Shoot apex priming, antioxidants, and mechanical injuries modulating regeneration and transformation potential of grain legume crop ricebean (Vigna umbellata (Thunb.) Ohwi & Ohashi)

Vigna umbellata is an unexplored and underutilized grain legume of high nutritional values. This crop can be improved for several agronomic attributes via genetic engineering. The presented study emphasizes the first-time standardization of parameters affecting regeneration and transformation using shoot apices. Various parameters comprise concentration of growth regulators, age of explants, explant's priming with 6-benzylaminopurine (BAP), genotype effect, modulation of explant's compatibility towards Agrobacterium infection via mechanical wounding, and assessment of the type and concentration of antioxidants (silver nitrate and thiourea). Out of various variables, the 2-d-old apices primed with 15 mu M BAP formed an average of 10.4 shoots per explant with 100% regeneration frequency on shoot regeneration medium [SRM2: MSB5V (MS - Murashige and Skoog salts and B-5 - Gamborg's vitamins), 2.5 mu M BAP and 5.0 mg L-1 silver nitrate]. Scanning electron microscopy revealed morphology of explant's meristem for suitability to Agrobacterium infection. Wounded explants pierced with copper metal particles did not affect the regeneration compared to longitudinal incisions or pierced with a needle. A transformation study was carried out using Agrobacterium tumefaciens (strain EHA105 harboring pCAMBIA2301 vector). The stable expression of uidA gene in tissues of transgenics (T-0) and seedlings (T-1) evidenced the success of the transformation process. Transgenic plants (T-0) were characterized by polymerase chain reaction and Southern hybridization and progeny plants via reverse transcriptase-polymerase chain reaction. The highest transformation efficiency (13%) was achieved via copper metal particles comparative to other methods of wounding in a short span of 6 to 7 wk from initiation of cultures to transplantation.

Automated summary

This study standardized the parameters affecting regeneration and transformation using shoot apices in Vigna umbellata. The results showed that by priming the explants with 6-benzylaminopurine (BAP) and optimizing the concentration of growth regulators and antioxidants, efficient shoot formation and successful transformation could be achieved.