Effect of genotype and explant on Agrobacterium mediated genetic transformation of chickpea (Cicer arietinum L.)

Agrobacterium mediated transformants of chickpea (Cicer arietinum L.) for insect resistant cry1Ac gene were generated using two different explants of embryo axes (EA) and embryo slices (ES) from two genotypes of Pusa 256 and PG 186. The transformation frequencies generated in each case were compared to determine the better explant type and genotype for transformation of chickpea. The explants, were treated with Agrobacterium suspension, cocultivated and subsequently selected on increasing kanamycin in three selection cycles of two-week duration each. After completion of selection cycles, 3.1 and 3.8 percent kanamycin resistant EA and ES explants were recovered respectively, whereas percentage survival was 3.05 and 3.8 for the two genotypes of Pusa 256 and PG 186 respectively. The putative transgenic shoots were regenerated on MS medium with B5 vitamins and rooted on MSR medium (1/2 MS with 1.0 mg/l IBA). Presence of transgene in putative transformed shoots was confirmed using PCR with cry1Ac and nptII gene specific primers. In a random sample of 30 putative transformed shoots, 27 were found to be PCR positive for both cry1Ac and nptII genes. The survival percent obtained was used to estimate the transformation responsiveness of 3.1 and 3.75 percent for EA and ES explants and 3.05 and 3.8 percent for the two genotypes of Pusa 256 and PG 186 giving an overall survival percent of 3.4 percent in the experiments carried out on chickpea transformation.