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Theoretical and Applied Genetics

, Volume 72, Issue 2, pp 279–286 | Cite as

Fusion of plant protoplasts: a study using auxotrophic mutants of Nicotiana plumbaginifolia, Viviani

  • I. Negrutiu
  • D. De Brouwer
  • J. W. Watts
  • V. I. Sidorov
  • R. Dirks
  • M. Jacobs
Article

Summary

Protoplast fusion studies between various auxotrophic mutants of Nicotiana plumbaginifolia were performed to optimize conditions for PEG-mediated fusion and to identify factors influencing the plant protoplast fusion process. Numerous parameters in the isolation, culture, and fusion of protoplasts were tested, and established fusion protocols were compared. Fusion rates, calculated on the basis of colony growth on selection medium (genetic complementation), ranged from 10−4 to 10−2. Conditions that allow rapid and reproducible fusions at the highest rates were established. Particular emphasis was given to fusion of mesophyll-derived protoplasts, for which the ability to regenerate fertile plants from fusion products was shown to be particularly high. Preliminary experiments using electric-field mediated fusion suggest that electrofusion may offer significant advantages over the traditional chemical fusion.

Key words

Protoplast Nicotiana plumbaginifolia Auxotrophs Electrofusion PEG 

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References

  1. Ashmore SE, Gould AR (1982) Protoplast fusion and the cell cycle. Plant Cell Rep 1:225–228Google Scholar
  2. Bates GW, Hasenkampf CA (1985) Culture of plant somatic hybrids following electrical fusion. Theor Appl Genet 70:227–233Google Scholar
  3. Berri SF (1983) Factors affecting hybridisation between sexually incompatible species of Petunia. In: Potrikus I, Harms Ct, Hinnen A, Hütter R, King PJ, Shillito RD (eds) Protoplasts 1983. Birkhäuser, pp 82–84Google Scholar
  4. Dirks R, Sidorov D, Negrutiu I, Jacobs M (1985) Complementation analysis of nitrate reductase-deficient mutants of N. plumbaginifolia: evidence for a new category of cnx mutants. Mol Gen Genet 201:339–343Google Scholar
  5. Gleba YY, Sytnik KM (1984) Protoplast fusion and parasexual hybridization of higher plants. In: Shoeman R (ed) Protoplast fusion. Genetic engineering in higher plants. Springer, Berlin Heidelberg New York, pp 36–61Google Scholar
  6. Glimelius K, Eriksson T, Grafe R, Müller AJ (1978) Somatic hybridization of nitrate reductase-deficient mutants of N. tabacum by protoplast fusion. Physiol Plant 44:273–277Google Scholar
  7. Glimelius K, Wallin A, Eriksson T (1978) Concavalin A improves the PEG method for fusing plant protoplasts. Physiol Plant 44:92–96Google Scholar
  8. Harms CT, Potrykus I, Widholm JM (1981) Complementation and dominant expression of amino acid analogue resistance markers in somatic hybrid clones from D. carota after protoplast fusion. Z Pflanzenphysiol 101:337–390Google Scholar
  9. Hartmann JX, Galla JD, Emma DA, Kao KN, Gamborg OL (1976) The fusion of erythrocytes by treatment with proteolytic enzymes and PEG. Can J Genet Cytol 18:503–512Google Scholar
  10. Hauptmann R, Kumar A, Widholm JM (1983) Carrot X tobacco somatic hybrids selected by amino acid analog resistance complementation. In: Potrykus I, Harms CT, Hinnen A, Hütter R, King PJ, Shillito RD (eds) Protoplast 1983. Birkhäuser, pp 92–94Google Scholar
  11. Hein T, Przewozny T, Schieder O (1983) Culture and selection of somatic hybrids using an auxotrophic cell line. Theor Appl Genet 64:119–122Google Scholar
  12. Installé P, Negrutiu I, Jacobs P (1985) Protoplast-derived plants in N. plumbaginifolia: improving the regeneration response of wild type and mutant cultures. J Plant Physiol 119:443–454Google Scholar
  13. Kao NN (1982) Plant protoplast fusion and isolation of heterokaryons. In: Wetter LR, Constabel F (eds) Plant tissue culture methods. NRCC 19876, Prairie Reg Laboratories, Saskatoon, Sask, pp 49–57Google Scholar
  14. Kao KN, Michayluk MR (1974) A method for high frequency intergeneric fusion of plant protoplasts. Planta 115:355–367Google Scholar
  15. Kao KN, Constabel F, Michayluk MR, Gamborg OL (1974) Plant protoplast fusion and growth of intergeneric hybrid cells. Planta 120:215–227Google Scholar
  16. Maliga P (1982) Cell culture procedures for N. plumbaginifolia. Plant Mol Biol Newslett 3:108–113Google Scholar
  17. Marton L, Manh Dung T, Mendel RR, Maliga P (1982) Nitrate reductase-deficient lines from haploid protoplast cultures of N. plumbaginifolia. Mol Gen Genet 186:301–304Google Scholar
  18. Menczel L, Nagy F, Kiss Z, Maliga P (1981) Streptomycinresistant and sensitive somatic hybrids of N. tabacum + N. knitiana: correlation of resistance to N. tabacum plastids. Theor Appl Genet 59:191–195Google Scholar
  19. Menczel L, Galiba G, Nady F, Maliga P (1982) Effect of radiation dosage on efficiency of chloroplast transfer by protoplast fusion in Nicotiana. Genetics 100:487–495Google Scholar
  20. Meyer Y, Herth W (1978) Chemical inhibition of cell wall formation and cytokinesis, but not of nuclear division, in protoplasts of N. tabacum. Planta 142:253–262Google Scholar
  21. Negrutiu I (1981) Improved conditions for large scale culture, mutagenesis, and selection of haploid protoplasts of N. plumbaginifolia. Z Pflanzenphysiol 104:431–442Google Scholar
  22. Negrutiu I, Dirks R, Jacobs M (1983) Regeneration of fully nitrate reductase-deficient mutants from protoplast cultures of haploid N. plumbaginifolia. Theor Appl Genet 66:341–347Google Scholar
  23. Negrutiu I, Jacobs M, Cattoir-Reynaerts A (1984) Progress in cellular engineering of plants: biochemical and genetic assessment of selectable markers from cultured cells. Plant Mol Biol 3:289–302Google Scholar
  24. Negrutiu I, De Brouwer D, Dirks R, Jacobs M (1985) Amino acid auxotrophs from protoplast cultures of N. plumbaginifolia. 1. BUdR enrichment selection, plant regeneration, and general characterisation. Mol Gen Genet 199:330–338Google Scholar
  25. Power GB, Pearson EM, Heyward C, George D, Evans PK, Berry SF, Cocking EC (1976) Somatic hybridization of Petunia hybrida and P. parodii. Nature 263:500–502Google Scholar
  26. Shillito RD, Paszkowsky J, Potrykus I (1983) Agarose plating and a bead type culture technique enable and stimulate development of p-derived colonies in a number of plant species. Plant Cell Rep 2:244–247Google Scholar
  27. Watts JW, King JM (1984) A simple method for large scale electrofusion and culture of plant protoplasts. Biosci Rep 4:335–342Google Scholar
  28. White DWR, Vasil IK (1979) Use of amino acid analogresistant cell lines for selection of N. sylvestris somatic cell hybrids. Theor Appl Genet 55:107–112Google Scholar
  29. Zimmermann U, Scheurich P (1981) High frequency fusion of protoplasts by electric fields. Planta 151:26–32Google Scholar
  30. Zimmermann U (1982) Electric-field mediated fusion and related electrical phenomena. Biochim Biophys Acta 694:227–277Google Scholar

Copyright information

© Springer-Verlag 1986

Authors and Affiliations

  • I. Negrutiu
    • 1
  • D. De Brouwer
    • 1
  • J. W. Watts
    • 1
  • V. I. Sidorov
    • 1
  • R. Dirks
    • 1
  • M. Jacobs
    • 1
  1. 1.Institute of Molecular BiologySt. Genesius-RodeBelgium

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