Development of transgenic resistance to virus diseases in Pisum sativum

This research examined utilizing electroporation for pollen-mediated transformation of pea, circumventing selectable markers, cultivar limitations, tissue culture manipulations, and shortening the time required generating transgenic peas. A two-dye electroporation optimization protocol provided the basis for the rapid generation of 99 independently generated transgenic pea lines from both germinated and non-germinated pollen. The efficiency of transformation ranged from a high of 12.4% for non-germinated pollen to a low of 5.9% for germinated pollen and 6% for germinated co-transformed pollen, depending on environmental conditions. The protocols developed and the results obtained using this transformation method are presented.; Data from the multiple year field studies of 27 independently transformed lines of peas. Eleven lines tested were transformed with Pea enation coat protein (PEMV-CP) and sixteen lines with a dsRNase gene, pac1 and mutants of this gene, derived from the yeast Schizosaccharomyces pombe. The data suggests that these transgenic peas possess a commercially useful level of virus resistance. The PEMV-CP pea lines possess a high level of resistance to PEMV in the field and the pac1 and the E251K mutation possess high levels of resistance to both PEMV and the Pea streak virus (PeSV). During these field tests there was no detection of any deleterious agronomic side effects by the presence transgene(s). The result of the field studies both virus-resistance and agronomic impacts are presented.; Post-transcriptional gene silencing is thought to be a specific virus-resistance mechanism in plants. Examinations of virus-resistance using both biological and molecular assays of the PEMV-CP construct and the pac1 and E251 K constructs suggest that the mechanism of resistance conferred by all of these genes is post-transcriptional gene silencing. In this research the evidence suggests that virus-resistance in PEMV-CP transgenic peas operates on a co-suppression level the virus-resistance in pac1 and E251K transgenic peas may operate by supplementing endogenous genes in the overall post-transcriptional gene silencing system. The protocols for assaying the mechanism and the results of these tests are presented and discussed.