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An Improved Leaf Protoplast System for Highly Efficient Transient Expression in Switchgrass (Panicum virgatum L.)

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Metabolic Pathway Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2096))

Abstract

As a robust perennial C4-type monocot plant and a native species to North America, switchgrass (Panicum virgatum) has been evaluated and designated as a strong candidate bioenergy crop by the U.S. DOE. Although genetic modifications of switchgrass have been used to successfully reduce the recalcitrance of switchgrass biomass for biofuel production, the generation of transgenic switchgrass is still a slow and laborious process. A transient protoplast system can provide an excellent platform to accelerate the selection of genes-of-interest for tailoring switchgrass biomass. However, partially due to the lack of the complete genomic information, the attempts to optimize the transient protoplast system for switchgrass remain scarce. In this chapter, we provide an improved protocol for switchgrass protoplast isolation, increased transformation efficiency using CsCl gradient ultracentrifugation-derived plasmid DNA and extended application of the transient switchgrass protoplast system to analyze protein expression using western blot.

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Acknowledgments

This work was supported by the Center for Direct Catalytic Conversion of Biomass to Biofuels (C3Bio), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0000997. This work was authored in part by Alliance for Sustainable Energy, LLC, the Manager and Operator of the National Renewable Energy Laboratory for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes.

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Authors and Affiliations

  1. Biosciences Center, National Renewable Energy Laboratory, Golden, CO, USA

    Chien-Yuan Lin, Hui Wei, Bryon S. Donohoe & Michael E. Himmel

  2. National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO, USA

    Melvin P. Tucker

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  1. Chien-Yuan Lin
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  2. Hui Wei
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  3. Bryon S. Donohoe
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  4. Melvin P. Tucker
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Correspondence to Michael E. Himmel .

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  1. National Renewable Energy Laboratory, Golden, CO, USA

    Michael E. Himmel

  2. National Renewable Energy Laboratory, Golden, CO, USA

    Yannick J. Bomble

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Lin, CY., Wei, H., Donohoe, B.S., Tucker, M.P., Himmel, M.E. (2020). An Improved Leaf Protoplast System for Highly Efficient Transient Expression in Switchgrass (Panicum virgatum L.). In: Himmel, M., Bomble, Y. (eds) Metabolic Pathway Engineering. Methods in Molecular Biology, vol 2096. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0195-2_6

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  • DOI: https://doi.org/10.1007/978-1-0716-0195-2_6

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