ABSTRACTSYSTEM DEVELOPMENT FOR IN VITRO REGENERATION AND GENE DELIVERY INTO COMMON BEAN (PHASEOLUS VULGARIS)ByKingdom Moses KwapataCommon bean is an important staple food source for many people worldwide. Given the social economic and nutritional importance of this crop, the research presented in this dissertation focused on development of a novel system for in vitro regeneration using apical shoot meristem primordia explant and a gene delivery system for common bean. The research investigated methods for reducing recalcitrance of common bean towards in vitro regeneration. The results showed that a ratio of 2.5 mg L-1 benzyladenine (BA) to 0.1 mg L-1 of naphthalene acetic acid (NAA) or indole-3-acetic acid (IAA) promoted robust multiple shoot regeneration. The addition of 30 mg L-1 of silver nitrate reduced the inhibitory effect of phenolic compounds.Standardized conditions for gene delivery into apical shoot meristem primordia were developed using both BiolisticTM bombardment and Agrobacterium tumefaciens with two marker genes, bar and gus. Results showed that transformation efficiency of the bar transgene with particle bombardment method was 8.4% using 7584 kPa helium pressure with a concentration of 1.5 μg of plasmid DNA per bombardment and bombarding the explants twice at a 24 hour interval. Effect of co-cultivation period for different strains of A. tumefaciens (EHA105, LBA4404 and GV3301) and genotypes of common bean were assessed. Transient and stable expression of the gus gene showed `Sedona' to be more amenable to Agrobacterium transformation than `Matterhorn'. A co-cultivation period of 15 days with Agrobacterium strain GV3301was most effective in producing the highest transient expression of 81% and stable expression of 0.68%. The above results show that the BiolisticTM gun delivery system is more efficient than Agrobacterium system for generating stable transgenes into common bean.Testing was conducted for stable integration and expression of two major agronomically valuable transgenes. The first was the barley (Hordeum vulgare) late embryogenesis abundant protein (HVA1) gene, which confers drought tolerance. Significant resilience of transformed plants versus wild type towards drought stress was observed with a corresponding increase in root length for transgenic genotypes `Matterhorn' and `Sedona'. The second gene tested was the wheat (Triticum aestivum) germin gene (gf2.8) that produces an oxalate oxidase that reduce pathogenicity of Sclerotinia sclerotiorum the causal agent of white mold of common bean. Transfer of this gene delayed the onset of lesions caused by S. sclerotiorum for a period of 72 hours in leaves of transgenic `Matterhorn'. In conclusion, the goals and objectives of the research were achieved by demonstrating the applicability of novel protocols that were developed for in vitro regeneration followed by gene delivery of two marker genes and two agronomically important genes into common bean. The novelty of this research is the utilization of apical shoot primordia cells that are actively dividing. The delivery of transgenes into these cells followed by their selection and regeneration resulted into stable transgenic common bean plants.
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