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

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Agricultural Science

Background:

• Cowpea (Vigna unguiculata) is a vital global legume crop.
• Conventional Agrobacterium-mediated transformation methods for cowpea are inefficient, facing challenges in gene delivery, selection, and regeneration.
• Improving cowpea transformation is essential for crop breeding and genetic enhancement.

Purpose of the Study:

• To establish a highly efficient and reliable transformation system for cowpea.
• To overcome existing limitations in Agrobacterium-mediated gene transfer, selection, and regeneration.
• To demonstrate the applicability of CRISPR/Cas-mediated gene editing in the developed system.

Main Methods:

• Utilized embryonic axis explants from imbibed mature cowpea seeds.
• Removed the shoot apical meristem to induce direct shoot organogenesis from cotyledonary nodes.
• Employed a ternary transformation vector system for efficient Agrobacterium-mediated gene delivery.
• Used spcN as a selectable marker for robust transgenic selection and recovery.

Main Results:

• Achieved direct multiple shoot organogenesis from cotyledonary node tissue.
• Demonstrated efficient Agrobacterium-mediated gene delivery and robust transgenic selection using spcN.
• Generated transgenic cowpea plantlets from cotyledonary nodes with frequencies ranging from 4% to 37% across diverse genotypes.
• Successfully performed CRISPR/Cas-mediated gene editing in the transgenic plants.
• Ensured transgenic plant generation without escapes or chimera formation.

Conclusions:

• The developed transformation system significantly enhances cowpea transformation efficiency.
• The method provides a reliable platform for generating transgenic cowpea plants and facilitating gene editing.
• The principles established may be applicable to improving transformation efficiencies in other legume species.