Preparation and Inoculation of Mesophyll Protoplasts from Monocotyledenous and Dicotyledenous Hosts

A.L.N. Rao1

1 University of California, Riverside, Riverside, California
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16D.2
DOI:  10.1002/9780471729259.mc16d02s4
Online Posting Date:  February, 2007
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Abstract

This unit describes a simple and efficient protocol for inoculating mesophyll protoplasts of either monocotyledonous or dicotyledenous host plants using polyethylene glycol. The unit covers isolation of biologically active protoplasts, their transfection with viral RNA, and conditions for the maintenance of transfected protoplasts. It also highlights critical aspects of these steps and discusses common problems and their solutions.

Keywords: Mesophyll protoplasts; barley protoplasts; tobacco protoplasts; infection with viral RNA

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of Mesophyll Protoplasts from Monocotyledenous Hosts
  • Basic Protocol 2: Isolation of Mesophyll Protoplasts from Tobacco (Nicotiana benthamiana) Leaves
  • Basic Protocol 3: Polyethylene Glycol–Mediated Inoculation of Protoplasts with Viral RNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Mesophyll Protoplasts from Monocotyledenous Hosts

  Materials
  • 5‐ to 6‐day‐old barley leaves
  • Enzyme solution for barley (see recipe)
  • 0.55 M sucrose (see recipe)
  • 0.55 M mannitol, pH 5.9 (see recipe)
  • 5 mg/ml fluorescein diacetate: dissolve 5 mg flourescein diacetate (Sigma) in 1 ml acetone; store in an opaque container for up to 6 months at 4°C
  • 28°C incubator
  • 100‐ml beakers
  • 300‐ to 350‐µm gauze
  • 15‐ and 50‐ml polypropylene tubes
  • Beckman Model J6B centrifuge (preferred) or clinical tabletop centrifuge
  • Large‐bore pipet
  • Microscope equipped with UV light source (also see unit 2.1)
  • Additional reagents and solutions for counting cells in a hemacytometer ( appendix 4A)

Basic Protocol 2: Isolation of Mesophyll Protoplasts from Tobacco (Nicotiana benthamiana) Leaves

  Materials
  • 3‐week‐old N. benthamiana leaves
  • 0.55 M mannitol, pH 5.9 (see recipe)
  • Carborundum powder (Fisher)
  • Enzyme solution for tobacco (see recipe)
  • 100 × 15‐mm petri dish
  • Cotton swabs
  • 28°C incubator
  • Additional reagents and equipment for preparing the leaves for protoplasts extraction and removing and counting the protoplasts ( protocol 1, steps 4 to 20)

Basic Protocol 3: Polyethylene Glycol–Mediated Inoculation of Protoplasts with Viral RNA

  Materials
  • Protoplasts in 15‐ml polypropylene tubes ( protocol 1 or protocol 22)
  • Viral RNA or transcripts (unit 16.2)
  • 40% polyethylene glycol (PEG; see recipe)
  • 0.55 M mannitol, pH 5.9 (see recipe)
  • Culture medium for mesophyll protoplasts (see recipe)
  • 24‐well plate (Corning)
  • Fluorescent lamp
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Figures

Videos

Literature Cited

Literature Cited
   Hall, R.D. 1991. The initiation and maintenance of plant cell suspension cultures. In Plant Tissue Culture Manual (K. Lindsey, ed.) A3:1‐21. Kluwer Academic Publishers, Dordrecht, Netherlands.
   Hibi, T., Rezelman, G., and Van Kamen, A. 1975. Infection of cowpea mesophyll protoplasts with cowpea mosaic virus. Virology 64:308‐318.
   Howell, S.H. and Hull, R. 1978. Replication of cauliflower mosaic virus and transcription of its genome in turnip leaf protoplasts. Virology 86:468‐481.
   Loesch‐Fires, L.S. and Hall, T.C., 1980. Synthesis, accumulation and encapsidation of individual brome mosaic virus RNA components in barley protoplasts. J. Gen. Virol. 47:323‐332.
   Maule, A.J., Boulton, M.I., and Wood, K.R. 1980. Poyethylene glycol mediated infection of cucumber protoplasts by cucumber mosaic virus and RNA. J. Gen. Virol. 47:199‐203.
   Motoyoshi, F. and Oshima, N. 1979. Standardization in inoculation procedure and effect of a resistance gene on infection of tomato protoplasts with tobacco mosaic virus RNA. J. Gen. Virol. 44:801‐806.
   Rao, A.L.N., Duggal, R., Lahser, F., and Hall, T.C. 1994. Analysis of RNA replication in plant viruses. In Methods in Molecular Genetics: Molecular Virology Techniques (K.W. Adolph, ed.) Vol. 4, pp. 216‐236. Academic Press, Orlando, Fla.
   Takabe, I. and Otsuki, Y. 1969. Infection of tobacco mesophyll protoplasts by tobacco mosaic virus. Proc. Natl. Acad. Sci. U.S.A. 64:843‐848.
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