Development of an Efficient and Reproducible Embryonic Axis-targeted Tissue Culture-based Transformation Protocol for Pigeonpea (Cajanus cajan)

Abstract

Background: Tissue culture-based transformation approach poses significant challenges for genetic modification of recalcitrant pigeonpea due to poor regeneration efficiency. In this study, we present a comprehensive high-frequency regeneration and transformation protocol using embryonic axesas explants. Methods: Optimization of transformation parameters and culture conditions for different stages of adventitious shoot regeneration, including shoot induction, elongation and rooting, significantly enhanced transformation efficiency. Agrobacterium-mediated transformation was carried out using embryonic axes explants with pCAMBIA 2301 vector. Culturing of explants on MS basal media supplemented with BAP 2.5 in Preculture, BAP 2.5 in Selection Medium I, IAA 0.5, GA 0.5 and Zeatin 1 in Selection Medium II resulted in formation of numerous multiple shoots from primordia-forming calli at axillary bud regions. Result: Shooting efficiency was 82% and 77%, for cultivars, Pusa 992 and Asha respectively. Rooting efficiency was nearly 100% for both when cultured on half-strength MScontaining 15 g/l sucrose and 0.5 mg/l IAA. Regenerated plants exhibited a high level ofsurvival of 90% upon transplantation into pots. Successful gene integration and expression were confirmed through GUS histochemical and molecular analyses, with approximately 57% and 65% transformation efficiencies for cultivars Pusa 992 and Asha, respectively. This study presents a robust and reproducible tissue culture-based transformation system for pigeonpea, applicable across diverse genotypes, offering new possibilities for genetic improvement in this crucial legume crop.