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Characterization of M1 mutant phenotypes induced by EMS mutagenesis in pepper (Capsicum annum) and screening M2 families for tolerance to salinity
Thesis Abstract:
Globally, pepper (Capsicum annuum L.) is one of the most important vegetables for its uses as a spice, culinary, as well as nutritional value. Climate change and altered salinity levels can significantly impact plant growth and development as salinity is a major issue that adversely influences crop productivity. Improving plant tolerance to salt stress would be critical to allow pepper cultivation in salinized areas. However, breeding for improved salinity tolerance in pepper is limited. Mutation breeding by artificial genetic mutagenesis is an important method for improving crops and creating new genetic resources, especially when diversity for a trait of interest is rare. Ethyl methanesulfonate (EMS) is a chemical agent widely used to induce mutations at particular loci of economically important traits. Therefore, the objective of the study was to develop an EMS mutation protocol for peppers, construct an EMS mutant population (M1 and M2 generation), identify mutants in M1 generation, and screen the M2 families for tolerance to salinity stress. Our hypothesis was the mutants might manifest tolerance to salinity compared to the wild type as a result of mutation. Ornamental pepper line "9852-123" obtained from the World Vegetable Center was used to develop the EMS mutant population of M1 and harvest the M2 seeds after self-pollination. The M1 populations were obtained from 0.23, 0.46, 0.69, and 0.93% of EMS treated for 12 and 24 h. Morphological changes associated with mutations in M1 plants were measured. Germination percentage, seedling survival, abnormal phenotype, as well as the incidence of chimera and leaf pigmentation were identified. Plant height, days to flowering, and seed weight were also measured. M1 mutant plants were self-pollinated and M2 seeds were produced. The M2 populations from 0.69 and 0.93% EMS were selected to use in the salinity tolerance screening because high concentration was likely to cause high mutation in the genome. Pilot experiment was conducted to identify the optimal NaCl concentration to cause salinity stress in plants. Control and two salt conditions were applied: 0 mM (control), 100 mM (condition 1), and 150 mM (condition 2) of NaCl. Salt stress affected root and shoot growth, despite 150 mM NaCl (condition 2) as comparable to the control was chosen as the plants had the most severe symptoms (wilt and wrinkle) at 3 days after treatment. A total of 24 M2 families were used in this experiment consisting of 371 and 388 individuals for batch 1 and batch 2 experiment, respectively. Interestingly, the mutants were able to survive in 150 mM NaCl treatment. Salt stress was then applied to 300 mM NaCl. We found 13 M2 plants survived the 300 mM NaCl screening from batch 1 and six M2 plants from batch 2 experiment. The potential mutant lines with salinity tolerance were kept to reach the productive stage and eventually harvest the M3 generation. Further investigation of the M2 plants with tolerance to salinity is needed to better characterize the tolerance and identify associated genes in the future. This experiment contributes to the pepper research community throughout the world to withstand the global issue of saline soil.
