Ascertaining Lethal Dose 50 (LD₅₀) and Simultaneous Effect of Ethyl Methane Sulphonate (EMS) and Sodium Azide (SA) On Seedling Characters in Mungbean Genotypes ‘Pusa 1031’ and ‘Pusa 1431’

Mungbean, one of India’s 13 dietary legumes, is the country’s third-most significant pulse after chickpea and pigeonpea (Singh et al., 2015). Mungbean and other pulses have historically been farmed on the minimal-fertility ground with low productivity with limited input (Khan and Goyal, 2009). Genetic reconstitution is required for these crops to evolve distinct plant kinds due to genetic improvement for enhanced production (Siddique Sadiq et al., 1999). Mutation breeding has emerged as one of the essential methods in mungbean for developing and disseminating novel genotypes and high-yielding cultivars (Pathirana, 2011). The fundamental advantage of utilizing induced mutations is the capacity to ameliorate a single or a small number of desirable traits in a crop without materially changing the remainder of its genetic makeup (Awan, 2005). Mutagenesis using Ethyl Methane Sulphonate (EMS) and Sodium azide (SA) has shown to produce a diverse range of mutations in mungbean viz., Chlorophyll mutants (Khan and Siddiqui, 1993), Pod mutants and seed mutants (Wani et al., 2017), High and lower yielding mutants (Wani et al., 2011) and Pollen Fertility (Kulthe, 2019). Chemical mutagens typically exert their effects through single base pair and single nucleotide polymorphisms (Sikora et al., 2011). By adding an ethyl group to the guanine bases at the O-6 position, EMS encourages DNA polymerase to insert thymine mismatches with O-6-ethyl guanines, resulting in point mutations at arbitrary loci (Suprasanna et al., 2014). Sodium azide is a potent mutagen that can cause various gene or point mutations by uncharged HN3 molecule found in acidic state enters the cells and increase their ability to cause mutations (Singh and Olejniczak, 1983).
 
The efficacy of the strategies employed will significantly influence the outcomes of mutation breeding programme. The fact that mutagens effects depend on their dose is well apparent. Establishing the appropriate dose is the most crucial step before beginning any mutagen treatment. In the lack of comprehensive data, it is occasionally required to calculate the LD₅₀ through a small-scale test to determine the ideal dose in the M1 population. However, the standardization of the lethal dose 50 estimates for the mungbean genotypes, viz., ‘Pusa 1031’ and Pusa 1431', has not been attempted in any prior studies. Because of this, the current study was conducted to determine the LD₅₀ dosage of sodium azide and ethyl methane sulphonate for two mungbean genotypes and to evaluate how differently seedlings respond to various mutagen concentrations.

Plant material
 
The genetically pure seeds of two mungbean genotypes, ‘Pusa-1031’ and ‘Pusa-1431’, used in this study were obtained from the ICAR-Indian Agricultural Research Institute (IARI), Pusa, New Delhi. Since these cultivars already have valuable genetic backgrounds, we chose them as the subject matter for this study and median lethal dose for two chemical mutagens namely ethyl methane sulphonate and sodium azide, which was not estimated prior to this.
 
Experimental site
 
The experiment was carried out during January 2023, in Plant Breeding Laboratory, College of Post Graduate Studies in Agricultural Sciences, CAU (I), Umiam, Meghalaya.
 
Ethyl methane sulphonate (EMS) treatment
 
One of the chemical mutagens employed in this experiment was Ethyl Methane Sulphonate (EMS). The investigation was carried out in a lab environment with three replications. To activate the seeds metabolism and boost the mutagen’s effectiveness, 50 uniform dry seeds of each genotype per replication were pre-soaked in distilled water for 3 hours. The seeds were then exposed to ten concentrations of EMS (10 mM to 100 mM with a ten mM difference) in 0.1 M phosphate buffer at pH 7.0 for six hours while being gently shaken occasionally. The seeds were then rinsed under running water thrice for 30 minutes to eliminate any remaining mutagens. Seeds for the control were submerged in distilled water for the duration of the treatment.
 
Sodium azide (SA) treatment
 
This experiment used sodium azide (SA) as another chemical mutagen studied in a controlled laboratory environment with three replications. Fifty uniform dry seeds of each genotype per replication were pre-soaked in distilled water for 3 hours to make the seeds metabolically active and increase the efficacy of mutagens. The seeds were then treated with different concentrations of SA (0.01 mM, 0.02 mM, 0.03 mM, 0.04 mM and 0.05 mM) prepared in 0.1 M phosphate buffer at pH 3.0 with occasional gentle shaking for six hours. Afterward, seeds were washed under running water thrice for 30 minutes to remove residual mutagen. Seeds for control were soaked in distilled water for the total treatment time without SA treatment. 
 
Treated seeds were germinated in trays and kept at room temperature. Following the International Seed Testing Association (ISTA) rules, observations on numerous seedling parameters were recorded. The methodology of the work is represented in (Fig 1). For the evaluation of LD₅₀, data were subjected to probit analysis and various parameters were determined using a mean of three replicates using the OPSTAT Statistical Software Package for Agricultural Research Workers (Sheoran et al., 1998).

What is the significance of finding the LD₅₀ values for different mutagens in different genotypes of plants? Lethal Dose 50 (LD₅₀) is the dose that causes a fifty percent reduction in viable plants or seeds (Gad, 2014). The ideal mutagen dose is one of the critical factors in the success of mungbean mutation breeding programme. Maximum mutation and minimal fatality are produced by a mutagen at its optimal dose (Kodym et al., 2012). Many scientists believe the ideal dose should resemble the Lethal Dose 50 (LD₅₀). Based on seed germination rates of treated seeds at various mutagen concentrations compared to untreated controls, LD₅₀ values were determined. Median lethal dose for the mutagen Ethyl Methane Sulphonate (EMS), of two mungbean genotypes namely ‘Pusa 1031’ and ‘Pusa 1431’ was determined as 58.81 mM and 45.04 mM, respectively (Fig 2). The finding mentioned above is consistent with past research in different pulse crops conducted by various authors, viz., chickpea (Khan and Kozgar, 2011), cowpea (Nair and Gayatri, 2022), greengram (Jeevi, 2020; Vairam, 2014; Singh and Kole, 2005) and blackgram (Jain and Khandelwal, 2009). For the mutagen sodium azide, the LD₅₀ was determined to be 0.047 mM for both genotypes (Fig 3). The above observation was confirmed by a previous study in chickpea by (Khan and Kozgar, 2011) and Linseed by Jahan et al., (2021). For EMS, “Pusa 1031” displayed a higher LD₅₀ value; however, both genotypes displayed comparable LD50 values for the mutagen sodium azide. For the mutagen ethyl methane sulphonate, the differences in LD₅₀ values between cultivars show that median lethal dose values vary from genotype to genotype, which variations in the genetic background and pedigree of the cultivars may bring about. Comparable LD50 values were obtained with sodium azide treatment for both genotypes, indicating that this may be the optimal LD₅₀ dose for both genotypes.




 
Effect of ethyl methane sulphonate and sodium azide on germination of two mungbean genotypes
 
The proportion of seeds that germinated following treatment compared to the control for two mungbean genotypes was used to calculate seed germination. Ethyl Methane Sulphonate substantially reduced the germination percentage for both genotypes. The germination percentage at the control and 100 mM, when treated with EMS, was observed as 89.66 % and 23.07% in ‘Pusa 1031’ and 89.83% and 15.78% in ‘Pusa 1431’ respectively (Table 1 and Fig 4a). The per cent reduction of germination over control at 100 mM EMS was noted as 74.26% and 82.42% for the genotypes’ Pusa 1031' and ‘Pusa 1431’ respectively (Table 1). With a corresponding rise in EMS concentration, both the genotypes displayed linear trends for germination percentage. Observing the percent reduction in germination of treated seed over control for the mutagen EMS, which has noticeably distinct values for both genotypes at various mutagen doses. Different genotype susceptibility to mutagen EMS or various innate seed germination abilities could cause this difference. For EMS, the maximum germination was recorded at a concentration of 10 mM, with “Pusa 1031” (83%) leading the way, followed by “Pusa 1431” (78.04%) (Table 1).



When the genotypes were treated with Sodium Azide, germination percentage values at the control and 0.05 mM SA were recorded as 89.66% and 34.61% in ‘Pusa 1031’ and 89.83% and 40% in ‘Pusa 1431’ respectively (Table 1). The percentage reduction of germination over control at 0.05 mM SA was observed as 61.39 % and 54.61% for the genotypes ‘Pusa 1031’ and ‘Pusa 1431’ respectively (Table 1), both the genotypes revealed linear trend with a matching increase in SA concentration (Fig 4b). The percentage drop of treated versus control for the mutagen Sodium azide has shown significantly different results for the two mungbean genotypes. At concentrations of 0.01 mM and 0.02 mM, a significant germination percentage drop over control, i.e., 6.39 to 25.28%, in the genotype Pusa 1431 (Table 1), demonstrating that mutagen activity at these concentrations is inhibiting germination. For the mutagen sodium azide, the highest germination was observed at a concentration of 0.01 mM, with ‘Pusa 1031’ (85%) leading the way followed by ‘Pusa 1431’ (82%) (Table 1).
 
Declining trends in germination percentage with a commensurate increase in mutagen dosage were reported by Lavanya et al. (2023), Wani et al. (2021), Jyothsna et al., (2022), Omosun et al. (2022), Chaudhary et al., (2021), Khan and Kozgar (2011), Shahwar et al., (2019), Nair and Gayathri (2022), Singh and Kole (2005). In seeds treated with EMS and SA, a five and four-days delayed germination was noticed in both genotypes. A similar pattern of post-chemical mutagen delayed germination was observed by Taziun et al. (2017) and Nair and Gayathri (2022). According to Kurobane et al., (1979), mutations impair the enzyme’s ability to function, which affects germination. The production of germination-related enzymes may be impacted by mutation. At specific dosages, different genotypes responded to seed germination differently from one another. The physiological effects of EMS and SA, which impede the metabolic activities required for germination and have a more noticeable effect at larger dosages, may explain why germination decreases with increasing mutagen dose. ‘Pusa 1431’, which illustrated the most considerable per cent reduction in germination over the control, is more affected than Pusa 1031, on the whole. The above finding has been confirmed by Pusa 1431 possessing a lower LD₅₀ for the EMS than ‘Pusa 1031’. Maximum germination reduction was seen at mutagen doses above LD₅₀ values for each genotype, indicating that LD₅₀ is the most effective dose for inducing viable mutations and any value above LD₅₀ results in plants with the most detrimental effects, which makes them challenging to advance for further generations.
 
Effect of ethyl methane sulphonate and sodium azide on seedling shoot length, root length, total seedling length and seedling vigour index of two mungbean genotypes
 
In the current study, the severity of these defects increased as the concentration of chemical mutagens increased. For both genotypes, 0.01 mM and 0.05 mM of Sodium Azide concentration and 10 mM and 100 mM of Ethyl Methane Sulphonate concentration have been determined to be the highest and lowest levels for seedling shoot length, seedling root length and total seedling length, respectively. (Table 2 and Table 3). Higher mutagenic concentrations of both chemical mutagens induced a drastic reduction in total seedling length, which ranged from 14.87 cm to 2.77 cm in ‘Pusa 1031’ and 8.17 cm to 3.11 cm in ‘Pusa 1431’ for EMS (Table 2) and for SA-treated seedlings, it was recorded as 11.20 cm to 5.10 in ‘Pusa 1031’ and 8.17 cm to 4.50 in “Pusa 1431,” respectively (Table 3). These results are consistent with those of Nilahayati et al., (2023), Omosun et al., (2022), Jyothsna et al., (2022) and Chaudhary et al., (2021). Seedling root length and shoot length was observed to be decreasing trend as the concentration of both the chemical mutagens increased (Table 2), similar trend was observed by Ravi et al., (2023). Genotype ‘Pusa 1431’ has shown similar root length at 0.01mM and 0.02 mM sodium azide concentration (Table 3).




 
The seed vigor index was calculated based on the formula proposed by Abdul Baki and Anderson (1973), viz.
 

Seed vigor index = Seed germination (%) x Seedling total length
 

With higher mutagen doses, the seedling vigor index showed a sharp decline for both chemical mutagens and it ranged from 1238.89 to 63.85 in ‘Pusa 1031’ and 637.40 to 48.95 in ‘Pusa 1431’ for EMS treatment (Table 4). However, for SA treatment, the seed vigor index ranged from 960.00 to 176.54 in “Pusa 1031” and 673.75 to 180.00 in ‘Pusa 1431’ (Table 4 ). While the higher concentrations of SA, i.e., 0.04 mM to 0.05 mM, genotype ‘Pusa 1431’ displayed a greater seed vigor index compared to ‘Pusa 1031’ (Table 4).


 
Sodium azide and ethyl methane sulphonate treatments had a detrimental effect on various seedling characteristics because they hindered physiological and enzymatic processes. In the current study, the severity of these defects increased as the concentration of chemical mutagens increased. Similar declining trends for various seedling characters were observed by Ravi et al., (2023), Omosun et al., (2022), Jyothsna et al., (2022) and Chaudhary et al., (2021).

The degree of reaction of a particular genotype to a certain mutagen differs from that of other genotype because each genotype has a distinct genetic makeup and relevant enzymatic and physiological responses. The statement is supported by the results of the present investigation, in which two different mungbean genotypes showed different LD₅₀ values for the chemical mutagen-Ethyl Methane Sulphonate. Despite having obtained similar LD₅₀ values for both genotypes when treated with sodium azide, both genotypes showed remarkable differences in seedling parameters like seedling vigor, which depicted genotype “Pusa 1431” having shown higher seedling vigor compared to “Pusa 1031” at higher concentrations when treated with sodium azide, which is indicative of having more significant potential for better seedling growth and survival at higher doses. In order to establish the best mutagenic doses, the study assessed the LD₅₀ for sodium azide and ethyl methane sulphonate on two mungbean genotypes. For “Pusa 1031” and “Pusa 1431,” the LD₅₀ for ethyl methane sulphonate was determined to be 58.81 mM and 45.04 mM, respectively. When treated with sodium azide, the LD₅₀ values for both genotypes was 0.047 mM. With varying dosages of SA and EMS, several seedling parameters responded differently in both genotypes. The results showed that sodium azide at lower concentrations is more effective than EMS for obtaining the median lethal dose in both genotypes. Conducting the required field testing is also encouraged to confirm the results and retrieve beneficial mutants with higher crop value in subsequent generations.

The authors duly acknowledge the School of Crop Improvement, Department of Genetics and Plant Breeding, CPGAS, CAU (I) for providing facilities and for the smooth conduct of the experiment.
 

There was no specific funding for this work.

The authors declare that there are no conflicts of interest.