An Improved Transient Expression System Using Arabidopsis Protoplasts

Yangrong Cao1, Hong Li1, An Q. Pham2, Gary Stacey2

1 State Key Lab of Agricultural Microbiology, College of Life Science Technology, Huazhong Agricultural University, Wuhan, Hubei, 2 Division of Plant Science, National Center for Soybean Biotechnology, University of Missouri, Columbia, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20013
Online Posting Date:  June, 2016
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Abstract

Transient gene expression in protoplasts provides a powerful tool to study protein expression, protein localization, protein‐protein association, and gene expression regulation, etc. There are several methods including electroporation, which have been reported to introduce DNA into protoplasts. However, one of the best methods used is polyethylene glycol (PEG)‐mediated transfection. Here, we describe an improved PEG‐mediated transformation method including preparation of protoplasts, PEG‐mediated transformation, and, by way of example, expression of the AtLYK5 gene (AT2G33580) in protoplasts. The protoplast transient expression system provides unique capabilities to support cell‐based experiments involved in plant biochemistry and physiology. © 2016 by John Wiley & Sons, Inc.

Keywords: Arabidopsis; protoplast; transient expression

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Protoplast Isolation
  • Basic Protocol 2: PEG Transformation of Protoplasts
  • Basic Protocol 3: Example: Protoplast Transformation and Expression of the AtLYK5 Gene
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Protoplast Isolation

  Materials
  • Arabidopsis Columbia (Col‐0) plants
  • Sunshine soil mix (or other suitable potting mix)
  • Enzyme solution (see recipe)
  • W5 solution (see recipe)
  • Growth chamber or greenhouse
  • Razor blades
  • 100 μm nylon mesh
  • 50‐ml Corning tubes
  • Swinging‐bucket centrifuge

Basic Protocol 2: PEG Transformation of Protoplasts

  Materials
  • Protoplast solution prepared in protocol 1
  • Mg/Ca solution (see recipe)
  • Plasmid DNA (see recipe)
  • PEG/Ca solution (see recipe)
  • W5 solution (see recipe)
  • 50‐ml Corning tubes
  • Centrifuge
  • 6‐well plate
  • Growth chamber

Basic Protocol 3: Example: Protoplast Transformation and Expression of the AtLYK5 Gene

  Materials
  • pUC‐GW14‐LYK5 (Cao et al., )
  • PureYield Plasmid Miniprep System (Promega)
  • Protoplasts solution prepared in protocol 1
  • Protoplasts transformation prepared in protocol 2
  • Protein lysis buffer (see recipe)
  • Ice
  • SDS‐PAGE gel running and transfer system (BioRad)
  • 5× SDS electrophoresis loading buffer (see recipe)
  • SDS electrophoresis running buffer (see recipe)
  • Transfer buffer (see recipe)
  • Anti‐HA‐peroxidase antibody (Roche)
  • SuperSignal Wes Pico Chemiluminescent Substrate (Thermo Scientific)
  • NanoDrop spectrophotometer (Thermo Scientific)
  • Growth chamber
  • Centrifuge
  • 2‐ml tubes
  • Electrophoresis apparatus
  • PVDF membrane
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Figures

Videos

Literature Cited

Literature Cited
  Abel, S. and Theologis, A. 1994. Transient transformation of Arabidopsis leaf protoplasts: A versatile experimental system to study gene expression. Plant J. 5:421‐427. doi: 10.1111/j.1365‐313X.1994.00421.x.
  Armstrong, C.L., Petersen, W.L., Buchholz, W.G., Bowen, B.A., and Sulc, S.L. 1990. Factors affecting PEG‐mediated stable transformation of maize protoplasts. Plant Cell Rep. 9:335‐339. doi: 10.1007/BF00232864.
  Bates, G.W. 1994. Genetic transformation of plants by protoplast electroporation. Mol. Biotech. 2:135‐145. doi: 10.1007/BF02824806.
  Cao, Y., Liang, Y., Tanaka, K., Nguyen, C.T., Jedrzejczak, R.P., Joachimiak, A., and Stacey, G. 2014. The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin‐induced complex with related kinase CERK1. Elife 3:e03766. doi: 10.7554/eLife.03766.
  Compton, M.E., Saunders, J.A., and Veilleux, R.E. 2000. Use of protoplasts for plant improvement. In Plant Tissue Culture Concepts and Laboratory Exercises, 2nd Ed. pp. 249‐261.
  Nishiguchi, M., Sato, T., and Motoyoshi, F. 1987. An improved method for electroporation in plant protoplasts: Infection of tobacco protoplasts by tobacco mosaic virus particles. Plant Cell Rep. 6:90‐93.
  Pröls, M., Töpfer, R., Schell, J., and Steinbib, H.H. 1988. Transient gene expression in tobacco protoplasts: I. time course of CAT appearance. Plant Cell Rep. 7:221‐224. doi: 10.1007/BF00272528.
  Zhang, Y., Su, J., Duan, S., Ao, Y., Dai, J., Liu, J., Wang, P., Li, Y., Liu, B., Feng, D., Wang, J., and Wang, H. 2011. A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast‐related processes. Plant Methods 7:30. doi: 10.1186/1746‐4811‐7‐30.
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