Inoculation of Plants Using Bombardment

Victor Gaba1, Amit Gal‐On1

1 The Volcani Center, Bet Dagan
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16B.3
DOI:  10.1002/9780471729259.mc16b03s00
Online Posting Date:  January, 2006
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This unit describes methods for the construction and use of handheld particle bombardment devices for high‐efficiency inoculation of intact plants with nucleic acids and viruses. The devices accelerate heavy metal particles coated with nucleic acids or viruses into plant tissues and are driven by pressurized gas. They are inexpensive to construct and use, and can be assembled in any laboratory. The equipment enables inoculation with full‐length infectious cDNA, PCR products, virus from sap or a virus preparation, and in vitro viral transcripts. The inoculation of some phloem‐limited RNA viruses is also possible. Additionally, this technology allows for inoculation of large numbers of plants (mass bombardment), inoculation of soft plants that do not survive bombardment inoculation by other means, inoculation in the greenhouse, study of viral recombination in plants, rapid promoter analysis, and monitoring of virus movement using an infectious clone bearing a reporter gene.

Keywords: particle bombardment; gene gun; inoculation of soft‐tissue plants; gold powder; tungsten powder; RNA viruses

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Table of Contents

  • Basic Protocol 1: Assembly of Bombardment Apparatus
  • Basic Protocol 2: Bombardment with cDNA or RNA
  • Alternate Protocol 1: Mass Bombardment Using a HandGun
  • Support Protocol 1: Purification and Preparation of cDNA or RNA Transcript for Bombardment
  • Support Protocol 2: Bombardment with Plant SAP, Virus Preparation, or Viral RNA Extract
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Assembly of Bombardment Apparatus

  • Materials for construction of the discharge assembly (Fig. ):
    • Luer adapter, male luer lock to 1/ 4‐in. (6.35‐mm) unified fine threads (UNF; e.g., Cole‐Parmer cat. no. 31507‐73; a)
    • Luer adapter, male (M) 1/ 4‐in. (6.35‐mm) fitting attached to a machined UNF female thread to accept the luer lock adapter (b)
    • 13‐mm‐diameter plastic filter holder (Pall‐Gelman cat. no. 4312; c)
  • Materials for construction of the Blowpipe apparatus (Fig. ):
    • 7‐mm rubber vacuum hose, ∼5 m (a)
    • Plastic two‐way tap, 8 to 10 mm (b)
    • Barbed fitting to connect rubber tubing to a metal fitting (c)
    • 1/ 4‐in. (6.35‐mm) coupling adapter female piping thread (FPT)/FPT, if required
    • Worm‐driven hose clips for fixing rubber tubing onto metal pipes (d)
  • Materials for construction of the HandGun apparatus:
  • Materials for assembling the HandGun device:
    • Electrically operated miniature solenoid valve (Dynamco cat. no. D3533KL0; Fig. a)
    • Base plate (Dynamco cat. no. B03B2B) with 1/ 4‐in. (6.35‐mm) apertures (Fig. b).
    • 1/ 4‐in. (6.35‐mm) plugs, nonprotruding (Fig. )
    • Pipe‐tubing elbow for connecting the pneumatic hose (Fig. c)
    • 1/ 4‐in. (6.35‐mm) adapter(s) FPT/M: either one ∼40 mm length or two short ones (Fig. d)
    • Clamp stand (Fig. f)
    • Miniature DIN plug (Fig. e)
  • Timer components (Fig. ):
    • Electrical wire, flexible, two‐conductor, ∼6 m
    • 220 V, variable (0 to 100 msec) timer with suitable base (Megatron Electronics and Controls cat. no. MSST‐700‐CPT; a)
    • Electrical push‐button switch (b)
    • Plastic box for mounting the timer with electrical connections (c)
  • Secondary gas regulator components:
    • Pipe‐tubing elbows (Fig. c)
    • Pressure regulator (∼0.05 to 0.85 MPa), with female 1/ 4‐in. (6.35‐mm) fittings (Fig. a)
    • T‐adapter, MFM, 1/ 4, 1/ 2, 1/ 4 in. (6.35, 12.7, 6.35 mm) (Fig. b)
    • 0‐ to 10‐bar (0‐ to 1000‐kPa) gas pressure gauge, 100 mm diameter, 1/ 2‐in. (12.7‐mm) male fitting (Fig. c)
    • 1/ 4‐in. (6.35‐mm) adapter M/M (Fig. b)
    • Two‐way tap; 1/ 4‐in. (6.35‐mm) female fittings (optional; Fig. d)
    • 20‐µm gas line filter with female 1/ 4‐in. (6.35‐mm) fittings (optional)
    • Wall‐mounting bracket
  • Gas cylinder and accessories:
    • Pipe tubing elbow (Fig. c) or fitting to adapt to gas line or a pipe‐tubing elbow fitted directly to the cylinder regulator
    • Gas cylinder (helium or alternate, e.g., nitrogen) with regulator
    • 6‐mm nylon pneumatic hose, ∼6 m
NOTE: Wind each male joint with Teflon tape prior to closing.

Basic Protocol 2: Bombardment with cDNA or RNA

  • 1.25 M Ca(NO 3) 2⋅4H 2O, pH 10.5 or 7.8: adjust pH slowly with HCl, autoclave, aliquot, and store up to 2 years at −20°C
  • Tungsten or gold particle stock (see recipes)
  • cDNA or RNA transcripts in water ( protocol 4) or
  • Plant sap, purified virus, viral RNA extract, or total RNA ( protocol 5)
  • Target plants at correct stage, e.g., cotyledon
  • Microcentrifuge tubes, sterile
  • Blowpipe or HandGun ( protocol 1)
  • Disposable gloves (unit 1.3)
  • 10‐ml syringe
NOTE: Use normal precautions for working with nucleic acids, including sterile solutions and autoclaved utensils.
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Literature Cited

   Engebrecht, J., Brent, R., and Kaderbhai, M.A. 1991. Minipreps of plasmid DNA. 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. 1.6.1‐1.6.10. John Wiley & Sons, Hoboken, N.J.
   Fakhfakh, H., Vilaine, F., Makni, M., and Robaglia, C. 1996. Cell‐free cloning and biolistic inoculation of an infectious cDNA of potato virus Y. J. Gen. Virol. 77:519‐523.
   Finer, J.J., Vain, P., Jones, M.W., and McMullen, M.D. 1992. Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Rep. 11:323‐328.
   Gal‐On, A., Meiri, E., Huet, H., Wu, J.H., Raccah, B., and Gaba, V. 1995. Particle bombardment drastically increases the infectivity of cloned cDNA of zucchini yellow mosaic potyvirus. J. Gen. Virol 76:3223‐3227.
   Gal‐On, A., Meiri, E., Elman, C., Gray, D.J., and Gaba, V. 1997. Simple handheld devices for the efficient infection of plants with viral encoding constructs by particle bombardment. J. Virol. Methods 64:103‐110.
   Gal‐On, A., Meiri, E., Raccah, B., and Gaba, V. 1998. Recombination of engineered defective RNA species produces infective potyvirus in planta. J. Virol. 72:5268‐5270.
   Gray, D.J., Hiebert, E., Lin, C.M., Compton, M.E., McColley, D.W., Harrison, R.J., and Gaba, V. 1994. Simplified construction and performance of a device for particle bombardment. Plant Cell, Tissue Org. Cult. 37: 179‐184.
   Heilig, S., Elbing, K.L., and Brent, R. 1998. Large‐scale preparation of plasmid DNA. 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. 1.7.1‐1.7.16. John Wiley & Sons, Hoboken, N.J.
   Kimalov, B., Gal‐On, A., Stav, R., Belausov, E., and Arazi, T. 2004. Maintenance of coat protein amino terminal net charge is essential for zucchini yellow mosaic virus systemic infectivity. J. Gen. Virol. 85:3421‐3430.
   Klein, T.M., Wolf, E.D., Wu, R., and Sanford, J.C. 1987. High‐velocity microprojectiles for delivering nucleic acids into living cells. Nature 327:70‐73.
   Russell, J.A., Roy, M.K., and Sanford, J.C. 1992. Physical trauma and tungsten toxicity reduce the efficiency of biolistic transformation. Plant Physiol. 98:1050‐1056.
   Shiboleth, Y.M., Arazi, T., Wang, Y., and Gal‐On, A. 2001. A new approach for weed control in a cucurbit field employing an attenuated potyvirus‐vector for herbicide resistance. J. Biotechnol. 92:37‐46.
   Takeuchi, Y., Dotson, M., and Keen, N.T. 1992. Plant transformation: A simple particle bombardment device based on flowing helium. Plant Mol. Biol. 18:835‐839.
   Yang, G., Mawassi, M., Ashoulin, L., Gafny, R., Gaba, V., Gal‐On, A., and Bar‐Joseph, M. 1997. A cDNA clone from a defective RNA of citrus tristeza virus is infective in the presence of the helper virus. J. Gen. Virol. 78:1765‐1769.
   Wang, Y., Gaba, V., Wolf, D., Xia, X.D., Zelcer, A., and Gal On, A. 2000. Identification of a novel plant virus promoter using a potyvirus infectious clone. Virus Genes 20:11‐17.
Key References
   Gal‐On, et al., 1995. See above.
  Particle bombardment as an inoculation method using a vacuum device.
   Gal‐On, et al., 1997. See above.
  Particle bombardment as an inoculation method using handheld devices.
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