Study of Chlorophyll and Macro Mutations Induced by Physical Mutagens in Black Gram [Vigna mungo (L.) Hepper]

Mutation breeding is relatively a quicker method for improvements of crops. It has been observed that induced mutations can increase yield as well as other quantitative traits in plants. Blackgram is an important kharif crop in India grown on about 2.7 lakh hectares. The seeds are mostly consumed by the people owing to its high protein content (Akhaury, 1991). The natural productivity of black gram is only 480 kg/ha (Chaturvedi and Ali, 2002). This low yield may be due to narrow genetic base and non availability of high yielding varieties with disease resistance. Natural variability is an essential pre-requisite for any successful breeding programme. Mutation breeding is a supplementary breeding programme to identify the mutants with high yield potential, early maturity, disease and pest resistance (Singh, 1981). The choice of mutagen holds great importance in changing the frequency and spectrum of chlorophyll mutations in a predictable manner.
        
Chlorophyll mutations although not useful for plant breeding purpose, may be used to assess the efficiency and effectiveness of mutagens in order to select suitable mutagen at appropriate concentration so as to use them in applied mutagenesis programme. Physical mutagens such as gamma rays and electron beam is used in induction of variability. Present investigations were undertaken to study the comparative effectiveness of these mutagens under similar treatment conditions. In the present study, the effect of gamma rays, electron beam and combined treatment (Electron beam + Gamma rays) employed singly or in combinations was studied on frequency and spectrum of chlorophyll mutations and viable macro mutations in M₂ generation of blackgram.

The dried seeds of the blackgram varieties ADT 3 and CO 6 were treated with 200Gy, 300Gy and 400Gy doses of gamma irradiation using ⁶⁰CO gamma source for appropriate time at the Bhabha Atomic Research Mumbai, India. For each treatment, well filled 500 seeds with uniform moisture content was used. For electron beam, the seeds of ADT 3 and CO 6 were treated with 200Gy, 300Gy and 400Gy doses using 10 MeV electron beam from electron accelerator facility at Electron Beam Centre, Bhabha Atomic Research Centre, Kharghar, Navi Mumbai, India.
        
Dry, healthy and uniform sized seeds of blackgram variety ADT 3 and CO 6 were exposed with Electron beam and then same seeds were treated with gamma rays for combined treatment. The treated seeds with control were sown in germination trays at germination room, Plant Breeding and Genetics Department, Agricultural College and Research Institute, Madurai. Lethal dose (50%) was calculated byprobit analysis using the germination data.
        
After completion of the treatment with gamma rays, electron beam and combined treatment (Electron beam + Gamma rays) and their respective control seeds were sown immediately to raise the M₁ generation in a randomized block design (RBD) with two replications. Different biological parameters like germination, survival of plant, pollen fertility, plant height on 30^th day, seed fertility was recorded in randomly selected plants in each treatment in M₁ generation and harvested on single plant basis.
        
For raising of M₂ generations, the seeds of M₁ generation were space planted in the field in two replications. M₂ generation was screened for lethal chlorophyll mutations during the first four weeks, after germination. Whereas, viable chlorophyll and macro mutants were scored throughout the crop duration. The population was screened for chlorophyll and macro mutations according to the procedure given by Gustafesson (1947) with suitable modifications. The spectrum was recorded as xantha, chlorina, viridis and albina. The xantha mutants displayed a bright yellow to deep golden yellow colour. Chlorina mutants were yellowish green in colour, the viridis mutants displayed light green colour and albinashows white colour. The chlorophyll mutants like xantha, chlorine and viridis. Mutations frequency was calculated by the following methods given by Gaul, (1957).

Frequency of chlorophyll mutations observed in the M₂ generation of the varieties ADT 3 and CO 6 is provided in Table 1. The chlorophyll mutation frequency on the M₂ plant basis increased with the increase in the dose of gamma rays, electron beam and their combination (electron beam + gamma rays). The increased cholorophyll mutation frequency at higher doses may be attributed to the chromosomal aberrations or saturation in the mutational events which may result in the elimination of mutant cells during growth (Brock, 1965). Electron beam is most effective compared to other treatment.
        

 
In ADT 3, the chlorophyll mutants occurred in all the treatments. In gamma irradiated population, 200Gy treatment recorded maximum frequency of chlorophyll mutation on M₁ plant basis (38.67 per cent) and M₂ plant basis (1.56 per cent). In electron beam treatment, 400Gy treatment exhibited the maximum frequency of chlorophyll mutation on M₁ plant basis (48.15 per cent) while 200Gy (1.70 per cent) registered higher frequency on M₂ seedling basis. In combined treatment (electron beam + gamma rays), the chlorophyll mutation frequencyin treatment 300Gy + 300Gy and 200Gy + 200Gy recorded maximum frequency of chlorophyll mutation on M₁ plant basis (40.51 per cent) and M₂ plant basis (1.66 per cent). In CO 6, the gamma irradiated population, 200Gy treatment recorded maximum frequency of chlorophyll mutation on M₁ plant basis (34.91 per cent) and M₂ plant basis (1.58 per cent). In electron beam treatment, 400Gy treatment exhibited the maximum frequency of chlorophyll mutation on M1 plant basis (43.59 per cent) while 200Gy (1.47 per cent) registered higher frequency on M₂ seedling basis. In combined treatment (electron beam + gamma rays), the treatment 400Gy + 400Gy and 200 Gy + 200Gy recorded maximum frequency of chlorophyll mutation on M₁ plant basis (43.75 per cent) and M₂ plant basis (1.44 per cent).
        
In the present study, chlorophyll mutants were scored on M₁ plant and M₂ seedling bases. Of the two methods of estimating the frequency of mutations, M₂ seedling basis was considered as the best index (Gaul, 1960). The concept was reflected in present investigation wherein the high frequency of chlorophyll mutants was found at lower doses in ADT 3 and CO 6 on M₁ plant basis and M₂ seedling basis in both gamma rays,electron beam and combined treatment (electron beam + gamma rays). Electron beam treatments produced maximum chlorophyll mutants than gammarays and combined treatment (electron beam + gamma rays) in both the varieties. Similar results were obtained in blackgram (Deepalakshmi and Ananda Kumar 2004; Thilagavathi and Mullinathan 2009) and in greengram (Singh et al., 2005; Awnindra and Singh 2007; Vairam et al., 2014).
        
The spectrum of chlorophyll mutants induced by gamma rays,electronbeam and their combination included albrina, chlorina, Xanthaviridis, virids and xanthan (Table 2). In gamma rays irradiated population of ADT 3, the occurrence of albino was very high followed by xantha and chlorina than the other types. The order of relative percentage of different chlorophyll mutants occurrence was Xanthaviridis (from 26.47 in 400 Gy to 29.21 per cent in 300Gy) > albino (from 8.99 in 300 Gy to 25.00 per cent in 400 Gy) >xantha (from 22.06 in 400 Gy to 23.68 per cent in 200Gy) > chlorina (from 16.18 in 400Gy to 20.22 per cent in 300Gy) > viridis (from 10.29 in 400Gy to 17.98 per cent in 300Gy). Albino mutants were more in higher doses, whereas xanthaviridis, xantha, chlorina and viridis were higher at lower doses. In electron beam irradiated population, the order of relative percentage of different chlorophyll mutants occurrence was xanthaviridis (from 33.33 in 200Gy to 35.21 per cent in 400Gy) > xantha (from 18.31 in 400Gy to 22.83 per cent in 300Gy) > chlorina (from 14.08 in 400Gy to 20.65 per cent in 300Gy) > viridis (from 14.13 in 300Gy to 17.78 per cent in 200Gy) > albino (from 8.70 in 300Gy to 15.49 per cent in 400Gy). Albino mutants were higher in higher doses. In combined treatment (electron beam +gamma ray) irradiated population, the order of relative percentage of different chlorophyll mutants occurrence was xanthaviridis (from 26.87 in 400Gy + 400Gy to 33.72 per cent in 300Gy + 300Gy) > xantha (from 22.09 in 300Gy + 300Gy to 23.36 per cent in 200Gy + 200Gy) > viridis (from 19.63 in 200Gy + 200Gy to 20.90 per cent in 400Gy +400Gy) > albino (from 8.14 in 300Gy + 300Gy to 14.93 per cent in 400Gy + 400Gy).               



In CO 6, Xanthaviridis and xantha occurred in higher proportion followed by viridis, albino and chlorinafor gamma rays, electron beam and combined treatments. The frequency of xanthaviridis ranged from 30.65 per cent (400Gy) to 35.90 per cent (300Gy). In electron beam, the frequency of xanthaviridis ranged from 28.24 per cent (300Gy) to 34.82 per cent (200Gy). In combined treatment (electron beam + gamma ray), the frequency of xanthaviridis ranged from 30.36 per cent (400Gy + 400Gy) to 31.18 per cent (200Gy + 200Gy)
               
In ADT 3, the chlorophyll mutants occurred in all the treatments. Gamma rays, electron beam and combined treatment (electron beam + gamma rays) exhibited maximum number of xanthaviridis mutants while xanthaand viridis were intermediate in its occurrence. Chlorina and albino occurred at least proportion. Similar finding was reported in cowpea (Devmani Bind et al., 2016). The occurrence of chlorophyll mutants was higher in electron beam while compared to gamma ray treatments. Similar results were reported in blackgram (Souframanien et al., 2016; Loyavar et al., 2017) and in greengram(Singh et al., 2005; Singh, 2007). In Co 6,xanthaviridis and xantha occurred in higher proportion followed by viridis, chlorina and albino in all the treatments of gamma rays, electron beam treatments and combined treatment (electron beam + gamma rays). Similar finding was observed by Gautam and Mittal, 1998 in blackgram; Devmani et al., 2016 in cowpea. To increase the mutation frequency and mutagenic rate of specific mutagen, appropriate concentrations will be effective (Auti, 2005). In the present study, chlorophyll mutants were high in electron beam treatment than Gamma rays and combined treatment. The efficiency was found to be highest at lower and intermediate doses of mutagenic treatments. These results suggest that high mutation rates could be obtained with moderate dose of mutagen in blackgram.