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Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.)

Genetic transformation of perennial ryegrass (Lolium perenne L.) is critical for fundamental and translational research in this important grass species. It often relies on Agrobacterium-mediated transformation of callus tissue. However, callus induction is restricted to a few genotypes that respond...

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Autores principales: Grogg, Daniel, Rohner, Marius, Yates, Steven, Manzanares, Chloe, Bull, Simon E., Dalton, Sue, Bosch, Maurice, Studer, Bruno, Broggini, Giovanni A. L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9370183/
https://www.ncbi.nlm.nih.gov/pubmed/35956532
http://dx.doi.org/10.3390/plants11152054
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author Grogg, Daniel
Rohner, Marius
Yates, Steven
Manzanares, Chloe
Bull, Simon E.
Dalton, Sue
Bosch, Maurice
Studer, Bruno
Broggini, Giovanni A. L.
author_facet Grogg, Daniel
Rohner, Marius
Yates, Steven
Manzanares, Chloe
Bull, Simon E.
Dalton, Sue
Bosch, Maurice
Studer, Bruno
Broggini, Giovanni A. L.
author_sort Grogg, Daniel
collection PubMed
description Genetic transformation of perennial ryegrass (Lolium perenne L.) is critical for fundamental and translational research in this important grass species. It often relies on Agrobacterium-mediated transformation of callus tissue. However, callus induction is restricted to a few genotypes that respond well to tissue culture. Here, we report callus induction from different perennial ryegrass genotypes and explants, such as shoot tips, seeds, and anthers, which were transformed with several plasmids for functional genomics. β-glucuronidase (GUS) histochemical staining showed the LmdsRNAbp promoter sequence was active in stigmas, spikelets, anthers, and leaves. We also transformed calli with plasmids allowing gene silencing and gene knock-out using RNA interference and CRISPR/Cas9, respectively, for which genotypic and phenotypic investigations are ongoing. Using 19 different constructs, 262 transgenic events were regenerated. Moreover, the protocol regenerated a doubled haploid transgenic event from anther-derived calli. This work provides a proof-of-concept method for expanding the range of genotypes amenable to transformation, thus, serving research and breeding initiatives to improve this important grass crop for forage and recreation.
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spelling pubmed-93701832022-08-12 Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.) Grogg, Daniel Rohner, Marius Yates, Steven Manzanares, Chloe Bull, Simon E. Dalton, Sue Bosch, Maurice Studer, Bruno Broggini, Giovanni A. L. Plants (Basel) Article Genetic transformation of perennial ryegrass (Lolium perenne L.) is critical for fundamental and translational research in this important grass species. It often relies on Agrobacterium-mediated transformation of callus tissue. However, callus induction is restricted to a few genotypes that respond well to tissue culture. Here, we report callus induction from different perennial ryegrass genotypes and explants, such as shoot tips, seeds, and anthers, which were transformed with several plasmids for functional genomics. β-glucuronidase (GUS) histochemical staining showed the LmdsRNAbp promoter sequence was active in stigmas, spikelets, anthers, and leaves. We also transformed calli with plasmids allowing gene silencing and gene knock-out using RNA interference and CRISPR/Cas9, respectively, for which genotypic and phenotypic investigations are ongoing. Using 19 different constructs, 262 transgenic events were regenerated. Moreover, the protocol regenerated a doubled haploid transgenic event from anther-derived calli. This work provides a proof-of-concept method for expanding the range of genotypes amenable to transformation, thus, serving research and breeding initiatives to improve this important grass crop for forage and recreation. MDPI 2022-08-05 /pmc/articles/PMC9370183/ /pubmed/35956532 http://dx.doi.org/10.3390/plants11152054 Text en © 2022 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
Grogg, Daniel
Rohner, Marius
Yates, Steven
Manzanares, Chloe
Bull, Simon E.
Dalton, Sue
Bosch, Maurice
Studer, Bruno
Broggini, Giovanni A. L.
Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.)
title Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.)
title_full Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.)
title_fullStr Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.)
title_full_unstemmed Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.)
title_short Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.)
title_sort callus induction from diverse explants and genotypes enables robust transformation of perennial ryegrass (lolium perenne l.)
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9370183/
https://www.ncbi.nlm.nih.gov/pubmed/35956532
http://dx.doi.org/10.3390/plants11152054
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