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Recombinant DNA technology called transgenesis is often used to add a foreign gene or remove a detrimental gene from an organism. Such genetically modified organisms are called transgenic organisms.
The first-ever transgenic plant was a tobacco plant developed in 1983 that showed resistance against the tobacco mosaic virus. Since then, many transgenic plants have been developed and commercialized for improving the agricultural, ornamental, and horticultural value of a crop plant. Transgenic...
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A Robotic Platform for High-throughput Protoplast Isolation and Transformation
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An Improved Leaf Protoplast System for Highly Efficient Transient Expression in Switchgrass (Panicum virgatum L.).

Chien-Yuan Lin1, Hui Wei1, Bryon S Donohoe1

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

Methods in Molecular Biology (Clifton, N.J.)
|July 29, 2020
PubMed
Summary

This study improves switchgrass (Panicum virgatum) protoplast isolation and transformation efficiency. The optimized transient system accelerates the selection of genes for bioenergy crop development.

Keywords:
CsCl gradient ultracentrifugationGFP expressionProtoplastSwitchgrassTransient expression system

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Area of Science:

  • Agricultural Science
  • Plant Biotechnology
  • Bioenergy Research

Background:

  • Switchgrass (Panicum virgatum) is a North American C4 monocot and a leading bioenergy crop candidate.
  • Genetic modification of switchgrass is crucial for biofuel production but remains slow and challenging.
  • Optimizing transient protoplast systems is vital for accelerating gene discovery in switchgrass.

Purpose of the Study:

  • To develop an improved protocol for switchgrass protoplast isolation and transformation.
  • To enhance the efficiency of transient gene expression analysis in switchgrass.
  • To facilitate the selection of genes for tailoring switchgrass biomass for bioenergy applications.

Main Methods:

  • Optimized protocol for switchgrass protoplast isolation.
  • Utilized CsCl gradient ultracentrifugation-derived plasmid DNA for increased transformation efficiency.
  • Applied western blot analysis to assess protein expression in transiently transfected protoplasts.

Main Results:

  • Successfully improved switchgrass protoplast isolation efficiency.
  • Achieved higher transformation efficiency using purified plasmid DNA.
  • Demonstrated the utility of the transient protoplast system for protein expression analysis.

Conclusions:

  • The enhanced transient protoplast system significantly accelerates gene function studies in switchgrass.
  • This optimized system provides a valuable platform for developing improved bioenergy crops.
  • Further research can leverage this protocol for rapid genetic screening and trait development in switchgrass.