GAMMA RAY AND EMS INDUCED EFFECTIVENESS AND EFFICIENCY OF CHLOROPHYLL MUTATIONS IN AROMATIC RICE (ORYZA SATIVA L.)

NSave Nature to Survive QUARTERLY 9(3&4): 975-979, 2015 (Supplement on Rice) GAMMA RAY AND EMS INDUCED EFFECTIVENESS AND EFFICIENCY OF CHLOROPHYLL MUTATIONS IN AROMATIC RICE (ORYZA SATIVA L.) SANJEEV SINGH, RISHI KUMAR SHARMA, PRAKASH SINGH 1 AND SATISH KUMAR CHAKRAVARTI 1 Department of Agricultural Botany, Udai Pratap Autonomous College, Varanasi - 221 002, INDIA Department of Genetics and Plant Breeding, I. Ag. Sc., B.H.U., Varanasi - 221 005, INDIA e-mail: ssinghupcv@gmail.com ABSTRACT INTRODUCTION In mutagenesis, the choice of the mutagen is most important and various methods have been developed to ascertain the most effective and efficient mutagen(s) and mutagenic treatment(s) for the induction of desirable characters in cultivated crops. Chlorophyll mutations are most convenient for evaluating the genetic effect of mutagens in plants (Gustafsson, 1951). It may serve as a good index for determining the doses of different mutagens. Mutation breeding has been used widely for the improvement of various plant characters from time to time. The usefulness of a mutagen depends both on its mutagenic effectiveness and efficiency. Efficient mutagenesis envisages the production of maximum desirable changes accompanied by the least possible undesirable changes. Experiences have shown that the frequency of desirable mutations depends upon the effectiveness and efficiency of the mutagens used ( Konzak et al.,1965; Nilan et al.,1965; Kumar and Mani,1997; Singh et al., 2001). The present paper deals with the effectiveness and efficiency of gamma rays and EMS alone or in combination with regard to chlorophyll mutations in two varieties of aromatic rice (Oryza sativa L.) in M 2. MATERIALS AND METHODS The two aromatic rice varieties viz., Pusa Basmati 1 and Kalanamak were employed as experimental materials under present investigation. Three hundred pure, uniform, healthy and dry (12% moisture) seeds for each treatment of aromatic rice Pusa Basmati 1 and Kalanamak were irradiated with gamma rays at five doses, viz., 10 kr, 20 kr, 30 kr, 40 kr and 50 kr at NBRI, Lucknow. EMS solution with different concentrations, i.e., 0.2%, 0.3%, 0.4% and 0.5% were prepared by mixing appropriate volume of ethyl methane sulphonate in phosphate buffer (ph 7.0). The 300 pure, uniform, healthy and dry seeds (12% moisture) were subjected to pre soaking in distilled water for 6 hours at room temperature. The soaked seeds were then transferred to EMS solution of different concentrations in research lab of the department. The seeds were kept in EMS solution for 6 hours and seeds were given intermittent shaking throughout the period of treatment to maintain uniformity. The mutagen solution was drained out. The treated seeds were then washed in running tap water for 1 hour to remove residual chemical from the seeds, if any. The combination treatment of gamma rays and ethyl methane sulphonate was also done as described earlier. For this, the gamma ray treated seeds were soaked in distilled water for six hours and then treated with EMS solution of 0.2% concentration for six hours followed by washing in running tap water for 1 hour. For each combination treatment, 300 seeds were used. The effectiveness and efficiency of gamma ray and EMS alone or in combination with regard to chlorophyll mutations in two varieties of aromatic rice (Oryza sativa L.) in M 2 were studied. In Pusa Basmati 1, the segregation of M 1 panicle progenies was observed maximally (60 per cent) at 50 kr gamma rays + 0.2% EMS, whereas in Kalanamak, it was found maximally (80 per cent) at 40 kr gamma rays. The highest frequency (1.472 per cent) of chlorophyll mutations in Pusa Basmati 1 was obtained at 50 kr gamma rays+0.2% EMS, whereas in Kalanamak the highest frequency (2.262 per cent) of chlorophyll mutations was obtained at 40 kr gamma rays. The chlorophyll mutations were induced by all the doses of mutagens alone or in combination relatively at a fair frequency in both the varieties in M 2. Gamma ray at 10 kr was found as the most efficient dose in both Pusa Basmati 1 and Kalanamak cultivars on sterility basis. Mutagenic effectiveness was observed maximum in Pusa Basmati 1 at 0.4% EMS and in Kalanamak at 0.3% EMS. KEY WORDS Chlorophyll mutation Efficiency Effectiveness Gamma rays, EMS Received : 10.06.2015 Revised : 18.08.2015 Accepted : 13.09.2015 *Corresponding author 975

SANJEEV SINGH et al., The M 1 was grown during Kharif 2011 followed by M 2 during Kharif 2012 at the Research Farm, Udai Pratap Autonomous College, Varanasi. For spikelet sterility, 10 plants from each replication of each treatment along with the control of both the varieties were randomly selected and tagged. The percentage of spikelet sterility was determined by estimating the proportion of completely unfilled spikelets out of the total number of spikelets of the main panicle. The 50 plants from each treatment were randomly selected and one panicle per plant was harvested separately from randomly selected plants to raise the M 1 progeny rows in M 2. The chlorophyll mutation frequency was calculated dose wise per 100 M 2 plants (Gaul, 1964). The chlorophyll mutants were classified at seedling stage following the classification of Gustafsson (1940).The mutagenic efficiency and effectiveness were estimated following the method of Konzak et al. (1965). Mutagenic efficiency = Msd/I or Msd/S Mutagenic effectiveness = Msd/tc or Msd/Gy Where, Msd = mutation frequency on M 2 seedling basis (%); t = period of treatment with chemical mutagen; c= concentration of chemical mutagen in terms of percentage; Gy=Gray of physical mutagen; S=spikelet sterility (%). RESULTS AND DISCUSSION The most extensive studies to alter the spectrum of mutations and to achieve some degree of mutagen specificity in higher plants have been carried out with the chlorophyll deficient mutations because of their ease in detection and frequent appearance following mutagenic treatment (Nilan, 1967). This was successfully procured through the following: The frequencies of the chlorophyll mutations computed on the basis of the M 1 panicle progenies and M 2 seedlings in cvs. Pusa Basmati 1and Kalanamak have been presented in Table 1. In Pusa Basmati 1 the segregation of M 1 panicle progenies was observed maximally (60 per cent) at 50 kr gamma rays + 0.2% EMS followed by 40 kr gamma rays, whereas in Kalanamak, it was found maximally (80 per cent) at 40 kr gamma rays followed by 10 kr gamma rays.the highest (1.472 per cent) and lowest (0.170 per cent) frequencies of chlorophyll mutations in Pusa Basmati 1 were obtained at 50 kr gamma rays+0.2% EMS and 0.2% EMS, respectively whereas in Kalanamak (Table 1), the highest (2.262 per cent) and lowest (0.215 per cent) frequencies of chlorophyll mutations were obtained at 40 kr gamma rays and 0.2% EMS, respectively. In general, the frequency of chlorophyll mutations was higher in both the varieties at higher doses. The chlorophyll mutants, such as, albina white leaves without chlorophyll (lethal), xantha complete yellow colour of leaves (lethal) and viridis uniform light yellow green colour of leaves, (viable) were scored in M 2 at the seedling stage. The albina and xantha mutants did not survive (lethal mutants) whereas viridis was observed as a viable mutant. The frequency of chlorophyll mutations in M 2 induced by gamma rays and EMS alone and/or in combination treatment is presented in Table 2. The chlorophyll mutants were induced by all the doses of mutagens alone or in combination at fair frequency in both the varieties in M 2. However, the spectrum of chlorophyll mutations was quite narrow as only three mutant types, viz., albina, xantha and viridis could be procured. The albina mutants were induced in both the varieties by all the treatments. Similarly Chakravarti et al., 2013 observed that among chlorophyll mutations, albina mutant was most frequent in both the genotypes of aromatic rice. The albina mutants were induced in both the varieties by all the treatments. The xantha mutants were recorded maximally at 30 kr gamma rays+0.2% EMS in Pusa Basmati 1 and at 30 kr gamma rays in Kalanamak; induction of viridis mutants to the maximum extent was produced in Pusa Basmati 1 at 10 kr gamma rays and in Kalanamak at 40 kr gamma rays. The induction of albina mutants was obtained maximally at 50 kr Table 1: Frequency of chlorophyll mutations induced by mutagenic treatments in aromatic rice in M 2 Mutagenic treatment Pusa Basmati 1 Kalanamak M 1 panicles M 2 seedlings M 1 panicles M 2 seedlings Studied Segregating Per Studied Segregating Per Studied Segregating Per Studied Segregating Per cent cent cent cent Control 50 0 0 3105 0 0 50 0 0 2900 0 0 Gamma rays 10 kr 50 15 30 2940 40 1.360 50 35 70 2816 50 1.775 20kR 50 16 32 2890 16 0.554 50 11 22 2763 11 0.398 30kR 50 20 40 2842 30 1.056 50 25 50 2703 35 1.295 40kR 50 25 50 2782 35 1.258 50 40 80 2652 60 2.262 50 kr 50 15 30 2600 15 0.557 50 10 20 2552 10 0.431 EMS (Ethyl methane sulphonate) 0.2% 50 05 10 2941 05 0.170 50 06 12 2792 06 0.215 0.3% 50 11 22 2893 11 0.380 50 30 60 2749 40 1.455 0.4% 50 16 32 2811 16 0.569 50 20 40 2703 26 0.740 0.5% 50 10 20 2793 10 0.358 50 06 12 2665 06 0.225 Gamma rays + EMS 10 kr+0.2% 50 22 44 2919 31 1.062 50 11 22 2860 11 0.385 20 kr+0.2% 50 11 22 2844 11 0.387 50 15 30 2756 20 0.724 30 kr+0.2% 50 20 40 2813 35 1.244 50 15 30 2651 15 0.566 40 kr+0.2% 50 10 20 2764 10 0.362 50 21 42 2590 21 0.776 50 kr+0.2% 50 30 60 2718 40 1.472 50 20 40 2523 20 0.832 976

GAMMA RAY AND EMS INDUCED EFFECTIVENESS AND EFFICIENCY Table 2: Frequency and spectrum of chlorophyll mutations induced by mutagenic treatments in aromatic rice in M 2 Mutagenic treatments Pusa Basmati 1 Kalanamak Total number Frequency (%) Total mutation Total number Frequency (%) Total mutation of seedlings albina xantha viridis frequency (%) of seedlings albina xantha viridis frequency (%) Control 3105 - - - - 2910 - - - - Gamma rays 10 kr 2940 0.850-0.510 1.360 2816 1.598-0.177 1.775 20kR 2890 0.554 - - 0.554 2763 0.398 - - 0.398 30kR 2842 0.704 0.352-1.056 2703 0.740 0.555-1.295 40kR 2782 1.078-0.180 1.258 2652 0.377-1.885 2.262 50 kr 2600 0.385-0.192 0.557 2552 0.431 - - 0.431 EMS (Ethyl methane sulphonate) 0.2% 2941 0.170 - - 0.170 2792 0.215 - - 0.215 0.3% 2893 0.380 - - 0.380 2749 1.455 - - 1.455 0.4% 2811 0.569 - - 0.569 2703 0.370 0.185 0.185 0.740 0.5% 2793 0.358 - - 0.358 2665 0.225 - - 0.225 Gamma rays + EMS 10 kr+0.2% 2919 1.062 - - 1.062 2860 0.385 - - 0.385 20 kr+0.2% 2844 0.387 - - 0.387 2756 0.543-0.181 0.724 30 kr+0.2% 2813 0.533 0.533 0.178 1.244 2651 0.377-0.189 0.566 40 kr+0.2% 2764 0.362 - - 0.362 2590 0.386 0.195 0.195. 0.776 50 kr+0.2% 2718 1.104 0.184 0.184 1.472 2523 0.832 - - 0.832 Table 3: Mutagenic efficiency and effectiveness of different mutagens in aromatic rice Treatment Pusa Basmati 1 Kalanamak M 1 spikelet Mutation Mutagenic Mutagenic M 1 spikelet Mutation Mutagenic Mutagenic sterility as frequency on efficiency effectiveness sterilty as frequency on efficiency effectiveness percent of M 2 seedling (Msd/S) percent of M 2 seedling (Msd/S) control(%)s basis (%)Msd control (%)S basis (%)Msd Control 0 0 0 0 0 0 0 0 Gamma rays 10 kr 12 1.360 0.113 0.136 13 1.775 0.136 0.177 20kR 17 0.554 0.032 0.028 22 0.398 0.018 0.020 30kR 39 1.056 0.027 0.035 32 1.295 0.040 0.043 40kR 42 1.258 0.030 0.031 46 2.262 0.049 0.056 50 kr 54 0.557 0.010 0.012 50 0.431 0.008 0.008 EMS (Ethyl methane sulphonate) 0.2% 16 0.170 0.010 0.142 15 0.215 0.014 0.179 0.3% 20 0.380 0.020 0.211 28 1.455 0.052 0.808 0.4% 30 0.569 0.017 0.237 28 0.740 0.026 0.308 0.5% 39 0.358 0.009 0.119 38 0.225 0.006 0.075 Gamma rays+ EMS 10 kr+0.2% 15 1.062 0.070 0.095 15 0.385 0.026 0.034 20 kr+0.2% 18 0.387 0.021 0.018 20 0.724 0.036 0.034 30 kr+0.2% 38 1.244 0.032 0.039 30 0.566 0.019 0.018 40 kr+0.2% 45 0.362 0.008 0.009 45 0.776 0.017 0.019 50 kr+0.2% 35 1.472 0.042 0.029 51 0.832 0.016 0.016 gamma rays + 0.2% EMS in Pusa Basmati 1 and at 10 kr gamma rays in Kalanamak (Table 2). Chlorophyll mutations provide one of the most dependable indices for the evaluation of genetic effects of mutagenic treatments and have been reported in rice and other crops by several workers (Reddi and Suneetha, 1992;Singh et al.,2001; Singh and Singh,2003;Singh,2006; Ahirwar et al., 2014). The frequency of chlorophyll mutants was found independent of mutagenic doses of gamma rays ( Reddi and Rao, 1988; Singh et al.., 1998).During the present study, albina mutant occurred in higher frequency than xantha or viridis. Earlier workers also reported a higher frequency of albina mutants (Rao and Rao, 1983; Singh et al., 1998). However, Awan et al., 1980; Reddi and Reddi 1984; Reddi and Rao, 1988; Reddi and Suneetha, 1992 observed a higher frequency of viridis than albina or xantha in their studies in rice by involving physical and/or chemical mutagens. In the present study, genotypic difference in response to induction of chlorophyll mutations was observed as the frequency of induced chlorophyll mutations in M 2. The experimental results demonstrated that the total frequency of chlorophyll mutations was higher in Pusa Basmati 1 than Kalanamak. Similar reports for genotypic difference in rice were reported by many workers (Reddi and Rao, 1988; Singh et al., 1998). The differential response of genotypes to induction of chlorophyll mutations is possibly due to difference in the genetic makeup of the varieties used for mutagenesis. 977

SANJEEV SINGH et al., 0.14 0.12 0.1 0.08 Pusa basmati 1 Kalanamak 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Pusa basmati 1 Kalanamak 0.06 0.04 0.02 0 Control 10 kr 20 kr 30 kr 40 kr 50 kr 0.2% EMS 0.3% EMS 0.4% EMS 0.5% EMS 10 kr+0.2% 20 kr+0.2% 30 kr+0.2% 40 kr+0.2% 50 kr+0.2% Control 10 kr 20 kr 30 kr 40 kr 50 kr 0.2% EMS 0.3% EMS 0.4% EMS 0.5% EMS 10 kr+0.2% 20 kr+0.2% 30 kr+0.2% 40 kr+0.2% 50 kr+0.2% Figure 1: Mutagenic efficiency Mutagenic efficiency and effectiveness of physical and chemical mutagens Mutagenic efficiency is a measure of the proportion of mutation in relation to undesirable changes (factor mutations/ sterility) like sterility, injury, survival etc. Mutagenic efficiency was observed maximum in Pusa Basmati1 at 10 kr gamma rays followed by 10kR gamma rays+0.2% EMS and 50kR gamma rays+0.2% EMS on sterility basis (Table 3; Fig. 1). On the other hand, mutagenic efficiency was recorded maximally in Kalanamak at 10 kr gamma rays followed by 0.3%EMS and 40kR gamma rays. However combination treatment of 40 kr gamma rays+0.2%ems in Pusa Basmati1 and 0.5% EMS in Kalanamak was found as the least efficient on sterility basis. Bordoloi and Talukdar (1999) reported that low and moderate doses gamma rays were more efficient and useful in inducing genetic variability in rice. On contemporary Reddi and Suneetha (1992) reported that alkylating agents were more efficient. Mutagenic effectiveness denotes the frequency of mutations induced by a unit dose of mutagen (factors mutation/ dose).mutagenic effectiveness was observed maximum in Pusa Basmati 1 at 0.4% EMS followed by 0.3% EMS and 0.2% EMS. On the other hand it was recorded maximally in Kalanamak at 0.3% EMS followed by 0.4% EMS and 0.2% EMS (Table 3 Fig.2). In general mutagenic effectiveness of different mutagens was observed in the order as EMS> gamma rays>gamma rays + EMS. Similarly Siddiq and Swaminathan (1968) found that EMS was most effective mutagen followed by gamma rays and NG. Kumar and Mani (1997) found that a highly efficient mutagen might not be highly effective one also. A somewhat parallel conclusion was drawn by Konzak et al., (1965) in barley using a variety of alkylating agents. The genetic background of the material, the intracellular condition and perhaps cell cycle play an important role in determining the effectiveness and efficiency of the mutagens. The mutation breeding has been widely used for the improvement of various plant characters from time to time. It has been experienced that the frequency of desirable mutations depends upon the effectiveness and efficiency of the mutagens used (Konzak et al., 1965; Nilan et al., 1965). ACKNOWLEDGMENT Figure 2: Mutagenic effectiveness The first author is grateful to the University Grants Commission, New Delhi for financial assistance by sanctioning a major research project. REFERENCES Gustafsson, A. 1951. Induction of changes in genes and chromosome. II. Mutations, environment and evolution. Cold Spring Harb. Quant. Biol. 4: 601-632. Konzak, C. F., Wagner, J. and Foster, R. J. 1965. Efficient chemical mutagenesis: The use of induced mutations in plant breeding. Radiat Bot (Suppl). 5: 49-70. Nilan, R. A., Konzak, C. F., Wagner, J. and Legault, R. R. 1965. Effectiveness and efficiency of radiations for inducing genetic and cytogenetic changes. The Use of Induced Mutations in Plant Breeding. Rad. Bot. 5(Suppl.): 71-91. Kumar, R. and Mani, S. C. 1997. Chemical mutagenesis in Manhar variety of rice (Oryza sativa L.). Indian J. Genet. 57(2): 120-126. Singh, Sanjeev, Singh, J. and Singh, R. K. 2001. Gamma ray, EMS and sodium azide induced effectiveness and efficiency of chlorophyll mutations in Basmati rice (Oryza sativa L.). Crop Res. 22(1): 113-120. Gaul, H. 1964. Mutation in plant breeding. Rad. Bot. 4: 155-232. Gustafsson, A. 1940. The mutation system of the chlorophyll apparatus. Lunds. Univ. Arrskr. N.F. Adv. 36: 1-40. Nilan, R. A. 1967. Nature of induced mutations in higher plants. In Induced mutations and their utilization. Proc. Symp. Erwin-Baur- Cedachtnisvoriesungen-IV, Gaterstleben, 1966. Akademie-Verlag. Berlin. pp. 5-8. Chakravarti, S. K., Singh, S., Kumar, H., Lal, J. P. and Vishwakarma, M. K. 2013. Study of induced polygenic variability in M 1 and chlorophyll mutations in M 2 in aromatic rice. The Bioscan. 8(1): 49-53. Reddi, T. V. V. S. and Suneetha, J. 1992. Chlorophyll deficient mutations induced in rice by alkylating agents and azide. Cytologia. 57(2):283-288. Singh, Sanjeev and Singh, J. 2003. Mutations in Basmati rice induced by gamma rays, ethyl methane sulphonate and sodium azide. Oryza. 40(1-2): 5-10. Singh, S. 2006. Gamma rays induced mutations in Basmati rice (Oryza sativa L.). Indian J. Genet. 66(2): 143-144. Ahirwar, R. N., Lal, J. P. and Singh, P. 2014. Gamma-rays and Ethyl Methane Sulphonate induced mutation in microsperma lentil (Lens culinaris L. Medikus). The Bioscan (Supplement on Genetics and 978

GAMMA RAY AND EMS INDUCED EFFECTIVENESS AND EFFICIENCY Plant Breeding): 9(2): 691-695. Reddi, T. V. V. S. and Rao, D. R. M. 1988. Relative effectiveness and efficiency of single and combination treatments using gamma rays and sodium azide in inducing chlorophyll mutations in rice. Cytologia. 53: 491-498. Singh, Sanjeev, Richharia, A. K. and Joshi, A. K. 1998. An assessment of gamma ray induced mutations in rice (Oryza sativa L.). Indian J. Genet. 58(4): 455-463. Rao, G. M. and Rao, V. M. 1983. Mutagenic efficiency, effectiveness and factor of effectiveness of physical and chemical mutagens in rice. Cytologia. 48: 427-436. Awan, M. A., Konzak, C. F., Rutger, J. N. and Nilan, R. A. 1980. Mutagenic effects of sodium azide in rice. Crop. Sci. 20: 663-668. Reddi, T. V. V. S. and Reddi, V. R. 1984. Frequency and spectrum of chlorophyll mutants induced in rice by chemical mutagens. Theor. Appl. Genet. 67: 231-233. Bordoloi, P.K. and Talukdar, P. 1999. Effect of gamma radiation on genetic variabilities of few quantitative characters in rice. Ann. Biol. (Ludhiana). 15(1): 25-28. Siddiq, E. A. and Swaminathan, M. S. 1968. Enhanced mutation induction and recovery caused by nitroso-guanidine in Oryza sativa. Indian J. Genet. 28(3): 297-300. 979

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