Pyramiding of modified cry1Ab and cry1Ac genes of Bacillus thuringiensis in transgenic chickpea (Cicer arietinum L.) for improved resistance to pod borer insect Helicoverpa armigera

The modified cry1Ab and cry1Ac insecticidal genes of Bacillus thuringiensis (Bt) under the control of two different constitutive promoters have been introduced into chickpea (Cicer arietinum L.) by Agrobacterium-mediated transformation of pre-conditioned cotyledonary nodes. 118 stable transformed T-0 plants as independent transformation events were obtained expressing individual cry1Ab, cry1Ac or both pyramided genes for their co-expression driven by either cauliflower mosaic virus 35S promoter with duplicated enhancer (CaMV35S) or synthetic constitutive promoter (Pcec) and their combinations. Integration and inheritance of transgenes in T-0 and T-1 population of transgenic chickpea plants were determined by PCR, RT-PCR and Southern hybridization. Results of Southern hybridization showed single copy integration of cry1Ab or cry1Ac genes in most of the transgenic plants developed with either single or pyramided genes and reflected Mendelian inheritance of transgenes in T-1 progeny. Real time PCR of pyramided transgenic plants clearly showed differential expression of transcripts for both the genes driven by CaMV35S and Pcec promoters. Quantitative assessment of Bt Cry toxins by ELISA of T-0 transgenic chickpea plants showed expression of toxin ranging from 5 to 40 ng mg(-1) of total soluble protein (TSP) in leaves of transgenic plants. Insect bioassay performed with transgenic plants showed relatively higher toxicity for plants expressing Cry1Ac protein as compared to Cry1Ab to Helicoverpa armigera. Pyramided transgenic plants with moderate expression levels (15-20 ng mg(-1) of TSP) showed high-level of resistance and protection against pod borer larvae of H. armigera as compared to high level expression of a single toxin. These results have shown the significance of pyramiding and co-expression of two Cry toxins for efficient protection against lepidopteran pests of chickpea.