Orchid Seeds Research Articles

Shifts in bacterial community composition during symbiotic seed germination of a terrestrial orchid and effects on protocorm development.

It is well known that orchid seeds depend on mycorrhizal fungi to supply the necessary nutrients that support germination in natural environments. Apart from fungi, bacteria may also be involved in the germination process of orchid seeds, but so far, their role has not been intensively studied. This research provides evidence that bacterial community composition changes during seed germination of the terrestrial orchid Gymnadenia conopsea. Interestingly, in vitro experiments showed that Pseudomonas spp., which were the most dominant bacteria in the later germination stages, improved protocorm growth. These results suggest that bacteria contribute to the germination of orchid seeds, which may open new perspectives to apply bacteria as a biofertilizer in the introduction and restoration of G. conopsea populations.

Integration of fungal transcriptomics and metabolomics provides insights into the early interaction between the ORM fungus Tulasnella sp. and the orchid Serapias vomeracea seeds

In nature, germination of orchid seeds and early plant development rely on a symbiotic association with orchid mycorrhizal (ORM) fungi. These fungi provide the host with the necessary nutrients and facilitate the transition from embryos to protocorms. Despite recent advances in omics technologies, our understanding of this symbiosis remains limited, particularly during the initial stages of the interaction. To address this gap, we employed transcriptomics and metabolomics to investigate the early responses occurring in the mycorrhizal fungus Tulasnella sp. isolate SV6 when co-cultivated with orchid seeds of Serapias vomeracea. The integration of data from gene expression and metabolite profiling revealed the activation of some fungal signalling pathways before the establishment of the symbiosis. Prior to seed contact, an indole-related metabolite was produced by the fungus, and significant changes in the fungal lipid profile occurred throughout the symbiotic process. Additionally, the expression of plant cell wall-degrading enzymes (PCWDEs) was observed during the pre-symbiotic stage, as the fungus approached the seeds, along with changes in amino acid metabolism. Thus, the dual-omics approach employed in this study yielded novel insights into the symbiotic relationship between orchids and ORM fungi and suggest that the ORM fungus responds to the presence of the orchid seeds prior to contact.

The potential of bacterial endophytes on orchids

Orchids are one of the most exquisite and diverse plant species in nature. The seeds of orchids are non-endospermic and, therefore, dependent on endophytes for germination, growth, and adaptability. Orchids are prized for their beauty and therapeutic and culinary qualities by naturalists and the general public. Many orchid species are now endangered or vulnerable due to collectors' eagerness to take them. Current studies on orchids have concentrated on isolating and identifying the mycorrhizal and non-mycorrhizal endophytes that either directly or indirectly help orchids to grow, develop, and produce beneficial secondary metabolites. In orchids, bacterial endophytes play an essential role in the formation of mycorrhizae and the stability of relationships between plants and fungi. Endophytic bacteria can grow in orchids by producing phytohormones, doing photosynthesis, fixing nitrogen, promoting the mineral nutrition cycle, forming siderophores, and producing diverse beneficial metabolites to improve biomass production, stress tolerance, and biocontrol of potential phytopathogenic fungal species. This article examines how endophytic bacteria are associated with orchids and their potential growth-promoting abilities.

Complexities and Innovations in Orchid Germination: A Review of Symbiotic and Asymbiotic Techniques

<i>Orchidaceae</i> is the second-largest family among flowering plants; one of the most fascinating characteristics is the morphology of the seed. One of the most important complications is that orchid seeds are tiny and lack endosperm, meaning they almost entirely depend on outside sources for nutrients while germinating or developing. The main objective of this review is to give a summary of the present methods being used in orchid germination, highlighting symbiotic and asymbiotic methods. Symbiotic germination requires interaction with mycorrhizal fungi, which provide the necessary nutrients and support for seedling growth. However, in asymbiotic approaches, controlled conditions are provided to promote germination by using nutrient-rich media. The article also highlights some of these advances that have taken place recently and ways that could be adopted to improve their effectiveness in the future. For example, the effectiveness and ability to grow orchids have improved due to recent developments in tissue culture and biotechnology. This study relies on applying the analytical approach to previous relevant studies on the subject. Nevertheless, further research is needed to refine these techniques and improve their usability in orchid conservation. To summarize, while significant progress has been achieved in understanding and enhancing orchid germination processes, further study and innovation are required. By resolving remaining issues and investigating new approaches, it may be possible to better assist orchid conservation and encourage the long-term cultivation of these wonderful plants.

Towards a standardised orchid germination protocol: determining the optimal duration of chemical scarification for orchid seeds by means of viability and permeability staining

Complex nutrient media are used for the germination of orchids in the laboratory. These substrates are also suitable for the growth of microbes and thus the sterilization of both the medium and the seeds prior to sowing is mandatory. The surface disinfection of orchid seeds with chemical substances eliminates possible infections and enhances germination in several cases, by chemically scarifying their seed coats. However, long durations of chemical treatments may result in viability loss, and consequently, precisely determining the optimal treatment duration is essential for developing effective germination protocols. In this study, the double staining technique with Tetrazolium Chloride and Trypan Blue is tested as a proxy method to determine the optimal chemical treatment duration for 10 orchid taxa of Greece. Various durations of chemical treatment with 5% Ca(OCl)2 were carried out on independent seed samples which were subsequently tested, pairwise, for both germination and viability. The results in the corresponding experiments are extraordinarily similar and we conclude that the double viability staining is a quick and accurate technique in determining the permeability and optimal duration for chemical scarification prior to germination experiments. The storability potential of the taxa studied is discussed in comparison with previous data of the NKUA Seed Bank.

A review: Molecular identification of orchid mycorrhiza

Orchids are a diverse and widespread family of flowering plants, with over 25,000 known species and more than 100,000 hybrids and cultivars. Orchids are characterised by their often showy and highly specialised flowers and have unique and intricate floral. Orchids are known to be highly dependent on their mycorrhizal fungi for nutrient uptake, especially during the early stages of their development. Orchid seeds lack the endosperm present in most other seeds, which means they cannot germinate without a source of nutrition. The relationship between orchids and mycorrhiza is known as orchid mycorrhizae or orchid mycorrhiza. In orchid mycorrhiza, the orchid plant forms a mutualistic relationship with certain species of fungi that are able to penetrate the orchid’s roots and colonise its tissues to provides the orchid with essential nutrients. Orchid mycorrhizal fungi are often highly specific, meaning that they can only form partnerships with certain orchid species, and vice versa. The importance of mycorrhizal fungi in the orchid life cycle is crucial from both evolutionary and ecological standpoints. Therefore, it is essential to acquire a thorough comprehension of this relationship and develop methodologies for isolating, identifying, and preserving significant fungal strains that are associated with different orchid species. In recent years, there has been a considerable increase in research concentration on mycorrhizal interactions in orchids. However, certain inquiries remain unresolved pertaining to the fungal communities associated with orchids as well as the divergences notices across different species and geographical locales. The present paper provides a through, and extensive analysis of the fungal life associated with orchids. This article presents a succinct overview of the molecular techniques utilised by researchers globally to isolate and identify peloton-forming fungi in both temperate terrestrial and tropical orchids. The review begins by proving a concise introduction to the background material regarding the wide range of fungal species that are linked with orchids. It then proceeds to explores the topic of orchid mycorrhizal fungi (OMF) and orchid non-mycorrhizal fungi (ONF). The subsequent analysis explores the crucial function that orchid mycorrhizal fungi play in the processes of seed germination and development. Moreover, the study elaborates on the methodologies utilised for isolating fungi, extracting fungal DNA, selecting primers, amplifying DNA and subsequent analysis sequence data. This article considers several molecular identification approaches that are used in studying orchid endophytic mycorrhizal. Using molecular approaches, orchid mycorrhizal can be further explored and identified.

The patterns of inbreeding depression in food-deceptive Dactylorhiza orchids.

Inbreeding depression (ID) in food-deceptive plants have been reported previously, however, it has not been often proven that selfed seeds germinate better than outbred ones or that selfing affects ID. To resolved these issues, food-deceptive related Dactylorhiza majalis, D. incarnata var. incarnata and D. fuchsii orchids were investigated. Hand pollination treatments and control pollination were conducted. Fruit set, number of seeds per fruit, seed length, number of well-developed seeds per fruit, and proportion of in vitro asymbiotic germination seeds, were analyzed in relation to inflorescence levels and used as fitness indicators for these orchids. The ID and pollen limitation were measured. The lowest ID (δ = -1.000) was in D. majalis, and present in combination with a high pollen limitation in its populations. D. fuchsii showed higher ID (δ = 0.366), and D. incarnata var. incarnata weak ID (δ = 0.065), although ID varied between its fitness components. The seed number per fruit differed significantly between the treatments and the inflorescence levels in treatments. This study emphasizes that the breeding system rather than the flower position on the inflorescence shaped the quality and quantity of reproductive output. The ID and its effect on germination of food-deceptive orchid seeds undoubtedly played an important role.

Orchid seeds are not always short lived in a conventional seed bank!

Orchid seeds are reputed to be short lived in dry, cold storage conditions, potentially limiting the use of conventional seed banks for long-term ex situ conservation. This work explores whether Cattleya seeds are long lived or not during conventional storage (predried to ~12 % relative humidity, then stored at -18 °C). We explored the possible interaction of factors influencing seed lifespan in eight species of the genus Cattleya using physiological (germination and vigour), biochemical (gas chromatography), biophysical (differential scanning calorimetry) and morphometric methods. Seeds were desiccated to ~3 % moisture content and stored at -18 °C for more than a decade, and seed quality was measured via three in vitro germination techniques. Tetrazolium staining was also used to monitor seed viability during storage. The morphometric and germination data were subjected to ANOVA and cluster analysis, and seed lifespan was subjected to probit analysis. Seeds of all Cattleya species were found to be desiccation tolerant, with predicted storage lifespans (P50y) of ~30 years for six species and much longer for two species. Cluster analysis showed that the three species with the longest-lived seeds had smaller (9-11 %) airspaces around the embryo. The post-storage germination method impacted the quality assessment; seeds equilibrated at room temperature for 24 h or in 10 % sucrose solution had improved germination, particularly for the seeds with the smallest embryos. Chromatography revealed that the seeds of all eight species were rich in linoleic acid, and differential scanning calorimetry identified a peak that might be auxiliary to selecting long-lived seeds. These findings show that not all orchids produce seeds that are short lived, and our trait analyses might help to strengthen prediction of seed longevity in diverse orchid species.

Asymbiotic germination, seedling establishment and fungal uptake of Pterostylis montana and P. paludosa, two orchid species endemic to New Zealand

ABSTRACT Asymbiotic germination of orchid seeds has been successful for many species, and this could be an important method to generate seedlings to restore threatened populations. However, successful reintroduction of orchids into wild habitats depends on establishment of mycorrhizal symbioses. This communication reports on asymbiotic methods used to propagate two greenhood species endemic to New Zealand, Pterostylis montana (Not Threatened) and P. paludosa (At Risk – Declining). We furthermore investigated whether asymbiotically generated seedlings were colonised with orchid mycorrhizal fungi (OMF) when introduced to soil collected from natural forest areas where these species are not known to occur. We found that seedlings transferred to natural soil hosted at least seven different endophytic fungi, but no known OMF. We caution against the use of asymbiotically germinated seeds for reintroductions before getting a better understanding of the dynamics of mycorrhizal infections and their distribution in the soil.

Invitro germination and optimization of basal media for protocorm-like bodies proliferation in Dieniaophrydis(J. Koenig) Seidenf

Dienia ophrydis is an endangered terrestrial orchid used for its ornamental and therapeutic purposes. This orchid possesses an anti-malarial alkaloid known as Malaxin. There is ongoing exploitation in the wild due to illegitimate herbal collection and climate change. To control the potential risk of extinction, there are no conservational measures or in vitro protocols established in this genus. The study focuses on addressing a germination protocol and enhancement of protocorm-like bodies (PLB) proliferation by optimizing the basal media and vitamins. It also aims to improve the misinterpretation of tetrazolium viability results in orchid testa along with a permeability test. Further, the seeds were subjected to 1% TTC staining to perform embryo viability test and 0.4% trypan blue (TB) dye for testa permeability. Ten basal media such as Gamborg's medium, Mitra medium, Thomale GD medium, Vacin and Went medium, Knudson C medium, Knop's medium, MS medium and ½ MS medium, ¼ MS medium, and modified MS medium (MMS) were tested for seed germination. The Half-strength MS medium responded with 92% of the highest germination and T-GD medium is not suitable for seed germination. The seed viability and permeability result showed 93% of permeability, 72% viability, and 9% dead seeds. Protocorms were transferred to the modified mMS medium containing half-strength macronutrients, full micronutrients, and 10 ppm of thiamine in vitamin formulation. PLB proliferation was observed and 12 shoots emerged from a single protocorm explant. Subsequently, the seedlings were acclimatized with 8-0-24 ratios of nitrogen, phosphorus, and potassium (NPK) as determined by soil testing. The study contributes in experimenting with seed germination of terrestrial orchid seeds asymbiotically and to establishing a protocol for conservation programs, especially in this genus.

Metagenomic Analyses of Viruses in the Orchid Mycorrhizal Interaction Using Improved Assemble Tools.

In nature, mycorrhizal association with soil-borne fungi is indispensable for orchid species. Compatible mycorrhizal fungi form endo-mycorrhizal structures in orchid cells, and the fungal structures are digested in orchid cells to be supplied to orchids as nutrition. Because orchid seeds lack the reserves for germination, they keep receiving nutrition through mycorrhizal formation from seed germination until nonphotosynthetic young seedlings develop leaves and become photoautotrophic. Seeds of all orchids germinate with the help of their own fungal partners, and therefore, specific partnership has been acquired in a long evolutionary history between orchids and fungi. Assuming that horizontal transmission of viruses may occur in such a close relationship, we are focusing on viruses that infect orchids and their mycorrhizal fungi. We prepared aseptically germinated orchid plants (i.e., fungi-free plants) together with pure-cultured fungal isolates and conducted transcriptome analyses (RNA-seq) by next-generation sequencing (NGS) approach. To reconstruct virus-related sequences that would have been present in the RNA sample of interest, de novo assembly process is required using short read sequences obtained from RNA-seq. In the previous version of our protocol (see Viral Metagenomics, first edition 2018), virus searches were conducted using contig sets constructed by a single assembler, but this time we devised a method to construct more reliable contigs using multiple assemblers and again reinvestigated that viruses could be detected. Because the virus detection efficiency and number of detected virus species clearly differed depending on the assembly pipeline and the number of the input data, multiple methods should be used to identify viral infection, if possible.

Evolution of Seed Dispersal Modes in the Orchidaceae: Has the Vanilla Mystery Been Solved?

Orchid seeds are predominantly wind-dispersed, often developed within dry, dehiscent fruits that typically release millions of dust-like seeds into the air. Animal-mediated seed dispersal is a lesser-known phenomenon in the family and predominantly occurs in groups belonging to early-diverging lineages bearing indehiscent, fleshy fruits with hard, rounded, dark seeds. In this review, we explore the evolutionary trends of seed dispersal mechanisms in Orchidaceae, focusing on the pantropical genus Vanilla. Notably, certain Neotropical species of Vanilla produce vanillin-aromatic compounds synthesized naturally in their fruits, which plays a pivotal role in seed dispersal. Ectozoochory occurs in dry, dehiscent fruits, whose seeds are dispersed by (i) male euglossine bees collecting the fruit’s vanillin aromatic compounds and (ii) female stingless bees collecting the fruit’s mesocarp. Endozoochory occurs in (iii) highly nutritious, indehiscent fruits consumed by terrestrial mammals or (iv) fleshy, dehiscent fruits whose mesocarp is consumed by arboreal mammals. Wind dispersal appears to be a derived state in Orchidaceae and, given its predominance, a trait likely associated with enhanced speciation rates. Zoochory primarily occurs in groups derived from early-diverging lineages; occasional reversions suggest a link between dispersal mode and fruit and seed traits. Interestingly, fruit dehiscence and fleshiness in Vanilla lack phylogenetic signal despite their role in determining dispersal modes, suggesting potential environmental adaptability.

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 and symbiotic seed culture of Polystachya concreta (Jacq.) Garay & H.R. sweet from Ecuador

The Orchidaceae is the most numerous and diverse family in Ecuador with more than 4200 species. Information on the interaction between these neotropical orchids seed and mycorrhizal fungi during germination and seedling development in situ is very limited. Symbiotic seed germination culture allows screening for compatible mycobionts resulting in mycorrhizal associations to develop under sterile and controlled environmental conditions. A previously developed symbiotic seed culture protocol was used to screen seed germination and seedling development in the neotropical orchid Polystachya concreta. Mature seeds collected from native populations in the northwest Andes Mountains of Ecuador display high viability (92%) and germination percentage (96%). Light was not a limiting factor for gemination and seedling development in this orchid species. Tulasnella isolates from roots of different orchid species in situ facilitated seed germination only, whereas Tulasnella calospora (UAMH 9824), isolated from Spiranthes brevilabris in Florida, facilitated both seed germination and seedling development in P. concreta. Ceratobasidium strains, isolated from roots of different orchid species in Ecuador, neither promoted seed gemination or seedling development. Symbiotic seed culture did not significantly improve germination compared to that observed in the absence of fungi. Higher germination rates and percentages of more advanced seedling development were observed on asymbiotic PhytoTechnology Orchid Seed Sowing Medium (P723). However, significantly larger seeds seedlings were observed when seeds were cocultured with certain fungal isolates. Information obtained from this study confirm that this symbiotic seed culture application will be both beneficial for screening potential mycobiont isolates and seedling production for conservation of native orchid species in Ecuador.

AN EXPLORATORY STUDY OF ORCHIDACEAE SPECIES FRUITS IN THE CENTRAL ZONE OF VERACRUZ STATE, MEXICO

The knowledge on Orchidaceae family fruits becomes useful in new studies, by helping to identify species that are not in flowering period and providing material for in vitro germination of orchid seeds. The hypothesis was that the number of orchid specimens with naturally pollinated fruits, from the central zone of the state of Veracruz, Mexico, would be more than the number of specimens that do not developed fruit naturally. The objectives of this work were: to record the flowering and fruiting periods, to characterize the orchid fruits pollinated naturally or manually. The fruit collection sites were located within the municipalities of Amatlán de los Reyes, Fortín and Tlaltetela. The work was performed between January 2019 and April 2022 (39 months). Ripe orchid fruits were collected. Morphometry was measured by recording weight (g), length (cm) and diameter of the central region (cm), complemented with a photographic record. The greatest fruit diversity of these orchids was observed and collected during the month of March. Forty-two species and their fruits were recorded, which were grouped into 8 subtribes and 32 genera. The genera with the highest number of species were: Epidendrum, Prosthechea and Oncidium, each with three species. Three species bloomed for 11 months: Platystele stenostachya, Specklinia digitale and S. tribuloides. The minor and major fruiting periods correspond to Specklinia digitale and Trichocentrum stramineum, respectively. Given the fact that in all three sites studied the greatest number of species and fruits is associated with natural pollination (27 species, 113 fruits), which exceeds the number of manually pollinated species (15 species, 51 fruits), it is possible to suggest that the largest contingent inhabits environments still compatible with the natural process of their reproduction in these sites.

Interspecific hybridization of Dendrobium mirbelianum x D. nindii or D. discolor, in vitro seed germination, seedling growth and plantlet acclimatization

Abstract. Maulida D, Yusnita Y, Hapsoro D, Agustiansyah A, Karyanto A. 2023. Interspecific hybridization of Dendrobium mirbelianum x D. nindii or D. discolor, in vitro seed germination, seedling growth and plantlet acclimatization. Biodiversitas 24: 3004-3011. Dendrobium mirbelianum Gaudich. (P1), D. nindii W.Hill (P2), and D. discolor Lindl. (P3) as a parent crossing are potentially resulting a new type of orchid as the novel breeding achievement. This study aimed to identify orchid crossing compatibility and evaluate the most effective and efficient media for hybrid seed germination and growth. The study consisted of three experiments, namely interspecific hybridization (experiment 1), in vitro hybrid seed germination (experiment 2), and in vitro growth of the hybrid seedlings (experiment 3). The first experiment was conducted by a simple modified line×tester mating design, and all experiments used a completely randomized design, with five replications in the first experiment and three replications in the second and third experiments. The first experiment presented all of the crossing emerging seed pods, but only hybrid seeds from cross D. mirbelianum x D. nindii and its reciprocal cross that potent to germinate in all tested media as the second experiment result. The third experiment showed FF+tryptone was the best media indicated by the highest and heaviest seedling after 8 months after sowing. All of the crossings resulted seed pods, however, only D. mirbelianum x D. nindii and its reciprocal cross that able to germinate in all tested media. Foliar fertilizer is the most recommended media for germinating hybrid orchid seeds, and FF+tryptone is the best for seedling growth.

Effect of different substrates on in vitro symbiotic seed germination for soilless production of Anacamptis laxiflora orchid

In recent years, the orchid species have become endangered due to overuse and habitat destruction. As with most flowering plants, seed production is the primary strategy for reproduction in orchids. Orchids produce tiny seeds consisting of a seed coat and a rudimentary embryo. However, it lacks the endosperm, which is generally required as the primary energy source during germination. The only way to germinate orchid seeds is to get nutrients from an external source. In nature, this is achieved by mycorrhizal symbiosis. This study used Ceratobasidium sp. inoculation of Anacamptis laxiflora (Lam.) seeds combined with media with various organic substrates to determine their effectiveness on germination and seedling development by in vitro culture. The highest germination rate (35.78%) was obtained in the medium with addition of young hazelnut leaves. Then, soilless ex vitro symbiotic germination was performed on young hazelnut leaves, the most effective organic substrate. Seed germination was determined to be 19.01% in this medium while 14.87% seedlings with developed leaves and roots were formed. For the first time, success was achieved by producing A. laxiflora from seed in ex vitro conditions without soil and adapting it to nature.

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants.

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

A novel method to produce massive seedlings via symbiotic seed germination in orchids.

Orchids produce large numbers of dust-like seeds that rely heavily on orchid mycorrhizal fungi (OMFs) for germination. Using OMFs to facilitate orchid proliferation is considered an effective method for orchid conservation but still presents challenges in practice. In this study, orchid seed-fungus complexes, in which orchid seeds and fungal mycelia were embedded together to form granules, were developed as platforms to facilitate seed germination and seedling production. Overall, seedlings were produced by seed-fungus complexes for five orchid species with large variations in the percentages of seedlings produced among species/treatments. For the different fungal treatments in Dendrobium officinale, Sebacinales LQ performed much better than the other fungal strains. At 90 days after sowing, 75.8±2.6% seedlings were produced in the LQ treatment, which was significantly higher than in the Tulasnella sp. JM (22.0±3.0%) and Tulasnella sp. TPYD-2 (5.3±1.0%) treatments, as well as in the LQ and TPYD-2 cocultured treatment (40.4±3.2%), while no seedlings were formed in the Tulasnella sp. SSCDO-5 or control treatments. For the other four orchid species, only one compatible fungus for each species was used, and the percentages of seedlings in epiphytic Dendrobium devonianum (67.2±2.9%) and D. nobile (38.9±2.8%) were much higher than those in terrestrial Paphiopedilum spicerianum (2.9±1.1%) and Arundina graminifolia (6.7±2.1%) at 90 days after sowing. Adding 1% polymer water-absorbent resin to the seed-fungus complexes of D. officinale seeds with fungal strain Sebacinales LQ significantly increased seedling formation, while other additional substances showed negative effects on seedling formation. For the storage of seed-fungus complexes, it is recommended to store the seed-fungus complexes in valve bags at room temperature for a short time and at a low temperature of 4°C for no more than 30 days. As a platform for symbiotic seed germination, the seed-fungus complex can facilitate seed germination, produce seedlings and support subsequent seedling growth, and its seedling productivity depends on seed germination characteristics, seed viability, and the efficiency of fungi. Seed-fungus complexes have great potential to be used as propagules in orchid conservation.

Temperature conditions optimization for storing tropical orchid seeds on the example of <i>Eulophia streptopetala</i> and <i>Stanhopea tigrina</i>

The paper presents the results of the study of temperature regime optimization for storing seeds of tropical orchids on the example of Eulophia streptopetala Lindley and Stanhopea tigrina Batem ex Lindl. Mature seeds were frozen at temperatures of 18C, 40C or 80C for a period of 1, 3, 6 or 12 months, the storage temperature was +4C. Before and after freezing, the viability of seeds was determined by visual and tetrazole methods. After defrosting, the seeds were sown and the plants grown from them were cultivated on a nutrient asymmetric medium, then the dynamics of plant development was recorded, the content of photosynthesis pigments (chlorophyll and carotenoids) was quantified. The analysis of the data obtained showed that the viability of seeds significantly decreases after storage, including in conditions of low temperatures. At the same time, in experimental groups of plants, the terms of early ontogenetic states were shortened, and the growth rate of protocorms increased. Freezing of seeds caused further restructuring of the photosynthetic apparatus. The optimal temperatures for storing seeds of the species Eulophia streptopetala were +4C, and for Stanhopea tigrina 80C and 40C.