Efficiency and Effectiveness of Gamma Rays and. G. M. Rao

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1 Cytologia 42: , 1977 Efficiency and Effectiveness of Gamma Rays and EMS in Rice Received December 18, 1975 G. M. Rao Department of Botany, Agricultural College, Bapatla (A. P.), India Mutation breeding has been very useful in inducing variability and became a potential tool in any plant breeding programme in the changing agricultural pattern. While ionizing radiations still remain a tour de force in inducing genetic variability, a number of chemicals have been found to be equally or even many more times effective and efficient mutagens. Ethyl methane sulphonate (EMS) has been reported to be the most potent among the chemical mutagens used in rice (Kawai and Sato 1965) and in other crops (Gaul et al. 1972, Jacob 1965). With the identification of potent chemical mutagens and with the growing understanding of numerous parameters which influence sensitivity to mutagens, the scope for increasing the frequency and spectrum of mutations has widened. The relative ease of application and the comparitive low cost of chemical mutagens has caused an unprecedented surge of interest in the artificial induction of mutation with the chemical agents. Hence it becomes necessary to evaluate the efficiency and effectiveness of EMS in contrast to gamma rays in rice. The present study apart from throwing light on efficiency and effectiveness of EMS over gamma rays will also yield information on spikelet fertility as criteria for selection and different injuries including cytological effects in M, generation. Materials and methods The following varieties, namely, TKM-6 (indica), Norin-27 (japonica), IARI and AR-4-87 (intermediates) and Oryza spontinae (wild species) were treated - with gamma rays and EMS. Gamma rays: Five hundred dry seeds of uniform size and moisture content (13% }1.0) arranged in mono layer were subjected to an acute dose of 25 KR. Ethyl methane sulphonate (EMS): Five hundred healthy seeds of uniform size presoaked in distilled water for 20 hours were treated with freshly prepared solution of EMS-0.6% concentration (Buffered at ph 7) for 6 hours with intermittant shaking at 25 Ž }2 Ž. Immediately after the treatment, the seeds were washed thoroughly in running water to reduce the residual effect of the mutagen sticking to the seed coat. The treated seeds were sown in pots and 25-day old seedlings were transplanted in rows with a spacing of 4 inches within the lines and 6 inches between lines. Ger mination counts were taken from the material germinated on moist filter paper in petridishes and emergence of coleoptile was taken as indication of germination. Survival counts were taken from 20-day old seedlings. All M1 plants were selfed

2 444 G. M. Rao Cytologia 42 to prevent crossing and each M1 plant was harvested individually and M2 progeny was raised as seperate rows. For estimation of seed fertility all the tillers of M, plants was considered as a unit. The number of full developed and empty spikelets were counted for each plant and expressed as percentage. The M1 plant were divided into two fertile groups, namely, high fertile (51 to 100%) and low fertile (0 to 50%) and about 100 plants were selected for each group in each treatment. Pollen fertility was recorded from 100 plants for each treatment in M1 generation. Darkly stained and unstained pollen grains were counted for each plant and expressed as percentage. Chlorophyll mutations were carefully recorded in 100 M2 families (M1 plant progeny) for each group of treatment. The mutation frequency in M2 generation was calculated as percentage of M2 families. In the present study, mutagenic ef ficiency and effectiveness were calculated by the formula suggested by Konzak et al. (1964). Results and discussion M, generation The varieties of indica, japonica, intermediate and wild species included in this study appeared to differ from each other in radio sensitivity for gamma treatment as measured by survival, pollen and seed fertility and meiotic abnormalities (Table 1). The wild species O. spontinae showed marked difference in survival percent for gam ma treatment compared to other varieties under study. The variety Norin-27 (japonica) recorded lowest survival rate of 52% and proved to be highly sensitive. The indica and intermediate types were intermediate in sensitivity between japonica and wild species. The results are in agreement of Siddiq and Swaminathan (1968). The survival percent was the highest in EMS (0.6%) treatment compared to gamma rays (25KR.) and there were no marked differences between varieties. Li (1965) and Swaminathan et al. (1970) studying the effect of gamma rays and EMS on rice observed similar effects. Gamma rays induced maximum seed and pollen sterility than EMS treatment. In gamma treatment the japonica variety Norin-27 recorded maximum spikelet and pollen sterility ie., 62% and 50.9% respectively, where as O. spontinae recorded the lowest spikelet (32,8%) and pollen (39.9%) sterility. The varieties TKM-6, IARI and AR-4-87 were intermediate between japonica and wild species. There were no marked differences between varieties in respect of spikelet and pollen sterility in EMS treated population. The present results support the findings of Sampath (1973), Siddiq and Swaminathan (1968) and Swaminathan et al. (1970). Sterility induced by chemical mutagens, more particularly alkalating agents in many cases was not found to be associated with chromosomal abnormalities (Bansal and Natarajan 1965, Gaul et al. 1966, Moutschen-Dahmen 1965, Prasad 1972). Where as radiation induced sterility in many instances was reported to be associated with chromosomal aberrations (Hsieh 1961, Li et al. 1964, Soriano 1960, Venkatanadha chari 1963, Verano and Soriano 1965, Yeh and Henderson 1963). Most of the radiation induced sterility in M1 generation and further generations is

3 1977 Efficiency and Effectiveness of Gamma Rays and EMS in Rice 445 Table 1. Effect of Gamma rays and EMS on germination (ger.), survival (sur.), spikelet fertility (spi. fer.), Pollen fertility (pollen fer.) and meiotic abnormalities (mei. abn.) in M, generation probably haplontic according to Muntzing (1930). In contrast to this EMS-induced sterility appears to have diplontic nature, as was concluded in a study of Sato and Gaul (1967). The actual origin of EMS-induced sterility might be gene mutations or more probably "invisible deficiencies", the frequency of which may be relatively higher than after ionizing radiation. In the present studies the frequency of chromo somal aberrations (translocations, bridges, fragments and laggards) in meiosis were high in gamma treatment and nil in EMS treated population (Table 1). M2 generation Scoring for chlorophyll mutations was done both on the basis of M1 plant progeny (100 Ml plants) and of M1 spike progeny (about 600 M1 spikes). The results obtained on chlorophyll mutation frequency by both these methods were almost same. Hence, the data from M1 plant progeny basis was presented in Table 2. The frequency of chlorophyll mutations in M1 generation revealed no marked dif ferences between gamma and EMS treated populations. Within gamma treated population the varieties TKM-6, AR-4-87 and wild species O. spontinae showed

4 446 G. M. Rao Cytologia 42 similar frequency of chlorophyll mutations in low and high fertility groups. How ever, Norin-27 and IARI recorded marked differences in chlorophyll mut ation frequency in both the groups. Individual varieties differed in mutation spect rum at low and high fertility classes but col lectively there were no marked differences. The EMS treated mutation frequency was remarkably higher in all varieties and wild species of high fertile group than the low fertile group. The spectrum of mutations Fig. 1. Histogram showing frequency in high fertile group was markedly different from that of the low fertile group (Fig. 1). For example, the percent of albinos were about six times more and xantha type of mutations which were absent in low fertile and spectrums of chlorophyll mutations class are present in considerable quantity in in M2 generation. high fertile group. Between the two treat ments, EMS recorded 100% more xantha mutants and about 7% less albino mut ants than gamma treated population. Several attempts have been made to establish correlations between different indices of M1 damage and M2 mutation frequency. Coldecott (1961) showed a close relationship between the decrease in M1 seedling height and chlorophyll mutation frequency in M2. Ehrenberg et al. (1961), Gaul (1958, 1964), Mesken and Van derveen (1968) and Sampath (1973) studying the effect of physical and chemical mutagens on seed fertility in relation to chlorophyll mutation frequency propos ed the frequency of factor mutation was completely independent of the degree of sterility. Kivi (1965), Sharma and Bansal (1970) and Walther (1969) however, demonstrated a definite relation between grade of M1 fertility and frequency of chlorophyll mutation. The present studies revealed that different varieties under study reacted independently in respect of the frequency of chlorophyll mutations at the two levels of fertility in gamma treatment. However, EMS treated population clearly showed high frequency of chlorophyll mutations in high fertile class. The spectrum of chlorophyll mutations produced by radiation was different from that of chemical mutagen. Gamma rays produced more albino type and less xantha compared to EMS treatment. Similar differences between mutation spectrum produced by radiation and chemical mutagens have been reported by Ehrenberg et al. (1959), Wettestein et al. (1959), Swaminathan et al. (1962) and Varughese and Swaminathan (1968). Chemical mutagens tended to be more specific in the mole cular reaction with genes compared to radiation. Mutagen efficiency and effectiveness Mutagenic efficiency is a measure of the proportion of mutations in relation to undesirable changes (factor mutation/sterility) like sterility, injury, survival, meiotic aberrations etc., while mutagenic effectiveness denotes the frequency of mutations induced by a unit dose of mutagen (factor mutation/dose). The effect iveness of a mutagen is of theoritical importance but does not have any immediate

5 Table 2. Spectrum and frequency of chlorophyll mutations in the M 2 generation at two levels of spikelet fertility

6 O 448 G. M. Rao Cytologia 42 practical implications. The data on efficiency and effectiveness at two levels of fertility was presented in Table 3. The efficiency of gamma rays and EMS differed markedly between varieties at low fertility level. In the varieties TKM-6, Norin-27 and wild species Table 3. Efficiency and effectiveness of gamma rays and EMS at two levels of spikelet fertility. spontinae, gamma treatment was efficient in low fertility group. Where as in intermediate varieties, EMS treatment proved better. The efficiency of gamma rays was equal at both levels of fertility in all varieties except IARI and Norin 27. Invariably EMS treatment is highly efficient over gamma treatment in high - fer tile group and over both the treatments of low fertile group. Gamma treatment was found to be equally effective at both levels of fertility in TKM-6, AR-4-87 and O. spontinae but the effectiveness was more prominent in IARI in high fertile group and in Norin-27 in low fertile group. Between the treatments gamma rays were more effective in Norin-27 and O. spontinae and EMS in IARI-10017, TKM-6 and AR-4-87 at low fertility level. However, at high fertility level EMS treatment was markedly effective over gamma treatment, which was also superior to both the treatments at low fertility level. Even though the rate of mutation was almost equal in gamma and EMS treat ments, the efficiency was markedly high in EMS treated population which might be due to high seed fertility in M1 generation. In view of the findings of Rao and Siddiq (in press) that EMS induced more heritable changes over gamma treatment, the present findings ie., EMS is both efficient and effective over gamma treatment is of immens value. The present findings are in confirmity with Konzak et al. (1965). Based on the results of the present investigations, it appears that EMS treatment is remarkably efficient and effective in producing chlorophyll mutations in M1 plant progeny showing high seed fertility (51 to 100%). Summary All varieties under study showed varied degrees of sensitivity for different characters, namely, survival, meiotic aberrations and pollen and spikeley sterility

7 1977 Efficiency and Effectiveness of Gamma Rays and EMS in Rice 449 to gamma irradiation and less sensitive to EMS treatment. In gamma treatment the wild species O. spontinae exhibited the highest resistance for M1 injury, whereas the japonica (Norin-27) recorded the lowest and the rest are recorded intermediary values. There were no marked differences in frequency of chlorophyll mutations in gamma and EMS treated population. Within gamma treatment, frequency and spectrum of chlorophyll mutations, it was almost same in high and low fertility groups. However, EMS treated population recorded remarkable differences in frequency and spectrum of chlorophyll mutations in both the fertility groups. In general EMS was found to be most efficient and effective mutagen at high fertility level than gamma treatment. References Bansal, H. C. and Natarajan, A. T Induction of variability for spikelet sterility in Hordeum. Indian J. Genet. 25: Caldecott, R. S Effects of ionizing radiations on seeds. Proc. of Symp., Karlsuha, Ehrenberg, L., Gustafsson, A. and Lundquist, U The mutagenic effects of ionizing radia tions and reactive ethylene derivatives in barley. Hereditas 45: , - and Viable mutants induced in barley by ionizing radiation and chemical mutagens. Hereditas 47: Gaul, H Present aspects of induced mutations in plant breeding. Euphytica 7: Mutations in plant breeding. Rad. Bot. 4: , Bender, K., Ulonska, E. and Saty, M EMS induced genetic variability in barley. The problem of EMS induced sterility and a method to increase the efficiency of EMS treatment. In "Mutations in Plant Breeding". Proc. of Symp. FAO/IAEA, Vienna. -, Frimmel, G., Gichner, T. and Ulonska, E Efficiency of mutagenesis. In "Induced Mutations and Plant Improvement". Proc. of Symp. FAO/IAEA, Vienna. Hsieh, S. C Effect of X-rays and thermal neutrons on chromosomes of rice IV. Mutations induced by X-rays. Bot. Bull. Acad. Sin. 2: Jacob, M Mutagenesis of some monofunctional alkylating agents. In "Arabidopsis Re search, Rep." Int. Symp. Univ. Gottingen. Kawai, T. and Sato, H Studies on artificial induction of mutations in rice by chemicals 1. Chlorophyll mutations by ethyl imine and ethylene oxide treatments. Bull. Natt. Inst. Agrl. Sci. 13: Kivi, E. I Some aspects of sterelity effect of radiation on the basis of a gamma and X-ray treated barley. In "The Use of Induced Mutations in Plant Breeding". Proc. of Symp. FAO/IAEA, Vienna. Konzak, C. F., Nilan, R. A., Wagner, J. and Foster, R. J Efficient chemical mutagenesis. In "The use of Induced Mutations in Plant Breeding". Proc. of Symp. FAO/IAEA, Vienna. Li, W. C A comparative study of effects between the treatment of gamma ray and methyl ethyl sulphonate in rice. J. Agric. For. (Taichung) 14: Li, H. W., Yang, K. K. S., Li, S. S. Y. and Ho, K. C Relation of association and orienta tion of chromosomes in interchange heterozygotes of rice and their fertility. Bot. Bull. Acad. Sin. 5: Mesken, M. and Vanderveen, J. H The problem of induced sterility: A comparison be tween EMS and X-rays in Arabidopsis thaliana. Euphytica 17: 363. Moutschen-Dahmen, J Effects cytogenetique set sterility des an methane sulfonate d'ethyl (EMS) chezlorge. Rev. Cytol. Biol. Veget. 28: Prasad, M.V.R A comparison of mutagenic effectiveness and efficiency of gamma rays, EMS, NMU an NG. Indian J. Genet. 32:

8 450 G. M. Rao Cytologia 42 Sampath, S Studies on chemical mutagenesis in rice. Final report (U.S. P. L. 480 Project.) Sato, M. and Gaul, H Effects of ethyl methane sulphonate on the fertility of barley. Rad. Bot. 7: Sharma, R. P. and Bansal, H. C Influence of radiation and chemical induced sterility on mutation frequency and spectrum in barley. Indian J. Genet. 30: Siddiq, E. A. and Swaminathan, M. S Rice breeding with induced mutations. Tech. Reports Series No. 86, IAEA, Vienna. Soriano, J. D Radiation induced inter changes and mutations in rice. Natural Appl. Sci. Bull. (Quesan city) 17: Swaminathan, M. S Mutation breeding in rice in India. In "Rice Breeding with Induced Mutations". Proc. of Symp. FAO/IAEA, Vienna. -, Chopra, V. L. and Bhaskaran, S Chromosome aberrations and the frequency and spectrum of mutations induced by ethyl methane sulphonate in barley. Indian J. Genet. 22: Venkatanadha chari, G, Cytogenetical abnormalities in irradiated rice. Andhra Agric. J. 10: Verano, L. M. and Soriano, J. D Cytogenetic effects of X-rays in rice. Natural Appl, Sci. Bull. 19: Walther, F Effectiveness of mutagenic treatments with ionizing radiation in barley. In "Induced Mutations in Plants". Proc. of Symp. IAEA/FAO, Vienna. Wettestein, D., Gustafsson, A. and Ehrenberg, L Mutations Forschung and Zuchtung Arb. gemeinseh. Forsch. des Landes nordrhein. Westfalen 73: Yeh, B. and Henderson, M. T Effects from irradiation of rice seed with gamma rays and neutrons on several chracterstics of the R1 generation. Bot. Bull. Acad. Sin. 4: