In Vitro Plant Regeneration and Recovery of Cowpea (Vigna unguiculata) Transformants Via Agrobacterium-mediated Transformation

In vitro regeneration of plants from mature seed-derived shoot apices and embryonal axes and their competence to Agrobacterium-mediated genetic transformation have been assessed in two cultivars of Vigna unguiculata. The explants produced multiple shoots on MS medium supplemented with 5 �M 6-benzylaminopurine (BAP) within 20 days of culture. BAP performed better when compared with other cytokinins. The efficacy of BAP on shoot multiplication was not improved with addition of inole-3-acetic acid (IAA). The various parameters, i.e. Agro strain, vector and method of concultivation have been optimized using transient gus expression. Among the three Agrobacterium strains EHA105 (pBin9Gusint), C58C1 (pIG121Hm) and LBA4404 (pTOK233) tested, EHA105 (pBin9Gusint) strain was found to be most virulent. Prewounding of the apical meristems with a fine needle was found to be essential for Agrobacterium infection of shoot apices. Application of bacterial suspension at the tip of the wounded apices was more effective as compare to the explants cocultivated in bacterial suspension. A total of 510 shoot apices were inoculated from which 14 putatively transformed shoots were recovered on MS + BAP (5�M) + kanamycin (100 mg/L). These shoots showed GUS staining in stem and/or occasionally in leaves. Transformed shoots were rooted on MS medium and grown to maturity for further analysis of gene expression from T0 and T1 progenies.