Development of a Method for Germinating Endangered Cypripedium guttatum and Efficacy of Seedling Production from Preserved Seeds Aimed at Ex Situ Conservation

Abstract

Cypripedium guttatum (common name: Spotted Lady’s Slipper) grows naturally in one location in Japan. However, the failure of conservation measures to increase the C. guttatum population has created an urgent need to supplement in situ conservation with ex situ conservation. We were contracted by the Ministry of the Environment (MOE) Tohoku Regional Environment Office to develop a method to germinate C. guttatum seeds collected from C guttatum’s natural habitat and stored by the MOE Shinjuku Gyoen National Garden Management Office and to evaluate the efficacy of using preserved seeds, which is essential for promoting ex situ conservation. Prior to attempting to germinate preserved seeds, we conducted a pilot study using 900 seeds collected from C. guttatum maintained in cultivation plots and Oyamada medium developed especially for Cypripedium spp. The 312 seedlings that germinated were subcultured in seedling growth medium; of these, the 99 seedlings that showed growth were removed from their flasks and transplanted to planting trays filled with horticultural soil and grown for 2 years. The 30 surviving seedlings were planted out in experimental plots and the first bloom was observed the following year. Based on these results, we sowed 290 seeds collected in 2014 and stored by the Shinjiku Gyoen National Park Management Office, which resulted in the germination of six seedlings. These seedlings were subcultured in seedling growth medium and ultimately yielded two plants. Based on the results of this pilot study, we conducted a germination experiment using 7,265 preserved seeds collected in 2015. Intermittent germination of 108 seedlings was observed between day 50 and day 800 after sowing. The resulting germination rate (1.5%) was significantly lower than the germination rate of seeds collected from cultivated plants (34.7%) conducted as a control experiment. Of these 108 seedlings, the 101 that exhibited differentiation were subcultured in seedling growth medium; of these, the 57 that showed growth were removed from their flasks and transplanted to planting trays filled with horticultural soil and grown for 2 years. The 22 surviving seedlings were submitted to MOE. This research, conducted as part of MOE’s effort to conserve and increase populations of endangered plants, has yielded the first report both inside and outside Japan of germination of C. guttatum from preserved seeds collected from wild C. guttatum and demonstrates the efficacy of using preserved seeds for ex situ conservation.