A method for isolating embryonic cells in order to establish primary cell cultures has been developed. Embryos, intact or cut into small pieces, were digested with different cell-separating enzymes and after 24h post-digestion in 0.6M mannitol they were sheared by using a hypodermic syringe. The resulting cell suspension consisted of morphologically intact cells. This work has been done using embryos of species belonging to widely differing families to show the general applicability of the method. In the procedure to macerate embryos into single cells it was found that embryos were surrounded by a cuticle. Primary cultures of embryonic cells of carrot, Daucus carota L., var. Chanteney Red Core and apple, Pyrus malus L., var. Phiriki and Golden Delicious, were established. Carrot embryo cells divided and approximately 30% of them gave direct growth to embryoids, while approximately 40% formed callus and 20% showed increase of their size when cultured in the Fujimura and Komamine (1975) agar medium. Apple embryo cells cultured in variously supplemented agar media showed only a few divisions but no colony formation was observed. In liquid cultures apple embryonic cells divided and showed the typical sigmoid curve. When they were transferred to low auxin-containing or auxin-deprived media they showed rare embryo formation. Adventitious roots, shoots or both and embryos were formed in apple embryonic tissues of the varieties Phiriki and Golden Delicious when cultured under light and at 25 +/- 2°C on the Murashige and Skoog (1962) medium as modified by Pech et al. (1975) containing various growth regulators or combinations of them. Of the different parts of the embryo tissue, the cotyledon was most responsive in morphogenesis. Embryos developed from structures formed mainly at the base of the cotyledon. The majority of these structures proliferated and formed callus, while very few of them continued their growth as embryos. The formation of shoots and roots was also localised at the base of the cotyledon. Adventitious shoots when transferred to the same medium containing 2mg/l IAA, rooted and formed plantlets.
|Date of Award||1983|