摘要
在5科8种具二细胞型花粉的植物中,通过'活体-离体技术'得到花粉管,从中分离出大量生活精细胞。应用低渗-低酶法可有效地释放精细胞并有利于其纯化。用聚乙二醇(PEG)的'小规模融合法'诱导了5种植物同种精细胞之间、唐菖蒲(Gladiolus gandavensis Van Houtte)与朱顶红(Hippeastrum vittatum Herb.)异种精细胞之间、黄花菜(Hemerocallis minorMill.)精细胞与同种小孢子原生质体之间、朱顶红精细胞与萱草(Hemerocallis fulva L.)小孢子原生质体之间以及唐菖蒲精细胞与同种花瓣原生质体之间的融合,均获得了具生活力的同核体或异核体。二甲基亚砜(DMSO)能促进唐菖蒲花瓣原生质体对精细胞的摄取。研究了影响分离和融合效果的若干因素。
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 shock-weak enzyme treatment' method could effectively release sperms from pollen tubes and favor subsequent 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-tum, 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 obtained 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.
基金
国家自然科学基金与国家教委博士学科点专项科研基金资助的课题
关键词
二细胞型
花粉
精细胞
分离
融合
Bicellular pollen
Sperm cell
Isolation
Fusion
