黄蝉生殖细胞的离体分裂

DIVISION IN ISOLATED GENERATIVE CELLS OF ALLEM-AN DA NERIIFOLIA

本文采用改进的二步渗透压法,分离黄蝉(Allemanda neriifolia)的生殖细胞。先将花粉放在含50%蔗糖的BK培养液内,28℃,往复振荡90次/分,培养2小时。在此条件下花粉不但能够萌发,且能形成长约300μm的花粉管。培养2小时后加入适量的不含蔗糖的BK培养液,使原培养液的蔗糖浓度由50%骤降至30%,大量花粉管受到渗透压冲击在末端破裂,释放出包括生殖细胞在内的内含物,经过滤、荧光素二醋酸酯鉴定,具有酯酶活性和完整的细胞膜,生殖细胞仍然保持生活力。生殖细胞从花粉管释出后,在很短的时间内(约30分钟)便全部由纺锤形变为圆球形。在含30%蔗糖的BK培养液内悬浮培养,离体的生殖细胞不但能保持生活力,而且还可以继续分裂,获得离体生殖细胞的各种分裂相。

An osmotic shock method of isolating generative cells from Allemanda neriifolia was described. Fresh pollen grains were first placed in a Brewbaker and Kwack's medium (BK medium) containing 50% sucrose, incubated at 28℃ for 2 hours. During this incubation period pollen grains germinated and produced pollen tubes measuring about 200 μrn long. After this initial incubation period, a fixed amount of BK medium without sucrose was added thus diluting, the original medium to a sucrose concentration of 30% - an optimum concentration for generative cell growth. The addition, of the BK medium without sucrose brought about an osmotic, shock effect on the pollen tubes and caused most of the tubes to burst at the tip region thus releasing the contents together with the generative cell from the tube into the 30% sucrose + BK medium. After rsolation and filtering into a fresh lot of 30% sucrose + BK medium, genera- tive cells changed from spindle into spherical-shaped cells. In the 30% sucrose + BK medium, the generative cells divided and within a short period of 3 to 5 hours a laege number of cells at various stages of mitosis was obtained.