Development and evaluation of triple gene transgenic cotton lines expressing three genes (Cry1Ac-Cry2Ab-EPSPS) for lepidopteran insect pests and herbicide tolerance
Abstract 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 EPSP...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-11-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-22209-w |
| _version_ | 1797984765693919232 |
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| author | Hamid Anees Siddiqui Shaheen Asad Rubab Zahra Naqvi Muhammad Asif Chengcheng Liu Xin Liu Muhammad Farooq Saifullah Abro Muhammad Rizwan Muhammad Arshad Muhammad Sarwar Imran Amin Zahid Mukhtar Shahid Mansoor |
| author_facet | Hamid Anees Siddiqui Shaheen Asad Rubab Zahra Naqvi Muhammad Asif Chengcheng Liu Xin Liu Muhammad Farooq Saifullah Abro Muhammad Rizwan Muhammad Arshad Muhammad Sarwar Imran Amin Zahid Mukhtar Shahid Mansoor |
| author_sort | Hamid Anees Siddiqui |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-04-11T07:06:50Z |
| format | Article |
| id | doaj.art-57cc8331f84e49e3be8648a4f953b941 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-11T07:06:50Z |
| publishDate | 2022-11-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-57cc8331f84e49e3be8648a4f953b9412022-12-22T04:38:22ZengNature PortfolioScientific Reports2045-23222022-11-0112111010.1038/s41598-022-22209-wDevelopment and evaluation of triple gene transgenic cotton lines expressing three genes (Cry1Ac-Cry2Ab-EPSPS) for lepidopteran insect pests and herbicide toleranceHamid Anees Siddiqui0Shaheen Asad1Rubab Zahra Naqvi2Muhammad Asif3Chengcheng Liu4Xin Liu5Muhammad Farooq6Saifullah Abro7Muhammad Rizwan8Muhammad Arshad9Muhammad Sarwar10Imran Amin11Zahid Mukhtar12Shahid Mansoor13Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, College Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS)Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, College Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS)Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, College Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS)Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, College Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS)Beijing Genomics Institute ShenzhenBeijing Genomics Institute ShenzhenAgricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, College Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS)Plant Breeding and Genetics Division, Nuclear Institute of Agriculture (NIA)Plant Breeding and Genetics Division, Nuclear Institute of Agriculture (NIA)Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, College Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS)Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, College Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS)Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, College Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS)Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, College Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS)Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, College Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS)Abstract 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.https://doi.org/10.1038/s41598-022-22209-w |
| spellingShingle | Hamid Anees Siddiqui Shaheen Asad Rubab Zahra Naqvi Muhammad Asif Chengcheng Liu Xin Liu Muhammad Farooq Saifullah Abro Muhammad Rizwan Muhammad Arshad Muhammad Sarwar Imran Amin Zahid Mukhtar Shahid Mansoor Development and evaluation of triple gene transgenic cotton lines expressing three genes (Cry1Ac-Cry2Ab-EPSPS) for lepidopteran insect pests and herbicide tolerance Scientific Reports |
| title | Development and evaluation of triple gene transgenic cotton lines expressing three genes (Cry1Ac-Cry2Ab-EPSPS) for lepidopteran insect pests and herbicide tolerance |
| title_full | Development and evaluation of triple gene transgenic cotton lines expressing three genes (Cry1Ac-Cry2Ab-EPSPS) for lepidopteran insect pests and herbicide tolerance |
| title_fullStr | Development and evaluation of triple gene transgenic cotton lines expressing three genes (Cry1Ac-Cry2Ab-EPSPS) for lepidopteran insect pests and herbicide tolerance |
| title_full_unstemmed | Development and evaluation of triple gene transgenic cotton lines expressing three genes (Cry1Ac-Cry2Ab-EPSPS) for lepidopteran insect pests and herbicide tolerance |
| title_short | Development and evaluation of triple gene transgenic cotton lines expressing three genes (Cry1Ac-Cry2Ab-EPSPS) for lepidopteran insect pests and herbicide tolerance |
| title_sort | development and evaluation of triple gene transgenic cotton lines expressing three genes cry1ac cry2ab epsps for lepidopteran insect pests and herbicide tolerance |
| url | https://doi.org/10.1038/s41598-022-22209-w |
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