Frequency and spectrum of chlorophyll mutations and induced variability in ricebean (Vigna umbellata Thunb, Ohwi and Ohashi)

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1 Legume Research, 40 (1) 2017 : Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Frequency and spectrum of chlorophyll mutations and induced variability in ricebean (Vigna umbellata Thunb, Ohwi and Ohashi) Madhu Patial*, S.R Thakur 1, K.P. Singh 2 and Anjana Thakur 3 Chaudhary Shrawan Kumar Himachal Pradesh Krishi Vishvavidyalaya, Palampur , Himachal Pradesh, India. Received: Accepted: DOI: /lr.v0iOF ABSTRACT A comparative study of frequency and spectrum of chlorophyll mutations induced by physical (gamma rays) and chemical mutagens (EMS) in relation to the effect of mutations in M 1 plant, induction of mutants in M 2 and variability studies in M 3 generation was made in two ricebean (Vigna umbellata Thunb, Ohwi and Ohashi) varieties; one local (Totru Local) and one widely cultivated one (BRS-1) during The treatments included three doses each of gamma rays (30, 40 and 50 kr) and EMS (0.50, 0.60 and 0.70%). Both gamma rays and EMS proved to be hazardous for germination and plant survival at higher dose and the comparative reduction in these characters was higher under EMS treatments than gamma rays. 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. Highest frequency of chlorophyll mutants were found under O.50% EMS in BRS-1 and 0.70% EMS in Totru Local. Genotypic differences were seen as highest frequency of mutations was observed in Totru Local which was more sensitive to mutagenic treatments than BRS-1. The coefficients of variation for all quantitative characters studied in M 3 generation were of higher magnitude compared to control in both the cultivars. The lower doses (30kR in gamma rays and 0.50% in EMS) were found 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 Local indicating that breeding for these traits can be achieved by phenotypic selection. Key words: Chlorophyll mutations, EMS, Frequency and spectrum, Gamma rays, Ricebean, Variability. INTRODUCTION Ricebean (Vigna umbellata Thunb, Ohwi and Ohashi) belongs to family Fabaceae, subfamily Papilionaceae. It is considered as a minor food and fodder crop and is often grown as intercrop or mixed crop with maize (Zea mays), sorghum (Sorghum bicolor) or cowpea (V. unguiculata), as well as a sole crop on a very limited area. Like other Asiatic Vigna species, ricebean is a fairly short-lived warm-season annual and is adapted to extremes or uncongenial ecological niches particularly in areas receiving less rainfall with erratic distribution. Production of ricebean varies greatly within India. It is a rich source of protein and its nutritional quality is higher as compared to many other legumes of Vigna family (Katoch, 2013). Ricebean has been considered as one of the best nutritionally balanced pulses in the world and has even been included in the school children s nutritional programmes in Philippines (NAS, 1979). Despite its multifarious importance, the systematic effort to improve the yielding ability of the crop has been undertaken by the simplest techniques of germplasm evaluation and hybridization. However, in general, a little work on mutation has been undertaken in this crop. Chlorophyll mutants are used as markers in genetic, physiological and biochemical investigations. They are the most frequently observed and easily identified factorial mutations in M 2 generation. The comparative study of induced chlorophyll mutation frequency in M 2 generation is the most dependable index for determining the extent of induced genetic changes in the mutagenic treated population and effectiveness of mutagenic treatments (Gustaffson, 1954). Improvement in the frequency and spectrum of mutations in a predictable manner and thereby achieving desired plant characteristics for their either direct or indirect exploitation in the breeding programme is an important goal *Corresponding author s mcaquarian@gmail.com and address: ICAR-IARI, Regional Station, Amartara Cottage, Shimla , India. 1 Department of Plant Breeding and Genetics, CSK HPKV, Palampur (H.P), India. 2 Department of Plant Physiology, CSK HPKV, Palampur (H.P), India. 3 Krishi Vigyan Kendra, Bara, CSK HPKV, Palampur (H.P), India.

2 40 LEGUME RESEARCH - An International Journal of mutation research. Although, studies on induced mutations have been undertaken in the past in some legumes (Sinha and Lal, 2007; Singh and Rao, 2007), limited reports are available on crops like ricebean. No systematic and comparative study of frequency and spectrum of chlorophyll mutations induced by physical and chemical mutagens on local and widely cultivated genotypes is available in literature. Variability is the pre-requisite for selection and varietal development in crop plants and mutation induction has become a proven way of creating variation within a crop species (Novak and Brunner, 1992). It offers the possibility of inducing desired attributes that either cannot be found in nature or have been lost during evolution. Shu (2009) reported the mutational enhancement of genetic diversity in seventeen plant species. The mutants obtained can become important genetic resources for breeding, gene discovery and functional analysis of various genes. The present study was therefore, undertaken to understand the response of local and widely cultivated ricebean genotypes to more than one type of mutagenic treatment with a view to identify the mutagen-treatment combination causing maximum chlorophyll mutations in M 2 generation and to induce variability in two ricebean genotypes. MATERIALS AND METHODS The material for the study comprised of two contrasting ricebean genotypes viz., BRS 1, having black grains (like urdbean), high yield and widely grown but, late maturing and Totru Local having creamish grains, low yield, early maturing and locally grown. Four hundred dry and healthy seeds with moisture content of 10-12% were used for each treatment. Three doses of physical mutagen (gamma rays) i.e 30kR, 40kR and 50kR and three doses of chemical mutagen [ethyl methane sulphonate (EMS)] i.e 0.50%, 0.60% and 0.70% were used. Gamma rays were secured from gamma Cell-200 having 2500 Curie 60 Co source installed in the Division of Genetics, at National Physical Laboratory, Indian Agricultural Research Institute, New Delhi. The treatments of EMS were given in 0.1M phosphate buffer adjusted to ph 7.0. The seeds were pre soaked in distilled water for 6 hours and then immersed in freshly prepared mutagen solution for 6 hours (with periodic shaking), followed by post treatment washing in gentle flow of tap water to remove the traces of chemical from the seed surface and then air dried on blotting paper at room temperature. Untreated dry seeds were used as a control (checks). Sowing of M 1 generation was done immediately after treatment with the mutagens during kharif and single plants were harvested individually and planted as M 2 family rows during the next crop season. In M 1 generation, the data on reduction in germination and subsequent survival (lethality) and seedling height reduction (injury) was recorded as per Sharma (1990) to estimate the damage caused by the mutagens. Half of the M 2 seeds of each plant were used for raising M 2 generation during kharif and half were kept for raising M 2 generation during kharif along with M 3 to avoid environmental effects. All M 2 family plots consisted of single 5 m long rows with spacing of 45 X 20 cm and 30 X 15 cm for BRS-1 and Totru Local, respectively. Different kinds of chlorophyll mutants were scored at different stages of growth by using modified classification of Kharkwal (1998) and mutation frequency was worked out as percent of mutated M 2 families/plants as per the method suggested by Konzak et al. (1965) and Kharkwal (1998). The next year, remaining M 2 seeds were sown along with M 3 seeds. M 2 generation was raised in single plant completely randomized design and M 3 generation was raised in RCBD with two replications. The details of number of families tested under each variety in M 2 and M 3 generations under each dose are given below: The number of families studied in M 2 and M 3 generations is as under: Mutagens Doses Number of families under each variety BRS-1 Totru Local M 2 M 3 M 2 M 3 Gamma rays 30kR kR kR EMS 0.50% % % Data in M 2 generation were recorded on a single plant basis dose-variety wise. Observations were recorded on 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 computed according to Sharma (1998). RESULTS AND DISCUSSION Both gamma rays and EMS proved to be hazardous for germination at higher dose i.e. under gamma rays, the reduction was maximum in 50 kr while under EMS it was maximum in 0.70% (Table 1). This reduction in germination may be attributed to the damage caused to the enzyme system

3 Volume 40 Issue 1, Table 1: Effect of mutagens on germination and plant survival in M 1 generation Treatments Germination % Survival % Reduction in survival Actual Relative Actual Relative over control (%) BRS-1 Gamma rays Control kr kr kr EMS Control % % % Totru Local Gamma rays Control kr kr kr EMS Control % % % encompassing repair mechanism or due to the production of toxic substances in the treated cells. Prakash and Shambulingappa (1999) in ricebean and Meena and Dwivedi (2015) in chickpea have also reported similar reduction in germination with increasing doses of mutagens. Like germination, mutagenic treatments also reduced plant survival drastically with increasing dose of both the mutagens. Lethal hits or relatively more chromatid or chromosomal breaks due to gamma rays and physical toxicity leading to lethality at higher doses of chemicals, have in general, been held responsible for drop in plant survival. Similar trend of reduction in survival with increasing dose has been reported by many workers (Ignacimuthu and Babu, 1988 in urdbean and mungbean; Karthika and Lakshmi, 2007 in soybean; Ambavane et al., 2015 in finger millet). Reduction in the germination and plant survival was higher under EMS treatments than gamma rays in both the varieties. Some plants were killed after germination also but, killing was not drastic and percentage of germination and plant survival at maturity showed linear and positive relationship. Five types of chlorophyll mutants were observed in M 2 generation. These were viridis, xantha, chlorina, albino, and albo-viridis (Fig. 1 and 2): i) The viridis were represented by light green colour in the early stages of growth. This colour gradually changed to the normal green colour during the subsequent period of growth of the plant and were viable. ii) The xantha mutants were yellow to yellow white, lethal (12-15 days), carotenoids present but, chlorophyll was absent. 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 or carotenoids were formed. v) Albo-viridis were classified as green with white apex. Swaminathan et al. (1962) in barley and wheat reported the randomness in the action of physical mutagens and specificity of EMS to certain loci for the production of chlorophyll mutations. The frequency of induced chlorophyll mutations ranged in order of viridis > xantha = albina > chlorina > albo-viridis in BRS-1 and viridis > xantha > albina > chlorina = albo-viridis in Totru Local (Table 2). Viridis appeared in every dose in both the genotypes while other mutants were present in some but, absent in other doses. This presence or absence of mutants in one or other dose is attributed to the polygenic control of chlorophyll formation alongwith high mutability of genes for chlorophyll (Gaul,

4 42 LEGUME RESEARCH - An International Journal Fig 1: Chlorophyll mutants in BRS-1, a: control, b: albina, c: xantha, d: viridis, e: chlorina, f: albo-viridis Fig 2: Chlorophyll mutants in Totru Local, a: control, b: albina, c: xantha, d: viridis, e: chlorina, f: albo-viridis

5 Table 2: Frequencies and spectrum of chlorophyll mutations in BRS-1 and Totru Local in M 2 generation Volume 40 Issue 1,

6 44 LEGUME RESEARCH - An International Journal 1964). Different types of chlorophyll mutants in different varieties and differential frequency spectrum could be due to differential genotypic response to the mutagens used. Devi et al. (2002) have also reported similar results whereby they got highest frequency of viridis. All the mutagenic treatments induced varying frequencies of chlorophyll mutations in M 2 generation. The decrease in total frequency on family basis (%) of different mutants from 30 kr to 40 kr and 0.50% to 0.60% EMS was distinct but, conversely it increased from 40 kr to 50 kr in both BRS-1 and Totru Local. Whereas, frequency from 0.60% to 0.70% EMS decreased in BRS-1 while it increased in Totru Local (Table 2). The frequency on plant basis (%) showed similar trend except that there was no decrease in frequency from 0.5%EMS to 0.6% EMS. In this case, therefore, response seems to be genotype-dose dependent and did not showed clear-cut trend of decrease or increase of frequencies. This may be due to the elimination of chlorophyll mutations at the higher doses because of lethality. Highest response to irradiation in both BRS-1 and Totru Local was shown at 30 kr, while in EMS highest response was at 0.50% in BRS-1 and at 0.70% in Totru Local. Thus, the differential response was shown by the two genotypes under the EMS. EMS was found to be more efficient in inducing chlorophyll mutations than gamma rays in ricebean. There is a general notion of chemicals inducing higher chlorophyll mutations than radiation (Kharkwal, 1998). Different workers have also reported similar results where EMS has shown more frequency than gamma rays (Prakash and Khanure, 1999; Waghmare and Mehra 2001). Of the two varieties used in the study, the average chlorophyll mutation frequency was higher in Totru Local than BRS-1 which indicated differential genotypic response of chlorophyll controlling genes to mutation, the genes in former being more sensitive. The low sensitivity of BRS-1 might be due to past exposure of its parents to physical radiations and acquisition of some level of resistance to mutagens. Similar behavior of varieties to mutagenic treatments has been reported in different crops (Deepalakshmi and Kumar, 2003; Sangsiri et al. 2005). Induced variability: In M 3 generation variability induced was reported by estimating the mean values, PCV, GCV, heritability and genetic advance for yield and yield components (Table 3). An increase in genetic variability was observed as there was shift in mean values due to the occurrence of extreme types, both on the positive and negative side of the control. In genotype BRS-1, 30kR proved to be most suitable dose in gamma rays where maximum traits showed moderate to very high PCV, GCV, h 2 and GA. EMS at 0.50% bs and 0.60% showed similar trend. In these doses all the selection parameters were recorded to be high to very high for the traits pods per cluster, seeds per pod and pod length indicating the relative importance of these traits for selection. In Totru Local, 30 kr and 0.50% alongwith 0.70% EMS were the most suitable doses for inducing maximum variability in the characters thereby showing high to very high values of selection parameters. The important traits which were most effective for making selection in Totru Local were clusters of pods per plant, pods per cluster, seeds per pod and pods length. In both BRS-1 and Totru Local; gamma rays doses of 30 kr and EMS dose of 0.50% followed by 0.60% EMS dose in BRS-1 and 0.70% EMS dose in Totru Local were the most suitable doses. These doses produced mutagenized populations with high magnitudes of four different parameters of variability. All the three characters suitable for making selection in BRS-1 were also suitable for Totru Local. Increase in the variability parameters in mutagenized populations of ricebean have also been reported by Lokesha et al. (1991) and Lokesha and Veeresh (1993). Mahla et al. (2010) in clusterbean, Singh et al. (2007) in cowpea and Kham et al. (2015) in sorghum also reported induction of variability through gamma rays and EMS. An increase in genetic variability might be due to the occurrence of extreme types, both on the positive and negative side of the control. This is due to the fact that the effect of mutagens on the quantitative traits has been interpreted as that owing to detrimental mutations which are supposed to occur more frequently than favorable ones. Brock (1965) reported that in the species which had previously been selected to breeding, random mutations resulted in an increase in variance and a shift in the mean away from the direction of previous selection.

7 p = Significant positive shift in mean n = Significant negative shift in mean Table 3: Estimates of mean values, shift in mean, coefficient of variation, heritability (h2bs) and genetic advance (GA) for different traits in M3 generations of BRS-1 and Totru Local. Volume 40 Issue 1,

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