Abstract:Since the enzymatic technique for isolating embryo sac (ES) has been established on fixed materials of several angiosperms as well as on fresh ovules of Antirrhinum majus in our lab, further works on isolation of viable ESs were carried on. Fresh ovules were macerated in a solution of enzymes, sucrose with or without potassium dextran sulphate by a microshaker at 28–30℃ for several hours. The enzymes included pectinase, cellulase, snailase and pectolyase Y-23, the combination and concentration of which varied with the plant species and the developmental stages of ESs. To date the mature ESs of Helianthus annuus, A. majus and Nicotiana tabacum and the ESs after fertilization with proembryo and endosperm cells in the two former species were well isolated. Nomarski interference contrast and Hoechst 33253 fluorescence microscopical observations revealed that the ESs retained their cell structure and were rich of ergastic substances. Fluorochromasia induced by fluorescein diacetate further proved that they were really viable.