Optimization of Agrobacterium-mediated transformation conditions for efficient Bacillus thuringiensis (Bt) cotton transformation

Muhammad Mahmood Ahmed; Samina Perveen; Rabia Shabir; Alishba Hadi; Noor ul Ain; Mirza Abid Mehmood; Zulfiqar Ali; Zulqurnain Khan

doi:10.55627/pbulletin.003.01.0750

Authors

Muhammad Mahmood Ahmed Institute of Plant Breeding PBB, MNS University of Agriculture, Multan & Institute of Biochemistry, Biotechnology and Department of Bioinformatics (IBBB), IUB, Bahawalpur
Samina Perveen Institute of Plant Breeding PBB, MNS University of Agriculture, Multan
Rabia Shabir Institute of Plant Breeding PBB, MNS University of Agriculture, Multan
Alishba Hadi Department of Biotechnology, Institute of Biochemistry, Biotechnology and Bioinformatics (IBBB), IUB, Bahawalpur
Noor ul Ain Department of Biotechnology, Institute of Biochemistry, Biotechnology and Bioinformatics (IBBB), IUB, Bahawalpur
Mirza Abid Mehmood Institute of Plant Protection (IPP), MNS University of Agriculture, Multan
Zulfiqar Ali Institute of Plant Breeding PBB, MNS University of Agriculture, Multan
Zulqurnain Khan Institute of Plant Breeding PBB, MNS University of Agriculture, Multan

DOI:

https://doi.org/10.55627/pbulletin.003.01.0750

Abstract

Cotton is one of the world’s largest fiber-producing crops. It is also known as the important cash crop of Pakistan. Cotton crop products contribute to economy as cottonseed oil, animal feed and the fiber support the textile industries, thus, playing a significant role in uplifting the economy of a country. But over time, bollworm attacks had escalated, severely reducing the yield of cotton and damaging cotton production. Bt (Bacillus thuringiensis) cotton is being widely used nowadays due to its enhanced resistance against cotton bollworms which are responsible for reducing crop production leading to high economic loss. It greatly affects the economy by increasing the cost of production and protection. Using resistant strains of Bt cotton not only makes it easier to combat bollworm attacks but also reduces the substantial expense issues that farmers face. Bt has many toxic proteins among which the two crystal proteins, Cry1Ac and Cry2Ab, are used to develop resistance against bollworms in cotton. This study aimed to optimize the developing transgenic callus containing Cry1Ac and Cry2Ab genes in cotton. The dual gene construct (Cry1Ac + Cry2Ab) provides more resistance to the cotton crop as compared to the single gene construct against whom the most bollworms have developed resistance. The vector was inoculated into the hypocotyl segments and the transformation process was preceded by shifting the hypocotyls at different mediums Agrobacterium tumefaciens strain LBA4404 was used for cotton transformation. The construct-containing vector was introduced into a standard cotton line. A molecular confirmation test was carried out utilizing PCR and gene-specific primers after the formation of the transgenic callus. As a result of the research an indigenous cotton line that expresses double Bt genes for insect resistance was developed.

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