Plant Molecular Biology

, Volume 8, Issue 5, pp 363–373 | Cite as

Hybrid genes in the analysis of transformation conditions

I. Setting up a simple method for direct gene transfer in plant protoplasts
  • I. Negrutiu
  • R. Shillito
  • I. Potrykus
  • G. Biasini
  • F. Sala


Direct gene transfer into plant protoplasts has been recently developed, and conditions for high frequency transformation of SR1 tobacco protoplasts established. In this paper we analyse numerous transformation parameters in a comparative study on SR1Nicotiana tabacum and N. plumbaginifolia, and report on a simple chemical technique for very efficient protoplast transformation. It is based on the synergistic interaction of MgCl2 and PEG. The technique yielded up to 1400 transformants per 3×105 treated N. tabacum protoplasts (up to 4.8% of the survivors, late selected clones). Using N. plumbaginifolia, the frequencies were 10-fold lower, indicating that the ‘competence’ for transformation has a species-specific component.

Key words

transformation MgCl2-PEG-electroporation competence protoplasts 





relative transformation frequency


absolute transformation frequency


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  1. 1.
    Caboche M: Nutritional requirements of protoplast-derived, haploid tobacco cells grown at low densities in liquid medium. Planta 149: 7–18 (1980).Google Scholar
  2. 2.
    Czernilofsky AP, Hain R, Herrera-Estrella L, Lörz H, Goyvaerts E, Baker BJ, Schell J: Fate of selectable marker DNA integrated into the genome of Nicotiana tabacum. DNA 5: 101–113 (1986).Google Scholar
  3. 3.
    Davey MR, Cocking EC, Freeman J, Pearce N, Tudor I: Transformation of Petunia protoplasts by isolated Agrobacterium plasmids. Plant Sci Lett 18: 307–313 (1980).Google Scholar
  4. 4.
    Fromm M, Taylor L, Walbot V: Expression of genes transferred into monocot and dicot plants cells by electroporation. Proc Natl Acad Sci USA 82: 5842–5828 (1985).Google Scholar
  5. 5.
    Fromm F, Taylor L, Walbot V: Stable transformation of maize after gene transfer by electroporation. Nature 319: 791–793 (1986).Google Scholar
  6. 6.
    Hain R, Stabel P, Czernilofsky P, Steinbiss H, Herrera-Estrella L, Schell J: Uptake, integration, expression, and genetic transmission of a selectable chimeric gene by plant protoplasts. Mol Gen Genet 199: 161–168 (1985).Google Scholar
  7. 7.
    Installé P, Negrutiu I, Jacobs M: Protoplast-derived plants in N. plumbaginifolia: improving the regeneration response of wild type and mutant cultures. J Plant Physiol 119: 443–454 (1985).Google Scholar
  8. 8.
    Klebe RJ, Harriss JV, Sharp ZD, Douglas MG: A general method for PEG-induced genetic transformation of bacteria and yeast. Gene 25: 333–341 (1983).Google Scholar
  9. 9.
    Krens FA, Molendijk L, Wullems GJ, Schilperoort RA: In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296: 72–74 (1982).Google Scholar
  10. 10.
    Menczel L, Nagy F, Kiss Z, Maliga P: Streptomycin-resistant and sensitive somatic hybrids of N. tabacum + N. knitiana: correlation of resistance to N. tabacum plastids. Theor Appl Genet 59: 191–195 (1981).Google Scholar
  11. 11.
    Meyer P, Walgenbach E, Sussmann K, Hombrechter G, Saedler H: Synchronized tobacco protoplasts are efficiently transformed by DNA. Mol Gen Genet 201: 513–518 (1985).Google Scholar
  12. 12.
    Mouras A, Saul M, Essad S, Potrykus I: Localisation by in situ hybridisation of a low copy chimeric resistance gene induced into plants by direct gene transfer. Mol Gen Genet, submitted (1986).Google Scholar
  13. 13.
    Negrutiu I, Dirks R, Jacbos M: Regeneration of fully nitrate reductase-deficient mutants from protoplast culture of N. plumbaginifolia. Theor Appl Genet 66: 341–347 (1983).Google Scholar
  14. 14.
    Negrutiu I, De Brouwer D, Watts J, Sidorov V, Dirks R, Jacobs M: Fusion of plant protoplasts: a study using auxotrophic mutants of N. plumbaginifolia. Theor Appl Genet 72: 279–286 (1986a).Google Scholar
  15. 15.
    Negrutiu I, Heberlee-Bors E, Potrykus I: Attempts to transform for Kanamycin-resistance in mature pollen of tobacco. In: Mulcahy DL, Mulcahy GB, Ottaviano E (eds) Biotechnology and Ecology of Pollen, pp 65–70. New York: Springer Verlag (1986b).Google Scholar
  16. 16.
    Paszkowski J, Shillito RD, Saul M, Mandak V, Hohn T, Hohn B, Potrykus I: Direct gene transfer to plants. EMBO J 3: 2717–2722 (1984).Google Scholar
  17. 17.
    Paszkowski J, Saul M: Direct gene transfer to plants. In: Weissbach A, Weissbach (eds) Methods in Enzymology, Vol. 118, pp 668–685. New York: Academic Press (1986).Google Scholar
  18. 18.
    Peerbolte R, Krens F, Mans R, Floor M, Hoge J, Wullems G, Schilperoort R: Transformation of plant protoplasts with DNA: cotransformation of non-selected calf thymus carrier DNA and meiotic segregation of transforming DNA sequences. Plant Mol Biol 5: 235–247 (1985).Google Scholar
  19. 19.
    Potrykus I, Shillito RD, Saul M, Paszkowski J: Direct gene transfer. State of the art and future perspectives. Plant Mol Biol Reporter 3: 117–128 (1985a).Google Scholar
  20. 20.
    Potrykus I, Paszkowski J, Saul M, Petruska J, Shillito R: Molecular and general genetics of a hybrid foreign gene introduced into tobacco by direct gene transfer. Mol Gen Genet 199: 169–177 (1985b).Google Scholar
  21. 21.
    Potrykus I, Paszkowski J, Saul M, Shillito R: Direct gene transfer for theoretical and applied genetics. Theor Appl Genet (in press) (1986).Google Scholar
  22. 22.
    Shillito R, Paszkowski J, Potrykus I: Agarose plating and bead-type culture technique enable and stimulate development of protoplast-derived colonies in a number of plant species. Plant Cell Rep 2: 244–247 (1983).Google Scholar
  23. 23.
    Shillito R, Saul M, Paszkowski J, Potrykus I: High efficiency direct gene transfer to plants. Biotechnology 3: 1099–1103 (1985).Google Scholar
  24. 24.
    Shillito R, Potrykus I: Protoplasts: isolation, culture, plant regeneration. In: Weissbach A, Weissbach (eds) Methods in Enzymology, Vol. 118, pp 549–578. New York: Academic Press (1986).Google Scholar
  25. 25.
    Waldron C, Malcolm SK, Murphy EB, Roberts JL: Method for high frequency DNA-mediated transformation of plant protoplasts. Plant Mol Biol Reporter 3: 169–173 (1985). (1985).Google Scholar

Copyright information

© Martinus Nijhoff Publishers 1987

Authors and Affiliations

  • I. Negrutiu
    • 1
  • R. Shillito
    • 1
  • I. Potrykus
    • 1
  • G. Biasini
    • 2
  • F. Sala
    • 2
  1. 1.Friedrich Miescher-InstitutBaselSwitzerland
  2. 2.Dipartimento di genetica e microbiologiaUniversità di PaviaPaviaItaly

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