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Induction of competent cells for Agrobacterium tumefaciens-mediated stable transformation of common bean (Phaseolus vulgaris L.)

Stable transformation of common bean (Phaseolus vulgaris L.) has been successful, to date, only using biolistic-mediated transformation and shoot regeneration from meristem-containing embryo axes. In this study, using precultured embryo axes, and optimal co-cultivation conditions resulted in a succe...

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Autores principales: Song, Guo-qing, Han, Xue, Wiersma, Andrew T., Zong, Xiaojuan, Awale, Halima E., Kelly, James D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058285/
https://www.ncbi.nlm.nih.gov/pubmed/32134988
http://dx.doi.org/10.1371/journal.pone.0229909
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author Song, Guo-qing
Han, Xue
Wiersma, Andrew T.
Zong, Xiaojuan
Awale, Halima E.
Kelly, James D.
author_facet Song, Guo-qing
Han, Xue
Wiersma, Andrew T.
Zong, Xiaojuan
Awale, Halima E.
Kelly, James D.
author_sort Song, Guo-qing
collection PubMed
description Stable transformation of common bean (Phaseolus vulgaris L.) has been successful, to date, only using biolistic-mediated transformation and shoot regeneration from meristem-containing embryo axes. In this study, using precultured embryo axes, and optimal co-cultivation conditions resulted in a successful transformation of the common bean cultivar Olathe using Agrobacterium tumefaciens strain EHA105. Plant regeneration through somatic embryogenesis was attained through the preculture of embryo axes for 12 weeks using induced competent cells for A. tumefaciens-mediated gene delivery. Using A. tumefaciens at a low optical density (OD) of 0.1 at a wavelength of 600 nm for infection and 4-day co-cultivation, compared to OD(600) of 0.5, increased the survival rate of the inoculated explants from 23% to 45%. Selection using 0.5 mg L(-1) glufosinate (GS) was effective to identify transformed cells when the bialaphos resistance (bar) gene under the constitutive 35S promoter was used as a selectable marker. After an 18-week selection period, 1.5% -2.5% inoculated explants, in three experiments with a total of 600 explants, produced GS-resistant plants through somatic embryogenesis. The expression of bar was confirmed in first- and second-generation seedlings of the two lines through reverse polymerase chain reaction. Presence of the bar gene was verified through genome sequencing of two selected transgenic lines. The induction of regenerable, competent cells is key for the successful transformation, and the protocols described may be useful for future transformation of additional Phaseolus germplasm.
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spelling pubmed-70582852020-03-13 Induction of competent cells for Agrobacterium tumefaciens-mediated stable transformation of common bean (Phaseolus vulgaris L.) Song, Guo-qing Han, Xue Wiersma, Andrew T. Zong, Xiaojuan Awale, Halima E. Kelly, James D. PLoS One Research Article Stable transformation of common bean (Phaseolus vulgaris L.) has been successful, to date, only using biolistic-mediated transformation and shoot regeneration from meristem-containing embryo axes. In this study, using precultured embryo axes, and optimal co-cultivation conditions resulted in a successful transformation of the common bean cultivar Olathe using Agrobacterium tumefaciens strain EHA105. Plant regeneration through somatic embryogenesis was attained through the preculture of embryo axes for 12 weeks using induced competent cells for A. tumefaciens-mediated gene delivery. Using A. tumefaciens at a low optical density (OD) of 0.1 at a wavelength of 600 nm for infection and 4-day co-cultivation, compared to OD(600) of 0.5, increased the survival rate of the inoculated explants from 23% to 45%. Selection using 0.5 mg L(-1) glufosinate (GS) was effective to identify transformed cells when the bialaphos resistance (bar) gene under the constitutive 35S promoter was used as a selectable marker. After an 18-week selection period, 1.5% -2.5% inoculated explants, in three experiments with a total of 600 explants, produced GS-resistant plants through somatic embryogenesis. The expression of bar was confirmed in first- and second-generation seedlings of the two lines through reverse polymerase chain reaction. Presence of the bar gene was verified through genome sequencing of two selected transgenic lines. The induction of regenerable, competent cells is key for the successful transformation, and the protocols described may be useful for future transformation of additional Phaseolus germplasm. Public Library of Science 2020-03-05 /pmc/articles/PMC7058285/ /pubmed/32134988 http://dx.doi.org/10.1371/journal.pone.0229909 Text en © 2020 Song et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Song, Guo-qing
Han, Xue
Wiersma, Andrew T.
Zong, Xiaojuan
Awale, Halima E.
Kelly, James D.
Induction of competent cells for Agrobacterium tumefaciens-mediated stable transformation of common bean (Phaseolus vulgaris L.)
title Induction of competent cells for Agrobacterium tumefaciens-mediated stable transformation of common bean (Phaseolus vulgaris L.)
title_full Induction of competent cells for Agrobacterium tumefaciens-mediated stable transformation of common bean (Phaseolus vulgaris L.)
title_fullStr Induction of competent cells for Agrobacterium tumefaciens-mediated stable transformation of common bean (Phaseolus vulgaris L.)
title_full_unstemmed Induction of competent cells for Agrobacterium tumefaciens-mediated stable transformation of common bean (Phaseolus vulgaris L.)
title_short Induction of competent cells for Agrobacterium tumefaciens-mediated stable transformation of common bean (Phaseolus vulgaris L.)
title_sort induction of competent cells for agrobacterium tumefaciens-mediated stable transformation of common bean (phaseolus vulgaris l.)
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058285/
https://www.ncbi.nlm.nih.gov/pubmed/32134988
http://dx.doi.org/10.1371/journal.pone.0229909
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