蓝猪耳精细胞的分离及两个精细胞群体的收集

Isolation and Collection of Two Groups of Sperm Cells from Torenia fournieri

摘要:

蓝猪耳是二细胞型花粉,生殖细胞在花粉管中分裂形成两个精细胞。用体内-体外技术培养出花粉管后,将其置于爆破液中即可释放出花粉管内含物,其中包括两个精细胞和营养细胞。在显微镜下两个精细胞具二型性:体积较大的精细胞与花粉管的营养核相连,体积较小的精细胞只与大精细胞连接。两个精细胞之间的连接比较结实,需用微量酶液将两个精细胞分开。用显微操作仪就可分别挑选出两个精细胞群体,分别有上百个细胞。蓝猪耳精细胞的成功分离为利用蓝猪耳开展离体受精研究打下了良好的基础。这种单一纯化的精细胞群体的获得为用分子生物学方法区分两个精细胞的特异基因和蛋白质创造了条件。

Abstract:

Pollen of Torenia fournieri is two-celled and the division of generative cell occurs in the pollen tube. In vivo - in vitro technique was applied to get isolated sperm cells. After growing in vivo for 5 h, the pollinated style was cut and transferred into a medium containing 5% (w/v) sucrose, 0.01% (w/v) H3BO3, 0.01% (w/v) CaCl2, 0.01% (w/v) KH2PO4, at pH 5.0. Numerous pollen tubes emerged from the cut end of the style after culture for 2-3 h. When the pollen tubes were transferred to a broken solution containing 5％ mannito1, some pollen tubes broke and the tube contents including two sperm cells were released in the broken solution．The two sperm cells of Torenia fournieri are dimorphism：one is big and the other small．The bigger one (Svn) associates with vegetative nucleus and the small one (Sua) associates with the bigger one．Two sperm cells can be divided into two individual groups using a micromanipulator．These tw o dimorphic sperm cells could provide male gametophytic cells for further in vitro studies and various biotechnological experiments，such as preferential fertilization and gametic recognition in higher plant.