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J Integr Plant Biol, 1985, 27 (6): -, Research Article
Isolation of Embryo Sac with the Enzyme-Squash Technique
Li Le-gong and Hu Shi-yi
doi:
Abstract
The enzyme-squash technique is especially suited for studying the development of megaspores and embryo sacs in angiosperms. Ovulles are fixed in FPA, FAA or Carnoy's for 2–24 hrs. After passing through a series of alcohol and distilled water, they are treated in an aqueous solution of 2% Driselase. for 3-6 hrs. at 28℃. Ovules are then transferred with a drop of laeto-phenol-glycerin fluid to a slide, covered with a cover glass. By the aid of gently tapping and pressing the cover glass, as that of ordinary squash method, cells of ovules are separated and the megaspores or embryo sacs are isolated, and then examined with phase contrast optics With this technique we have successfully isolated the megaspores and embryo sacs in different developmental stages from fixed ovules of several plant species, including Atropa belladonha, Vanilla fragrans, Belamcanda chinensis, Platycodon grandifIorus and Oenothera odorata. The rosults of the experiments indicate that this technique for isolation of embryo sac has several advantages it is suitable both in tenuinucellate and crassinucellate ovules the manipulation of this technique is rather simple and the special instruments are net required the difficulties of the separation of the embryo sac from its nucellar epidermis or tissues of the chalazal end can be overcome by this method. The initial results of isolation of vital embryo sacs from living ovules has been gotten with this technique.
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