Characterization of embryo and protocorm development of Paphiopedilum spicerianum

Paphiopedilum spicerianum (P. spicerianum) is a rare orchid species with high ornamental value. Asymbiotic germination is the most efficient propagation method for conservation and commercial purposes because clonal propagation is very difficult and the separation of native species of Paphiopedilum through aseptic seeding is uncommon owing to their conservatism. However, a high protocorm developmental arresting rate during the asymbiotic germination is the major obstacle for seedling establishment. The fundamental understanding of embryo and protocorm developmental mechanisms will guide the development of an effective propagation method. The morphological and physiological characterization of the key developmental process of embryos and protocorms shows that the mature seeds of P. spicerianum consist of a spherical embryo without an endosperm. Seed coats become heavily lignified once the embryo is mature. Embryo cell size is relatively uniform, and significant structure polarity and cell size gradients occur at the early protocorm stage. The high level of auxin and cytokinin accumulation at the early stage of embryo development and protocorm stage may help to facilitate cell division. The transcriptome profiles of protocorms at three different developmental stages were compared to explore the regulatory mechanism of protocorm development. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that differentially expressed genes were implicated in secondary metabolite metabolism, plant hormone signal transduction and photosynthesis. The temporal expression patterns of candidate genes related to embryo and shoot development were analyzed to reveal their roles in protocorm development: in the early stage of protocorm development, embryonic development related genes such as SERKs and BBM1 were active, while in the late stage of protocorm, shoot apical meristem related genes such as WOX8, CLAVATA2, CUC2, and SCR were active.