A comprehensive method to generating and identifying transgenic tobacco lines with a single transgene integration locus for functional analysis
Tobacco is a popular model plant used for studying gene function. The generation of transgenic tobacco is tremendously essential in functional genomics. The generated transgenic plants must undergo careful selection and analysis before being used. However, most published protocols for generating tra...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Universiti Putra Malaysia
2025
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| Online Access: | http://psasir.upm.edu.my/id/eprint/120950/ http://psasir.upm.edu.my/id/eprint/120950/1/120950.pdf |
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| author | Hilman, Mohamad Shafek Nawawi, Omar Azhari, Mohd Farhan Bai, Tianqi Zhang, Cuixian Abdullah, Mohd Puad Masani, Mat Yunus Abdul Yusuf, Chong Yu Lok |
| author_facet | Hilman, Mohamad Shafek Nawawi, Omar Azhari, Mohd Farhan Bai, Tianqi Zhang, Cuixian Abdullah, Mohd Puad Masani, Mat Yunus Abdul Yusuf, Chong Yu Lok |
| author_sort | Hilman, Mohamad Shafek |
| building | UPM Institutional Repository |
| collection | Online Access |
| description | Tobacco is a popular model plant used for studying gene function. The generation of transgenic tobacco is tremendously essential in functional genomics. The generated transgenic plants must undergo careful selection and analysis before being used. However, most published protocols for generating transgenic tobacco for functional genomics are not comprehensive and involve sophisticated equipment. This study demonstrates an efficient and comprehensive method for developing and selecting transgenic tobacco lines without involving sophisticated equipment. Transgene was delivered into the genome of a tobacco plant via Agrobacterium tumefaciens. Polymerase Chain Reaction (PCR) was performed to verify the integration of transgenes in the putative primary transformants. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) examined transgene expressions. The number of transgene integration loci (TIL) was determined by transgene segregation analysis. PCR results revealed that ≈97% of the primary transformants were positive. The transgene was highly expressed in the transgenic plants. Segregation analysis showed that 47.6%–66.7% of the transgenic plants contained a single TIL, and the T1 and T2 progenies inherited the transgene. Homozygous transgenic lines with a single TIL were successfully developed by using our method. This manuscript encompasses detailed guidance on genetic transformation, molecular analysis, seed production, and transgene segregation analysis. It serves as a guideline for the researchers to produce transgenic tobacco lines that can be used for functional analysis. The procedures described here can be conducted in standard laboratories as they require no high-end equipment. This comprehensive and efficient method for generating transgenic tobacco will foster functional genomics. |
| first_indexed | 2025-11-15T14:49:29Z |
| format | Article |
| id | upm-120950 |
| institution | Universiti Putra Malaysia |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-15T14:49:29Z |
| publishDate | 2025 |
| publisher | Universiti Putra Malaysia |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | upm-1209502025-10-16T07:20:04Z http://psasir.upm.edu.my/id/eprint/120950/ A comprehensive method to generating and identifying transgenic tobacco lines with a single transgene integration locus for functional analysis Hilman, Mohamad Shafek Nawawi, Omar Azhari, Mohd Farhan Bai, Tianqi Zhang, Cuixian Abdullah, Mohd Puad Masani, Mat Yunus Abdul Yusuf, Chong Yu Lok Tobacco is a popular model plant used for studying gene function. The generation of transgenic tobacco is tremendously essential in functional genomics. The generated transgenic plants must undergo careful selection and analysis before being used. However, most published protocols for generating transgenic tobacco for functional genomics are not comprehensive and involve sophisticated equipment. This study demonstrates an efficient and comprehensive method for developing and selecting transgenic tobacco lines without involving sophisticated equipment. Transgene was delivered into the genome of a tobacco plant via Agrobacterium tumefaciens. Polymerase Chain Reaction (PCR) was performed to verify the integration of transgenes in the putative primary transformants. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) examined transgene expressions. The number of transgene integration loci (TIL) was determined by transgene segregation analysis. PCR results revealed that ≈97% of the primary transformants were positive. The transgene was highly expressed in the transgenic plants. Segregation analysis showed that 47.6%–66.7% of the transgenic plants contained a single TIL, and the T1 and T2 progenies inherited the transgene. Homozygous transgenic lines with a single TIL were successfully developed by using our method. This manuscript encompasses detailed guidance on genetic transformation, molecular analysis, seed production, and transgene segregation analysis. It serves as a guideline for the researchers to produce transgenic tobacco lines that can be used for functional analysis. The procedures described here can be conducted in standard laboratories as they require no high-end equipment. This comprehensive and efficient method for generating transgenic tobacco will foster functional genomics. Universiti Putra Malaysia 2025 Article PeerReviewed text en cc_by_nc_nd_4 http://psasir.upm.edu.my/id/eprint/120950/1/120950.pdf Hilman, Mohamad Shafek and Nawawi, Omar and Azhari, Mohd Farhan and Bai, Tianqi and Zhang, Cuixian and Abdullah, Mohd Puad and Masani, Mat Yunus Abdul and Yusuf, Chong Yu Lok (2025) A comprehensive method to generating and identifying transgenic tobacco lines with a single transgene integration locus for functional analysis. Pertanika Journal of Tropical Agricultural Science, 48 (1). pp. 31-58. ISSN 1511-3701; eISSN: 2231-8542 http://pertanika2.upm.edu.my/pjtas/browse/regular-issue?article=JTAS-3068-2024 10.47836/pjtas.48.1.03 |
| spellingShingle | Hilman, Mohamad Shafek Nawawi, Omar Azhari, Mohd Farhan Bai, Tianqi Zhang, Cuixian Abdullah, Mohd Puad Masani, Mat Yunus Abdul Yusuf, Chong Yu Lok A comprehensive method to generating and identifying transgenic tobacco lines with a single transgene integration locus for functional analysis |
| title | A comprehensive method to generating and identifying transgenic tobacco lines with a single transgene integration locus for functional analysis |
| title_full | A comprehensive method to generating and identifying transgenic tobacco lines with a single transgene integration locus for functional analysis |
| title_fullStr | A comprehensive method to generating and identifying transgenic tobacco lines with a single transgene integration locus for functional analysis |
| title_full_unstemmed | A comprehensive method to generating and identifying transgenic tobacco lines with a single transgene integration locus for functional analysis |
| title_short | A comprehensive method to generating and identifying transgenic tobacco lines with a single transgene integration locus for functional analysis |
| title_sort | comprehensive method to generating and identifying transgenic tobacco lines with a single transgene integration locus for functional analysis |
| url | http://psasir.upm.edu.my/id/eprint/120950/ http://psasir.upm.edu.my/id/eprint/120950/ http://psasir.upm.edu.my/id/eprint/120950/ http://psasir.upm.edu.my/id/eprint/120950/1/120950.pdf |