In vitro Regeneration and Genetic Transformation for Enhanced Stress Resistance in Chickpea Plants

Abstract

Abiotic stresses, including drought, salinity, and heat, and biotic stresses such as Fusarium wilt substantially reduce the productivity of chickpea (Cicer arietinum L.), an important grain legume for human nutrition. Efficient regeneration and genetic transformation systems are essential for accelerating crop improvement and generating climate-resilient cultivars. In this study, an optimised in vitro regeneration system combined with Agrobacterium-mediated transformation was developed to transfer the stress-responsive AtDREB1A gene into chickpea. Cotyledonary nodes and cuttings produced 7.2 ± 0.5 and 7.0 ± 0.3 shoots per plant, respectively. Shoots cultured on MS medium supplemented with 2.0 mg L⁻¹ 6-benzylaminopurine (BAP), 0.5 mg L⁻¹ kinetin, and 0.2 mg L⁻¹ α-naphthaleneacetic acid (NAA) showed the highest regeneration frequency (94.7 ± 1.8%). The highest rooting frequency (91.6 ± 1.7%) was obtained on half-strength MS medium supplemented with 1.0 mg L⁻¹ indole-3-acetic acid (IAA) and 0.2 mg L⁻¹ NAA. A transformation efficiency of 10.25 ± 0.8% was obtained using A. tumefaciens strain EHA105 carrying the construct pCAMBIA1301::AtDREB1A. Stable transgene integration and expression were confirmed by PCR, Southern blot hybridisation, and quantitative real-time PCR, with the highest transgene accumulation detected in transgenic line T3 (8.11 ± 0.48-fold). Under drought, salinity, and Fusarium wilt stress, transgenic plants performed significantly better than wild-type plants in terms of relative water content, drought survival, salinity survival, membrane stability index, and disease incidence (p ≤ 0.05). The optimised regeneration and transformation platform offers a robust and reproducible approach for chickpea genetic improvement and supports molecular breeding, functional genomics, CRISPR/Cas genome engineering, and the development of climate-smart chickpea cultivars for sustainable crop production.