Genetic Engineering of COWPEA For Storage Pest Resistance

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Cowpea (Vigna unguiculata L. Walp) is an important grain legume widely consumed by 120 million people. Cowpea seeds and fresh peas are a rich source of protein, certain minerals and vitamins. However, cowpea seeds are highly susceptible to storage pests, bruchid species causing massive damage to the stored seeds and seriously limiting its yield potential. Success through conventional breeding methods for developing resistant varieties is limited due to the natural genetic barriers in cowpea germplasm. Consequently, the transfer of insect pest resistance genes by genetic engineering could potentially aid plant breeders in overcoming these constraints. A critical step in the development of robust Agrobacterium tumefaciens-mediated transformation system in recalcitrant grain legume is the establishment of optimal conditions for efficient T-DNA delivery into target tissue and recovery of transgenic plants. We report a dramatic increase in efficiency of T-DNA delivery by constitutive expression of additional vir genes in resident pSB1 vector in Agrobacterium strain LBA4404. A geneticin based selection system permitted rapid and efficient identification of transgenic shoots without interfering with their regeneration, and eliminated the bulk of escapes. Supplementation of 0.5 µM kinetin to medium containing 5.0 µM benzyl aminopurine after 1week of culture followed by 3 weeks of culture were found critical for optimal multiplication and elongation of transformed shoots from cotyledonary node explants. Combining these three developments, we recovered fertile transgenic plants at a frequency of 1.64%, significantly higher than previous reports. The presence, integration, expression and inheritance of transgenes were confirmed by molecular analysis. We employed the developed Agrobacterium-mediated cowpea transformation method for introduction of the bean (Phaseolus vulgaris) α-amylase inhibitor-1 (αAI-1) gene into a commercially important Indian cowpea cultivar, Pusa Komal and generated fertile transgenic plants at a frequency of 1.67%. The presence, integration, expression and inheritance of αAI-1gene was confirmed by molecular analysis. Expression of αAI-1 gene under bean phytohemagglutinin promoter resulted in accumulation of αAI-1 in transgenic seeds. The transgenic protein was active as an inhibitor of porcine α-amylase in vitro. Transgenic cowpeas expressing αAI-1 strongly inhibited the development of C. maculatus and C. chinensis in insect bioassays.
Supervisor: Lingaraj Sahoo