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Improving Agrobacterium tumefaciens−Mediated Genetic Transformation for Gene Function Studies and Mutagenesis in Cucumber (Cucumis sativus L.)

In the post−genomics era, Agrobacterium tumefaciens−mediated genetic transformation is becoming an increasingly indispensable tool for characterization of gene functions and crop improvement in cucumber (Cucumis sativus L.). However, cucumber transformation efficiency is still low. In this study, we...

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Autores principales: Liu, Hanqiang, Zhao, Jianyu, Chen, Feifan, Wu, Zhiming, Tan, Junyi, Nguyen, Nhien Hao, Cheng, Zhihui, Weng, Yiqun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10048676/
https://www.ncbi.nlm.nih.gov/pubmed/36980873
http://dx.doi.org/10.3390/genes14030601
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author Liu, Hanqiang
Zhao, Jianyu
Chen, Feifan
Wu, Zhiming
Tan, Junyi
Nguyen, Nhien Hao
Cheng, Zhihui
Weng, Yiqun
author_facet Liu, Hanqiang
Zhao, Jianyu
Chen, Feifan
Wu, Zhiming
Tan, Junyi
Nguyen, Nhien Hao
Cheng, Zhihui
Weng, Yiqun
author_sort Liu, Hanqiang
collection PubMed
description In the post−genomics era, Agrobacterium tumefaciens−mediated genetic transformation is becoming an increasingly indispensable tool for characterization of gene functions and crop improvement in cucumber (Cucumis sativus L.). However, cucumber transformation efficiency is still low. In this study, we evaluated the effects of several key factors affecting the shoot−regeneration rate and overall transformation efficiency in cucumber including genotypes, the age and sources of explants, Agrobacterium strains, infection/co−cultivation conditions, and selective agents. We showed that in general, North China cucumbers exhibited higher shoot−regeneration rate than US pickling or slicing cucumbers. The subapical ground meristematic regions from cotyledons or the hypocotyl had a similar shoot−regeneration efficiency that was also affected by the age of the explants. Transformation with the Agrobacterium strain AGL1 yielded a higher frequency of positive transformants than with GV3101. The antibiotic kanamycin was effective in selection against non−transformants or chimeras. Optimization of various factors was exemplified with the development of transgenic plants overexpressing the LittleLeaf (LL) gene or RNAi of the APRR2 gene in three cucumber lines. The streamlined protocol was also tested in transgenic studies in three additional genes. The overall transformation efficiency defined by the number of verified transgenic plants out of the number of seeds across multiple experiments was 0.2–1.7%. Screening among T(1) OE transgenic plants identified novel, inheritable mutants for leaf or fruit color or size/shape, suggesting T−DNA insertion as a potential source of mutagenesis. The Agrobacterium−mediated transformation protocol from this study could be used as the baseline for further improvements in cucumber transformation.
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spelling pubmed-100486762023-03-29 Improving Agrobacterium tumefaciens−Mediated Genetic Transformation for Gene Function Studies and Mutagenesis in Cucumber (Cucumis sativus L.) Liu, Hanqiang Zhao, Jianyu Chen, Feifan Wu, Zhiming Tan, Junyi Nguyen, Nhien Hao Cheng, Zhihui Weng, Yiqun Genes (Basel) Article In the post−genomics era, Agrobacterium tumefaciens−mediated genetic transformation is becoming an increasingly indispensable tool for characterization of gene functions and crop improvement in cucumber (Cucumis sativus L.). However, cucumber transformation efficiency is still low. In this study, we evaluated the effects of several key factors affecting the shoot−regeneration rate and overall transformation efficiency in cucumber including genotypes, the age and sources of explants, Agrobacterium strains, infection/co−cultivation conditions, and selective agents. We showed that in general, North China cucumbers exhibited higher shoot−regeneration rate than US pickling or slicing cucumbers. The subapical ground meristematic regions from cotyledons or the hypocotyl had a similar shoot−regeneration efficiency that was also affected by the age of the explants. Transformation with the Agrobacterium strain AGL1 yielded a higher frequency of positive transformants than with GV3101. The antibiotic kanamycin was effective in selection against non−transformants or chimeras. Optimization of various factors was exemplified with the development of transgenic plants overexpressing the LittleLeaf (LL) gene or RNAi of the APRR2 gene in three cucumber lines. The streamlined protocol was also tested in transgenic studies in three additional genes. The overall transformation efficiency defined by the number of verified transgenic plants out of the number of seeds across multiple experiments was 0.2–1.7%. Screening among T(1) OE transgenic plants identified novel, inheritable mutants for leaf or fruit color or size/shape, suggesting T−DNA insertion as a potential source of mutagenesis. The Agrobacterium−mediated transformation protocol from this study could be used as the baseline for further improvements in cucumber transformation. MDPI 2023-02-27 /pmc/articles/PMC10048676/ /pubmed/36980873 http://dx.doi.org/10.3390/genes14030601 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Liu, Hanqiang
Zhao, Jianyu
Chen, Feifan
Wu, Zhiming
Tan, Junyi
Nguyen, Nhien Hao
Cheng, Zhihui
Weng, Yiqun
Improving Agrobacterium tumefaciens−Mediated Genetic Transformation for Gene Function Studies and Mutagenesis in Cucumber (Cucumis sativus L.)
title Improving Agrobacterium tumefaciens−Mediated Genetic Transformation for Gene Function Studies and Mutagenesis in Cucumber (Cucumis sativus L.)
title_full Improving Agrobacterium tumefaciens−Mediated Genetic Transformation for Gene Function Studies and Mutagenesis in Cucumber (Cucumis sativus L.)
title_fullStr Improving Agrobacterium tumefaciens−Mediated Genetic Transformation for Gene Function Studies and Mutagenesis in Cucumber (Cucumis sativus L.)
title_full_unstemmed Improving Agrobacterium tumefaciens−Mediated Genetic Transformation for Gene Function Studies and Mutagenesis in Cucumber (Cucumis sativus L.)
title_short Improving Agrobacterium tumefaciens−Mediated Genetic Transformation for Gene Function Studies and Mutagenesis in Cucumber (Cucumis sativus L.)
title_sort improving agrobacterium tumefaciens−mediated genetic transformation for gene function studies and mutagenesis in cucumber (cucumis sativus l.)
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10048676/
https://www.ncbi.nlm.nih.gov/pubmed/36980873
http://dx.doi.org/10.3390/genes14030601
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