Seed production and storage for endangered Morus boninensis using an ex-situ living collection.

Meaningful conservation techniques for the endangered Morus boninensis require seed production and storage, since in situ purebred seed production is infrequent in Ogasawara Islands (World Natural Heritage Site) in Japan. Ex situ living M. boninensis specimens produced seeds by open- and artificial-pollination in a greenhouse. Seed desiccation tolerance and storage characteristics were examined. Open-pollinated seeds were dried at 75, 33 and 8% relative humidity (RH) for 5 days then cryopreserved at -170 °C. Artificially pollinated seeds were dried at 8% RH then stored cryogenically at -170 °C and in freezers at -80 or -20 °C for 6 months. Germination rates were compared among seeds before and after drying and storage. Germination rates of undried seeds were 83% for open-pollinated seeds and 65% for artificially pollinated seeds. M. boninensis seeds exhibited the characteristic high desiccation tolerance of orthodox seeds and maintained a high germination rate after drying to a moisture content of approximately 4%. Cryopreservation of open-pollinated dry seeds with moisture contents of ca. 12, 7 and 4% maintained germination rates, while cryopreservation of undried seeds (moisture content ca. 44%) resulted in no germinability. Cryogenic and freezer storage of artificially pollinated dry seeds for 6 months resulted in maintenance of germination rates comparable to undried seeds before storage. Seed production was enabled by ex situ M. boninensis collection. The present study also developed a method for low temperature seed storage. Ex situ living tree collection represents a feasible seed resource for reintroduction and restoration programmes for M. boninensis.

Somatic embryogenesis and plant regeneration from immature zygotic embryos of Hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.).

We established a plant regeneration system for Hinoki cypress (Chamaecyparis obtusa) via somatic embryogenesis. Embryogenic tissues were successfully induced on three kinds of Smith media from megagametophyte explants containing pre-cotyledonary embryos of C. obtusa plus-trees. Factors affecting somatic embryo maturation were examined. The concentration of polyethylene glycol 4000 in the medium was a critical factor for embryo maturation and its effective concentration was 150 g/l. The addition of 30 g/l maltose to the medium had a positive effect on embryo maturation, but sucrose was ineffective. The mature somatic embryos germinated at a germination frequency of approximately 60%, and the presence of activated charcoal was effective in stimulating plantlet growth. The plantlets acclimatized successfully in a greenhouse. To our knowledge, this is first report describing details of a plant regeneration method for C. obtusa via somatic embryogenesis.