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Electroporation of Tobacco Leaf Protoplasts Using Plasmid DNA or Total Genomic DNA

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Plant Cell Electroporation and Electrofusion Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 55))

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Abstract

Direct gene transfer into tobacco leaf protoplasts via electroporation is used in many laboratories for transient-expression studies. Protoplasts in general are a convenient model to study events that occur in planta rapidly. Transient expression provides answers in a matter of days instead of the several months that would be needed if the same experiment was carried out with transgenic plants. Examples include: elicitor regulation of a plant defense gene (1), UV induction of chalcone synthase (2), induction of an Adh1 promoter by low concentrations of oxygen (3), hormone response (4,5), transactivation of a promoter by cotransfection with two plasmids, one encoding a transcription factor and the other one carrying its target promoter in front of a reporter gene (6), homologous recombination (7), and nuclear targeting (8,9). However it must be noted that although in the above examples transient expression is mimicking what can be observed in stable transformants, there are cases reported where there are noticeable differences between the two approaches. For example, the strictly seed-specific gene encoding soybean β-conglycinin is expressed in tobacco mesophyll protoplasts, but the sequences responsible for that expression are different from those controlling expression in stable-transformation experiments (10). The same situation has been suggested for the promoter of the pathogenesis-related 1a protein gene (11).

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References

  1. Dron, M., Cloux, S. D., Dixon, R. A., Lawton, M. A., and Lamb, C. J. (1988) Glutathione and fungal elicitor regulation of a plant defense gene promoter in electroporated protoplasts. Proc. Natl. Acad. Sci. USA 85, 6738–6742.

    Article  PubMed  CAS  Google Scholar 

  2. Shulze-Lefert, P., Becker, A. M., Shulze, W., Hahlbrock, K., and Dangl, J. (1989) Functional architechture of the light-responsive chalcone synthase promoter from parsley. Plant Cell 1, 707–714.

    Article  Google Scholar 

  3. Walker, J. C., Howard, E. S., Dennis, E. S., and Peacock, W. J. (1987) DNA sequences required for anaerobic expression of the maize alcohol dehydrogenase 1 gene. Proc. Natl. Acad. Sci. USA 84, 6624–6628.

    Article  PubMed  CAS  Google Scholar 

  4. Marcotte, W. R., Bayley, C. C., and Quatrano, R. S. (1988) Regulation of a wheat promoter by abscissic acid in rice protoplasts. Nature 335, 454–457.

    Article  CAS  Google Scholar 

  5. Huttly, A. K. and Baulcombe, D. C. (1989) A wheat alpha-amylase2 promoter is regulated by gibberellin in transformed oat aleurone protoplasts. EMBO J. 8, 1907–1913.

    PubMed  CAS  Google Scholar 

  6. Hattori, T., Vasil, V., Rosenkrans, L., Hannah, L. C., Mc Carthy, D. R., and Vasil, I. K. (1992) The viviparous-1 gene and abscisic acid activate the Cl regulatory gene for anthocyanin biosynthesis during seed maturation in maize. Genes Dev. 6, 609–618.

    Article  PubMed  CAS  Google Scholar 

  7. Wirtz, U., Schell, J., and Czernilofski, A. (1987) Recombination of selectable marker DNA in Nicotiana tabacum. DNA 6, 245–253.

    Article  PubMed  CAS  Google Scholar 

  8. Restrepo, M. A., Freed, D. D., and Carrington, J. C. (1990) Nuclear transport of polyviral proteins. Plant Cell 2, 987–998.

    Article  PubMed  CAS  Google Scholar 

  9. Carrington, J. C., Freed, D. D., and Lemicke, A. J. (1991) Bipartite signal sequence mediates nuclear translocation of the plant polyviral Nla protein. Plant Cell 3, 953–962.

    Article  PubMed  CAS  Google Scholar 

  10. Fujiwara, T., Naito, S., Chino, M., and Nagata, T. (1991) Electroporated protoplasts express seed specific gene promoters. Plant Cell Reports 9, 602–606.

    Article  CAS  Google Scholar 

  11. Beilmann, A., Pfitzner, A. J., Goodmann, H. M., and Pfitzner, M. (1991) Functional analysis of the pathogenesis-related 1a protein gene minimal promoter region. Eur. J. Biochem. 196, 415–421.

    Article  PubMed  CAS  Google Scholar 

  12. Negrutiu, I., Shillito, R., Potrykus, I., Biasini, G., and Sala, F. (1987) Hybrid genes for the analysis of transformation conditions I—setting up a simple method for direct gene transfer in plant protoplasts. Plant Mol. Biol. 8, 363–373.

    Article  CAS  Google Scholar 

  13. Installe, P., Negrutiu, I., and Jacobs, M. (1985) Protoplasts-derived plants in N. plumbaginifolia: improving the regeneration response of wild type and mutant cultures. J. Plant Physiol. 119, 443–454.

    CAS  Google Scholar 

  14. Shewry, P. R., Tatham, A., Halford, N. G., Barker, J. H. A., Hannapel, U., Gallois, P., Thomas, M., and Kreis, M. (1994) Opportunities for manipulating the seed protein composition of wheat and barley in order to improve quality. Transgenic Res. 3, 3–12.

    Article  PubMed  CAS  Google Scholar 

  15. Gallois, P., Lindsey, K., Malone, R., Kreis, M., and Jones, M. G. K. (1992) Gene rescue in plants by direct gene transfer of total genomic DNA into protoplasts. Nucleic Acids Res. 20(15), 3977–3982.

    Article  PubMed  CAS  Google Scholar 

  16. Giraudat, J., Hauge, B. M., Valon, C., Smalle, I., Parcy, F., and Goodman, H. M. (1992) Isolation of the Arabidopsis ABI3 gene by positional cloning. Plant Cell 4, 1251–1261.

    Article  PubMed  CAS  Google Scholar 

  17. Coen, E. S., Romero, J. M., Doyle, S., Elliot, R., Murphy, G., and Carpenter, R. (1990) Floricaula: a homeotic gene required for flower development in Antirrhinum majus. Cell 63, 1311–1322.

    Article  PubMed  CAS  Google Scholar 

  18. Feldmann, K. A. (1991) T-DNA insertion mutagenesis in Arabidopsis: mutational spectrum. Plant J. 1-1, 71–82.

    Article  Google Scholar 

  19. Simoens, C., Alliotte, Th., Mendel, R., Muller, A., Schiemann, J., Lijsebettens, M., Schell, J., Van Montagu, M., and Inzé, D. (1986) A binary vector for transferring genomic libraries to plants. Nucleic Acids Res. 14, 8073–8090.

    Article  PubMed  CAS  Google Scholar 

  20. Prosen, D. E. and Simpson, R. B.(1987) Transfer of a ten-number genomic library to plants using Agrobacterium tumefaciens. BioTechnology 5, 966–971.

    Article  CAS  Google Scholar 

  21. Klee, H. J., Hayford, M. B., and Rogers, S. G. (1987) Gene rescue in plants a model system for “shotgun” cloning by retransformation. Mol. Gen. Genet. 210, 282–287.

    Article  CAS  Google Scholar 

  22. Olszewski, N. E., Martin, F. B., and Ausubel, F. M. (1988) Specialised binary vector for plant transfomation expression of the Arabidopsis thaliana ALS gene in Nicotiana tabacum. Nucleic Acid Res. 16-22, 10,765–10,782.

    Article  Google Scholar 

  23. Lazo, G. R., Stem, P. A., and Ludwig, R. A. (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. BioTechnol. 9, 963–967.

    Article  CAS  Google Scholar 

  24. Gallois, P., Lindsey, K., Malone, R., Kreis, M., and Jones, M. G. K. (1992) Gene rescue in plants by direct gene transfer of total genomic DNA into protoplasts. Nucleic Acids Res. 20, 3977–3982.

    Article  PubMed  CAS  Google Scholar 

  25. Arumuganathan, K. and Earle, E. D. (1991) Nuclear DNA content of some important plant species. Plant Mol. Biol. Rep. 9-3, 208–218.

    Article  Google Scholar 

  26. Bennett, M. D., Smith, J. B., and Heslop-Harrison, J. S. (1982) Nuclear DNA amounts in angiosperm Proc. R. Soc. Lond. B216, 179–199.

    Article  Google Scholar 

  27. Bennett, M. D. and Smith, J. B. (1976) Nuclear DNA amounts in angiosperm. Phil. Trans. R. Soc. Lond. B274, 227–274.

    Google Scholar 

  28. Lowy, I., Pellicer, A., Jackson, J. F., Sim, G. K., Silverstein, S., and Axel, R. (1980) Isolation of transforming DNA. cloning the hamster aprt gene. Cell 22, 817–823.

    Article  PubMed  CAS  Google Scholar 

  29. Perucho, M., Hanahan, D., Lipsich, L., and Wigler, M. (1980) Isolation of the chicken thymidine kinase gene by plasmid rescue. Nature 285, 207–210.

    Article  PubMed  CAS  Google Scholar 

  30. Goldfarb, M., Shimizu, K., Perucho, M., and Wigler, M. (1982) Isolation and preliminary characterisation of the human transforming gene from T24 bladder carcinoma cells. Nature 296, 404–409.

    Article  PubMed  CAS  Google Scholar 

  31. Littman, D. R., Thomas, Y., Maddon, P. J., Chess, L., and Axel, R. (1985) The isolation and sequence of the gene encoding T8 a molecule defining functional classes of T lymphocytes. Cell 40, 237–246.

    Article  PubMed  CAS  Google Scholar 

  32. Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15, 473–497.

    Article  CAS  Google Scholar 

  33. Nagy, J. I. and Maliga, P. (1976) Callus induction and plant regeneration from mesophyll protoplasts of N. Sylvestris. Z. Pflanzenphysiol. 78, 453–455.

    Google Scholar 

  34. Kao, K. N., Constabel, F., Michayluk, R., and Gamborg, O. L. (1974) Plant protoplast fusion and growth on intergeneric hybrid cells. Planta. 120, 215–217.

    Article  CAS  Google Scholar 

  35. Caboche, M. (1986) Nutritional requirement of protoplasts derived, haploid tobacco cells grown at low densities in liquid medium. Planta 149, 7–18.

    Article  Google Scholar 

  36. Dellaporta, S. L., Wood, J., and Hicks, J. B. (1983) A plant DNA minipreparation version II. Plant Mol. Biol. Rep. 1, 19–21.

    Article  CAS  Google Scholar 

  37. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 545.

    Google Scholar 

  38. Paszkowski, J., Baur, M., Bogucki, A., and Potrykus, I. (1988) Gene targeting in plants. EMBO J. 7, 4021–4026.

    PubMed  CAS  Google Scholar 

  39. Jefferson, R. A., Kavangh, T. A., and Bevan, M. W. (1987) GUS: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901–3907.

    PubMed  CAS  Google Scholar 

  40. Torres, J. T., Block, A., Hahlbrock, K., and Somssich, I. E. (1993) Influence of bacterial strain genotype on transient expression of plasmid DNA in plant protoplasts. Plant J. 4(3), 587–592.

    Article  CAS  Google Scholar 

  41. Etzold, T., Fritz, C. C., Shell, J., and Schreier, P. H. (1987) A point mutation in the chloroplast 16S rRNA gene of a streptomycin resistant Nicotiana tabacum FEBS Lett. 219, 343–346.

    Article  CAS  Google Scholar 

  42. Chapel, M. and Glimelius, K. (1990) Temporary inhibition of the cell wall synthesis improves the transient expression of the GUS gene in Brassica napus mesophyll protoplasts. Plant Cell Rep. 9, 105–108.

    Article  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Laboratoire de Physiologie et Biologie Moleculaire Vegetale, Université de Perpignan, France

    Patrick Gallois

  2. Depatment of Botany, University of Leicester, UK

    Keith Lindsey

  3. Waksman Institute, Rutgers University, Piscataway, NJ

    Renee Malone

Authors
  1. Patrick Gallois
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  2. Keith Lindsey
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  3. Renee Malone
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Editor information

Editors and Affiliations

  1. Harvard University, Boston, MA

    Jac A. Nickoloff

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© 1995 Humana Press Inc., Totowa, NJ

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Gallois, P., Lindsey, K., Malone, R. (1995). Electroporation of Tobacco Leaf Protoplasts Using Plasmid DNA or Total Genomic DNA. In: Nickoloff, J.A. (eds) Plant Cell Electroporation and Electrofusion Protocols. Methods in Molecular Biology™, vol 55. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-328-7:89

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  • DOI: https://doi.org/10.1385/0-89603-328-7:89

  • Publisher Name: Springer, Totowa, NJ

  • Print ISBN: 978-0-89603-328-3

  • Online ISBN: 978-1-59259-542-6

  • eBook Packages: Springer Protocols

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