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Callus Cultures of Arabidopsis
Publication Name:
Current Protocols in Microbiology
Unit Number:
UNIT 16D.1
DOI:
10.1002/9780471729259.mc16d01s00
Online Posting Date:
February, 2006 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
Table of Contents
- Unit Introduction
- 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
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
2 O, 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; Basic 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 16D.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 (Basic 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
2 O, sterile - Microcentrifuge, refrigerated
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Figures
-
Figure 16D.1.1Appearance of callus cultures in sterile 100 × 15–mm petri dishes at several stages of growth. (A) Plant tissue appears green and has not undergone dedifferentiation at 2 weeks into the first passage. (B) At 1 week into the second passage, callus appears green with tinges of yellow and becomes undifferentiated. (C) Callus appears mostly yellow and friable at 1½ weeks into the third passage. This plate contains ~1 g callus and is the earliest time point for digestion of callus into protoplasts. (D) Callus clumps become larger as tissue grows within each passage. This plate is 3 weeks into the third passage and is ready to be split to begin the next passage. Appearance of callus in the fourth through sixth stages should not change.
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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