Gametophyte and embryonic ontogeny: understanding the reproductive calendar ofCypripedium japonicumThunb. (Cypripedoideae, Orchidaceae), a lady's slipper orchid endemic to East Asia

Background The genusCypripediumL. is one of the five genera of the subfamily Cypripedioideae, members of which are commonly known as lady's slipper orchids.Cypripedium japonicumis a perennial herb native to East Asia, specifically China, Japan, and Korea. Due to its limited distribution, the species is included in the Endangered category of the IUCN Red List. Results We investigated gametophyte development, including complete embryogenesis, inC. japonicum. The complete reproductive cycle is presented based on our observations. Anther development begins under the soil, and meiosis of pollen mother cells begins 3 weeks before anthesis, possibly during early April. The megaspore mother cells develop just after pollination in early May and mature in mid-late June. The pattern of embryo sac formation is bisporic, and there are six nuclei: three forming the egg apparatus, two polar nuclei, and an antipodal cell in the mature embryo sac. Triple fertilization results in the endosperm nucleus, which degenerates when the proembryo reaches the eight-to-sixteen-cell stage. Conclusion Our overall comparisons of the features of gametophyte and embryo development inC. japonicumsuggest that previous reports on the embryology ofCypripediumare not sufficient for characterization of the entire genus. Based on the available information, a reproductive calendar showing the key reproductive events leading to embryo formation has been prepared.