| Abstract: | Living sperm cells were isolated in large quantities from the pollen tubes, grown by the in vivo-in vitro technique in 8 bicellular pollen species belonging to 5 families. An “osmotic shook weak enzyme treatment” method could effectively release sperms from pollen tubes and favor sub sequent purification. The viable sperm yields were up to 82.9% in Zephyranthes candida and 78.2% in Hemerocallis minor. Fusions were successfully induced by polyethylene glycol (PEG) according to the "small-scale fusion" procedure in various combinations, viz., between the same sperm cells in 5 species, between sperm cells of Gladiolus gandavensis and Hippeastrum vitta turn, between sperm cells and microspore protoplasts in Hemerocallis minor, and between sperm cells of H. vittatum and microspore protoplasts of Hemerocallis fulva. Test with fluorochrome reaction, more than 85% of the fusion products of sperm cells in Z. candida were viable. The yieid of viable fusion products between sperm cells and microspore protoplasts in Hemerocallis minor was about 75% and half of them could survive after culture for 24h. The induction of fusion between sperm cells and petal protoplasts in G. gandavensis by a combined PEG-dimethyl sulfoxide (DMSO) treatment was investigated in detail. About 90% of the fusion products thus obtamed were viable. Several critical factors affecting the fusion efficiency were studied. These included the ratio of sperm cell number to petal protoplast number in the mixture, concentrations of PEG and DMSO, and duration of incubation in the inducing solution. It appeared that addition of DMSO could significantly increase the fusion frequency, and that there may be a synergistic effect between PEG and DMSO. This is the first attempt to use isolated sperm cells for fusion studies in bicellular pollen species. |
