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Protoplast isolation and transient transformation system for Ginkgo biloba L.

Ginkgo biloba L. has a unique evolutionary status. Owing to its high medicinal and ornamental value, ginkgo has also recently become a research hotspot. However, the large genome and long juvenile period, as well as the lack of an effective genetic transformation system, have hindered gaining a full...

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Autores principales: Han, Xin, Rong, Hao, Feng, Yining, Xin, Yue, Luan, Xiaoyue, Zhou, Qi, Xu, Meng, Xu, Li-an
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10099357/
https://www.ncbi.nlm.nih.gov/pubmed/37063206
http://dx.doi.org/10.3389/fpls.2023.1145754
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author Han, Xin
Rong, Hao
Feng, Yining
Xin, Yue
Luan, Xiaoyue
Zhou, Qi
Xu, Meng
Xu, Li-an
author_facet Han, Xin
Rong, Hao
Feng, Yining
Xin, Yue
Luan, Xiaoyue
Zhou, Qi
Xu, Meng
Xu, Li-an
author_sort Han, Xin
collection PubMed
description Ginkgo biloba L. has a unique evolutionary status. Owing to its high medicinal and ornamental value, ginkgo has also recently become a research hotspot. However, the large genome and long juvenile period, as well as the lack of an effective genetic transformation system, have hindered gaining a full understanding of the comprehensive functions of ginkgo genes. At present, heterologous expression of genes in model plants is the primary method used in ginkgo-related research; however, these distant plant model relatives limit reliable interpretation of the results for direct applications in ginkgo breeding. To overcome these limitations, in this study, an efficient isolation and transient expression system for ginkgo protoplasts was established. A large number of intact and homogeneous ginkgo mesophyll protoplasts were isolated using 2% cellulase and 0.25% pectinase in 0.4 M mannitol. The activity of these protoplasts remained above 90% even after 24 h. Furthermore, when the concentration of the polyethylene glycol 4000 solution was 30%–40% (w/v), the transformation efficiency of the protoplasts reached 40%. Finally, the reliability of the system was verified using subcellular localization, transient overexpression, and protein interaction experiments with ginkgo genes, thereby providing a technical platform for the identification and analysis of ginkgo gene functions. The proposed method partially compensates for the limitations associated with the lack of a genetic transformation system and provides technical support to expand research on elucidating the functions of ginkgo genes.
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spelling pubmed-100993572023-04-14 Protoplast isolation and transient transformation system for Ginkgo biloba L. Han, Xin Rong, Hao Feng, Yining Xin, Yue Luan, Xiaoyue Zhou, Qi Xu, Meng Xu, Li-an Front Plant Sci Plant Science Ginkgo biloba L. has a unique evolutionary status. Owing to its high medicinal and ornamental value, ginkgo has also recently become a research hotspot. However, the large genome and long juvenile period, as well as the lack of an effective genetic transformation system, have hindered gaining a full understanding of the comprehensive functions of ginkgo genes. At present, heterologous expression of genes in model plants is the primary method used in ginkgo-related research; however, these distant plant model relatives limit reliable interpretation of the results for direct applications in ginkgo breeding. To overcome these limitations, in this study, an efficient isolation and transient expression system for ginkgo protoplasts was established. A large number of intact and homogeneous ginkgo mesophyll protoplasts were isolated using 2% cellulase and 0.25% pectinase in 0.4 M mannitol. The activity of these protoplasts remained above 90% even after 24 h. Furthermore, when the concentration of the polyethylene glycol 4000 solution was 30%–40% (w/v), the transformation efficiency of the protoplasts reached 40%. Finally, the reliability of the system was verified using subcellular localization, transient overexpression, and protein interaction experiments with ginkgo genes, thereby providing a technical platform for the identification and analysis of ginkgo gene functions. The proposed method partially compensates for the limitations associated with the lack of a genetic transformation system and provides technical support to expand research on elucidating the functions of ginkgo genes. Frontiers Media S.A. 2023-03-15 /pmc/articles/PMC10099357/ /pubmed/37063206 http://dx.doi.org/10.3389/fpls.2023.1145754 Text en Copyright © 2023 Han, Rong, Feng, Xin, Luan, Zhou, Xu and Xu https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Han, Xin
Rong, Hao
Feng, Yining
Xin, Yue
Luan, Xiaoyue
Zhou, Qi
Xu, Meng
Xu, Li-an
Protoplast isolation and transient transformation system for Ginkgo biloba L.
title Protoplast isolation and transient transformation system for Ginkgo biloba L.
title_full Protoplast isolation and transient transformation system for Ginkgo biloba L.
title_fullStr Protoplast isolation and transient transformation system for Ginkgo biloba L.
title_full_unstemmed Protoplast isolation and transient transformation system for Ginkgo biloba L.
title_short Protoplast isolation and transient transformation system for Ginkgo biloba L.
title_sort protoplast isolation and transient transformation system for ginkgo biloba l.
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10099357/
https://www.ncbi.nlm.nih.gov/pubmed/37063206
http://dx.doi.org/10.3389/fpls.2023.1145754
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