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J Integr Plant Biol, 1982, 24 (4): -, Research Article
Embryological Observations on Ovary Culture of Unpollinated Young Flowers in Hordeum vulgare L.
Huang Qun-fei, Yang Hong-yuan and Zhou Chang
Young barley flowers of various stages (from megaspore tetrad to mature embryo sac) were used as materials for culture and subsequent embryological observation. Two culture methods, vertical flower culture and horizontal ovary culture, were adopted. The inocula were cultured at nearly 25 ℃ in dark on N6 medium solidified with agar (0.8%) and supplemented with sucrose (3%–12%), MCPA (0.5–2 ppm), NAA or IAA (lppm) and KT or BAP (0.5–1 ppm). After inoculation, ovaries were sampled at 2–3 day intervals, fixed in aceto-methanol (1:3), stained in toto in diluted Ehrlich’s hematoxylin and sectioned by paraffin method. In all three cultivars tested, embryogenesis within unfertilized embryo sac was observed. The gynogenetic embryos, totally 59 in number, derived mostly from egg apparatus, but some of them came from antipodals too. Usually only one embryo was located in an embryo sac, but in a few cases, two embryos within one embryo sac were observed. The first embryogenie division was transverse in direction, resulting in a basal cell and a terminal cell. The basal cell elongated strikingly and thus pushed the terminal cell toward the center of the embryo sac. Subsequent divisions often led to the formation of a proembryo with peculiar linear shape. Later, multicel- lular embryoids with various sizes and shapes were observed. Some of them showed organ differentiation. Most of the differentiating embryoids were similar to the ordinary zygotic embryo of barley, with a terminal scutellum and a lateral coleoptile. However, some of them showed some abnormal appearance. Ovaries inoculated at megaspore tetrad stage could not be induced to gynogenesis, although in a few cases probable nucellus embryos were observed. Instead, ovaries inoculated at later stages (from uninucleate to mature embryo sacs) did give rise to gynogenetic embryogenisis without the occurence of adventitious embryogeny. The induction-frequency was higher in materials inoculated at 8-nucleate or mature embryo sac stages than at earlier stages. In the latter cases, triggering of embryogenesis could take place only when the embryo sacs were well-differentiated after a period of game tophytie development during culture. Gynogenetic embryos could be induced by both vertical flower culture or horizontal ovary culture, but the former was superior in providing better conditions for growth of ovaries and embryo sacs and thus yielded more embryoids. Divisions of unfertilized polar nuclei leading to endosperm-like free nuclei were also found in cultured ovaries. However, such structure was not likely to play a similar role of nurse tissue as in vivo for the gynogenetic embryos in vitro, since it did not often accompany the occurence of embryoids within the same embryo sacs.
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