Callus Cultures of Arabidopsis

John C. McCormack1, Anne E. Simon1

1 University of Maryland, College Park, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16D.1
DOI:  10.1002/9780471729259.mc16d01s00
Online Posting Date:  January, 2006
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Abstract

Protoplasts are plant cells lacking cell walls. They can be generated from stationary callus cultures derived from Arabidopsis thaliana seedlings. After treatment of the callus with cellulase and pectinase, protoplasts are inoculated with viral RNAs using polyethylene glycol. After various times postinoculation, total RNA is extracted and subjected to electrophoresis on nondenaturing agarose gels for further analysis. Stationary callus cultures are simpler to maintain than more traditional suspension cultures and yield high‐quality, uniform protoplasts. Protoplasts prepared in this fashion can also be used for uptake of DNA.

Keywords: protoplasts; callus cultures; Arabidopsis thaliana; RNA virus replication; plant viruses

     
 
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Table of Contents

  • Basic Protocol 1: Culturing of Arabidopsis Callus
  • Basic Protocol 2: Preparation and Inoculation of Callus Culture Protoplasts with Infectious Viral RNA using Polyethylene Glycol
  • Basic Protocol 3: Extraction of Total RNA from Arabidopsis Protoplasts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Culturing of Arabidopsis Callus

  Materials
  • Arabidopsis thaliana (ecotype Col‐0) seeds
  • 70% ethanol
  • Bleach containing 4% to 6% sodium hypochlorite
  • 10% (w/v) sodium dodecyl sulfate (SDS; appendix 2A)
  • H 2O, sterile
  • Callus maintenance medium (CM) 1% agar plates (see recipe)
  • 1.5‐ml microcentrifuge tubes, sterile
  • Forceps, sterile
  • 20°C incubator with light control (e.g., Percival Scientific I‐36LL; http://www.percival‐scientific.com)

Basic Protocol 2: Preparation and Inoculation of Callus Culture Protoplasts with Infectious Viral RNA using Polyethylene Glycol

  Materials
  • 0.6 M mannitol, room temperature and 4°C
  • Arabidopsis callus (in the third to sixth passage; protocol 1)
  • Protoplast inoculation medium (PIM; see recipe)
  • Cellulase, Trichoderma viride (10 KU/g dry weight; Calbiochem)
  • Pectinase, Rhizopus sp. (3 KU/g dry weight; Calbiochem)
  • RNA of interest: transcribe in vitro (see unit 16.4)
  • 1.0 M CaCl 2
  • 50% polyethylene glycol (PEG; see recipe)
  • 0.6 M mannitol containing 1 mM CaCl 2, 4°C
  • Protoplast culture medium (PCM; see recipe)
  • 14.6‐cm Pasteur pipet: melt into L‐shape
  • Rotating shaker
  • 50‐ml polypropylene centrifuge tubes, sterile
  • 125‐ml glass bottles: sterilize by autoclaving
  • Refrigerated centrifuge with bucket rotor appropriate for 50‐ml tubes
  • Funnel: sterilze by autoclaving
  • 53‐µm nylon mesh (Small Parts): sterilize by autoclaving

Basic Protocol 3: Extraction of Total RNA from Arabidopsis Protoplasts

  Materials
  • Inoculated protoplasts ( protocol 2)
  • 1:1 phenol/chloroform (Tris‐buffered phenol; appendix 2A)
  • RNA extraction buffer (see recipe)
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 100% and 70% ethanol
  • H 2O, sterile
  • Microcentrifuge, refrigerated
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Figures

Videos

Literature Cited

Literature Cited
   Brown, T., Mackey, K., and Du, T. 2004. Analysis of RNA by northern and slot blot hybridization. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 4.9.1‐4.9.14. John Wiley & Sons, Hoboken, N.J.
   Cocking, E.C. 1960. Method for the isolation of plant protoplasts and vacuoles. Nature 187:927‐929.
   Gallagher, S.R. 2004. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. A.3D.1‐A.3D.12. John Wiley & Sons, Hoboken, N.J.
   Kreps, J.A. and Simon, A.E. 1997. Environmental and genetic effects on circadian clock‐regulated gene expression in arabidopsis. Plant Cell 9:297‐304.
   Nagy, P.D., Pogany, J., and Simon, A.E. 1999. RNA elements required for RNA recombination function as replication enhancers in vitro and in vivo in a plus strand RNA virus. EMBO J. 18:5653‐5665.
   Takebe, I., Otsuki, Y., and Aoki, S. 1968. Isolation of tobacco mesophyll cells in intact and active state. Plant Cell Physiol. 9:115‐124.
   Wang, J. and Simon, A.E. 1997. Analysis of the two subgenomic RNA promoters for turnip crinkle virus in vivo and in vitro. Virology 232:174‐186.
   Zhang, F. and Simon, A.E. 2003. A novel procedure for the localization of viral RNAs in protoplasts and whole plants. Plant J. 35:665‐673.
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