蓝猪耳卵细胞和合子的分离

蓝猪耳(Torenia fournieri)胚囊部分裸露出胚珠,在光学显微镜下能清楚观察到卵细胞和助细胞的形态结构.用解剖和酶解-解剖两种方法都能分离出生活卵细胞.用前种方法机械分离出的卵细胞数量较少(5%),但避免了酶对配子识别研究的干扰.在后种方法中加入0.1%纤维素酶和0.1%果胶酶既能使分离更加容易操作,又对卵细胞没有致命伤害,能在短时间内分离出较多的卵细胞(18%).用酶解-解剖方法也可分离出授粉14 h后的合子细胞.

Isolation of Egg Cells and Zygotes from Torenia fournieri

Torenia fournieri is a special plant with embryo sac partly protruding through the micropyle of ovule, and its egg cell, two synergids and part of the central cell can be clearly observed using light microscope (Plate I-1, 2). The feature of embryo sac of T. fournieri makes it easy to isolate egg cell and zygote, especially for physiologically mature and vigorous egg cell to study its fertilization mechanism. Egg cells of T. fournieri were isolated from embryo sacs 2 d after anthesis using enzymatic digestion or mechanical dissection after mild osmotic shock (Plate I-3, 4). About 5% egg cells (2-3 from 50 ovules) could be mechanically dissected within 2 h. When 0.1% cellulase and 0.1% pectinase were added into the solution, about 18% egg cells (8-10 from 50 ovules) could be isolated within 2 h (Plate I-5). Although egg cells could be isolated by mechanical dissection, they can be used in in vitro fertilization to probe fertilization mechanism without deleterious effect of enzymatic action on the surface of egg cell. But it is easier to isolate egg cells by using enzymatic digestion, and more egg cells can be obtained for the research of molecular biology. Using enzymatic digestion method, the zygote of Torenia fournieri was also isolated from the pollinated ovules (Plate I-7-10). Basing on our successful isolation of mature sperm cells, the isolation of egg cells of T. fournieri will make in vitro fertilization possible for the first time in a dicot plant.