Summary
Plant heterokaryons were identified and isolated from a population of protoplasts following fusion. Endogenous chlorophyll autofluorescence and exogenously supplied fluorochromes were utilized to differentiate between parental and heterokaryon populations. Flow cytometric analysis detected heterokaryons based upon the simultaneous presence of both chlorophyll and an exogenously supplied fluorochrome within one cell. These parameters were utilized to sort large numbers of heterokaryons from the fusion mixture using modified flow instrumentation. Modifications to the instrumentation which allowed this sorting are discussed.
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Arndt-Jovin, D. J., Jovin, T. M., 1978: Automated cell sorting with flow systems. Annu. Rev. Biophys. Bioeng.7, 527–558.
Bergounioux, C.,Perennes, C.,Tabaeizadeh, Z.,Prevot, P.,Gadal, P., 1983: Characterization and separation ofPetunia hybrida andLycopersicon esculentum protoplasts based on natural fluorescence or FITC added to label antibodies in immunoreaction with the protoplast membrane. Sixth Int. Protoplast Symp., Basel, Switzerland.
Brown, S., Renaudin, J.-P., Prevot, C., Guern, J., 1984: Flow cytometry and sorting of plant protoplasts: Technical problems and physiological result from a study of pH and alkaloids inCatharanthus roseus. Physiol. Veget.27, 541–554.
Chein, Y. C., Kao, K. N., Wetter, L. R., 1982: Chromosomal and isozyme studies ofNicotiana tabacum—Glycine max Hybrid Cell Lines. Theor. appl. Genet.62, 301–304.
Cocking, E. C., Davey, M. R., Pental, D., Power, J. B., 1981: Aspects of plant genetic manipulation. Nature (Lond.)293, 265–270.
Davey, M. R., Kumar, A., 1983: Higher plant protoplasts—retrospect and prospect. Int. Rev. Cytol. Suppl.1b, 219–299.
Fellner-Feldegg, H., Hurlbut, P., Glimelius, K., 1984: Sorting of miniprotoplasts by flow cytometry. Tenth Int. Conf. Anal. Cytol., Pacific Grove, California, U.S.A.
Frearson, E. M., Power, J. B., Cocking, E. C., 1973: The isolation, culture, and regeneration ofPetunia leaf protoplasts. Dev. Biol.33, 130–137.
Galbraith, D. W., Afonso, C. L., Harkins, K. R., 1984: Flow sorting and culture of protoplasts: Conditions for high-frequency recovery, growth, and morphogenesis from sorted protoplasts of suspension cultures ofNicotiana. Plant Cell Rep.3, 151–155.
Gleba, Y. Y., Hoffmann, F., 1978: Hybrid cell linesArabidopsis thaliana andBrassica campestris: no evidence for specific chromosome elimination. Mol. gen. Genet.165, 257–264.
—,Momot, V. P., Cherep, N. N., Skarzynskaya, M. V., 1982: Intertribal hydrid cell lines ofAtropa belladona xNicotiana chinensis obtained by cloning individual protoplast fusion products. Theor. appl. Genet.62, 75–79.
Griffith, J. K., Cram, L. S., Crawford, B. D., Jackson, P. J., Schilling, J., Schimke, R. T., Walters, R. A., Wilder, M. E., Jett, J. H., 1984: Construction and analysis of DNA sequence libraries from flow-sorted chromosomes: Practical and theoretical considerations. Nucl. Acids Res.12, 4019–4034.
Hamill, J. D., Patnaik, G., Pental, D., Cocking, E. C., 1984: The culture of manually isolated heterokaryons ofNicotiana rustica. Proc. Indian Acad. Sci. (Plant Sci.)93, 317–327.
Harkins, K. R., Galbraith, D. W., 1984: Flow sorting and culture of plant protoplasts. Physiol. Plant60, 43–52.
Harms, C. T., Potrykus, I., 1978: Enrichment for heterokaryocytes by the use of iso-osmotic density gradients after plant protoplast fusion. Theor. appl. Genet.53, 49–55.
Jackson, P. J., Roth, E. J., McClure, P. R., Naranjo, C. M., 1984: Selection, isolation and characterization of cadmium-resistantDatura innoxia suspension cultures. Plant Physiol.75, 914–918.
Kao, K. N., 1977: Chromosomal behaviour in somatic hybrids of soybean—Nicotiana glauca. Mol. gen. Genet.150, 225–230.
Kruth, H. S., 1982: Flow cytometry: Rapid biochemical analysis of cells. Anal. Biochem.125, 225–242.
Loken, M. R., Stall, A. M., 1982: Flow cytometry as a analytical and preparative tool in immunology. J. immunol. Meth.50, R 85.
Melamed, M. R..Mullaney, P. F., Mendelsohn, M. I., 1979: Flow cytometry and sorting. New York: J. Wiley and Sons.
Patnaik, G., Cocking, E. C., Hamill, J., Pental, D., 1982: A simple procedure for the manual isolation and identification of plant heterokaryons. Plant Sci. Lett.24, 105–110.
Redenbaugh, K., Ruzin, S., Bartholomew, J., Bassham, J. A., 1982: Characterization and separation of plant protoplasts via flow cytometry and cell sorting. Z. Pflanzenphysiol.107, 65–80.
Rotman, B. R., Papermaster, R. O., 1966: Membrane properties of living cells as studies enzymatic hydrolysis of fluorogenic esters. Proc. Natl. Acad. Sci. U.S.A.55, 134–141.
Stovel, R. T., 1977: The influence of particles on jet breakoff. J. Histochem. Cytochem.25, 813–829.
Thorell, B., 1981: Flow-cytometric analysis of cellular endogenous fluorescence simultaneously with emission from exogenous fluorochromes, light scatter and absorption. Cytometry2, 39–43.
—, 1983: Flow-cytometric monitoring of intracellular flavins simultaneously with NAD(P)H levels. Cytometry4, 61–65.
Wetter, L. R., Kao, K. N., 1980: Chromosome and isoenzyme studies on cells derived from protoplast fusion ofNicotiana withGlycine max xNicotiana glauca cell hybrids. Theor. appl. Genet.57, 273–277.
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Alexander, R.G., Cocking, E.C., Jackson, P.J. et al. The characterization and isolation of plant heterokaryons by flow cytometry. Protoplasma 128, 52–58 (1985). https://doi.org/10.1007/BF01273235
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DOI: https://doi.org/10.1007/BF01273235