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*Corresponding author’s e-mail: mcaquarian@gmail.com and address: ICAR-IARI, Regional Station, Amartara Cottage, Shimla-171004, India.1 Department of Plant Breeding and Genetics, CSK HPKV, Palampur-176062 (H.P), India.2 Department of Plant Physiology, CSK HPKV, Palampur-176062 (H.P), India.3Krishi Vigyan Kendra, Bara, CSK HPKV, Palampur-176062 (H.P), India.

Legume Research, 40 (1) 2017 : 39-46Print ISSN:0250-5371 / Online ISSN:0976-0571

Frequency and spectrum of chlorophyll mutations and induced variabilityin ricebean (Vigna umbellata Thunb, Ohwi and Ohashi)Madhu Patial*, S.R Thakur1, K.P. Singh2 and Anjana Thakur3

Chaudhary Shrawan Kumar Himachal Pradesh Krishi Vishvavidyalaya,Palampur-176062, Himachal Pradesh, India.Received: 19-06-2015 Accepted: 08-04-2016 DOI:10.18805/lr.v0iOF.10757

ABSTRACTA comparative study of frequency and spectrum of chlorophyll mutations induced by physical (gamma rays) and chemicalmutagens (EMS) in relation to the effect of mutations in M1 plant, induction of mutants in M2 and variability studies in M3generation was made in two ricebean (Vigna umbellata Thunb, Ohwi and Ohashi) varieties; one local (Totru Local) andone widely cultivated one (BRS-1) during 2005-2008. The treatments included three doses each of gamma rays (30, 40 and50 kR) and EMS (0.50, 0.60 and 0.70%). Both gamma rays and EMS proved to be hazardous for germination and plantsurvival at higher dose and the comparative reduction in these characters was higher under EMS treatments than gammarays. The frequencies and spectrum of five different kind of induced chlorophyll mutations was in the order; viridis >xantha = albina > chlorina > albo-viridis in BRS-1 and viridis > xantha > albina > chlorina = albo-viridis in Totru Local.EMS was found to be more effective in inducing maximum frequencies of chlorophyll mutants than gamma rays. Highestfrequency of chlorophyll mutants were found under O.50% EMS in BRS-1 and 0.70% EMS in Totru Local. Genotypicdifferences were seen as highest frequency of mutations was observed in Totru Local which was more sensitive to mutagenictreatments than BRS-1. The coefficients of variation for all quantitative characters studied in M3 generation were of highermagnitude compared to control in both the cultivars. The lower doses (30kR in gamma rays and 0.50% in EMS) werefound to be the most important doses for inducing desirable variability in ricebean and three traits i.e. pods per cluster,seeds per pod and pod length showed high heritability coupled with high genetic advance in both BRS-1 and Totru Localindicating that breeding for these traits can be achieved by phenotypic selection.

Key words: Chlorophyll mutations, EMS, Frequency and spectrum, Gamma rays, Ricebean, Variability.

INTRODUCTIONRicebean (Vigna umbellata Thunb, Ohwi and

Ohashi) belongs to family Fabaceae, subfamily Papilionaceae.It is considered as a minor food and fodder crop and is oftengrown as intercrop or mixed crop with maize (Zea mays),sorghum (Sorghum bicolor) or cowpea (V. unguiculata), aswell as a sole crop on a very limited area. Like other AsiaticVigna species, ricebean is a fairly short-lived warm-seasonannual and is adapted to extremes or uncongenial ecologicalniches particularly in areas receiving less rainfall with erraticdistribution. Production of ricebean varies greatly withinIndia. It is a rich source of protein and its nutritional qualityis higher as compared to many other legumes of Vigna family(Katoch, 2013). Ricebean has been considered as one ofthe best nutritionally balanced pulses in the world andhas even been included in the school children’s nutritionalprogrammes in Philippines (NAS, 1979). Despite its

multifarious importance, the systematic effort to improvethe yielding ability of the crop has been undertaken by thesimplest techniques of germplasm evaluation andhybridization. However, in general, a little work onmutation has been undertaken in this crop.

Chlorophyll mutants are used as markers in genetic,physiological and biochemical investigations. They are themost frequently observed and easily identified factorialmutations in M2 generation. The comparative study ofinduced chlorophyll mutation frequency in M2 generation isthe most dependable index for determining the extent ofinduced genetic changes in the mutagenic treated populationand effectiveness of mutagenic treatments (Gustaffson,1954). Improvement in the frequency and spectrum ofmutations in a predictable manner and thereby achievingdesired plant characteristics for their either direct or indirectexploitation in the breeding programme is an important goal

40 LEGUME RESEARCH - An International Journal

of mutation research. Although, studies on induced mutationshave been undertaken in the past in some legumes(Sinha and Lal, 2007; Singh and Rao, 2007), limited reportsare available on crops like ricebean. No systematicand comparative study of frequency and spectrum ofchlorophyll mutations induced by physical and chemicalmutagens on local and widely cultivated genotypes isavailable in literature.

Variability is the pre-requisite for selection andvarietal development in crop plants and mutation inductionhas become a proven way of creating variation within a cropspecies (Novak and Brunner, 1992). It offers the possibilityof inducing desired attributes that either cannot be found innature or have been lost during evolution. Shu (2009)reported the mutational enhancement of genetic diversity inseventeen plant species. The mutants obtained can becomeimportant genetic resources for breeding, gene discovery andfunctional analysis of various genes.

The present study was therefore, undertaken tounderstand the response of local and widely cultivatedricebean genotypes to more than one type of mutagenictreatment with a view to identify the mutagen-treatmentcombination causing maximum chlorophyll mutations in M2

generation and to induce variability in two ricebeangenotypes.

MATERIALS AND METHODSThe material for the study comprised of two

contrasting ricebean genotypes viz., BRS 1, having blackgrains (like urdbean), high yield and widely grown but, latematuring and Totru Local having creamish grains, low yield,early maturing and locally grown. Four hundred dry andhealthy seeds with moisture content of 10-12% were usedfor each treatment. Three doses of physical mutagen (gammarays) i.e 30kR, 40kR and 50kR and three doses of chemicalmutagen [ethyl methane sulphonate (EMS)] i.e 0.50%, 0.60%and 0.70% were used.

Gamma rays were secured from gamma Cell-200having 2500 Curie 60Co source installed in the Division ofGenetics, at National Physical Laboratory, IndianAgricultural Research Institute, New Delhi. The treatmentsof EMS were given in 0.1M phosphate buffer adjusted topH 7.0. The seeds were pre soaked in distilled water for 6hours and then immersed in freshly prepared mutagensolution for 6 hours (with periodic shaking), followed bypost treatment washing in gentle flow of tap water to removethe traces of chemical from the seed surface and then airdried on blotting paper at room temperature. Untreated dryseeds were used as a control (checks).

Sowing of M1 generation was done immediatelyafter treatment with the mutagens during kharif 2005-2006and single plants were harvested individually and planted asM2 family rows during the next crop season. In M1

generation, the data on reduction in germination andsubsequent survival (lethality) and seedling height reduction(injury) was recorded as per Sharma (1990) to estimate thedamage caused by the mutagens.

Half of the M2 seeds of each plant were used forraising M2 generation during kharif 2006-2007 and half werekept for raising M2 generation during kharif 2007-2008 alongwith M3 to avoid environmental effects. All M2 family plotsconsisted of single 5 m long rows with spacing of 45 X 20cm and 30 X 15 cm for BRS-1 and Totru Local, respectively.Different kinds of chlorophyll mutants were scored atdifferent stages of growth by using modified classificationof Kharkwal (1998) and mutation frequency was workedout as percent of mutated M2 families/plants as per themethod suggested by Konzak et al. (1965) and Kharkwal(1998). The next year, remaining M2 seeds were sown alongwith M3 seeds. M2 generation was raised in single plantcompletely randomized design and M3 generation was raisedin RCBD with two replications. The details of number offamilies tested under each variety in M2 and M3 generationsunder each dose are given below:

Data in M2 generation were recorded on a singleplant basis dose-variety wise. Observations were recordedon days to flowering, days to maturity, clusters of pod /plant,pods/cluster, seeds/pod, pod length (cm) and seed yield/plant (g).Estimates of genetic parameters were computedaccording to Sharma (1998).

RESULTS AND DISCUSSIONBoth gamma rays and EMS proved to be hazardous

for germination at higher dose i.e. under gamma rays, thereduction was maximum in 50 kR while under EMS it wasmaximum in 0.70% (Table 1). This reduction in germinationmay be attributed to the damage caused to the enzyme system

The number of families studied in M2 and M3 generations is asunder:

Mutagens Doses Number of families under each variety BRS-1 Totru Local

M2 M3 M2 M3

Gamma rays 30kR 32 33 22 1940kR 31 27 32 2850kR 29 16 15 11

EMS 0.50% 18 17 21 230.60% 26 21 12 140.70% 12 11 10 7

Volume 40 Issue 1, 2017 41

encompassing repair mechanism or due to the production oftoxic substances in the treated cells. Prakash andShambulingappa (1999) in ricebean and Meena and Dwivedi(2015) in chickpea have also reported similar reduction ingermination with increasing doses of mutagens. Likegermination, mutagenic treatments also reduced plantsurvival drastically with increasing dose of both themutagens. Lethal hits or relatively more chromatid orchromosomal breaks due to gamma rays and physical toxicityleading to lethality at higher doses of chemicals, have ingeneral, been held responsible for drop in plant survival.Similar trend of reduction in survival with increasing dosehas been reported by many workers (Ignacimuthu and Babu,1988 in urdbean and mungbean; Karthika and Lakshmi, 2007in soybean; Ambavane et al., 2015 in finger millet).Reduction in the germination and plant survival washigher under EMS treatments than gamma rays in both thevarieties. Some plants were killed after germination also but,killing was not drastic and percentage of germination andplant survival at maturity showed linear and positiverelationship.

Five types of chlorophyll mutants were observed in M2

generation. These were viridis, xantha, chlorina, albino, andalbo-viridis (Fig. 1 and 2):i) The viridis were represented by light green colour in the

early stages of growth. This colour gradually changed tothe normal green colour during the subsequent period ofgrowth of the plant and were viable.

ii) The xantha mutants were yellow to yellow white, lethal(12-15 days), carotenoids present but, chlorophyll wasabsent.

iii) The chlorina mutants were yellowish green in colour.iv) The albina mutants were completely white in colour and

were completely lethal (6-8 days), no chlorophyll orcarotenoids were formed.

v) Albo-viridis were classified as green with white apex.

Swaminathan et al. (1962) in barley and wheatreported the randomness in the action of physical mutagensand specificity of EMS to certain loci for the production ofchlorophyll mutations.

The frequency of induced chlorophyll mutationsranged in order of viridis > xantha = albina > chlorina >albo-viridis in BRS-1 and viridis > xantha > albina >chlorina = albo-viridis in Totru Local (Table 2). Viridisappeared in every dose in both the genotypes while othermutants were present in some but, absent in other doses.This presence or absence of mutants in one or other dose isattributed to the polygenic control of chlorophyll formationalongwith high mutability of genes for chlorophyll (Gaul,Ta

ble

1: E

ffect

of

mut

agen

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atio

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ver

cont

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%)

BR

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rays

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trol

93.3

410

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91.9

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30 k

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66.7

261

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40 k

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48.8

139

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43.3

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629

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32.6

067

.40

EMS

Con

trol

91.4

410

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89.9

410

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0.50

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61.8

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42.6

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ocal

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ma

rays

Con

trol

84.7

810

0.00

81.9

810

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kR

67.7

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65.6

180

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19.9

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54.9

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7

42 LEGUME RESEARCH - An International Journal

Fig 2: Chlorophyll mutants in Totru Local, a: control, b: albina, c: xantha, d: viridis, e: chlorina, f: albo-viridis

Fig 1: Chlorophyll mutants in BRS-1, a: control, b: albina, c: xantha, d: viridis, e: chlorina, f: albo-viridis

Volume 40 Issue 1, 2017 43

Tabl

e 2:

Fre

quen

cies

and

spe

ctru

m o

f ch

loro

phyl

l mut

atio

ns in

BR

S-1

and

Totru

Loc

al in

M2 g

ener

atio

n

44 LEGUME RESEARCH - An International Journal

1964). Different types of chlorophyll mutants in differentvarieties and differential frequency spectrum could be dueto differential genotypic response to the mutagens used. Deviet al. (2002) have also reported similar results whereby theygot highest frequency of viridis.

All the mutagenic treatments induced varyingfrequencies of chlorophyll mutations in M2 generation. Thedecrease in total frequency on family basis (%) of differentmutants from 30 kR to 40 kR and 0.50% to 0.60% EMS wasdistinct but, conversely it increased from 40 kR to 50 kR inboth BRS-1 and Totru Local. Whereas, frequency from0.60% to 0.70% EMS decreased in BRS-1 while it increasedin Totru Local (Table 2). The frequency on plant basis (%)showed similar trend except that there was no decrease infrequency from 0.5%EMS to 0.6% EMS. In this case,therefore, response seems to be genotype-dose dependentand did not showed clear-cut trend of decrease or increaseof frequencies. This may be due to the elimination ofchlorophyll mutations at the higher doses because of lethality.Highest response to irradiation in both BRS-1 and TotruLocal was shown at 30 kR, while in EMS highest responsewas at 0.50% in BRS-1 and at 0.70% in Totru Local.Thus, the differential response was shown by the twogenotypes under the EMS. EMS was found to be moreefficient in inducing chlorophyll mutations than gamma raysin ricebean. There is a general notion of chemicalsinducing higher chlorophyll mutations than radiation(Kharkwal, 1998). Different workers have also reportedsimilar results where EMS has shown more frequency thangamma rays (Prakash and Khanure, 1999; Waghmare andMehra 2001).

Of the two varieties used in the study, the averagechlorophyll mutation frequency was higher in TotruLocal than BRS-1 which indicated differential genotypicresponse of chlorophyll controlling genes to mutation,the genes in former being more sensitive. The low sensitivityof BRS-1 might be due to past exposure of its parents tophysical radiations and acquisition of some level ofresistance to mutagens. Similar behavior of varieties tomutagenic treatments has been reported in differentcrops (Deepalakshmi and Kumar, 2003; Sangsiri et al.2005).

Induced variability: In M3 generation variability inducedwas reported by estimating the mean values, PCV, GCV,

heritability and genetic advance for yield and yieldcomponents (Table 3). An increase in genetic variability wasobserved as there was shift in mean values due to theoccurrence of extreme types, both on the positive andnegative side of the control. In genotype BRS-1, 30kR proved to be most suitabledose in gamma rays where maximum traits showed moderateto very high PCV, GCV, h2

bs and GA. EMS at 0.50%and 0.60% showed similar trend. In these doses all theselection parameters were recorded to be high to very highfor the traits pods per cluster, seeds per pod and podlength indicating the relative importance of these traits forselection.

In Totru Local, 30 kR and 0.50% alongwith 0.70%EMS were the most suitable doses for inducing maximumvariability in the characters thereby showing high to veryhigh values of selection parameters. The important traitswhich were most effective for making selection in Totru Localwere clusters of pods per plant, pods per cluster, seeds perpod and pods length.

In both BRS-1 and Totru Local; gamma rays dosesof 30 kR and EMS dose of 0.50% followed by 0.60% EMSdose in BRS-1 and 0.70% EMS dose in Totru Local werethe most suitable doses. These doses produced mutagenizedpopulations with high magnitudes of four differentparameters of variability. All the three characters suitablefor making selection in BRS-1 were also suitable for TotruLocal.

Increase in the variability parameters in mutagenizedpopulations of ricebean have also been reported by Lokeshaet al. (1991) and Lokesha and Veeresh (1993). Mahla et al.(2010) in clusterbean, Singh et al. (2007) in cowpea andKham et al. (2015) in sorghum also reported induction ofvariability through gamma rays and EMS. An increase ingenetic variability might be due to the occurrence of extremetypes, both on the positive and negative side of the control.This is due to the fact that the effect of mutagens on thequantitative traits has been interpreted as that owing todetrimental mutations which are supposed to occur morefrequently than favorable ones. Brock (1965) reported thatin the species which had previously been selected tobreeding, random mutations resulted in an increase invariance and a shift in the mean away from the direction ofprevious selection.

Volume 40 Issue 1, 2017 45

p =

Sign

ifica

nt p

ositi

ve s

hift

in m

ean

n

= S

igni

fican

t neg

ativ

e sh

ift in

mea

n

Tabl

e 3:

Esti

mat

es o

f mea

n va

lues

, shi

ft in

mea

n, c

oeffi

cient

of

varia

tion,

her

itabi

lity

(h2 bs

) and

gen

etic

adv

ance

(GA

) fo

r diff

eren

t tra

its in

M3 g

ener

atio

ns of

BR

S-1

and

Totru

Loc

al.

46 LEGUME RESEARCH - An International Journal

REFERENCESAmbavane, A.R., Sawardekar, S.V., Sawantdesai, S.A. and Gokhale N.B. (2015). Studies on mutagenic effectiveness and

efficiency of gamma rays and its effect on quantitative traits in finger millet (Eleusine coracana L. Gaertn).Journal of Radiation Research and Applied Sciences, 8: 120–125.

Brock, R. D. (1965). Induced mutations affecting quantitative characters. Radiation Botany, 5:451-464.Deepalakshmi, A. J. and Kumar, A.C. R. (2003). Efficiency and effectiveness of physical and chemical mutagens in

urdbean (Vigna mungo (L.) Hepper). Madras Agricultural Journal, 90: 485-489.Devi, T. R., Prodhan, H. S., Brajendra, N. and Rohinikumar, M. (2002). Effectiveness and efficiency of gamma rays in

inducing chlorophyll mutations in M2 generation of three ricebean genotypes (Vigna umbellata (L.) Thunb, Ohwiand Ohashi). Environment and Ecology, 20 : 890-893.

Federer, W.T. (1956) . Augmented (or honnuiaku) designs. Hawaii Plant Research 2 : 191-208.Gaul, H. (1964). Mutations in plant breeding. Radiation Botany, 4 :155-232.Gustaffson, A. (1954). Mutation, viability and population structure. Acta Agric Scand, 4: 601-31.Ignacimuthu, S. and Babu, C. R. (1988). Radio sensitivity of the wild and cultivated urd and mungbean. Indian Journal

of Genetics and Plant Breeding, 48 : 331-342.Karthika, R. and Lakshmi, B. S. (2007). Fixation of lethal dose50 and the performance of M1 population in two soybean

varieties. Legume Research, 30 : 49-52.Katoch, R. (2013). Nutritional potential of rice bean (Vigna Umbellata): An Underutilized legume. Journal of Food

Science, 78: 8-16.Kham, N. H., Win N. C., Minn, M. (2015). Study on the variability of induced mutation for improvement of local cultivar

sorghum (Shweni-15). International Journal of Technical Research and Applications, 3: 139-144.Kharkwal, M. C. (1998). Induced mutations in chickpea (Cicer arietinum L.) II. Frequency and spectrum of chlorophyll

mutations. Indian Journal of Genetics and Plant Breeding, 58 : 465-474.Konzak, C. F., Nilan, R. A., Wanger, J. and Foster, R. J. (1965). Efficient chemical mutagenesis. Radiation Botany, 5: 49-70.Lokesha, R. and Veeresh, L. C. (1993). Induced mutagenesis and genetic improvement of rice bean (Vigna umbellata

(Thunb) Ohwi and Ohashi. Legume Research, 16: 37-40.Lokesha, R., Vasanth, K. E., Veeresh, L. C. and Shivashankar, G. (1991). Gamma-ray induced genetic divergence in rice bean

(Vigna umbellata Thumb Ohwi and Ohashi). In: Golden Jubilee Symposium on Genetic Research and Education:Current trends and the next fifty years, Indian Society of Genetics and Plant Breeding. IARI, New Delhi, p.719.

Mahla, H. R., Kumar, D. and Shekhawat, A. (2010). Effectiveness and efficiency of mutagens and induced variability inclusterbean (Cyamopsis tetragonoloba). The Indian Journal of Agricultural Sciences, 80:1033-7.

Meena, G. S. and Dwivedi, P. (2015). Effect of gamma rays and nitroso methyl urea on seed germination, seedling heightand survivability of chickpea (Cicer arietinum L.) var. RSG-963. Trends in Life Sciences, 4: 397-399.

NAS. (1979). Tropical legume : Resources for the future. National Academy of Sciences, Washington DC, pp. 331.Novak, F. J. and Brunner, H. (1992). Plant breeding: induced mutation technology for crop improvement. In: IAEA

BULLETIN, 4/1992. pp. 25-33.Prakash, B. G. and Khanure, S. K. (1999). Isolation of mutants and their frequencies under M2 generation in ricebean

(Vigna umbellata L. Thunb). Madras Agriculture Journal, 86 : 568-572.Prakash, B. G. and Shambulingappa, K. G. (1999). Estimation of chlorophyll and viable mutation under M2 generation in

ricebean (Vigna umbellata L. Thunb). Karnataka Journal of Agriculture Sciences, 12 : 38-42.Sangsiri, C., Sarajjapinun, W. and Srinives, P. (2005). Gamma radiation induced mutations in mungbean. Science Asia,

31 : 251-255.Sharma, J. R. (1998). Statistical and Biometrical Techniques in Plant Breeding. New Age International (P) Limited, p. 429.Sharma, S.K. (1990). Mutagenic effectiveness and efficiency in microsperma lentil. Cytologia, 55:243-247.Shu, Q. Y. (2009). Induced plant mutations in the genomics era. In: IAEA. Food and Agriculture Organization of United

Nationals, Rome, pp. 455-458.Singh, B. and Rao, G. T. (2007). Induced chlorophyll mutations in green gram (Vigna radiata (L.) Wilczek). Legume

Research, 30 : 308-310.Singh, S. P., Singh, R. P., Singh, N. K., Prasad, J. P. and Sahi, J. P. (2007). Mutagenic efficiency of gamma rays, EMS and

its combination on microsperma lentil. International Journal of Agricultural Sciences, 3: 113-118.Sinha, A. and Lal, J. P. (2007). Effect of mutagens on M1 parameters and qualitative changes induced in M2 generation in

lentil. Legume Research, 30 : 180-185.Swaminathan, M.S., Chopra, V.L. and Bhaskaran, S. (1962). Chromosome aberrations, the frequency and spectrum of

mutations induced by EMS in barley and wheat. Indian Journal of Genetics and Plant Breeding, 22: 190-207Waghmare, V. N. and Mehra, R. B. (2001). Induced chlorophyll mutations, mutagenic effectiveness and efficiency in

Lathyrus sativus L. Indian Journal of Genetics and Plant Breeding, 61: 53-56.