Skip to main content
SpringerLink
Log in

Transient gene expression in electroporated Solanum protoplasts

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts. The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv. Désirée. Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation. Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S. brevidens mesophyll protoplasts −250 V/cm; Désirée mesophyll protoplasts −225 V/cm; Désirée suspension culture protoplasts −225 V/cm; and Désirée tuber protoplasts −150 V/cm. The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory. In time courses, maximum CAT activity (in Désirée mesophyll protoplasts) occurred 36–48 h after electroporation. Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the β-glucuronidase (gus) gene, showed expression (at DNA concentrations between 0–10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts. At higher DNA concentrations (20–30 pmol/ml) the patatin promoter directed 4–5 times higher levels of gus expression. Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Bevan M, Barker R, Goldsbrough A, Jarvis M, Kavanagh T, Iturriaga G: The structure and transcription start site of a major potato tuber protein gene. Nucleic Acids Res 14: 4625–4638 (1986).

    PubMed  Google Scholar 

  2. Chu G, Hayakawa H, Berg P: Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res 15: 1311–1326 (1987).

    PubMed  Google Scholar 

  3. Fish N, Karp A, Jones MGK: Production of somatic hybrids by electrofusion in Solanum. Theor Appl Genet 76: 260–266 (1988).

    Google Scholar 

  4. Foulger D, Jones MGK: Improved efficiency of genotype-dependent regeneration from protoplasts of important potato cultivars. Plant Cell Rep 5: 72–76 (1986).

    Article  Google Scholar 

  5. Frearson EM, Power JB, Cocking EC: The isolation culture and regeneration of Petunia leaf protoplasts. Devl Biol 33: 130–137 (1973).

    Article  Google Scholar 

  6. Fromm M, Taylor LP, Walbot V: Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci USA 82: 5824–5828 (1985).

    PubMed  Google Scholar 

  7. Fromm M, Taylor LP, Walbot V: Stable transformatigh of maize after gene transfer by electroporation. Nature 319: 791–793 (1986).

    PubMed  Google Scholar 

  8. Fromm M, Walbot V: Transient expression of DNA in plant cells. In: Hohn Th, Schell J (eds) Plant DNA Infectious Agents, chapter 13. Springer-Verlag, Wien New York (1978).

    Google Scholar 

  9. Gorman CM, Moffat LF, Howard BH: Recombinant genomes which express chloramphenicol acetyltrans-ferase in mammalian cells. Mol Cell Biol 2: 1044–1051 (1982).

    PubMed  Google Scholar 

  10. Howard EA, Walker JC, Dennis ES, Peacock WJ: Regntlated expression of an alcohol dehydrogenase 1 chimeric gene introduced into maize protoplasts. Planta 170: 535–540 (1987).

    Article  Google Scholar 

  11. Huisman MJ, Linthorst HJM, Bol JF, Cornelissen BJC: The complete nucleotide sequence of potato virus X reveals homologies at the amino acid level with various plus-stranded RNA viruses. J Gen Virol 69: 1789–1798 (1988).

    PubMed  Google Scholar 

  12. Hussey G, Stacey NJ: Factors affecting the formation of in vitro tubers of potato (Solanum tuberosum L.). Ann Bot 53: 565–578 (1984).

    Google Scholar 

  13. Jefferson RA, Kavanagh TA, Vevan MW: GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).

    PubMed  Google Scholar 

  14. Lörz H, Baker B, Schell J: Gene transfer to cereal cells mediated by protoplast transformation. Mol Gen Genet 199: 178–182 (1985).

    Article  Google Scholar 

  15. Logemann J, Mayer JE, Schell J, Willmitzer L: Differential expression of genes in potato tubers after wounding. Proc Natl Acad Sci USA 85: 1136–1140 (1988).

    Google Scholar 

  16. Maniatis T, Goodbourn S, Fischer JA: Regulation inducable and tissue-specific gene expression. Science 236: 1237–1245 (1987).

    PubMed  Google Scholar 

  17. Mignery GA, Pikaard CS, Park WD: Molecular characterization of the patatin multigene family of potato. Gene 62: 27–44 (1988).

    Article  PubMed  Google Scholar 

  18. Nelson RS, Criessen GP, Bright SWJ: Plant regeneration from protoplasts of Solanum brevidens. Plant Sci Lett 30: 355–362 (1983).

    Google Scholar 

  19. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH: Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1: 841–845 (1982).

    PubMed  Google Scholar 

  20. Okada K, Takebe I, Nagata T: Expression and integration of genes introduced into highly synchronised protoplasts. Mol Gen Genet 205: 398–403 (1986).

    Google Scholar 

  21. Paszkowski J, Shillito RD, Saul MW, Mandak V, Hohn T, Hohn B, Potrykus I: Direct gene transfer to plants. EMBO J 3: 2717–2722 (1984).

    Google Scholar 

  22. Potrykus I, Saul MW, Petruska J, Paskzowski J, Shillito RD: Direct gene transfer of cells of a graminaceous monocot. Mol Gen Genet 199: 183–188 (1985).

    Article  Google Scholar 

  23. Pröls M, Töpfer R, Schell J, Steinbiß H-H: Transient gene expression in tobacco protoplasts: I. Time course of CAT appearance. Plant Cell Rep 7: 221–224 (1988).

    Google Scholar 

  24. Rosahl S, Schmidt R, Schell J, Willmitzer L: Isolation and characterization of a gene from Solanum tuberosum encoding patatin, the major storage protein of potato tubers. Mol Gen Genet 203: 214–220 (1986).

    Article  Google Scholar 

  25. Sanchez-Serrano J, Schmidt R, Schell J, Willmitzer L: Nucleotide sequence of protinase inhibitor II encoding cDNA of potato (Solanum tuberosum) and its mode of expression. Mol Gen Genet 203: 15–20 (1986).

    Article  Google Scholar 

  26. Schöler HR, Gruss P: Specific interaction between enhancer-containing molecules and cellular components. Cell 36: 403–411 (1984).

    Article  PubMed  Google Scholar 

  27. Shillito RD, Saul MW, Paszkowski J, Muller M, Potrykus I: High efficiency direct gene transfer to plants. Bio/technology 3: 1099–1103 (1985).

    Article  Google Scholar 

  28. Tempelaar MJ, Jones MGK: Fusion characteristics of plant protoplasts in electric fields. Planta 165: 205–216 (1985).

    Google Scholar 

  29. Twell D, Ooms G: The 5′ flanking DNA of a patatin gene directs tuber specific expression of a chimaeric gene in potato. Plant Mol Biol 9: 365–375 (1987).

    Google Scholar 

  30. Twell D, Ooms G: Structural diversity of the patatin gene family in potato cv. Désirée. Mol Gen Genet 212: 325–336 (1988).

    Article  PubMed  Google Scholar 

  31. Widholm JM: The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol 47: 189–194 (1972).

    PubMed  Google Scholar 

  32. Winterbourne DJ, Thomas S, Hermon-Taylor J, Hussain I, Johnstone AP: Electric shock mediated transfection of cells: Characterization and optimization of electrical parameters. Biochem J 251: 427–434 (1988).

    PubMed  Google Scholar 

  33. Zimmermann U: Electrical breakdown, electropermeabilization and electrofusion. Rev Physiol Biochem Pharmacol 105: 175–256 (1986).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Rothamsted Experimental Station, Biochemistry Department, AFRC Institute of Arable Crops Research, AL5 2JQ, Harpenden, Herts., U.K.

    Heddwyn Jones, Gert Ooms & Michael G. K. Jones

Authors
  1. Heddwyn Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gert Ooms
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael G. K. Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jones, H., Ooms, G. & Jones, M.G.K. Transient gene expression in electroporated Solanum protoplasts. Plant Mol Biol 13, 503–511 (1989). https://doi.org/10.1007/BF00027310

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00027310

Key words

Search

Navigation