Induced Genetic Variability through Physical and Chemical Mutagens in M₂ Generation of Greengram

India is the largest grower as well as consumer of mungbean with an annual production of 20.70 lakh tonnes and average productivity of 481 kg/ha (Department of Pulses Development, GoI, Annual Report 2016-17). Green gram is one of the significant Kharif and summer pulses of our country, which contains 25% protein and is an excellent and cheap source of high-quality protein as compared to meat, fish, eggs etc. (Kumar et al., 2013). Seeds are effectively edible and furthermore excellent source of minerals (calcium, iron (4-7 mg/100 g), zinc (3 mg/100 g), potassium and phosphorus) and vitamins (folate and vitamin K) and dietary fibres. Seeds of mungbean are nutritionally enhanced as ascorbic acid is incorporated with an increase in riboflavin and thiamine (Keerthiga et al., 2018).
        
Induced mutagenesis has been utilized to create desired genetic variability. Mutation can be induced by physical mutagens, namely X-rays, gamma rays, fast neutrons, ultraviolet and beta radiations. Apart from physical mutagens, several chemical mutagens are frequently used to induce mutagenesis namely Ethyl Methane Sulphonate (EMS), Ethylene amine (EI), Methyl Nitroso Urea (MNU), N-nitroso-N-methyl Urea (NMU) and Ethyl Nitroso Urea (ENU) (Tah, 2006).
        
The extent of improvement in green gram is very slow, owing to the lower amount of variability in its existing germplasms (Sushmita and Jayamani, 2018). Thus in such cases, mutation breeding can be considered as one of the driving forces of evolution as it leads to the creation of desired genetic variability, which in turn leads to crop improvement. Accordingly, in the present study, an attempt was made to study the extent of variability produced in M₂ generation as a result of induced mutation and to evaluate the correlation coefficients and path coefficients to formulate selection criteria for evolving high yielding mutant lines.

The experiment to evaluate M₂ generation was conducted at the Field Experimentation Center, Department of Plant Breeding and Genetics, Assam Agricultural University (AAU), Assam. Uniform and healthy seeds of green gram cv. Pratap (SG-1) were treated with two mutagens, gamma rays as physical mutagen and EMS as chemical mutagen as well as in combination. For physical treatment, two thousand uniform, healthy and dry seeds of mungbean genotype Pratap (SG-1) with 12% moisture were treated with 100, 200 and 300 Gray of gamma rays for each dose (Source- Cobalt 60) at Bidhan Chandra Krishi Bishwavidyalay (BCKV), West Bengal (WB). For combination treatment, genetically pure two thousand uniform, healthy seeds were first exposed to gamma rays (Source-Cobalt-60) of 100 Gy and 200 Gy for each dose at BCKV, WB. After pre-soaking, the seeds were blotted dry and treated with freshly prepared chemical mutagen solution of EMS of concentration of 0.1%, 0.2% and 0.3%. The seeds were treated with the mutagenic solution for 6 hours at room temperature 26°C±2°C with intermittent shaking over, the seeds were thoroughly washed in running tap water for two hours and then sown in the field to raise the M₁ generation. The two hundred forty M₁ harvested progenies were laid down in randomized block design with three replications during Kharif, 2017 to raise the M₂ generation. Test plots were managed following the recommended site-specific standard agronomic practices. The test plot size was 24 sq m and spacing between rows and plants was 30 and 10 cm, respectively.
        
Observations were recorded on 20 randomly chosen plants from each plot for 8 characters viz., days to maturity, branches per plant, number of clusters per plant, number of pods per plant, pod length (cm), number of seeds per plant, and seed yield per plant (g/plant). Means were computed, and data were subjected to analysis of variance, phenotypic and genotypic coefficients of variation and heritability (broad sense), genetic advance as per cent of the mean, genotypic correlation, phenotypic correlation and path analysis.

The analysis of variance showed highly significant differences among the 7 treatments (100 Gy, 200 Gy, 300 Gy, 0.1% EMS, 0.2% EMS, 0.3% EMS and 200 Gy + 0.2% EMS) including control for all the 8 traits studied. The treatment 200 Gy+0.2% EMS recorded the highest values for the traits branches per plant (4.75 ), number of pods per plant (14.53), pod length (6.55 cm), number of seeds per pod (10.63) and seed yield per plant (7.75 g/plant) (Table 1.1 and Table 1.2). Khan and Wani (2006) reported that during mutagenesis if mutations occur at random for the quantitative traits, no significant change is expected in the mean values. However, the shift in mean values in the positive direction indicates that more positive mutations have occurred for these traits, which are presented in Table 2.1 and Table 2.2. In accordance with the present study, maximum positive shift against the control was shown by the combined dose (200 Gy + 0.2% EMS) for almost all the characters except days to heading in which the combined dose showed negative shift from the control that is generally preferred as it depicts earliness as discussed by Goyal et al., (2019) for urad bean. This indicates that most of the variability has been recorded in the combined dose of 200 Gy + 0.2% EMS than other mutagenic treatments suggesting that this treatment can be considered as the most effective mutagenic treatment to raise mutant population. Mutations affecting quantitative characters can best be inferred by the estimation of genetic parameters in the mutagen treated populations that are presented in Table 3. High magnitude of phenotypic coefficient of variation (PCV) as well as genotypic coefficient of variation (GCV) were recorded for seed yield per plant (38.48% and 38.32% respectively), number of pods per plant (25.79% and 25.64% respectively) and number of clusters per plant (25.79% and 24.42% respectively) indicating the presence of ample variation among plants belonging to the different mutagenic treatments for these characters as supported by the findings of Paul et al., (2017), Sushmita and Jayamani, (2018). The highest magnitude of heritability in broad sense coupled with high genetic advance was registered for seed yield per plant (99.15% and 78.61%) followed by other characters which were in agreement with the findings provided by Ahmed et al., (2012). The higher values of heritability and genetic advance suggests the predominance of additive gene action and the variability so evolved can be effectively exploited for further genetic improvement of green gram as suggested by Khan and Wani (2006). The phenotypic and genotypic correlation coefficient is presented in Table 4. In the present study, correlation coefficient among morphological characteristics and yield and its component showed that at both the genotypic and phenotypic level, seed yield per plant had a significant and positive correlation with almost all the characters except for two characters, i.e., number of branches and clusters per plant which showed non- significant results. Moreover, at both the genotypic and phenotypic level, seed yield per plant had maximum positive correlation with number of pods per plant (0.87 and 0.87 respectively) followed by pod length (0.83 and 0.77 respectively) which indicates that the selection based on these traits in the treated populations can be made for screening high yielding mutants, as suggested by Laskar and Khan, (2017). Positive and significant correlation for the number of pods and number of clusters per plant with seed yield was observed by Saxena et al., (2007) and for a number of pods with seed yield was recorded by Rajan et al., (2000) and Kumar et al., (2013).
 

 

 

 

 

 

        
Path analysis presented in Table 5 revealed that at a genotypic level, number of pods per plant (5.68) had the maximum direct effect on seed yield per plant followed by pod length (1.32) and number of branches per plant (0.02) indicating that these characters may be considered as prime traits during the course of selection for enhancing the seed yield of green gram. The number of pods per plant showed a positive and direct correlation with seed yield per plant in accordance with the results given by Rao et al., (2006), Srivastava and Singh (2012) and Anand et al., (2016). The highest positive indirect effects registered on seed yield per plant by number of pods per cluster (5.18) via pod length (4.15) and number of pods per plant indicating that such characters can be indirectly selected for crop improvement rather than direct selection of yield as the basic character. The residual effect at the genotypic level of -0.32 indicates the influence of environmental factors on the creation of variability among the characters.
 

This study revealed that the enormous magnitude of variation in the characters among the different mutagenic treatments present in M₂ generation describes the effectiveness of the mutagens in the study. The existence of this large genetic variation can be exploited in the further breeding programme in the development of desirable mutant lines. In the above discussion, it was observed that in general, the combination dose, i.e., 200 Gy+0.2% EMS gave superior results in almost all the yield attributing characters than the other doses over the control which in turn shows that such combined doses of both physical and chemical mutagens can be exploited in breeding programmes to generate sufficient as well as desirable amount of variability.