Study on chemical mutagenesis in cotton (Gossypium sp.) using ethyl methane Sulfonate (EMS)

Introduction

Cotton (Gossypium spp.), belonging to the Malvaceae family, is one of the most important fibre crops globally which thrives in tropical and subtropical regions, requiring warm, dry conditions and sufficient water, often through irrigation. Cotton is an often cross-pollinated crop, predominantly self-pollinated but with 5-50% cross-pollination influenced by species, floral traits, and pollinators. India leads the world in cotton cultivation area, with around 9.6 million hectares, approximately 25% of the global total, and contributes about 16% to global cotton production. Cotton plays a vital role in India's agricultural economy, contributing nearly 30% to the agricultural GDP and supporting millions of farmers and textile workers. Mutation breeding is favoured over hybridisation for cotton improvement due to its efficiency in creating variability, preserving desirable traits, and overcoming hybridisation barriers (Waghmare et al., 2000 and Oladosu et al., 2016) [11,12,7] . Mutation breeding involves altering a plant's DNA sequence, either naturally or artificially, to enhance traits such as disease resistance, yields, or stress tolerance. Chemical mutagenesis, particularly using agents like ethyl methane sulfonate (EMS), is widely applied in agriculture (Waghmare & Koranne, 2000) [11,12] . EMS is an alkylating agent that causes point mutations by ethylating guanine bases in DNA, leading to GC to AT transitions. It is effective in inducing high-frequency mutations without major chromosomal damage, making it a preferred tool in plant breeding and genetic research (Atiq et al., 2023) [1] . A study was conducted to examine the effects of different concentrations of EMS and to find out the optimum mutagenic dose (LD₅₀) of EMS on seedling and growth related traits in selected cotton varieties. Ultimately, the superior EMS-induced mutant lines with improved germination and early vigour traits were identified with enough variability induction without drastically reducing its viability.

It induces point mutations by ethylating guanine, causing G:C to A:T transitions. Though EMS is efficient in inducing mutations, it is highly toxic and requires careful handling under strict safety measures. Uniform and healthy seeds of MCU 5 and MCU 12 were treated with EMS at concentrations of 0.2%, 0.4%, and 0.6% for two durations of 8 and 14 hours each. Each treatment used 100 seeds per variety. A control set (T 13 and T 14 ) was soaked in distilled water for 12 hours. Posttreatment, seeds were thoroughly washed and sown in sterilized pot mixtures under controlled conditions.

Treatment details: The experiment included 14 treatments (T 1 -T 14 ) and was laid out in three replications of 100 seeds each. T 1 -MCU 5 @ 0.2 percent concentration for 8 hours T 2 -MCU 5 @ 0.2 percent concentration for 14 hours T 3 -MCU 5 @ 0.4 percent concentration for 8 hours T 4 -MCU 5 @ 0.4 percent concentration for 14 hours T 5 -MCU 5 @ 0.6 percent concentration for 8 hours T 6 -MCU 5 @ 0.6 percent concentration for 14 hours T 7 -MCU 12 @ 0.2 percent concentration for 8 hours T 8 -MCU 12 @ 0.2 percent concentration for 14 hours T 9 -MCU 12 @ 0.4 percent concentration for 8 hours T 10 -MCU 12 @ 0.4 percent concentration for 14 hours T 11 -MCU 12 @ 0.6 percent concentration for 8 hours T 12 -MCU 12 @ 0.6 percent concentration for 14 hours T 13 -MCU 5 soaked for 12 hrs (Control) T 14 -MCU 12 soaked for 12 hrs (Control)

Observations noted: Data were recorded for germination percentage (%), seedling survival rate (%), root length (cm), mean seedling length (cm), seedling fresh and dry weight (mg), and seedling vigour indices I and II. Germination was recorded on the 7th day, and biometric observations followed. Seedling Vigour Index I was calculated by multiplying germination percentage with mean seedling length, while Index II used seedling dry weight.

Statistical approach:

In the present experiment, the Completely Randomized Design (CRD) was adopted, as it provides a simple and effective layout where treatments are assigned to experimental units entirely at random. For the statistical analysis of the data obtained under CRD, INDOSTAT software was employed. The software facilitates precise computation of various statistical parameters, analysis of variance (ANOVA), and comparison of treatment means, thereby enabling accurate interpretation of the experimental results.

Results and Discussion

Analysis of variance:

The mean square values for eight seedling related traits are mentioned in the table 1. The mean square value of germination percentage (65.821), survival rate (22.453), root length (3.965), shoot length (6.223), fresh weight (0.200), dry weight (0.004), seed vigour index I (119.155) and seed vigour index II (80.80). The maximum mean square value is seed vigour index I (119.155) and the lowest mean square value is dry weight (0.004). The results of ANOVA revealed a significant treatment effect, indicating that the differences among the treatment means were not due to experimental error but rather to the actual influence of the treatments. This suggests that at least one treatment mean differed significantly from the others (Badigannavar et al., 2000 and Winkler et al., 2023) [2,13,14] . It is inferred that the treatments imposed in the experiment had a measurable and meaningful impact on the response variable.

Mean Performance of EMS Treatments:

The mean performance of treatments, including treated and untreated, evaluated for eight seedling related traits was represented in Table 2.

In respective of all the traits, the mutagen-treated treatment T 1 (MCU 5 @ 0.2 percent concentration for 8 hours) marked the highest scores for germination percentage (79.74%), seedling survival rate (62.45%), root length (6.67 cm), mean seedling length (14.74 cm), seedling fresh and dry weight (1.51 mg and 0.20 mg respectively), and seedling vigour indices I and II (616.79 and 15.99 respectively) whereas the mutagen-treated treatment T 12 (MCU 12 @ 0.6 percent concentration for 14 hours) secures the least scores for germination percentage (42.00 %), seedling survival rate (25.54 %), root length (3.91cm), mean seedling length (9.25 cm), seedling fresh and dry weight (0.89 mg and 0.11 mg respectively), and seedling vigour indices I and II (178.55 and 4.77 respectively). Apart from the treatment T 1 , the treatment T 2 (MCU 5 @ 0.2% concentration for 14 hours) exhibited consistently superior performance across all the eight seedling-related traits, recording appreciable improvements without any noticeable deviations, thereby highlighting its effectiveness in enhancing seedling vigour. Among the control, T 13 (MCU 5 soaked for 12 hrs) recorded the highest overall germination at 97.00% with more survival rate of 92.64%, mean seedling length (17.23 cm) and seedling vigour indices I and II (931.10 and 25.64 respectively). For the traits, root length (8.25 cm), seedling fresh weight (1.85 mg) and seedling dry weight (0.243 mg), T 14 (MCU 12 soaked for 12 hrs) registered its highest measures. The present investigation highlights the contrasting responses of MCU 5 and MCU 12 genotypes to EMS mutagenesis. Among the treatments, T 1 (MCU 5 @ 0.2% for 8 hours) consistently recorded higher values for germination percentage, seedling survival, root length, seedling length, and vigour indices, indicating that this treatment represents an optimum mutagenic dose capable of inducing variability without severely affecting physiological processes. Similar observations were reported by Gaul (1964) [3] , who emphasized the importance of identifying a balanced mutagenic dose to ensure effectiveness with minimum lethality. Interestingly, T 2 (MCU 5 @ 0.2% for 14 hours) also exhibited superior seedling performance, ranking next to T 1 . This highlights the relative tolerance of MCU 5 to prolonged mutagen exposure, particularly at lower concentrations, which aligns with the concept of a biologically effective dose as suggested by Jain (2010) [5] . In contrast, T 12 (MCU 12 @ 0.6% for 14 hours) resulted in severe reductions in all seedling traits, which may be attributed to the cytotoxic and genotoxic effects of higher EMS concentrations. This is mainly attributed to mitotic arrest and abnormal cell division in meristematic zones, reducing elongation and root architecture development (Muthusamy & Jayabalan, 2001) [6] . At elevated levels, EMS is known to cause excessive chromosomal aberrations and physiological damage. EMS affects protein synthesis; membrane permeability and chlorophyll content of the cotton seedlings that often show delayed leaf emergence, reduced chlorophyll index and slower seedling vigour. (Girija & Dhanavel, 2009) [4] . This clearly demonstrates the dose-and genotype-dependent response of mutagens, wherein MCU 12 exhibited greater sensitivity compared to MCU 5. MCU 12 exhibited high sensitivity to mutagenic stress and would require cautious dosage standardization in future breeding experiments (Winkler et al., 2023) [13] . Among the controls, T 13 (MCU 5 soaked for 12 hours) displayed maximum germination (97.00%) and vigour indices, confirming the innate vigour of MCU 5 under untreated conditions. Conversely, T 14 (MCU 12 soaked for 12 hours) recorded maximum values for root length and seedling biomass, indicating inherent differences in genotype-specific growth attributes. It is clearly evident that the untreated seeds recorded the highest results because they were free from the cytotoxic and genotoxic effects of EMS (Shim et al., 2019) [9] . In the absence of mutagenic stress, the physiological and biochemical processes such as enzymatic activation, hormonal regulation, and mobilization of stored reserves proceed normally, ensuring higher germination, better seedling survival, and superior vigour indices (Roychowdhury & Tah, 2011) [8] . Since no DNA alkylation or chromosomal damage occurs in untreated seeds, the genetic stability is maintained and the seedlings are able to express their full inherent growth potential (Atiq et al., 2023) [1] .

Variability measures of CRD:

The variability factors calculated under CRD such as standard error (SE), critical difference (CD), and coefficient of variation (CV %) provide valuable insights into the reliability and precision of the experiment were demonstrated in the Table 3. In this study, SE for mean across traits was ranged from 0.007 (dry weight) to 14.865 (vigour index I). Root length had the next lowest SE (0.026). Standard error difference was lowest in dry weight (0.011), followed by fresh weight (0.036), and highest in vigour index I (21.022). Critical difference values ranged from 0.023 (dry weight) to 8.526 (vigour index I). CV was lowest for germination percentage (1.906%), followed by root length (2.496%), suggesting stable expression. The highest CV was noted in seed vigour index II (8.448%). In this study, the acceptable range of SE and CD values, together with comparatively low CV percentages, indicate that the experimental error was minimal and the experiment achieved strong precision and reliability. The small SE and SE (d) values across most traits, along with moderate CVs and significant CD values, confirm that the observed differences among treatments are genuine and attributable to treatment effects rather than random error (Ul-Allah et al., 2019) [10] . Collectively, these variability measures demonstrate that the experiment not only ensured statistical precision but also generated substantial variability, which can be effectively exploited through appropriate selection strategies for the genetic improvement of cotton.

Conclusion

Overall, it is concluded that the mutagen-treated seedlings showed reduced growth performance compared to untreated controls, highlighting inhibitory effects at higher doses.

From these results, it can be inferred that MCU 5 is more amenable to EMS mutagenesis than MCU 12, particularly at lower concentrations and shorter durations. Treatment T 1 (0.2% for 8 hours) may be considered as the optimum/LD₅₀ level treatment for MCU 5, where sufficient mutagenic effectiveness was achieved without compromising viability. Excessive mutagen dosage (0.6% for 14 hours) proved detrimental, reinforcing the importance of selecting an effective but safe mutagenic dose. Therefore, MCU 5 emerges as a promising genotype for mutation breeding programmes, and EMS treatment at 0.2% for 8 hours may be recommended for generating variability in seedling vigour-related traits.

References

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