Summary
An optimized protocol for the electroporation-based transfection of tobacco protoplasts is described that routinely results in transgene expression frequencies approaching 90%. The overall efficiency of the procedure depends collectively on numerous key parameters, including protoplast viability; DNA concentration, purity, and topology; carrier DNA; and electrical conditions such as ionic strength of the electroporation buffer, electric field strength, pulse duration, and capacitance. Individual methodologies that address each one of these parameters are presented in sufficient detail to enable successful reproduction of this method along with notes that describe helpful tips.
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References
Fromm, M., Callis, J., Taylor, L. P., and Walbot, V. (1987) Electroporation of DNA and RNA into plant protoplasts, in Methods in Enzymology, Vol. 153 (Wu, R. and Grossman, L., eds.), Academic Press, London, UK, pp. 351–366.
Joersbo, M. and Brunstedt, J. (1991) Electroporation: mechanism and transient expression, stable transformation and biological effects in plant protoplasts. Physiol. Plant. 81, 256–264.
Bates, G. W. (1994) Genetic transformation of plants by protoplast electroporation. Mol. Biotech. 2, 135–145.
Dillen, W., Van Montagu, M., and Angenon, G. (1998) Electroporation-mediated DNA transfer to plant protoplasts and intact plant tissues for transient gene expression assays, in Cell Biology, Vol. 4 (Celis, J. E., ed.), Academic Press, London, UK, pp. 92–99.
Chen, G. Y., Conner, A. J., Wang, J., Fautrier, A. G., and Field, R. J. (1998) Energy dissipation as a key factor for electroporation of protoplasts. Mol. Biotech. 10, 209–216.
Joersbo, M., Brunstedt, J., and Floto, F. (1990) Quantitative relationship between parameters of electroporation. J. Plant Physiol. 137, 169–174.
Maccarrone, M., Veldink, G. A., Agro, A. F., and Vliegenthart, J. F. G. (1995) Lentil root protoplasts—a transient expression system suitable for coelectroporation of monoclonal antibodies and plasmid molecules. Biochim. Biophys. Acta 1243, 136–142.
Valat, L., Toutain, S., Courtois, N., et al. (2000) GFLV replication in electroporated grapevine protoplasts. Plant Sci. 155, 203–212.
Higo, K. and Higo, H. (1996) Cloning and characterization of the rice CatA catalase gene, a homologue of the maize Cat3 gene. Plant Mol. Biol. 30, 505–521.
Kao, C.-Y., Cocciolone, S. M., Vasil, I. K., and McCarty, D. R. (1996) Localization and interaction of the cis-acting elements for abscisic acid, VIVIPAROUS1, and light activation of the C1 gene of maize. Plant Cell 8, 1171–1179.
Ecker, J. R. and Davis, R. W. (1986) Inhibition of gene expression in plant cells by expression of antisense RNA. Proc. Natl. Acad. Sci. USA 83, 5372–5376.
Teeri, T. H., Patel, G. K., Aspegren, K., and Kauppinen, V. (1989) Chloroplast targeting of neomycin phosphotransferase II with a pea transit peptide in electroporated barley protoplasts. Plant Cell Rep. 8, 187–190.
Fisk, H. J. and Dandekar, A. M. (1998) Nuclear localization of a foreign gene product in tobacco results in increased accumulation due to enhanced stability. Plant Sci. 133, 177–189.
Nagata, T., Okada, K., Kawazu, T., and Takebe, I. (1987) Cauliflower mosaic virus 35 S promoter directs S phase specific expression in plant cells. Mol. Gen. Genet. 207, 242–244.
Salmenkallio, M., Hannus, R., Teeri, T. H., and Kauppinen, V. (1990) Regulation of α-amylase promoter by gibberellic acid and abscisic acid in barley protoplasts transformed by electroporation. Plant Cell Rep. 9, 352–355.
Kao, K. N. and Michayluk, M. R. (1974) A method for high-frequency intergeneric fusion of plant protoplasts. Planta 115, 355–367.
Tyagi, S., Spörlein, B., Tyagi, A. K., Herrmann, R. G., and Koop, H. U. (1989) PEG-and electroporation-induced transformation in Nicotiana tabacum: influence of genotype on transformation frequencies. Theor. Appl. Genet. 78, 287–292.
Fisk, H. J. and Dandekar, A. M. (1993) The introduction and expression of transgenes in plants. Sci. Hortic. 55, 5–36.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15, 473–497.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edit., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 1.21–1.39.
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Fisk, H.J., Dandekar, A.M. (2005). Electroporation: Introduction and Expression of Transgenes in Plant Protoplasts. In: Peña, L. (eds) Transgenic Plants: Methods and Protocols. Methods in Molecular Biology™, vol 286. Humana Press. https://doi.org/10.1385/1-59259-827-7:079
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DOI: https://doi.org/10.1385/1-59259-827-7:079
Publisher Name: Humana Press
Print ISBN: 978-1-58829-263-6
Online ISBN: 978-1-59259-827-4
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