葱卵细胞的分离

将大葱(Allium fistulosum)胚珠置于酶液中30分钟可将其外珠被去掉。可清楚地看到由内珠被包裹的胚珠中胚囊的轮廓。将胚珠转移至不含酶的相同溶液中, 用解剖针从胚珠中部切割, 然后挤压胚珠的珠孔部位, 卵器细胞从胚珠的切口处逸出。再用显微操作仪将卵细胞和2个助细胞分开, 达到葱卵细胞分离的目的。酶对分离卵细胞具有重要的作用, 经0.2%果胶酶Y23、0.8%果胶酶、0.8%纤维素酶和0.5%半纤维素酶的处理, 可在2小时内从30 个胚珠中分离出18个卵细胞。随着胚囊的发育, 2个助细胞的体积出现明显差异。生活的葱卵细胞的成功分离, 为建立葱离体受精体系创造了条件。

Isolation of Egg Cells from Allium fistulosum

Viable egg cells of Allium fistulosum were isolated by enzymatic digestion and mechanical dissection. The ovules were incu-bated in an enzymatic solution containing 0-0.8% pectolyse Y-23, 0-1.2% pectinase (Serva), 0-1.2% cellulase (Onozuka R-S), 0-1% hemicellulase (Sigma) and 6%-12% mannitol for 20-40 min to remove the outer integument. Then ovules were tr-ansferred into an isolating solution without enzymes for mechanical dissection. The ovules were cut from the middle part, and the micropylar part was gently extruded by use of a dissection needle. In general, the three cells of the egg apparatus spilled out from the cut end of the ovule. The enzymatic solution containing 0.2% pectolyse Y-23, 0.8% pectinase (Serva), 0.8% cellulase (Onozuka RS), 0.5%hemicellulase (Sigma) and 10% mannitol was best for isolating about 18 egg cells from 30 ovules in 2 h. The isolated egg cells could be used for in vitro fertilization to explore the fertilization mechanism and for egg development with molecular biological methods.