Abstract: Orchid plants develop protocorms upon germination and produce protocorm-like structures called protocorm-like bodies (PLBs) from protocorms and somatic cells via tissue culture. Protocorm-like bodies have broad technical application potential in the orchid industry and their regeneration is a distinct developmental process in the plant kingdom. However, little is known about this unparalleled developmental program. In this study, we identified a PLB-abundant gene, ethylene response factor (ERF), and a transcription factor named DoERF5, and determined its important role in PLB regeneration in Dendrobium orchid. Overexpression of DoERF5 in Dendrobium greatly enhanced the PLB regeneration from PLB and stem explants, and upregulated the expression of WOUND-INDUCED DEDIFFERENTIATION (DoWIND) homologs and SHOOT MERISTEMLESS (DoSTM), as well as the genes involved in cytokinin biosynthesis (DoIPT) and the cytokinin response factors (DoARRs). However, silencing DoERF5 reduced the regeneration rate of PLBs, and downregulated the expression of DoWIND homologs, DoSTM and DoARRs. We demonstrated that DoERF5 is directly bound to the DoSTM promoter and regulates its expression. In addition, overexpression of DoSTM in Dendrobium orchid resulted in favorable regeneration of PLBs. Our results clarify that DoERF5 regulates the regeneration of PLB by enhancing DoSTM expression. Our findings provide new insights into how DoERF5 mediates PLB regeneration and offers technical potential in improving clonal propagation, preservation, and the bioengineering of orchids.

Key words: EMSA, ERF transcription factor, orchid, protocorm-like body, STM