Summary The Orchidaceae is of economic and ecological importance and constitutes ˜10% of all seed plant species. Here, we report a genome physical map for Cymbidium sinense, a well‐known species belonging to genus Cymbidium that has thousands of natural variation varieties of flower organs, flower and leaf colours and also referred as the King of Fragrance, which make it arose into a unique cultural symbol in China. The high‐quality chromosome‐scale genome assembly was 3.52 Gb in size, 29 638 protein‐coding genes were predicted, and evidence for whole‐genome duplication shared with other orchids was provided. Marked amplification of cytochrome‐ and photosystem‐related genes was observed, which was consistent with the shade tolerance and dark green leaves of C. sinense. Extensive duplication of MADS‐box genes, and the resulting subfunctional and expressional differentiation, was associated with regulation of species‐specific flower traits, including wild‐type and mutant‐type floral patterning, seasonal flowering and ecological adaption. CsSEP4 was originally found to positively regulate gynostemium development. The CsSVP genes and their interaction proteins CsAP1 and CsSOC1 were significantly expanded and involved in the regulation of low‐temperature‐dependent flowering. Important genetic clues to the colourful leaf traits, purple‐black flowers and volatile trait in C. sinense were also found. The results provide new insights into the molecular mechanisms of important phenotypic traits of Cymbidium and its evolution and serve as a powerful platform for future evolutionary studies and molecular breeding of orchids.

Versatile protoplast platforms greatly facilitate the development of modern botany. However, efficient protoplast-based systems are still challenging for numerous horticultural plants and crops. Orchids are globally cultivated ornamental and medicinal monocot plants, but few efficient protoplast isolation and transient expression systems have been developed. In this study, we established a highly efficient orchid protoplast isolation protocol by selecting suitable source materials and optimizing the enzymatic conditions, which required optimal D-mannitol concentrations (0.4–0.6 M) combined with optimal 1.2% cellulose and 0.6% macerozyme, 5 μM of 2-mercaptoethanol and 6 h digestion. Tissue- and organ-specific protoplasts were successfully isolated from young leaves [∼3.22 × 106/g fresh weight (FW)], flower pedicels (∼5.26 × 106/g FW), and young root tips (∼7.66 × 105/g FW) of Cymbidium orchids. This protocol recommends the leaf base tissues (the tender part of young leaves attached to the stem) as better source materials. High yielding viable protoplasts were isolated from the leaf base of Cymbidium (∼2.50 × 107/g FW), Phalaenopsis (1.83 × 107/g FW), Paphiopedilum (1.10 × 107/g FW), Dendrobium (8.21 × 106/g FW), Arundina (3.78 × 106/g FW) orchids, and other economically important monocot crops including maize (Zea mays) (3.25 × 107/g FW) and rice (Oryza sativa) (4.31 × 107/g FW), which showed marked advantages over previous mesophyll protoplast isolation protocols. Leaf base protoplasts of Cymbidium orchids were used for polyethylene glycol (PEG)-mediated transfection, and a transfection efficiency of more than 80% was achieved. This leaf base protoplast system was applied successfully to analyze the CsDELLA-mediated gibberellin signaling in Cymbidium orchids. We investigated the subcellular localization of the CsDELLA-green fluorescent protein fusion and analyzed the role of CsDELLA in the regulation of gibberellin to flowering-related genes via efficient transient overexpression and gene silencing of CsDELLA in Cymbidium protoplasts. This protoplast isolation and transient expression system is the most efficient based on the documented results to date. It can be widely used for cellular and molecular studies in orchids and other economically important monocot crops, especially for those lacking an efficient genetic transformation system in vivo.

Background Manipulation of flowering time and frequency of blooming is key to enhancing the ornamental value of orchids. Arundina graminifolia is a unique orchid that flowers year round, although the molecular basis of this flowering pattern remains poorly understood. Results We compared the A. graminifolia transcriptome across tissue types and floral developmental stages to elucidate important genetic regulators of flowering and hormones. Clustering analyses identified modules specific to floral transition and floral morphogenesis, providing a set of candidate regulators for the floral initiation and timing. Among candidate floral homeotic genes, the expression of two FT genes was positively correlated with flower development. Assessment of the endogenous hormone levels and qRT-PCR analysis of 32 pathway-responsive genes supported a role for the regulatory networks in floral bud control in A. graminifolia. Moreover, WGCNA showed that flowering control can be delineated by modules of coexpressed genes; especially, MEgreen presented group of genes specific to flowering. Conclusions Candidate gene selection coupled with hormonal regulators brings a robust source to understand the intricate molecular regulation of flowering in precious orchids.

Protoplast systems have been proven powerful tools in modern plant biology. However, successful preparation of abundant viable protoplasts remains a challenge for Cymbidium orchids. Herein, we established an efficient protoplast isolation protocol from orchid petals through optimization of enzymatic conditions. It requires optimal D-mannitol concentration (0.5 M), enzyme concentration (1.2 % (w/v) cellulose and 0.6 % (w/v) macerozyme) and digestion time (6 h). With this protocol, the highest yield (3.50 × 107/g fresh weight of orchid tissue) and viability (94.21%) of protoplasts were obtained from flower petals of Cymbidium. In addition, we achieved high transfection efficiency (80%) through the optimization of factors affecting polyethylene glycol (PEG)-mediated protoplast transfection including incubation time, final PEG4000 concentration and plasmid DNA amount. This highly efficient protoplast-based transient expression system (PTES) was further used for protein subcellular localization, bimolecular fluorescence complementation (BiFC) assay and gene regulation studies of flowering related genes in Cymbidium orchids. Taken together, our protoplast isolation and transfection protocol is highly efficient, stable and time-saving. It can be used for gene function and molecular analyses in orchids and other economically important monocot crops.

BackgroundCymbidium goeringii is one of the most horticulturally important and popular ornamental plants in the orchid family (Orchidaceae). It blooms in winter during January–March and a period of low temperature is necessary for its normal flowering, otherwise there is flower bud abortion, which seriously affects the economic benefits. However, the molecular mechanism underlying winter-blooming behavior in C. goeringii is unclear.ResultsIn this research, we firstly study the flowering physiology of C. goeringii by cytobiology observations and physiological experiments. Using comparative transcriptome analysis, we identified 582 differentially expressed unigenes responding to cold treatment that were involved in metabolic process, flowering time, hormone signaling, stress response, and cell cycle, implying their potential roles in regulating winter-blooming of C. goeringii. Twelve MADS-box genes among them were investigated by full-length cDNA sequence analysis and expression validation, which indicated that three genes within the SHORT VEGETATIVE PHASE (SVP) sub-group had the most significant repressed expression after cold treatment. Further analysis revealed that the SVP genes showed population variation in expression that correlated with cold-regulated flowering and responded to low temperature earlier than the flowering pathway integrators CgAP1, CgSOC1, and CgLFY, suggesting a potential role of CgSVP genes in the early stage of low-temperature-induced blooming of C. goeringii. Moreover, a yeast two-hybrid experiment confirmed that CgSVP proteins interacted with CgAP1 and CgSOC1, suggesting that they may synergistically control the process of C. goeringii flowering in winter.ConclusionsThis study represents the first exploration of flowering physiology of C. goeringii and provides gene expression information that could facilitate our understanding of molecular regulation of orchid plant winter-flowering, which could provide new insights and practical guidance for improving their flowering regulation and molecular breeding.Electronic supplementary materialThe online version of this article (10.1186/s12864-019-5425-7) contains supplementary material, which is available to authorized users.