Isolation, Culture, and Transient Transformation of Plant Protoplasts

Jinbo Shen1, Jiaxin Fu2, Jin Ma3, Xiangfeng Wang1, Caiji Gao1, ChuXiong Zhuang2, Jianmin Wan3, Liwen Jiang4

1 School of Life Sciences, Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong, 2 State Key Laboratory for Conservation and Utilization of Subtropical Agro‐Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou, 3 National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, 4 Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 2.8
DOI:  10.1002/0471143030.cb0208s63
Online Posting Date:  June, 2014
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Abstract

Transient gene expression in protoplasts, which has been used in several plant species, is an important and versatile tool for rapid functional gene analysis, protein subcellular localization, and biochemical manipulations. This unit describes transient gene expression by electroporation of DNA into protoplasts of Arabidopsis or tobacco suspension‐cultured cells and by polyethylene glycol (PEG)‐mediated DNA transformation into protoplasts derived from rice leaf sheaths. PEG‐mediated DNA transformation for transient gene expression in rice protoplasts in suspension culture is also described as an alternative technique. Methods for collecting intracellular and secreted proteins are also provided. Curr. Protoc. Cell Biol. 63:2.8.1‐2.8.17. © 2014 by John Wiley & Sons, Inc.

Keywords: Arabidopsis; tobacco; rice; protoplast isolation; transient expression; electroporation; PEG

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Electroporation into Protoplasts of Arabidopsis and Tobacco Suspension‐Cultured Cells
  • Alternate Protocol 1: PEG‐Mediated Transformation into Protoplasts of Rice Suspension‐Cultured Cells
  • Basic Protocol 2: Generation, Culture, and Transformation of Protoplasts Derived from Rice Plants
  • Support Protocol 1: Harvesting Proteins from the Protoplasts and Culture Medium
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Electroporation into Protoplasts of Arabidopsis and Tobacco Suspension‐Cultured Cells

  Materials
  • 4‐ to 5‐day‐old Arabidopsis PSB‐D suspension‐cultured cells: Arabidopsis Biological Resource Center (ARBC), cat. no. CCL84840 or 2‐ to 3‐day‐old tobacco BY‐2 suspension‐cultured cells: Reiken Bioresource Center (RBC), Experimental Plant Division, cat. no. rpc00001; see Critical Parameters for considerations in growing healthy cells
  • Arabidopsis MS culture medium (see recipe)
  • BY‐2 MS culture medium (see recipe)
  • Arabidopsis and tobacco BY‐2 enzyme solution (see recipe)
  • Electroporation buffer (see recipe)
  • 10 to 40 µg plasmid DNA with transforming DNA of interest (e.g., aleurain‐GFP (Shen et al., 2013), prepared as in Hellig et al. (1998); also see Anticipated Results)
  • Protoplast culture medium (see recipe)
  • Laminar flow hood (e.g., class II biological safety cabinet, Nuaire)
  • 50‐ml conical tubes (e.g., Falcon; BD)
  • Microcentrifuge (e.g., Eppendorf model 5418)
  • 0.22‐µm syringe filter (e.g., Millex‐GP, Millipore)
  • 250‐ml glass flask
  • 25°, 26°, and 27°C incubators
  • Platform shaker (e.g., Innova 49, New Brunswick)
  • Light microscope
  • Centrifuge with swinging bucket rotor (e.g., Eppendorf model 5810R)
  • 1‐ml pipettor and 1‐ml pipet tips (e.g., Axygen Scientific)
  • Pasteur pipet
  • Peristaltic pump (e.g., Millivac, Millipore)
  • 0.4‐cm gap electroporation cuvettes and electroporation apparatus (e.g., Gene Pulser Xcell, Bio‐Rad)
  • 35 × 10–mm petri dishes, tissue culture–treated (Corning)
  • Additional reagents and solutions for performing cell counts with a hemacytometer (unit 1.1)

Alternate Protocol 1: PEG‐Mediated Transformation into Protoplasts of Rice Suspension‐Cultured Cells

  Additional Materials (also see protocol 1)
  • 3‐ to 5‐day‐old rice suspension‐cultured cells (He et al., ), growing in Arabidopsis MS culture medium (see recipe); see Critical Parameters for considerations in growing healthy cells
  • Rice enzyme solution (see recipe)
  • PEG solution (see recipe)
  • W5 solution (see recipe)
  • R2S medium (see recipe)
  • 27° and 30°C incubators
  • 50‐µm nylon mesh (e.g., Energy Filtration Co.)
  • 90‐mm petri dishes (e.g., Sterilin, Thermo Scientific)

Basic Protocol 2: Generation, Culture, and Transformation of Protoplasts Derived from Rice Plants

  Materials
  • Rice seedlings: National Institute of Agrobiological Sciences (NAIS) Genebank, kitaake variant (Oryza sativa ssp. japonica) recommended (see Critical Parameters)
  • W5 solution (see recipe)
  • Rice leaf sheaths enzyme solution (see recipe)
  • MMg solution (see recipe)
  • 1 µg/µl plasmid DNA with transforming DNA of interest [e.g., aleurain‐GFP (Shen et al., 2013), prepared as in Hellig et al. (1998); also see Anticipated Results]
  • PEG solution (see recipe)
  • W5 solution (see recipe)
  • WI solution (see recipe)
  • Razor blade
  • Whatman qualitative filter paper, Grade 1 (e.g., Fisher Scientific)
  • 25‐ml glass flasks
  • Fine plastic Pasteur pipet (e.g., Samco, Thermo Scientific)
  • 28°C incubator
  • 50‐ml conical tubes (e.g., Falcon; BD)
  • 50‐µm nylon mesh (e.g., Energy Filtration Co.)
  • Centrifuge with swinging bucket rotor for 50‐ml conical tubes (e.g., Eppendorf 5810)
  • 1‐ml top‐cut pipet tips (Axygen Scientific)
  • 35 mm × 10–mm petri dishes, tissue culture–treated (Corning)
  • Additional reagents and equipment for counting cells with a hemacytometer (unit 1.1)

Support Protocol 1: Harvesting Proteins from the Protoplasts and Culture Medium

  Materials
  • 100% trichloroacetic acid (TCA; e.g., Sigma‐Aldrich)
  • 10 mg/ml bovine serum albumin (BSA; e.g., Sigma‐Aldrich)
  • SDS sample buffer ( appendix 2A)
  • Acetone (e.g., VWR)
  • 250 mM NaCl
  • 5× extraction buffer (see recipe)
  • 5× extraction buffer with appropriate denaturing (e.g., sodium dodecyl sulfate) or nondenaturing (e.g., Triton X‐100) detergents
  • 15‐ml conical, plastic centrifuge tube (e.g., Falcon, BD)
  • Centrifuge with swinging bucket rotor for 15‐ml conical tubes (e.g., Eppendorf 5810)
  • Glass needle, sharpened by pulling the end of a Pasteur pipet to a fine tip on a small gas burner or spirit lamp
  • 1.5‐ml centrifuge microcentrifuge tube
  • Pasteur pipet
  • Peristaltic pump
  • 1‐ml syringe and 25‐gauge 5/8‐in. needle (e.g., Terumo, cat. no. SS‐01T2516)
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Figures

Videos

Literature Cited

Literature Cited
  Andreason, G.L. and Evans, G.A. 1989. Optimization of electroporation for transfection of mammalian cell lines. Anal. Biochem. 180:269‐275.
  Assmann, S.M., Simoncini, L., and Schroeder, J.I. 1985. Blue‐light activates electrogenic ion pumping in guard‐cell protoplasts of Vicia faba. Nature 318:285‐287.
  Boston, R.S., Becwar, M.R., Ryan, R.D., Goldsbrough, P.B., Larkins, B.A., and Hodges, T.K. 1987. Expression from heterologous promoters in electroporated carrot protoplasts. Plant Physiol. 83:742‐746.
  Cai, Y., Jia, T., Lam, S.K., Ding, Y., Gao, C., San, M.W.Y., Pimpl, P., and Jiang, L. 2011. Multiple cytosolic and transmembrane determinants are required for the trafficking of SCAMP1 via an ER–Golgi–TGN–PM pathway. Plant J. 65:882‐896.
  Calvin, N.M. and Hanawalt, P.C. 1988. High‐efficiency transformation of bacterial cells by electroporation. J. Bacteriol. 170:2796‐2801.
  Delorme, E. 1989. Transformation of Saccharomyces cerevisiae by electroporation. Appl. Environ. Microbiol. 55:2242‐2246.
  Ding, Y., Wang, J., Chun Lai, J.H., Ling Chan, V.H., Wang, X., Cai, Y., Tan, X., Bao, Y., Xia, J., Robinson, D.G., and Jiang, L. 2013. Exo70E2 is essential for exocyst subunit recruitment and for EXPO formation in both plants and animals. Mol. Biol. Cell 25:412‐426.
  Feher, A., Felfoldi, K., Preiszner, J., and Dudits, D. 1991. PEG‐mediated transformation of leaf protoplasts of Solanum tuberosum L cultivars. Plant Cell Tissue Org. Cult. 27:105‐114.
  Fromm, M.E., Taylor, L.P., and Walbot, V. 1986. Stable transformation of maize after gene‐transfer by electroporation. Nature 319:791‐793.
  Gao, C.J., Yu, C.K.Y., Qu, S., San, M.W.Y., Li, K.Y., Lo, S.W., and Jiang, L.W. 2012. The golgi‐localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and golgi retention signals at its C terminus. Plant Cell 24:2086‐2104.
  Harrison, M.J., Choudhary, A.D., Dubery, I., Lamb, C.J., and Dixon, R.A. 1991. Stress responses in alfalfa (Medicago Sativa L) .8. Cis‐elements and trans‐acting factors for the quantitative expression of a bean chalcone synthase gene promoter in electroporated alfalfa protoplasts. Plant Mol. Biol. 16:877‐890.
  Hayashimoto, A., Li, Z.J., and Murai, N. 1990. A polyethylene glycol‐mediated protoplast transformation system for production of fertile transgenic rice plants. Plant Physiol. 93:857‐863.
  He, C., Wang, L., Liu, J., Liu, X., Li, X., Ma, J., Lin, Y., and Xu, F. 2013. Evidence for ‘silicon’ within the cell walls of suspension‐cultured rice cells. New Phytol. 200:700‐709.
  Heilig, J.S., Karen, L., Elbing, K.L., and Brent, R. 1998. Large‐scale preparation of plasmid DNA. Curr. Protoc. Mol. Biol. 41:1.7.1‐1.7.16.
  Jacobsen, J.V. and Beach, L.R. 1985. Control of transcription of alpha‐amylase and ribosomal‐RNA genes in barley aleurone protoplasts by gibberellin and abscisic acid. Nature 316:275‐277.
  Jia, T.R., Gao, C.J., Cui, Y., Wang, J.Q., Ding, Y., Cai, Y., Ueda, T., Nakano, A., and Jiang, L.W. 2013. ARA7(Q69L) expression in transgenic Arabidopsis cells induces the formation of enlarged multivesicular bodies. J. Exp. Bot. 64:2817‐2829.
  Kost, B., Galli, A., Potrykus, I., and Neuhaus, G. 1995. High‐efficiency transient and stable transformation by optimized DNA microinjection into Nicotiana tabacum protoplasts. J. Exp. Bot. 46:1157‐1167.
  Lam, S.K., Siu, C.L., Hillmer, S., Jang, S., An, G.H., Robinson, D.G., and Jiang, L.W. 2007. Rice SCAMP1 defines clathrin‐coated, trans‐Golgi‐located tubular‐vesicular structures as an early endosome in tobacco BY‐2 cells. Plant Cell 19:296‐319.
  Li, J.F., Chung, H.S., Niu, Y.J., Bush, J., McCormack, M., and Sheen, J. 2013. Comprehensive protein‐based artificial microRNA screens for effective gene silencing in plants. Plant Cell 25:1507‐1522.
  Lipphardt, S., Brettschneider, R., Kreuzaler, F., Schell, J., and Dangl, J.L. 1988. UV‐inducible transient expression in parsley protoplasts identifies regulatory cis‐elements of a chimeric Antirrhinum majus chalcone synthase gene. EMBO J. 7:4027‐4033.
  Manavella, P.A. and Chan, R.L. 2009. Transient transformation of sunflower leaf discs via an Agrobacterium‐mediated method: Applications for gene expression and silencing studies. Nat. Protoc. 4:1699‐1707.
  Mathur, J., Koncz, C., and Szabados, L. 1995. A simple method for isolation, liquid culture, transformation and regeneration of Arabidopsis thaliana protoplasts. Plant Cell Rep. 14:221‐226.
  Miao, Y. and Jiang, L. 2007. Transient expression of fluorescent fusion proteins in protoplasts of suspension‐cultured cells. Nat. Protoc. 2:2348‐2353.
  Miao, Y., Yan, P.K., Kim, H., Hwang, I., and Jiang, L. 2006. Localization of green fluorescent protein fusions with the seven Arabidopsis vacuolar sorting receptors to prevacuolar compartments in tobacco BY‐2 cells. Plant Physiol. 142:945‐962.
  Mitra Mazarei, H.A.‐A., Rudis, M.R., and Stewart, C.N. Jr. 2008. Protoplast isolation and transient gene expression in switchgrass, Panicum virgatum L. Biotechnol. J. 3:354‐359.
  Ohnuma, M., Yokoyama, T., Inouye, T., Sekine, Y., and Tanaka, K. 2008. Polyethylene glycol (PEG)‐mediated transient gene expression in a red alga, Cyanidioschyzon merolae 10D. Plant Cell Physiol. 49:117‐120.
  Paris, N., Saint‐Jean, B., Faraco, M., Krzeszowiec, W., Dalessandro, G., Neuhaus, J.M., and Di Sansebastiano, G.P. 2010. Expression of a glycosylated GFP as a bivalent reporter in exocytosis. Plant Cell Rep. 29:79‐86.
  Qi, P., Lin, Y.S., Song, X.J., Shen, J.B., Huang, W., Shan, J.X., Zhu, M.Z., Jiang, L.W., Gao, J.P., and Lin, H.X. 2012. The novel quantitative trait locus GL3.1 controls rice grain size and yield by regulating Cyclin‐T1;3. Cell Res. 22:1666‐1680.
  Rasmussen, J.O. and Rasmussen, O.S. 1993. PEG mediated DNA uptake and transient gus expression in carrot, rapeseed, and soybean protoplasts. Plant Sci. 89:199‐207.
  Sheen, J. 2001. Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol. 127:1466‐1475.
  Shen, J., Suen, P.K., Wang, X., Lin, Y., Lo, S.W., Rojo, E., and Jiang, L. 2013a. An in vivo expression system for the identification of cargo proteins of vacuolar sorting receptors in Arabidopsis culture cells. Plant J. 1003‐1017.
  Shen, J., Zeng, Y., Zhuang, X., Sun, L., Yao, X., Pimpl, P., and Jiang, L. 2013b. Organelle pH in the Arabidopsis endomembrane system. Mol. Plant 1419‐1437.
  Sparkes, I.A., Runions, J., Kearns, A., and Hawes, C. 2006. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protoc. 1:2019‐2025.
  Stefano, G., Renna, L., Chatre, L., Hanton, S.L., Moreau, P., Hawes, C., and Brandizzi, F. 2006. In tobacco leaf epidermal cells, the integrity of protein export from the endoplasmic reticulum and of ER export sites depends on active COPI machinery. Plant J. 46:95‐110.
  Ueki, S., Lacroix, B., Krichevsky, A., Lazarowitz, S.G., and Citovsky, V. 2009. Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment. Nat. Protoc. 4:71‐77.
  Wang, H. and Jiang, L.W. 2011. Transient expression and analysis of fluorescent reporter proteins in plant pollen tubes. Nat. Protoc. 6:419‐426.
  Wang, J., Ding, Y., Wang, J., Hillmer, S., Miao, Y., Lo, S.W., Wang, X., Robinson, D.G., and Jiang, L. 2010. EXPO, an exocyst‐positive organelle distinct from multivesicular endosomes and autophagosomes, mediates cytosol to cell wall exocytosis in Arabidopsis and tobacco cells. Plant Cell 22:4009‐4030.
  Wang, S.C., Tiwari, S.B., Hagen, G., and Guilfoyle, T.J. 2005. AUXIN RESPONSE FACTOR7 restores the expression of auxin‐responsive genes in mutant Arabidopsis leaf mesophyll protoplasts. Plant Cell 17:1979‐1993.
  Worley, C.K., Zenser, N., Ramos, J., Rouse, D., Leyser, O., Theologis, A., and Callis, J. 2000. Degradation of Aux/IAA proteins is essential for normal auxin signalling. Plant J. 21:553‐562.
  Wu, F.S. and Feng, T.Y. 1999. Delivery of plasmid DNA into intact plant cells by electroporation of plasmolyzed cells. Plant Cell Rep. 18:381‐386.
  Yanagisawa, S., Yoo, S.D., and Sheen, J. 2003. Differential regulation of EIN3 stability by glucose and ethylene signalling in plants. Nature 425:521‐525.
  Yoo, S.D., Cho, Y.H., and Sheen, J. 2007. Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nat. Protoc. 2:1565‐1572.
  Zhuang, X., Wang, H., Lam, S.K., Gao, C., Wang, X., Cai, Y., and Jiang, L.W. 2013. A BAR‐domain protein SH3P2, which binds to phosphatidylinositol 3‐phosphate and ATG8, regulates autophagosome formation in Arabidopsis. Plant Cell 25:4596‐4615.
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