In vitro proliferation and genetic diversity of Cypripedium macranthos var. rebunense

In vitro culture from seeds to full maturity of Cypripedium macranthos var. rebunense, one of the most endangered species in Japan, was studied. Immature seeds from wild plants germinated efficiently in Tsutsumi-Tomita medium and 1/3 strength Murashige-Skoog medium both supplemented with 1% sucrose. Formed protocorms were transferred onto the same Murashige-Skoog medium developed into seedlings, which were subjected to a cold-treatment. The seedlings were further successively cultured in artificial soils (Florialite and then Cryptomoss) under artificially controlled photoperiod and temperature conditions. After 7 years, fully matured plants successfully flowered. The genetic diversity of the wild colony of this orchid in Rebun Island was examined. Leaf samples randomly collected were subjected to random amplified polymorphic DNA analysis. Initially examined 31 samples were classified into independent 5 groups, indicating that the colony consists of genetically different individuals. This suggested that plants in the habitat are heterogenous as to the genetic structure. Introduction of artificially propagated plants with a similar genetic background to this habitat would match the genetic diversity of the existing population. The results of this study may be helpful in planning the rescue of other endangered plant species.