Epiphytic Orchid Research Articles

Host suitability for germination differs from that of later stages of development in a rare epiphytic orchid

We explore phorophyte suitability for germination and establishment of the epiphytic orchid, Psychilis kraenzlinii. We found that the orchid grows on a subset of the available tree species and shows preference for the endemic Machaonia portoricensis (Rubiaceae). The orchid preferred trees with smoother bark with high water holding capacity and low water retention capacity. Microclimatic conditions under which embryos began pre-germination stages mirrored that of the adult orchid, but germination did not, suggesting that suitable germination sites are not necessarily the best sites for later stages of development. 

Associations between Epiphytic Orchids and Their Hosts and Future Perspectives of These in the Context of Global Warming

Epiphytic species are ecologically important and a significant component of biodiversity. To ensure their efficient conservation, we need to understand their ecology and host plant associations. It is also important to investigate how the predicted temperature change will affect their future distribution. Here, we use data collected in Nepal to investigate how epiphytic orchids are associated with host species, their distribution patterns, and how they may be threatened by the predicted increase in temperature towards the end of the 21st century. We used the phi coefficient (Φ) of association to calculate the associations of epiphytic orchid species with plants and rarefaction to describe the diversity of orchids associated with a particular host species. We used interpolation to estimate the distribution of epiphytic orchids and their host species along altitudinal gradients. The phi (Φ) coefficient of association revealed that 30 species of host plants showed more association with different orchid species than expected. The number of epiphytic orchids increased with the number of host individuals. We predict that an increase in temperature by ~3 °C, which is a more moderate value of temperature increase by the end of the 21st century, will affect at least 52 narrow-ranged species of orchids and 58 narrow-ranged species of host plants. Therefore, we should make efforts to prevent many plant species from becoming extinct, as an increase in temperature is likely to affect their existence.

Phorophyte preference of an epiphytic orchid (Dendrobium wangliangii) is independent of orchid mycorrhizal fungi that promote seed germination

Abstract Epiphytism is an example of an obligate interspecific interaction between plants, where a plant species grows on another plant species for physical support. In epiphytic orchids that rely on suitable host tree species (phorophytes) and mycorrhizal fungi for germination and growth, a distinct preference has been observed for particular host tree species. However, the factors driving this host preference remain unclear. In this study, we investigated whether mycorrhizal fungi that promote seedling development determine the preference of phorophytes in the epiphytic orchid Dendrobium wangliangii. First, we identified the preferred host tree species for this orchid. Next, in vitro and in situ germination experiments were conducted to identify potential fungi that promote seed germination. Finally, high‐throughput sequencing was used to assess the distribution of these fungi on both colonized host trees and potential host trees, including individual trees of the host species not yet colonized by the orchid and trees of another species growing in the vicinity of a colonized tree. Dendrobium wangliangii exhibited a preference for trees within the Fagaceae family. Two Fusarium species from the bark of host trees were identified as the primary fungi that promote seed germination. Successful protocorm formation and seedling development were observed exclusively on preferred phorophytes. Fungal community analyses further indicated that both Fusarium spp. occurred on all potential host trees, suggesting that the host preference of this epiphytic orchid is not constrained by the distribution of suitable mycorrhizal fungi. Synthesis: Regarding the epiphytes that depend on mycorrhizal fungi for successful seedling recruitment, we conclude that strong epiphyte–phorophyte associations do not arise from the limited distribution of mycorrhizal fungi but are more likely the result of other factors inherent in the phorophyte environment.

Mycorrhizal Fungi of Phalaenopsis japonica (Orchidaceae) and Their Role in Seed Germination and Seedling Development

Epiphytic orchids comprise 68% of vascular epiphytes globally; nevertheless, many are endangered. One such epiphytic orchid is Phalaenopsis japonica, which is widely used in the floricultural industry. This study aimed to identify the mycorrhizal fungi of adult P. japonica and their roles in seed germination and seedling development. Root samples were collected from 32 adults across 4 sites in southern Japan, and mycorrhizal fungi were identified using Sanger and high-throughput sequencing (HTS). The results show phylogenetically diverse mycobionts, mainly Ceratobasidiaceae (CE) and Tulasnellaceae (TU), with dominant OTUs designated CE6 and CE22. Sanger sequencing found 9 OTUs, 4 CE, and 5 TU; HTS detected 22 OTUs, 4 CE, 16 TU, and 2 Serendipitaceae. Seeds inoculated with CE6 improved germination and protocorm development compared with other strains. In addition, asymbiotic seedlings inoculated with CE6, CE22, and TU18 displayed varying effects in growth, with CE6 being the most notable. While TU18 did not promote seed germination, it effectively promoted leaf development in seedlings. Overall, Ceratobasidiaceae was predominantly associated with seed germination, seedling growth, and the adult stages, with CE6 and CE22 becoming the primary partners throughout the life history of P. japonica. Our findings illuminate mycorrhizal symbiosis in epiphytic habitats, offering conservation and commercial production insights.

Auxin production and plant growth promotion by Microbacterium albopurpureum sp. nov. from the rhizoplane of leafless Chiloschista parishii Seidenf. orchid.

The strains of the genus Microbacterium, with more than 150 species, inhabit diverse environments; plant-associated bacteria reveal their plant growth-promoting activities due to a number of beneficial characteristics. Through the performance of diverse techniques and methods, including isolation of a novel Microbacterium strain from the aerial roots of leafless epiphytic orchid, Chiloschista parishii Seidenf., its morphological and biochemical characterization, chemotaxonomy, phylogenetic and genome analysis, as well as bioassays and estimation of its auxin production capacity, a novel strain of ET2T is described. Despite that it shared 16S rRNA gene sequence similarity of 99.79% with Microbacterium kunmingense JXJ CY 27-2T, so they formed a monophyletic group on phylogenetic trees, the two strains showed clear divergence of their genome sequences. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values of ET2T differed greatly from phylogenetically close JXJ CY 27-2T. Based on the differences being below the threshold for species similarity, together with the unique chemotaxonomic characteristics, strain ET2T represents a novel species of the genus Microbacterium. Several genes, putatively involved in auxin biosynthesis were predicted. This strain revealed obvious plant growth-promoting activities, including diazotrophy and biosynthesis of tryptophan-dependent auxins (indole-3-acetic and indole-3-pyruvic acids). Microbial auxins directly stimulated the rhizogenesis, so that the ET2T-inoculated seeds of wheat, cucumber and garden cress showed evident promotion in their growth and development, both under optimal and under cold stress conditions. Based on phenotypic, chemotypic and genotypic evidences, the strain ET2T belongs to the genus Microbacterium, order Micrococcales, class Actinomycetes, and it represents a novel species, for which the name Microbacterium albopurpureum sp. nov. is proposed, with strain ET2T (VKPM Ac-2212, VKM Ас-2998) as the type strain.

Effects of Climate Change on the Distribution of Prosthechea mariae (Orchidaceae) and within Protected Areas in Mexico.

The impact of climate change on the distribution of native species in the Neotropics remains uncertain for most species. Prosthechea mariae is an endemic epiphytic orchid in Mexico, categorized as threatened. The objective of this study was to assess the effect of climate change on the natural distribution of P. mariae and the capacity of protected areas (PAs) to safeguard optimal environmental conditions for the species in the future. Historical records were obtained from herbaria collections and through field surveys. We utilized climate variables from WorldClim for the baseline scenario and for the 2050 period, using the general circulation models CCSM4 and CNRM-CM5 (RCP 4.5). Three sets of climate data were created for the distribution models, and multiple models were evaluated using the kuenm package. We found that the species is restricted to the eastern region of the country. The projections of future scenarios predict not only a substantial reduction in habitat but also an increase in habitat fragmentation. Ten PAs were found within the current distribution area of the species; in the future, the species could lose between 36% and 48% of its available habitat within these PAs. The results allowed for the identification of locations where climate change will have the most severe effects, and proposals for long-term conservation are addressed.

Root photosynthesis prevents hypoxia in the epiphytic orchid Phalaenopsis.

Orchids (Phalaenopsis spp.) growing in tropical and subtropical regions are epiphytes. As such, they grow on trees with the root system utilised to anchor themselves to tree branches. These roots are highly specialised, display a large diameter and are often green, suggesting the ability to carry out photosynthesis. However, the role of photosynthesis in orchid roots is controversial. Orchids that are leafless can photosynthesise in their roots, thus indicating that some orchid roots carry out photosynthesis in a similar manner to leaves. However, the primary site of photosynthesis in orchids are in their leaves, and the roots of epiphytic orchids may mostly conduct internal refixation of respiratory CO2 . Besides contributing to the overall carbon metabolism of orchid plants, oxygen produced through root photosynthesis may also be important by alleviating potential root hypoxia. The bulky tissue of most epiphytic orchid roots suggests that oxygen diffusion in these roots can be limited. Here, we demonstrate that the bulky roots of a widely commercially cultivated orchid belonging to the genus Phalaenopsis are hypoxic in the dark. These roots are photosynthetically active and produce oxygen when exposed to light, thus mitigating root hypoxia.

Effect of substrates of transplantation of the rare epiphytic orchid Dendrobium farmeri for conservation

Abstract. Nuammee A, Pingyot T, Foowan S, Pumikong S, Rujichaipimon W, Sornpood S, Panyadee P. 2024. Effect of substrates of transplantation of the rare epiphytic orchid Dendrobium farmeri for conservation. Biodiversitas 25: 708-715. Dendrobium farmeri Paxton is an epiphytic orchid, one of the most celebrated for its beauty. However, its natural population is dwindling, and it faces the risk of extinction. Although tissue culture techniques are successful for orchid propagations, the transplantation of in vitro-derived plantlets to ex-vitro conditions is often restricted by high mortality, which is considered a challenging and crucial stage in orchid conservation. This study investigated the effect of substrate mixtures on the survival and growth of D. farmeri plantlets under greenhouse conditions. The substrates for acclimatization contained coconut husk, charcoal chips, brick chips, sphagnum moss, fern fiber, and leaf mold in various ratios. Survival percentage and vegetative growth characteristics of plantlets were the main observations recorded. The result showed that the substrate mixture containing brick chips, charcoal chips, and sphagnum moss in a 1:1:2 ratio was the most effective, yielding 100% at 30 days and 96.7% at 180 days post-transplantation, fostering improved development of plantlets under greenhouse conditions. The findings suggest that this optimized substrate mixture could be a standard approach for acclimating D. farmeri and potentially other epiphytic orchids, enhancing conservation efforts and commercial cultivation.

Phytochemical analysis of root extracts of Rhynchostylis retusa (L.) Blume from the Eastern Ghats of India

Rhynchostylis retusa (Orchidaceae) is an important ethnomedicinal herb in Indian systems of medicine. Tribal healers commonly employ the roots of this epiphytic orchid to treat various ailments. In the present study, the shade-dried root powder of R. retusa was subjected to cold extraction with four solvents, such as n-hexane, acetone, ethyl acetate, and methanol. The crude root extracts were then taken for qualitative phytochemical screening. Furthermore, GC-MS analysis of ethyl acetate and methanol root extracts was carried out. The methanol root extract of R. retusa showed a positive result for all the phytochemicals tested except for saponins. GC-MS analysis of R. retusa methanol root extract revealed the presence of 16 phytoconstituents. Major phytoconstituents such as 9-Hexadecenoic acid, methyl ester, [Z] Heptadecanoic acid, 16-methyl-, methyl ester, Ethanone,1-[4-(4-morpholyl benzylidene amino)phenyl]-, are recorded in the methanol root extract. In the present study, both methanol and ethyl acetate root extracts showed the presence of9-Hexadecenoic acid, methyl ester, [Z] as the major phytocompound. The phytocompounds identified in methanol and ethyl acetate root extracts exhibit various biological activities, including anti-inflammatory, antibacterial, antifungal, antioxidant, and anticancer properties. Based on the findings of the current study, it can be inferred that the roots of R. retusa contain diverse bioactive compounds with medicinal properties. Further investigation of these R. retusa root extracts holds the potential for the discovery and development of innovative pharmaceuticals.

Inventory of Orchid Species in Customary Forest Area of Cipta Karya Village, Sungai Betung District, Bengkayang Regency

Orchids are one of the flowering plants that have many species and are widely distributed, as well as having different attractions and characteristics in each species and habitat. The aim of this research to inventory the types of orchids in Customary Forest Area of Cipta Karya Village, Bengkayang Regency. The research was conducted using the cruise method. The characteristics observed were the morphology of orchids such as roots, pseudobulbs, stems, leaves, and flowers. The inventory results found 13 species of orchids. Epiphytic orchids were found more commonly in this area, with a total of  9 species, while terrestrial orchids were found with 4 species. The epiphytic orchid species foundware composed of Appendicula sp., Bulbophyllum sp., Coelogyne speciosa, Cymbidium sp., Dendrobium crumenatum, Dendrobium anosmum, Luisia sp., Micropera pallida and Oberonia sp. The terrestrial orchid species found ware composed of Acanthephippium javanicum, Malaxis latifolia, Nephelaphyllum pulchrum, and Spathoglottis plicata.

KEANEKARAGAMAN JENIS ANGGREK DI JALUR PENDAKIAN WIRAYANA GUNUNG CAKRABUANA KABUPATEN MAJALENGKA

The existence of orchids in nature continues to decline due to habitat destruction and overexploitation. Conservation efforts that have been carried out so far have often experienced difficulties due to insufficient data and information as a basis for management, including conservation areas. The more widespread forest destruction will result in these species being threatened with extinction. This study aims to determine the diversity of orchid species, on the Wirayana Mount Cakrabuana Climbing Path. The information is expected to provide an initial contribution to the area management and orchid conservation efforts on the Wirayana Mount Cakrabuana Climbing Path. This research was conducted by means of a vegetation survey, sampling and documentation by exploring the existence of orchids. The sampling technique used the path transect method. The number of transects used is one line transect with a line length of 1500 m with a total of 22 sample plots. Data analysis was carried out by calculating the Important Value Index (INP), Species Dominance Index (Di) and Species Diversity Index (H'). The results showed that in the natural forest area of ​​Mount Cakrabuana there were 22 species of individuals which included 7 species of terrestrial orchids and 15 species of epiphytic orchids with a total of 243 individuals found. From the results of the calculation of species diversity, it is stated that the diversity of orchid species in the natural forest area of ​​Mount Cakrabuana is classified as moderate (2,973). This information is very important, especially for area managers in preserving the biodiversity of Mount Cakrabuana, especially orchids in the future.

Compuestos bioactivos en plántulas de Laelia speciosa (Orchidaceae) cultivados en biorreactor de inmersión temporal

Laelia speciosa is an epiphytic orchid endemic to Mexico, enjoying special protected status due to illegal sales and clandestine field extraction. Until now, in vitro cultivation of this plant has not been explored using temporary immersion bioreactor (TIB). L. speciosa seedlings were grown in a TIB, and the presence of bioactive compounds released into the liquid culture medium was determined. Bioactive compounds such as antioxidants, total polyphenols, and flavonoids were identified. The maximum concentration of total polyphenols was 2.29 ± 0.10 mg GAE/g, and the antioxidant capacity reached 33.7 %. Colorimetric tests revealed the presence of flavanones and flavonols. Gas chromatography-mass spectrometry identified components of the culture medium as 1,3-dihydroxyacetone dimer and 5-(hydroxymethyl)-2-furancarbaldehyde. Fourier transform infrared spectroscopy confirmed the presence of polyphenols in the culture medium. This work provides an alternative method for obtaining bioactive compounds while conserving L. speciosa seedlings.

Characterization of the complete chloroplast genome of Gastrochilus sinensis (Orchidaceae, Epidendroideae), a beautiful epiphytic orchid from China

Gastrochilus sinensis is a beautiful epiphytic orchid with high ornamental value. In this study, the first complete chloroplast genome sequence of G. sinensis was determined using next-generation sequencing (NGS). The de novo assembled chloroplast genome was 148,020 bp in length, including a pair of inverted repeat regions (IRs; 25,987 bp), a small single-copy region (SSC; 11,045 bp), and a large single-copy region (LSC; 85,001 bp). The chloroplast genome encodes 109 unique genes, including 75 protein-coding genes (PCGs), 30 tRNA genes, and four rRNA genes. The total GC content of the chloroplast genome was 36.8%. The phylogenetic analysis showed a close relationship between G. sinensis and G. formosanus species. The complete chloroplast genome provides fundamental information for genetic diversity and phylogenetic relationships in Gastrochilus.

Antimicrobial and Phytochemistry study of Dendrobium linearifolium Teijsm. & Binn. from Gumitir, Jember, Indonesia

Dendrobium linearifolium Teijsm. & Binn. is an epiphytic orchid growing in Mount Gumitir, Jember, Indonesia and had been used by indigenous people of Bali to treat earaches. Previous research has identified its DNA barcode and the presence of alkaloids and flavonoids with the highest concentration found in the leaves. However, an extensive analysis of their chemical constituents and their potential medicinal properties from this plant is not yet available. This study aims to determine the antimicrobial activity and phytochemical contents of the methanol extract of D. linearifolium leaves. The antimicrobial test was conducted using the agar diffusion method against two fungi: Aspergillus niger and Aspergillus falvus and three pathogenic bacteria i.e., Streptococcus aureus, Salmonella typhi, and Escherichia coli. Phytochemical analysis was performed using GC-MS. The results of the antimicrobial test indicate that the extract of D. linearifolium leaves possessed antimicrobial activity with the highest level against A. niger and the gram-positive bacteria S. aureus. The phytochemical analysis detected a total of 47 compounds with a total of 26 having medicinal potential. The five most dominant compounds with antimicrobial activity, i.e., 1-propanol, 2-(2-hydroxypropoxy)- (CAS) 2-(2-hydroxypropoxy)-1-propanol; 4-methyl-2,5-dimethoxybenzaldehyde; phenol, 2-methoxy- (CAS) guaiacol; 2-propanol, 1,1′-oxybis- (CAS) dipropylene glycol; and acetic acid (CAS) ethylic acid.

Physiological and transcriptome analysis reveal the nitrogen preference and regulatory pathways of nitrogen metabolism in an epiphytic orchid, Cymbidium tracyanum

Epiphytic orchids acquire nutrients elements, including nitrogen, from the atmosphere. However, the nitrogen form preferred by epiphytic orchids Cymbidium tracyanum and adaptive mechanisms that underlie nitrogen metabolism remain unclear. In this study, we determined which nitrogen form the C. tracyanum prefers, and identified adaptive strategies and molecular pathways activated during nitrogen metabolism. For this purpose, we measured the physiological and morphological responses to different ratios of nitrate and ammonium (NO3−/NH4+ = 1:0, 3:1, 1:1, 1:3, 0:1) and used transcriptome analysis to identify genes differentially expressed in response to these nitrogen forms. Physiological and morphological measures of plant growth (i.e., net photosynthetic rate, biomass accumulation, leaf area, root elongation and branching) were highest in plants treated with nitrate. Furthermore, C. tracyanum plants treated with nitrate had a higher root/mass ratio and a lower leaf/mass ratio. Transcriptome analysis identified that several key genes (NRT1.1–2, NRT1.4–4, NRT1.7–2, and CLC-G-2) highly expressed in plants treated with pure nitrate. These genes may be involved in the absorption and transportation of nitrate, and in auxin transportation from leaf to root in C. tracyanum. Our findings suggest that the nitrogen form preferred by C. tracyanum is nitrate and that nitrate alters both root and leaf traits via several molecular pathways, including the plant hormone signal transduction pathway and MAPK signaling pathway. We speculate that the preference of C. tracyanum for nitrate as a nitrogen form may be a result of long-term adaptation to epiphytic habitat via the regulation of leaf and root traits.

Ancient tea gardens play important role on in situ conservation of epiphytic orchids in southwest Yunnan, China

Southwest Yunnan of China is the homeland of famous Pu’er tea and preserved the largest area of ancient tea gardens in the world. The ancient tea gardens keeping a large number of shade trees have prevailed for hundreds of years persist and formed typical agroforestry systems. To assess the potential of the ancient tea gardens for in situ orchid conservation, in this study, three typical ancient tea gardens located at different elevations, JiNuo Mountain ancient tea garden (JNM, 1150 m asl.), JingMai Mountain ancient tea garden (JMM, 1345 m asl.), and LaoBanZhang ancient tea garden (LBZ, 1787 m asl.), were selected to investigate species diversity and composition, and conservation status of all orchids. Floral visitors, reproductive success, and seedling recruitment of selected epiphytic orchids were also investigated from 2019 to 2021. We identified 88 orchid species (85 epiphytic and 3 terrestrial orchids) in 33 genera in the three ancient tea gardens, including one critically endangered species (CR), four endangered species (EN) and 16 vulnerable species (VU). Although the orchid species diversity was not significantly different, the species composition varied greatly among the three ancient tea gardens. For the randomly selected 40 epiphytic orchid species, all species could set fruits naturally, and the natural fruit-sets ranged from 1.11 ± 6.0% to 62.07 ± 15.4% with great variations among species and years. For 60 epiphytic orchid species observed, seedling recruitment for 26 species (43.3%) were observed and confirmed. Among 42 insect visitors observed, 21 species including bees, flies and ants, were confirmed as effective pollinators to 16 epiphytic orchids. These results revealed that the ancient tea gardens maintained high species diversity of epiphytic orchids and provided pollinator services for reproductive success and suitable micro-environment for seed germination and seedling recruitment. We strongly suggested that the ancient tea gardens in the area could serve as important supplements for in situ conservation of epiphytic orchids.

KARAKTERISTIK POHON INANG DAN KEANEKARAGAMAN ANGGREK (Orchidaceae) EPIFIT DI HUTAN LINDUNG LUNUNIANAM, DESA FATU’ULAN, KECAMATAN KI’E, KABUPATEN TIMOR TENGAH SELATAN, PROVINSI NUSA TENGGARA TIMUR

This study was conducted to determine the characteristics of orchid host trees, the diversity of epiphytic orchids (Orchidaceae), and the preservation of host trees and epiphytic orchids in the Lununianam protected forest area. This research was conducted in Lununianam Protection Forest, Fatu'ulan Village, Ki'e District, South Central Timor Regency, East Nusa Tenggara Province. Which is carried out for 1 month, from November to December 2022. This study used purposive sampling methods, surveys, and cruising paths with data analysis using qualitative descriptive and quantitative descriptive analysis. Host tree data was processed using vegetation analysis formulas and orchid data was processed using the Shannon wiener (H') diversity index formula.
 The results showed that there were 5 species of host trees found in the study site, namely forest guava (Syzygium pycnanthum), Matani (Pterocarpus indicus), Tahi (Celtis wightii planch), Kamala (Mallotus philippensis), Jenitri (Elaeocarpus ganitrid). Host trees found in Lununianam Protection forest have the characteristics of large hard and rough bark, have a height range of 12-30 m, and bark overgrown with moss, The results of vegetation analysis of host tree species with the highest Important Value Index namely Syzygium Pycnanthum and Elaeocarpus ganitrus have the lowest Important Value Index. Orchids found in the sampling plot in the Lununianam Protection Forest contained 8 species of epiphytic orchids, namely Dendrobium Sp, Vanda insignis, Eria multiflora, Flickingeria grandiflora, Trichoglottis bipenicillata, Pholidota carnea, Appendicula reflexa Blume, and Ceologyne speciosa. The calculation of Shannon Wiener's diversity index of 1.57 (H') is classified as medium (1<H'<3). The most common type of orchid found is the type Eria multifora with a total of 168 individuals and the type that is less commonly found is Appendicula reflexa Blume with 4 individuals.

Phylogenomics reveal DcTPS-mediated terpenoid accumulation and environmental response in Dendrobium catenatum

Terpenoids are essential for plants to adapt for specific ecological niches which are also extensively used in industries of flavor, fragrance, spice, and medicine. Dendrobium catenatum, belonging to epiphytic orchids, is a traditional medicinal plant of high economic value due to its natural abundance in secondary metabolites beneficial for humans. However, knowledge is lacking about the genetic structure of terpenoids and terpenes-mediated stress response in D. catenatum. In this study, terpene synthase DcTPS was mined as an evolutionary gene using phylogenomics in D. catenatum and relative species, and the function of this gene was characterized by transient overexpression in tobacco leaves. Subsequently, co-expression network analysis and machine learning were used to identify transcription factors closely related to DcTPS. Expression changes of DcTPS in response to environmental stresses were also conducted. All these results provided new insights into DcTPS-mediated molecular mechanism of terpenoid accumulation and environmental adaptation in D. catenatum, which will be valuable to guide the cultivation of high-quality medicinal plants and development of terpenoids-related industrial products.

Gibberellic Acid Inhibits Dendrobium nobile-Piriformospora Symbiosis by Regulating the Expression of Cell Wall Metabolism Genes.

Orchid seeds lack endosperms and depend on mycorrhizal fungi for germination and nutrition acquisition under natural conditions. Piriformospora indica is a mycorrhizal fungus that promotes seed germination and seedling development in epiphytic orchids, such as Dendrobium nobile. To understand the impact of P. indica on D. nobile seed germination, we examined endogenous hormone levels by using liquid chromatography-mass spectrometry. We performed transcriptomic analysis of D. nobile protocorm at two developmental stages under asymbiotic germination (AG) and symbiotic germination (SG) conditions. The result showed that the level of endogenous IAA in the SG protocorm treatments was significantly higher than that in the AG protocorm treatments. Meanwhile, GA3 was only detected in the SG protocorm stages. IAA and GA synthesis and signaling genes were upregulated in the SG protocorm stages. Exogenous GA3 application inhibited fungal colonization inside the protocorm, and a GA biosynthesis inhibitor (PAC) promoted fungal colonization. Furthermore, we found that PAC prevented fungal hyphae collapse and degeneration in the protocorm, and differentially expressed genes related to cell wall metabolism were identified between the SG and AG protocorm stages. Exogenous GA3 upregulated SRC2 and LRX4 expression, leading to decreased fungal colonization. Meanwhile, GA inhibitors upregulated EXP6, EXB16, and EXP10-2 expression, leading to increased fungal colonization. Our findings suggest that GA regulates the expression of cell wall metabolism genes in D. nobile, thereby inhibiting the establishment of mycorrhizal symbiosis.

Asymbiotic Seed Germination and In Vitro Seedling Development of the Endangered Orchid Species Cypripedium guttatum.

Cypripedium guttatum is a highly restricted terrestrial orchid that faces increasing endangerment owing to its habitat destruction and illegal collection. Compared to epiphytic orchids, terrestrial orchids such as C. guttatum have harder seed coats and more demanding in vitro germination conditions. This study aimed to develop an effective in vitro propagation system for C. guttatum to aid in its conservation. Seeds from mature capsules were subjected to various conditions, including sterilization using 1% sodium hypochlorite (NaOCl) and different light conditions, culture media, hormones, and organic supplements, to assess germination and early seedling development in vitro. Sterilization with 1% NaOCl significantly improved the germination rate, especially under dark conditions. Germination initiation occurred at 2 and 3 months in orchid seed sowing medium (OSM) and Murashige and Skoog (MS) medium, respectively. The addition of 1 mg/L naphthaleneacetic acid (NAA) further enhanced germination. However, the inclusion of organic supplements, such as apple and banana homogenates, in the culture medium led to substantial growth inhibition after 12 months. Notably, orchid maintenance medium (OMM) without organic additives proved to be the most suitable for seedling growth. The results of this study show that sterilization, appropriate light, and optimal NAA concentrations are beneficial for seed germination.