Assessment on Genetic Diversity in Proso Millet (Panicum miliaceum L.) Mutants for Yield and Yield Parameters Raised during M₂ Generation

Proso millet (Panicum miliaceum L.), an ancient cereal crop, is highly adaptable to marginal environments and exhibits excellent drought tolerance. Despite its nutritional benefits and climate resilience, proso millet remains underutilized in modern breeding programs. Conventional breeding in this crop is constrained by a narrow genetic base, making mutation breeding a viable strategy to enhance genetic variability.
       
Ethyl methanesulfonate (EMS) is a widely used chemical mutagen known to induce point mutations, primarily through guanine alkylation, resulting in base substitutions during DNA replication (Action et al., 2021). EMS has been effectively employed in cereals and millets to generate desirable traits such as early maturity, dwarfism and increased yield components (Kumar et al., 2025).
               
Previous studies in foxtail millet have demonstrated that EMS-induced mutants exhibit significant phenotypic variation in plant height, flowering duration and grain weight (Action et al., 2021). This study extends that approach to proso millet, aiming to assess the effectiveness of EMS in inducing beneficial mutations in the M₂ generation and identifying superior lines for breeding programs.

Plant material and treatment details
 
The current study was conducted at SRM College of Agricultural Sciences. The M₁ generation which was cultivated during 2024 and was harvested to proceed for the next generation. The M₂ generation was grown in the rabi season of 2025.
       
Uniformly healthy seeds of proso millet were pre-soaked in distilled water for 6 hours and then treated with EMS at concentrations of (T₂) 0.1%, (T₃) 0.2%, (T₄) 0.3%, (T₅) 0.4%, (T₆) 0.5%, (T₇) 0.75% and (T₈) 1.0% of 250 seed for each treatment for 6 hours. Treated seeds were thoroughly washed in running water to eliminate residual EMS and sown in the field using a randomized block design (RBD) with three replications. Untreated seeds (T₁) served as the control.
       
The lethal dose (LD₅₀) of the chemical T7 was determined and established as the fixed dose for subsequent treatments. This fixed LD₅₀ dose was applied to the seeds at both pre- and post-exposure stages of M₁ generation. The harvested seeds from 50 randomly selected plants of each treatment were then allowed to grow and mature and the subsequent generations (M₂) were cultivated for further analysis.
 
Data recorded in M₂  generation
 
In the M₂ generation, a comprehensive phenotypic evaluation was conducted to assess the mutagenic impact of EMS on agronomic and morphological traits of proso millet. Plants were grown under uniform agronomic conditions with proper spacing and cultural practices to avoid environmental variation. Observations were recorded from ten randomly selected healthy plants per treatment per replication and the mean values were computed for each trait.
       
The following ten quantitative traits were recorded based on their relevance to plant development, productivity and breeding significance: days to 50% flowering, plant height, number of productive tillers per plant, panicle length (cm), number of leaves per plants, internode length (cm), flag leaf length (cm), flag leaf width (cm), days to maturity, 1000 seed weight. Each trait was carefully recorded using standard procedures to ensure accuracy.
 
Statistical analysis
 
The collected data were analyzed statistically to assess the significance of variation among the EMS treatments. The analysis of variance (ANOVA) conducted on these parameters demonstrates the mutagenic efficiency of EMS in generating genetic diversity within the crop. This variability analysis was performed using R Studio version 4.4.1.

Ten agronomic traits were evaluated in eight treatments, presumably mutagenic or varietal, to determine genetic variation, heritability and treatment effects. The data are expressed as means, standard deviations and ANOVA-based significance levels (Table 1), supported by genetic parameters (Table 2). The findings indicate significant genetic variation and differential responses to treatments among traits.




 
Days to 50% flowering
 
Days to 50% flowering varied considerably from 46.87 days (T₁) to 58.53 days (T₃). ANOVA revealed that there was a significant treatment difference (F = 5.15; P = 0.004) for which critical LSD was 6.85. Treatments 1, 2 and 7 had significantly earlier flowering, indicating that these treatments are earliness inducers. Genetically, the character was under moderate heritability (0.83) and low genetic progress (10.44%), which denoted partial environmental impact and probable dominance or epistatic gene action (Kumari et al., 2019).
 
Plant height
 
While the height of the plant varied between 92.00 cm and 111.93 cm within treatments, the variations were not statistically significant (P = 0.27). However, genetic analysis revealed high heritability (0.95) and high GA (% mean) of 23.26%, indicating that the variability observed is primarily genetic in origin. Treatment 6 had the highest height of plants, which may be investigated for enhancing biomass or lodging tolerance in future breeding programs Trivedi et al. (2017).
 
Number of productive tillers
 
This character had no significant treatment effect (P = 0.15), even though the highest mean was recorded in Treatment 8 (11.47), closely followed by T₇ and T₂. Genetically, the character was highly heritable (0.96) and had the highest GA (% mean) (25.25%), both supporting its potential for effective selection though at present statistically not significant. The extensive diversity and high genetic progress signify the potential of mutagenesis for improving tiller number (Bedis et al., 2006).
 
Panicle length
 
Panicle length ranged between 26.80 cm (T₅) and 32.60 cm (T₇), but no significant variation was noted (P = 0.16). The character was highly heritable (0.91) and had moderate genetic advance (13.01%), suggesting the dominance of additive gene action with potential minor environmental regulation. T₇ recorded the maximum panicle length and should be assessed for its yield potential in further studies Muduli and Misra, (2008).
 
Number of leaves
 
Leaf number was significantly different among treatments (P = 0.04), with Treatment 7 (6.47) and Treatment 6 (6.07) being superior to the rest. The LSD test revealed significant differences at 0.76 units. Genetically, this characteristic revealed high heritability (0.91) and genetic advance (20.60%), reflecting the dominant role of additive genes. Treatments 6 and 7 can therefore be given priority in selection.
 
Internode length
 
Internode length was greatly affected by treatment (P = 3.09) and the greatest value was observed in Treatment 6 (13.80 cm) that differed significantly from others (LSD = 0.88). The characteristic had high PCV (12.30%) and GCV (12.09%), heritability (0.96) and genetic advance (24.48%), indicating it as the most suitable character for selection and a target for enhancing plant architecture.
 
Flag leaf traits (length and width)
 
Flag leaf length also revealed highly significant treatment differences (P = 0.01). Treatments 2 to 8 were significantly greater than Treatment 1, with the maximum length being for Treatment 2 (27.01 cm). The trait possessed extremely high heritability (0.95) and GA of 17.16%, which favors selection for photosynthetic potential (Dhanalakshmi et al., 2019).
       
Flag leaf width, however, wasn’t significantly different across treatments (P = 0.39), though T₅ and T₈ had the highest values (1.37 cm). Genetic parameters also continue to give support to the utility of selection for the trait due to high heritability (0.92) and GA (15.20%).
 
Days to maturity
 
Notwithstanding a variation between 72.93 and 81.13 days, the variation in days to maturity between treatments was statistically not significant (P = 0.29). Genetically, this character had moderate heritability (0.82) and lower GA (9.63%), reflecting lesser gain from selection and greater environmental influence (Muduli and Misra, 2007).
 
1000-Grain weight
 
Treatment differed in 1000-grain weight from 2.51 g (T₁, T₅) to 3.00 g (T₆), but not statistically significant (P = 0.23). The trait was, however, highly heritable (0.93) and having a genetic advance of 20.03%, making it an important selection trait to enhance grain yield Bhave et al. (2016).

The study revealed that several traits exhibited high genetic variability and heritability, implying good potential for improvement through selection. Traits such as internode length, number of tillers, plant height and flag leaf traits were particularly responsive to mutagenic treatments and showed high genetic advance, indicating the effectiveness of the induced variation.
               
Among treatments, Treatment 6 stood out with the tallest plants, highest internode length and highest 1000-grain weight. Treatment 7 excelled in panicle length and number of leaves, while Treatment 2 showed superior flag leaf length. These findings suggest that specific treatments have enhanced desirable traits and should be considered for further advancement in breeding programs.

The present study was supported by the faculty of department of Genetics and Plant Breeding, Agronomy and physiology from SRM College of Agricultural Sciences, SRM Institute of Science and Technology.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.