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Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium-mediated transfer DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium-mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and transgenic plant generation without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at frequencies of 4% to 37% across a wide range of cowpea genotypes. CRISPR/Cas-mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies.

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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.