Abstract
Cotton is the main fiber producing crop globally, with a significant impact on the economy of Pakistan. Bt cotton expressing a Cry1Ac gene is grown over a large area in Pakistan, however, there is a major concern that bollworms may develop resistance. Here we have used a durable resistance strategy against bollworms by developing a double gene construct containing Cry1Ac and Cry2Ab (pGA482-12R) for cotton transformation. Both Cry toxin genes have been cloned in the same T-DNA borders and transferred successfully into cotton via Agrobacterium-mediated transformation. Both genes are expressed in transgenic cotton plants and is likely to help breeders in developing new cotton cultivars by incorporating these genes in cotton lines having no Bt genes or expressing Cry1Ac gene (Mon 531). Positive transgenic cotton was identified by PCR using specific primers for the amplification of both Cry1Ac and Cry2Ab genes. Cry1Ac and Cry2Ab expression was confirmed with an immunostrip test and quantified using ELISA that showed significant spatio-temporal expression of Cry2Ab ranging from 3.28 to 7.72 µg/g of the tissue leaf. Insect bioassay with army worm (Spodoptera litura) was performed to check the efficacy of NIBGE (National Institute for Biotechnology and Genetic Engineering) double gene transgenic cotton plants and up to 93% insect mortality was observed.
Highlights
Cotton is the main fiber producing crop globally, with a significant impact on the economy of Pakistan
Farmers use chemical insecticides for controlling this menace of insect pests, but most of the pesticides applied by farmers are carcinogenic and neurotoxic in nature and are harmful to humans and other beneficial organisms
The main objective of this study was the development of insect-resistant cotton having double gene construct harboring Cry1Ac and Cry2Ab genes, and evaluation of these double gene cotton lines for Bt gene expression in different parts of the plant including leaves, boll rind and developing seeds at different stages of plant growth
Summary
Cotton is the main fiber producing crop globally, with a significant impact on the economy of Pakistan. Bt cotton expressing a Cry1Ac gene is grown over a large area in Pakistan, there is a major concern that bollworms may develop resistance. We have used a durable resistance strategy against bollworms by developing a double gene construct containing Cry1Ac and Cry2Ab (pGA48212R) for cotton transformation. Both Cry toxin genes have been cloned in the same T-DNA borders and transferred successfully into cotton via Agrobacterium-mediated transformation. Bt cotton was formally approved for commercial cultivation in Pakistan during 2010 It has provided better protection against bollworms, most of the available Bt cotton varieties grown have relatively low expression of Cry1Ac and www.nature.com/scientificreports/. Squares, boll rinds and maturing seeds are shown to have the lowest toxin expression[15], and while these are the most vulnerable parts for bollworms attack especially pink bollworm (P. gossypiella) which mainly feeds on the flower and seeds
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