Preparation and Electroporation of Oat Protoplasts from Cell Suspension Culture

Aurelie M. Rakotondrafara1, Jacquelyn R. Jackson1, Elizabeth Pettit Kneller1, W. Allen Miller1

1 Iowa State University, Ames, Iowa
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16D.3
DOI:  10.1002/9780471729259.mc16d03s05
Online Posting Date:  June, 2007
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Protoplasts provide a convenient system for introduction of nucleic acids into plant cells. Protoplasts allow rapid assays of gene expression and virus replication, with advantages for plant biology similar to those of cultured animal cells for the study of animal systems. Traditionally, preparation and handling of protoplasts has been as much art as science, requiring a special touch by the user. The purpose of this unit is to lay out in clear detail all the methods and nuances involved in protoplast preparation using a robust, reliable system that does not require skills beyond those expected of an unspecialized molecular biologist. Because dicots and monocots differ in many biological properties, and because different procedures may work better for different plants, separate units in this chapter are devoted to protoplast preparation from dicots (Arabidopsis, tobacco; refer to UNITS 16D.1 & 16D.4) and from a monocot (oat). This unit describes methods for preparation and transfection by electroporation of protoplasts derived from an oat suspension culture.

Keywords: cell suspension culture; oat protoplast; electroporation; transient expression; RNA virus replication

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Table of Contents

  • Basic Protocol 1: Preparation and Electroporation of Oat Protoplasts from Cell Suspension Culture
  • Support Protocol 1: Subculture Oat Suspension Culture
  • Basic Protocol 2: Long‐Term Storage and Shipping of Cell Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
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Basic Protocol 1: Preparation and Electroporation of Oat Protoplasts from Cell Suspension Culture

  • 7‐day‐old oat suspension culture (Avena sativa cv. Stout; see protocol 2)
  • Oat protoplast enzyme solution (see recipe)
  • Artificial sea water (ASW)/0.6 M mannitol (1:1 ratio; see recipe)
  • Oat protoplast electroporation buffer (see recipe)
  • 1 M spermidine: filter sterilize using a 0.2‐µm filter and divide into 1‐ml aliquots (store up to 1 year at –20°C)
  • Murashige and Skoog (MS) medium with 0.4 M mannitol (see recipe)
  • RNA sample
  • 50‐ml conical centrifuge tubes
  • 145 × 20− or 100 × 15–mm sterile plastic petri dishes
  • Gyrotory shaker (e.g., New Brunswick Scientific Model G2)
  • Motorized pipet filler/dispenser (e.g., Eppendorf EasyPet)
  • 10‐ml serological pipets
  • Centrifuge (e.g., Sorvall RC‐5C Plus) with SH‐3000 or comparable swinging‐bucket rotor with inserts for 15‐ml conical tubes
  • 4‐mm electroporation cuvettes
  • 1‐ml wide‐bore polypropylene pipet tips
  • 6‐well cell culture plates
  • Electroporator with square wave pulse (e.g., BTX T820 ElectroSquare Porator or Bio‐Rad GenePulser Xcell with CE module)
  • Additional reagents and equipment for counting cells with a hemacytometer ( appendix 4A)
NOTE: All work should be carried out under sterile conditions. Unless otherwise noted, all solutions should be autoclaved and then stored at 4°C. Equipment which will come in direct contact with the protoplasts should be sterilized before use. Keep solutions sterile at all times.

Support Protocol 1: Subculture Oat Suspension Culture

  • Oat suspension culture (available from the authors)
  • 40 ml Murashige and Skoog (MS) medium (see recipe) in 150‐ml Erlenmeyer flask sealed with cotton and wrapped in aluminum foil
  • Platform shaker (e.g., New Brunswick Scientific Classic Series C24 incubator/shaker)
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Literature Cited

Literature Cited
   Barnett, A., Hammond, J., and Lister, R.M. 1981. Limited infection of Cereal Leaf Protoplasts by Barley yellow dwarf virus J. Gen. Virol. 57:397‐401.
   Birsin, M., Onde, S., and Ozgen, M., 2001. Callus induction and plant regeneration from mature embryos of oat (Avena sativa L.). Turk. L. Biol. 25:427‐434.
   Chen, Z., Klockare, R., and Sundqvist, C. 1994. Origin of somatic embryogenesis is proliferating root primordia in seed derived oat callus. Hereditas 120:211‐216.
   Cocking, E.C. 1960. A method for the isolation of plant protoplasts and vacuoles. Nature 187:962‐963.
   Cocking, E.C. and Pojnar, E. 1969. An electron microscope study of the infection of isolated tomato fruit protoplasts by tobacco mosaic virus. J. Gen. Virol. 4:305‐312.
   Dinesh‐Kumar, S.P., Brault, V., and Miller, W.A. 1992. Precise mapping and in vitro translation of a trifunctional subgenomic RNA of barley yellow dwarf virus. Virology 187:711‐722.
   Fromm, M., Taylor, L.P., and Walbot, V. 1985. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Natl. Acad. Sci. U.S.A. 82:5824‐5828.
   Fromm, M., Callis, J., Taylor, L.P., and Walbot, V. 1987. Electroporation of DNA and RNA into plant protoplasts. Methods Enzymol. 153:351‐366.
   Furusawa, I. and Okuno, T. 1978. Infection with BMV of mesophyll protoplasts isolated from five plant species. J. Gen. Virol. 40:489‐492.
   Hibi, T. 1989. Electrotransfection of plant protoplasts with viral nucleic acids. Adv. Virus Res. 37:329‐342.
   Owens, L.D. 1979. Binding of ColE1‐Kan plasmid DNA by tobacco protoplasts. Plant Physiol. 63:683‐686.
   Samac, D.A., Nelson, S.E., and Loesch‐Fries, S.L. 1983. Virus protein synthesis in alfalfa mosaic virus infected alfalfa protoplasts. Virology 131:455‐462.
   Sander, E. and Mertes, G. 1984. Use of protoplasts and separate cells in plant virus research. Adv. Virus Res. 29:215‐263.
   Shen, R. and Miller, W.A. 2004. Subgenomic RNA as a riboregulator: Negative regulation of RNA replication by Barley yellow dwarf virus subgenomic RNA 2. Virology 327:196‐205.
   Young, M.J., Larkin, P.J., Miller, W.A., Waterhouse, P.M., and Gerlach, W.L. 1989. Infection of Triticum monococcum protoplasts with Barley Yellow Dwarf Virus. J. Gen. Virol. 70:2245‐2251.
   Zheng, Y.Z. and Edwards, M.C. 1990. Expression of resistance to barley stripe mosaic virus in barley and oat protoplasts. J. Gen. Virol. 71:1865‐1868.
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