Plant protoplast fusion and growth of intergeneric hybrid cells

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 Ca(2+) 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.