Selection of Somatic Hybrid Plants in Nicotiana Through Fluorescence-Activated Sorting of Protoplasts

Abstract

We have developed general techniques for the successful selection of plant heterokaryons from parental populations of plant protoplasts subjected to somatic fusion treatments. These techniques involve a combination of artificial fluorescence labeling, natural chlorophyll autofluorescence and fluorescence-activated cell sorting. We have recovered purified heterokaryons at high rates and viabilities, and these were regenerated into plants. This quick and routine technique has allowed the recovery and unambiguous characterization of three different interspecific somatic hybrids of Nicotiana.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1

    Harms, C.T. 1983. Somatic hybridization by plant protoplast fusion. Experientia Supplementum 46: 69–84.

  2. 2

    Galbraith, D.W. and Galbraith, J.E.C. 1979. A method for identification of fusion of plant protoplasts derived from tissue cultures. Zeit. Pflanzenphysiol. 93: 149–158.

  3. 3

    Galbraith, D.W. and Mauch, T.J. 1980. Identification of fusion of plant protoplasts II: conditions for the reproducible fluorescence labelling of protoplasts derived from mesophyll tissue. Zeit. Pflanzenphysiol. 98: 129–140.

  4. 4

    Galbraith, D.W. 1984. Selection of somatic hybrid cells by fluorescence-activated cell sorting, 433–447. In: Cell Culture and Somatic Cell Genetics of Plants. I. K. Vasil, (ed). Academic Press, New York.

  5. 5

    Harkins, K.R. and Galbraith, D.W. 1984. Flow sorting and culture of plant protoplasts. Physiol. Plant. 60: 43–52.

  6. 6

    Galbraith, D.W., Afonso, C.L. and Harkins, K.R. 1984. Flow sorting and culture of protoplasts: conditions for high-frequency recovery, growth and morphogenesis from sorted protoplasts of suspension cultures of nicotiana. Plant Cell Reports 3: 151–155.

  7. 7

    Kinsara, A.M. and Cocking, E.C. 1983. Assessment of somatic hybridization between Lycopersicon esculentum and L. peruvianum. Experientia Supplementum 45: 80–81.

  8. 8

    Hein, T. and Schieder, O. 1983. Production of somatic hybrids using auxotrophic nurse cultures. Experientia Supplementum 45: 104–105.

  9. 9

    Junker, S. and Pedersen, S. 1981. A universally applicable method of isolating somatic cell hybrids by two-color flow sorting. Bioch. Biophys. Res. Commun. 102: 977–984.

  10. 10

    Galbraith, D.W., Harkins, K.R., Maddox, J.M., Ayres, N.M., Sharma, D.P. and Firoozabady, E. 1983. Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220: 1049–1051

  11. 11

    Freeling, M. and Cheng, D.S.K. 1978. Radiation-induced alcohol dehydrogenase mutants in maize following allyl alcohol selection of pollen. Genet. Res. Camb. 31: 107–129.

  12. 12

    Evans, D.A. and Reed, S.M. 1981. Cytogenetic techniques, p. 213–240. In: Plant Tissue Culture: methods and applications in agriculture. T. A. Thorpe (ed). Academic Press, New York.

  13. 13

    Cardy, B.J., Stuber, C.W. and Goodman, M.M. 1980. Techniques for starch gel electrophoresis of enzymes from maize (Zea mays L.). Institute of Statistics Mimeograph Ser. 1317, North Carolina State University, Raleigh, North Carolina.

  14. 14

    Hamill, D.E. 1968. Peroxidase isozymes in maize; designation of locus Px. Maize Genetics Cooperative Newsletter 42: 36–37.

  15. 15

    Davis, B.J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121: 404–427.

  16. 16

    Vindelov, L.L., Christensen, I.J., Jensen, G. and Nissen, N.I. 1983. Limits of detection of nuclear DNA abnormalities by flow cytometric DNA analysis. Results obtained by a set of methods for sample storage, staining and internal standardization. Cytometry 3: 332–339.

  17. 17

    Durbin, R.D. and Uchytil, T.F. 1977. A survey of plant insensitivity to tentoxin. Phytopathol. 67: 602–603.

  18. 18

    Zelcer, A., Aviv, D. and Galun, E. 1978. Interspecific transfer of cytoplasmic male sterility by fusion between protoplasts of normal Nicotiana sylvestris and X-ray irradiated protoplasts of male-sterile N. tabacum. Zeit. Pflanzenphysiol. 90: 397–407.

  19. 19

    Gleba, Y.Y. and Sytnik, K.M. 1984. Protoplast Fusion, p. 98–110. Springer-Verlag, Berlin.

  20. 20

    Evans, D.A., Flick, C.E. and Jensen, R.A. 1981. Disease resistance:incorporation into sexually-incompatible somatic hybrids of the genus Nicotiana. Science 213: 907–909.

  21. 21

    Evans, D.A., Flick, C.E., Kut, S.A. and Reed, S.M. 1982. Comparison of Nicotiana tabacum and Nicotiana nesophila hybrids produced by ovule culture and protoplast fusion. Theor. Appl. Genet. 62: 193–198.

  22. 22

    Reed, S.M. and Collins, G.B. 1978. Interspecific hybrids in Nicotiana through in vitro culture of fertilized ovules. J. Hered. 69: 311–315.

  23. 23

    Chupeau, Y., Missonier, C., Hommel, M.-C. and Goujard, J. 1978. Somatic hybrids of plants by fusion of protoplasts: observations on the model system “Nicotiana glauca—Nicotiana langsdorffii”. Molec. Gen. Genet. 165: 239–245.

  24. 24

    Kao, K.N. 1977. Chromosomal behavior in somatic hybrids of soybean-Nicotiana glauca. Molec. Gen. Genet. 150: 225–230.

  25. 25

    Caboche, M. 1980. Nutritional requirements of protoplast-derived, haploid tobacco cells grown at low cell densities in liquid medium. Planta 149: 7–18.

Download references

Author information

Rights and permissions

Reprints and Permissions

About this article

Further reading