The Integration of the Metabolome and Transcriptome for Dendrobium nobile Lindl. in Response to Methyl Jasmonate.

Dendrobium nobile Lindl., as an endangered medicinal plant within the genus Dendrobium, is widely distributed in southwestern China and has important ecological and economic value. There are a variety of metabolites with pharmacological activity in D. nobile. The alkaloids and polysaccharides contained within D. nobile are very important active components, which mainly have antiviral, anti-tumor, and immunity improvement effects. However, the changes in the compounds and functional genes of D. nobile induced by methyl jasmonate (MeJA) are not clearly understood. In this study, the metabolome and transcriptome of D. nobile were analyzed after exposure to MeJA. A total of 377 differential metabolites were obtained through data analysis, of which 15 were related to polysaccharide pathways and 35 were related to terpenoids and alkaloids pathways. Additionally, the transcriptome sequencing results identified 3256 differentially expressed genes that were discovered in 11 groups. Compared with the control group, 1346 unigenes were differentially expressed in the samples treated with MeJA for 14 days (TF14). Moreover, the expression levels of differentially expressed genes were also significant at different growth and development stages. According to GO and KEGG annotations, 189 and 99 candidate genes were identified as being involved in terpenoid biosynthesis and polysaccharide biosynthesis, respectively. In addition, the co-expression analysis indicated that 238 and 313 transcription factors (TFs) may contribute to the regulation of terpenoid and polysaccharide biosynthesis, respectively. Through a heat map analysis, fourteen terpenoid synthetase genes, twenty-three cytochrome P450 oxidase genes, eight methyltransferase genes, and six aminotransferase genes were identified that may be related to dendrobine biosynthesis. Among them, one sesquiterpene synthase gene was found to be highly expressed after the treatment with MeJA and was positively correlated with the content of dendrobine. This study provides important and valuable metabolomics and transcriptomic information for the further understanding of D. nobile at the metabolic and molecular levels and provides candidate genes and possible intermediate compounds for the dendrobine biosynthesis pathway, which lays a certain foundation for further research on and application of Dendrobium.

[Effects of mycorrhizal planting on small molecular chemical components of Dendrobium officinale].

This study aimed to provide a scientific basis for the application of the mycorrhizal planting technology of Dendrobium officinale by investigating the effects of mycorrhizal planting on the fingerprints of D. officinale and the content of six chemical components. Seventeen samples of D. officinale under mycorrhizal and conventional planting were collected from four regions, such as Jinhua of Zhejiang. The HPLC fingerprints were established to evaluate the similarity of the samples. The content of six chemical components of the samples was determined by HPLC. There were 15 common peaks in the fingerprints, and five of them were identified by marker compounds, which were naringenin, 4,4'-dihydroxy-3,5-dimethoxybibenzyl, 3,4'-dihydroxy-5-methoxybibenzyl, 3',4-dihydroxy-3,5'-dimethoxybibenzyl(gigantol), and 3,4-dihydroxy-4',5-dimethoxybibenzyl(DDB-2). The similarities of the fingerprints of mycorrhizal and conventional planting samples and the control fingerprint were in the ranges of 0.733-0.936 and 0.834-0.942, respectively. The influences of mycorrhizal planting on fingerprints were related to planting regions, the germplasm of D. officianle, and the amount of fungal agent. The content of six chemical components in the samples varied greatly, and the content of DDB-2 was the highest, ranging from 69.83 to 488.47 µg·g~(-1). The mycorrhizal planting samples from Chongming of Shanghai and Taizhou of Jiangsu showed an increase in the content of 5-6 components, while samples from Zhangzhou of Fujian and Jinhua of Zhejiang showed an increase in the content of 1-2 components. The results showed that mycorrhizal planting technology did not change the chemical profile of small molecular chemical components of D. officinale, but affected the content of chemical components such as bibenzyls, which has a good application prospect.

Hallmarks of Comparative Transcriptome between Rhizomorphs and Hyphae of Armillaria sp. 541 Participating in Fungal Symbiosis with Emphasis on LysM Domains.

Armillaria sp. 541, a genus of root-infecting fungi, forms a symbiosis with traditional Chinese medicine Gastrodia elata (Orchid) and Polyporus umbellatus via extensive networks of durable rhizomorphs. It is not clear the hallmarks of comparative transcriptome between the rhizomorphs and hyphae of Armillaria sp. 541. In the present study, transcriptomic analysis of Armillaria sp. 541 identified 475 differentially expressed genes (DEGs) between Armillaria rhizomorphs (AR) and hyphae (AH). Of them, 285 genes were upregulated and 190 were downregulated. Bioinformatics analyses and tests demonstrated DEGs involved in oxidoreductase activity and peptidoglycan binding were significantly enriched in this process when rhizomorph formed from hyphae. We accordingly obtained 14 gene-encoding proteins containing the LysM domain, and further consensus pattern and phylogenetic analysis indicated that their amino acid sequences were conserved and their biological functions may be peptidoglycan binding for recognition between the fungus and host. Among these genes, one, named Armillaria LysM domain recognition gene (aLDRG), was expressed significantly when rhizomorphs were differentiated from hyphae. It was located in the cortical cells of the rhizomorph by in situ hybridization. Furthermore, biolayer interferometry (BLI) assay demonstrated that aLDRG can bind specifically to chitin oligosaccharide of the fungal cell wall, including N,N',N″-Triacetylchitotriose (CO3) and N,N',N″,N'″,N″″-Pentaacetylchitopentaose (CO5). Therefore, we deduced that Armillaria sp. 541 expressed higher levels of LysM protein aLDRG for better binding of oligosaccharide after rhizomorphs were generated. This study provides functional genes for further studies on the interaction between Armillaria sp. 541 and its host.

[Comparison and health risk assessment of mineral elements in stems and leaves of Dendrobium officinale cultivated with conventional method and mycorrhizal fungi].

This study aims to analyze the variation of the content of mineral elements in stems and leaves of Dendrobium officinale cultivated with conventional method and mycorrhizal fungi, which is expected to lay a basis for safety of stems and leaves of D. officinale. A total of 7 samples from Jiangsu, Fujian, Shanghai, and Zhejiang were collected, which were then cultivated with conventional method and mycorrhizal fungi, separately. The content of 17 mineral elements in stems and leaves was measured by inductively coupled plasma-mass spectrometry(ICP-MS), and the content changes of the mineral elements were analyzed. The health risks of Pb, Cd, Hg, and As in stems were assessed by target hazard quotient(THQ). The results showed that the content of polluting elements in stems and leaves of D. officinale was low, and the content in the plants cultivated with mycorrhizal fungi was reduced. The content of K, Ca, Mg, and P was high in stems and leaves of the species, suggesting that cultivation with mycorrhizal fungi improved the content of other elements irregularly. According to the THQ, the safety risk of stems of D. officinale cultivated with either conventional method or mycorrhizal fungi was low, particularly the D. officinale cultivated mycorrhizal fungi. The results indicated that cultivation with mycorrhizal fungi influenced the element content in stems and leaves of D. officinale. It is necessary to study the culture substrate, processing technology, and the mechanism of the increase or decrease in mineral elements of D. officinale in the future.

Functional characterization of a farnesyl diphosphate synthase from Dendrobium nobile Lindl.

Dendrobium nobile Lindl. has been used as a traditional Chinese medicine for a long time, in which the most important compound is dendrobine functioning in a variety of pharmacological activities. Farnesyl diphosphate synthase (FPPS) is one of the key enzymes in the biosynthetic pathway of dendrobine. In this work, we found the expression profiles of DnFPPS were correlated with the contents of dendrobine under the methyl jasmonate (MeJA) treatments at different time. Then, the cloning and functional identification of a novel FPPS from D. nobile. The full length of DnFPPS is 1231 bp with an open reading frame of 1047 bp encoding 348 amino acids. The sequence similarity analysis demonstrated that DnFPPS was in the high homology with Dendrobium huoshanense and Dendrobium catenatum and contained four conserved domains. Phylogenetic analysis showed that DnFPPS was the close to the DhFPPS. Then, DnFPPS was induced to express in Escherichia coli, purified, and identified by SDS-PAGE electrophoresis. Gas chromatography-mass spectrometry analysis indicated that DnFPPS could catalyze dimethylallyl pyrophosphate and isopentenyl pyrophosphate to produce farnesyl diphosphate. Taken together, a novel DnFPPS was cloned and functionally identified, which supplied a candidate gene for the biosynthetic pathway of dendrobine.

Acetylome analysis of acetylation providing new insight into sclerotial generation in medicinal fungus Polyporus umbellatus.

Sclerotium-forming fungi are ecologically diverse and possess notable pathogenic or medicinal properties. The sclerotial generation mechanism is still elusive though Polyporus umbellatus sclerotia are typical Traditional Chinese Medicine with diuretic and antitumor effects. Protein acetylation displays a crucial role in several biological processes, but the functions of acetylation in this valuable fungus are unknown at present. In this study, acetylome of P. umbellatus was studied using nano LC-Triple TOF mass spectrometry system following immune-affinity-based enrichment. Totally, 648 acetylated sites in 342 proteins were identified and nine motifs were found to be conserved in P. umbellatus including KacY, KacA, KacL, KacG, MacS, MacA, RacA, RacL, and RacG. Acetylated proteins taken part in types of biological processes, particularly to those in biological processes associated with reactive oxygen species (ROS) metabolism. Inhibitors complement tests were carried out to verify the role of ROS in acetylation modification. It was concluded that oxidative stress regulated sclerotial generation via proteins acetylation in P. umbellatus. The present study presents new insight into the essential roles of acetylation in sclerotial formation, which may also be applicable for other sclerotium-forming fungi.

Utilization of functional agro-waste residues for oyster mushroom production: Nutritions and active ingredients in healthcare.

A large amount of agro-industrial residues are produced from the planting, production and processing of traditional Chinese herbs. As a tonic, edible, and economical herb, Codonopsis pilosula root has been extensively developed into medicine and functional food. However, thousands of tons of aerial parts (stems, leaves, flowers and fruits) have been directly discarded after harvest each year. To utilise agro-wastes, Pleurotus ostreatus was cultivated on a basal substrate supplemented with C. pilosula stems and leaves (CSL). Physicochemical analyses revealed that the basal substrate mixed with CSL was more abundant in cellulose, hemicellulose, and most of micronutrients such as K, Ca, Mg, S, Fe, Zn and Mo. After the first flush, the fruit bodies in CSL group exhibited a higher fresh weight, a wider average pileus diameter and a lower moisture level. Nutrition analyses presented a higher protein content and a lower fat content in mushrooms from CSL group compared with control group. Interestingly, 14 amino acids (glutamine, arginine, valine, leucine, and etc.) and 3 micronutrients (Se, Fe and Zn) were increased after CSL addition to the substrate. Based on untargeted metabolomics, a total of 710 metabolites were annotated. Compared with control group, there were 142 and 117 metabolites significantly increased and decreased in the CSL group. Most of them were grouped into classes of amino acids and peptids, fatty acids, carbohydrates, terpenoids, and etc. Moreover, an abundance of phytometabolites from Codonopsis were detected in P. ostreatus from CSL group, including polyacetylenes or polyenes, flavonoids, alkaloids, terpenoids, organic acids, and etc. UPLC-MS/MS results demonstrated that lobetyolin content in the CSL group samples was 0.0058%. In summary, the aerial parts of C. pilosula processed for use in the production of edible mushroom is an emerging strategy to converting agricultural waste into functional foods.

Dengratiols A-D, four new bibenzyl derivatives from Dendrobium gratiossimum.

Dengratiol A (1), an unprecedented bibenzyl derivative bearing a tropolone unit together with three pairs of bibenzyl enantiomers (±)-dengratiols B-D [(±)-2-(±)-4], were isolated from Dendrobium gratiossimum Rchb.f. The resolution of enantiomers was performed with chiral HPLC. Their structures were characterized by extensive spectroscopic data analysis and calculated electronic circular dichroism (ECD). A hypothetical biosynthetic pathway for 1 is proposed. Biological assay revealed that (-)-2 showed moderate antiviral effect against IAV with IC50 value of 6.3 µM, and (±)-2 displayed cytotoxic activities against five human tumor cell lines with IC50 values ranging from 15.5 to 42.5 µM.

Untargeted Metabolomics Reveals Multiple Phytometabolites in the Agricultural Waste Materials and Medicinal Materials of Codonopsis pilosula.

Codonopsis pilosula has been used in traditional Chinese medicine for hundreds of years, where it has been used to treat anaemia, fatigue, a weak spleen, and stomach problems, among other ailments. The roots of C. pilosula are considered medicinal, while the aerial parts are always directly discarded after harvest in autumn or winter. Some studies have shown that the stems and leaves of C. pilosula also contain a variety of active metabolites, including saponins, flavonoids, terpenoids, and polysaccharides. To efficiently utilise resources, waste products from C. pilosula leaves and stems were analysed by untargeted metabolomics and chemometrics. A total of 1508 metabolites were detected and annotated, of which 463 were identified as differentially expressed metabolites (DEMs). These DEMs were grouped into classes, such as carboxylic acids and derivatives, steroids, organic oxygen compounds, fatty acyls, prenol lipids, and flavonoids. Metabolic profiling of C. pilosula tissues showed that the contents of polyacetylenes, polyenes, flavonoids, some alkaloids, steroids, terpenoids, and organic acids were higher in stems and leaves, whereas the contents of the main lignans and some alkaloids were more enriched in roots. Moreover, C. pilosula stems and leaves also contained a lobetyolin, syringin and atractylenolide III, which were detected by LC-MS/MS and HPLC-UV. The extracts of C. pilosula aerial parts also showed stronger antioxidant properties than roots. C. pilosula stems and leaves were rich in active ingredients and might have great value for development and utilisation.

Do the Main Components of the Sclerotia of Umbrella Polypore Mushroom, Polyporus umbellatus (Agaricomycetes), Correlate with Armillaria Associates?

Polyporus umbellatus is a traditional Chinese medicinal mushroom. The growth of P. umbellatus sclerotia requires the rhizomorphs of Armillaria spp. to supply nutrition. Whether the main components (MC) of sclerotia of P. umbellatus are related to the phylogeny of Armillaria associates or other environmental factors is largely unknown. In this study, we collected 17 sclerotia and soil samples from northeast to southwest China. In total, 17 Armillaria associates were isolated, and sclerotial MC contents and soil characteristics (total N, P, K, and organic matter) were determined. The analysis revealed that the MC content of P. umbellatus did not resemble a Brownian motion process in phylogeny of Armillaria associates, but were significantly influenced by the total N content of the soil. These results provide clear evidence that sclerotia of P. umbellatus associating with phylogenetic related Armillaria associates possess differing MC content. The mechanisms of nutrient exchange in P. umbellatus-Armillaria associations now require further elucidation.

Identification and expression of DoCCaMK during Sebacina sp. symbiosis of Dendrobium officinale.

Dendrobium officinale Kimura et Migo is a famous precious medicinal plant in China. Seed and seedling were cultivated with the mycorrhizal fungus Sebacina sp. CCaMK was initially cloned from D. officinale based on a SSH cDNA library of symbiotically germinated seeds with Sebacina sp. Phylogenetic analysis was performed among DoCCaMK and other CCaMKs. The particle bombardment technique was used to visualize DoCCaMK-GFP. qRT-PCR and western blot analysis were conducted to determine the tissue expression patterns of DoCCaMK with (SGS) and without (UGS) Sebacina sp. Furthermore, the effect of KN-93 on CCaMK expression was also examined. Using NMT the net Ca2+ fluxes and the CCaMK concentration were measured during D. officinale seed germination. DoCCaMK had the highest homology with Lilium longiflorum CCaMK. The DoCCaMK-GFP protein localized in the nucleus and cell membrane. CCaMK expression was significantly upregulated after symbiosis with Sebacina sp. KN-93 could be used as an inhibitor of CCaMK to inhibit D. officinale seed germination. Ca2+ influx and the concentration of the CCaMK in the SGS group was significantly more than that of the UGS group. The characterization of CCaMK provides certain genetic evidence for the involvement of this gene during seed germination and mycorrhizal cultivation in D. officinale.

Ultrastructural changes during the symbiotic seed germination of Gastrodia elata with fungi, with emphasis on the fungal colonization region.

BACKGROUND: Gastrodia elata is a fully mycoheterotrophic orchid and has long been used in traditional Chinese medicine. The life cycle of G. elata requires an association with two different fungi-Mycena for seed germination and Armillaria for tuber growth. The association with Armillaria is representative of the phytophagous type of orchid mycorrhiza: the intracellular hyphae are lysed without forming condensed pelotons. However, whether the association with Mycena during seed germination belongs to the same type of orchid mycorrhiza is unknown. RESULTS: Histological and ultrastructural studies revealed several notable features in different developmental stages. First, a thickened cell wall with papillae-like structures appeared during fungal penetration in the suspensor end cell, epidermal cells and cortical cells of germinating embryos. In addition, the formation of two distinctive cell types in the colonized region of a protocorm (i.e., the passage canal cell filled with actively growing fungal hyphae) can be observed in the epidermal cell, and the distinctive digestion cell with a dense cytoplasm appears in the cortex. Finally, within the digestion cell, numerous electron-dense tubules form a radial system and attach to degrading fungal hyphae. The fungal hyphae appear to be digested through endocytosis. CONCLUSIONS: The present study provides important structural evidence for the phytophagous type of orchid mycorrhiza in the symbiotic germination of G. elata with Mycena. This case demonstrates a particular nutrient transfer network between G. elata and its litter-decaying fungal partner.

Effects of the mycorrhizal fungus Ceratobasidium sp. AR2 on growth and flavonoid accumulation in Anoectochilus roxburghii.

BACKGROUND: Anoectochilus roxburghii is a traditional Chinese medicine with potent medicinal activity owing to the presence of secondary metabolites, particularly flavonoids. A. roxburghii also maintains a symbiotic relationship with mycorrhizal fungi. Moreover, mycorrhizal fungi can induce metabolite synthesis in host plants. However, little is known about the role of mycorrhizal fungi in promoting the accumulation of flavonoid metabolites in A. roxburghii. METHODS: A. roxburghii and the isolated fungus Ceratobasidium sp. AR2 were cocultured. The portion of A. roxburghii above the medium treated with or without AR2 was studied by transcriptome and target metabolome analyses. RESULTS: AR2 promoted the growth and development of A. roxburghii. The contents of total flavonoid, rutin, isorhamnetin, and cyanidin-3-glucoside chloride were increased compared with those in uninoculated cultures. Transcriptome analysis suggested that 109 unigenes encoding key enzymes were potentially associated with changes in flavonoids. Quantitative real-time polymerase chain reaction of fourteen flavonoid-related unigenes showed that most flavonoid biosynthetic genes were significantly differentially expressed between inoculated and uninoculated plantlets. CONCLUSION: The isolate AR2 could significantly promote the growth and development of A. roxburghii and the accumulation of flavonoids. Overall, our findings highlighted the molecular basis of the effects of mycorrhizal fungi on flavonoid biosynthesis in A. roxburghii and provided novel insights into methods to improve the yield and quality of A. roxburghii.

Aquilariaenes A-H, eight new diterpenoids from Chinese eaglewood.

Aquilariaenes A-H (1-8), eight new diterpenoids and nor-diterpenoids (1-8) belonging to abietane or pimarane, were isolated from the petroleum ether extract of Chinese eaglewood. Their structures were elucidated on the basis of extensive spectroscopic methods including HRESIMS, IR, 1D and 2D NMR spectroscopic data analyses. Antidepressant activities of isolates for in vitro inhibition of serotonin and norepinephrine reuptake in rat brain synaptosomes were evaluated.

[Isolation and expression analysis of a hexose transporter gene DoHT1 in Dendrobium officinale].

In this study, RACE technology was employed to isolate the full length cDNA of DoHT1 in Dendrobium officinale, followed by bioinformatics analysis of the sequence characteristics. And the expression pattern of the gene was also analyzed by quantitative PCR. The full length cDNA of DoHT1 was 1 586 bp in length, containing a 1 536 bp ORF, which encoded a 511-aa protein with molecular weight of 56.18 kD and isoelectric point of 9.08. The deduced DoHT1 protein had the major facilitator superfamily conserved domain (22-483), SUGAR₋TRANSPORT₋1 (139-164), and SUGAR₋TRANSPORT₋2 (338-355), typical for sugar transporter; DoHT1, without a single peptide had 11 transmembrane regions, and was predicted to locate in the plasma membrane; DoHT1 had high identities (54.7%-80.7%) with HTs proteins from various plants. DoHT1 belonged to the MST (monosaccharide transporter) group of the evolutionary tree, and was closely related to the Phalaenopsis equestris. DoHT1 was differentially expressed in the three included organs. The transcripts were significantly the most abundant in the leaves with 19.36 fold than roots, then 1.82 fold in the stems than the roots. The identification and molecular characterization of the full length DoHT1 will be essential for further function study of the gene during the regulation of sugar metabolism of D. officinale.

Revealing proteins associated with symbiotic germination of Gastrodia elata by proteomic analysis.

BACKGROUND: Gastrodia elata, a mycoheterotrophic orchid, is a well-known medicinal herb. In nature, the seed germination of G. elata requires proper fungal association, because of the absence of endosperm. To germinate successfully, G. elata obtains nutrition from mycorrhizal fungi such as Mycena. However, Mycena is not able to supply nutrition for the further development and enlargement of protocorms into tubers, flowering and fruit setting of G. elata. To date, current genomic studies on this topic are limited. Here we used the proteomic approach to explore changes in G. elata at different stages of symbiotic germination. RESULTS: Using mass spectrometry, 3787 unique proteins were identified, of which 599 were classified as differentially accumulated proteins. Most of these differentially accumulated proteins were putatively involved in energy metabolism, plant defense, molecular signaling, and secondary metabolism. Among them, the defense genes (e.g., pathogenesis-/wound-related proteins, peroxidases, and serine/threonine-protein kinase) were highly expressed in late-stage protocorms, suggesting that fungal colonization triggered the significant defense responses of G. elata. CONCLUSIONS: The present study indicated the metabolic change and defensive reaction could disrupt the balance between Mycena and G. elata during mycorrhizal symbiotic germination.

[DNA marker-assisted selection of medicinal plants (â ) .Breeding research of disease-resistant cultivars of Panax notoginseng].

SSR is one of the most important molecular markers used in molecular identification and genetic diversity research of Dendrobium nobile. In order to enrich the library of SSR and establish a method for rapid identification of D. nobile, the SSR information was analyzed in the transcriptome of D. nobile. A total of 32 709 SSRs were obtained from the transcriptome of D. nobile, distributed in 26 742 unigenes with the distribution frequency of 12.90%. SSR loci occurred every 3 748 bp. Mono-nucleotide repeat was the main type, account for as much as 72.18% of all SSRs, followed by di-nucleotide (15.97%) and tri-nucleotide (11.19%). Among all repeat types, A/T was the predominant one followed by AG/CT. Finally a total of 62 157 primer pairs were designed for marker development. Randomly 20 pairs of primers were selected for PCR amplification, 17 amplified on clear and reproducible bands, the amplification rate was 85.0%.Thirteen pairs were polymorphic among the 3 Dendrobium plants. The results indicated that the unigenes generated from transcriptome sequencing in D. nobile can be used as effective source to develop SSR markers. The SSR loci in the transcriptome of D. nobile have the characteristics of type riches, high density and high potential of polymorphism, and these characteristics might applied in the study of molecular identification, genetic diversity and marker-assisted breeding of D. nobile and its closely related species.

[Interaction between endophytes and host plant and the role of endophytes in genuineness analysis of medicinal plant].

Research of plant development and metabolism has drawn lots of attention with the fast development of science of mycorrhizal biology, molecular biology and metabonomics technology. It has become one of hot fields in the study of endophytes and plant, which would affect plant 's metabolite composition. This would provide opportunity for appraising and modifying traits to medicinal plant, and would also perfect the tranditional standpoint on forming reason of medicinal plant genuineness. Here we provide a review of theory and mechanism, research and application of interaction between plant and endophyte. This review may enhance understanding of medicinal plant, and evaluating the quality of herbs in production.

[Effect of endophytic fungi on modernization of traditional Chinese medicine].

The modernization of traditional Chinese medicine (TCM) is a foundation of TCM to go abroad and get international recognition. It is the only way to revitalize the TCM industry. But in the process of it, we are facing various challenges: heavy metal contamination, low content of active ingredients, less innovation, the contradiction between resource utilization and protection, and so on. How to apply new technology and new theory of life science to solve these problems becomes an urgent matter. In recent years, the studies found that endophytic fungi played an irreplaceable positive role in the growth and development of herbal medicine, and had great impact on the quality of traditional Chinese medicine. Therefore this paper introduces the effect of endophytic fungi on genuine traditional Chinese medicines, cultivation of TCM, development and protection of TCM, et al, and explores its applicative prospect, providing new idea and new power for promoting the development of modernization of TCM.

Embryology of two mycoheterotrophic orchid species, Gastrodia elata and Gastrodia nantoensis: ovule and embryo development.

BACKGROUND: Gastrodia elata, a famous herbal medicine, has been received great attention on its treatments of headache, vertigo and epilepsy. Gastrodia nantoensis is a newly described species from central Taiwan with potential medicinal value. Gastrodia species are fully mycoheterotrophic orchids, and the courses of their seed development are more rapid as compared to the chlorophyllous orchids. A better understanding of their reproductive biology would provide insights into the propagation and conservation of the mycoheterotrophic orchid species. RESULTS: Based on the histological and histochemical investigations, we observed some notable features in ovule and embryo development. First, only the archesporial cell and/or megasporocyte are present within their ovaries at the time of anthesis. Second, their suspensors consist of a single cell and their mature embryos consist of a gradient of small to large cells. Nile red staining of a globular embryo reveals the presence of cuticular material in the surface wall of embryo proper and the lateral walls of suspensor cell, indicating that the basal wall of suspensor cell is the major route for nutrient supply from maternal tissues to embryo proper. Third, their seed coats are derived from a single integument, and lignin but not cuticular material is present in the outer most layer of seed coat and persists through seed maturation. CONCLUSIONS: The faster seed development of Gastrodia species is due to the speedy courses of ovule and embryo development. In the mature seeds, the presence of a differentiated apical zone in embryo proper suggests the easy-to-germinate character. This study provides basic knowledge for further molecular studies on embryo development and symbiotic germination of Gastrodia species.