Agrobacterium-mediated Transformation of Cotton (Gossypium hirsutum) Using a Synthetic cry1Ab Gene for Enhanced Resistance Against Heliothis armigera

Abstract

Cotton (Gossypium hirsutum L.) is an important fiber crop in Iran, cultivated on 150000-200000 ha of land. In Iran the estimated loss due to the insect pest is more than 30%. Traditionally, pests are controlled by 10-12 times spraying per growing season of environmentally harmful chemical insecticides (e.g. endosulfan and/or methosystox). In order to produce transgenic cotton resistance to insects, hypocotyl explants were transformed with Agrobacterium tumefaciens strain LBA4404 harboring the recombinant binary vector pBI121 containing the cry1Ab gene under the control of CaMV 35S promoter. Neomycin phosphotransferase (nptII) gene was used as a selectable marker. Inoculated tissue sections were placed onto co-cultivation medium. Transformed calli were selected on MS medium containing 50 mg/l of kanamycin and 200 mg/l of cefotaxime. Plantlets were subsequently regenerated from putative transgenic calli. Polymerase chain reaction (PCR) and southern blot analysis were used to confirm the integration of cry1Ab and nptII transgenes into the plant genome. Western immunoblot analysis of proteins extracted from leaves of transgenic plants revealed the presence of an immunoreactive band with a molecular weight (MW) of approximately 67kDa in transgenic cotton lines using the anti-Cry1Ab polyclonal anti-serum. Homozygous T2 plants (Line 61) for the cry1Ab gene showed significantly higher levels of insect resistance against Heliothis armigera larvae compared with the control plants. Transgenic plants are currently grown in the greenhouse and will be crossed with Iranian cotton breeding lines.