Mutagenic Effectiveness and Efficiency in [Vigna unguiculata (L.) Walp.] Variety Phule Phandari (PP) and VCM-8

Among the legumes, cowpea is important cultivated legume crop in the world. Cowpea grows well in poor dry conditions, thrive in soils up to 85% sand (Obatolu, 2003). The optimum temperature for cowpea growth is 30°C (86°F), making it the only available one as a summer crop for most of the world. Cowpeas are grown mostly for their edible beans like other legumes. Cowpea seeds consist of 25% protein as well as minerals and vitamins and have very low fat content and are usually cooked to make them edible. The seed is a rich source of folic acid, some anti-nutritional elements, notable phytic acid and protease inhibitors, which reduce the nutritional value of the crop. In India Cowpea is cultivated in Rajasthan, Gujarat, Punjab, Maharashtra and Tamilnadu with an average  yield of cowpea 7089 kg/ha (2017)  which is very low; roughly was about two-third of the world’s average of 10,095 kg/ha. Thus, the efforts should be made to enhance the production and productivity of cowpea by adopting any breeding method. Mutation breeding might be the effective alternate to induce genetic variation, particularly for traits having low level of genetic variation (Szarejko and Forster 2007). The chlorophyll deficient sectors formed in M₁ generation and chlorophyll mutants formed in M₂ generation are being used conveniently as preliminary index of the mutagenic effectiveness and efficiency. The improvement aspects like quality, quantity and morphological characters along with chlorophyll mutations have been successfully attained by several researchers through mutation breeding in different crops like mustard (Julia et al., 2018), chickpea (Gaur et al., 2007) and sesame (Savant and Kothekar 2011). The usefulness of a mutagen in mutation breeding depends on its mutagenic effectiveness and efficiency (Savant et al., 2010). The selection of effective and efficient mutagen is very essential to recover a high frequency and spectrum of desirable mutations (Mahabatra 1983; Solanki and Sharma 1994). The success in mutation breeding program for any crop can be achieved by increasing the spectrum and frequency  in viable mutations. Therefore the present investigation was undertaken to study the mutagenic response of EMS and gamma rays in cowpea.

In the present investigation the seeds of two varieties of cowpea, phulepandhari and VCM-8 were obtained from pulse crop research substation, Pandharpur, Maharashtra, India. The two mutagens used for mutagenic treatment are Ethyl methanesulphonate (EMS) and Gamma rays. Initially, a pilot experiment were conducted to know LD₅₀. LD₅₀ is the dose in which half of the individuals among the treated population dies. It is a parameter to decide the effective dose for a mutagen treatment in any crop species. Suitable concentrations and dosage of mutagens were decided on the basis of  LD₅₀.

The chemical mutagenic treatments were carried out at room temperature of 25±2^oC. Healthy seeds of uniform size were selected and treated with 0.20%, 0.25% and 0.30% concentrations of EMS. In case of physical mutagen the dry seeds with seed moisture content of 08 per cent were exposed to gamma rays at three different doses viz., at 250 Gy (gray), 350 Gy. and 450 Gy. at department of Biophysics, Govt. Inst. Of Science, Aurangabad, India. Untreated seeds stock of the respective genotypes were used as control. 300 seeds of each treatment were sown in the field following randomized block design (RBD) with six replications each consisting of 50 seeds along with control for raising the M₁ generation. The seeds were sown keeping distance of 25 cm between the plants and 50 cm between the rows.

The germination percentage was counted after 10 days. The biological damage (lethality, seedling injury and pollen sterility) was computed as the percentage reduction in plant survival, seedling height and fertility of the pollen respectively. At maturity, seeds of M₁ plants from each treatment were harvested separately and used these seeds to raise M₂ generation in the next season. The M₂ generation were raised in randomized block design (RBD) on plant to row basis. Chlorophyll mutants were recorded during 06 to 15 days after sowing of M₂ generation. Chlorophyll mutants viz. Albina, Chlorina, Xantha and Viridis, were classified  according to the classification of Gustafsson (1940) and Blixit (1961). Different kinds of chlorophyll mutations were identified on the basis of amount of chlorophyll synthesized and distributed in the plants in accordance with the classification of Gustafsson (1940) and Blixit (1961). The mutant Albina  was found to be Lethal mutant and characterized by entirely white leaves of seedlings. It  survived for 08-10 days after emergence. The mutant Chlorina was characterized yellowish green (pale green) seedlings in colour and they survived for reasonably longer period some of theme produced flowers and seeds as well. The leaves of mutant Xantha were bright yellow in colour and their seedlings were survived for 22-28 days. Viridis were viable mutants characterized by mixed pattern of  light green leaves which become normal green colour at later stages. However, this mutant died before maturity in the present study. The spectrum of chlorophyll mutations was determined as the relative proportion of different types of chlorophyll mutants to the total number of chlorophyll mutations. The M₂ population were also screened several times for morphological mutations throughout the crop season. Mutation frequency was calculated as percentage of mutated M₂ progenies for both chlorophyll and morphological mutations in each treatment as per the following formula.
 

         

 
The mutagenic effectiveness is the measure of the frequency of mutation induced by unit dose of mutagen. The mutagenic efficiency narrates the proportion of mutation in relation to biological damage induced. Mutagenic effectiveness and efficiency were calculated on the basis of formula suggested by Konzak et al., (1965). The mutagenic efficiency expressed in terms of lethality, seedling injury and pollen sterility is given as follows:
 

The tolerance level of the biological material to a particular mutagen are manifested in M₁ generation itself in terms of germination, seedling injury, pollen sterility etc. (Gaul 1970). In present investigation the estimated LD₅₀ doses for seed germination are 600 Gy (gray) for gamma ray and 0.50% concentration for EMS. The dose below 200 Gy for gamma ray and 0.15% concentration for EMS will definitely increase the availability of the population of M₂ generation but the mutation spectrum induced will not be satisfying. However if the dose is more than 600 Gy and 0.50% concentration, then, enough population may not be available to grow M₂ generation. Thus, doses just below 600 Gy and 0.50% concentration for EMS are suggested for further mutation breeding program for inducing good mutations while insuring ample amount of individuals for screening those mutations.

Chlorophyll deficient sectors produced in M₁ generation and Chlorophyll mutations produced in M₂ generation provide one of the most dependable indices for the evaluation of genetic effects of different dosses of mutagen in a particular plant. Van Harten (1998) reported that collective effect of several genes located on different loci of many chromosomes is responsible for chlorophyll synthesis. Mutations in these genes may lead to chlorophyll mutations which could be considered as the most dependable indices for evaluation of different mutagenic efficiencies. These mutagenic efficiencies insures the induction of the genetic variability applicable for crop improvement.

Mutation frequency and relative percentage of chlorophyll mutants were presented in Table 1. Mutation frequency has been used as the indicator of mutagenic effect. The analysis of the mutation frequency revealed that in general, the mutation frequency increased with the increase in dose and was irrespective of the variety. The maximum amount of mutation frequencies were recorded at 450 Gy gamma ray treatment which was followed by 0.3% EMS treatment in both of verities viz. variety phulepandhari (pp) and variety VCM-8.



The present study revealed that the cowpea varieties responded differentially to gamma ray and EMS for the production of chlorophyll mutations. The performance of chlorophyll mutations and their relative frequencies are recorded in Table 1. Almost all the dosage of both of the mutagens are succeeded in inducing all types of the chlorophyll mutants except treatment EMS 0.20% which failed to produce Albina in variety VCM-8 whereas in case of variety phulepandhari, the mutagenic treatments 250 Gy, EMS 0.20% and EMS 0.25% are failed to produce Albina this could be because these lower dosage are not sufficient to induce mutation in all of the genes  necessary for the synthesis of chlorophylls. Wide range of chlorophyll mutants are observed in soybean (Karthika and Subbalakshmi 2006); sesame (Savant et al., 2010) and finger millet (Ambavane et al., 2015).

All types of chlorophyll mutations were observed in both of the varieties at higher dosage of both EMS and gamma rays which are known potent mutagens in inducing point mutations and chromosomal aberrations suggesting their preferential action on genes for chlorophyll development. Higher concentrations of mutagens became more efficient in inducing chlorophyll mutations, these doses/concentrations have induced wider spectrum of chlorophyll mutations compared to lower doses of mutagens in both of the varieties. The results of the effectiveness and efficiency of the two mutagens are given in Table 2 and Table 3 which indicates the response of the varieties to mutagen were varying.





It is found that mutagenic effectiveness and efficiency is dependent upon the nature of induced mutations. In order to obtain high effectiveness and efficiency, the mutation effect must greatly surpass other effects in the cell such as physiological and toxic effects which results in seedling injury and eliminate mutation. 

In the present study, the EMS treatments in both of the verities did not follow exact trend for mutagenic effectiveness. Reduction in effectiveness from 0.725 (EMS 0.20%) to 0.68 (EMS 0.25%) was recorded and then it is increased to 1.02 at EMS 0.30% treatment in variety phulepandhari. Similar observations in chickpea var. Pusa-372 were reported by Wani (2009). In case of variety VCM-8, initially the effectiveness is increased with increase in dose from 0.866 at 0.20% to 1.56 at 0.25% and then it is decreased to 1.34 at higher treatment 0.30%. Initial increase in effectiveness with increase in dose followed by decrease in effectiveness with increase in dose was reported in earlier studies in cluster bean (Bhosle and Kothekar 2010).  

In variety phule pandhari, the mutagenic effectiveness is decreased with increasing dosage of gamma ray. The highest mutagenic effectiveness (0.736) was found at lowest dose 250 Gy and the lowest value of mutagenic effectiveness (0.604) is recorded at higher dose 450 Gy. Similar results of higher mutagenic effectiveness at lower mutagen doses were reported by Kulthe et al., (2013) in winged bean treated with gamma rays. In case of variety VCM-8 the lowest effectiveness (0.56) is recorded at higher dose 450 Gy, which increased to (0.70) at moderate dose 350 Gy. and again reduced to 0.66 at 250 Gy (Table 3). Among two mutagens used, EMS proved to be more effective than gamma ray and its response was at a higher frequency in variety VCM-8. Decrease in effectiveness at higher concentration of EMS and gamma ray may be due to the biological damage which increases with increase in dose at a rate greater than the frequency of mutation indicating that the mutagenic effectiveness and efficiency may depend upon the nature of induced mutation or aberrations.

Mutagenic efficiency may differ for different plant tissues or individuals because of the differential test conditions influencing the expression of the true potential of the mutagenic agents and his coworkers (Konzak et al., 1965). Mutagenic efficiency observed in both varieties was generally higher on sterility basis as compared to the lethality and seedling injury basis (Table 2 and 3). Variety phulepandhari showed the highest mutagenic efficiency at lowest dose of EMS for lethality and pollen sterility however for seedling injury it is increased from 0.854 (EMS 20%) to 1.227 at EMS 25%, further it is decreased up to 0.694 at higher dose. Dube et al., (2011) also recorded similar results in cluster bean treated with gamma rays where increase was observed up to 20 kR (kilo Rad) and again decreased at higher dose. Gamma ray succeeded in inducing the highest mutagenic efficiency at lowest doses and the lowest mutagenic efficiency at highest dose for pollen sterility, lethality and seedling injury in variety VCM-8 (Table 3). It is observed that the lower dose of gamma ray proved to be most efficient instead of lower dose of EMS on seedling injury basis in both the varieties.

The total mutagenic efficiency calculated on the basis of lethality, seedling injury and pollen sterility in both varieties was found to be highest at lowest doses of both mutagens. Further it is reduced with increasing dose. Higher efficiency at lower concentration/dose of the mutagen is reported by Savant et al., (2010) in sesame and Wani (2009) in chickpea. Thilagavathi and Mullainathan (2009) reported higher mutagenic effectiveness and efficiency at lower doses of EMS in black gram. Decreased mutagenic effectiveness and efficiency at higher doses/concentrations observed in present investigation was also reported by Dhanavel et al., (2008) and Nair et al. (2014) in cowpea. In the present study, VCM-8 proved to be more sensitive to mutagen sensitive than phulephandari as seen elsewhere. Variations in sensitivity within crops and even within genotype may depend on their genetic architecture and the mutagens employed. EMS concentration induced high chromosomal aberrations than gamma ray treatments which indicate greater efficiency of EMS for inducing mitotic abnormalities in the cells of treated population.

In present investigation the optimum response of both of the mutagens were observed at lower doses. The frequency of chlorophyll mutations may be included as an indicator to determine the capacity, power, efficacy, influence, potency of plant mutagen. However, an effective mutagen may not necessarily show high efficiency and vice versa. The effectiveness does not increase with increase in dose and was found lower at higher doses. The mutagenic doses inducing mutations with less biological damage are proved to be more effective and efficient.  Efficiency of a mutagenic agent depends on a number of attributes like the reactivity of the agent with the biological material, its applicability to the biological system, the level of physiological damage, chromosomal aberration, pollen sterility, lethality seedling injury and alteration in genes necessary for chlorophyll synthesis.