Possibilities of diagnosis of parthenogenesis by culture in vitro of unpollinated ovaries
--Alatortseva, TA; Tyrnov, VS

For seven years we have carried out comparative investigations of lines reproducing by sexual mode, and forms able to undergo parthenogenesis (AT-1 and F1 from AT-1 x sexual forms) (Alatortseva and Tyrnov, Biol Cult Cell and Biotechnology, Proc Int Conf, Alma-Ata, p.139, 1989 (in Russian); Reproductive Biol and Plant Breeding, XIII EUCARPIA Congr, p. 329-330, 1992; Apomixis in Angiosperms: problems and perspectives of investigation, Proc Int Symp, Saratov, Russia, p. 8-9, 1994 (in Russian); Alatortseva, Tyrnov, and Suchanov, Embryology and Seed Reproduction, Proc XI Int Symp Leningrad, USSR, 1990, St.Petersburg Nauka, 1992, p. 29-30; Tyrnov and Alatortseva, Devel Genet, Tashkent, p.164-166, 1990 (in Russian). It has been ascertained, that in the same conditions in vitro on nutrient medium MS with addition of sucrose (9%), 2,4-D (2.0 mg/l) and vitamins the difference was distinct in the reaction of ovaries, which were taken from the donors with different modes of reproduction. The difference is expressed in transition of the egg to division with formation of a proembryo in apomicts, while in female sexual cells of amphimictic plants (16 lines) there are not visible changes, and soon they degenerate. In ovaries of apomictic lines the beginning of autonomous division of the egg without pollination falls 8-10 days after the moment of silk appearance, including the time as explants in nutrient medium. In 3-4 week ovaries, on the surface of apomictic proembryo numerous embryoids appear, which either produce daughter embryoids or form haploid regenerants after 2-3 months of cultivation. Egg division and formation of globular proembryo can take place on hormoneless medium with lower concentrations of sucrose (2-3%).

The influence of endosperm on the fate of the proembryo was also investigated. In unpollinated ovaries of apomictic lines in vivo either autonomous development of proembryo and defective endosperm or only proembryo or only endosperm occur. By cultivation in vitro, regeneration of plantlets happens through embryo- or embryoidogenesis in the absence of endosperm. In the presence of endosperm the process of differentiation of embryos and reproduction of embryoids in early developmental stages is inhibited. Thus, the presence of endosperm in unpollinated ovaries is not a positive factor for formation of apomictic proembryos and regeneration. It can not be excluded that endosperm can play a negative role in sexual forms, as in cases of its development without fertilization its ploidy and other traits will be similar with characteristics of apomictic lines. The absence of endosperm in ovaries is compensated by nutrient medium. This explains the ability of the embryo for further development in vitro, whereas in vivo it is excluded. Under cultivation of unpollinated ovaries, formation of plant-regenerants happens by two ways: through direct embryogenesis or by means of embryoidogenesis. However, in the case of direct embryogenesis the developing embryo frequently degenerates. Regenerants form mainly from embryoids. Sometimes in tissues of the maternal embryo, side by side with centres of embryoidogenesis, formation of absolutely autonomous elements of the conductive system, roots, leaf-like structures and also teratologous structures, giving ugly plants, can be observed. However, they do not inhibit production of normal embryoids and plants.

It has been determined that autonomous parthenogenesis can be manifested in vitro in hybrids between line AT-1 and common amphimictic lines, although with lower frequency than in pure line AT-1. Thus, use of in vitro techniques in parthenogenetic lines allows the possibility of producing haploid plants in mass quantity, and of carrying out diagnosis and selection of apomictic forms. This work was supported in part by a grant from the Russian Foundation for Basic Research. 

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