Bioassay and molecular tests of transgenic chickpea (Cicer arietinum L.) resistant to Helicoverpa armigera Hub.

Pod borer is one of the main causes for yield loss of chickpea. Therefore, breeding of chickpea for resistance to this pest is important. The use of Cry toxin from Bacillus thuringiensis is an effective strategy for producing of transgenic resistant chickpea to this pest. These toxins are able to become active in the midgut of larvae and disrupt the insect's digestive system. We studied the stability and expression of cry1Ac gene obtained as T2 transgenic chickpea with cry1Ac gene and nptII gene with binary T-DNA in T3 and T4 generations of transgenic plants and observed the transgenic lines with cry1Ac gene and no nptII gene suggesting that separation between cry1Ac and nptII genes was occurred by recombination between two T-DNAs. In T3, PCR results showed that 6 of 25 putative transgenic plants had cry1Ac gene but all of them showed the nptII gene. From six samples with positive PCR in cry1Ac gene, five of them had positive results in RT-PCR reaction, confirming the expression of cry1Ac gene in transgenic lines. PCR results in T4 plants showed that 73 of 94 plants had cry1Ac gene and 81 of 94 samples included the nptII gene. In 10 samples cry1Ac gene separated from nptII gene. According ELISA results, in all samples tested, Cry1Ac protein was expressed in different concentrations. Bioassay tests showed that all pod borer larvae fed by leaves of transgenic plants, were dead, but all survived when the larvae were fed with leaves of non-transgenic plants. So, expected phenotype was observed successfully.