Creation of borer pests resistance genetically engineering peach (Prunus persica L.) plants by constitutively overexpressing the cry1Ab gene

Abstract

The plasmid pBI121 cry1Ab used to transform peach explants to produce insect resistant plants was constructed by cloning the synthetic cryIAb gene with the intron of the castor bean catalase-1 gene into the pBI121 binary vector, under the control of the CaMV35S promoter. Leaf discs of peach (Prunus persica L.) were co-cultivated for two days with this construct in A. tumefaciens strain LBA 4404. Explants were plated on WPM medium supplemented with 125 mg L−1 kanamycin, 3% sucrose, 2.5 mg L−1 2,4-Dichlorophenoxyacetic acid, and 0.5 mg L−1 6-benzyladenine in darkness for callus formation. The calli were then selected on WPM medium supplemented with 125 mg L−1 kanamycin, 3% sucrose, 3.00 mg L−1 Thidiazuron, 1.0 mg L−1 kinetin, and 0.5 mg L−1 indoleacetic acid in the light for at least five subcultures (with a regeneration efficiency of 91.8%). The integration of the cry1Ab gene into the peach genome was confirmed by polymerase chain reaction (PCR) and northern blot analysis. A transformation efficiency of 28% was obtained when leaves were incubated for 15 min with A. tumefaciens. The cry1Ab gene expression was confirmed using RT-PCR, northern blot hybridization, immune-strip test, and insect bioassays, respectively. For insect bioassay, it was evident that the protein cryIAb expressed in transformed peach plants showed 100% mortality at 1000 ppm against Synanthedon exitiosa larvae after 96 h. The obtained results demonstrated a significant improvement of cryIAb toxin protein against lepidopteron larvae in peach.