Developing a rapid and highly efficient cowpea regeneration,transformation and genome editing system using embryonic axis explants |
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Authors: | Ping Che Shujun Chang Marissa K. Simon Zhifen Zhang Ahmed Shaharyar Jesse Ourada Dennis O’Neill Mijael Torres-Mendoza Yinping Guo Kathleen M. Marasigan Jean-Philippe Vielle-Calzada Peggy Ozias-Akins Marc C. Albertsen Todd J. Jones |
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Affiliation: | 1. Corteva Agriscience, Johnston, Iowa, 50131 USA;2. Department of Horticulture and Institute of Plant Breeding, Genetics & Genomics, University of Georgia Tifton Campus, Tifton, GA, 31973 USA;3. Group of Reproductive Development and Apomixis, UGA Laboratorio Nacional de Genómica para la Biodiversidad, CINVESTAV Irapuato, Guanajuato, 36821 México |
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Abstract: | 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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Keywords: | cowpea transformation embryonic axis cotyledonary node (cot-node) shoot organogenesis spectinomycin Agrobacterium CRISPR/Cas |
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