Introduction of cry1Ab gene into cotton (Gossypium hirsutum) enhances resistance against Lepidopteran pest (Helicoverpa armigera)

G. A. Khan, A. Bakhsh, S. Riazuddin, T. Husnain

Abstract


The significant breakthrough in plant biotechnology is the development of techniques to transform genes from unrelated sources into commercially important crop plants to develop resistance against insect pests. A local cotton cultivar MNH-93 was transformed through Agrobacterium tumefaciens strain C58C1 assisted by bombardment with tungsten particles. The Agrobacterium strain contained the recombinant binary vector pKMAB harboring cry1Ab under 35S promoter. Neomycin phosphotransferase (nptII) gene was used as a selectable marker at a concentration of 50 mg L–1. The transformation efficiency remained 0.26%. The primary transformants were analyzed for transgene integration and expression through PCR and Southern Blotting and Western dot blot. The gene copy number was determined by Southern analysis in order to find out the cry1Ab integration sites. The Bt protein being produced in the transgenic plants was quantified using ImageQuant software, which ranged from 0.00 to 1.35% of the total protein. The positive plant seeds obtained from T0 progeny were further raised under greenhouse and field conditions to evaluate their field performance. Leaf biotoxicity assays were performed to determine the efficacy of introduced gene. The results showed that transgenic lines in T1 progeny have appreciable level of resistance (40-60%) against lepidopteran pests in both green house and field conditions.


Keywords


gene copy number; leaf toxicity bioassay; MS medium; transformation

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References


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DOI: 10.5424/sjar/20110901-136-10