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Development of a miniaturized Ti-plasmid and helper plasmid system for Agrobacterium-mediated plant transformation

Tumor-inducing (Ti) plasmid is the requisite for Agrobacterium-mediated plant transformation. Over decades, continuous efforts have been made to improve the efficiency of Agrobacterium-mediated plant transformation and most of them focused on the binary vector system. A binary vector system comprise...

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Main Authors: Yuh, Leng Teo, Shu, Ting Chang, Wai, Keat Toh, Xin, Yen Tor, Chai, Ling Ho, Pek, Chin Loh, Hann, Ling Wong
Format: Article
Published: Malaysian Society of Molecular Biology and Biotechnology 2023
Online Access:http://psasir.upm.edu.my/id/eprint/100596/
https://www.msmbb.my/index.php/archive-issues/18-apjmbb/461-archive-issue-30
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spelling my.upm.eprints.1005962023-10-09T18:37:19Z http://psasir.upm.edu.my/id/eprint/100596/ Development of a miniaturized Ti-plasmid and helper plasmid system for Agrobacterium-mediated plant transformation Yuh, Leng Teo Shu, Ting Chang Wai, Keat Toh Xin, Yen Tor Chai, Ling Ho Pek, Chin Loh Hann, Ling Wong Tumor-inducing (Ti) plasmid is the requisite for Agrobacterium-mediated plant transformation. Over decades, continuous efforts have been made to improve the efficiency of Agrobacterium-mediated plant transformation and most of them focused on the binary vector system. A binary vector system comprises of a binary vector of which transferred DNA (T-DNA) resided on and a Ti plasmid to carry those essential virulence genes. In this study, we constructed a miniaturized helper Ti plasmid, designated as pYL102, with the aim to enhance the overall Agrobacterium-mediated transformation rate. The size of pYL102 was reduced to ~60% of the original plasmid pCAMBIA5105. Subsequently, pYL102 was coupled with the broad host range (BHR) bacterial expression vector, pYL101C, of which the key regulatory virulence gene, virG-N54D, was cloned in and expressed under the control of a strong constitutive PINTc promoter. To test the functionality of the constructed vector system, A. tumefaciens C58C1 carrying pYL102, pYL101C::virG-N54D and the transformation vector pGWB2::e35S-sfGFP was used to transform Nicotiana benthamiana leaves by agroinfiltration. Green fluorescence was observed in spots infiltrated with Agrobacterium carrying the test plasmids. The fluorescence intensity from the test agroinfiltrated leaves was significantly higher than those of the mock-infiltrated leaves (p<0.01), indicating the vector system can be used for plant transformation. Malaysian Society of Molecular Biology and Biotechnology 2023-01-19 Article PeerReviewed Yuh, Leng Teo and Shu, Ting Chang and Wai, Keat Toh and Xin, Yen Tor and Chai, Ling Ho and Pek, Chin Loh and Hann, Ling Wong (2023) Development of a miniaturized Ti-plasmid and helper plasmid system for Agrobacterium-mediated plant transformation. Asia Pacific Journal of Molecular Biology and Biotechnology, 30 (3). 23 - 32. ISSN 0128-7451; ESSN:2672-7277 https://www.msmbb.my/index.php/archive-issues/18-apjmbb/461-archive-issue-30 10.35118/apjmbb.2022.030.3.03
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
description Tumor-inducing (Ti) plasmid is the requisite for Agrobacterium-mediated plant transformation. Over decades, continuous efforts have been made to improve the efficiency of Agrobacterium-mediated plant transformation and most of them focused on the binary vector system. A binary vector system comprises of a binary vector of which transferred DNA (T-DNA) resided on and a Ti plasmid to carry those essential virulence genes. In this study, we constructed a miniaturized helper Ti plasmid, designated as pYL102, with the aim to enhance the overall Agrobacterium-mediated transformation rate. The size of pYL102 was reduced to ~60% of the original plasmid pCAMBIA5105. Subsequently, pYL102 was coupled with the broad host range (BHR) bacterial expression vector, pYL101C, of which the key regulatory virulence gene, virG-N54D, was cloned in and expressed under the control of a strong constitutive PINTc promoter. To test the functionality of the constructed vector system, A. tumefaciens C58C1 carrying pYL102, pYL101C::virG-N54D and the transformation vector pGWB2::e35S-sfGFP was used to transform Nicotiana benthamiana leaves by agroinfiltration. Green fluorescence was observed in spots infiltrated with Agrobacterium carrying the test plasmids. The fluorescence intensity from the test agroinfiltrated leaves was significantly higher than those of the mock-infiltrated leaves (p<0.01), indicating the vector system can be used for plant transformation.
format Article
author Yuh, Leng Teo
Shu, Ting Chang
Wai, Keat Toh
Xin, Yen Tor
Chai, Ling Ho
Pek, Chin Loh
Hann, Ling Wong
spellingShingle Yuh, Leng Teo
Shu, Ting Chang
Wai, Keat Toh
Xin, Yen Tor
Chai, Ling Ho
Pek, Chin Loh
Hann, Ling Wong
Development of a miniaturized Ti-plasmid and helper plasmid system for Agrobacterium-mediated plant transformation
author_facet Yuh, Leng Teo
Shu, Ting Chang
Wai, Keat Toh
Xin, Yen Tor
Chai, Ling Ho
Pek, Chin Loh
Hann, Ling Wong
author_sort Yuh, Leng Teo
title Development of a miniaturized Ti-plasmid and helper plasmid system for Agrobacterium-mediated plant transformation
title_short Development of a miniaturized Ti-plasmid and helper plasmid system for Agrobacterium-mediated plant transformation
title_full Development of a miniaturized Ti-plasmid and helper plasmid system for Agrobacterium-mediated plant transformation
title_fullStr Development of a miniaturized Ti-plasmid and helper plasmid system for Agrobacterium-mediated plant transformation
title_full_unstemmed Development of a miniaturized Ti-plasmid and helper plasmid system for Agrobacterium-mediated plant transformation
title_sort development of a miniaturized ti-plasmid and helper plasmid system for agrobacterium-mediated plant transformation
publisher Malaysian Society of Molecular Biology and Biotechnology
publishDate 2023
url http://psasir.upm.edu.my/id/eprint/100596/
https://www.msmbb.my/index.php/archive-issues/18-apjmbb/461-archive-issue-30
_version_ 1781706664421883904
score 13.211869

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