
variable temperature and ionic concentrations (Chopra and Pai, 1979).
Ramana and Natrajan (1965) studied the mutagenic efficiency of
certain alkylating agents under different treatment conditions of
temperature and pH concentrations in barley. They concluded that
factors such as concentration and diffusion of mutagens, rate of
hydrolysis and influence of alkylating and non-alkylating groups of
chemicals play a considerable role in determining the mutagenicity of a
compound.
2.3.2. Mutagenic sensitivity
It is well known that the same mutagen dose can cause different
degrees of affect in different species. Varied mutagenic sensitivity in
different genotypes was first reported by Gregory (1955) in groundnut
and by Lamprechet (1956) in peas.
Prasad and Das (1980b) studied mutagenic sensitivity of gamma
rays and methylmethane sulphonate (MMS) in different varieties of
Lathyrus sativus. They observed differential mutagenic response in
terms of chlorophyll mutations. Similar varietal differences were
17
recorded in production of viable and chlorophyll mutations in Nigella
sativa (Mitra and Bhowmik, 1999) following gamma rays and EMS
treatments.
Sharma and Sharma (1981a) observed differential mutagenic
response of gamma rays and NMU in microsperma and macrosperma
lentils. They observed better viability of chlorophyll mutations like
Xantha and Chlorina in the microsperma than in the macrosperma
varieties.
Venkatachalam and Jayabalan (1995) while using EMS, SA and
gamma rays found distinct varietal differences in groundnut (Arachis
hypogea). Distinct varietal differences to NMU and gamma rays in
Vigna radiata was observed by Singh and Chaturvedi (1980). Geeta and
Vaidyanthan (1997) observed different phenotypic response of two
soyabean cultivars to ethidium bromide and gamma rays.
Difference to radiosensitivity were also reported by Khan (1999)
in black gram and Nerker (1976) in Lathyrus sativus. Akbar et al.
(1976) concluded that differences in radiosensitivity may be due to
differences in their recovery process including enzyme activity. In
chickpea, Kharkwal (1998b) reported that varieties of desi type were
more resistant towards mutagenic treatments than kabuli and green
seeded type.
Mutagenic response to cytological aberrations has been reported
by many workers (Rao and Laxmi, 1980; Suganthi and Reddy, 1992).
Mitra and Bhowmik (1996) observed no varietal differences with
18
regard to mitotic index as well as to meiotic abnormalities in Nigella
sativa. Both cultivars of Nigella sativa were found equally
radiosensitive. Ahmad (1978) and Ahmad and Godward (1981) reported
radiosensitivity in nine cultivars of chickpea. Out of these nine, two
cultivars CSIMF and FIO were identified as the most radioresistant and
radiosensitive, respectively Kharakwal (1981a) reported mutagenic
sensitivity in four varieties of chickpea on the basis of total
germination rate, seedling damage, pollen sterility and plant survival.
In general, the varieties with large assortment of recessive
alleles governing trait(s) show greater sensitivity and frequency of M2
mutants than the varieties having more dominant alleles governing a
trait (Gelin et al., 1958; Blixt, 1970). The mechanism controlling
sensitivity to chemical mutagens and X-rays have been reported to be
different from those determining sensitivity to gamma rays (Sokolov
and Balchunene, 1977).
2.3.3. Biological damage
There are many reports to demonstrate the effect of physical and
chemical mutagens and their combination treatments on different
biological parameters such as germination, survival, injury and sterility
(Khan, 1990; Khan, 1999; Sareen and Kaul, 1999; Mitra and Bhowmik,
1999). Reduction in seedling height following treatments with gamma
rays and EMS was observed in barley (Sharma, 1970). Gupta and
Yashvir (1975) reported a radioprotective effect of EMS in
Abelmoschus esculantum. The combined treatments of gamma rays and
19
EMS showed higher germination percentage than in corresponding EMS
treatments. Choudhry (1983) reported a symmetric reduction in
germination in different varieties of wheat with higher doses of gamma
rays.
Khalatkar and Batia (1975) studied the effect of gamma rays,
EMS and their combinations on Mi parameters in barley. They
observed that the seedling injury, chromosomal aberrations, pollen and
seed sterility were less in combined treatments than in separate
treatments. Gamma rays were reported to inhibit the uptake of EMS
due to the generalized action of radiation on metabolic processes in the
cells. Singh and Chaturvedi (1980) reported mutagen induced damage
such as plant injury and lethality in Mi generation arising due to
physiological, chromosomal and factor mutations.
Gautam et al. (1992) observed a direct relationship of pollen and
ovule sterility with gamma rays and EMS doses in Vigna mungo, the
maximum occurring at higher doses. Increase in pollen sterility and
decrease in germination with increasing doses of gamma rays in
Capsicum annum was reported by Rao and Laxmi (1980).
Based on plant survival and sterility, the mutation rate of NMU
was found to be 1.5-2.0 times higher than gamma rays (Sharma and
Sharma, 1981a) in microsperma and macrosperma lentils. Rapoport
(1966) has called the super mutagens in view of their higher mutagenic
effect. Mutagenic efficiency based on injury and lethality was found
higher in combined treatments of gamma rays and NMU than their
20
respective individual treatments (Dixit and Dubey, 1986). Combined
treatments also showed greater reduction in seedling survival than the
individual treatments.
Bhatnagar (1984) reported the adverse affect of combined
treatments on germination and survival of plants in chickpea. The
pollen sterility increased in combined treatments indicating the
additive or synergistic effect. Reduction in seed germination with the
increase in dose of gamma rays in chickpea was reported by Khanna
(1981, 1991). The EMS treatment was found to cause higher sterility
than gamma rays in chickpea (Kharakwal, 1981b).
2.3.4. Induction of cytological abnormalities
Estimation of cytological abnormalities and their magnitude
during mitosis or meiosis is most convenient for evaluating the effect
of mutagen. It also provides a considerable clue to assess the
radiosensitivity of plants to both physical and chemical mutagens.
Mutagen induced chromosomal aberrations have been reported by many
workers in different plants such as chilli (Mesharam and Humme,
1984), pea (Kallo, 1972), triticale (Pushpalatha et al., 1992), lentil
(Reddy and Annaduri, 1991), barley and wheat (Swaminathan et al.,
1962; Reddy et al, 1991), fenugreek (Anis and Wani, 1997) and
Capsicum annum (Anis et ai, 2000). Most of these workers observed
dose dependent increase in frequency and chromosomal abnormalities
with respect to mutagenic treatments.
21
Subhash and Nizam (1977) reported that increasing the dose of
X-rays resulted in the formation of increased number of multivalents,
fragments, bridges and micronuclei in Capsicum annum. Katiyar (1978)
has reported chromosomal aberrations like stickiness, clumping, altered
association, breakage, laggards and abnormal microspores after gamma
irradiation in chilli. Pollen sterility increased with increase in dose of
gamma rays and abnormalities were comparatively more in Mi than in
M2 generations. Similar results were also reported by many workers
(Rao and Laxmi, 1980; Tarar and Dnyansagar, 1980; Subhash and
Venkatarajan, 1983).
Laxmi et al. (1975) reported different meiotic abnormalities like
chromatin bridges, laggards, fragments, cytomixis, tripolar divisions,
inversions, micronuclei and unequal separation of chromosomes in
pearlmillet following treatments with gamma rays and EMS. Lagging
chromosomes and unequal separation of chromosomes were more
frequent than other anomalies. They further reported that gamma rays
were more effective than EMS or the combination treatments in
inducing chromosomal anomalies. Increase in the frequency of meiotic
anomalies with the increase in dose and duration of mutagen were
reported by Suganthi and Reddy (1992). Similar results were also
reported in Turnera ulmifolia (Tarar and Dnyansagar, 1980) after
treatments with gamma rays and EMS.
Mitra and Bhowmik (1996) reported radiosensitivity in two
cultivars of black cumin (Nigella sativa) after treatments with gamma
22
rays and EMS. Mitotic index was found to decrease with increasing
dose of mutagens but the mitotic and meiotic abnormalities showed
increasing trend with mutagen doses. They observed no varietal
differences with regard to mitotic index as well as cytological
abnormalities.
Venkateshwarlu et al. (1988) studied the effect of single and
combined treatments with gamma rays, EMS and hydroxyl amine (HA)
in Catharanthus roseus, besides various meiotic aberrations, tetrad
abnormalities like monads, dyads, triads and polyads were also
observed. In tomato, meiotic abnormalities and pollen sterility were
found more in combination than in individual treatments (Jayabalan
and Rao, 1987b). Dose dependent decrease in pollen fertility was
reported in Vigna radiata (Ignacimuthu and Sakthivel, 1989) following
treatments with gamma rays and EMS. They observed significant
positive correlations between chromosomal abnormalities and pollen
sterility. Gamma rays induced meiotic abnormalities have also been
reported in chickpea (Ahmad, 1993). The meiotic abnormalities
increased with increase in dose of gamma rays. Decrease in mitotic
index with increase in dose of gamma rays has also been reported
(Khanna, 1991).
2.4. Mutagenic effectiveness and efficiency
The usefulness of any mutagen in plant breeding depends not
only on its effectiveness but also upon its efficiency. Mutagenic
effectiveness is a measure of frequency of mutations induced by unit
23
mutagen dose, whereas mutagenic efficiency is the measure of
proportion of mutation in relation to undesirable changes like lethality,
injury, sterility, mitotic and meiotic chromosome aberrations. The
methods of calculating mutagenic efficiency and effectiveness were
suggested by Konzak et al. (1965).
Effectiveness and efficiency of different mutagens vary
distinctly. Ethyline imine has been reported to be superior to gamma
rays in its effectiveness and efficiency (Debelyi et al, 1975). MMS
recorded the higher mutagenic effectiveness in rice (Rao and Rao,
1983) whereas gamma rays were found to be more effective than EMS
in chilli (Rao et al, 1991). Prasad and Das (1980a) reported 0.2% MES
to be more affective than lowest dose (lOkR) of gamma rays while as
MMS was found more efficient than EMS in inducing mutations
(Minocha and Arnason, 1962). Dixit and Dubey (1986) observed that
NMU treatment was 2-5 times more efficient in comparison with
gamma rays, whereas combined treatments showed a higher efficiency
than respective individual treatments. Higher efficiency of combination
treatments has also been reported in barley (Khalatkar and Bhatia,
1975). Khan (1999) studied the effectiveness and efficiency of EMS,
gamma rays and their combinations in black gram. Lower doses of
mutagens were found more effective, while gamma rays treatments
were more efficient than EMS and combined treatments in producing
chlorophyll mutations. Lower doses of physical and chemical mutagens
and their combinations were found more effective and efficient by
many workers (Prasad, 1972; Sharma and Sharma, 1981a).
24
Chemical mutagens have been reported to be more effective in
causing mutations as compared to gamma rays and combined treatments
by many workers (Swaminathan et al., 1962; Khan, 1990). Jagtap and
Das (1976) studied the effectiveness and efficiency of four
monofunctional alkylating agents (EMS, dES, MES and EI) in barley.
dES was found more efficient than EMS, MES, EI in relation to
lethality only. Whereas MES was most efficient in relation to sterility
as well as in producing high frequency of mutants per mutation. On the
other hand, the factor of effectiveness i.e., mutation per 100 treated
seeds was highest in ethyline imine. It has been reported that among
the monofunctional mutagens, methylating agents were more toxic and
thus, need to be used only at lower concentrations (IAEA, 1970) as
against ethylating agents that are reported to be less toxic and can be
applied in relatively higher concentrations to yield more mutations at
equimolar concentrations.
Comparative mutagenic effectiveness and efficiency of physical
and chemical mutagens in chickpea has been reported by Kharakwal
(1998a). Chemical mutagens have been found to be more efficient in
inducing chlorphyll as well as viable mutations. NMU in particular,
was found to be very affective and efficient than gamma rays and EMS.
2.5. Chlorophyll mutations
A study of the relative frequency of chlorophyll mutations
following mutagenic treatment helps in determining the relative
efficiency of a mutagen. In chickpea, different workers have reported
25
higher chlorophyll mutation frequency around 200-400 Gy gamma rays
and 0.3% EMS (Nerkar and Mote, 1978a; Singh, 1988a). In general,
EMS treatments induced more mutations relative to treatments with
gamma rays as in case of many other plant materials. This is due to the
preferential action of EMS on genes for chlorophyll development
located near the centromere (Swaminathan et ai, 1962; Varghese and
Swaminathan, 1968; Khan and Siddiqui, 1993b). Chlorophyll mutations
affected not only colour but also variability and fertility (Ivannikov et
ai, 1970). There may be variations in the incidence of chlorophyll
mutations (Nerker and Mote, 1978a) which is attributed to the
differences in number of genes controlling the chlorophyll development
in different varieties.
Many types of chlorophyll mutations viz. albina, xantha',
'chlorina, viridis, alboviridis, xanthoviridis, virescens and redina were
isolated in different studies (Athwal, 1963; Lysikov et ai, 1967;
Nerker and Mote, 1978a; Kharkwal, 1980; Singh, 1988a; Khan and
Siddiqui, 1993b). It is generally believed that ionizing radiations
produce high frequency of 'albina' type of chlorophyll mutations and
the chemical mutagens produce other type of chlorophyll mutations
(Gustafsson, 1963). However, in chickpea, all mutants including
'albina' type were in general more frequent in EMS treatment than in
gamma rays (Singh, 1988a). An earlier study in chickpea (Lysikov et
ai, 1967) reported higher frequencies of chlorophyll mutations in
combined treatments of physical and chemical mutagens. The
26
frequencies of the various types of chlorophyll mutations in different
varieties with different mutagens have been found markedly different.
2.6. Morphological mutations
Several induced morphological mutations have been reported in
literature showing alterations in the morphology of various plant parts.
Singh (1988a) isolated 25 types of morphological mutations for plant
height, stem, leaf, flower and seed characters of chickpea. Generally,
physical mutagens induce more morphological mutations than chemical
mutagens (Gaul, 1960, 1964). Contrary to this, Singh (1988a) observed
that EMS induced marginally more morphological mutations than
gamma rays. Pleiotropic effect of morphological mutations was
reported by Deshmukh et al. (1972) in chickpea and Khan and Siddiqui
(1996) in mungbean. According to Blixt (1972) morphological changes
are either as a result of pleiotropic gene action or of cryptic
chromosomal deletions.
Variation in size, texture, type and modification of leaf parts
have been reported by many workers (Vesileva, 1978; Venkatarajan and
Subhash, 1986; Khan and Siddiqui, 1996). Singh et al. (1999) isolated
several macromutations affecting different morphological characters in
Vigna mungo after treatment with gamma rays and EMS. Gamma rays
induced bold seeded mutant was reported in Vigna mungo (Singh,
1996). Singh et al. (2000a) reported that some of the morphological
mutations like foliage and growth habit appeared more frequently than
other types in mungbean. The frequency of viable mutations has been
27
found to increase with increase in the dose of EMS, SA and their
combinations with gamma rays (Thakur and Sethi, 1995). Sharma
(1970) reported that the combination treatments induced the wider
spectrum of viable mutants than the individual mutagen treatment by
inducing more mutation types.
A wide range of morphological mutants affecting leaf shape
(Kharkwal, 1981a), plant height (Kharkwal,1981b), growth habit
(Khanna,1981; Dekov and Radkov, 1982) were isolated in chickpea
after seed treatment with physical and chemical mutagens.
2.7. Induction of polygenic variability
The availability of ample genetic variability is prerequisite for
attempting selection in plant breeding to develop desired plant types in
any crop. In crop improvement programme, it is the quantitative
variation for yield and its component traits that is important to plant
breeder. In recent years, the role of mutation breeding in increasing the
genetic variability for polygenic traits in number of crops have been
proved beyond doubt (Khan, 1984; Chopra and Sharma, 1985;
Ignacimuthu and Babu,1993; Solanki and Sharma, 1999; Waghmare and
Mehra, 2000).
The significance of micromutations in evolution was first
recognized and emphasized by Baur (1924) and later it has been
studied by many workers in different crop plants. Gaul (1965)
emphasized the significance of micromutations in plant breeding by
stating that micromutations may effect all morphological and
28
physiological characters like macromutations and they might have
higher mutation rates than the macromutations. Several workers have
so far reported encouraging results about the induction of useful
quantitative variability in different crop plants viz. Gustaffson (1963)
in barley, Gaul (1966) in wheat, Ramulu (1974) in sorghum, Sharma
and Sharma (1982) in lentil, Shah et al. (1986) in chilli, Khan (1984),
Mahetra et al. (1990), Tickoo and Chandra (1999) in mungbean and
Singh et al. (2000b) in urdbean. In chickpea, different workers have
reported increased variability for different agronomic characters in
mutagen treated populations as evident by significant changes in mean
and coefficient of variability in comparison to control. Majority of the
results suggested a negative shift (Nerker and Mote, 1978b; Singh,
1988a; Khan and Siddiqui, 1993a) although in some cases positive shift
was also observed (Mandal, 1974; Kumar et al., 1981). Increased
variability in the form of high heritability and genetic advance for
different quantitative characters has been reported by many workers
(Sharma and Sharma, 1982; Sarkar et al, 1987; Khan, 1988b; Rao et
al., 1988; Nayeem and Ghasim, 1990; Sharma et al., 1990; Ignacimuthu
and Babu, 1993; Srivastava and Singh, 1993). Gamma rays induced
mutagenic variability in chickpea was reported by Kale et al. (1980).
He reported that variability was considerably high for most of the traits
and high heritability estimates with high genetic advance was found for
yield and 100-seed eight.
29
2.8. Desirable mutants
Isolation of desirable mutants showing improvement over parent
genotypes for different characters of interest is one of the important
aspects of induced mutagenesis. Several workers have reported induced
variability for protein content in wheat (Singhal et al., 1978), in rice
(Siddiq et al, 1970), in barley (Doll, 1972), in maize (Balint et al,
1970; Singh and Axtel, 1973) and in chickpea (Farooq and Nizam,
1979b and Mehrajuddin, 2001). Kharkwal (1998c) induced wide range
of variability for crude protein content in chickpea through treatments
with physical and chemical mutagens. Sheikh et al (1982) isolated
high yielding and high protein mutants of chickpea following gamma
ray treatments. Increased seed protein content due to mutagenic
treatment was also reported by many workers (Rafiov and
Gasanov,1977; Abo-Hegazi, 1980).
Since there is ever increasing demand for improvement in yield
of pulses including the chickpea, mutants for increased yield have also
been reported by several workers. These mutants showed higher yield
in comparison to normal cultivars (Kharakwal, 1983; Khan, 1984; Rao,
1988; Hassan and Khan, 1991). Besides the gamma-irradiation derived
mutants, Ivannikov and Moraru (1968) isolated the mutants for
increased yield in chemical induced mutagenesis. Some high yielding
mutants in chickpea after treatment with physical and chemical
mutagens have been reported by Kharakwal (1981a).
30
Chapter 3
MATERIALS AND METHODS
3.1. Materials
3.1.1. Varieties used
Two varieties of chickpea (Cicer arietinum L.) namely Pusa-212
and BG-256 were used in the present study. Seeds of variety Pusa-212
were procured from the Government Seed Store, Aligarh (U.P.) and the
seeds of variety BG-256 were obtained from Dr. S.K. Chaturvedi,
Senior Scientist, Indian Institute of Pulses Research, Kanpur. Both the
varieties are popular for cultivation in this region. A brief description
of both the varieties is given below:
3.1.1.1. Variety Pusa-212
It is a cross of P-340 XG-130 and was released in 1982, seeds
are medium bold and light brown, mature in 114-120 days, average
yield is 16-17 q/ha.
3.1.1.2. Variety BG-256
Plant is erect with medium height, seeds bold, mature in 130-135
days, average yield is 19-20 q/ha.
3.1.2. Mutagens used
Hydrazine hydrate (HZ), NH2-NH2-SO2, a base analogue, is
manufactured by Sigma Chemical Company, USA.
31
Methylmethane sulphonate (MMS), CH3OSO2CH3, Monofunctional
alkylating agent, manufactured by Sissco Research Laboratories Pvt.
Ltd., Mumbai, India.
Sodium azide (SA), NaNa, A respiratory inhibitor, manufactured by
Indian Drugs and Pharmaceuticals Ltd., Hyderabad, India.
3.2. Experimental procedures
3.2.1. Preparation of mutagenic solutions
All solutions of the chemical mutagens were prepared in
phosphate buffer of pH-7. Only freshly prepared solutions were used
for all the treatments.
3.2.2. Pretreatment
Healthy seeds of uniform size of each variety were used in the
present experiments. The seeds were soaked in distilled water for 9
hours prior to the treatment with mutagens.
3.2.3. Mutagen administration
Concentrations used: Four different concentrations viz. 0.01, 0.02,
0.03 and 0.04% of HZ, MMS and SA were used for treating the
presoaked seeds.
Treatment time: The treatments were given at temperature of 25±1°C
for 6 hours.
Sample size: 255 seeds were used for each treatment.
Controls: For each variety 255 pre-soaked seeds were again soaked in
phosphate buffer for 6 hours to serve as controls.
32
3.3. Ml generation
Three replications of seventy-five seeds each, were sown for
every treatment in each variety in the pots.
The remaining lot of thirty seeds of each treatment with their
respective controls of both the varieties were spread over moist cotton
in petriplates, in order to determine percentage of seed germination and
seedling height i.e. root and shoot length. The petriplates were kept in
the B.O.D. incubator at 25±1°C temperature.
3.3.1. Observations recorded in Mi generation
Following parameters were studied in Mi 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 petriplates. Seeds which gave rise to both
radical and plumule were considered as germinated.
_, . . ...^ No. of seed germinated ,^. Germmation (%) = x 100
Total no. of seeds 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 of fifteen
randomly selected seedlings for each treatment seedling injury as
measured by the reduction in the root and shoot length was calculated
in terms of percentage of root and shoot injury.
33
3.3.1.3. Plant survival
The surviving plants in different treatments were counted at the
time of maturity and the survival was computed as percentage of the
germinated seeds.
3.3.1.4. Pollen fertility
Pollen fertility was estimated from fresh pollen samples. From
mature anthers, some amount of pollen was dusted on a slide
containing a drop of 1% acetocarmine solution. Pollen grains, which
took stain and had a regular outline were considered as fertile, while
empty and unstained ones as sterile.
The following formula was used to calculate the percentage
inhibition or injury or reduction:
Percentage inhibition
or . . Control - treated ,„^
Percentage mjury = x 100 Control
or
Percentage reduction
3.3.2. Morphological variants
Some induced morphological variants affecting plant form, plant
height and leaf were isolated in Mi generation.
Observations were also made on 25-30 normal-looking plants in
each treatment with their controls.
The following eight quantitative characters were studied in Mi
generation.
34
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 up to the date of harvesting of the plant.
4. Number of fertile branches: Number of fertile branches were
counted at maturity as the number for fertile branches which had more
than one pods.
5. Number of pods: Number of pods were counted at maturity as
the number of pods borne on the whole plant.
6. Seeds per pod: Twenty best pods were threshed and number of
seeds per pod was counted. The mean was calculated for each plant.
7. 100-seed weight (g): It was the weight of a random sample of
hundred seeds from each plant.
8. Total plant yield: Plant yield was the weight of total number of
seeds harvested per plant and the yield of each plant was recorded in
grams.
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 affective
selection. The variability present in breeding populations can be
assessed in the following three ways:
35
(i) using simple measures of variability
(ii) by estimating the various components of variance
(iii) by studying the genetic diversity
Simple measures of variability
Data collected for eight quantitative characters in Mi generation
were subjected to statistical analysis to find out ranged, mean, standard
error, standard deviation and coefficient of variability.
3.4.1.1. Range
It is the difference between the lowest and highest values present
in the observations included in a sample.
3.4.1.2. Mean X
The mean is computed by taking the sum of the number of values
(Xi, X2, .... Xn) and dividing by the total number of values involved,
thus
(X, + X2 + X3 Xn) X =
N or
N
where, Xi, X2, X3, .... Xn = Observations
N = Total number of observations involved
3.4.1.3. Standard error (S.E.)
It is the measure of the uncontrolled variation present in a
sample. It is estimated by dividing the estimate of standard deviation
by the square root of the number of observations in the sample and is
denoted by S.E., thus
36
S.D. of the sample o.b. = p=
where, S.D. = Standard deviation
N = Number of observations
3.4.1.4. Standard deviation (S.D.)
The standard deviation is calculated by the following formula for
each parameter of study.
SD _ V(X-X,)^+(X-X,)^ ( X ^
N
Where, X= Mean of observations involved
Xi, X2 Xn = Observations
N = Number of observations
3.4.1.5. Coefficient of variability (C.V.)
It measures the relative magnitude of variation present in
observations relative to magnitude of their arithmetic mean. It is
defined as the ratio of standard deviation to the arithmetic mean
expressed as percentage and is a unit less number. The following
formula is applied to compute coefficient of variability (C.V.).
C.V.(%) = ^^^"^^^^^^^^^^^°"xlOO 100
or
= ^ x l O O X
where, S.D. = Standard deviation of sample
X = Arithmetic mean
37
Chapter 4
EXPERIMENTAL RESULTS
Seed germination, plant survival, seedling growth and pollen
fertility are widely used as indices in determining biological effects of
various mutagens. Data on the effects of hydrazine hydrate (HZ),
methylmethane sulphonate (MMS) and sodium azide (SA) on Mi
parameters are described below:
4.1. Seed germination
The data recorded on seed germination are presented in Table 4;
Fig. 1. Chemical mutagens did not show drastic effects on germination
percentage in both the varieties viz. Pusa-212 and BG-256. In all the
mutagen treatments, only moderate and gradual decline in germination
percentage with increased concentrations was recorded. In the var.
Pusa-212, the control gave 96.00 percent seed germination. The seed
germination percentage was 82.00 with 0.04% HZ and 85.00 and 86.00
with 0.04% MMS and 0.04% SA, respectively. The other var. (BG-256)
behaved more or less identically. In the var. Pusa-212, the highest
percentage inhibition in seed germination was 14.58 with 0.04% HZ
whereas it was 14.28 with 0.04% of HZ treatment in the var. BG-256.
HZ treatments were most effective in inhibiting seed germination
followed by MMS and SA in both the varieties. Variety Pusa-212 was
found to be more sensitive than the var. BG-256.
38
In general, seed germination started on second day after sowing
in controls of both the varieties. However, it was delayed by 2-3 days
in the lots treated with higher concentrations of the mutagens.
4.2. Seedling height
The study of seedling height in petriplate experiments after
seven days of sowing showed a decline over control in the mutagen
treated populations. The decrease coincides with increase in the
concentration of the mutagen in both the varieties (Tables 5 and 6; Fig.
2). The seedling injury was more drastic at the highest concentrations
of mutagens and it was (42.64, 19.12 and 25.73 percent) in the var.
Pusa-212, whereas in the var. BG-256, seedling injury was (21.97,
12.86 and 18.47 percent) with HZ, MMS and SA treatments,
respectively. Among three different mutagens used in the present
study, the reduction in seedling height was more prominent in HZ
followed by SA and MMS treatments.
In the present study, root appears to be more sensitive than the
shoot in both the varieties Pusa-212 and BG-256. In the var. Pusa-212,
the length of the root in the control is 7.70 cm and it reduces to 4.70
cm (i.e. 3 cm reduction) with a treatment of 0.04% HZ whereas in the
var. BG-256, the root length in control plant is 8 cm and it reduces to
5.60 cm (i.e. 2.40 cm reduction) with 0.04% of HZ.
4.3. Pollen fertility
Although some amount of pollen sterility was also observed in
control plants of both the varieties, but pollen fertility decreased and
Table-4: Effect of mutagens on seed germination, plant survival and pollen fertility in two varieties of chickpea {Cicer arietinum L.)
Treatment
Seed germination (%)
Actual
Plant —— survival at Percentage maturity (%) inhibition
Pollen fertility (%)
Actual Percentage reduction
Control 0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
Control 0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
96.00 93.00 91.00 84.00 82.00
94.00 92.00 87.00 85.00
94.00 92.00 88.00 86.00
98.00 95.00 94.00 88.00 84.00
94.00 92.00 90.00 89.00
96.00 94.00 92.00 91.00
Variety
-
3.12 5.20 12.50 14.58
2.08 4.16 9.37 11.45
2.08 4.16 8.33 10.41
Pusa-212
93.32 92.00 89.30 77.34 80.00
85.34 80.00 82.67 84.00
86.67 78.65 82.66 81.33
Variety BG-256
-
3.06 4.08 10.20 14.28
2.08 6.12 8.16 9.18
2.04 4.08 6.12 7.14
96.00 88.00 92.00 92.00 82.67
86.67 90.65 86.65 88.00
78.65 82.67 89.33 88.00
97.00 93.15 93.00 90.35 89.00
92.25 90.00 88.50 87.00
94.25 93.10 91.60 90.00
98.00 94.80 94.00 92.60 90.00
93.50 92.00 90.15 89.00
97.00 95.80 94.10 93.00
3.96 4.12 6.85 8.24
4.89 7.21 8.76 10.30
2.83 4.02 5.56 7.21
3.26 4.08 5.51 8.16
4.59 6.12 8.01 9.18
1.02 2.24 3.97 5.10
100 -
90 -
80 -
S? 70 H
1 '° 1 50
5 40 30
20
Control 0.01 0.02
HZ%
0.03
- - • -
• - Pusa212
— BG-256
0.04
c o n c 1 o O
100
90
80
70
60
50
40
30-1
20
10
Control 0.01 0.02
MMS%
0.03 0.04
100
90 -
80
^ 70 -
1 60 -
1 ̂ °] 5 «
30
20 -
in 1 , Control 0.01 0.02
SA%
0.03
— •
- - • • - - Pusa-212
— • — B G - 2 5 6
1 ^
0.04
Fig.l: EfTect of mutagens on seed germination in Mi generation of chickpea {Cicer arietinum L.).
100 -90 -
80 -
| ™ -f 60 H a c 50 -
1 4o^ "• 3 0 -
20 -
Control 0.01 0.02
HZ%
0.03
- - • • - - Pusa212
0.04
100 -
90 -
80 -
i . Toil 60 4)
t 50 1
= 40 ^
"̂ 30 ^ 20
*-—-^r-,r-^
Control
- — - .
0.01
, 0.02
MMS%
0.03
- - • • - - Pusa212
, 0.04
100-
90 -
^ 70 -
1 60
- 50 0 = 40-°- 30
20-
10-1
Control 0.01
, 0.02
SA%
0.03
- - • - - Pusa212
, 0.04
Fig. 3: Effect of mutgens on poUen fertility in Mj generation of chickpea (Cicer arietinum L.).
SS
i
100
90
80
70
60
50
40
30
20
10
••- - Pusa212
- • BG-256
Control 0.01 0.02
HZ%
0.03 0.04
100 -
9 0 -
80 -
70 -
w 60 ->
£ 50
« 40 30
20
i n
• -^~r~rr
Control 0.01 0.02
MMS%
0.03
- - • • - - Pusa212
0.04
100 -
90
80
70
•5 6 0 ->
"E 50
« 40-30
20
lU -
•-"^^>x,^
Control
c ^ - - • . . ,
0.01 0.02
SA%
0.03
- - • • - -
_
0.04
Pusa212
-BG-256
Fig.4: Effect of mutagens on plant survival at maturity in Mi generation of chickpea (Cicer arietinum L.).
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d +1 o NO ON
00
d +1 o o d
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NO d +1 o o 00
d +1 o 00 f — 1
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d +1 o ON
d CN
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q d
<
m o d
< oo
o d
? u £ Ol
I O) £ ^ ,?! (0
40
35
30
25
20
15
10
5
0
Control 0.01 0.02
HZ%
0.03
Pusa212
-BG-256
0.04
E u ^ 1 ^
^ JZ S J O)
:§ ^
£
40
35
30
25
20
15
10
5
0
Pusa212
-BG-256
Control 0.01 0.02
MMS %
0.03 0.04
40 -
35-
f 30-o £ 2 5 -U)
1 20 H a : i 15-•o * 10 -
5 -
(\
• . ,
Control
~-«
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t —
0.02
SA%
0.03
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•
0.04
Fig.2: Effect of mutagens on seedling height in M| generation of chickpea (Cicer arietinum L.).
39
gave a dose dependent relationship in both the varieties (Table 4; Fig.
3). Pollen sterility induced by MMS was found to be more in
comparison to HZ and SA. The fertility was lowest (87.00 and 89.00
percent) at 0.04% MMS in Pusa-212 and BG-256, respectively. It
ranged from 97.00 to 87.00 percent in the var. Pusa 212 and 98.00 to
89.00 in the var. BG-256. Var. Pusa-212 was found to be more
sensitive than the var. BG-256 based on the reduction in pollen
fertility.
4.4. Plant survival
Data on plant survival in Mi generation recorded at maturity are
given in Table 4; Fig. 4. Percentage of plant survival was noted to
decrease gradually in all mutagen treatments. However, it was dose-
independent. The highest plant survival was observed in the controls of
both the varieties. Both the varieties responded more or less in the
same manner.
4.5. Morphological variations
Different types of morphological variants with altered characters
affecting plant form, plant height and leaf were isolated in Mi
generation of chickpea populations of the two varieties, Pusa-212 and
BG-256 (Table 7; Plate I and II). Frequency of morphological
variations, on mutagen basis, showed almost equal frequency in HZ
(5.57 percent) and MMS (5.06 percent) while lowest (2.00 percent)
frequency was recorded in SA treated populations (Table 8). Frequency
of morphological variations, on variety basis, indicated that both the
40
varieties of chickpea responded differently to thie mutagen treatments.
In the var. Pusa-212, frequency was 4.80 percent whereas it was 3.70
percent in the var. BG-256 (Table 7).
The characteristics of each variant, isolated in Mi, are as
follows:
1. Dwarf: These were characterized by reduced plant height (Plate-
II; Fig. 5). Their mean height was 20.00 cm against control
(50.00 cm).
2. Tall: These plants were grown upto an average height of 76.00
cm with few branches; pods were not produced in these plants
(Plate-I; Fig. 2).
3. Prostrate: These plants were spread and occupy more area due
to their spread habit; leaves were modified into needle like
structures; plants possessed small pods containing shriveled
seeds (Plate-I; Fig. 3).
4. One sided branches: Branches were produced on one side of
stem; reduced plant height; plants were late in flowering. These
plants were screened from the var. Pusa-212 (Plate-I; Fig. 4).
5. Narrow leaves: Plants were characterized by the leaves modified
into narrow leaflets. Such plants were more frequent at the
higher concentrations of mutagens in both the varieties (Plate-II;
Fig. 1).
41
6. Bushy: These plants were isolated from the higher
concentrations of mutagens in both the varieties (Plate-II; Fig.
2). Plants produced numerous branches at soil level.
7. Axillary branches: These plants had axillary branches which
were late in maturing; plant growth was vigorous; yield was
higher in comparison to control plants (Plate-II; Fig. 4).
4.6. Effect of mutagens on quantitative characters in Mi
Data on the effect of various treatments with HZ, MMS and SA
are given in Tables 9-16. Statistical analysis was done to find out
range, mean, standard error, shift inX, standard deviation for eight
quantitative characters namely plant height, days to flowering, days to
maturity, fertile branches per plant, pods per plant, seeds per pod, 100-
seed weight (g) and total plant yield (g) in two varieties of chickpea.
In the present study, means for all the quantitative characters
shifted in both positive as well as in the negative direction, being more
in the positive side for the characters like fertile branches per plant,
pods per plant, seeds per pod, 100-seed weight and total plant yield.
Though increase in coefficient of variation (CV) of the mutagens
treated populations was of low magnitude, yet it differed from
character to character. The highest increase in CV over the control was
recorded for seeds per pod. Moderate values of CV were observed for
plant height, fertile branches per plant, 100-seed weight and total plant
yield whereas the characters days to flowering, days to maturity and
pods per plant gave lower values of CV.
42
Table 7: Frequency and spectrum of morphological variants induced by mutagens in chiclipea (Cicer arietinum L.) varieties Pusa-212 and BG-256.
X7«v.
Varianis
Dwarf
Tall
Prostrate
One sided branches
Narrow leaves
Bushy
Axillary branches
Total number of morphological variants
Total number of Mi
Frequency (%)
plants
Number observed in
Pusa-212
6
6
5
2
10
4
3
36
750
4.80
BG-256
5
8
3
-
8
3
2
29
784
3.70
Table 8: Frequency of morphological variants in various mutagens in Mi.
Mutagen
HZ
MMS
SA
Noumber of Mi plants studied
520
513
501
Number of variants scored
29
26
10
Frequency (%)
5.57
5.06
2.00
Plate-I: Morphological variants isolated in M| generation.
Fig.
Fig.
Fig.
Fig.
1.
2.
3.
4.
Control plant
Tall plant
Prostrate variant
Plant showing or
%
^ 7
'iL'-V-^s^
^ .̂ 5̂ ' \L 7
1 D PLATE -I
Plate - I I : Morphological variants isolated in M| generation.
Fig. 1. Narrow leaves
Fig. 2. Bushy plant
Fig. 3. and 4. Control and axillarx branches
Fig. 5. Dwarf plant
TabIe-9: Range, mean, shift in X, standard deviation (SD) and coefficient of variation (CV) for plant height (cm) in two varieties of chickpea (Cicer arietinum L.)
Treatment
Control
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0,01% SA 0.02% SA 0.03% SA 0.04% SA
Control
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
Range
49-52
43-48 40-43 38-42 38-41
46-52 46-51 46-51 46-50
46-51 45-50 43-48 42-45
48-51
45-47 45-47 45-47 45-46
47-51 47-48 46-50 46-48
46-48 46-48 45-47 45-47
Mean±S.E. Shift in X
Variety Pusa-212
50.70±0.42
45.30±0.53 41.8010.39 40.00±0.47 39.60±0.49
49.4010.53 49.2010.44 48.9010.49 48.7010.51
48.8010.54 47.4010.61 45.3010.53 43.7010.42
-
-5.40 -8.90 -10.70 -11.10
-1.30 -1.50 -1.80 -2.00
-1.90 -3.30 -5.40 -7.00
Variety BG-256
49.7010.39
46.6010.15 45.7010.20 45.6010.20 45.5010.61
48.6010.47 47.8010.19 47.6010.47 47.0010.28
47.2010.23 46.8010.31 46.2010.23 45.90+0.22
-
-3.10 -4.00 -4.10 -4.20
-1.10 -1.90 -2.10 -2.70
-2.50 -2.90 -3.50 -3.80
S.D.
1.34
1.68 1.24. 1.48 1.49
1.68 1.40 1.57 1.62
1.72 1.91 1.68 1.34
1.00
0.48 0.64 0.66 0.50
1.49 0.60 1.49 0.89
0.74 0.98 0.74 0.70
CV (%)
2.65
3.70 2.90 3.70 3.78
3.41 2.84 3.23 3.31
3.52 4.02 3.70 3.07
2.02
1.05 1.40 1.45 1.09
3.07 1.25 3.14 1.90
1.58 2.09 1.61 1.52
Table-10: Range, mean, shift in X, standard deviation (SD) and coefflcient of variation (CV) for days to flowering in two varieties of chickpea {Cicer arietinum L.)
Treatment
Control
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
Control
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
Range
79-81
75-78 75-77 75-77 75-77
77-78 76-78 76-78 76-79
79-80 78-79 76-79 77-80
78-80
76-77 75-77 75-77 75-77
76-77 76-78 76-78 75-77
78-79 78-79 77-79 77-78
Mean+S.E. Shift in X
Variety Pusa-212
79.8010.23
76.2010.31 ' 76.0010.20
76.60+0.15 76.4010.15
77.5010.15 77.2010.27 76.9010.26 78.7010.25
79.4010.15 78.4010.15 77.8010.31 79.7010.25
-
-3.60 -3.80 -3.20 -3.40
-2.30 -2.60 -2.90 -1.10
-0.40 -1.40 -2.00 -0.10
Variety BG-256
78.7010.20
76.4010.15 75.9010.17 75.8010.23 76.1010.25
77.2010.18 77.1010.22 76.6010.20 76.0010.24
78.3010.14 78.3010.14 77.8010.24 77.5010.16
-
-2.30 -2.80 -2.90 -2.60
-1.50 -1.60 -2.10 -2.70
-0.40 -0.40 -0.90 -1.20
S.D.
0.75
0.98 0.63 0.48 0.49
0.50 0.87 0.83 0.78
0.49 0.49 0.97 0.78
0.64
0.48 0.54 0.75 0.78
0.60 0.70 0.66 0.77
0.45 0.45 0.75 0.50
CV (%)
0.93
1.29 0.83 0.62 0.64
0.65 1.13 1.08 0.99
0.62 0.62 1.26 0.97
0.81
0.64 0.71 0.98 1.02
0.77 0.90 0.86 1.01
0.58 0.58 0.96 0.64
Table-11: Range, mean, shift in X, standard deviation (SD) and coefficient of variation (CV) for days to maturity in two varieties of chickpea (Cicer arietinum L.)
Treatment
Control
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
Control
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
Range
116-118
110-113 112-114 109-113 112-14
112-114 112-114 113-115 113-115
113-115 114-116 115-117 114-116
128-130
127-130 125-128 125-127 124-126
124-128 124-127 124-128 123-128
124-129 123-129 120-129 120-128
Mean±S.E. Shift in X
Variety Pusa-212
116.50±0.40
112.7010.14 113.2010.23 112.6010.25 113.5010.21
114.5010.20 113.5510.14 114.9510.24 114.9010.36
115.8510.14 115.6010.19 116.5010.22 115.1010.14
-
-3.80 -3.30 -3.90 -3.00
-2.00 -2.95 -1.55 -1.60
-0.65 -0.90 -0.00 -1.40
Variety BG-256
129.0010.28
128.3010.37 126.4010.40 126.0010.20 125.1010.26
127.2010.23 126.5010.26 127.8010.26 128.0010.28
128.8010.27 128.3010.37 128.6010.68 127.5010.53
-
-0.70 -2.60 -3.00 -3.90
-1.80 -2.50 -1.20 -1.00
-0.20 -0.70 -0.40 -1.50
S.D.
0.75
0.45 0.75 0.80 0.67
0.63 0.46 0.78 1.14
0.46 0.60 0.70 0.46
0.89
1.18 1.28 0.63 0.83
0.74 0.83 0.83 0.89
0.87 1.19 2.15 1.68
CV (%)
0.64
0.39 0.66 0.71 0.59
0.55 0.40 0.68 0.99
0.39 0.52 0.60 0.40
0.69
0.92 1.01 0.50 0.66
0.58 0.65 0.64 0.69
0.67 0.92 1.67 1.31
Table-12: Range, mean, shift in X, standard deviation (SD) and coefficient of variation (CV) for fertile branches per plant in two varieties of chickpea (Cicer arietinum L.)
Treatment
Control
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
Control
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
Range
20-22
26-27 25-27 24-27 24-26
23-26 23-26 23-24 22-24
20-24 21-23 20-23 20-22
22-24
24-26 24-26 22-25 23-25
22-24 20-22 22-24 22-24
24-27 25-27 24-26 24-26
Mean+S.E. Shift in X
Variety Pusa-212
20.9010.26
26.50+0.15 25.90±0.26 25.6010.32 25.0010.24
25.0010.40 24.2010.36 23.6010.15 22.9010.26
21.9010.47 21.9010.22 21.6010.42 21.1010.22
-
+5.60 +5.00 +4.70 +4.10
+4.10 +3.30 +2.70 +2.00
+1.00 +1.00 +0.70 +0.20
Variety BG-256
22.8010.27
25.0010.28 24.9010.22 23.9010.33 23.7010.24
23.2010.27 21.4010.28 22.8010.30 23.0010.28
25.8010.39 26.3010.20 25.2010.23 25.1010.29
-
+2.20 +2.10 +1.10 +0.90
+0.40 -1.40 0.00
+0.20
+3.00 +3.50 +2.40 +2.30
S.D.
0.83
0.50 0.83 1.01 0.77
1.26 1.16 0.48 0.83
1.51 0.83 1.35 0.70
0.87
0.89 0.70 1.04 0.78
0.87 0.91 0.97 0.89
0.39 0.64 0.75 0.94
CV (%)
3.97
1.88 3.20 3.98 3.09
5.05 4.81 2.07 3.62
6.90 3.79 6.25 3.31
3.82
3.57 2.81 4.35 3.29
3.74 4.28 4.29 3.88
1.51 2.43 2.96 3.75
Table-13: Range, mean, shift variation (CV) for arietinum L.)
Treatment
Control
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
Control
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
Range
90-93
95-97 95-98 95-97 94-96
94-97 93-96 93-95 93-95
93-95 90-95 92-95 91-93
89-92
94-97 93-96 90-93 90-96
90-94 90-95 91-93 89-95
90-92 90-92 89-90 89-92
in X, standard deviation (SD) and coefficient of pods per plant in two varieties of chickpea {Cicer
Mean+S.E. \ Shift in X
Variety Pusa-212
91.20+0.34
96.40±0.20 96.0010.32 95.9010.22 94.7010.24
95.6010.33 94.7010.35 94.2010.23 94.0010.28
93.9010.22 93.2010.57 93.1010.35 91.8010.23
Variety BG-256
90.3010.40
95.5010.40 93.7010.28 91.8010.46 93.0010.58
92.7010.45 92.1010.64 92.0010.24 91.8010.46
91.2010.24 90.8010.27 89.5010.16 90.6010.38
-
+5.20 +4.80 +4.70 +3.50
+4.40 +3.50 +3.00 +2.80
+2.70 +2.00 +1.90 +0.60
-
+5.20 +3.40 +1.50 +2.70
+2.40 +1.80 +1.70 +1.50
+0.90 +0.50 -0.80 -0.30
S.D.
1.07
0.66 0.97 0.70 0.78
1.01 1.09 0.74 0.89
0.70 1.83 1.13 0.74
1.26
1.28 0.90 1.46 1.84
1.42 2.02 0.78 1.46
0.75 0.87 0.50 1.20
CV (%)
1.18
0.69 1.02 0.72 0.82
1.06 1.16 0.79 0.95
0.74 1.97 1.21 0.81
1.40
1.34 0.96 1.60 1.98
1.53 2.19 0.84 1.60
0.82 0.96 0.55 1.32
Table-14: Range, mean, shift in X, standard deviation (SD) and coefficient of variation (CV) for seeds per pod in two varieties of chickpea (Cicer arietinum L.)
Treatment Range Mean±S.E. Shift in X S.D. CV (%)
Variety Pusa-212
Control 1-2 1.2010.12 0.40 33.33
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
Control
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
1-2 1-3 1-3 1-2
1-3 1-2 1-2 1-2
1-2 1-3 1-3 1-2
1-2
1-3 1-3 1-3 1-3
1-2 1-3 1-3 1-2
1-2 1-3 1-3 1
1.4010.15 1.8010.24 1.6010.20 1.5010.15
1.5010.21 1.3010.14 1.3010.14 1.3010.14
1.2010.13 1.3010.20 1.3010.20 1.1010.09
+0.20 +0.60 +0.40 +0.30
+0.30 +0.10 +0.10 +0.10
0.00 +0.10 +0.10 -0.10
Variety BG-256
1.20+0.13
1.5010.21 1.8010.24 1.5010.20 1.4010.20
1.2010.13 1.3010.20 1.3010.20 1.40+0.15
1.2010.13 1.3010.20 1.3010.20 1.0010.00
-
+0.30 +0.60 +0.30 +0.20
0.00 +0.10 +0.10 +0.20
0.00 +0.10 +0.10 -0.20
0.49 0.75 0.66 0.50
0.67 0.46 0.46 0.46
0.40 0.64 0.64 0.30
0.40
0.67 0.75 0.64 0.66
0.40 0.64 0.64 0.49
0.40 0.64 0.64 0.00
34.99 41.57 41.45 33.33
44.72 35.25 35.25 35.25
33.33 49.25 49.25 27.27
33.33
44.72 41.57 42.66 47.38
33.33 49.25 49.25 34.99
33.33 49.23 49.23 0.00
Table -15: Range, mean, shift in X, standard deviation (SD) and coefficient of variation (CV) for 100-seed weight (g) in two varieties of chickpea (Cicer arietinum L.)
Treatment Range Mean+S.E. Shift in X S.D. CV (%)
Variety Pusa-212
Control 16.50-18.36 17.24+0.20 0.63 3.69
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
Control
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
18.00-21.00 19.00-21.05 17.00-20.10 17.00-19.45
17.50-19.10 17.00-20.85 16.00-18.50 15.50-18.80
16.00-19.00 15.50-18.00 16.90-19.00 17.00-19.00
16.00-18.63
18.18-20.00 18.18-21.20 15.00-17.10 15.65-17.00
17.00-19.86 18.00-20.00 16.00-17.85 16.00-17.85
16.00-18.00 16.00-18.00 16.20-19.20 17.00-19.92
19.45±0.30 20.00±0.25 18.20±0.32 17.50±0.23
18.4810.22 18.87±0.33 17.2010.30 16.4810.33
17.6010.42 16.65+0.24 17.77+0.24 18.0010.42
Variety BG-
17.3210.27
19.4410.20 19.6210.40 16.1010.28 16.0610.15
18.7310.31 18.9210.25 16.4810.20 16.9810.20
17.2910.22 17.0410.25 17.6110.37 18.3110.33
+2.21 +2.76 +0.96 +0.26
+1.24 +1.63 +0.04 -0.76
+0.36 -0.59 +0.53 +0.76
•256
-
+2.12 +2.30 -1.22 -1.26
+1.41 +1.60 -0.84 -0.34
-0.03 -0.28 +0.29 +0.99
0.95 0.79 0.66 0.74
0.69 1.06 0.95 1.05
1.35 0.76 0.75 0.77
0.86
0.66 1.29 0.91 0.49
0.98 0.79 0.63 0.63
0.72 0.82 1.18 1.06
4.89 3.95 3.62 4.22
3.72 5.62 5.54 6.36
7.70 4.59 4.22 4.30
4.97
3.40 6.58 5.65 3.08
5.34 4.18 3.87 3.87
4.16 4.81 6.71 5.81
Table-16: Range, mean, shift in X, standard deviation (SD) and coefficient of variation (CV) for yield per plant in two varieties of chickpea {Cicer arietinum L.)
Treatment
Control
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
Control
0.01% HZ 0.02% HZ 0.03% HZ 0.04% HZ
0.01% MMS 0.02% MMS 0.03% MMS 0.04% MMS
0.01% SA 0.02% SA 0.03% SA 0.04% SA
Range
28-30
32-34 33-36 30-32 30-32
30-34 31-34 31-34 32-35
30-34 29-30 29-32 28-30
30.00-34.00
30.10-32.58 32.10-34.61 32.10-35.56 31.38-35.80
31.00-33.00 30.20-32.78 31.16-33.00 30.60-33.20
30.00-32.60 31.00-33.00 30.00-32.56 32.00-34.00
MeanlS.E. Shift in X
Variety Pusa-212
29.3010.20
32.80+0.27 34.1010.33 31.9010.26 31.7010.62
32.1010.46 32.3010.28 31.5010.35 31.5510.29
31.8010.48 30.0010.24 30.3010.37 29.0010.28
-
+3.50 +4.80 +2.60 +2.40
+2.80 +3.00 +2.20 +2.25
+2.50 +0.70 +1.00 -0.30
Variety BG-256
31.7111.20
31.2810.28 33.4310.27 33.5910.39 34.0010.31
31.9210.21 31.4210.27 32.1210.20 32.1210.33
31.5410.28 31.9210.21 31.4410.26 32.7410.22
-
+0.43 +1.72 +1.88 +2.29
+0.21 +0.29 +0.41 +0.41
+0.17 +0.21 +0.27 +1.03
S.D.
0.64
0.87 1.04 0.83 0.65
1.46 0.90 0.60 0.94
0.94 0.77 1.18 0.89
0.38
0.88 0.85 1.26 1.34
0.68 0.85 0.65 1.04
0.88 0.68 0.83 0.69
CV (%)
2.18
2.65 3.06 2.60 2.05
4.57 2.78 1.90 2.97
2.78 2.58 3.91 3.08
1.19
2.87 2.55 3.76 3.94
2.14 2.85 2.02 3.26
2.81 2.14 2.65 2.11
43
Chapter 5
DISCUSSION
Mutation breeding is an efficient tool to amend and/or rectify
certain character(s) without altering the other traits of the crop plants,
in a relatively short span, especially when the characters under study
show simple Mendalian inheritance. Basic information on the
frequency and spectrum of mutations, treatment procedures and
methods of handling the treated population, would be highly desirable
for an effective use of this technique in the improvement of chickpea.
The study of biological damage in terms of lethality, seedling growth
depression, survival at maturity, frequency of chimeric plants, pollen
and seed fertility in Mi generation will give an opportunity to assess
mutagenic sensitivity of the biological system under study. The
sensitivity of any biological system to a particular mutagenic treatment
depends on various factors such as: (1) properties of biological system,
(2) physical and chemical properties of the mutagen, (3) concentration
of the mutagen, (4) duration of treatment, (5) temperature during
treatment, (6) hydrogen ion concentration, (7) pre and post treatment
conditions.
The sensitivity of chickpea varieties to various mutagenic
treatments was assessed by studying the biological damage induced in
Ml, in terms of seed germination, seedling growth, plant survival at
maturity and pollen fertility, besides frequencies of morphological
44
variations in Mi. Mutagenic treatments cause reduction in various Mi
parameters, which in turn can be used as indices to test the mutagenic
sensitivity of an organism (Nilan et al., 1968). In the present study,
reduction in seed germination was dose dependent and linear. Similar
observations were made in Oryza sativa (Kumar and Mani, 1997),
triticale (Pushpalatha et al, 1992), Vigna radiata (Khan and AH, 1987;
Khan et al, 1998 a,b). Capsicum annum (Raghuvanshi and Singh,
1979) and chickpea (Kharakwal, 1998a). Inhibition of germination after
irradiation has been attributed to chromosome deletion (Sparrow and
Evans, 1961) and changes in variety of biochemical and physiological
systems (Sparrow and Woodwell, 1962). Delayed and reduced seed
germination caused by various mutagens in the present study, may be
as a result of depression in the rate of mitotic proliferations or altered
enzyme activity. The denatured DNA after sometime may be repaired
resulting in the activation of biological processes involved in
germination and thus delayed germination is observed (Hutterman et
al, 1978).
The growth of seedlings were observed after the first seven days
of germination. There is a definite trend towards the decrease of
seedling height with the increasing concentrations of mutagens in both
the varieties. Reduced growth after mutagen treatment has been
reported by Goud et al. (1970) in Sorghum vulgare, Rajput (1970) in
Triticum aestivum, Reddy (1974) and Chakrabarti (1975) in Oryza
sativa and Mehrajuddin (2001) in Cicer arietinum. Mutagen induced
reduction in seedling height and growth inhibition may be due to
45
destruction or damage to apical meristems (Patel and Shah, 1974),
partial failure of the internodes to elongate, decrease in the number of
proliferating cells (Van't Hof and Sparrow, 1965) and chromosome
structural damage in meristematic cells (Gray and Scholes, 1951). Root
is found to be more sensitive than the shoot in both the varieties
studied in the present investigation. This shows that the shoot and root
respond differently to the mutagen treatment. A great deal of shoot
growth is due to the cell elongation whereas the root growth is more
dependent on cell division (Sinha and Godward, 1972).
Sterile pollen observed in both control as well as in mutagenic
population of both the varieties of chickpea used in the present study.
However, the percentage sterility increased considerably in mutagen
treatments. A depression in pollen fertility was also reported in Vigna
radiata (Rajput, 1973; Chandra et al, 1978; Khan and Hashim, 1978;
Ganguli and Bhaduri, 1980; Khan et al, 2000), in triticale
(Pushpalatha et al, 1992), in Hordeum vulgare (Tiwari, 1999).
Maximum reduction in fertility was observed in MMS, followed by HZ
and SA. Fahmy and Fahmy (1957) reported that alkylating agents have
a high ability to produce deficiencies of cryptic nature. A high
frequency of pollen sterility in EMS treated barley was attributed by
Sato and Gaul (1967) to gene mutations. Chromosomal aberrations
following EMS treatment have also been demonstrated (Nerker, 1977;
Mehrajuddin, 2001). Ali and Siddiq (1999) in Oryza sativa reported
that among the three mutagens, gamma rays, EMS and SA, the male
fertility was affected the most by SA treatments. Contrary to these
46
findings, in the present study, SA proved to be less toxic with regard to
pollen sterility in chickpea.
Plant survival at maturity, in both the varieties, decreased over
control but it was dose independent. These results are contrary to the
earlier findings of Raghuvanshi and Singh (1979) in chilli, Anwar and
Reddy (1981) in rice. Khan and Ali (1987) in mungbean and
Mehrajuddin (2001) in chickpea, who reported positive relationship
between the dose of the mutagen and final plant survival. The mutagens
are capable of creating chromosomal damage leading to mitotic arrest
and have lethal effects on different stages of plant growth.
Morphological variations affecting different plant parts were
isolated on screening of Mi populations. A number of mutagen induced
mutations in stem, leaf, flower, pod and other characters have also
been reported in Cicer arietinum (Nerker and Mote, 1978b; Ahmad and
Godward, 1993; Kharakwal 1998c; Mehrajuddin, 2001), in pigeon pea
(Rao and Reddy, 1984), in Vigna radiata (Khan and Siddiqui, 1996)
and in Vigna mungo (Singh et al., 1999). The frequency of
morphological variants was almost equal with HZ and MMS treatment
followed by SA. These results are in agreement with the earlier
findings of Kak and Kaul (1975) in Hardeum vulgare and Reddy and
Smith (1984) in Sorghum bicolor. Some workers have reported that
these macromutations were monogenic recessive in nature. Var. Pusa-
212 gave higher frequency of morphological variants than the var. BG-
256. This reflects to differences in their mutagenic sensitivity.
47
-'' The practical utiliy of induced mutations for improvement of
polygenic traits is well established, since most of the economic
characters in crop species are quantitatively inherited. Mutagenesis has
provided a handy tool to enhance the natural mutational rate and
thereby enlarging the genetic variability and increasing the scope for
obtaining the desired selections particularly, induction of
micromutations in polygenic system, controlling quantitative characters
are important for crop improvement. This is well established that
ionizing radiations and certain chemicals can successfully induce
mutations for polygenic traits in various crops (Gregory, 1965;
Swaminathan, 1969; Blixt and Gottschalk, 1975; Kaul, 1977; Farooq
and Nizam, 1979b; Kharakwal, 1983; Kaul and Kumar, 1983; Khan,
1990).
Range, mean, coefficient of variation (CV%) for eight attributes
of chickpea provided ample evidence that mutagenic treatments could
alter mean values and create additional genetic variability for
polygenic traits. Khan (1990) reported variable response of
quantitative characters to different mutagenic treatments in mungbean.
A positive relationship between the dose of the mutagen and M]
biological parameters, with an exception of survival, was evident from
the present study in chickpea. However, the extent of decrease was not
same in the two varieties showing the varietal differences. In the
present study, based on most of the Mi parameters, var. Pusa-212 was
found to be more sensitive than BG-256. The difference in mutagen
sensitivity varied not only between crop plants of unrelated families
48
but also between genera, species and varieties. The sensitivity of an
organism depends upon the mutagen employed, genetic make up (Blixt,
1968), amount of DNA and its replication time in the initial stages
(Varghees and Swaminathan, 1968) physiological state of tissue (Ilivea
Staneva, 1971), ability to repair damage (Auerbach, 1967) besides
physiological factors such as pH, moisture, oxygen, temperature
(Brock, 1965; Gelin, 1968). Differential response of two chickpea
varieties to the same concentration of a mutagen under similar
treatment conditions may be attributed to the differences in the degree
of heterochromatization at the varietal level. Genetic differences even
though very small (as single gene difference) can induce significant
changes in the mutagen sensitivity which influence various plant
characters in M] generation (Borojevic, 1970).
49
Chapter 6
SUMMARY
The objective of this study was to explore the possibility of
inducing variability for quantitative characters, viz., plant height (cm),
days to flowering days to maturity, fertile branches per plant, pods per
plant, seeds per pod, 100—seed weight (g) and total plant yield (g) by
using three chemical mutagens namely hydrazine hydrate (HZ) - a base
analogue, methhylmethane sulphonate (MMS) - an alkylating agent and
sodium azide (SA) - a respiratory inhibitor in two varieties (Pusa-212
and BG-256) of chickpea. The other aspects of this study were:
(1) biological damage in Mi generation;
(2) frequency of morphological variations and its spectrum.
The present study of germination, seedling height, plant survival at
maturity and pollen fertility indicates varying effects of the mutagen
treatments. When a critical assessment was made for the average of
biological damage done by the mutagen treatment in the two varieties,
greater biological damage was observed in the var. Pusa-212 in
comparison to the var. BG-256. Based on inhibition in seed
germination, HZ was found to be more effective followed by MMS and
SA whereas based on seedling injury, the order was HZ>SA> MMS in
both the varieties. Based on pollen sterility in both the varieties MMS
was found to be more effective in both the varieties.
50
Various types of morphological variants with alterations in plant
form, plant height and leaf were isolated and characterized. Frequency
of morphological variations induced by HZ and MMS was more or less
equal while SA showed lowest frequency of morphological variations
in both the varieties. The spectrum of induced variant differed not only
between varieties but also with in a variety. The spectrum was narrow
in the var. BG-256 than the var. Pusa-212.
In the present study, means for all the quantitative characters
shifted in both positive as well as in the negative direction of the
control mean. Coefficient of variation (CV%) different from trait to
trait and the highest CV over the control was recorded for seeds per
pod.
Based on most of the Mi parameters, var. Pusa-212 was found to
be more sensitive than BG-256.
51
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