Tolerance enhancement of Dendrobium officinale by salicylic acid family-related metabolic pathways under unfavorable temperature.

BACKGROUND: Unfavorable temperatures significantly constrain the quality formation of Dendrobium officinale, severely limiting its food demand. Salicylic acid (SA) enhances the resistance of D. officinale to stress and possesses various analogs. The impact and mechanism of the SA family on improving the quality of D. officinale under adverse temperature conditions remains unclear. RESULTS: Combined with molecular docking analysis, chlorophyll fluorescence and metabolic analysis after treatments with SA analogues or extreme temperatures are performed in this study. The results demonstrate that both heat and cold treatments impede several main parameters of chlorophyll fluorescence of D. officinale, including the ΦPSII parameter, a sensitive growth indicator. However, this inhibition is mitigated by SA or its chemically similar compounds. Comprehensive branch imaging of ΦPSII values revealed position-dependent improvement of tolerance. Molecular docking analysis using a crystal structure model of NPR4 protein reveals that the therapeutic effects of SA analogs are determined by their binding energy and the contact of certain residues. Metabolome analysis identifies 17 compounds are considered participating in the temperature-related SA signaling pathway. Moreover, several natural SA analogs such as 2-hydroxycinnamic acid, benzamide, 2-(formylamino) benzoic acid and 3-o-methylgallic acid, are further found to have high binding ability to NPR4 protein and probably enhance the tolerance of D. officinale against unfavorable temperatures through flavone and guanosine monophosphate degradation pathways. CONCLUSIONS: These results reveal that the SA family with a high binding capability of NPR4 could improve the tolerance of D. officinale upon extreme temperature challenges. This study also highlights the collaborative role of SA-related natural compounds present in D. officinale in the mechanism of temperature resistance and offers a potential way to develop protective agents for the cultivation of D. officinale.

Genome-wide identification of Ankyrin (ANK) repeat gene families in three Dendrobium species and the expression of ANK genes in D. officinale under gibberellin and abscisic acid treatments.

BACKGROUND: Dendrobium Sw. represents one of the most expansive genera within the Orchidaceae family, renowned for its species' high medicinal and ornamental value. In higher plants, the ankyrin (ANK) repeat protein family is characterized by a unique ANK repeat domain, integral to a plethora of biological functions and biochemical activities. The ANK gene family plays a pivotal role in various plant physiological processes, including stress responses, hormone signaling, and growth. Hence, investigating the ANK gene family and identifying disease-resistance genes in Dendrobium is of paramount importance. RESULTS: This research identified 78 ANK genes in Dendrobium officinale Kimura et Migo, 77 in Dendrobium nobile Lindl., and 58 in Dendrobium chrysotoxum Lindl. Subsequently, we conducted comprehensive bioinformatics analyses on these ANK gene families, encompassing gene classification, chromosomal localization, phylogenetic relationships, gene structure and motif characterization, cis-acting regulatory element identification, collinearity assessment, protein-protein interaction network construction, and gene expression profiling. Concurrently, three DoANK genes (DoANK14, DoANK19, and DoANK47) in D. officinale were discerned to indirectly activate the NPR1 transcription factor in the ETI system via SA, thereby modulating the expression of the antibacterial PR gene. Hormonal treatments with GA3 and ABA revealed that 17 and 8 genes were significantly up-regulated, while 4 and 8 genes were significantly down-regulated, respectively. DoANK32 was found to localize to the ArfGAP gene in the endocytosis pathway, impacting vesicle transport and the polar movement of auxin. CONCLUSION: Our findings provide a robust framework for the taxonomic classification, evolutionary analysis, and functional prediction of Dendrobium ANK genes. The three highlighted ANK genes (DoANK14, DoANK19, and DoANK47) from D. officinale may prove valuable in disease resistance and stress response research. DoANK32 is implicated in the morphogenesis and development of D. officinale through its role in vesicular transport and auxin polarity, with subcellular localization studies confirming its presence in the nucleus and cell membrane. ANK genes displaying significant expression changes in response to hormonal treatments could play a crucial role in the hormonal response of D. officinale, potentially inhibiting its growth and development through the modulation of plant hormones such as GA3 and ABA.

Underlying mechanism of Dendrobium huoshanense resistance to lead stress using the quantitative proteomics method.

Lead affects photosynthesis and growth and has serious toxic effects on plants. Here, the differential expressed proteins (DEPs) in D. huoshanense were investigated under different applications of lead acetate solutions. Using label-free quantitative proteomics methods, more than 12,000 peptides and 2,449 proteins were identified. GO and KEGG functional annotations show that these differential proteins mainly participate in carbohydrate metabolism, energy metabolism, amino acid metabolism, translation, protein folding, sorting, and degradation, as well as oxidation and reduction processes. A total of 636 DEPs were identified, and lead could induce the expression of most proteins. KEGG enrichment analysis suggested that proteins involved in processes such as homologous recombination, vitamin B6 metabolism, flavonoid biosynthesis, cellular component organisation or biogenesis, and biological regulation were significantly enriched. Nearly 40 proteins are involved in DNA replication and repair, RNA synthesis, transport, and splicing. The effect of lead stress on D. huoshanense may be achieved through photosynthesis, oxidative phosphorylation, and the production of excess antioxidant substances. The expression of 9 photosynthesis-related proteins and 12 oxidative phosphorylation-related proteins was up-regulated after lead stress. Furthermore, a total of 3 SOD, 12 POD, 3 CAT, and 7 ascorbate-related metabolic enzymes were identified. Under lead stress, almost all key enzymes involved in the synthesis of antioxidant substances are up-regulated, which may facilitate the scavenging of oxygen-free radical scavenging. The expression levels of some key enzymes involved in sugar and glycoside synthesis, the phenylpropanoid synthesis pathway, and the terpene synthesis pathway also increased. More than 30 proteins involved in heavy metal transport were also identified. Expression profiling revealed a significant rise in the expression of the ABC-type multidrug resistance transporter, copper chaperone, and P-type ATPase with exposure to lead stress. Our findings lay the basis for research on the response and resistance of D. huoshanense to heavy metal stress.

Genome-Wide Identification of Glutathione S-Transferase Family from Dendrobium officinale and the Functional Characterization of DoGST5 in Cadmium Tolerance.

Glutathione S-transferases (GSTs) are members of a protein superfamily with diverse physiological functions, including cellular detoxification and protection against oxidative damage. However, there is limited research on GSTs responding to cadmium (Cd) stress. This study classified 46 GST genes in Dendrobium officinale (D. officinale) into nine groups using model construction and domain annotation. Evolutionary analysis revealed nine subfamilies with diverse physical and chemical properties. Prediction of subcellular localization revealed that half of the GST members were located in the cytoplasm. According to the expression analysis of GST family genes responding to Cd stress, DoGST5 responded significantly to Cd stress. Transient expression of DoGST5-GFP in tobacco leaves revealed that DoGST5 was localized in the cytoplasm. DoGST5 overexpression in Arabidopsis enhanced Cd tolerance by reducing Cd-induced H2O2 and O2- levels. These findings demonstrate that DoGST5 plays a critical role in enhancing Cd tolerance by balancing reactive oxygen species (ROS) levels, offering potential applications for improving plant adaptability to heavy metal stress.

JA signal-mediated immunity of Dendrobium catenatum to necrotrophic Southern Blight pathogen.

BACKGROUND: Dendrobium catenatum belongs to the Orchidaceae, and is a precious Chinese herbal medicine. In the past 20 years, D. catenatum industry has developed from an endangered medicinal plant to multi-billion dollar grade industry. The necrotrophic pathogen Sclerotium delphinii has a devastating effection on over 500 plant species, especially resulting in widespread infection and severe yield loss in the process of large-scale cultivation of D. catenatum. It has been widely reported that Jasmonate (JA) is involved in plant immunity to pathogens, but the mechanisms of JA-induced plant resistance to S. delphinii are unclear. RESULTS: In the present study, the role of JA in enhancing D. catenatum resistance to S. delphinii was investigated. We identified 2 COI1, 13 JAZ, and 12 MYC proteins in D. catenatum genome. Subsequently, systematic analyses containing phylogenetic relationship, gene structure, protein domain, and motif architecture of core JA pathway proteins were conducted in D. catenatum and the newly characterized homologs from its closely related orchid species Phalaenopsis equestris and Apostasia shenzhenica, along with the well-investigated homologs from Arabidopsis thaliana and Oryza sativa. Public RNA-seq data were investigated to analyze the expression patterns of D. catenatum core JA pathway genes in various tissues and organs. Transcriptome analysis of MeJA and S. delphinii treatment showed exogenous MeJA changed most of the expression of the above genes, and several key members, including DcJAZ1/2/5 and DcMYC2b, are involved in enhancing defense ability to S. delphinii in D. catenatum. CONCLUSIONS: The findings indicate exogenous MeJA treatment affects the expression level of DcJAZ1/2/5 and DcMYC2b, thereby enhancing D. catenatum resistance to S. delphinii. This research would be helpful for future functional identification of core JA pathway genes involved in breeding for disease resistance in D. catenatum.

Genome-Wide Analysis of the TCP Transcription Factor Genes in Dendrobium catenatum Lindl.

Teosinte branched1/cycloidea/proliferating cell factor (TCP) gene family members are plant-specific transcription factors that regulate plant growth and development by controlling cell proliferation and differentiation. However, there are no reported studies on the TCP gene family in Dendrobium catenatum Lindl. Here, a genome-wide analysis of TCP genes was performed in D. catenatum, and 25 TCP genes were identified. A phylogenetic analysis classified the family into two clades: Class I and Class II. Genes in the same clade share similar conserved motifs. The GFP signals of the DcaTCP-GFPs were detected in the nuclei of tobacco leaf epidermal cells. The activity of DcaTCP4, which contains the miR319a-binding sequence, was reduced when combined with miR319a. A transient activity assay revealed antagonistic functions of Class I and Class II of the TCP proteins in controlling leaf development through the jasmonate-signaling pathway. After different phytohormone treatments, the DcaTCP genes showed varied expression patterns. In particular, DcaTCP4 and DcaTCP9 showed opposite trends after 3 h treatment with jasmonate. This comprehensive analysis provides a foundation for further studies on the roles of TCP genes in D. catenatum.

DoRWA3 from Dendrobium officinale Plays an Essential Role in Acetylation of Polysaccharides.

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.

Comparative analysis of B-BOX genes and their expression pattern analysis under various treatments in Dendrobium officinale.

BACKGROUND: Studies have demonstrated that BBX (B-BOX) genes play crucial roles in regulatory networks controlling plant growth, developmental processes and stress response. Nevertheless, comprehensive study of BBX genes in orchids (Orchidaceae) is not well studied. The newly released genome sequences of Dendrobium officinale and Phalaenopsis equestris have allowed a systematic analysis of these important BBX genes in orchids. RESULTS: Here we identified 19 (DoBBX01-19) and 16 (PeBBX01-16) BBX genes from D. officinale and P. equestris, respectively, and clustered into five clades (I-V) according to phylogenetic analysis. Thirteen orthologous, two DoBBXs paralogous and two PeBBXs paralogous gene pairs were validated. This gene family mainly underwent purifying selection, but five domains experienced positive selection during evolution. Noteworthy, the expression patterns of root, root_tips, stem, leaf, speal, column, lip, and flower_buds revealed that they might contribution to the formation of these tissues. According to the cis-regulatory elements analysis of BBX genes, qRT-PCR experiments were carried out using D. officinale PLBs (protocorm-like bodies) and displayed that these BBX genes were differentially regulated under AgNO3, MeJA (Methyl Jasmonate), ABA (abscisic acid) and SA (salicylic acid) treatments. CONCLUSIONS: Our analysis exposed that DoBBX genes play significant roles in plant growth and development, and response to different environmental stress conditions of D. officinale, which provide aid in the selection of appropriate candidate genes for further functional characterization of BBX genes in plants.

Metabolic Profiling of Dendrobium officinale in Response to Precursors and Methyl Jasmonate.

Alkaloids are the main active ingredients in the medicinal plant Dendrobium officinale. Based on the published genomic and transcriptomic data, a proposed terpenoid indole alkaloid (TIA) biosynthesis pathway may be present in D. officinale. In this study, protocorm-like bodies (PLBs) with a high-yielding production of alkaloids were obtained by the optimization of tryptophan, secologanin and methyl jasmonate (MeJA) treatment. The results showed that the total alkaloid content was 2.05 times greater than that of the control group when the PLBs were fed with 9 µM tryptophan, 6 µM secologanin and 100 µM MeJA after 36 days. HPLC analysis showed that strictosidine synthase (STR) activity also increased in the treated plants. A total of 78 metabolites were identified using gas chromatography-mass spectrometry (GC-MS) in combination with liquid chromatography-mass spectrometry (LC-MS) methods; 29 differential metabolites were identified according to the multivariate statistical analysis. Among them, carapanaubine, a kind of TIA, exhibited dramatically increased levels. In addition, a possible underlying process of the metabolic flux from related metabolism to the TIA biosynthetic pathway was enhanced. These results provide a comprehensive view of the metabolic changes related to alkaloid biosynthesis, especially TIA biosynthesis, in response to tryptophan, secologanin and MeJA treatment.

Exogenous Catalase and Pyruvate Dehydrogenase Improve Survival and Regeneration and Affect Oxidative Stress in Cryopreserved Dendrobium nobile Protocorm-like Bodies.

BACKGROUND: Reactive oxygen species (ROS)-induced oxidative damage is responsible for viability loss in plant tissues following cryopreservation. Antioxidants may improve viability by preventing or repairing the injury. OBJECTIVE: This work aimed at studying the effect of catalase (CAT) and pyruvate dehydrogenase (PDH), which are involved in ROS metabolism and are differentially expressed during pollen cryopreservation, for cryopreservation of Dendrobium nobile Lindl. 'Hamana Lake Dream' protocorm-like bodies (PLBs). MATERIALS AND METHODS: Different concentrations of exogenous CAT or PDH were added at the loading, PVS2 treatment, unloading steps during vitrification-cryopreservation of PLBs. Their survival and regeneration were evaluated and correlated with physiological oxidative indexes. RESULTS: PLB survival increased significantly when CAT and PDH were added separately to the unloading solution at a suitable concentration. CAT at 400 U·ml-1 increased PLB survival and regeneration by 33.5 and 14.6 percent respectively. It had no impact on the production of superoxide anion radical (·O2-) and on superoxide dismutase (SOD) activity, but it reduced the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents and enhanced ascorbic acid (AsA) and endogenous CAT levels compared to PLBs cryopreserved using the standard vitrification protocol (CK1). PDH at 0.1 U·ml-1 significantly improved PLB survival (by 2.5 percent), but it had no marked effect on regeneration compared to the CK1 group. It induced the same variations in ·O2-, AsA and endogenous CAT levels that were observed following CAT addition. However, PDH did not affect the H2O2 and MDA content but significantly increased SOD activity. CONCLUSION: These results indicate that the addition of 400 U·ml-1 CAT and 0.1 U·ml-1 PDH at the unloading step increased survival of cryopreserved PLBs and that this improvement was associated with scavenging of H2O2 and the repair of oxidative damage. Exogenous CAT also significantly improved PLB regeneration after cryopreservation, while PDH had no obvious effect. The effect of exogenous CAT on PLB survival and regeneration was stronger than that of PDH, which may be due to the increased SOD activity by PDH addition.

Evaluation of genetic variability in in vitro sodium azide-induced Dendrobium 'Earsakul' mutants.

In vitro mutagenesis of Dendrobium 'Earsakul' was carried out by incubating the protocorm-like bodies in 0-5 mM sodium azide for 1 h. Twenty-eight putative mutants were evaluated for genetic variability compared to untreated control plants using inter-simple sequence repeat (ISSR) analysis. Polymorphic fragments were produced by 9 of 12 ISSR primers. A total of 173 amplified ISSR fragments varying in size from 140 to 5000 bp were obtained, 39 of which were polymorphic (22.5%). Of the 28 putative mutants, 15 (53.6%) showed altered genetic profiles compared to control and were identified as mutants. These results suggest that sodium azide can be effectively utilized to generate mutants in Dendrobium 'Earsakul', and ISSR provides a powerful tool that allows efficient early detection of these mutants. The identified mutants are currently being multiplied for further evaluation of their horticultural characteristics.

[Effect of ABA on transcriptionally active Ty1-copia retrotransposons in Dendrobium officinale].

Using universal primer Tyl-copia retrotransposon RT, the conserved reverse transcriptase domain of about 260 bp was amplified by RT-PCR from the Dendrobium officinale which induced by 100 micromol x L(-1) abscisic acid (ABA), indicating these retrotransposons activated by 100 micromol x L(-1) ABA. The amplicons were recovered and cloned,then sequenced and analyzed by related bioinformatics software. Forty-two Ty1-copia like retrotransposon RT transcriptionally activated were obtained with high heterogeneity. The length of these sequences varied from 247 to 266 bp, and was rich in AT and homology ranged from 46.3% to 98.9%. The same to Ty1-copia like retrotransposon RT of genome, different c/s-acting regulatory elements induced by stress conditions and the starting transcription signals, corresponding to CAAT box, TATA box conserved sequences and some other regulatory elements. The c/s-acting regulatory elements induced by stress conditions of reverse transcriptase transcriptionally activated of Tyl-copia retrotransposons were significantly increased than that of Ty1-copia like retrotransposon RT of genome. When being translated into amino acids, fifteen sequences presented stop codon mutation, nineteen sequences presented frameshift mutation, and all sequences presented conserved sequence "SLYGKQ" mutation. Five categories were identified through phylogenic analysis after alignment analyses of their amino acid sequences, and with Ty1-copia like retrotransposon RT of genome having low homology, which indicated that reverse transcriptase transcriptionally activated of Ty1-copia retrotransposons which induced by ABA had Significantly differences with Ty1-copia like retrotransposon RT of genome.

Physiological and transcriptomic analyses of cadmium stress response in Dendrobium officinale seedling.

Dendrobium officinale is an economically important Chinese herb with ornamental and medicinal values. However, the mechanisms by which D. officinale adapts to cadmium (Cd) stress is unknown. Here, physiological changes in D. officinale roots and leaves exposed to increasing levels of Cd stress (CdSO4 concentration of 2, 5, 9, 14 mg L-1) were analyzed at 7, 15, 30, and 45 days after treatment. The Cd stress of 14 mg L-1 significantly increased the levels of antioxidants and induced malondialdehyde and proline accumulation (P < 0.05). Cd subcellular distribution showed that Cd sequestration into soluble fraction is the major detoxification mechanism in D. officinale roots. Subsequently, the transcriptome profile of D. officinale roots treated with 14 mg L-1 Cd for 15 and 30 days was analyzed. Compared to control, 2,469 differentially expressed genes (DEGs) were identified, comprising 1,486 up-regulated genes and 983 down-regulated genes. The DEGs associated with metabolic pathways for Cd uptake, transportation and detoxification were analyzed. Several processes such as metal transporter, sulfate glutathione metabolism, cell wall metabolism, phenylpropanoid metabolism were identified to be important for Cd stress adaptation. More genes were expressed at 15 days after treatment compared to 30 days. WRKY, Trihelix, NF-YC, MYB, bZIP and bHLH transcription factors were over-expressed at both time points. Furthermore, candidate genes from the glutathione metabolism pathway were identified, and qRT-PCR analysis of ten DEGs indicated a high coorelation with RNA-seq expression profiles. Our findings provide significant information for further research of Cd stress responsive genes functions in D. officinale, especially the genes from the glutathione metabolism pathway.

Interaction between a dark septate endophytic isolate from Dendrobium sp. and roots of D. nobile seedlings.

Interactions between an isolate of dark septate endophytes (DSE) and roots of Dendrobium nobile Lindl. seedlings are reported in this paper. The isolate was obtained from orchid mycorrhizas on Dendrobium sp. in subtropical forest. The fungus formed typical orchid mycorrhiza in aseptic co-culture with D. nobile seedlings on modified Murashige-Skoog (MMS) medium. Anatomic observations of the infected roots showed that the DSE hyphae invaded the velamen layer, passed through passage cells in exodermis, entered the cortex cells, and then formed fungal pelotons of orchid mycorrhiza. D. nobile seedlings' plant height, stem diameter, new roots number and biomass were greatly enhanced by inoculating the fungus to seedlings. The fungus was identified as Leptodontidium by sequencing the polymerase chain reaction-amplified rDNA ITS1-5.8S-ITS2 (internal transcribed spacer (ITS)) regions and comparison with similar taxa.

L-methionine sulfoximine as a novel selection agent for genetic transformation of orchids.

A key challenge in molecular breeding of orchids is the creation of efficient and reproducible gene transformation systems. In this study, we report a new transformation method utilizing L-methionine sulfoximine (MSO) as a novel agent for selection of transgenic Dendrobium hybrids D. Madame Thong-In and D. Chao Praya Smile with the bialaphos resistance (bar) gene as a selectable marker. Gene transformation was performed by biolistic bombardment with a 4-day recovery period on MSO-free medium and two selection stages on media with increasing amounts of selection agent, using concentrations of 5 and 10 microM MSO for D. Madame Thong-In, and 0.5 and 2 microM MSO for D. Chao Praya Smile. Independent transgenic orchid lines were obtained and the presence of the transgene was confirmed by PCR and Southern blot analysis. Because of substantial time and economic savings, the new transformation system using MSO as a selection agent will facilitate functional studies on orchid genes and genetic engineering of orchids with commercially valuable traits.

Enhancement of growth and polysaccharide production in suspension cultures of protocorm-like bodies from Dendrobium huoshanense by the addition of putrescine.

Putrescine at 0.6 mM stimulated protocorm-like body growth and polysaccharide synthesis in suspension cultures of Dendrobium huoshanense. The specific growth rate of protocorm-like body increased from 0.047 to 0.056 day(-1), and the maximum dry weight and polysaccharide production reached 33.2 and 2.94 g l(-1), respectively, while they were 24.6 and 2.12 g l(-1), respectively, in the control. The administration of polyamine inhibitor, alpha-DL-difluoromethylarginine, at 1 mM, decreased protocorm-like body growth and polysaccharide production to 21.4 and 1.76 g l(-1), respectively.

[Experiment on seeding induction from seed of Demdrobium candidum].

Demdrobium candidum seed should be used as the explant to induce a great deal of seedings. The optimal medium for seeds germination was VW + NAA 0. 18 mg/L + 6-BA 0. 53 mg/L + GA 0. 28 mg/L + sucrose 20 g/L + banana mud 100 g/L + active carbon 10 g/L. The VW + NAA 0.18 mg/L + 6-BA 0.53 mg/L + GA 0.28 mg/L + sucrose 20 g/l + banana mud 100 g/L + active carbon 10 g/L was best for rooting of the plantlets.

Comparative Transcriptomic Analysis of Vernalization- and Cytokinin-Induced Floral Transition in Dendrobium nobile.

Vernalization is required for floral initiation in Dendrobium. Interestingly, those beneficial effects can also be achieved by exogenous cytokinin application in greenhouses. Thus, an as yet unknown crosstalk/interaction may exist between vernalization and cytokinin signaling pathways. In this study, we showed, by de novo transcriptome assembly using RNA-seq data from both vegetative and reproductive tissue samples, that some floral transition-related genes-DnVRN1, FT, SOC1, LFY and AP1-were differentially expressed in low-temperature-challenged (LT) or thidiazuron (TDZ)-treated plants, compared to those mock-treated (CK). Both LT and TDZ upregulated SOC1, LFY and AP1, while the upregulation of DnVRN1 and FT was only LT-induced. We further found that LT promoted the upregulation of some key cytokinin signaling regulators, including several cytokinin biosynthesis-related genes and type-B response regulator (RR)-encoding genes, and that both LT and TDZ triggered the significant upregulation of some marker genes in the gibberellin (GA) signaling pathway, indicating an important low temperature-cytokinin-GA axis in flowering. Our data thus have revealed a cytokinin-GA signal network underlying vernalization, providing a novel insight into further investigation of the molecular mechanism of floral initiation in Dendrobium.

Effects of Melatonin on Colchicine-Treated PLBs of Dendrobium sonia-28 Orchid.

Dendrobium hybrid orchid is popular in orchid commercial industry due to its short life cycle and ability to produce various types of flower colours. This study was conducted to identify the morphological, biochemical and scanning electron microscopy (SEM) analysis in the Dendrobium sonia-28 orchid plants. In this study, 0.05 and 0.075 % of colchicine-treated Dendrobium sonia-28 (4-week-old culture) protocorm-like bodies (PLBs) were treated in different concentrations of melatonin (MEL) posttreatments (0, 0.05, 0.1, 0.5, 1, 5 and 10 µM). Morphological parameters such as number of shoots, growth index and number of PLBs were determined. In the 0.05 and 0.075 % of colchicine-treated PLBs which were posttreated with 0.05 µM MEL resulted in the highest value of the morphological parameters tested based on the number of shoots (84.5 and 96.67), growth index (16.94 and 12.15) and number of PLBs (126.5 and 162.33), respectively. SEM analysis of the 0.05 µM MEL posttreatment on both the colchicine-treated regenerated PLBs showed irregular cell lineages, and some damages occurred on the stomata. This condition might be due to the effect of plasmolyzing occurred in the cell causing irregular cell lineages.

[The influence of abscisic acid treatment on desiccation tolerance in Dendrobium candidum protocorm-like bodies].

Effect of ABA on desiccation tolerance in the protocorm-like bodies (PLBs) of D. candidum was studied. The best result was obtained while ABA precultured at 5 micromol/L for 24 h (Fig.2). PLBs vitality rate was 10 times of the control after 2 h desiccation (Table 1). Detection of cytoplasmic osmoticums showed that ABA preculture could not induce the accumulations of soluble sugar, proline, etc (Tables 3, 4 and 7). But the content of soluble polysaccharide rose conspicuously (Table 5). So we think that the accumulation of soluble polysaccharide may play an important role in desiccation tolerance induced by ABA preculture.