Development of a High-Efficiency Hairy Root Transformation System for Diverse Cowpea (Vigna unguiculata) Genotypes

Cowpea (Vigna unguiculata (L.) Walp.) is a globally cultivated leguminous crop, but an efficient and stable genetic transformation system for cowpea is lacking. Thus, in this study, using different cowpea accessions, the main factors (genotype, explant, Agrobacterium strain for infection, and vector) affecting genetic transformation efficiency were systematically screened. Among the 43 cowpea accessions, two accessions (JD-0212 and A132) whose in vitro regeneration frequencies were high (propagation coefficient > 0.8 and adventitious bud induction index > 0.7) were identified. A system with a high infection rate for the two cowpea accessions was subsequently constructed, including cotyledonary nodes with cotyledons as the optimal explants, the Agrobacterium rhizogenes strain K599 and the rbcs-RUBY vector. Next, the system was optimized for its transformation conditions, such as infection duration, vacuum infiltration parameters and cocultivation time. The maximum transformation efficiency of genotype JD-0212 reached 82.79% under the optimal transformation conditions: 60 min of infection combined with 30 s of vacuum infiltration (-0.08 MPa), followed by four days of cocultivation. Furthermore, the transformation efficiency was validated in 86 cowpea accessions using two distinct vectors (rbcs-RUBY and bs-EGFP), indicating an average transformation efficiency of 42.09% (ranging between 4.04% and 82.79%). An efficient hairy root genetic transformation system for cowpea was established in this study.