Renanthera imschootiana Rolfe is an endangered tropical epiphytic orchid that is threatened with extinction due to over-collection and the loss of suitable habitats. In vitro propagation is a useful way to mass produce plants for re-establishment in the wild and for commercial propagation. Seeds collected 150 days after pollination (DAP) were the optimum stage for in vitro culture. Seed germination reached 93.1% on quarter-strength MS (i.e., MS containing a quarter of macro- and micronutrients) medium containing 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), 20% coconut water (CW), 1.0 g l⁻¹ peptone, 10 g l⁻¹ sucrose and 1.0 g l⁻¹ activated charcoal (AC). Quarter-strength MS medium supplemented with 1.0 mg l⁻¹ BA, 0.5 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 10 g l⁻¹ sucrose and 20% CW was suitable for the sub-culture of protocorm-like bodies (PLBs) in which the PLB proliferation ratio was 2.88. Quarter-strength MS medium containing 1.0 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 100 g l⁻¹ banana homogenate (BH), and 1.0 g l⁻¹ AC was suitable for plantlet formation and 95.67% of plantlets developed from PLBs within 60 days of culture. Hyponex N016 medium supplemented with 0.5 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 20 g l⁻¹ sucrose, 150 g l⁻¹ BH, and 1.0 g l⁻¹ AC was suitable for the in vitro growth of plantlets about 2-cm in height. Plantlets 3-cm in height or taller were transplanted to Chilean sphagnum moss, and 95% of plantlets survived after 60 days in a greenhouse. Three hundred transplanted of seedlings 360-days old were reintroduced into three natural habitats. Highest percentage survival (79.67%) was observed in Yuanjiang Nature Reserve two years after reintroduction, followed by Huolu Mountain forest park (71.33%). This protocol is an efficient means for the large-scale propagation and in vitro and in vivo germplasm conservation of R. imschootiana.

Backgrounds: Paphiopedilum is an important genus of the orchid family Orchidaceae and has high horticultural value. The wild populations are under threat of extinction because of overcollection and habitat destruction. Mature seeds of most Paphiopedilum species are difficult to germinate, which severely restricts their germplasm conservation and commercial production. The factors inhibiting germination are largely unknown. Results: In this study, large amounts of non-methylated lignin accumulated during seed maturation of Paphiopedilum armeniacum (P. armeniacum), which negatively correlates with the germination rate. The transcriptome profiles of P. armeniacum seed at different development stages were compared to explore the molecular clues for non-methylated lignin synthesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that a large number of genes associated with phenylpropanoid biosynthesis and phenylalanine metabolism during seed maturation were differentially expressed. Several key genes in the lignin biosynthetic pathway displayed different expression patterns during the lignification process. PAL, 4CL, HCT, and CSE upregulation was associated with C and H lignin accumulation. The expression of CCoAOMT, F5H, and COMT were maintained at a low level or down-regulated to inhibit the conversion to the typical G and S lignin. Quantitative real-time RT-PCR analysis confirmed the altered expression levels of these genes in seeds and vegetative tissues. Conclusions: This work demonstrated the plasticity of natural lignin polymer assembly in seed and provided a better understanding of the molecular mechanism of seed-specific lignification process.