Enhancing the resilience of Bt Cowpea [Vigna unguiculata (l.) Walp] for insect resistance management

Cowpea is an important food security crop in many countries but its production is constrained by the legume pod borer which can cause yield losses of up to 50%. In this thesis, a molecular approach was used to generate genetically modified cowpea plants which were resistant to this insect pest. Several candidate genes were initially identified and the insecticidal activity of their expressed proteins against the pod borer was tested in insect bioassays. The most promising gene was then used to generate GM cowpea plants, some of which were shown to be resistant to pod borer in insect feeding trials

An Improved Transformation System for Cowpea (Vigna unguiculata L. Walp) via Sonication and a Kanamycin-Geneticin Selection Regime

An improved cowpea transformation method utilizing Agrobacterium-mediated gene delivery to explants derived from the cotyledonary nodes of imbibed cowpea seed is described. The explants were regenerated following a sonication procedure and a stringent selection comprising alternating regimes of kanamycin and geneticin. The method was reproducible and led to the recovery of independent fertile transgenic plants in the greenhouse at a level of about one per cent of starting explants. A transgene encoding an insecticidal protein from Bacillus thuringiensis was used to demonstrate the efficacy of the system

Transgenic cowpeas (Vigna unguiculata L. Walp) expressing Bacillus thuringiensis Vip3Ba protein are protected against the Maruca pod borer (Maruca vitrata)

Although cowpea is an important food security crop in Africa, its production is constrained by insect pests such as the legume pod borer (Maruca vitrata) (Lepidoptera). Potential control strategies have focussed on using insecticidal toxins such as the crystal Cry proteins and vegetative insecticidal proteins (Vips) encoded by the cry and vip genes, respectively, of the bacterium Bacillus thuringiensis (Bt). This study sought to identify vip genes encoding toxins active against Maruca Pod Borer (MPB), from Australian Bt isolates. A collection of 224 Bt isolates was screened with gene-specific primers to identify those containing target vip genes namely vip3Aa35, vip3Af1, vip3Ag, vip3Ca2 and vip3Ba1. The coding sequences of the vip3 genes were cloned and over-expressed in Escherichia coli to produce Vip3 protein. The proteins were incorporated into Maruca artificial diets for use in insect bioassays with MPB larvae to screen for toxicity. Of these, Vip3Ba1 protein was found to strongly inhibit larval growth and was selected as the candidate gene for cowpea transformation. A vip3Ba gene reconstructed for plant expression was used in transforming cowpea via Agrobacterium tumefaciens. Transgenic lines expressing Vip3Ba protein were used in insect feeding trials to assess protection against Maruca and were found to be completely protected from this pest. We propose that the vip-cowpea lines could be combined with existing cry-transgenic cowpea to introgress this additional resistance trait and thus avoid or greatly delay the development of resistance in Maruca