Protoplast-Based Transient Expression and Gene Editing in Shrub Willow (Salix purpurea L.)

Brennan Hyden; Guoliang Yuan; Yang Liu; Lawrence B. Smart; Gerald A. Tuskan; Xiaohan Yang

doi:10.3390/plants11243490

Protoplast-Based Transient Expression and Gene Editing in Shrub Willow (Salix purpurea L.)

Brennan Hyden, Guoliang Yuan, Yang Liu, Lawrence B. Smart, Gerald A. Tuskan, Xiaohan Yang

Research output: Contribution to journal › Article › peer-review

Abstract

Shrub willows (Salix section Vetrix) are grown as a bioenergy crop in multiple countries and as ornamentals across the northern hemisphere. To facilitate the breeding and genetic advancement of shrub willow, there is a strong interest in the characterization and functional validation of genes involved in plant growth and biomass production. While protocols for shoot regeneration in tissue culture and production of stably transformed lines have greatly advanced this research in the closely related genus Populus, a lack of efficient methods for regeneration and transformation has stymied similar advancements in willow functional genomics. Moreover, transient expression assays in willow have been limited to callus tissue and hairy root systems. Here we report an efficient method for protoplast isolation from S. purpurea leaf tissue, along with transient overexpression and CRISPR-Cas9 mediated mutations. This is the first such report of transient gene expression in Salix protoplasts as well as the first application of CRISPR technology in this genus. These new capabilities pave the way for future functional genomics studies in this important bioenergy and ornamental crop.

Original language	English
Article number	3490
Journal	Plants
Volume	11
Issue number	24
DOIs	https://doi.org/10.3390/plants11243490
State	Published - Dec 2022

Bibliographical note

Publisher Copyright:
© 2022 by the authors.

Funding

This work was supported by the U.S. Department of Energy (DOE) Office of Science Graduate Student Research (SCGSR) Program and the Center for Bioenergy Innovation, a DOE Bioenergy Research Center supported by the Biological and Environmental Research program. Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the U.S. Department of Energy under Contract Number DE-AC05-00OR22725. A Pre-doctoral Research Fellowship from the United States Department of Agriculture National Institute supported BLH for Food and Agriculture (award #2021-67034-35116). This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan ( http://energy.gov/downloads/doe-public-access-plan ).

Funders	Funder number
BLH for Food and Agriculture	2021-67034-35116
DOE Bioenergy Research Center
Office of Science Graduate Student Research
SCGSR
United States Department of Agriculture National Institute
U.S. Department of Energy
Biological and Environmental Research
Oak Ridge National Laboratory	DE-AC05-00OR22725
Center for Bioenergy Innovation

Keywords

Salix
gene editing
protoplasts

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.3390/plants11243490

Cite this

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title = "Protoplast-Based Transient Expression and Gene Editing in Shrub Willow (Salix purpurea L.)",

abstract = "Shrub willows (Salix section Vetrix) are grown as a bioenergy crop in multiple countries and as ornamentals across the northern hemisphere. To facilitate the breeding and genetic advancement of shrub willow, there is a strong interest in the characterization and functional validation of genes involved in plant growth and biomass production. While protocols for shoot regeneration in tissue culture and production of stably transformed lines have greatly advanced this research in the closely related genus Populus, a lack of efficient methods for regeneration and transformation has stymied similar advancements in willow functional genomics. Moreover, transient expression assays in willow have been limited to callus tissue and hairy root systems. Here we report an efficient method for protoplast isolation from S. purpurea leaf tissue, along with transient overexpression and CRISPR-Cas9 mediated mutations. This is the first such report of transient gene expression in Salix protoplasts as well as the first application of CRISPR technology in this genus. These new capabilities pave the way for future functional genomics studies in this important bioenergy and ornamental crop.",

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author = "Brennan Hyden and Guoliang Yuan and Yang Liu and Smart, {Lawrence B.} and Tuskan, {Gerald A.} and Xiaohan Yang",

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doi = "10.3390/plants11243490",

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volume = "11",

journal = "Plants",

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AU - Hyden, Brennan

AU - Yuan, Guoliang

AU - Liu, Yang

AU - Smart, Lawrence B.

AU - Tuskan, Gerald A.

AU - Yang, Xiaohan

PY - 2022/12

Y1 - 2022/12

N2 - Shrub willows (Salix section Vetrix) are grown as a bioenergy crop in multiple countries and as ornamentals across the northern hemisphere. To facilitate the breeding and genetic advancement of shrub willow, there is a strong interest in the characterization and functional validation of genes involved in plant growth and biomass production. While protocols for shoot regeneration in tissue culture and production of stably transformed lines have greatly advanced this research in the closely related genus Populus, a lack of efficient methods for regeneration and transformation has stymied similar advancements in willow functional genomics. Moreover, transient expression assays in willow have been limited to callus tissue and hairy root systems. Here we report an efficient method for protoplast isolation from S. purpurea leaf tissue, along with transient overexpression and CRISPR-Cas9 mediated mutations. This is the first such report of transient gene expression in Salix protoplasts as well as the first application of CRISPR technology in this genus. These new capabilities pave the way for future functional genomics studies in this important bioenergy and ornamental crop.

AB - Shrub willows (Salix section Vetrix) are grown as a bioenergy crop in multiple countries and as ornamentals across the northern hemisphere. To facilitate the breeding and genetic advancement of shrub willow, there is a strong interest in the characterization and functional validation of genes involved in plant growth and biomass production. While protocols for shoot regeneration in tissue culture and production of stably transformed lines have greatly advanced this research in the closely related genus Populus, a lack of efficient methods for regeneration and transformation has stymied similar advancements in willow functional genomics. Moreover, transient expression assays in willow have been limited to callus tissue and hairy root systems. Here we report an efficient method for protoplast isolation from S. purpurea leaf tissue, along with transient overexpression and CRISPR-Cas9 mediated mutations. This is the first such report of transient gene expression in Salix protoplasts as well as the first application of CRISPR technology in this genus. These new capabilities pave the way for future functional genomics studies in this important bioenergy and ornamental crop.

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Protoplast-Based Transient Expression and Gene Editing in Shrub Willow (Salix purpurea L.)

Abstract

Bibliographical note

Funding

Keywords

UN SDGs

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