Figure 1 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
Preparation of in vitro symbiotic germination assay. (a) A plain oatmeal agar (OMA) plate. (b) A piece of fungal inoculum is put at the center of the plate. Hyphae will appear as a thin, whitish layer on the medium. (c) Surface-sterilized seeds (arrowhead) sown on top of the hyphal layer. (d) Symbiotically germinating Bletilla striata seed during inoculation with Tulasnella sp. after two weeks. Scale bars, 500 µm.
Source publication
As one of the largest families of flowering plants, Orchidaceae is well-known for its high diversity and complex life cycles. Interestingly, such exquisite plants originate from minute seeds, going through challenges to germinate and establish in nature. Alternatively, orchid utilization as an economically important plant gradually decreases its na...
Contexts in source publication
Context 1
... in vitro studies do not always represent realistic environmental conditions, these act as a microenvironment replica and may function as a tool for extensive studies [47,49]. The most convenient way to conduct in vitro study is by simply inoculating seeds and a suitable fungus on a solid agar medium (Figure 1a-c). This study requires two elements: (1) putative fungal symbiont and (2) a medium containing a nutrient source that is available to the fungus but not the orchid seed by assuming that the seeds can only absorb water, not nutrients. ...
Context 2
... a study on Cyrtosia septentrionalis, a small box chamber was used for inoculation with no direct contact between the fungus and seeds [33]. Depending on the species, seed viability, OMF compatibility, or medium components, seeds can germinate in a month (Figure 1d) or less [48] or even up to a year [20,55]. According to Table 1, many orchids are considered generalist-or anecdotally, promiscuous-forming OM with fungi isolated from another orchid species [6,48,[56][57][58][59][60]. ...
Citations
... La germinación asimbiótica in vitro de diferentes especies de orquídeas, ha permitido introducir y germinar semillas con una alta tasa de germinación, obtención de plantas sanas y vigorosas en algunas de ellas (Velázquez et al., 2016); sin embargo, las respuestas de cada una de éstas en los medios nutritivos es variable (Flores-Hernández et al., 2017); también, tiene que ver el estado de madurez fisiológica y el estado indehiscente de las cápsulas (Pérez-Martinez & Castañeda-Garzón, 2016). Los embriones se transforman en estructuras diferenciadas denominadas protocormo (Pujasatria et al., 2020), donde sucede la morfogénesis que a veces demora años (Mendoza, 2016); Eulophia flava (Lindl.) Hook.f. ...
The application of techniques is important for the massive and efficient propagation of orchids, without affecting the natural environment and its conservation.The objective was to evaluate the effects of six culture media: t1, Knudson; t2, MS 75% + NAA (1,0 mgL-1) + GA3 (3,0 mgL-1); t3, MS 75% + 100 mLL-1coconut water; t4, MS 100% + GA3 (3,0 mgL-1); t5, MS 100% + NAA (1,0 mgL-1) + GA3 (5,0 mgL-1); t6, MS 100% + 100 mLL-1coconut water on in vitro seed germination and protocorms formation of the orchid E. catillus. Seeds were obtained from a capsule in stage of physiological maturity from sector Grapanazú, Huancabamba, province of Oxapampa-Pasco, Peru. A randomized complete block design was used. It was found that, at 64 and 71 days, the treatments t1, t3, t4 and t6 had the same seed germination and development of in vitro protocorms, however, all of them were higher than t2 y t5. We concluded that Knudson media, MS 75% and 100% + 100 mLL-1coconut water and MS 100% + GA3 (3,0 mgL-1), enhanced seed germination.
... A high proportion of orchid species are projected to have short-lived seeds (Hay et al. 2010;Merritt et al. 2014), and as such could be dried and stored in liquid nitrogen directly without in vitro methods (Popova et al. 2016). However, orchid seeds are unusual in that they are routinely germinated in vitro, whether asymbiotically or symbiotically (Koene et al. 2020;Pujasatria et al. 2020), and many germinate readily, although some species require modifications in the media or conditions (Zale et al. 2022). The family Orchidaceae consists of over 27,000 species, mostly from the tropics and subtropics, but fewer than 2,000 have been assessed for their conservation status. ...
The conservation of threatened exceptional plants, which cannot be conserved by seed banking, requires in vitro technologies for many of the approaches needed for their long-term ex situ conservation. This study evaluated the current in vitro plant literature, as represented in Web of Science, to determine its taxonomic overlap with the families and genera of the 775 species currently listed as exceptional. Web of Science was searched using the terms micropropagation, somatic embryogenesis, zygotic embryo, and cryopreservation, and the target genera and families were identified in the more than 19,000 articles evaluated. There were five families with significant overlap between the in vitro literature and exceptional species: Fabaceae, Asteraceae, Orchidaceae, Arecaceae, and Rutaceae. However, there was less overlap at the level of genus, with Citrus, Coffea, and Quercus having the most articles . Significant gaps were also found, with 14 exceptional families and half of the exceptional genera having no representation in the Web of Science search results. The 20 exceptional species with the most articles were all economically important species, and these had 343 threatened congeners that could be prioritized for research. A highly important group of exceptional plants that was significantly under-represented in the literature was tropical woody species, which form the backbone of the diversity of the world’s threatened rainforests. Overall, there are areas of strength upon which to build future work, but significant gaps where research should be prioritized for effectively conserving exceptional plants.
... Esta técnica ha permitido la conservación La conservación in vitro por mínimo crecimiento implica estrategias específicas centradas en la reducción de nutrientes en el medio de cultivo y la aplicación de inhibidores del crecimiento (Chauhan et al., 2019). Entre estos inhibidores se incluyen sustancias como el ácido abscísico (ABA), ancimidol (ANC), paclobutrazol (PBZ), polietilenoglicol (PEG) y otros osmorreguladores como sorbitol y manitol, modificando (Mancilla-Álvarez et al., 2019;Pujasatria et al., 2020). ...
La relevancia ornamental de las especies de orquídeas radica en sus características morfológicas. Notylia barkeri Lindl. está catalogada en el Apéndice II de la Convención sobre el Comercio Internacional de Especies Amenazadas de Fauna y Flora Silvestres. Por tanto, resulta imperativo instituir métodos de preservación para salvaguardar su viabilidad a largo plazo. El objetivo fue establecer un protocolo de conservación in vitro de N. barkeri. Se evaluaron diferentes concentraciones de ácido abscísico (0,1 y 2 mg L-1) y ancimidol (0,1 y 2 mg L-1) en medio Murashige y Skoog. Después de 180 días se evaluó el porcentaje de supervivencia, número y longitud de brotes, número de hojas, número y longitud de raíces. El mayor porcentaje de supervivencia (85.71 %) se observó en tratamiento testigo. Mientras que en 1 mg L-1 de ácido abscísico se observó 57.14 %. Los menores porcentajes se observaron en ancimidol. Se observó que en 1 mg L-1 de ácido abscísico se reduce la longitud de brotes sin afectar su supervivencia a diferencia de cuando se utilizó ancimidol. Además, en 1 mg L-1 de ácido abscísico se redujo el número y longitud de raíces. Nuestros resultados pueden contribuir a la conservación de esta especie con interés ornamental.
... Based on the evaluations made on a total of 1770 species of orchids, it has been determined that approximately 46.5% of these species are classified under the categories of vulnerability, endangered, or critical endangered as reported by the International Union for Conservation of Nature (IUCN, 2021). This precarious state remains due to a variety of variables, including their difficult germination process and human intervention, such as overcollection caused by economic and horticultural needs (Pujasatria et al., 2020;Suresh et al., 2023). ...
... Orchids are extraordinarily important for biodiversity, conservation, and producing a vast array of therapeutic substances, nutritious foods, and ornamental plants (Hinsley et al., 2018). Orchid conservationists strive to manage market needs and biodiversity on a global scale, which would need largescale production (Pujasatria et al., 2020). Numerous species encounter the peril of extinction; however, orchids adopt two distinct evolutionary strategies, namely sympodial growth and monopodial development, which are regulated by a diverse array of endophytic fungus species. ...
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.
... Furthermore, the existence of compatible mycorrhizal fungi in Paphiopedilum seedlings enhances the adaptability of plants to the environment to increase plant survival and growth rate (Zhang et al., 2018). However, Paphiopedilum seedlings do not simply form a symbiotic relationship with any fungus, and germination can only happen when an appropriate fungus is present (Pujasatria et al., 2020), which includes Tulasnellaceae, Ceratobasidiaceae, Serendipitaceae, Mycena, Helicogloea, Fusarium, or other fungi (Xu and Guo, 1989;Kottke et al., 2010;Sebastián et al., 2014;Jiang et al., 2019). Understanding the interaction between Paphiopedilum and mycorrhizal fungi is a significant for generative reproduction, augmenting existing populations, and maintaining species diversity in orchids . ...
... Our results were similar to previous studies. For example, significantly upregulated expression of mannose-specific lectin genes was found in several studies of orchid-fungi symbiotic relationships, such as between a Cymbidium hybrid and six different fungal strains (Reinhold-Hurek et al., 2015), Platanthera sikkimensis, T. calospora and Epidendrum secundum (Pujasatria et al., 2020). In addition, a mannose-specific lectin was reported to be involved in the establishment of the interaction with microorganisms during Gastrodia elata seed germination (Zeng et al., 2017). ...
... After the mycorrhizal fungi colonize the orchid seeds, the mycelium passes through the seed coat and produces amylase to hydrolyze starch in the embryo, which promotes the growth of the embryo protoplasm and absorbs nutrients Frontiers in Microbiology 11 frontiersin.org from the surrounding environment to accelerate the further germination and growth of seeds (Pujasatria et al., 2020). In this study, transcriptomics showed that two pathways (starch and sucrose metabolism, and glycolysis/gluconeogenesis) related to energy metabolism were significantly enriched, involving a total of 72 DEGs, all of which were upregulated in the CK in comparison with the FQXY019 treatment groups (Supplementary Tables S13, S14). ...
Introduction
Paphiopedilum barbigerum is currently the rarest and most endangered species of orchids in China and has significant ornamental value. The mature seeds of P. barbigerum are difficult to germinate owing to the absence of an endosperm and are highly dependent on mycorrhizal fungi for germination and subsequent development. However, little is known about the regulation mechanisms of symbiosis and symbiotic germination of P. barbigerum seeds.
Methods
Herein, transcriptomics and proteomics were used to explore the changes in the P. barbigerum seeds after inoculation with (FQXY019 treatment group) or without (control group) Epulorhiza sp. FQXY019 at 90 days after germination.
Results
Transcriptome sequencing revealed that a total of 10,961 differentially expressed genes (DEGs; 2,599 upregulated and 8,402 downregulated) were identified in the control and FQXY019 treatment groups. These DEGs were mainly involved in carbohydrate, fatty acid, and amino acid metabolism. Furthermore, the expression levels of candidate DEGs related to nodulin, Ca²⁺ signaling, and plant lectins were significantly affected in P. barbigerum in the FQXY019 treatment groups. Subsequently, tandem mass tag-based quantitative proteomics was performed to recognize the differentially expressed proteins (DEPs), and a total of 537 DEPs (220 upregulated and 317 downregulated) were identified that were enriched in processes including photosynthesis, photosynthesis-antenna proteins, and fatty acid biosynthesis and metabolism.
Discussion
This study provides novel insight on the mechanisms underlying the in vitro seed germination and protocorm development of P. barbigerum by using a compatible fungal symbiont and will benefit the reintroduction and mycorrhizal symbiotic germination of endangered orchids.
... Orchids are able to grow in diverse climates and ecosystems, spanning from sea level to temperate and tropical mountains (Oktalira et al., 2019;Steinfort et al., 2010). Orchids have both terrestrial and epiphytic growth habits and produce a large number of tiny seeds (Pujasatria et al., 2020). Orchids hold a prominent place in the list of endangered plant species in certain countries like Türkiye (León-Yánez et al., 2011;Qin et al., 2017). ...
(1) Background: Anacamptis coriophora (Orchidaceae) is a highly endangered orchid in Turkey due to its excessive collection and the continuing deterioration of its habitat. In this study, the cultivation conditions of A. coriophora were determined. (2) Methods: a sterile soil mixture was filled into jars, and the fungal isolate (previously isolated from A. coriophora roots), Ceratobasidiaceae MG762693, was inoculated in separate glass jars, producing fungal compost when hyphae were developed. This fungal compost was then filled into pots where A. coriophora seed packs (0.001 g) were placed and subsequently moistened with sterile liquid nutrient medium. After 45 days of germination, fifty seedlings of approximately equal size were transferred directly to a natural environment, and after 6 months of development, the measuring of the tubers was done. The phenological process was then monitored until flowering. (3) Results: After 45 days, germination and developmental stage rates were determined from the seed packs in the pots inoculated with the Ceratobasidiaceae MG762693 fungal isolate, and 64.3% germination and 11.75% leaf-rooted seedlings (stage 4) occurred. Plants flowered in June the following year, and the seeds ripened in July. The largest tuber in adult individuals was about 3 times the weight of first-year tubers. Each individual formed 2 or 3 tubers, thus increasing the number of tubers approximately 2.5 times in 2 years. (4) Conclusions: In this study, ex vitro symbiotic seedlings were planted in the natural environment, and a small population was formed over a 2-year period. The results revealed that orchids can be grown on a large scale with this method, both economically and for conservation and reintroduction.
... To date, in vitro shoot propagation or asymbiotic seed germination is considered a reliable method for the conservation of orchids (Hoang et al. 2016;Pujasatria et al. 2020;Santos-Díaz et al. 2022). Especially the number of explants that are available for in vitro conservation projects can be seasonal or very limited in quantity (Sarasan 2010). ...
... Mycorrhizal dependency is the largest hurdle to overcome in orchid propagation as the mycorrhizae provide most of the minerals, nutrients, vitamins, and water needed for germination and seedling development (Herrera et al., 2019) and vary in the specificity of their relationship with their orchid hosts (McCormick et al., 2018;Li et al., 2021). In vitro symbiotic germination has been used for orchid propagation (Pujasatria et al., 2020), however, successful propagation requires extensive knowledge of the specific orchid-mycorrhizal interaction, as well as the ability to isolate and grow the fungus, which can take considerable time and effort. Mycorrhizae isolated from the roots of mature plants may not be suitable for inducing seed germination (McCormick et al., 2021;Zhao et al., 2021), and many orchid mycorrhizal fungi are unculturable axenically, making symbiotic germination impossible (Li et al., 2021). ...
Lewis Knudson first successfully germinated orchid seeds asymbiotically on artificial medium in 1922. While many orchid species have since been grown asymbiotically, the tremendous variation in how species respond to artificial medium and growth conditions ex situ has also become apparent in the past century. In this study, we reviewed published journal articles on asymbiotic orchid seed germination to provide a summary of techniques used and to evaluate if these differ between terrestrial and epiphytic species, to identify areas where additional research is needed, and to evaluate whether asymbiotic germination could be used more often in ex situ conservation. We found articles reporting successful asymbiotic germination of 270 species and 20 cultivars across Orchidaceae. Researchers often used different techniques with epiphytic versus terrestrial species, but species-specific responses to growth media and conditions were common, indicating that individualized protocols will be necessary for most species. The widespread success in generating seedlings on artificial media suggests that asymbiotic techniques should be another tool for the conservation of rare orchid species. Further advances are needed in understanding how to introduce mycorrhizae to axenically grown orchids and to maximize the viability of seedlings reintroduced into natural habitats to fully utilize these methods for conservation.
... However, many orchid species are listed as endangered owing to habitat degradation and dependence on other organisms, i.e., pollinators (Suetsugu et al. 2015;Freitas et al. 2020). Moreover, establishing orchids in the natural habitat is always complicated, even in suitable conditions, since orchids' seeds are nearly impossible to germinate without external nutritional support (Pujasatria et al. 2020). For seeds to successfully germinate and grow into mature plants, they must establish orchid mycorrhizal (OM) symbiosis (Arditti and Ghani 2000;Barthlott et al. 2014;Yeh et al. 2019;Pujasatria et al. 2020). ...
... Moreover, establishing orchids in the natural habitat is always complicated, even in suitable conditions, since orchids' seeds are nearly impossible to germinate without external nutritional support (Pujasatria et al. 2020). For seeds to successfully germinate and grow into mature plants, they must establish orchid mycorrhizal (OM) symbiosis (Arditti and Ghani 2000;Barthlott et al. 2014;Yeh et al. 2019;Pujasatria et al. 2020). Colonization by orchid mycorrhizal fungi (OMF) starts at seed germination and may persist into adulthood. ...
... Most orchids associate with Rhizoctonia-like fungi, a broad term for several genera resembling the anamorphic morphology of Rhizoctonia. As symbionts, there are three teleomorphic genera in this group: Ceratobasidium, Serendipita (often associated with Sebacina sensu lato), and Tulasnella (Rasmussen et al. 2015;Yeh et al. 2019;Pujasatria et al. 2020). Originally, those fungi are saprophytic, endophytic, or even pathogenic. ...
Epiphytic orchids are commonly found in exposed environments, which plausibly lead to different root fungal community structures from terrestrial orchids. Until recently, few studies have been conducted to show the fungal community structure during the growth of a photosynthetic and epiphytic orchid in its natural growing site. In this study, the Vanda falcata (commonly known as Neofinetia falcata), one of Japan’s ornamental orchids, was used to characterize the fungal community structure at different developmental stages. Amplicon sequencing analysis showed that all development stages contain a similar fungal community: Ascomycota dominate half of the community while one-third of the community belongs to Basidiomycota. Rhizoctonia-like fungi, a polyphyletic basidiomycetous fungal group forming mycorrhizas in many orchids, exist even in a smaller portion (around one-quarter) compared to other Basidiomycota members. While ascomycetous fungi exhibit pathogenicity, two Ceratobasidium strains isolated from young and adult plants could initiate seed germination in vitro. It was also found that the colonization of mycorrhizal fungi was concentrated in a part of the root where it directly attaches to the phorophyte bark, while ascomycetous fungi were distributed in the velamen but never colonized cortical cells. Additionally, the root parts attached to the bark have denser exodermal passage cells, and these cells were only colonized by mycorrhizal fungi that further penetrated into the cortical area. Therefore, we confirmed a process that physical regulation of fungal entry to partition the ascomycetes and mycorrhizal fungi results in the balanced mycorrhizal symbiosis in this orchid.
... PTC, defined as the aseptic culture of cells, organs, and their components under controlled in vitro conditions [44], is a biotechnological tool that has been successfully applied to recovery, conservation and clonal propagation of orchids [45][46][47][48]. Since the development of a method for asymbiotic germination by Lewis Knudson in 1921 [49], PTC has become highly diversified in orchid species. ...
... Asymbiotic germination under in vitro culture conditions has been used for eight Laelia species thus far, showing this to be an effective alternative to increased germination and survival rates while maintaining genetic diversity. Furthermore, asymbiotic germination is a powerful technique for establishing aseptic cultures for other in vitro downstream purposes [18,48,66,67]. ...
... High multiplication rate of somatic embryos after 8 weeks of subculturing. PTC, defined as the aseptic culture of cells, organs, and their components under controlled in vitro conditions [44], is a biotechnological tool that has been successfully applied to recovery, conservation and clonal propagation of orchids [45][46][47][48]. Since the development of a method for asymbiotic germination by Lewis Knudson in 1921 [49], PTC has become highly diversified in orchid species. ...
Orchids (Orchidaceae) are plants that are highly appreciated by their beautiful flowers worldwide. Moreover, they represent a source of metabolites with applications in medicine and biotechnology. Within the Orchidaceae family, the Laelia genus is a group of orchid species from the Neotropics and is probably one of the most representative genera of America. Laelia orchids are cultivated by their splendid flowers and are widely used in orchid breeding. Here, we revise the use of the Laelia genus in orchid breeding and metabolite bioprospecting. We also analyze the use of plant tissue culture (PTC) as an alternative to conventional propagation and as a strategy for the recovery of those Laelia species threatened with extinction. We summarize and discuss the recent advances in the application of different PTC techniques for mass multiplication based on asymbiotic germination, organogenesis, protocorm-like bodies development, and somatic embryogenesis, and the advances of in vitro conservation by cryoconservation and the use of slow-growth promoting hormones. Finally, we suggest future directions and venues in research for Laelia species.
