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Fast-track transformation and genome editing in Brachypodium distachyon

BACKGROUND: Even for easy-to-transform species or genotypes, the creation of transgenic or edited plant lines remains a significant bottleneck. Thus, any technical advance that accelerates the regeneration and transformation process is welcome. So far, methods to produce Brachypodium distachyon (Bd)...

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Autores principales: Soulhat, Camille, Wehbi, Houssein, Fierlej, Yannick, Berquin, Patrick, Girin, Thomas, Hilson, Pierre, Bouchabké-Coussa, Oumaya
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10053978/
https://www.ncbi.nlm.nih.gov/pubmed/36991448
http://dx.doi.org/10.1186/s13007-023-01005-1
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author Soulhat, Camille
Wehbi, Houssein
Fierlej, Yannick
Berquin, Patrick
Girin, Thomas
Hilson, Pierre
Bouchabké-Coussa, Oumaya
author_facet Soulhat, Camille
Wehbi, Houssein
Fierlej, Yannick
Berquin, Patrick
Girin, Thomas
Hilson, Pierre
Bouchabké-Coussa, Oumaya
author_sort Soulhat, Camille
collection PubMed
description BACKGROUND: Even for easy-to-transform species or genotypes, the creation of transgenic or edited plant lines remains a significant bottleneck. Thus, any technical advance that accelerates the regeneration and transformation process is welcome. So far, methods to produce Brachypodium distachyon (Bd) transgenics span at least 14 weeks from the start of tissue culture to the recovery of regenerated plantlets. RESULTS: We have previously shown that embryogenic somatic tissues grow in the scutellum of immature zygotic Bd embryos within 3 days of in vitro induction with exogenous auxin and that the development of secondary embryos can be initiated immediately thereafter. Here, we further demonstrate that such pluripotent reactive tissues can be genetically transformed with Agrobacterium tumefaciens right after the onset of somatic embryogenesis. In brief, immature zygotic embryos are induced for callogenesis for one week, co-cultured with Agrobacterium for three days, then incubated on callogenesis selective medium for three weeks, and finally transferred on selective regeneration medium for up to three weeks to obtain plantlets ready for rooting. This 7-to-8-week procedure requires only three subcultures. Its validation includes the molecular and phenotype characterization of Bd lines carrying transgenic cassettes and novel CRISPR/Cas9-generated mutations in two independent loci coding for nitrate reductase enzymes (BdNR1 and BdNR2). CONCLUSIONS: With a short callogenesis stage and streamlined in vitro regeneration following co-cultivation with Agrobacterium, transgenic and edited T0 Bd plantlets can be produced in about 8 weeks, a gain of one to two months compared to previously published methods, with no reduction in transformation efficiency and at lower costs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13007-023-01005-1.
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spelling pubmed-100539782023-03-30 Fast-track transformation and genome editing in Brachypodium distachyon Soulhat, Camille Wehbi, Houssein Fierlej, Yannick Berquin, Patrick Girin, Thomas Hilson, Pierre Bouchabké-Coussa, Oumaya Plant Methods Methodology BACKGROUND: Even for easy-to-transform species or genotypes, the creation of transgenic or edited plant lines remains a significant bottleneck. Thus, any technical advance that accelerates the regeneration and transformation process is welcome. So far, methods to produce Brachypodium distachyon (Bd) transgenics span at least 14 weeks from the start of tissue culture to the recovery of regenerated plantlets. RESULTS: We have previously shown that embryogenic somatic tissues grow in the scutellum of immature zygotic Bd embryos within 3 days of in vitro induction with exogenous auxin and that the development of secondary embryos can be initiated immediately thereafter. Here, we further demonstrate that such pluripotent reactive tissues can be genetically transformed with Agrobacterium tumefaciens right after the onset of somatic embryogenesis. In brief, immature zygotic embryos are induced for callogenesis for one week, co-cultured with Agrobacterium for three days, then incubated on callogenesis selective medium for three weeks, and finally transferred on selective regeneration medium for up to three weeks to obtain plantlets ready for rooting. This 7-to-8-week procedure requires only three subcultures. Its validation includes the molecular and phenotype characterization of Bd lines carrying transgenic cassettes and novel CRISPR/Cas9-generated mutations in two independent loci coding for nitrate reductase enzymes (BdNR1 and BdNR2). CONCLUSIONS: With a short callogenesis stage and streamlined in vitro regeneration following co-cultivation with Agrobacterium, transgenic and edited T0 Bd plantlets can be produced in about 8 weeks, a gain of one to two months compared to previously published methods, with no reduction in transformation efficiency and at lower costs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13007-023-01005-1. BioMed Central 2023-03-29 /pmc/articles/PMC10053978/ /pubmed/36991448 http://dx.doi.org/10.1186/s13007-023-01005-1 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Methodology
Soulhat, Camille
Wehbi, Houssein
Fierlej, Yannick
Berquin, Patrick
Girin, Thomas
Hilson, Pierre
Bouchabké-Coussa, Oumaya
Fast-track transformation and genome editing in Brachypodium distachyon
title Fast-track transformation and genome editing in Brachypodium distachyon
title_full Fast-track transformation and genome editing in Brachypodium distachyon
title_fullStr Fast-track transformation and genome editing in Brachypodium distachyon
title_full_unstemmed Fast-track transformation and genome editing in Brachypodium distachyon
title_short Fast-track transformation and genome editing in Brachypodium distachyon
title_sort fast-track transformation and genome editing in brachypodium distachyon
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10053978/
https://www.ncbi.nlm.nih.gov/pubmed/36991448
http://dx.doi.org/10.1186/s13007-023-01005-1
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