This study successfully established an efficient in vitro propagation system for Paphiopedilum SCBG Huihuang90 via protocorm-like body (PLB) formation from seed-derived calluses, PLB proliferation and differentiation, root induction and greenhouse acclimatization. Furthermore, 1/2 Murashige and Skoog (MS) + 0.025 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) was suitable for the proliferation of PLBs, and 1/2MS + 10% coconut water (CW, v/v) + 0.5 g/L activated carbon (AC) was suitable for PLB differentiation. PLBs at different developmental stages required different kinds of sugars. This study provided a reference for research on the propagation techniques of other Paphiopedilum.

Background: Paphiopedilum, commonly known as slipper orchid, is an important genus of orchid family with prominent horticultural value. Compared with conventional methods such as tillers and in vitro shoots multiplication, induction and regeneration of protocorm-like bodies (PLBs) is an effective micropropagation method in Paphiopedilum. The PLB initiation efficiency varies among species, hybrids and varieties, which leads to only a few Paphiopedilum species can be large-scale propagated through PLBs. So far, little is known about the mechanisms behind the initiation and maintenance of PLB in Paphiopedilum. Results: A protocol to induce PLB development from seed-derived protocorms of Paphiopedilum SCBG Huihuang90 (P. SCBG Prince × P. SCBG Miracle) was established. The morphological characterization of four key PLB developmental stages showed that significant polarity and cell size gradients were observed within each PLB. The endogenous hormone level was evaluated. The increase in the levels of indoleacetic acid (IAA) and jasmonic acid (JA) accompanying the PLBs differentiation, suggesting auxin and JA levels were correlated with PLB development. Gibberellic acid (GA) decreased to a very low level, indicated that GA inactivation may be necessary for shoot apical meristem (SAM) development. Comparative transcriptomic profiles of four different developmental stages of P. SCBG Huihuang90 PLBs explore key genes involved in PLB development. The numbers of differentially expressed genes (DEGs) in three pairwise comparisons (A vs B, B vs C, C vs D) were 1455, 349, and 3529, respectively. KEGG enrichment analysis revealed that DEGs were implicated in secondary metabolite metabolism and photosynthesis. DEGs related to hormone metabolism and signaling, somatic embryogenesis, shoot development and photosynthesis were discussed in detail. Conclusion: This study is the first report on PLB development in Paphiopedilum using transcriptome sequencing, which provides useful information to understand the mechanisms of PLB development.

Orchid seeds lack endosperms and depend on mycorrhizal fungi for germination and nutrition acquisition under natural conditions. Piriformospora indica is a mycorrhizal fungus that promotes seed germination and seedling development in epiphytic orchids, such as Dendrobium nobile. To understand the impact of P. indica on D. nobile seed germination, we examined endogenous hormone levels by using liquid chromatography–mass spectrometry. We performed transcriptomic analysis of D. nobile protocorm at two developmental stages under asymbiotic germination (AG) and symbiotic germination (SG) conditions. The result showed that the level of endogenous IAA in the SG protocorm treatments was significantly higher than that in the AG protocorm treatments. Meanwhile, GA₃ was only detected in the SG protocorm stages. IAA and GA synthesis and signaling genes were upregulated in the SG protocorm stages. Exogenous GA₃ application inhibited fungal colonization inside the protocorm, and a GA biosynthesis inhibitor (PAC) promoted fungal colonization. Furthermore, we found that PAC prevented fungal hyphae collapse and degeneration in the protocorm, and differentially expressed genes related to cell wall metabolism were identified between the SG and AG protocorm stages. Exogenous GA₃ upregulated SRC2 and LRX4 expression, leading to decreased fungal colonization. Meanwhile, GA inhibitors upregulated EXP6, EXB16, and EXP10-2 expression, leading to increased fungal colonization. Our findings suggest that GA regulates the expression of cell wall metabolism genes in D. nobile, thereby inhibiting the establishment of mycorrhizal symbiosis.

Flower color is a key ornamental trait in Nobile-type Dendrobium, yet the molecular basis underlying purple flower formation remains poorly understood. In this study, a white-flowered paternal cultivar and its purple-flowered filial line were used to investigate the regulatory mechanism of purple floral pigmentation. Comparative phenotypic analysis showed that floral color divergence was established early during flower development, and anthocyanidin profiling of full-bloom petals revealed that purple flowers accumulated substantially higher levels of most anthocyanidins than white flowers, with delphinidin- and cyanidin-derived anthocyanidins together accounting for 80.26% and 94.56% of the total anthocyanidins in white- and purple-flowered materials, respectively. Transcriptome profiling identified a total of 21,235 differentially expressed genes (DEGs), with significant enrichment of phenylpropanoid- and flavonoid-related pathways, and MYB transcription factors prominently represented among the candidate regulators across the three comparison groups. Among them, DntMYB1 was identified as a C1 subgroup R2R3-MYB associated with floral pigmentation, and transient overexpression assays in Phalaenopsis hybrid V3 and a Nobile-type Dendrobium hybrid supported its positive role in visible purple pigmentation. By integrating RNA-seq and DAP-seq analyses, we identified 3205 candidate downstream targets of DntMYB1 and established a DntMYB1-centered regulatory module. Among these candidates, one Dnt4CL1 gene and one DntF3′H1 gene were validated as robust direct targets of DntMYB1 through yeast one-hybrid, EMSA, and dual-luciferase assays. These findings suggest that DntMYB1 is associated with purple flower formation by coordinately regulating both upstream precursor metabolism and downstream anthocyanin biosynthesis, providing new insight into the molecular regulation of flower color in Nobile-type Dendrobium and useful candidate genes for ornamental trait improvement.