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.
Highlights
The application of biotechnology tools to agriculture has allowed scientists to transform plants without the need for sexual compatibility between species, establishing the possibility of rapidly producing new crop varieties with traits beneficial to human health and the environment
The cotton crop was sown on the area of 2,820 thousand hectares; the production is estimated at 11.8 million bales (Economic Survey of Pakistan, 2008-09)
Polymerase chain reaction (PCR) analysis of newly transformed plants was performed for the amplification of cry1Ab using specific primers
Summary
The application of biotechnology tools to agriculture has allowed scientists to transform plants without the need for sexual compatibility between species, establishing the possibility of rapidly producing new crop varieties with traits beneficial to human health and the environment. Cotton (Gossypium hirsutum L.) is an important non-food cash crop in Pakistan and a significant source of foreign exchange earning. 13% of the cotton crop is lost due to insects (Gatehouse and Hilder, 1994) depending upon the weather conditions and other environmental factors every year. These insects are being controlled by chemical insecticides, which otherwise have serious environmental and human health threats. During the campaign 2008-09, 16,495 thousand tons of agricultural pesticides were imported in Pakistan with a cost of 68.7 millions US dollars (Economic Survey of Pakistan, 2008-09). Over 500 species of insects have become resistant to one or multiple synthetic chemical insecticides (Schnepf et al, 1998)
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