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Optimization of callus induction and proliferation of Paeonia lactiflora Pall. and Agrobacterium-mediated genetic transformation

Paeonia lactiflora Pall. is an important ornamental plant with high economic and medicinal value, which has considerable development prospects worldwide. The lack of efficient tissue culture techniques and genetic transformation systems has become a master obstacle for P. lactiflora research. The pu...

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Autores principales: Duan, Siyang, Xin, Rujie, Guan, Shixin, Li, Xueting, Fei, Riwen, Cheng, Wan, Pan, Qing, Sun, Xiaomei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9800923/
https://www.ncbi.nlm.nih.gov/pubmed/36589115
http://dx.doi.org/10.3389/fpls.2022.996690
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author Duan, Siyang
Xin, Rujie
Guan, Shixin
Li, Xueting
Fei, Riwen
Cheng, Wan
Pan, Qing
Sun, Xiaomei
author_facet Duan, Siyang
Xin, Rujie
Guan, Shixin
Li, Xueting
Fei, Riwen
Cheng, Wan
Pan, Qing
Sun, Xiaomei
author_sort Duan, Siyang
collection PubMed
description Paeonia lactiflora Pall. is an important ornamental plant with high economic and medicinal value, which has considerable development prospects worldwide. The lack of efficient tissue culture techniques and genetic transformation systems has become a master obstacle for P. lactiflora research. The purpose of the present study focuses on obtaining an efficient and stable genetic transformation method using callus as the receptor and exploring an efficient protocol for callus induction and proliferation associated with P. lactiflora. Callus induction and proliferation were performed using MS medium with various concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D), 1-Naphthaleneacetic acid (NAA), 6-Benzylaminopurine (6-BA) and thidiazuron (TDZ). The sensitivity of callus to kanamycin and cefotaxime was determined. Several parameters such as Agrobacterium cell density, infection time and co-culture duration were studied to optimize transformation efficiency. Agrobacterium strains EHA105 and pBI121 binary vector harboring the β-glucuronidase (GUS) gene were used for transformation. Expression of the GUS reporter gene was detected by GUS assay, polymerase chain reaction (PCR) and Quantitative Real-time PCR (RT-qPCR). The MS medium containing 1.0 mg·L(-1) NAA, 0.5 mg·L(-1) 2,4-D and 0.5 mg·L(-1) TDZ was optimal for callus induction and MS medium containing 0.5 mg·L(-1) NAA, 1.0 mg·L(-1) 2,4-D and 0.5 mg·L(-1) TDZ was the best for callus proliferation. The concentrations of kanamycin and cefotaxime used for screening positive callus were 125 mg·L(-1) and 200 mg·L(-1), respectively. Among various combinations analyzed, the best transformation result was obtained via the 25 min of infection of Agrobacterium at 0.6 OD(600) and 3 d of co-culture. Overall, this study provided technical support and theoretical guidance for improving the callus induction and proliferation efficiency and the study of gene function in P. lactiflora.
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spelling pubmed-98009232022-12-31 Optimization of callus induction and proliferation of Paeonia lactiflora Pall. and Agrobacterium-mediated genetic transformation Duan, Siyang Xin, Rujie Guan, Shixin Li, Xueting Fei, Riwen Cheng, Wan Pan, Qing Sun, Xiaomei Front Plant Sci Plant Science Paeonia lactiflora Pall. is an important ornamental plant with high economic and medicinal value, which has considerable development prospects worldwide. The lack of efficient tissue culture techniques and genetic transformation systems has become a master obstacle for P. lactiflora research. The purpose of the present study focuses on obtaining an efficient and stable genetic transformation method using callus as the receptor and exploring an efficient protocol for callus induction and proliferation associated with P. lactiflora. Callus induction and proliferation were performed using MS medium with various concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D), 1-Naphthaleneacetic acid (NAA), 6-Benzylaminopurine (6-BA) and thidiazuron (TDZ). The sensitivity of callus to kanamycin and cefotaxime was determined. Several parameters such as Agrobacterium cell density, infection time and co-culture duration were studied to optimize transformation efficiency. Agrobacterium strains EHA105 and pBI121 binary vector harboring the β-glucuronidase (GUS) gene were used for transformation. Expression of the GUS reporter gene was detected by GUS assay, polymerase chain reaction (PCR) and Quantitative Real-time PCR (RT-qPCR). The MS medium containing 1.0 mg·L(-1) NAA, 0.5 mg·L(-1) 2,4-D and 0.5 mg·L(-1) TDZ was optimal for callus induction and MS medium containing 0.5 mg·L(-1) NAA, 1.0 mg·L(-1) 2,4-D and 0.5 mg·L(-1) TDZ was the best for callus proliferation. The concentrations of kanamycin and cefotaxime used for screening positive callus were 125 mg·L(-1) and 200 mg·L(-1), respectively. Among various combinations analyzed, the best transformation result was obtained via the 25 min of infection of Agrobacterium at 0.6 OD(600) and 3 d of co-culture. Overall, this study provided technical support and theoretical guidance for improving the callus induction and proliferation efficiency and the study of gene function in P. lactiflora. Frontiers Media S.A. 2022-12-16 /pmc/articles/PMC9800923/ /pubmed/36589115 http://dx.doi.org/10.3389/fpls.2022.996690 Text en Copyright © 2022 Duan, Xin, Guan, Li, Fei, Cheng, Pan and Sun 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
Duan, Siyang
Xin, Rujie
Guan, Shixin
Li, Xueting
Fei, Riwen
Cheng, Wan
Pan, Qing
Sun, Xiaomei
Optimization of callus induction and proliferation of Paeonia lactiflora Pall. and Agrobacterium-mediated genetic transformation
title Optimization of callus induction and proliferation of Paeonia lactiflora Pall. and Agrobacterium-mediated genetic transformation
title_full Optimization of callus induction and proliferation of Paeonia lactiflora Pall. and Agrobacterium-mediated genetic transformation
title_fullStr Optimization of callus induction and proliferation of Paeonia lactiflora Pall. and Agrobacterium-mediated genetic transformation
title_full_unstemmed Optimization of callus induction and proliferation of Paeonia lactiflora Pall. and Agrobacterium-mediated genetic transformation
title_short Optimization of callus induction and proliferation of Paeonia lactiflora Pall. and Agrobacterium-mediated genetic transformation
title_sort optimization of callus induction and proliferation of paeonia lactiflora pall. and agrobacterium-mediated genetic transformation
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9800923/
https://www.ncbi.nlm.nih.gov/pubmed/36589115
http://dx.doi.org/10.3389/fpls.2022.996690
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