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Development and evaluation of double gene transgenic cotton lines expressing Cry toxins for protection against chewing insect pests.

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.

SUBMITTER: Siddiqui HA

PROVIDER: S-EPMC6692333 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Development and evaluation of double gene transgenic cotton lines expressing Cry toxins for protection against chewing insect pests.

Siddiqui Hamid Anees HA Asif Muhammad M Asad Shaheen S Naqvi Rubab Zahra RZ Ajaz Sobia S Umer Noroza N Anjum Naveed N Rauf Imran I Sarwar Muhammad M Arshad Muhammad M Amin Imran I Saeed Muhammad M Mukhtar Zahid Z Bashir Aftab A Mansoor Shahid S

Scientific reports 20190813 1

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 transfe ...[more]

PMID: 31409859

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Project description:Cotton is an international agricultural commodity and the main cash crop of Pakistan of which quality and quantity are subject to various whims of nature. Climate change, insect pest complex, and weeds are reducing its productivity. Here, we have developed triple gene cotton containing EPSPS gene along with two Bt toxin genes Cry1Ac and Cry2Ab using a strategy where all three genes are cloned in the same T-DNA, followed by successful cotton transformation via Agrobacterium-mediated transformation. This strategy has been developed to help cotton breeders in developing new cultivars by incorporating these genes into the non-transgenic or single Bt (Cry1Ac) gene cotton background where all three genes will inherit together. The expression of all three proteins was confirmed through immunostrips and was quantified through enzyme-linked immunosorbent assay (ELISA). The spatio-temporal expression of Bt protein in different parts of triple gene NIBGE cotton plants was determined. Maximum expression was found in leaves followed by seeds and boll rinds. Insect bioassays with cotton bollworms (Helicoverpa armigera), armyworms (Spodoptera litura), and pink bollworms (Pectinophora gossypiella) showed more than 90% mortality. The best performing line (NIBGE-E2) on the basis of spatiotemporal expression, glyphosate assays, and insect mortality data, was used for event characterization by using the genome sequencing approach. The event was successfully characterized and named NIBGE 20-01. A diagnostics test based on event-specific PCR was developed and its ability to distinguish NIBGE 20-01 event from other commercial transgenic cotton events was confirmed. To confirm stable expression of all three proteins in the field conditions, homozygous transgenic lines were grown in the field and the expression was confirmed through immunostrip assays. It was found that all three genes are expressed under field conditions. To show that all three genes are inherited together upon crossing with local elite cotton lines, the F1 generation was grown under glasshouse and field conditions. The expression of all three genes was confirmed under field conditions. Our results showed that transgenic cotton with three genes cloned in the same T-DNA can express all genes and can be conveniently transferred into elite cotton lines through a single cross.

Dataset Information

Development and evaluation of double gene transgenic cotton lines expressing Cry toxins for protection against chewing insect pests.

Publications

Development and evaluation of double gene transgenic cotton lines expressing Cry toxins for protection against chewing insect pests.

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