Developing a rapid and highly efficient cowpea regeneration and transformation system using embryonic axis explants

Cowpea is one of the most important legume crops planted worldwide, especially in Sub-Saharan Africa and Asia. Despite decades of effort, genetic engineering of cowpea is still challenging due to inefficient in vitro shoot regeneration, Agrobacterium-mediated T-DNA delivery and transgenic selection. Here, we report a rapid and highly efficient cowpea transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem by cutting through the middle of the epicotyl stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. Furthermore, the application of a ternary transformation vector system using an optimized pVIR accessory plasmid provided high levels of Agrobacterium-mediated gene delivery. The utilization of spectinomycin as the selection agent enabled more efficient transgenic selection and plant recovery. Transgenic cowpea shoots developed exclusively from the cotyledonary nodes at high frequencies of 4.5 to 37% across a wide range of cowpea genotypes. We believe that the transformation principles established in this study could also be applied to other legumes to increase transformation efficiencies.