Effect of ethyl methane sulphonate on germination, survival, fertility and mutagenic efficiency in Lentil (Lens culinaris Medik.)

Introduction

Lentil (Lens culinaris Medik.) is an important food legume crop cultivated widely in many parts of the world, particularly in South Asia. It is a rich source of dietary protein, carbohydrates, vitamins, minerals and dietary fibre. Lentil also improves soil fertility through biological nitrogen fixation and therefore plays an important role in sustainable cropping systems. In India, lentil is mainly cultivated during the rabi season and contributes significantly to nutritional security, especially among vegetarian populations. Despite its importance, the productivity of lentil remains comparatively low due to several biotic and abiotic stresses, limited availability of high-yielding varieties and a narrow genetic base in cultivated germplasm. Conventional breeding methods such as selection and hybridization have contributed to lentil improvement; however, the creation of sufficient genetic variability remains a major challenge. Therefore, induced mutagenesis is considered a useful supplementary approach for broadening genetic variability and developing desirable plant types. Chemical mutagens, particularly ethyl methane sulphonate (EMS), are widely used in plant breeding because they induce point mutations and create heritable variation without drastically disturbing the overall genetic background of an established variety. EMS has been successfully used in several pulse crops to generate variation for traits such as plant height, maturity duration, branching, pod number, seed size, yield and stress tolerance. However, the response to EMS varies according to genotype, concentration and treatment conditions. Higher concentrations may cause excessive biological damage, including poor germination, reduced survival and reduced reproductive fertility. Therefore, the present investigation was undertaken to study the effect of different EMS concentrations on seed germination, plant survival, pollen fertility and ovule fertility in two lentil varieties, Markanda and Vipasha. The study also aimed to identify an appropriate EMS dose for inducing useful variability with minimum biological damage in lentil.

Materials and Methods

The present investigation was conducted during the Rabi season of 2024-25 at the Research Farm and laboratories of the School of Agriculture, Abhilashi University, Chail Chowk, Mandi, Himachal Pradesh, India. The experimental site is located at approximately 31°33′12″ N latitude and 76°59′18″ E longitude, at an altitude of about 1450 m above mean sea level. Two lentil (Lens culinaris Medik.) varieties, namely Markanda and Vipasha, were used as experimental material. Healthy, uniform and undamaged seeds of both varieties were selected for mutagenic treatment. Ethyl methane sulphonate (EMS) was used as the chemical mutagen. Seeds were pre-soaked in distilled water for six hours and then treated with five EMS concentrations: 0.1, 0.2, 0.3, 0.4 and 0.5%. EMS solutions were prepared in phosphate buffer of pH 7.0. The pre-soaked seeds were treated with respective EMS solutions for six hours at room temperature with intermittent stirring. Untreated seeds soaked in distilled water served as the control. After treatment, seeds were thoroughly washed under running water to remove residual EMS. For laboratory evaluation, 100 seeds from each treatment were placed in Petri dishes lined with moist filter paper. Seed germination percentage was recorded after germination, while plant survival percentage was recorded on the basis of surviving seedlings. For field evaluation, treated and untreated seeds were sown in a randomized block design with three replications. Seed germination and plant survival were recorded in the M₁ generation under field conditions. Pollen fertility was estimated by staining pollen grains using acetocarmine stain. Well-stained, round and fully developed pollen grains were considered fertile, whereas shrivelled or unstained pollen grains were considered sterile. Ovule fertility was calculated based on the proportion of fertile ovules. The LD₅₀ dose was estimated from seedling survival percentage using probit analysis. Mutation frequency, mutagenic effectiveness and mutagenic efficiency were calculated in the M₂ generation according to the method suggested by Konzak et al. (1965) [1] . The collected data were statistically analysed to determine the effect of EMS concentrations on the studied characters.

Results and Discussion

Effect of EMS on Seed Germination and Plant Survival under Laboratory Conditions

The effect of different concentrations of EMS on seed germination and plant survival of lentil varieties Markanda and Vipasha under laboratory conditions is presented in Table 1. A progressive decline in seed germination and plant survival was observed with increasing EMS concentration in both varieties. In Markanda, seed germination decreased from 96.00% in the control to 92.00, 86.00, 79.00, 70.00 and 58.00% at 0.1, 0.2, 0.3, 0.4 and 0.5% EMS, respectively. The maximum reduction in germination over control was recorded at 0.5% EMS (39.58%), whereas the minimum reduction was observed at 0.1% EMS (4.17%). Plant survival also declined from 95.00% in the control to 93.68% at 0.1% EMS and 67.37% at 0.5% EMS. The highest reduction in plant survival was observed at 0.5% EMS (29.08%). A similar trend was observed in Vipasha. Seed germination declined from 96.00% in the control to 90.00, 82.00, 73.00, 62.00 and 48.00% at increasing EMS concentrations from 0.1 to 0.5%. The reduction in germination was highest at 0.5% EMS (50.00%). Plant survival in Vipasha decreased from 93.00% in the control to 88.17% at 0.1% EMS and 55.91% at 0.5% EMS. The maximum reduction in plant survival over control was recorded at 0.5% EMS (39.88%). The results indicated that EMS caused dose-dependent biological damage in both lentil varieties. Higher EMS concentrations adversely affected seed viability, seedling growth and survival, possibly due to mutagen-induced physiological disturbances and chromosomal damage. Vipasha showed greater reduction in germination and survival than Markanda, indicating comparatively higher sensitivity to EMS treatment. Similar reductions in germination and survival with increasing mutagen concentration have been reported in lentil and other pulse crops by Konzak et al. (1965) [1] , Singh et al. (2014) [3] , and Sood et al. (2023) [2] .

Effect of EMS on Seed Germination and Plant Survival under Field Conditions

The effect of different EMS concentrations on seed germination and plant survival of lentil varieties Markanda and Vipasha under field conditions is presented in Table 2. Similar to laboratory observations, seed germination and plant survival decreased progressively with increasing EMS concentration in both varieties. The reduction in germination and survival under field conditions was comparatively greater than that observed under laboratory conditions, indicating that treated seedlings were further affected by environmental stress after sowing. Vipasha showed greater sensitivity to EMS than Markanda, as reflected by its lower germination and survival percentages at higher concentrations. The findings confirm that increasing EMS concentration causes greater biological damage, resulting in poor seedling establishment and survival. Similar dose-dependent effects of EMS on germination and survival have been reported in several pulse crops.

References

1. Konzak CF, Nilan RA, Wagner J, Foster RJ. Efficient chemical mutagenesis. In: The use of induced mutations in plant breeding. Rome: FAO/IAEA; 1965. p. 49-70.
2. Sood S, Kumar R, Sharma P. Induced mutagenesis for genetic improvement of pulse crops: A review. Legume Research. 2023;46(4):401-409.
3. Singh A, Singh S, Kumar P. Effect of gamma rays and ethyl methane sulphonate on germination, survival and pollen fertility in lentil (Lens culinaris Medik.). International Journal of Agricultural Sciences. 2014;10(2):567-571.