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

SUMMARY 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. Significance Statement The tissue culture and transformation technology developed herein represents a significant advance in Agrobacterium‐mediated transformation and CRISPR/Cas‐mediated genome editing of an important orphan crop, cowpea (Vigna unguiculata (L.) Walp.). The principles established in this study have the potential to improve the transformation and editing efficiencies not only for cowpea, but also for other legume species, such as soybean (Glycine max) and common bean (Phaseolus vulgaris).