Isolation of embryo sacs from Dianthus ovules by enzymatic treatments and microdissection

Mature ovules of Dianthus (Caryophyllaceae) were histologically observed by clearing and serial sectioning to characterize the cells of the embryo sac . The results show that the mature embryo sac was located deep inside the h emitropous ovule due to thick nucellar tissue at the micropylar region. For the isolation of the embryo sacs, ovules were collected from ovaries of fl owers 1 day after anthesis, and treated with an enzyme solution for digesti ng cell walls on a gyratory shaker. After 12h of enzyme treatment, these ov ules were dissected using a glass needle under an inverted microscope to re lease the embryo sacs. The embryo sacs, characterized by their specific siz e, were successfully released by these successive treatments. The viability of the embryo sacs was more than 80% as assessed with fluorescein diacetat e staining. Fluorescent staining with 4,6-diamidino-2-phenylindole. reveale d the nuclei of the egg apparatus in the isolated embryo sacs. The procedur e for isolating embryo sacs established in this study will offer a new appr oach to further in vitro studies on fertilization in Dianthus.