Optimized Selection and Regeneration Conditions for Agrobacterium-mediated Transformation of Chickpea Cotyledonary Nodes

An efficient selection system and Agrobacterium mediated transformation for chickpea (Cicer arietinum L.) cotyledonary nodes were investigated. Effect of selective agents and antibiotics on multiple shoot and root induction of cotyledonary nodes and effects of mechanical injury and vacuum infiltration on transformation efficiency were evaluated. Selective agents and antibiotics were applied to explants at different concentrations for one month and numbers of regenerated shoots and roots were recorded. Kanamycin at 100 mg dm, hygromycin at 20 mg dm, phosphinotricin at 3 mg dm and glyphosate at 5 mg dm were found to be appropriate to select chickpea transformants. Lowest concentrations of all selective agents (50 mg dm kanamycin, 10 mg dm hygromycin, 3 mg dm phosphinotricin, 1 mg dm glyphosate) totally inhibited rooting of the regenerated shoots. Among the Agrobacterium-eliminating-antibiotics, timentin significantly increased and carbenicillin significantly decreased shoot induction after 4 weeks of culture. On the other hand, cefotaxime at all concentrations significantly decreased root induction. Sulbactam, a βlactamase inhibitor, in combination with carbenicillin and cefotaxime displayed effective inhibition of Agrobacterium tumefaciens growth. Furthermore, selection procedure formulated in this study was used in chickpea transformation studies. Histochemical GUS staining was performed 4 and 16 days after transformation to analyze putative transgenics. On the other hand, efforts were exerted on transformation to increase the efficiency. Mechanical injury prior to transformation and vacuum infiltration at 200 mm Hg for 40 min., during bacterial inoculation might be employed to increase the efficiency of chickpea transformation.