Development of Transgenic Cowpea Overexpressing Btcry 1 Ac and Btcry 1 Ab

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Cowpea (Vigna unguiculata L. Walp) is an important grain and fodder legume widely cultivated in Africa, India, Middle East and South America. Insect pest infestation, prevalently perennial damage of cowpea pods by pod borers, Maruca vitrata and Heliothis armigera is most severe. Breeders lack resistance background for incorporation to cultivated varieties to control the diseases. It has been demonstrated that the introduction and expresssion of crystalline toxin genes (cry) derived from Bacillus thuringiensis (Bt) through transgenic approaches are effective mechanisms for protecting crops against insect infestations. In cowpea, absence of an efficient system for shoot multiplication and plant regeneration amenable to Agrobacterium-mediated transformation, mechanisms to increase T-DNA transfer to regenerating cells and strategy for efficient selection of transformed shoots have been identified as major bottlenecks for implementing transgenic approaches for genetic improvement. This investigation was carried out to find out the role of seedling preconditioning with cytokinin on shoot proliferation efficiency of cotyledonary node explants. A significantly higher shoot proliferation (7.1 shoots per explants), and mean shoot length (2.6 cm) were obtained with cotyledonary node explants derived from seedlings preconditioned in 10 DM TDZ for 4 days. The preconditioned explants were employed for efficient regeneration of transgenic cowpea plants. Btcry1Ac and cry1Ab overexpression constructs were prepared and mobilized to Agrobacterium tumefaciens vir helper strain EHA105. Method for efficient recovery of transgenic cowpea plants using an improved kanamycin selection regime was established that showed 46.1% increase in efficiency as compared to the existing transformation methods. Agrobacterium-cocultivated cotyledonary node explants were selected on medium containing 150 mg/l kanamycin for 20 days and surviving explants were shifted to kanamycin-free media supplemented with reduced dosage of BAP (2.5 DM) that resulted in profuse proliferation of kanamycinresistant shoots with 63.6% increase in shoot length within 15 days of culture. Transgenic cowpea plants with introduced cry1Ac were recovered that showed presence, integration, expression and inheritance of transgene. The role of sonication and vacuum infiltration on increase in T-DNA transfer efficiency to cotyledonary node explants were investigated. A combination of 20 s sonication followed by 5 min vacuum infiltration treatments were found to significantly...
Supervisor: Lingaraj Sahoo