Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research
  • Published:

Selecting Somatic Hybrid Plants Using Magnetic Protoplast Sorting

Bio/Technology volume 12, pages 511–515 (1994)Cite this article

Abstract

We have used a magnetic cell sorter (MACS) technique designed for animal cells to produce large samples of potato somatic hybrids. Selected biotinylated lectins are used to mediate, via streptavidin, the binding of superparamagnetic microbeads to the protoplasts of one fusion partner. The other partner contains the selectable marker neomycinphosphotransferase II (NPT II). After fusion, the protoplast mixture is sorted by MACS, which retains protoplasts labeled with magnetic microbeads. This fraction is incubated on kanamycin-containing medium, where only hybrid cells grow into calli. We have optimized the protoplast staining with lectins, the protoplast separation with MACS and the regeneration of hybrid calli into plants. With MACS treatment, among regenerated plants the hybrids between two clones of Solatium tuberosum increased from 8 to 36 percent. In the interspecific somatic hybridization S. tuberosum x S. bulbocastanum the increase was from 28 to 82 percent. Hybrid plants were identified by DNA fingerprinting and had the morphology and cytology expected for fusion products. The large number of plants which can be obtained should enable selection of hybrids with desired chromosome number, morphology and seed fertility.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Austin, S., Baer, M.A. and Helgeson, J.O. 1985a. Transfer of resistance to potato leaf roll virus from Solatium brevidens into Solarium tuberosum by somatic fusion. Plant Sci. 39: 75–82.

    Article  Google Scholar 

  2. Fish, N., Karp, A. and Jones, M.G.K. 1988. Production of somatic hybrids by electrofusion in Solanum. Theor. Appl. Genet. 76: 260–266.

    Google Scholar 

  3. Pehu, E., Karp, A., Moore, K., Steele, S., Dunckley, R. and Jones, M.G.K. 1989. Molecular, cytogenetic and morphological characterization of somatic hybrids of dihaploid S. tuberosum and diploid S. brevidens, Theor. Appl. Genet. 78: 696–704.

    Article  CAS  Google Scholar 

  4. Preiszner, J., Feher, A., Veisz, O., Sutka, J. and Dudits, D. 1991. Characterization of morphological variation and cold resistance in interspecific somatic hybrids between potato (Solanum tuberosum) and S. brevidens Phil. Euphytica 57: 37–49.

    Google Scholar 

  5. Binding, H., Jain, S., Finger, J., Mordhorst, G., Nehls, R. and Gressel, J. 1982. Somatic hybridization of an atrazine resistant biotype of Solanum nigrum with Solanum tuberosum. Theor. Appl. Genet. 63: 273–277.

    Article  CAS  Google Scholar 

  6. Butenko, R.G. and Kuchko, A. 1980. Somatic hybridization of Solanum tuberosum and Solanum chacoense Bitt. by protoplast fusion, p. 293–330. In: Advances in Protoplast Research, Ferency, L. and Farkas, G. L. (Eds. ). Pergamon Press, Oxford.

    Google Scholar 

  7. Puite, K.J., Roest, S. and Pijnacker, L. 1986. Somatic hybrid potato plants after electrofusion of diploid Solanum tuberosum and Solanum phureja. Plant Cell Reports 5: 262–265.

    Article  CAS  Google Scholar 

  8. Pijnacker, L.P., Ferwerda, M.A., Puite, K.J. and Roest, S. 1987. Elimination of S. phureja chromosomes in S. tuberosum + S. phureja somatic hybrids. Theor. Appl. Genet. 73: 878–882.

    Article  CAS  Google Scholar 

  9. Mattheij, W.M., Eijlander, R., de Koning, J.R.A. and Louwes, K.M. 1992. Interspecific hybridization between the cultivated potato Solanum tuberosum subspecies tuberosum L. and the wild species S. circaeifolium subsp. circaeifolium Bitter exhibiting resistance to Phytophtora infestans (Mont. ) de Bary and Globodera pallida (Stone) Behrens. Theor. Appl. Genet. 83: 459–466.

    Article  CAS  Google Scholar 

  10. Seraaf, I., Sikachkr, D., Ducreux, G., Brown, S., Allot, M., Barghi, N. and Rossigol, L. 1991. Interspecific somatic hybridization in potato by protoplast electrofusion. Plant Science 76: 115–126.

    Article  Google Scholar 

  11. Matteij, W.M. and Puite, K.J. 1992. Tetraploid potato hybrids through protoplast fusions and analysis of their performance in field. Theor. Appl. Genet. 83: 807–812.

    Article  Google Scholar 

  12. Helgeson, J.P., Hunt, G.J., Haberlach, G.T. and Austin, S. 1986. Somatic hybrids between Solanum brevidens and Solanum tuberosum: Expression of a light blight resistance gene and potato leaf roll resistance. Plant Cell Reports 3: 212–214.

    Article  Google Scholar 

  13. Austin, S., Pohimann, J.D., Brown, C.R., Mojtahedi, H., Santo, G.S., Douches, D.S. and Helgeson, J.P. 1993. Interspecific somatic hybridization between Solanum tuberosum L. and Solanum bulbocastanum Dun. as a means of transferring nemetode resistance. American Potato Journal 70: 485–495.

    Article  Google Scholar 

  14. Pauls, K.P. and Chuong, P.V. 1987. Flow cytometric identification of Brassica napus protoplast fusion products. Can. J. Bot. 65: 334–338.

    Article  Google Scholar 

  15. Cocking, E.C., George, D., Price-Jones, M.J. and Power, J.B. 1977. Selection procedures for the production of interspecies somatic hybrids of Petunia hybrida and Petuniaparodii II. Albino complementation selection. Plant Science Lettres 10: 7–12.

    Article  Google Scholar 

  16. Masson, J., Lancelin, D., Bellini, C., Lecerf, M., Guerche, P. and Pelletier, G. 1989. Selection of somatic hybrids between diploid clones of potato (S. tuberosum L. ) transformed by direct gene transfer. Theor. Appl. Genet. 78: 153–159.

    Article  CAS  Google Scholar 

  17. Lo Schiavo, P., Giovinazzo, G. and Terzi, M. 1983. 8-Azaguanine resistant carrot cell mutants and their use as universal hybridizers. Mol. Gen. Genet. 192: 326–329.

    Article  CAS  Google Scholar 

  18. Waara, S.H.A. and Erikson, T. 1989. Somatic hybridization between anther derived dihaploid clones of potato (S. tuberosum L. ) and the identification of hybrid plants by isozyme analysis. Theor. Appl. Genet. 77: 49–56.

    Article  CAS  Google Scholar 

  19. Galbraith, D.W. and Mauch, T.J. 1979. Identification of fusion of plant protoplasts II: Conditions for the reproducible fluorescence labelling of protoplasts derived from mesophyll tissue. Z. Pflanzenphysiol. 98: 129–140.

    Article  Google Scholar 

  20. Afonso, C., Harkins, K., Thomas-Compton, M., Krejci, A. and Galbraith, D. 1985. Selection of somatic hybrid plants in Nicotiana through fluorescence activated sorting of protoplasts. Bio/Technology 3: 811–816.

    Google Scholar 

  21. Hammatt, N., Lister, A., Blackhall, N.W., Gartland, J., Ghose, T.K., Gilmour, D.M., Power, J.B., Davey, M.R. and Cocking, E.C. 1990. Selection of plant heterokaryons by flow cytometry. Protoplasma 154: 34–44.

    Article  Google Scholar 

  22. Miltenyi, S., Müller, W., Weichel, W. and Radbruch, A. 1990. High gradient magnetic cell separation with MACS. Cytometry 11: 231–238.

    Article  CAS  Google Scholar 

  23. Glimelius, K., Djupsjöbacka, M. and Fellner-Feldegg, H. 1986. Selection and enrichment of plant protoplast heterokaryons of Brassicaceae by flow sorting. Plant Science 45: 133–141.

    Article  CAS  Google Scholar 

  24. Binding, H. and Nehls, R. 1977. Regeneration of isolated protoplasts to plants in Solanum dulcamara L.Z. Pflanzenphysiologie 85: 279–280.

    Google Scholar 

  25. Puite, K., Ten Broeke, W. and Schaart, J. 1988. Inhibition of cell wall synthesis improves flow cytometric sorting of potato heterofusions resulting in hybrid plants. Plant Science 56: 61–66.

    Article  CAS  Google Scholar 

  26. Fahleson, J., Rahlen, L. and Glimelius, K. 1988. Analysis of plants regenerated from protoplast fusions between Brassica napus and Eruca saliva. Theor. Appl. Genet. 76: 507–512.

    Article  CAS  Google Scholar 

  27. Novy, R.G. and Hannemann, R.E. 1991. Hybridization between gp. Tuberosum haploids and 1 EBN wild potato species. Am. Pot. J. 68: 151–169.

    Article  Google Scholar 

  28. Ramana, N.S. and Hermsen, J.G. 1979. Genome relationship in tuber bearing Solanums, p. 647–655. In: The Biology and Taxonomy of the Solanaceae, Hawkes, J. G., Lester, R. N. and Skelding, A. D. (Eds.). Academic Press, London.

    Google Scholar 

  29. SreeRamulu, K. 1983. Case histories of genetic variability in vitro: potato, p. 449–473, Vol. 3. In: Cell culture and somatic Cell genetics of plants, Vasil, I. K. (Ed. ). Academic Press, Orlando, FL.

    Google Scholar 

  30. Knapp, S., Coupland, G., Uhrig, H., Starlinger, P. and Salamini, F 1988. Transposition of the maize transposable element Ac in Solanum tuberosum. Mol. Gen. Genet. 213: 285–290.

    Article  CAS  Google Scholar 

  31. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 15: 473–497.

    Article  CAS  Google Scholar 

  32. Shahin, E.A. 1984. Isolation, culture and regeneration of potato leaf protoplasts from plants preconditioned in vitro, p. 381–390, Vol I. In: Cell Culture and Somatic Cell Genetics of Plants. Vasil, I. K. (Ed. ). Academic Press, NY.

    Google Scholar 

  33. Foulger, D. and Jones, M.G.K. 1986. Improved efficiency of genotype dependent regeneration of important potato cultivars. Plant Cell Rep. 5: 72–76.

    Article  CAS  Google Scholar 

  34. Fish, N. and Karp, A. 1986. Improvement in regeneration from protoplasts of potato and studies on chromosome stability. 1. The effect on initial culture media. Theor. Appl. Genet. 72: 405–412.

    Article  CAS  Google Scholar 

  35. Tempelaar, M.J. and Jones, M.G.K. 1985. Fusion characteristics of plant protoplasts in electric fields. Planta 165: 205–216.

    Article  CAS  Google Scholar 

  36. Mehrle, W., Naton, B. and Hampp, R. 1990. Determination of physical membrane properties of plant cell protoplasts via the electrofusion technique: prediction of optimal fusion yields and protoplast viability. Plant Cell Rep. 8: 687–691.

    Article  CAS  Google Scholar 

  37. Saghai-Maroof, M.A., Solimann, K.M., Jorgensen, R.A. and Allard, R.W. 1984. Ribosomal spacer length polymorphism in barley: mendelian inheritance, chromosomal location, and population dynamics. Proc. Natl. Acad. Sci. USA 81: 8014–8018.

    Article  CAS  Google Scholar 

  38. Maniatis, T., Fritsch, E. and Sambrock, J. 1982. Molecular Cloning. A Laboratory Manual. Cold Spring Harbour Laboratory Press, NY.

    Google Scholar 

  39. Geitler, L. 1949. Schnellmethoden der Kem-und Chromosomenuntersuchung. 3. Aufl. Wien, Springer Verlag.

    Book  Google Scholar 

Download references

Author information

Author notes

  1. Helmut Uhrig: Corresponding author.

Authors and Affiliations

  1. Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, 50829, Köln, Germany

    Inca Dörr, Francesco Salamini & Helmut Uhrig

  2. Miltenyi Biotec GmbH, Friedrich-Ebert-Str. 68, 51429, Bergisch Gladbach, Germany

    Stefan Miltenyi

Authors
  1. Inca Dörr
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stefan Miltenyi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Francesco Salamini
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Helmut Uhrig
    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

Dörr, I., Miltenyi, S., Salamini, F. et al. Selecting Somatic Hybrid Plants Using Magnetic Protoplast Sorting. Nat Biotechnol 12, 511–515 (1994). https://doi.org/10.1038/nbt0594-511

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0594-511

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing