Isolation of Egg Cells and Zygotes from Torenia fournieri
蓝猪耳(Toreniafournieri)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14h后的合子细胞。Torenia fournieri is a special plant withembryo sac partly protruding through the micropyleof ovule, and its egg cell, two synergids and part ofthe central cell can be clearly observed using lightmicroscope (Plate I-1, 2). The feature of embryosac of T. fournieri makes it easy to isolate egg celland zygote, especially for physiologically mature andvigorous egg cell to study its fertilization mechanism.Egg cells of T. fournieri were isolated from em-bryo sacs 2 d after anthesis using enzymatic diges-tion or mechanical dissection after mild osmoticshock (Plate I-3, 4). About 5% egg cells (2–3 from50 ovules) could be mechanically dissected within2 h. When 0.1% cellulase and 0.1% pectinase wereadded into the solution, about 18% egg cells (8–10from 50 ovules) could be isolated within 2 h (PlateI-5). Although egg cells could be isolated by me-chanical dissection, they can be used in in vitro fer- tilization to probe fertilization mechanism withoutdeleterious effect of enzymatic action on the surfaceof egg cell. But it is easier to isolate egg cells byusing enzymatic digestion, and more egg cells canbe obtained for the research of molecular biology.Using enzymatic digestion method, the zygote ofTorenia fournieri was also isolated from the polli-nated ovules (Plate I-7–10). Basing on our success-ful isolation of mature sperm cells, the isolation ofegg cells of T. fournieri will make in vitro fertiliza-tion possible for the first time in a dicot plant.