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Optimization of selection conditions and agrobacterium mediated transformation of chickpea (cicer arietinum L. cv. Gökçe)

Öz, M. Tufan
The objective of this study was to optimize an efficient selection system and Agrobacterium mediated transformation of chickpea (Cicer arietinum L.). Cotyledonary node explants of Turkish chickpea cultivar Gökçe were used to determine the effects of selective agents, two antibiotics (Kanamycin, Hygromycin) and two herbicides (PPT, Glyphosate) as well as four antibiotics (Augmentin, Carbenicillin, Cefotaxime, Timentin) for eliminating Agrobacterium on multiple shoot and root induction. 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/L, Hygromycin at 20 mg/L, PPT at 3 mg/L and Glyphosate at 5 mg/L were found to be appropriate to select chickpea transformants. Lowest concentrations of all selective agents (50 mg/L Kanamycin, 10 mg/L Hygromycin, 3 mg/L PPT, 1 mg/L Glyphosate) totally inhibited rooting of the regenerated shoots. Among the Agrobacterium-eliminating antibiotics, Cefotaxime and Augmentin each up to 600 mg/L had no adverse effect on shoot induction, whereas Timentin (300 mg/L) significantly increased and Carbenicillin (300 mg/L) significantly decreased shoot induction after four weeks of culture. Augmentin was determined to have no effect on rooting capacities of chickpea shoots. However Cefotaxime at all concentrations significantly decreased root induction. On the other hand only high concentrations of Carbenicillin (300 mg/L) and Timentin (200 mg/L) significantly decreased rooting. Sulbactam in combination with Carbenicillin and Cefotaxime displayed effective inhibition of bacterial growth. Furthermore, Agrobacterium mediated transformation procedure for cotyledonary node explants of Gökçe, was also optimized by monitoring transient uidA expression on 4th, 9th, and 16th days after transformation. Transformation procedure was