Mutagenesis-induced bacterial wilt resistance in bell pepper and multi-year validation of resistance by in vivo, ex vivo, serological, and molecular approaches.
PURPOSE Bacterial wilt caused by Ralstonia solanacearum is a major constraint to bell pepper (Capsicum annuum L. var. grossum Sendt.) production worldwide, with no stable resistance available in commercially cultivated varieties. This study explored induced mutagenesis as a strategy to develop durable bacterial wilt resistance in the widely grown but highly susceptible cultivar 'California Wonder.' MATERIALS AND METHODS Genetic variability was induced using gamma irradiation and ethyl methane sulfonate (EMS). Mutant populations were advanced from M1 to M8 generations over 12 years (2012-2023). A total of 1,804 M2 progenies were screened under wilt-sick field conditions to identify putative resistant lines. Resistance was validated through an integrated approach including in vivo field screening, ex vivo seedling assays in Hoagland's nutrient solution, and serological confirmation using DAS-ELISA. Stable mutants were further evaluated across multiple generations and five cropping years for resistance stability, horticultural performance, and fruit quality traits. Molecular validation was performed using the SSR marker CAMS451 to confirm the association between resistance-linked alleles and phenotypic expression. RESULTS Screening of M2 progenies identified 22 putative resistant lines, of which 16 consistently expressed stable resistance across generations and environments. Six mutants DPCBWR-12 (1.5% EMS), DPCBWR-4 (1.75% EMS), DPCBWR-15 (1.75% EMS), DPCBWR-5 (1.75% EMS), DPCBWR-8 (16.0 kR gamma rays), and DPCBWR-3 (2.0% EMS) significantly outperformed the parent cultivar for yield-related traits and nutritional quality parameters. SSR marker analysis using CAMS451 confirmed the genetic association of resistance-linked alleles with observed phenotypic resistance. CONCLUSIONS This study provides the first evidence of successful induction of bacterial wilt resistance in bell pepper through gamma irradiation and EMS mutagenesis. The identified elite mutant lines possess durable resistance, improved yield, and enhanced fruit quality, making them promising candidates for multi-location testing, varietal release, and use as valuable genetic resources in sustainable bell pepper breeding programs.