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Overall growth form and different stages of the seed germination process of Gymnadenia conopsea. a Flowering stalk of G. conopsea. b Fruit set in G. conopsea; c One seed of G. conopsea. d, e After 60 days of incubation, protocorm differentiation was only observed in GS2 and HH1. f First leaf emerged after 120 days of dual cultured with GS2. g Trypan blue staining showing partial colonization of protocorm cells after incubation with fungal associates of the co-occurring orchid species. h Trypan blue staining showing fungal strain GS2 forming pelotons in most of the protocorm cells. a, b, e, f Scale bar = 1 cm, c scale bar = 100 μm, d scale bar = 500 μm, g, h scale bar = 50 μm
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Interactions with mycorrhizal fungi have been increasingly recognized as one of the most important ecological factors determining the distribution and local abundance of orchids. While some orchid species may interact with a variety of fungal associates, others are more specific in their choice of mycorrhizal partners. Moreover, orchids that co-occ...
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... conopsea is a terrestrial, photosynthetic orchid (Fig. 1a) that is widely distributed across Europe and Asia ( Meekers et al. 2012;Xing et al. 2020). Populations of G. conopsea can be found in a wide range of habitats, including forests, grasslands, and waterlogged meadows at altitudes varying between 0 and 4700 m throughout Europe and temperate and subtropical zones of Asia ( Meekers et al. ...
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... of G. conopsea can be found in a wide range of habitats, including forests, grasslands, and waterlogged meadows at altitudes varying between 0 and 4700 m throughout Europe and temperate and subtropical zones of Asia ( Meekers et al. 2012). Leaves emerge in spring, and flowering takes place between the end of June and the beginning of August (Fig. 1a). The species is self-compatible but relies on pollinators for successful pollination and fruit set. Fruit set is generally very high (> 90%) (Meekers et al. ...
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... and those with fungal mycelia on the surface were selected and surface cleaned several times with sterile water to minimize the detection of soil fungi. Roots were stored at 4 °C for isolation of mycorrhizal associates. Capsules of G. conopsea were collected in the middle of August, 2018. One capsule contains around ten thousand minute seeds (Fig. 1b, c). The capsules were surface sterilized and kept in plastic tubes at 4 °C for further ...
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... dishes containing 20 ml of sterile oat meal agar (OMA) medium (5 g rolled oats +12 g agar/L) were inoculated with a 1 cm × 1 cm plug of each fungal strain taken from fresh cultures on PDA and cultured for 3 to 5 days. Seeds of G. conopsea were surface sterilized following Dutra et al. (2009) (2002) (Fig. 1, Table 1). To determine whether a mycorrhizal symbiosis had been established, visualization of the mycobiont structures (pelotons) inside germinating seeds was evaluated following Phillips and Hayman ...
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... eight fungal strains were used to test their ability to promote seed germination of G. conopsea. After 23 days of incubation, seeds in all the eight treatments had swollen and the testa were broken (Fig. 1d). The highest (80.70 ± 3.52%) and the lowest (15.62 ± 1.25%) embryo swelling rates were observed for strains GS2 and DW5 (Fig. 4), respectively. The percentage of seed germination differed significantly between fungal isolates (Kruskal-Wallis test P < 0.001). After 45 days of incubation, all fungal strains except DW5 supported seed ...
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... strains except DW5 supported seed germination up to stage 2 (protocorm formation) (Fig. 4). The percentage of protocorm formation differed significantly (P < 0.001) between fungal isolates and was highest for Seedling with emergence of first leaf GS2. After 60 days of incubation, protocorm differentiation was only observed for strains GS2 and HH1 (Fig. 1e), and the percentage of protocorm differentiation with GS2 was also significantly higher than with HH1 (P < 0.001) (Fig. 4). Finally, only strain GS2 supported seed germination until the stage of first leaf emergence, indicating that protocorms gradually grew into seedlings after 4 months of incubation (Fig. 1f). The final seed ...
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... observed for strains GS2 and HH1 (Fig. 1e), and the percentage of protocorm differentiation with GS2 was also significantly higher than with HH1 (P < 0.001) (Fig. 4). Finally, only strain GS2 supported seed germination until the stage of first leaf emergence, indicating that protocorms gradually grew into seedlings after 4 months of incubation (Fig. 1f). The final seed germination rate supported by GS2 was 13.65 ± 1.53%. Fungal colonization was checked in inoculated treatments after 30 days. In all cases, pelotons were observed in the cortex cells of the protocorms (Fig. 1g, h), but colonization rates were much higher in the GS2 (78.52 ± 3.40%) treatment than in the other fungal ...
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... the stage of first leaf emergence, indicating that protocorms gradually grew into seedlings after 4 months of incubation (Fig. 1f). The final seed germination rate supported by GS2 was 13.65 ± 1.53%. Fungal colonization was checked in inoculated treatments after 30 days. In all cases, pelotons were observed in the cortex cells of the protocorms (Fig. 1g, h), but colonization rates were much higher in the GS2 (78.52 ± 3.40%) treatment than in the other fungal treatments, which varied between 15.31 ± 2.94 and 30.42 ± 4.60%. No germination was observed in the pure cultures without ...
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... f. (Xi et al., 2020;Shi et al., 2023), Cremastra appendiculata (D. Don) Makino and Gymnadenia conopsea (L.) R. Br. (Gao et al.,2020) (Fig. 2). Studies on the mechanism through which mycorrhizal fungi promote seed germination have focused on the effects of the immune defence response, nutrient supply and the role of plant hormones. ...
The family Orchidaceae is of the most diverse taxon in the plant kingdom, and most of its members are highly valuable herbal medicines. Orchids have a unique mycorrhizal symbiotic relationship with specific fungi for carbohydrate and nutrient supplies in their whole lifecycle. The large-scale cultivation of the medicinal plant Gastodia elata is a successful example of using mycorrhizal symbiotic technology. In this review, we adopted G. elata and Dendrobium officinale as examples to describe the characteristics of orchid mycorrhiza and mycorrhizal benefits for host plants’ growth and health (e.g. biotic and abiotic stress and secondary metabolite accumulation). The challenges in applying mycorrhizal technology to the cultivation of orchid medicinal plants in the future were also discussed. This review aims to serve as a theoretical guide for the cultivation of mycorrhizal technology in medicinal orchid plants.
... In orchid conservation, the isolation of the mycorrhizal fungi is a key step in understanding their mycorrhizal associations and facilitating effective mass propagation of endangered orchids through in vitro symbiotic germination (Liu et al., 2010). Mycorrhizal fungi are most frequently isolated from adult plant roots (e.g., Gao et al., 2020;Nontachaiyapoom et al., 2010;Otero et al., 2002). Such isolates from adult plants may be effective for seed germination in orchids that show continuous mycorrhizal fungus associations from the juvenile to the adult stage, but not in orchids that switch mycorrhizal fungi during ontogeny, as observed in V. falcata. ...
Vanda falcata is an epiphytic orchid native to East Asia with high horticultural and ornamental value. However, its wild populations are at risk of extinction due to overcollection and habitat loss. Orchids are highly dependent on mycorrhizal fungi for their nutrient acquisition from seed germination to adulthood; therefore, understanding their mycorrhizal associations throughout the life cycle is essential for their conservation. We investigated the mycorrhizal fungi of adult V . falcata plants through the molecular identification of root samples from 40 individual plants from 13 sites, covering a distance of 1200 km across Japan. To identify the fungi that promote V . falcata seed germination, we performed seed germination tests in vitro with fungal isolates from V . falcata and other epiphytic orchids. In adult plants, we detected a total of 11 operational taxonomic units (OTUs) within two fungal families, Ceratobasidiaceae and Tulasnellaceae, and a single Tulasnellaceae OTU (TU11) was found in samples from 12 sites. However, Vanda falcata seed germination and early development were induced in vitro by Ceratobasidiaceae fungi rather than Tulasnellaceae fungi, including TU11. These results suggest that different mycorrhizal fungi may be predominantly associated with V . falcata in the adult and seed germination stages. This mycorrhizal switching suggests that effective mycorrhizal fungi for conservation may differ between the adult and juvenile stages, even within a single orchid species. Our findings clarify the mycorrhizal associations of endangered epiphytic orchid species and could improve conservation efforts for these rare plants.
... However, Shao et al. (2020b) found that OrM co-cultures did not result in higher seed germination rates and seedling development of Dendrobium nobile compared to monocultures. Likewise, Gao et al. (2020) found that one Ceratobasidium taxon was responsible for germination to stage four (i.e., photosynthetic plant), despite other OrM taxa being able to initiate seed germination to stage two. In A. papilionacea, there was a clear difference between ceratobasidioid fungus and tulasnelloind fungi in seed germination rates, with the Ceratobasidium isolate less efficient in germinating seeds compared with different isolates of T. calospora, particularly the isolate tul7.1. ...
Orchids depend on mycorrhizal fungi to germinate from seed. While multiple orchid mycorrhizal (OrM) taxa are often found associated with adult orchids, the relative contribution of particular OrM taxa to germination and early orchid development is poorly understood. We isolated 28 OrM fungi associated with the Mediterranean orchid Anacamptis papilionacea and tested the efficiency of five isolates on germination and early development, four belonging to the Tulasnella calospora species complex and one belonging to Ceratobasidium. Co-cultures of varying two-way and three-way combinations of OrM isolates were used in vitro to compare the simultaneous effect on seed germination rate with monocultures. We then tested whether, when given initial priority over other fungi, particular OrM taxa were more effective during the early stages of development. Seedlings germinated with different isolates were transferred to a growth chamber, and either the same or different isolate was added 45 days later. After 3 months, the number of roots, length of the longest root, and tuber area were measured. All OrM fungi resulted in seed germination; however, lower germination rates were associated with the Ceratobasidium isolate compared to the tulasnelloid isolates. There was significant decreased germination in co-culture experiments when the Ceratobasidium isolate was added. Despite being associated with reduced germination rates, the addition of the Ceratobasidium isolate to the seedlings germinated with tulasnelloid strains resulted in significant increased tuber size. Although A. papilionacea associates with many OrM taxa, these results show that OrM fungi may play different roles during orchid germination and early development. Even when given initial priority, other fungi may colonize developing orchids and interact to influence early orchid development.
... Mycoheterotrophic and parasitic orchids exist with the great majority of ornamental orchids. The loss and survival of symbiotic genes connected to the evolution of particular symbionts spans from the ancestral arbuscular mycorrhiza to the recent ericoid and orchid mycorrhizae (Barrett et al., 2019;Gao et al., 2020). Here, nine tandem duplication gene pairs of DoWRKY were confirmed in peusodochromosomes. ...
With the rapid advancement of high-throughput sequencing technology, it is now possible to identify individual gene families from genomes on a large scale in order to study their functions. WRKY transcription factors are a key class of regulators that regulate plant growth and abiotic stresses. Here, a total of 74 WRKY genes were identified from Dendrobium officinale Kimura et Migo genome. Based on the genome-wide analysis, an in-depth analysis of gene structure and conserved motif was performed. The phylogenetic analysis indicated that DoWRKYs could be classified into three main groups: I, II, and III, with group II divided into five subgroups: II-a, II-b, II-c, II-d, and II-e. The sequence alignment indicated that these WRKY transcriptional factors contained a highly conserved WRKYGQK heptapeptide. The localization analysis of chromosomes showed that WRKY genes were irregularly distributed across several chromosomes of D. officinale. These genes comprised diverse patterns in both number and species, and there were certain distinguishing motifs among subfamilies. Moreover, the phylogenetic tree and chromosomal location results indicated that DoWRKYs may have undergone a widespread genome duplication event. Based on an evaluation of expression profiles, we proposed that DoWRKY5, 54, 57, 21, etc. may be involved in the transcriptional regulation of the JA signaling pathway. These results provide a scientific reference for the study of DoWRKY family genes.
... conditions. The key factors namely culture conditions, culture media, and seed maturity affect the efficiency of in vitro propagation (Bezerra et al., 2019;Gao et al., 2020;Teixeira da Silva, Dobranszki, et al., 2016). The decrease in the duration of the breeding cycle and hybrid innovations hold the key to efficient propagation . ...
One of the most exotic and ubiquitous plant species, the cultivation of orchids continues to gain momentum worldwide. The growing popularity of orchids and their cultivation worldwide is attributed to substantial progress in approaches like classical breeding, plant tissue culture and biotechnological interventions. In addition to ornamental value, orchids are extensively studied for their pharmacological properties and are widely used as food supplements, flavours, and medicine in different regions of the world. The unprecedented advances in whole-genome sequencing and omics technologies have significantly improved our knowledge of orchid biology, leading to translational success in the production of exotic varieties. With the emerging investigations into the cultivation and global trade popularity of orchids in the present era, this thematic article provides comprehensive insights into the existing and emerging trends in orchid cultivation, diverse ethnomedicinal uses, and multi-faceted applications, and the need for the legalisation of orchids for conservation and trade in the present era. In this way, advances in breeding and molecular approaches aim to significantly improve orchid cultivation and its socio-economic attributes. However, a detailed understanding of challenges in orchid conservation and implementation of domestic/global legislative guidelines are necessary for the protection of endangered species and their legalised trade across the globe.
... It is generally accepted that germination and PLB formation are more challenging and complex in terrestrial orchids than in epiphytic species because of different factors such as physical and physiological barriers and dependency on the mycorrhiza fungi [23]. The last factor is needed to provide essential nutrients, including macro and micro-elements, hydrocarbons, and amino acids, and also to adjust osmotic potential [48,49]. For the successful establishment of symbiotic cultures, mycorrhizal isolate preparation and the development of symbionts are required, while asymbiotic germination could alternatively provide an in vitro propagation platform for terrestrial orchids without symbiosis. ...
Himantoglossum affine is a threatened terrestrial orchid. We aimed to optimize asymbiotic seed germination and direct embryogenesis and to analyze the phytochemical profile and physico-biochemical analysis of leaf and tuber. The individual use of organic nitrogen compounds resulted in higher germination efficiencies, while the shortest times to germination were observed using coconut water plus casein hydrolysate. Plantlets grown on media supplemented with pineapple juice and peptone had the highest plantlet length and weight. For embryogenesis, the highest regeneration rate (44%) and embryo number/explant (10.12 ± 2.08) were observed in young protocorm-like body (PLB) explants with 0.5 mg/L naphthalene acetic acid (NAA) and 1 mg/L thidiazuron (TDZ). During the acclimatization process, the scattered vascular tubes converted to fully developed vascular tissues, ensuring maximum sap flux. Gas chromatography–mass spectrometry analysis identified 1,2,3-propanetriol, monoacetate, 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl, and 2-butenedioic acid, 2-methyl-, (E)- as the most prevalent compounds. We reported higher contents of total phenolics and flavonoids and antioxidant activity compared to other terrestrial orchids. The glucomannan content (36.96%) was also higher than starch content (31.31%), comparable to those reported in other tuberous orchids. Based on the fragmentation of H. affine populations in the Middle East and Euro-Mediterranean countries due to over-harvesting, climate change, and/or human impact, our procedure offers a tool for the re-introduction of in vitro-raised plants to threatened areas.
... By providing the required macro and micronutrients and hydrocarbons, and also by regulating osmotic status, the symbiotic relationship with mycorrhizal fungi promotes seed germination and enhances the growth and establishment of terrestrial orchid plantlets (Chen et al., 2012;Figura et al., 2021). Mycorrhizal isolate preparation and symbiont development are required extra perquisites for the successful establishment of symbiotic cultures, while asymbiotic procedure provides an alternative in vitro multiplication and conservation platform without the need for symbiosis relationships (Arcidiacono et al., 2021;Dowling and Jusaitis, 2012;Gao et al., 2020). Nonetheless, creating optimal formulations for asymbiotic germination of corresponding temperate species or sub-species has remained a challenge that deserves further investigation. ...
The present study examined the seed morphometric and viability, asymbiotic seed germination, protocorm formation, and plantlet growth in Orchis simia Lam., an at-risk Euro-Mediterranean terrestrial orchid species. Furthermore, tissue histology pattern in vitro grown plantlets and field-established was compared. After one month, shoot and root primordia started to grow on developing protocorms and small plantlets became visible after 2–3 subculturing. Asymbiotic in vitro seed germination and plantlet growth attributes were significantly (P<0.05) impacted by both type of main organic constituent and nitrogen sources. The seeds sown on media supplemented with pineapple juice (PJ) and casein hydrolysate (CH) had the highest germination percentage. In terms of required days to see the first signs of germination, CW+AV treatment brings about the fastest germination (6.8 ± 0.20 days). Protocorms grown on the media comprising PJ in combination with Aminoven (AV) or casein hydrolysate (CH) or Pep showed higher dimensions and therefore higher weight (% 450 increase) compared to protocorms grown on other media. Altogether, treatments with higher germination percentage, faster germination, and bigger protocorms finally resulted in better plantlet growth. Acclimatization of in vitro grown plantlets occurred by converting scattered vascular tubes into fully developed xylem and phloem vessels facilitating sap flow. Taking into account the fragmentation of O. simia populations in the Middle East and the Euro-Mediterranean basin mainly due to tuber overharvesting and climate change, the present procedure could be used for large-scale multiplication of in vitro raised seedlings to re-introduce into endangered regions.
... In order to realize the sustainable utilization of G. conopsea resource, an effective way was to obtain the fungal strain with the ability to support seed germination and further used in recovery of wild populations. One fungal strain GS2, initially isolated from roots of G. conopsea, was able to support G. conopsea seeds germination under both artificial and natural conditions (Gao et al., 2019(Gao et al., , 2020Jiang et al., 2022). The fungal strain GS2 was identified as Ceratobasidium sp. ...
... The fungal strain GS2 was identified as Ceratobasidium sp. (Gao et al., 2020). Some secondary metabolites of the symbiotic fungus may play a role in the interaction between host plants and fungi. ...
... The fungal strain GS2 (NCBI accession no.: OK655751.1) was previously isolated from the roots of G. conopsea and shown to promote seed germination and early seedling development under in vitro conditions (Gao et al., 2020). The strain was deposited in the Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences (accession number CGMCC no. ...
Five furanoids including a new analog ( S )-1,4-di(furan-2-yl)-2-hydroxybutane-1,4-dione ( 1 ) together with four known ones, rhizosolaniol ( 2 ), 5-hydroxymethylfurfural ( 3 ), 2-furoic acid ( 4 ) and (2-furyl) oxoacetamide ( 5 ), were isolated from the fungal strain Ceratobasidium sp. (GS2) inducing seed germination of the endangered medicinal plant Gymnadenia conopsea of Orchidaceae. The structure of new furanoid 1 was determined mainly based on HR-ESI-MS and NMR spectral data. Modified Mosher’s reactions were used to establish the stereochemistry of the hydroxyl group in 1 , which was not stable in Mosher’s reagents and transformed into four analogs 6 – 9 . These degraded products ( 6 – 9 ) were elucidated based on UPLC-Q-TOF-MS/MS analysis, and compound 8 was further isolated from the degraded mixture and its structure was characterized through NMR experiments. Therefore, the absolute configuration of compound 1 was determined by electronic circular dichroism combined with quantum-chemical calculations adopting time-dependent density functional theory. Compounds ( 1 – 5 ), and 8 showed weak antioxidant activities, and compounds ( 2 – 4 ) displayed phytotoxicity on punctured detached green foxtail leaves. In addition, compounds 3 and 4 strongly showed inhibition activities on the seed germination of G. conopsea . This was the first chemical investigation of the symbiotic fungus of G. conopsea .
... The dependence of orchids on mycorrhizal fungi in the earliest stages of development is well known (Smith & Read 2008), and researchers have studied the symbionts diversity in adult phase, as well as the specificity of interaction (Zettler et al. 2005, Meng et al. 2019, Thixton et al. 2020, Gao et al. 2020. However, most studies have focused on temperate regions and terrestrial orchids, while works on tropical and epiphytic orchids occur in less frequency. ...
Species from Ceratobasidium are known as orchid mycorrhizal, plant pathogens or saprophytic. In mycorrhizal association with orchids, Ceratobasidium can establish a highly specific interaction. Gomesa recurva is an epiphytic orchid occurring in the Brazilian Atlantic Forest. In the present study mycorrhizal fungi were isolated from roots of G. recurva and its phylogenetic positions was investigated. A total of nine isolates were obtained directly from pelotons and submitted to phylogenetic analysis based on alignments of sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal DNA. Phylogenetic analyzes, along with morphological data, revealed that isolates are distinct from all known Ceratobasidium species and a new species, namely Ceratobasidium gomesae sp. nov., is proposed. Given the dependence on mycorrhizae for germination and protocorm development, knowledge about orchid symbionts is an important factor in managing the conservation of these plants, and future studies may investigate the ability of C. gomesae to promote seed germination.
... In contrast, the in situ germination was supported only poorly by the inoculated OTUs (Fig. 3), except for Cer2, and the protocorms associated with multiple naturally occurring rhizoctonia (Appendix C: Table 3). These results fit the view of this species being a mycorrhizal generalist with the ability to engage numerous rhizoctonia strains both at the seedling and adult stage (e.g., Jacquemyn et al., 2012;T e sitelov a et al., 2013;Vogt-Schilb et al., 2020; but see Gao et al., 2020 for Asian populations). Yet, the inconsistent efficacy of the rhizoctonia OTUs in vitro and in situ may complicate the OTU selection for a generalist species restoration. ...
... The practical application of this method in orchid restoration actions should consider: 1) Level of orchid-fungus specificity of the target species -Species with high mycorrhizal specificity may be the most suitable for the restoration using the inoculation approach. Appraisal of the fungal ability to support symbiotic growth in situ is appropriate as not all putatively mycorrhizal fungi isolated from adults or even advanced protocorms may allow germination (this study; Gao et al., 2020;Zhao et al., 2021). In our study, all orchidfungus combinations that enabled in vitro germination also led to in situ germination, but the fungal efficacy varied in situ in the mycorrhizal generalist species; 2) Using inoculated agar for fungus transfer and local organic matter for soil amelioration -We used hay, the most common organic material in grasslands. ...
Grasslands restored on arable land often retain high residual nutrients, modified soil biota, and lower plant species diversity. Establishment of rare plant species with complex multitrophic interactions, typical of undisturbed nutrient-poor environments, may be hindered by the absence of interacting organisms. We hypothesised that the addition of a mycorrhizal symbiont improves the seed germination of orchids that crucially depend on fungi. We focused on grasslands restored on arable land 1–15 years ago featuring residual mineral nutrients and low organic matter contents compared to semi-natural grasslands and on four orchid species differing in the level of mycorrhizal specificity: high – Anacamptis pyramidalis and Orchis mascula – and low – Platanthera bifolia and Gymnadenia conopsea. Five fungal isolates obtained from non-green underground mycorrhizal orchid seedlings (protocorms) or adults' roots were tested for orchid-fungus compatibility under conditions in vitro. Orchid seeds inserted in retrievable seed packets were subsequently co-introduced with selected fungal isolates grown either on agar or sterilized hay into the soil of nine restored grasslands and incubated for twelve months. The identity of mycorrhizal fungi inside protocorms was verified by molecular methods. The isolates that supported protocorm establishment in vitro enabled also protocorm formation in situ, but success rates differed among orchid species. While mycorrhizal specialists produced most protocorms after inoculation, the mycorrhizal generalists took advantage of naturally occurring fungi and produced some protocorms both in inoculated and uninoculated treatments. We showed that the addition of mycorrhizal fungi enhanced protocorm formation regardless of the modified soil environment, especially in mycorrhizal specialist orchids. This method may help to restore populations of native orchid species in their former distribution ranges, including farming-altered habitats.
