Comparison of different in vitro regeneration and genetic transformation strategies for chile pepper (Capsicum annuum)

The study involved a comparison of different regeneration systems for selected chile pepper (Capsicum annuum) cultivars and attempts at genetic transformation using a variety of DNA delivery methods.; Regeneration experiments using cotyledon explants isolated from cultivars California Wonder, Jalapeno M, Jalapeno Hot, New Mexico 6-4, Sunbright Sweet, Sweet Banana, Serrano, Joe E. Parker, Crispy Hybrid Sweet and Jamaican Red showed that cvs. New Mexico 6-4 and Joe E. Parker out performed the others.; Cotyledons explants of cv. New Mexico 6-4 were co-cultivated with Agrobacterium tumefaciens harboring the co-integrate vector pGG2 containing the reporter gene glucuronidase (Gus) driven by Cauliflower mosaic virus 35S promoter and neomycin phosphotransferase (Npt II) driven by Nos promoter. Transient Gus activity was present in a large number of explants that were assayed, but few calluses and no shoots were positive for stable/long term Gus expression.; Factorial experiment showed that cv. New Mexico 6-4 to be better than cv. Golden Tower and cotyledon explants were better than hypocotyls for regeneration.; A variety of plasmid constructs that had the Gus gene driven by different promoters, were coated on gold microprojectiles and bombarded into suspension culture derived cells using PDS-1000/He. Transient assays showed that carrot late embryogenic promoter and 35S promoter were the best while wheat embryogenic promoter was the worst among the 5 that were tested.; Microprojectile bombardment of cotyledons and hypocotyls showed excellent transient Gus activity. More than 250 hypocotyls were positioned close together in a plate and bombarded. One of the experiments showed 3 hypocotyls with newly initiated roots to be positive for Gus activity. Bombardment of mature and immature embryos were not successful, as none of the embryos expressed long term Gus activity.; Genetic transformation of cv. New Mexico 6-4 mediated by A. rhizogenes yielded better results. Strain K 599 harboring various plasmid binary vectors were inoculated at the hypocotyl region of seedling explants. Hairy roots were initiated from 24% of the seedlings inoculated with pZP 200 to a maximum of 65% by p35S GusInt. Of these, 2-17% of the inoculated seedlings had roots testing positive for Gus expression. Hairy roots did not have to grow into the selection medium in order to be Gus positive. The pattern of expression of the introduced gene varied greatly. Seedlings with transformed roots had a different morphology.; Transformed hairy roots were excised and cultured on liquid medium under selection for several cycles. Hairy roots were found to express Gus even after 18 months. ELISA's performed on hairy root samples showed that samples from 3 independent transformation events were positive for Npt II protein expression. Roots are currently being cultured on various media to evaluate shoot or plantlet regeneration potential.