Calanthe tsoongiana is a rare orchid species native to China. Asymbiotic seed germination is of great importance in the ex situ conservation of this species. Based on morphological characteristics and anatomical structures, the C. tsoongiana developmental process from seeds to seedlings was divided into four stages (SA, PB, PC and PD), and subsequently, changes in endogenous hormone contents and gene expression were assessed using RNA-seq analysis.

K-means analysis divided the DEGs into eight clusters. The gene expression decreased markedly between the imbibed seed and globular protocorm stages, with this being the most notably enriched cluster. During the seed germination period, DEGs were dominated by ATP metabolic processes, respiration and photosynthesis. A small change in gene expression was found in the globular protocorm versus the finger-like protocorm stages. During the last developmental stage, DEGs were significantly enriched in lignin catabolic processes and plant-type secondary cell wall biogenesis.

DEG homologs, such as TSA1, DAO, NCED1, STM, and CUC2, were related to phytohormones and the morphogenesis of shoots, leaves and roots. Particularly, interactions between CUC2 and STM as well as AS1 and STM were likely involved in protocorm formation and development. Furthermore, TSA1 and DAO were distinctly validated and implicated in the synthesis and metabolism of auxin, which has a pivotal role in plant development.

Our study is the first to combine morphological and transcriptome analysis to examine the process of protocorm formation and development. The results provide a foundation for understanding the mechanisms of seed germination and protocorm development of C. tsoongiana.