Search Results (24)

An efficient in vitro propagation protocol for Eulophia bicallosa was developed using asymbiotic seed germination and protocorm proliferation. The effect of light on seed germination and development was evaluated on Vacin and Went (VW) medium under five conditions: darkness, white, green, red, and blue light for 24 weeks. Blue and red light significantly accelerated seed development, allowing progression to stage 5 within 24 weeks. For protocorm proliferation, six semi-solid culture media were tested. Half-strength Murashige and Skoog (½MS) medium yielded the best results after 8 weeks, producing the highest numbers of shoots (1.0), leaves (1.1), and roots (4.2) per protocorm, with 100% survival. The effects of organic additives were also evaluated using coconut water and potato extract. A combination of 200 mL L⁻¹ coconut water and 50 g L⁻¹ potato extract enhanced shoot formation (1.7 shoots), while 150 mL L⁻¹ coconut water with 50 g L⁻¹ potato extract increased both leaf (1.9) and root (8.8) numbers. The effects of cytokinins (benzyladenine (BA), kinetin (6-furfurylaminopurine), and thidiazuron (TDZ)) and auxins (indole-3-acetic acid (IAA), α-naphthalene acetic acid (NAA), indole-3-butyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D)) were investigated using ½MS medium supplemented with each plant growth regulator individually at concentrations of 0, 0.1, 0.5, 1.0, and 2.0 mg L⁻¹. Among the cytokinins, 0.1 mg L⁻¹ BA produced the highest survival rate (96%), while 1.0 mg L⁻¹ BA induced the greatest shoot formation (93%, 2.3 shoots). Among the auxins, 0.1 mg L⁻¹ IAA resulted in the highest survival (96%), and 1.0 mg L⁻¹ IAA significantly enhanced root induction (4.2 roots per protocorm). Acclimatization in pots containing a 1:1:1 (v/v) mixture of pumice, sand, and soil resulted in 100% survival. This protocol provides a reliable and effective approach for the mass propagation and ex situ conservation of E. bicallosa. Full article

In nature, orchid seed germination is extremely low, making in vitro asymbiotic seed germination essential for the propagation and conservation of endangered Vanda coerulea. This study optimized a micropropagation protocol and evaluated the genetic homogeneity of regenerated orchids. The synergistic effect of kinetin (KN) with auxins in the Mitra (M) medium best supported protocorm formation and seedling development. The highest shoot multiplication (5.62 ± 0.09) was achieved with 1.2 mg L⁻¹ KN and 0.6 mg L⁻¹ IBA (indole-3-butyric acid) in the medium. Enhanced leaf production (4.81 ± 0.37) was observed when 3.2 mg L⁻¹ KN was combined with 1.8 mg L⁻¹ IAA (indole-3-acetic acid), while root development was superior when 3.2 mg L⁻¹ KN together with 2.4 mg L⁻¹ IAA was incorporated in the medium. Anatomical sections confirmed well-developed leaf and root structures. Genetic fidelity assessment using random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), inter-primer binding site (iPBS), and start codon targeted (SCoT) markers revealed 97.17% monomorphism (240/247 bands) and low Nei’s genetic distances (0.000–0.039), indicating high similarity among the regenerants. Dendrogram clustering was supported by a high cophenetic correlation coefficient (CCC = 0.806) and strong resolution in Principal Coordinate Analysis (PCoA) (44.03% and 67.36% variation on the first two axes). The Mantel test revealed a significant correlation between both ISSR and SCoT markers with the pooled marker data. Flow cytometry confirmed the genome stability among the in vitro-propagated orchids, with consistently low CV (FL2-A) values (4.37–4.94%). This study demonstrated the establishment of a reliable in vitro protocol for rapidly propagating genetically identical V. coerulea via asymbiotic seed germination. Full article

This study aimed to determine the dynamics of dormancy using triphenyl tetrazolium chloride (TTC) and asymbiotic germination in Laelia speciosa (Kunth) Schltr. seeds stored for three years and one year at different temperatures. This is the first report of a circannual rhythm in L. speciosa seeds under controlled storage conditions. Two experiments were carried out: (a) with seeds from wild populations of L. speciosa collected at two different times and dehydrated to 4% relative humidity (RH) and stored for three years at 25 °C, 6 °C, −20 °C, and −80 °C, and (b) with seeds from six fruits stored in liquid nitrogen (LN2; −196 °C) at 25 °C for 12 months. The germination conditions were 25 ± 1 °C with 16 h of light (23 μmol m⁻² s⁻¹) and 8 h of darkness for both trials. Because they have a rudimentary embryo, orchids are attributed a morphological latency; however, the staining of the embryo with TTC (>92%) in all the evaluations carried out throughout a year and the decrease in asymbiotic germination in the intermediate evaluations record a circannual biological cycle or clock, under temperature and humidity control (4% RH). Full article

Orchids are crucial for the horticulture industry. Mycorrhizal fungi benefit crops by improving nutrition, plant growth, and disease resistance. However, the mycorrhizal association of horticultural hybrid orchids is poorly understood. To address this, we investigated mycorrhizal colonization in the entire root system and assessed the mycorrhizal community using a Dendrobium cultivar, D. Stardust ‘Firebird’, obtained from three nurseries. Additionally, we isolated and tested mycorrhizal fungi in symbiotic culture to assess their role in the seed germination and growth of Dendrobium species. All plants were colonized by mycorrhizal fungi, with a higher colonization rate in mature than in juvenile plants. Molecular identification of mycorrhizal fungi by Sanger and high-throughput sequencing revealed that the cultivar was associated with a phylogenetically diverse group of fungi, including mycorrhizal fungi from Tulasnellaceae, and several wood-decaying fungi. The Tulasnellaceae isolates significantly enhanced the seed germination of three Dendrobium species and increased the survival rate and growth of asymbiotic seedlings of D. moniliforme. This study is the first comprehensive examination of mycorrhizal associations in horticultural orchid hybrids, providing valuable insights for commercial production. Full article

Coriolopsis spp. are wood-decaying fungi that inhabit forests. They are mainly distributed in tropical and subtropical areas. Strain Epi910 was isolated from the asymbiotically germinated protocorm of Epidendrum sp. and identified as Coriolopsis strumosa. Symbiotic germination and high-throughput sequencing of the endophytic fungal communities of different parts were performed to characterize the function and spatial distribution of the Epi910 isolate. Under symbiotic germination, Epi910 promoted seed germination and seedling formation as an endophytic native fungus of Epidendrum sp. Endophytic fungal communities from seven different parts of Epidendrum sp. were characterized. In total, 645 OTUs were identified; 30 OTUs were shared among all seven parts. The internal transcribed spacer sequence of Epi910 was identical to that of a dominant shared OTU (OTU6). The relative abundance of OTU6 in the seven parts was identified as follows: capsule pericarp > seed > root > asymbiotically germinated protocorm > epiphytic root > ovary > rachis. Our results suggest that the isolate belonging to Coriolopsis strumosa could promote the germination of Epidendrum sp. There may, therefore, be endophytic fungi other than common orchid mycorrhizal fungi with the ability to enhance germination in orchids. Full article

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. Full article

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. Full article

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. Full article

Paraphalaenopsis labukensis Shim, A. Lamb & C.L. Chan (P. labukensis) is an endangered monopodial epiphytic orchid threatened due to habitat fragmentation and overharvesting. Consequently, this research aimed to achieve in vitro propagation of P. labukensis through asymbiotic seed germination and seedling development. This study focused on identification and optimizing capsule maturity, basal media and nutrient requirements. In addition, after hand pollinating the plant, their capsules’ flowers, length, girth, and colour variations were recorded weekly to ascertain morphological maturity characters of this species. Murashige and Skoog (MS), Knudson C (KC), and Vaccin and Went (VW) basal media were used to determine seed vegetation at 60, 90, and 120 days. Subsequently, for the proliferation and development of protocorms, the best basal media were those supplemented with different organic additives (coconut water (CW) and banana homogenate (BH)) and plant growth regulators (PGRs). As a result, 120 days after pollination (DAP) was chosen as the best capsule age for germination since the capsules had reached maturity. The highest germination rate was attained after 90 days of culture in Knudson C (KC) medium, with 98.78 ± 0.89%, followed by Murashige and Skoog (MS) medium, with 92.80 ± 3.26%. The KC supplemented with 0.5 mg/L NAA media documented a maximum percentage of 17.25 ± 0.96%. The 5 g/L (w/v) BH recorded a higher protocorm proliferation percentage than CW. Additionally, after 150 days of culture (DOC), this medium combination produced the most leaves, averaging four or five. The result of this present study has successfully established an effective in vitro propagation protocol for this species. Full article

A greater understanding of the relationship between native orchids and their mycorrhizal symbionts is needed to ensure more effective orchid conservation strategies. A protocol for symbiotic seed germination and seedling development was developed for E. geminiflorum. Mature seeds were collected from a naturally occurring orchid population in Ecuador. Putative mycorrhizal fungi isolated from other native orchid species were used to screen their ability to facilitate germination and seedling development in vitro in either a 0/24 h or 12/12 h light/dark photoperiod at 20 °C. The mycorrhizal fungus Tulasnella calospora (UAMH 9824) isolated from Spiranthes brevilabris in Florida, USA, was also included in this study. Sterilization treatments using 0.3%, 0.5% sodium hypochlorite/ethanol or 2% calcium hypochlorite were tested for their effectiveness as sterilant and their subsequent effects on seed germination percentage. Effective surface seed sterilization was achieved with either 0.5% NaClO/ethanol or 2% calcium hypochlorite. However, significantly higher percentages of germinated embryos developed into protocorms when NaOCl solutions were used compared to the other treatments. Seed germination occurred in both photoperiods tested; however, delayed germination was observed under complete darkness. Seeds of E. geminiflorum germinated without fungal inoculation; however, co-culture with Tulasnella strains improved germination significantly. Seedling development was only observed when seeds were cultured in asymbiotic medium or co-cultured with T. caloscopa (UAMH 9824). Significantly longer seedlings were obtained when T. calospora was present in the culture compared with seedlings cultured in asymbiotic medium. The establishment of mycorrhizal associations was confirmed by the presence of pelotons in the roots of E. geminiflorum seedlings. Full article

Cymbidium erythrostylum Rolfe is one of the most beautiful species of the genus cymbidium which is used as a cut flower or indoor plant. However, it was registered as a rare species even in its original habitat. This study was carried out to develop a protocol for in vitro propagation of C. erythrostilum. We used protosomes obtained by the asymbiotic germination of seed on 1/2 of Murashige and Skoog nutrient medium (MS) supplemented with 1 mg/L 2-Isopentenyl adenine (2iP) as objects of study. During the multiplication stage, the number of formed protosomes on a culture medium containing 0.8 mg/L 6-Benzylaminopurine (6-BAP) was higher (3.8 ± 0.2 protosomes). During the subsequent cultivation on ½ MS medium supplemented with 2 mg/L 6-BAP and 0.5 mg/L a-naphthaleneacetic acid (NAA), the highest numbers of shoots (4.00 ± 0.19 shoots/plantlet) and leaves (4.50 ± 0.14 leaves/plantlet) were obtained. At the rooting stage under in vitro conditions, the most effective was the use of ½ MS nutrient medium with the addition of 0.5 mg/L of indolyl-3-butyric acid IBA, 1 g/l charcoal, and 50 g/l banana puree. The obtained plants were successfully adapted to a substrate consisting of bark, perlite, and peat in a ratio of 1:1:1. Full article

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. Full article

Mycorrhizal specificity, i.e., the range of fungi allowing mycorrhizal partnerships, differs among orchid species, but that at early developmental stages is unclear. We investigated whether mycorrhizal specificity during seed germination and seedling development differs among three Dendrobium species, D. officinale, D. okinawense and D. moniliforme, in vitro. Nine mycorrhizal fungal strains were obtained from the roots of these species and cultured with a seed of each Dendrobium species. Five to eight fungal strains stimulated seed germination, whereas one to four fungal isolates significantly promoted protocorm development in the three species. To evaluate effects on leafy seedling growth, seedlings obtained from asymbiotic culture were cultured with nine fungal isolates. D. officinale and D. okinawense showed specificity for a single Serendipitaceae or Tulasnellaceae isolate, whereas D. moniliforme exhibited specificity for three isolates of Serendipitaceae and Tulasnellaceae. Therefore, the three Dendrobium species had a growth bottleneck from seed germination to the protocorm stage, and mycorrhizal specificity of protocorm growth and seedling development in vitro varied among the species. Our findings imply divergent mycorrhizal specificity in Dendrobium species at early developmental stages. This study provides insights into the diversity of orchid mycorrhizal specificity, as well as valuable information for conservation of endangered orchids. Full article

Cremastra appendiculata var. variabilis (Blume) I.D. Lund), also known as single-leaf cremastra (SLC), is a rare and threatened species native to Korea, and it has the potential to be grown as a beautiful flowering pot or garden plant. There is still no reliable strategy to multiply SLC. Thus, an effective method for propagating single-leaf cremastra was needed for its conservation and mass production. In the present study, we examined the effects of culture media, seed age, activated charcoal, and plant growth regulators on in vitro asymbiotic seed germination, secondary protocorm induction, and seedling formation. Asymbiotic seed germination of SLC was influenced by culture media, seed age, and their interaction. The addition of activated charcoal (500 mg/L) to the Murashige and Skoog (MS) medium increased the rate of germination. The seeds were best germinated (91.9%) by culturing on MS medium supplemented with activated charcoal (500 mg/L), α-naphthaleneacetic acid (3 µM), and kinetin (1 µM). The highest number (28.9) of secondary protocorms were produced when protocorms were cultured on MS medium containing 6-benzyladenine (4 µM) and kinetin (2 µM). When the protocorms were cultivated in a medium containing gibberellic acid (1 µM), they were able to transform into SLC with the highest success rate (78.7%). The propagation protocol described here may be helpful for SLC restoration programs and large-scale production. Full article

In the valuable orchid genus Cypripedium, the section Irapeana consists of a distinctive group of Mesoamerican species that is formed by Cypripedium dickinsonianum Hágsater, C. irapeanum Lex., and C. molle Lindl. All lady slipper orchids exhibit different distributions and abundances. Data analysis that used herbarium accessions and field investigations indicated that the habitats of these three species have been dramatically reduced. Prospecting for suitable habitats based on climatic, vegetation, and soil parameters allows us to predict potential distributions. Conservation strategies, such as ex situ propagation by asymbiotic and symbiotic approaches, have indicated that the culture media used are a determining factor for seedling development. Mycorrhizal isolates play a main role in the compatibility and further development of germinated seeds. The fungi isolated from adult plants belong to two different families, which makes it possible that widely distributed C. irapeanum populations will be fungal-specific as well as restricted for C. molle. Root mycorrhization patterns occur high on the secondary roots. In contrast with other species of the genus, in situ germination can occur over a short period of two months, but we have documented periods as long as ten years. Cypripedium is a highly problematic genus for ex situ conservation because the germination requirements and cultures are poorly documented, and there is great urgency for in situ conservation to develop strategies for identifying hotspot habitats and actualize the protection status to avoid extinction of this genus. Full article