Asymbiotic germination of immature seeds and the seedling development of Cypripedium macranthos Sw., an endangered lady's slipper orchid

Cypripedium macranthos is a lady's slipper orchid with high horticultural value. We document a reproducible protocol for asymbiotic seed germination based on a defined time frame. The optimum germination percentage was obtained from immature seeds collected at 6 weeks after pollination (WAP). At this time, the embryo reached the early globular stage. As seeds matured, the germination percentage declined sharply. Histological observation revealed that two distinct layers of seed coats enveloped the embryos tightly at maturity. The histochemical staining by Nile red and TBO indicated the presence of hydrophobic substance and phenolic compounds (e.g. lignin and cuticular material) in the seed coats. These features reflected the strong hydrophobic nature of seed coat that may cause the seed dormancy. Among the surveyed media, the modified Harvais medium was the optimum for germination. Among the surveyed organic supplements, potato homogenate (50 g l −1 ) or banana homogenate (25 or 50 g l −1 ) was suitable for the growth of seedlings.