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Title: Optimization of Agrobacterium-mediated transformation conditions for efficient Bacillus thuringiensis (Bt) cotton transformation
Authors: Muhammad Mahmood Ahmed, Samina Perveen, Rabia Shabir, Alishba Hadi, Noor ul Ain, Mirza Abid Mehmood, Zulfiqar Ali, Zulqurnain Khan
Journal: Plant Bulletin
Publisher: Airanam Research
Country: Pakistan
Year: 2024
Volume: 3
Issue: 1
Language: English
DOI: 10.55627/pbulletin.003.01.0750
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.
To optimize Agrobacterium-mediated gene transformation procedures in local cotton cultivars for the development of Bt cotton in Pakistan.
The study involved using four local cotton varieties (Coker312, Cyto124, CIM554, and MNH786). Seeds were sterilized and germinated. Hypocotyl segments were cut and inoculated with Agrobacterium tumefaciens strain LBA4404 containing a dual gene construct (Cry1Ac + Cry2Ab). These segments were then cultured on various media (non-selective and selective) to induce callus formation. Genomic DNA was extracted from the developed callus, and PCR was used with gene-specific primers to confirm the presence of the Cry1Ac and Cry2Ab genes.
graph TD
A[Seed Sowing and Sterilization] --> B[Root Stretching];
B --> C[Hypocotyl Cutting];
C --> D[Agrobacterium Inoculation];
D --> E[Callus Induction on Non-selective Media];
E --> F[Transfer to Selective Media for Callus Formation];
F --> G[Callus Maturation];
G --> H[Genomic DNA Extraction];
H --> I[PCR Confirmation of Genes];
I --> J[Development of Transgenic Cotton Callus];
The study highlights the challenges of using the Coker312 variety in Pakistan due to its poor adaptation to local arid to semi-arid environments. The development of transgenic cotton using local cultivars like CIM554 is crucial for improving insect resistance and economic stability in Pakistan. The dual gene construct (Cry1Ac + Cry2Ab) is expected to provide enhanced resistance against lepidopteran insects.
The CIM554 cotton variety showed the highest response (85%) for induced callus formation and regeneration efficiency on IBA selective media. Coker312 showed a 66% regeneration efficiency. PCR analysis confirmed the presence of both Cry1Ac and Cry2Ab genes in the CIM554 callus.
The research successfully optimized Agrobacterium-mediated transformation procedures for local cotton cultivars, leading to the development of an indigenous cotton line expressing dual Bt genes (Cry1Ac and Cry2Ab) for insect resistance, with CIM554 showing the most promising results.
1. Cotton Area in Pakistan (2023-24): The text states the cotton area under cultivation grew to 2.4 million hectares in 2023-24. (Confirmed by Reference [1] which mentions Pakistan Economic Survey 2023-24).
2. Bt Cotton Cultivation Start in Pakistan: The text mentions that cultivation of Bt cotton started in Pakistan in 2010. (Confirmed by Reference [21] and [22] which discuss the adoption of GM crops).
3. Dual Gene Construct: The study utilized a dual gene construct containing Cry1Ac and Cry2Ab genes. (Confirmed by the abstract and methodology sections).
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