Validation of nucellus-based direct somatic embryogenesis mutagenesis and phenotypic assessment of M1 mutants in kinnow mandarin (Citrus nobilis × Citrus deliciosa)

In ovulo nucellus based direct somatic embryogenesis (DSE) is an efficient tool to assess mutagenic efficiency in perennial crops. A study was carried out in 2022–23 and 2023–24 at ICAR-Indian Agricultural Research Institute, New Delhi to compare the influence of 0.1% ethyl methane sulphonate (EMS) for 5 h and gamma irradiation (80 Gy) on embryogenesis, plantlet regeneration, and growth traits in kinnow mandarin (Citrus nobilis × Citrus deliciosa). Both treatments significantly reduced ovule survival compared with control (86.67%). The survival declined to 48.00% under EMS (0.1% for 5 h) and 35.78% following gamma irradiation (80 Gy for 10.9 min). The frequency of direct somatic embryogenesis declined to 49.07% under EMS and 43.85% under gamma irradiation. In contrast, post embryogenic parameters, including germination and bipolar conversion efficiency, were more severely affected, decreasing to 39.06–41.89% under EMS and 32.87–30.14%, under gamma treatment. Regenerants displayed short stature, reduced internodal length, and smaller leaves, though leaf number remained unaffected, suggesting that stress mainly altered expansion and elongation rather than leaf initiation. Correlation analysis indicated that biomass under EMS was closely linked to leaf area, while gamma treatment weakened relationships between height related traits and biomass. Cluster analysis revealed moderate variability under EMS but greater divergence among gamma irradiated populations. Overall, EMS generated point mutations with moderate phenotypic effects, whereas gamma rays exerted stronger disruptive impacts on regeneration and variability. These findings demonstrate that in ovulo nucellus-derived DSE is a robust system for evaluating mutagenic efficiency and optimizing treatments for generating targeted variability in perennial fruit crops.