Isolation of egg cells and zygotes of Torenia fournieri L. and determination of their surface charge

Egg cells of Torenia fournieri were isolated from embryo sacs 1 day after anthesis using enzymatic digestion or mechanical dissection. About 5% of the egg cells and zygotes (2-3 from 50 ovules) could be mechanically dissected within 2 h. When 0.1% cellulase and 0.1% pectinase were added to the mannitol isolation solution, about 18% of the egg cells (8-10 from 50 ovules) could be isolated within 2 h. The egg cells isolated by mechanical dissection could be used for in vitro fertilization studies without any of the potentially deleterious effects of the enzymes on the plasma membrane of egg cell. The egg cells isolated using enzymatic digestion could be used in the study of the molecular biology of female gamete because more egg cells could be isolated with this technique. Using enzymatic digestion, over 10 zygotes from 50 ovules (over 20%) were isolated from the pollinated ovules. Coupled with our successful isolation of mature sperm cells, the isolation of egg cells of T. fournieri will make in vitro fertilization possible in a dicotyledon plant.