Plant protoplast fusion and growth of intergeneric hybrid cells
- 461 Downloads
Interspecific and intergeneric fusions of plant protoplasts were induced by polyethylene glycol (PEG) 1540 or 4000. The frequency of heterokaryocyte formation (or rate of fusion) was much higher when PEG was eluted with a high pH-high Ca2+ solution or a salt solution than when it was eluted with a protoplast culture medium. The frequency of heterokaryocyte formation was also affected by the types of enzymes used for wall degradation, duration of enzyme incubation and molality of the PEG solutions.
The maximum frequency of heterokaryocyte formation was 23% for V. hajastana Grossh.-soybean (Glycine max L.) and barley (Hordeum vulgare L.)-soybean, 35% for pea (Pisum sativum L.)-soybean, 20% for pea-V. hajastana, 14% for corn (Zea mays L.)-soybean and 10% for V. villosa Roth-V. hajastana.
40% of the barley-soybean, corn-soybean and pea-soybean heterokaryocytes divided at least once. Some divided many times and formed clusters of up to 100 cells in 2 weeks. The heterokaryocytes of soybean-V. hajastana, V. villosa-V. hajastana also divided. Of the PEG-treated protoplasts of N. langsdorffii and N. glauca 13.5% developed into tumor-like calli. The morphology of these calli was very much like that of the tumors produced on amphidiploid plants of N. langsdorffii x glauca.
Nuclear staining indicated that heterokaryocytes of V. hajastana-soybean, pea-soybean, corn-soybean and barley-soybean could undergo mitosis. Nuclear divisions in a heterokaryocyte were usually synchronized or almost synchronized. Nuclear fusion and true hybrid formation usually occurred during the first mitotic division after protoplast fusion. A hybrid of barley-soybean in third cell division was observed. The frequency of heterokaryocytes which underwent nuclear fusion has not been determined. Multipole formation and chimeral cell colonies were also observed.
KeywordsPolyethylene Glycol Hybrid Cell Hordeum Vulgare Pisum Sativum Cell Coloni
Unable to display preview. Download preview PDF.
- Carlson, P. S., Smith, H. H., Dearing, R. D.: Parasexual interspecific plant hybridization. Proc. nat. Acad. Sci. (Wash.) 69, 2292–2294 (1972)Google Scholar
- Constabel, F., Kao, K. N.: Agglutination and fusion of plant protoplasts by polyethylene glycol. Canad. J. Bot. 1603-1606 (1974)Google Scholar
- Hartmann, J. X., Kao, K. N., Gamborg, O. L., Miller, R. A.: Immunological methods for the agglutination of protoplasts from cell suspension cultures of different genera. Planta (Berl.) 112, 45–56 (1973)Google Scholar
- Kao, K. N., Gamborg, O. L., Michayluk, M. R., Keller, W. A., Miller, R. A.: The effects of sugars and inorganic salts on cell regeneration and sustained division in plant protoplasts. In: Protoplastes et fusion de cellules somatiques végétales, p. 207–213 (Tempe, J. ed.). Paris: CNRS 1973Google Scholar
- Kao, K. N., Gamborg, O. L., Miller, R. A., Keller, W. A.: Cell divisions in cells regenerated from protoplasts of soybean and Haplopappus gracilis. Nature (Lond.) New Biol. 232, 124 (1971)Google Scholar
- Kao, K. N., Michayluk, M. R.: A method for high-frequency intergeneric fusion of plant protoplasts. Planta (Berl.) 115, 355–367 (1974)Google Scholar
- Karr, A. L., Albersheim, P.: Polysaccharide-degrading enzymes are unable to attack plant cell walls without prior action by a “wall-modifying enzyme”. Plant Physiol. 46, 69–80 (1970)Google Scholar
- Keller, W. A., Harvey, B. L., Kao, K. N., Miller, R. A., Gamborg, O. L.: Determination of the frequency of interspecific protoplast fusion by differential staining. In: Protoplastes et fusion de cellules somatiques végétales, p. 456–463 (Tempe, J., ed.). Paris: CNRS 1973Google Scholar
- Keller, W. A., Melchers, G.: The effect of high pH and calcium on tobacco leaf protoplast fusion. Z. Naturforsch. 28c, 737–741 (1973)Google Scholar
- Miller, R. A., Gamborg, O. L., Keller, W. A., Kao, K. N.: Fusion and division of nucleic in multinucleated soybean protoplasts. Canad. J. Genet. Cytol. 13, 347–353 (1971)Google Scholar
- Murashige, T., Skoog, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497 (1962)Google Scholar
- Poste, G., Allison, A. C.: Membrane fusion. Biochim. biophys. Acta (Amst.) 300, 421–465 (1973)Google Scholar
- Singh, B. D., Harvey, B. L., Kao, K. N., Miller, R. A.: Selection pressure in cell populations of Vicia hajastana cultured in vitro. Canad. J. Genet. Cytol. 14, 65–70 (1972)Google Scholar
- Smith, H. H.: Genetic tobacco tumors and the problem of differentiation. Brookhaven Nat. Lab. Upton, N. Y., USA, Publ. BNL 967 (T-405) 1965Google Scholar