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These YABBY genes are transcription factors (TFs) that play crucial roles in various developmental processes in plants. There is no comprehensive characterization of YABBY genes in a valuable Chinese orchid herb, Dendrobium officinale. In this study, a total of nine YABBY genes were identified in the D. officinale genome. These YABBY genes were divided into four subfamilies: CRC/DL, FIL, INO, and YAB2. Expression pattern analyses showed that eight of the YABBY genes were strongly expressed in reproductive organs (flower buds) but weakly expressed in vegetative organs (roots, leaves, and stems). DoYAB1, DoYAB5, DoDL1, and DoDL3 were abundant in the small flower bud stage, while DoDL2 showed no changes throughout flower development. In addition, DoDL1-3 genes were strongly expressed in the column, tenfold more than in sepals, petals, and the lip. DoYAB1 from the FIL subfamily, DoYAB2 from the YAB2 subfamily, DoYAB3 from the INO subfamily, and DoDL2 and DoDL3 from the CRC/DL subfamily were selected for further analyses. Subcellular localization analysis showed that DoYAB1-3, DoDL2, and DoDL3 were localized in the nucleus. DoYAB2 and DoYAB3 interacted strongly with DoWOX2 and DoWOX4, while DoYAB1 showed a weak interaction with DoWOX4. These results reveal a regulatory network involving YABBY and WOX proteins in D. officinale. Our data provide clues to understanding the role of YABBY genes in the regulation of flower development in this orchid and shed additional light on the function of YABBY genes in plants., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Background: Gibberellins (GAs) are widely involved in plant growth and development. DELLA proteins are key regulators of plant development and a negative regulatory factor of GA. Dendrobium officinale is a valuable traditional Chinese medicine, but little is known about D. officinale DELLA proteins. Assessing the function of D. officinale DELLA proteins would provide an understanding of their roles in this orchid's development., Results: In this study, the D. officinale DELLA gene family was identified. The function of DoDELLA1 was analyzed in detail. qRT-PCR analysis showed that the expression levels of all DoDELLA genes were significantly up-regulated in multiple shoots and GA 3 -treated leaves. DoDELLA1 and DoDELLA3 were significantly up-regulated in response to salt stress but were significantly down-regulated under drought stress. DoDELLA1 was localized in the nucleus. A strong interaction was observed between DoDELLA1 and DoMYB39 or DoMYB308, but a weak interaction with DoWAT1., Conclusions: In D. officinale, a developmental regulatory network involves a close link between DELLA and other key proteins in this orchid's life cycle. DELLA plays a crucial role in D. officinale development., (© 2023. The Author(s).)

Dendrobium officinale, which is a medicine food homology plant, contains many metabolites, especially polysaccharides and flavonoids. Unlike flowers and stems, which are the most frequently harvested organs for a variety of uses, leaves tend to be discarded. This study assessed main metabolites in leaves to identify the most appropriate timing of collection during harvest, which was divided into three stages (S1-S3: 8, 10, and 11 months after sprouting, respectively). Metabolomic and transcriptomic analyses of S1-S3 were performed. Water-soluble polysaccharides (WSPs), flavonoids and free amino acids (FAAs) were detected in leaves. WSPs decreased from S1 to S3 but flavonoids and some FAAs (e.g., phophoserine) increased from S1 to S2, then decreased from S2 to S3. In all three stages, mannose was the dominant monosaccharide among WSPs, followed by glucose. In S2, 35 flavonoids were identified, the most abundant being rutin, schaftoside and vitexin, while 34 FAAs were identified in all three stages, the most abundant being tyrosine, phosphoserine and alanine. A total of 2584, 3414 and 2032 differentially expressed genes (DEGs) were discovered in S1 vs S2, S1 vs S3 and S1 vs S3, respectively. Correlation analysis revealed that five DEGs (DoSUS, DoXYLA, DoFRK, DoGMP, and DoCSLA), two DEGs (DoDFR, and DoANS) and a single DEG (DoPGAM) were involved in the metabolism of WSPs, flavonoids and phosphoserine, respectively. The findings of this study lay a foundation for the commercial exploitation of metabolites in the harvested leaves of D. officinale, and the use of detected DEGs in applied genetic studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Mannan polysaccharides (MPs), which contain substituted O-acetyl groups in their backbone, are abundant in the medicinal plant Dendrobium officinale. Acetyl groups can influence the physiological and biochemical properties of polysaccharides, which mainly accumulate in the stems of D. officinale at four developmental stages (S1-S4), showing an increasing trend and a link with water-soluble polysaccharides (WSPs) and mannose. The genes coding for enzymes that catalyze O-acetyl groups to MPs are unknown in D. officinale. The TRICHOME BIREFRINGENCE-LIKE (TBL) gene family contains TBL and DUF231 domains that can transfer O-acetyl groups to various polysaccharides. Based on an established D. officinale genome database, 37 DoTBL genes were identified. Analysis of cis-elements in the promoter region showed that DoTBL genes might respond to different hormones and abiotic stresses. Most of the genes with MeJA-responsive elements were upregulated or downregulated after treatment with MeJA. qRT-PCR results demonstrated that DoTBL genes had significantly higher expression levels in stems and leaves than in roots. Eight DoTBL genes showed relatively higher expression at S2-S4 stages, which showed a link with the content of WSPs and O-acetyl groups. DoTBL35 and its homologous gene DoTBL34 displayed the higher mRNA level in different organs and developmental stages, which might participate in the acetylation of MPs in D. officinale. The subcellular localization of DoTBL34 and DoTBL35 reveals that the endoplasmic reticulum may play an important role in the acetylation of MPs., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Linalool is an aromatic monoterpene produced in the Chinese medicinal plant Dendrobium officinale, but little information is available on the regulation of linalool biosynthesis. Here, a novel basic helix-loop-helix (bHLH) transcription factor, DobHLH4 from D. officinale, was identified and functionally characterized. The expression profile of DobHLH4 was positively correlated with that of DoTPS10 (R 2 = 0.985, p < 0.01), which encodes linalool synthase that is responsible for linalool production, during floral development. DobHLH4 was highly expressed in petals, and was significantly induced by methyl jasmonate. Analysis of subcellular localization showed that DobHLH4 was located in the nucleus. Yeast one-hybrid and dual-luciferase assays indicated that DobHLH4 bound directly to the DoTPS10 promoter harboring the G-box element, and up-regulated DoTPS10 expression. A yeast two-hybrid screen confirmed that DobHLH4 physically interacted with DoJAZ1, suggesting that DobHLH4 might function in the jasmonic acid-mediated accumulation of linalool. Furthermore, transient overexpression of DobHLH4 in D. officinale petals significantly increased linalool production by triggering linalool biosynthetic pathway genes, especially DoTPS10. We suggest a hypothetical model that depicts how jasmonic acid signaling may regulate DoTPS10 by interacting with DobHLH4 and DoJAZ1. In doing so, the formation of linalool is controlled. Our results indicate that DobHLH4 is a positive regulator of linalool biosynthesis and may be a promising target for in vitro-based metabolic engineering to produce linalool., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Flavonols are important active ingredients that are found in abundance in Dendrobium officinale. Research on flavonol biosynthesis currently focuses on the more ubiquitous kaempferol and quercetin, but little is known on the biosynthesis of myricetin. Notably, flavonol synthase (FLS), which is responsible for the biosynthesis of flavonols, has not yet been identified. In this study, we isolated a flavonol synthase, DoFLS1, from Dendrobium officinale. DoFLS1 harbors conserved 2-oxoglutarate-dependent dioxygenase-specific and FLS-specific motifs. DoFLS1 is a cytoplasmic protein. DoFLS1 was universally expressed in roots, stems, and leaves of juvenile and adult D. officinale plants. DoFLS1 expression was strongly correlated in juvenile and adult D. officinale plants (R 2  = 0.86 and 0.98, respectively; p < 0.01) with the average of corresponding flavonol levels. Transgenic Arabidopsis thaliana expressing DoFLS1 exhibited a 1.24-fold increase in flavonol content and a 25.78% decrease in anthocyanin content compare to wild-type plants, possibly resulting from a 78.61% increase in myricetin level. Moreover, the loss of anthocyanin was attributed to decreased expression of dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS) genes in transgenic A. thaliana that expressed DoFLS1. DoFLS1 also complemented the deficiency in flavonol of the A. thaliana fls1-3 mutant, which had reduced anthocyanin but increased flavonol content relative to the fls1-3 mutant. In addition, DoFLS1 was significantly upregulated after treatment with cold, drought or salicylic acid. These findings provide genetic evidence for the involvement of DoFLS1 in the biosynthesis of flavonol and in response to abiotic stresses.

The APETALA2 (AP2) transcription factors (TFs) play crucial roles in regulating development in plants. However, a comprehensive analysis of the AP2 family members in a valuable Chinese herbal orchid, Dendrobium officinale , or in other orchids, is limited. In this study, the 14 DoAP2 TFs that were identified from the D. officinale genome and named DoAP2-1 to DoAP2-14 were divided into three clades: euAP2, euANT, and basalANT. The promoters of all DoAP2 genes contained cis -regulatory elements related to plant development and also responsive to plant hormones and stress. qRT-PCR analysis showed the abundant expression of DoAP2-2 , DoAP2-5 , DoAP2-7 , DoAP2-8 and DoAP2-12 genes in protocorm-like bodies (PLBs), while DoAP2-3 , DoAP2-4 , DoAP2-6 , DoAP2-9 , DoAP2-10 and DoAP2-11 expression was strong in plantlets. In addition, the expression of some DoAP2 genes was down-regulated during flower development. These results suggest that DoAP2 genes may play roles in plant regeneration and flower development in D. officinale . Four DoAP2 genes ( DoAP2-1 from euAP2, DoAP2 - 2 from euANT, and DoAP2-6 and DoAP2-11 from basal ANT) were selected for further analyses. The transcriptional activation of DoAP2-1, DoAP2-2, DoAP2-6 and DoAP2-11 proteins, which were localized in the nucleus of Arabidopsis thaliana mesophyll protoplasts, was further analyzed by a dual-luciferase reporter gene system in Nicotiana benthamiana leaves. Our data showed that pBD-DoAP2-1, pBD-DoAP2-2, pBD-DoAP2-6 and pBD-DoAP2-11 significantly repressed the expression of the LUC reporter compared with the negative control (pBD), suggesting that these DoAP2 proteins may act as transcriptional repressors in the nucleus of plant cells. Our findings on AP2 genes in D. officinale shed light on the function of AP2 genes in this orchid and other plant species.

Background: DNA methylation is a conserved and important epigenetic modification involved in the regulation of numerous biological processes, including plant development, secondary metabolism, and response to stresses. However, no information is available regarding the identification of cytosine-5 DNA methyltransferase (C5-MTase) and DNA demethylase (dMTase) genes in the orchid Dendrobium officinale., Results: In this study, we performed a genome-wide analysis of DoC5-MTase and DodMTase gene families in D. officinale. Integrated analysis of conserved motifs, gene structures and phylogenetic analysis showed that eight DoC5-MTases were divided into four subfamilies (DoCMT, DoDNMT, DoDRM, DoMET) while three DodMTases were divided into two subfamilies (DoDML3, DoROS1). Multiple cis-acting elements, especially stress-responsive and hormone-responsive ones, were found in the promoter region of DoC5-MTase and DodMTase genes. Furthermore, we investigated the expression profiles of DoC5-MTase and DodMTase in 10 different tissues, as well as their transcript abundance under abiotic stresses (cold and drought) and at the seedling stage, in protocorm-like bodies, shoots, and plantlets. Interestingly, most DoC5-MTases were downregulated whereas DodMTases were upregulated by cold stress. At the seedling stage, DoC5-MTase expression decreased as growth proceeded, but DodMTase expression increased., Conclusions: These results provide a basis for elucidating the role of DoC5-MTase and DodMTase in secondary metabolite production and responses to abiotic stresses in D. officinale.

Key Message: This research provides comprehensive insight into the molecular networks and molecular mechanisms underlying D. officinale flower development. Flowers are complex reproductive organs and play a crucial role in plant propagation, while also providing sustenance for insects and natural bioactive metabolites for humans. However, knowledge about gene regulation and floral metabolomes in flowers is limited. In this study, we used an important orchid species (Dendrobium officinale), whose flowers can be used to make herbal tea, to perform transcriptome sequencing and metabolic profiling of early- and medium-stage flower buds, as well as opened flowers, to provide comprehensive insight into the molecular mechanisms underlying flower development. A total of 8019 differentially expressed genes (DEGs) and 239 differentiated metabolites were found. The transcription factors that were identified and analyzed belong exclusively to the MIKC-type MADS-box proteins and auxin responsive factors that are known to be involved in flower development. The expression of genes involved in chlorophyll and carotenoid biosynthesis strongly matched the metabolite accumulation patterns. The genes related to flavonoid and polysaccharide biosynthesis were active during flower development. Interestingly, indole-3-acetic acid and abscisic acid, whose trend of accumulation was inverse during flower development, may play an important role in this process. Collectively, the identification of DEGs and differentiated metabolites could help to illustrate the regulatory networks and molecular mechanisms important for flower development in this orchid.

The acetylation or deacetylation of polysaccharides can influence their physical properties and biological activities. One main constituent of the edible medicinal orchid, Dendrobium officinale , is water-soluble polysaccharides (WSPs) with substituted O -acetyl groups. Both O -acetyl groups and WSPs show a similar trend in different organs, but the genes coding for enzymes that transfer acetyl groups to WSPs have not been identified. In this study, we report that REDUCED WALL ACETYLATION (RWA) proteins may act as acetyltransferases. Three DoRWA genes were identified, cloned, and sequenced. They were sensitive to abscisic acid (ABA), but there were no differences in germination rate and root length between wild type and 35S::DoRWA3 transgenic lines under ABA stress. Three DoRWA proteins were localized in the endoplasmic reticulum. DoRWA3 had relatively stronger transcript levels in organs where acetyl groups accumulated than DoRWA1 and DoRWA2 , was co-expressed with polysaccharides synthetic genes, so it was considered as a candidate acetyltransferase gene. The level of acetylation of polysaccharides increased significantly in the seeds, leaves and stems of three 35S::DoRWA3 transgenic lines compared to wild type plants. These results indicate that DoRWA3 can transfer acetyl groups to polysaccharides and is a candidate protein to improve the biological activity of other edible and medicinal plants.

Terpene synthase (TPS) is a critical enzyme responsible for the biosynthesis of terpenes, which possess diverse roles in plant growth and development. Although many terpenes have been reported in orchids, limited information is available regarding the genome-wide identification and characterization of the TPS family in the orchid, Dendrobium officinale . By integrating the D. officinale genome and transcriptional data, 34 TPS genes were found in D. officinale . These were divided into four subfamilies (TPS-a, TPS-b, TPS-c, and TPS-e/f). Distinct tempospatial expression profiles of DoTPS genes were observed in 10 organs of D. officinale . Most DoTPS genes were predominantly expressed in flowers, followed by roots and stems. Expression of the majority of DoTPS genes was enhanced following exposure to cold and osmotic stresses. Recombinant DoTPS10 protein, located in chloroplasts, uniquely converted geranyl diphosphate to linalool in vitro. The DoTPS10 gene, which resulted in linalool formation, was highly expressed during all flower developmental stages. Methyl jasmonate significantly up-regulated DoTPS10 expression and linalool accumulation. These results simultaneously provide valuable insight into understanding the roles of the TPS family and lay a basis for further studies on the regulation of terpenoid biosynthesis by DoTPS in D. officinale .

Stigmatic mucilage plays a crucial role in pollen-grain adhesion on the stigma in flowering plants. Little information is available regarding mucilage biosynthesis in orchid plants. In the present study, stigmatic mucilage is rich in galactose-containing polysaccharides, mainly consisted of galactose and arabinose in Dendrobium officinale. Thirteen galactosyltransferases involved in biosynthesis of the β-1,3-galactose linkage polysaccharides, belonging to the CAZY GT31 family, were identified from D. officinale genome. A positive correlation between the mucilage content and the DoGALT2 expression at different stages was observed. DoGALT2 expressed overall sampled tissues with the highest in D. officinale stigmatic mucilage that contributes to pollen adhesion and elongation. DoGALT2 was targeted to Golgi, and had a GALT domain (PF01762) that was homologous to the characterized GALT2 in Arabidopsis. Compared to wild-type Arabidopsis, DoGALT2 overexpressing plants showed a higher content of galactose and galactose-containing alcohol-insoluble residues, and enhanced tolerance to abiotic stress. DoGALT2 complemented Arabidopsis GALT2 mutant (galt2-1), with an equivalent galactose with wild-type Arabidopsis but significantly higher than galt2-1. These findings provide evidence that DoGALT2 might be involved in regulating the biosynthesis of galactose-containing polysaccharides during D. officinale pollen development., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Dendrobium officinale is a precious traditional Chinese medicinal herb because it is abundant in mannose-containing polysaccharides (MCPs). GDP-mannose transporter (GMT), which translocates GDP-mannose into the Golgi lumen, is indispensable for the biosynthesis of MCPs. In this study, we found that the dominant polysaccharides in D. officinale were MCPs in a range of varieties and different physiological phases. After a positive correlation between the accumulation of mannose and the transcript levels of candidate GMT genes was found, three GMT genes (DoGMT1-3) were identified in D. officinale. DoGMT1, DoGMT2 and DoGMT3 exhibited the highest transcript level in stem that an organ for MCPs storage. All three DoGMT proteins were targeted to Golgi apparatus, and had a GDP binding domain (GXL/VNK) that was homologous to a specially characterized GMT protein GONST1 in Arabidopsis thaliana. Moreover, DoGMT1, DoGMT2 and DoGMT3 complemented a GDP-mannose transport-defective yeast mutant (vrg4-2), meanwhile they also demonstrated a higher GDP-mannose uptake activity. Therefore, we conclude that DoGMT1, DoGMT2 and DoGMT3 are able to transport GDP-mannose while the expression patterns of these genes correspond to the accumulation of MCPs in D. officinale. These findings support the importance of GMT genes from D. officinale in the biosynthesis of MCPs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Dendrobium officinale stems, including red and green stems, are widely used as a dietary supplement to develop nutraceutical beverages and food products. However, there is no detailed information on pigment composition of red and green stems. Here, we investigated the content and composition of pigments in red and green stems by Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry and assessed the differential accumulation of anthocyanins at the molecular level. The color of peels in red stems was caused by the presence of anthocyanins in epidermal cells unlike the peels of green stems. The glucoside derivatives delphinidin and cyanidin are responsible for the red color. Within the D. officinale anthocyanidin biosynthetic pathway, DoANS and DoUFGT , coding for anthocyanidin synthase and UDP-glucose flavonoid-3- O -glucosyltransferase, respectively, are critical regulatory genes related to the differential accumulation of anthocyanidin. These findings provide a more complete profile of pigments, especially anthocyanin, in D. officinale stems, and lay a foundation for producing functional foods.

The WRKY family, one of the largest families of transcription factors, plays important roles in the regulation of various biological processes, including growth, development and stress responses in plants. In the present study, 63 DoWRKY genes were identified from the Dendrobium officinale genome. These were classified into groups I, II, III and a non-group, each with 14, 28, 10 and 11 members, respectively. ABA-responsive, sulfur-responsive and low temperature-responsive elements were identified in the 1-k upstream regulatory region of DoWRKY genes. Subsequently, the expression of the 63 DoWRKY genes under cold stress was assessed, and the expression profiles of a large number of these genes were regulated by low temperature in roots and stems. To further understand the regulatory mechanism of DoWRKY genes in biological processes, potential WRKY target genes were investigated. Among them, most stress-related genes contained multiple W-box elements in their promoters. In addition, the genes involved in polysaccharide synthesis and hydrolysis contained W-box elements in their 1-k upstream regulatory regions, suggesting that DoWRKY genes may play a role in polysaccharide metabolism. These results provide a basis for investigating the function of WRKY genes and help to understand the downstream regulation network in plants within the Orchidaceae.

Phosphomannomutase (PMM, EC 5.4.2.8) catalyzes the interconversion of mannose-6-phosphate to mannose-1-phosphate, the precursor for the synthesis of GDP-mannose. In this study, the complementary DNA (cDNA) of the Phosphomannomutase (PMM) gene was initially cloned from Dendrobium officinale by RACE method. Transient transform result showed that the DoPMM protein was localized in the cytoplasm. The DoPMM gene was highly expressed in the stems of D. officinale both in vegetative and reproductive developmental stages. The putative promoter was cloned by TAIL-PCR and used for searched cis-elements. Stress-related cis-elements like ABRE, TCA-element, and MBS were found in the promoter regions. The DoPMM gene was up-regulated after treatment with abscisic acid, salicylic acid, cold, polyethylene glycol, and NaCl. The total ascorbic acid (AsA) and polysaccharide content in all of the 35S::DoPMM Arabidopsis thaliana transgenic lines #1, #2, and #5 showed a 40, 39, and 31% increase in AsA and a 77, 22, and 39% increase in polysaccharides, respectively more than wild-type (WT) levels. All three 35S::DoPMM transgenic lines exhibited a higher germination percentage than WT plants when seeded on half-strength MS medium supplemented with 150 mM NaCl or 300 mM mannitol. These results provide genetic evidence for the involvement of PMM genes in the biosynthesis of AsA and polysaccharides and the mediation of PMM genes in abiotic stress tolerance during seed germination in A. thaliana.

Plants of the Dendrobium genus, one of the largest in the Orchidaceae, manifest a diversity of medicinal effects encompassing antiangiogenic, immunomodulating, antidiabetic, cataractogenesis-inhibiting, neuroprotective, hepatoprotective, anti-inflammatory, antiplatelet aggregation, antifungal, antibacterial, antiherpetic, antimalarial, aquaporin-5 stimulating, and hemagglutininating activities and also exert beneficial actions on colonic health and alleviate symptoms of hyperthyroidism. The active principles include a wide range of proteinaceous and non-proteinaceous molecules. This mini-review discusses the latest advances in what is known about the medicinal and pharmaceutical properties of members of the Dendrobium genus and explores how biotechnology can serve as a conduit to mass propagate valuable germplasm for sustainable exploration for the pharmaceutical industry.

GDP-mannose pyrophosphorylase (GMP) catalyzed the formation of GDP-mannose, which serves as a donor for the biosynthesis of mannose-containing polysaccharides. In this study, three GMP genes from Dendrobium officinale (i.e., DoGMPs) were cloned and analyzed. The putative 1000 bp upstream regulatory region of these DoGMPs was isolated and cis-elements were identified, which indicates their possible role in responses to abiotic stresses. The DoGMP1 protein was shown to be localized in the cytoplasm. To further study the function of the DoGMP1 gene, 35S:DoGMP1 transgenic A. thaliana plants with an enhanced expression level of DoGMP1 were generated. Transgenic plants were indistinguishable from wild-type (WT) plants in tissue culture or in soil. However, the mannose content of the extracted water-soluble polysaccharides increased 67%, 96% and 92% in transgenic lines #1, #2 and #3, respectively more than WT levels. Germination percentage of seeds from transgenic lines was higher than WT seeds and the growth of seedlings from transgenic lines was better than WT seedlings under salinity stress (150 mM NaCl). Our results provide genetic evidence for the involvement of GMP genes in the biosynthesis of mannose-containing polysaccharides and the mediation of GMP genes in the response to salt stress during seed germination and seedling growth., Competing Interests: The authors declare no competing financial interests.

Orchids of the genus Dendrobium are of great economic importance in global horticultural trade and in Asian traditional medicine. For both areas, research yielding solid information on taxonomy, phylogeny, and breeding of this genus are essential. Traditional morphological and cytological characterization are used in combination with molecular results in classification and identification. Markers may be useful when used alone but are not always reliable in identification. The number of species studied and identified by molecular markers is small at present. Conventional breeding methods are time-consuming and laborious. In the past two decades, promising advances have been made in taxonomy, phylogeny and breeding of Dendrobium species due to the intensive use of molecular markers. In this review, we focus on the main molecular techniques used in 121 published studies and discuss their importance and possibilities in speeding up the breeding of new cultivars and hybrids., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The ability to germinate orchids from seeds in vitro presents a useful and viable method for the propagation of valuable germplasm, maintaining the genetic heterogeneity inherent in seeds. Given the ornamental and medicinal importance of many species within the genus Dendrobium, this review explores in vitro techniques for their asymbiotic seed germination. The influence of abiotic factors (such as temperature and light), methods of sterilization, composition of basal media, and supplementation with organic additives and plant growth regulators are discussed in context to achieve successful seed germination, protocorm formation, and further seedling growth and development. This review provides both a basis for the selection of optimal conditions, and a platform for the discovery of better ones, that would allow the development of new protocols and the exploration of new hypotheses for germination and conservation of Dendrobium seeds and seedlings.

The genus Dendrobium is one of the largest genera of the Orchidaceae Juss. family, although some of its members are the most threatened today. The reason why many species face a vulnerable or endangered status is primarily because of anthropogenic interference in natural habitats and commercial overexploitation. The development and application of modern techniques and strategies directed towards in vitro propagation of orchids not only increases their number but also provides a viable means to conserve plants in an artificial environment, both in vitro and ex vitro, thus providing material for reintroduction. Dendrobium seed germination and propagation are challenging processes in vivo and in vitro, especially when the extreme specialization of these plants is considered: (1) their biotic relationships with pollinators and mycorrhizae; (2) adaptation to epiphytic or lithophytic life-styles; (3) fine-scale requirements for an optimal combination of nutrients, light, temperature, and pH. This review also aims to summarize the available data on symbiotic in vitro Dendrobium seed germination. The influence of abiotic factors as well as composition and amounts of different exogenous nutrient substances is examined. With a view to better understanding how to optimize and control in vitro symbiotic associations, a part of the review describes the strong biotic relations of Dendrobium with different associative microorganisms that form microbial communities with adult plants, and also influence symbiotic seed germination. The beneficial role of plant growth-promoting bacteria is also discussed.

Dendrobium officinale is a traditional Chinese medicinal plant. The stems of D. officinale contain mannan polysaccharides, which are promising bioactive polysaccharides for use as drugs. However, the genes involved in the biosynthesis of mannan polysaccharides in D. officinale have not yet been identified. In this study, four digital gene expression profiling analyses were performed on developing stems of greenhouse-grown D. officinale to identify such genes. Based on the accumulation of mannose and on gene expression levels, eight CELLULOSE SYNTHASE-LIKE A genes (CSLA), which are highly likely to be related to the biosynthesis of bioactive mannan polysaccharides, were identified from the differentially expressed genes database. In order to further analyze these DoCSLA genes, a full-length cDNA of each was obtained by RACE. The eight genes, belonging to the CSLA family of the CesA superfamily, contain conserved domains of the CesA superfamily. Most of the genes, which were highly expressed in the stems of D. officinale, were related to abiotic stress. Our results suggest that the CSLA family genes from D. officinale are involved in the biosynthesis of bioactive mannan polysaccharides.

Dendrobium is a large genus in the family Orchidaceae that exhibits vast diversity in floral characteristics, which is of considerable importance to orchid breeders, biotechnologists and collectors. Native species have high value as a result of their medicinal properties, while their hybrids are important as ornamental commodities, either as cut flowers or potted plants and are thus veritable industrial crops. Thus, preservation of Dendrobium germplasm is valuable for species conservation, breeding programs and the floriculture industry. Cryopreservation represents the only safe, efficient and cost-effective long-term storage option to facilitate the conservation of genetic resources of plant species. This review highlights 16 years of literature related to the preservation of Dendrobium germplasm and comprises the most comprehensive assessment of thorough studies performed to date, which shows reliable and reproducible results. Air-drying, encapsulation-dehydration, encapsulation-vitrification, vitrification and droplet-vitrification are the current cryopreservation methodologies that have been used to cryopreserve Dendrobium germplasm. Mature seeds, pollen, protoplasts, shoot primordia, protocorms and somatic embryos or protocorm-like bodies (PLBs) have been cryopreserved with different levels of success. Encapsulation-vitrification and encapsulation-dehydration are the most used protocol, while PLBs represent the main explant explored.

Establishing an aseptic in vitro culture for Dendrobium, or for any plant in fact, is the most important step towards developing an effective in vitro tissue culture including micropropagation protocol. Success in initial aseptic culture will contribute to the successful production of in vitro cultures that may involve the initiation or formation of callus and/or protocorm-like bodies (PLBs), the induction, regeneration or multiplication of shoots, and the preparation and proliferation of plantlets suitable for acclimatization. The initiation of an aseptic culture is closely related to the appropriate selection of an explant source and its preparation, including its (in vivo) pre-treatment if necessary and subsequent disinfection procedures. Care in the choice of explant and the application of an appropriate disinfection protocol can successfully reduce, or eliminate, contamination in in vitro cultures while reducing the negative impact on plant tissues and plantlet regeneration. Many unique aseptic culture procedures for Dendrobium genus have been reported in the literature, very often specific to particular tissues or genotypes, and this review not only highlights the details of such protocols, but also provides practical advice for novice – and even seasoned – orchidologists who wish to research Dendrobium in vitro, although it is cautioned that there is currently no universal aseptic culture procedure that can be applied to all conditions, all explants or all genotypes.

Flavonoids are important bioactive components in Dendrobium officinale, a medicinal orchid. They are involved in many biological activities, including protecting plants against biotic and abiotic stresses. Research on the key genes related to flavonoid biosynthesis in D. officinale is limited. In this study, one of the key flavonoid biosynthesis genes, flavanone 3-hydroxylase (F3H), was characterized from D. officinale. The open reading frame of DoF3H was 1 134 bp long and it encoded a 377-amino acid protein. The DoF3H protein showed considerably high homology with F3H proteins from other plant species and shared a common evolutionary ancestor with other F3Hs. DoF3H transcripts were detected in different organs of adult plants and mainly accumulated in flowers, followed by roots, stems and leaves, a pattern that was similar to the content of flavonoids. Recombinant DoF3H protein, which was localized in the cytosol, could convert naringenin to dihydrokaempferol. The mRNA levels of DoF3H were significantly induced by salt and cold stresses. Furthermore, the heterologous expression of DoF3H in Escherichia coli conferred it higher tolerance to salt and cold stresses. These results provide insight into the molecular function of DoF3H in the biosynthesis of flavonoids, and provide a new application for improvement of abiotic tolerance in D. officinale. [ABSTRACT FROM AUTHOR]

The deacetylation of core histones controlled by the action of histone deacetylases (HDACs) plays an important role in the epigenetic regulation of plant gene transcription. However, no systematic analysis of HDAC genes in Dendrobium officinale, a medicinal orchid, has been performed. In the current study, a total of 14 histone deacetylases in D. officinale were identified and characterized using bioinformaticsbased methods. These genes were classified into RPD3/HDA1, SIR2, and HD2 subfamilies. Most DoHDAC genes in the same subfamily shared similar structures, and their encoded proteins contained similar motifs, suggesting that the HDAC family members are highly conserved and might have similar functions. Different cis-acting elements in promoters were related to abiotic stresses and exogenous plant hormones. A transient expression assay in onion epidermal cells by Agrobacterium-mediated transformation indicated that all of the detected histone deacetylases such as DoHDA7, DoHDA9, DoHDA10, DoHDT3, DoHDT4, DoSRT1 and DoSRT2, were localized in the nucleus. A tissue-specific analysis based on RNA-seq suggested that DoHDAC genes play a role in growth and development in D. officinale. The expression profiles of selected DoHDAC genes under abiotic stresses and plant hormone treatments were analyzed by qRT-PCR. DoHDA3, DoHDA8, DoHDA10 and DoHDT4 were modulated by multiple abiotic stresses and phytohormones, indicating that these genes were involved in abiotic stress response and phytohormone signaling pathways. These results provide valuable information for molecular studies to further elucidate the function of DoHDAC genes. [ABSTRACT FROM AUTHOR]

The original article can be found online at https://doi.org/10.1007/s00709-022-01790-x.Correction to: Protoplasma (2023) 260:483–495https://doi.org/10.1007/s00709-022-01790-xThe correct given name of the 5th Author is Jun not Juan.Publisher's noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.By Danqi Zeng; Can Si; Jaime A. Teixeira da Silva; Guangyi Dai; Jun Duan and Chunmei HeReported by Author; Author; Author; Author; Author; Author [Extracted from the article]

Dendrobium is one of the largest and most important (ornamentally and medicinally) orchid genera. Tissue culture is now an established method for the effective propagation of members of this genus. This review provides a detailed overview of the Dendrobium micropropagation literature. Through a chronological analysis, aspects such as explant, basal medium, plant growth regulators, culture conditions and final organogenic outcome are chronicled in detail. This review will allow Dendrobium specialists to use the information that has been documented to establish, more efficiently, protocols for their own germplasm and to improve in vitro culture conditions based on the optimized parameters detailed in this review. Not only will this expand the use for mass propagation, but will also allow for the conservation of important germplasm. Information on the in vitro responses of Dendrobium for developing efficient protocols for breeding techniques based on tissue culture, such as polyploidization, somatic hybridization, isolation of mutants and somaclonal variants and for synthetic seed and bioreactor technology, or for genetic transformation, is discussed in this review. This is the first such review on this genus and represents half a decade of literature dedicated to Dendrobium micropropagation. [ABSTRACT FROM AUTHOR]

For ornamental plants, in particular orchids, the flower is central to their beauty and commercial value. The ability to manipulate the floral transition from the vegetative to the reproductive phase and floral traits requires an understanding of the underlying molecular genetic mechanisms and robust transgenic protocols. Using Dendrobium species and hybrids, this review explores the advances that have been made in the genetics of flower development, color and senescence. Although the homologs of several MADS-box genes are still to be found, those that have already been cloned and analyzed bring promise to what has yet to be unraveled about the control of flower color and development. Recent advances in orchid transformation and the introduction of Dendrobium homologs into Arabidopsis thaliana have shed new light on the complexities of flower color and the ABCDE model of flower development in orchids, or the 'orchid code'. [ABSTRACT FROM AUTHOR]

Flowering is the most elusive and fascinating of all plant developmental processes. The ability to induce flowering in vitro in orchids would reduce the relatively long juvenile phase and provide deeper insight into the physiological, genetic and molecular aspects of flowering. This review synthesizes all available studies that have been conducted on in vitro flowering of orchids with the objective of providing valuable clues as to the mechanism(s) that is possibly taking place. [ABSTRACT FROM AUTHOR]

In plant tissue culture research, there is a constant need to search for novel substances that could result in better or more efficient growth in vitro. A relatively unknown compound, phloroglucinol (1,3,5-trihydroxybenzene), which is a degradation product of phloridzin, has growth-promoting properties. Phloroglucinol increases shoot formation and somatic embryogenesis in several horticultural and grain crops. When added to rooting media together with auxin, phloroglucinol further stimulates rooting, most likely because phloroglucinol and its homologues act as auxin synergists or auxin protectors. Of particular interest is the ability of phloroglucinol-a precursor in the lignin biosynthesis pathway-to effectively control hyperhydricity through the process of lignification, thus maximizing the multiplication rate of woody species and other species that are difficult to propagate. Phloroglucinol has also been used to improve the recovery of cryopreserved Dendrobium protocorms, increasing the potential of cryopreservation for application in ornamental biotechnology. Phloroglucinol demonstrates both cytokinin-like and auxin-like activity, much like thidiazuron, and thus has considerable potential for application in a wide range of plant tissue culture studies. [ABSTRACT FROM AUTHOR]

Abstract: Breeding orchids through traditional means is a lengthy process. Therefore, it would be advantageous if transgenic technologies could be applied for orchids to improve important traits such as novel flower colour, fragrance and shape, cut-flower longevity and flowering control, abiotic stress tolerance and resistance to pests and diseases. Even though there are several genetic transformation techniques available to orchid breeders, only two (Agrobacterium-mediated transformation and particle bombardment) have been successfully and consistently used thus far. This review aims to capture the full range of studies conducted on orchid transformation with a view of providing new perspectives for future molecular breeding programmes. [Copyright &y& Elsevier]

Members of the MYB superfamily act as regulators in a wide range of biological processes in plants. Despite this, the MYB superfamily from the Orchidaceae has not been identified, and MYB genes related to bioactive water-soluble polysaccharide (WSP) biosynthesis are relatively unknown. In this study, we identified 159 and 165 MYB genes from two orchids, Phalaenopsis equestris and Dendrobium officinale, respectively. The MYB proteins were classified into four MYB classes in both orchids: MYB-related (MYBR), R2R3-MYB, 3R-MYB and atypical MYB proteins. The MYBR proteins in both orchids were classified into five subfamilies and 12 genes were strongly up-regulated in response to cold stress in D. officinale. The R2R3-MYB proteins were both divided into 31 clades in P. equestris and D. officinale. Among these clades, nine contained MYB TFs related to secondary cell wall biosynthesis or testa mucilage biosynthesis in Arabidopsis thaliana. In D. officinale, 10 candidate genes showed an expression pattern corresponding to changes in the WSP content. Overexpression of one of these candidate genes (DoMYB75) in A. thaliana increased seed WSP content by about 14%. This study provides information about MYB genes in two orchids that will further help to understand the transcriptional regulation of WSP biosynthesis in these orchids as well as other plant species. [ABSTRACT FROM AUTHOR]

Background: MADS-box genes play crucial roles in plant floral organ formation and plant reproductive development. However, there is still no information on genome-wide identification and classification of MADS-box genes in some representative plant species. A comprehensive investigation of MIKC-type genes in the orchid Dendrobium officinale is still lacking. Results: Here we conducted a genome-wide analysis of MADS-box proteins from 29 species. In total, 1689 MADS-box proteins were identified. Two types of MADS-box genes, termed type I and II, were found in land plants, but not in liverwort. The SQUA, DEF/GLO, AG and SEP subfamilies existed in all the tested flowering plants, while SQUA was absent in the gymnosperm Ginkgo biloba, and no genes of the four subfamilies were found in a charophyte, liverwort, mosses, or lycophyte. This strongly corroborates the notion that clades of floral organ identity genes led to the evolution of flower development in flowering plants. Nine subfamilies of MIKCC genes were present in two orchids, D. officinale and Phalaenopsis equestris, while the TM8, FLC, AGL15 and AGL12 subfamilies may be lost. In addition, the four clades of floral organ identity genes in both orchids displayed a conservative and divergent expression pattern. Only three MIKC-type genes were induced by cold stress in D. officinale while 15 MIKC-type genes showed different levels of expression during seed germination. Conclusions: MIKC-type genes were identified from streptophyte lineages, revealing new insights into their evolution and development relationships. Our results show a novel role of MIKC-type genes in seed germination and provide a useful clue for future research on seed germination in orchids. [ABSTRACT FROM AUTHOR]

Highlights • Low temperature delayed ROS accumulation, and catabolism/polysaccharide degradation. • D. officinale polysaccharides consisted mainly of mannose and glucose. • Low temperature storage increased the accumulation of active polysaccharides. • An increase in polysaccharides was closely related to a decrease in starch. Abstract Polysaccharides are the most important group of active ingredients in the orchid Dendrobium officinale. Previous studies have focused on D. officinale polysaccharides during the pre-harvest period, but little is known about the changes in the amounts and activities of polysaccharides in D. officinale stems during the postharvest period. In this study, we evaluated the physiological properties of D. officinale stems during postharvest storage for 90 days at ambient temperature (25 °C) and at low temperatures (4 °C and 0 °C). The contents of polysaccharides, mannose, and glucose first increased then decreased during all storage conditions (storage at 25 °C, 4 °C and 0 °C). The carbohydrate and starch contents decreased considerably as the storage period extended, while cellulose content showed the opposite trend. Polysaccharides from D. officinale stored at ambient temperature exhibited lower antioxidant activity in vitro than polysaccharides from D. officinale stored at low temperatures. The transcript levels of genes encoding α-amylases and β-amylases were related to the degree of starch degradation, and application of an amylase inhibitor prevented starch degradation and polysaccharide accumulation. These results suggested that the increase in polysaccharide content in D. officinale stems stored under low temperatures was related to the decrease in starch content. Our results show that low-temperature storage is a useful approach to enhance the content of polysaccharides and extend the shelf-life of D. officinale. [ABSTRACT FROM AUTHOR]

• Genome-wide identification of LEA genes orchids, P. equestris and D. officinale. • LEA_1, LEA_4 and SAM group genes were abundant in seeds and PLBs. • Many D. officinale LEA genes modulated by stress hormones or abiotic stress. • DoLEA43 (LEA_1 group) was localized in both cytoplasm and nucleus. • DoLEA43 genes stimulated callus formation in transgenic Arabidopsis. Late embryogenesis abundant (LEA) proteins are widely involved in plant stress responsive, while their involvement in callus formation is largest unknown. In this study, we identified and conducted expression analysis of the LEA genes from Phalaenopsis equestris and Dendrobium officinale , and characterized a LEA gene from D. officinale. A total 57 and 59 LEA genes were identified in P. equestris and D. officinale , respectively. A phylogenetic analysis showed that AtM, LEA_5 and Dehydrin groups were absent in both orchids. LEA_1 group genes were strongly expressed in seeds, significantly down-regulated in flowers, and absent in vegetative organs (leaves, stems and roots) in both orchids. Moreover, LEA_1 and LEA_4 group genes from D. officinale were abundant in the protocorm-like body stage and were dramatically up-regulated in response to abscisic acid and salinity stress. A LEA_1 gene (DoLEA43) was selected for further functional analysis. DoLEA43 protein was localized in the cytoplasm and nucleus, and its promoter contained a WUN-motif that was modulated by wounding. Overexpression of DoLEA43 in Arabidopsis enhanced callus induction, causing changes to callus formation-related genes such as WIND1. Our results indicate the involvement of LEA genes in the induction of callus, which provide insights into plant regeneration. [ABSTRACT FROM AUTHOR]

Highlights: [•] A mass propagation protocol for Dendrobium ‘Zahra FR 62’ is described. [•] PLBs could be induced in half-strength MS, including 1mg/L TDZ and 0.5mg/L BA. [•] Most PLBs formed in a 3-L bioreactor containing half-strength MS+0.05mg/L BA. [•] PLBs formed with an average of 247.5 new PLBs per subculture. [•] Rooted plantlets were acclimatized (90–100% survival) on Cycas rumphii bulk. [ABSTRACT FROM AUTHOR]