Studies On Propagation & Breeding Biology Of Adiantm Reniforme Var. Sinense Through In Vitro Culture

Adiantum reniforme var. sinense is an endemic and endangered fern in China only distributed in a narrow belt along the Yangtze River. Due both to the Three Gorges Dam project and the over extra exploration by local people, its natural habitat was destroyed and the number of it was decreased largely. Since it has been found in Wanxian, it not only arouses the concern of botanists as a relict Quaternary plant, but also arouses the attention of Jurists garden as a new type of ornamental plants. Breeding studies, would not only benefit the production, protection and understanding of the endangered mechanism of this plant, but also enrich the landscaping materials, biological garden-sik diversity. Basing on the in vitro culture of A. reniforme var. sinense, studies of gametophyte development, sporophyte formation and effects of different factors on sporophyte regeneration had been carried out. The main findings are as follows:1) The spore is typical trilete, tetrachedral, yellowish brown in colour, and had an outer skin-like region of the sporoderm to peel off in bits. Spore germinated in Vittaria type and developed in Adiantum type. In nature, sex organs appeared from 40 to 60 days in succession. Most gametophytes were female, hermaphroditic gametophyte was few. Each gametophyte had several archegonia, and only one sporophyte was produced per gametophyte. The sporophyte was formed by inter gametophytic selfing hybridization.2) An efficient method for sterilizing and sowing spores was established. Through this method, contamination and loss of spores is minimized, and can be sowed in adjustable, even densities.3) The result of in vitro culture of spores showed that light was required for spore germination and gametophyte growth. MS medium with 1.5-3% （w/v） sucrose benefit the further development of gametophyte and sex organs formation. Most gametophytes were females, few gametophytes were hermaphroditic, were very few and no male gametophyte. Higher concentration of sucrose （4.5-6%, w/v） disturbed gametophyte growth and development. In vitro culture, sporophyte formation rate was extremely low in MS medium.4) In in vitro culture, appropriate amount of exogenous Ca²⁺could promote gametophytes to incrassate, produce more meristems and archegonia, and stimulate some gametophytes to induce 3-5 sporophytes from one gametophyte. However, long term treatment of exogenous Ca²⁺, inhibited the formation of sporophytes. The antheridia and archegonia number would increase by a proper period of growth regulators treatment, and the sporophytes were tetraploidy, which indicates the sporophytes formed by sexual propagation. But long term treatment of growth regulators would inhibit the sporophyte formation. Many sporophytes formed by asexual propagation from the gametophyte cell directly, after 5 subcultures in MS medium without growth regulators. And the sex organs were decreased greatly.5) Effects of different growth regulators on sporophyte are different. BA, KT and ZT could promote GGB formed form sporophytes at the beginning of in vitro culture. However, BA was the optimal cytokinin for GGB division and enlargement. During the later in vitro culture process, BA promoted GGB propagation, KT and ZT promoted GGB develop into sporophytes, and ZT was better than KT on sporophyte formation. Lower concentration of IBA and NAA could promote the rhizomes elongation and fronds spread. Higher concentration of IBA and NAA inhibited the rhizomes elongation, but stimulated the sporophyte propagation. 2,4-D could only stimulate callus formation.