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• Paphiopedilum spp. is known as lady slipper orchid. • Embryogenesis in Paphiopedilum spp. can be induced with plant growth regulator. • Development of somatic embryos in Paphiopedilum spp. is the same as zygotic embryos. • The homologous embryo gene RKD4 is present and functional in Paphiopedilum spp. Paphiopedilum spp. is one of the terrestrial orchids that is interesting as an ornamental plant. This orchid flower has a distinctive characteristic, namely its labellum resembles a sac, so it is sought after by ornamental flower collectors because of its uniqueness. This orchid is included in the Appendix I group according to CITES, namely a critical species that is threatened with extinction, so it needs to be conserved both in situ and ex situ. Ex situ conservation can be done by propagating orchids using in vitro culture techniques. The objective of this research is to carry out mass propagation of 3 species of Paphiopedilum spp orchids: P. supardii, P. primulinum, P. glaucophyllum through somatic embryogenesis and to characterize the existence of embryo gene RKD4 homolog in Paphiopedilum orchids. The method is to culture the nodes slices in dark conditions for 2 weeks, and then changed to photoperiod mode 16:8 hours (dark:light) on ½ MS media plus a combination of 2,4-D or NAA with TDZ. The somatic embryo were analyzed anatomically. This method is an improvement over previously reported methods for Paphiopedilum spp. due to its efficiency and reproducibility for conservation. We concluded that ½ MS media with a combination of NAA or 2,4D with TDZ were able to induce somatic embryogenesis. The result show that 3 mg L−1 NAA was the best PGRs for PLBs induction in P. supardii (50 %), 1 mg L−1 2,4-D + 0.5 mg L−1 TDZ was the best PGRs for callus embryogenic induction in P. primulinum (20 %), and 1 mg L−1 NAA was the best PGRs for PLBs induction in P. glaucophyllum (33 %). [ABSTRACT FROM AUTHOR]

Orchids produce millions of dust-like and reserveless tiny seeds per capsule and are unable to germinate on their own in nature. Orchid seeds have developed strategies to exploit seed germination-promoting orchid mycorrhizal fungi (sgOMFs) for acquisition of exogenous nutrients to fuel germination. Once colonized by sgOMFs, orchid seeds can digest intracellular sgOMF hyphae to acquire fungal nutrients, especially those for carbon nutrition. However, the underlying carbon delivery pathway from sgOMFs to orchid cells remains elusive. Clarification of the identities of fungally derived carbohydrates and the main molecular players responsible for the sgOMF-to-orchid carbon flow is of great significance for better understanding of symbiotic germination. To germinate in nature, orchid seeds strictly rely on seed germination-promoting orchid mycorrhizal fungi (sgOMFs) for provision of carbon nutrients. The underlying delivery pathway, however, remains elusive. We develop here a plausible model for sugar transport from sgOMFs to orchid embryonic cells to fuel germination. Orchids exploit sgOMFs to induce the formation of pelotons, elaborate intracellular hyphal coils in orchid embryos. The colonized orchid cells then obtain carbon nutrients by uptake from living hyphae and peloton lysis, primarily as glucose derived from fungal trehalose hydrolyzed by orchid-specific trehalases. The uptake of massive fungally derived glucose is likely to be mediated by two classes of membrane proteins, namely, sugars will eventually be exported transporters (SWEETs) and H+-hexose symporters. The proposed model serves as a launch pad for further research to better understand and improve orchid seed germination and conservation. [ABSTRACT FROM AUTHOR]

The beneficial effects of orchids as a medicinal plant have been highlighted in a number of widely used medical practices. The current study has deciphered the gastro-protective effects of Acampe ochracea methanol extract (AOCE), Aerides odorata methanol extract (AOE), Cymbidium aloifolium methanol extract (CAE), and Papilionanthe teres methanol extract (PTE) in ethanol-induced ulcer. Long-Evan rats fasted; different treatment groups were administered the samples as mentioned earlier (250 and 500 mg/kg) orally; distilled water and esomeprazole (20 mg/kg p.o., b.w.) were used as normal control and standard drugs; thereafter, they received 0.5 mL of an 80 % (v/v) ethanol solution an hour later. By suppressing ulceration of the mucosal layer and altering the biochemical features of gastric juice, including the levels of carbohydrate, protein, and pepsin, the findings showed that therapy with treatments (500 mg/kg) remarkably (p < 0.001) modulated the ulcer and protected the integrity of gastric mucosa. Furthermore, treatments notably (p < 0.001, and p < 0.01) elevated the levels of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH) and markedly (p < 0.001, and p < 0.01) decreased malondialdehyde (MDA) levels. Moreover, protecting the normal architecture of the stomach section from EtOH toxicity, the treatments unveiled notable protection from disruption of the membrane of epithelial cells and limited edema in the submucosal layer. Hence, the outcome of the investigation deciphered that the following orchids: A. ochracea, A. odorata, C. aloifolium, and P. teres might be therapeutic candidates for managing peptic ulcer disorders (PUD). [Display omitted] • Four orchid varieties were investigated for antiulcer effects. • Orchid extracts prevented ethanol-induced alcoholic ulcer generation. • Antioxidant enzymes CAT, SOD and GSH upregulated in the experimental model. • Protein and pepsin contents reflected the ulceration in the gastric mucosal layer. • Limited edema in the submucosal layer showed improvement by histopathological assay. [ABSTRACT FROM AUTHOR]

• The orchids plant diversity is assessed in the western Himalaya of Pakistan. • Orchids plant species are linked to seven distinct vegetation types. • Relatively moist and moderately warm microhabitats are important. • The higher relative abundance of woody plant species support orchids. • Increasing deforestation will affect the orchids diversity and abundance. Orchid species require unique microhabitat conditions and are globally distributed from the tropics to alpine regions. These plant species are important both ecologically and economically but are facing multiple threats, especially habitat destruction, climate change, and overexploitation. Therefore, documenting species richness, diversity, distribution, and important driving factors is crucial for biodiversity conservation. The orchid species distribution patterns and order of importance of the main driving environmental factors in the western Himalayas of Azad Jammu and Kashmir remain unclear. The main aims of this study were to explore the richness and distribution of orchids and neighboring vascular flora and to identify the principal driving environmental factors, as no study has yet targeted these plant species specifically in the study area. Field data collection surveys were conducted from August 2018 to July 2021 using the vegetation sampling method. The presence of ≥ two individuals belonging to any orchid species in a 20 × 20 m² land area criterion was used to select the study sites along the elevation gradient for data collection. Multivariate statistical tools, such as hierarchical classification and ordination, were used to analyze the data. A total of 32 orchid species belonging to 18 different Orchidaceae genera were recorded at the 57 study sites. Only one individual each of Herminium monorchis, Habenaria furcifera , and Malaxis muscifera was collected, depicting these orchids as extremely rare in the study area. A total of 324 vascular plant species (including orchids and their neighboring plant species in the studied plots) were classified into seven significantly (p < 0.05) different plant associations, each with a unique species composition. The results of canonical correspondence analysis showed that temperature variability was the most influential among the 28 environmental factors considered. Different microhabitats with an elevation range of ≥1500–3500 m a.s.l. in the central part of the study area are moister and richer in organic matter and support high orchid diversity. It was observed that a higher density of co-existing tree and shrub species and a higher geographic slope were supporting the growth and survival of orchid species as well. Conversely, higher deforestation activities and potassium levels in the soil were observed as negatively influencing factors. The influence of non-native plant species on orchid species distribution was not significant, indicating that the local orchid species were not remarkably affected when growing in microhabitats with optimal conditions. This study concluded that the central part of the study area is richer in orchid abundance and diversity and needs effective conservation and management planning. [ABSTRACT FROM AUTHOR]

The study involves devising an efficient method for in vitro protocorm-based plant production, as well as ex vitro establishment of Aerides multiflora and Rhynchostylis retusa. Both the orchids fall prey to the unscrupulous collection from the wild as they are widely used in the traditional medicinal system. Tissue culture technology can be effectively used for mass production of both the orchid species if the planned methodology is used for transplanting the regenerated plantlets into field conditions. MS medium supplemented with natural additives proved to be most optimal for protocorm development from seed explants (giving an average of 94 % to 97 % germination) and further multiplication while Mitra medium was best suited for complete plantlet regeneration from cultured protocorms. In A. multiflora an average of 3.33 shoots and 2.67 roots per protocorm, and in R. retusa mean number of 4.33 shoots and 3.10 roots per protocorm could be obtained on Mitra medium. Tissue culture raised plantlets of both the orchids were hardened in vitro and then shifted under polyhouse conditions. Peat moss, coconut husk, bark shavings, charcoal, and brick pieces, in varying combinations, were tried for optimal growth. Based on the best plantlet survival rate, 90 % in A. multiflora and 100 % in R. retusa , the growth medium combination of peat moss with charcoal, and bark shavings was selected to be the most optimal for orchid growth when kept at a temperature of 25–30 °C, relative humidity 60–70 %, and periodic watering only to avoid drying. The standardized methodology can be utilized for the field plantation of these two important orchids as a means of conservation and propagation strategy. [Display omitted] [ABSTRACT FROM AUTHOR]

• Xylocopid bees were the only pollinators of four commercially important Cyclopia species (C. maculata, C. subternata, C. intermedia and C. genistoides). • Honey bees were ineffective pollinators of Cyclopia owing to their inability to trip Cyclopia flowers and gain access to the floral reproductive parts. • Nectar measurements revealed low volume of nectar production with varied sugar concentration across species of Cyclopia. • The value of native non- Apis insects for crop pollination in a changing world is highlighted in Cyclopia. Pollination is an important ecosystem service. Animal-mediated pollination (mostly insects) increases the production of 35% of global crops. Honey bees (Apis mellifera) are considered important crop pollinators globally, but are under pressure and therefore the value of wild pollinators for crop production is receiving more attention. The importance of pollinators (including non- Apis) has been extensively studied in the agricultural sector of South Africa, however little research is available on the pollination of indigenous crops. With the considerable value of the indigenous honeybush (Cyclopia Vent.) in the tea industry, it is important to determine the pollinators since Cyclopia is widespread across the fynbos biome and the pollinators are largely unknown. Here we ask whether carpenter bees (xylocopid bees) are the only pollinators of commercially important Cyclopia species, or if honey bees contribute to pollination. Floral observations and camera trapping confirmed that six species of xylocopid bees were the only pollinators of four commercially important Cyclopia species. Honey bees were observed to be ineffective pollinators of Cyclopia owing to their inability to trip Cyclopia flowers and gain access to the floral reproductive parts. Similarly, an additional seven species including Diptera, Apidae and Lema sp. were unable to gain access to Cyclopia flowers, although were observed visiting. Nectar measurements revealed the highest nectar volume in C. genistoides, while C. intermedia had the highest nectar sugar concentration (above 35% for all species). The value of native non- Apis insects for crop pollination in a changing world is highlighted in Cyclopia, an indigenous legume gaining traction in the global tea market and therefore in cultivation. [ABSTRACT FROM AUTHOR]

• Orchids constitute one of the largest families of flowering plants. • The exotic beauty, aromatic, and long-lasting qualities of orchids make them highly prized flowers. • This paper showed vanda stangeana and Phalaenopsis hygrochila were compatible for hybridization. • Our findings shows MS medium at half-strength without any plant growth regulators gave best response in seed germination of V. stangeana x P. hygrochila. • Molecular markers like SSRs and SCoT could be used for quick and easy confirmation of orchid hybrids. Vanda stangeana Rchb. f. is an endemic species of Manipur, India. Its flowers are greenish-yellow, 5 cm in size with a pink mid-lobe, tessellated in chestnut brown color and tough texture with a long shelf life. Phalaenopsis hygrochila J.M.H. Shaw is a large-sized, hot to cool growing, epiphytic orchid with sturdy, 20 to 40 cm, more or less flattened stem with elliptic-oblong leaves. The flowers are fragrant, pale greenish yellow, more or less dotted with brown or brown-red, with a whitish or white base, waxy, long-lasting, up to 6 cm in diameter, column white, labellum slightly purplish purple, paler towards the margins. Both plants are included under CITES' Appendix II. The current study concentrates on a hybridization program involving two vandaceous orchid genera, namely Vanda stangeana and Phalaenopsis hygrochila to investigate their cross-ability and molecular confirmation of the synthesized hybrid using SSR and SCoT marker system. MS medium at half-strength enriched with 0.5 mg/L NAA + 0.5 mg/L Kn was the optimal medium for the development of the synthesized hybrid seedlings with the longest shoot (2.6 cm), the maximum number of leaves (4.5), leaf length (2.5 cm) and highest number of root (4.4) after 150 days of culture on the medium. In total, 65 amplified bands were revealed by seven SCoT primers (SCoT4, SCoT6, SCoT9, SCoT10, SCoT34, SCoT35 and SCoT36), of which 38 (58.46%) were polymorphic with the percentage ranging from 22.22% to 85.75%, thereby convincingly demonstrated the hybridity. Out of 8 pairs of SSR primers used, 3 primers (C106, MOK26 and C268) showed complementary banding patterns. Our study demonstrated that an intergeneric hybrid can be successfully produced by the hybridization of vandaceous orchids belonging to two different genera. [ABSTRACT FROM AUTHOR]

Invasive vespids are able to disrupt native species assemblages, modify ecological dynamics, and degrade ecosystem services. However, it is often difficult to quantify such effects within invaded ranges, principally due to the complexity of interactions, and a lack of comparative pre-invasion controls. In this study, we thus examine the effects of an invasive hornet, Vespa velutina , upon native species densities and pollination in a major food plant, Hedera hibernica. Using the highly heterogeneous distribution of V. velutina in a coastal area of the northwestern Iberian Peninsula, we assessed the impact of differing hornet abundance on insect diversity, flower visitation frequency, and predator-prey interactions. We then examined resultant effects upon the pollination success of H. hibernica , in the form of fruit and seed set. Our results demonstrated that in areas with high V. velutina abundance, the floral visitation frequencies and durations of insect pollinators were significantly altered. Effects varied widely across insect families, reflected in the differing predation success rates of V. velutina upon various native pollinators, in tandem with competitive exclusion. Interestingly, V. velutina was itself a frequent floral visitor, becoming the most common nectar forager in areas where it was abundant. In spite of this, H. hibernica reproductive success was significantly degraded in these areas, resulting in reduced seed set. As such, V. velutina appears to have multidirectional effects upon pollination services, first as an insect predator, and second as a nectar competitor and pollinator. Crucially, our findings suggest that V. velutina is an inferior pollinator when compared to the native species that it displaces, resulting in a net reduction in pollination efficacy, and hence reproductive success in H. hibernica. This study thus reveals the profound effects of an invasive vespid on native species through both competitive and predatory interactions. [Display omitted] [ABSTRACT FROM AUTHOR]

• Organic compounds in media improved in vitro asymbiotic seed germination and plantlet growth. • Somatic embryos were induced and regenerated on protocorm explants successfully growing to healthy plantlets. • A broad range of phytochemical compounds belonged to glycols, phenolics, and carbohydrates were identified in high ratios. • High contents TP and TF, and TAA values may explain diverse use of D. umbrosa in folk herbal medicines. We aimed to explore in vitro seed germination optimizing and somatic embryogenesis induction as well as to identify phytochemical constituents of endangered terrestrial orchid, Dactylorhiza umbrosa (Kar. & Kir.) Nevski. The highest germination rate (93.04±0.90%) was achieved with the combination of pineapple juice (PJ) and casein hydrolysate (CH). Moreover, the individual application of organic nitrogen compounds (Vamin, peptone, and casein hydrolysate) led to the higher germination efficiencies, with Pep being the most effective in shortening the germination time (the number of days from seed sown to embryo enlargement and seed testa rupture) (2.9 times faster compared to PJ+Pep+CH). Young protocorm explants treated with 1 mg L −1 2,4-D and 2 mg L −1 Kin showed the highest embryogenesis rate (39.99±5.72%), while this explant treated with 0.5 mg L −1 2,4-D and 2 mg L −1 Kin produced the highest embryo number (5.37±0.21). Morpholine, 4-(4-methyl-1-cyclohexen-1-yl) was the predominant phytochemical in tuber methanolic extract (60.63±3.23%), while 1,2,3-propanetriol was found at the highest percentage in leaf methanolic extract (47.48±3.23%). The extracts derived from the tubers had more content of total phenolics (TP) and total flavonoids (TF), as well as a greater antioxidant activity (TAA) compared to the extract obtained from the leaves. The recorded glucomannan content (31.31±0.60%) was lower than that of starch (32.65±1.22%), however both carbohydrates were responsible for the recorded viscosities of Salep samples. Due to high viscosity of products from Salep powder obtained from D. umbrosa , its tubers are used as superior crude materials in Iranian herbal markets. Taking into account the endangered status of D. umbrosa populations in the Euro-Mediterranean and the Middle East basins caused by climate change and human activities, our method may be exploited for large-scale propagation of in vitro raised plants and reintroduce them into their natural habitats. [ABSTRACT FROM AUTHOR]

• Novel and efficient symbiotic seed gemination and seedling development protocols were established for P. concreta. • Putative mycorrhizal fungi were isolated from native Ecuadorian orchids. • Tullasnella strains from different orchids enhanced seed germination of P. concreta. • T. calospora (UAMH9824) promote seed germination and seedling development of P. concreta. 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. [ABSTRACT FROM AUTHOR]

• This is the first report that analyses the genetic diversity of Aerides multiflora and Rhyncostylis retusa from a variety of locations in Uttarakhand. • The study indicating a genetic connection between the orchids that were collected from host trees and altitudes that were similar to one another. • Data can help in developing sustainable natural resource management, as well as conservation and hybridization strategies for A. multiflora and R. retusa. Aim of the study was to analyze genetic variability among natural populations of two endemic orchids (Aerides multiflora and Rhynchostylis retusa) of Uttarakhand, India using inter-simple sequence repeat (ISSR) makers. Both the species, known for their ornamental and medicinal potential, were collected from different locations in Uttarakhand region, and isolated DNA samples were amplified using fourteen ISSR primers. In the case of A. multiflora , eight of the fourteen primers tested showed amplification products with an average polymorphism of 35.39% and in the case of R. retusa , the average polymorphism was 63.32%. Cluster analysis for both species showed that samples collected from sites of similar latitudes and elevations were clustered together. Another key observation in the case of R. retusa was that the sub-clusters were formed on the basis of host tree association. This is the first report on the diversity analysis of A. multiflora and R. retusa in Uttarakhand. This study suggests that the orchids collected from locations with similar altitudes and host trees are genetically related to each other. This genetic profiling can be used as the basis to develop conservation protocols for these orchids to prevent them from entering the threatened category, as the epiphytic orchids are more susceptible to natural and anthropogenic pressures. Besides, the genetic variation analysis can be used to select the parent plants for developing commercial hybrids in both species. [ABSTRACT FROM AUTHOR]

Crop plants do not always reproduce at their full potential, which provides opportunities to increase crop yield through improved pollination services. Wild pollinator species make an important contribution to crop pollination and their distribution across the globe is uneven, creating a geographical template for adapting crop flowers to local pollinator assemblages, analogous to common evolutionary processes in wild plants. Flowers that attract many pollinator species are most resilient to pollinator failure and, therefore, such generalists are suitable models for modifying crop flowers. Using advances in genetic engineering and precision breeding in crop flowers to locally optimize pollination services and ultimately crop yield provides a novel solution for the pollination crisis that is complementary to focusing on safeguarding pollinator health. The global decline in insect diversity threatens pollination services, potentially impacting crop production and food security. Here, we argue that this looming pollination crisis is generally approached from an ecological standpoint, and that consideration of evolutionary principles offers a novel perspective. First, we outline that wild plant species have overcome 'pollination crises' throughout evolutionary history, and show how associated principles can be applied to crop pollination. We then highlight technological advances that can be used to adapt crop flowers for optimal pollination by local wild pollinators, especially by increasing generalization in pollination systems. Thus, synergies among fundamental evolutionary research, genetic engineering, and agro-ecological science provide a promising template for addressing a potential pollination crisis, complementing much-needed strategies focused on pollinator conservation. [ABSTRACT FROM AUTHOR]

• Reports long term clonal fidelity validation of M. acuminata. • Determination of quercetin, p -coumaric acid and eugenol within various tissues using HPTLC. • Assessment of anti- bacterial activity of various plant parts against prominent strains. • Validation of genetic conformity using SCoT and CBDP-gene targeted markers. • Model approach for other terrestrial orchids. Malaxis acuminata D. Don [ = Crepidium acuminatum (D. Don) Szlach.] is one of the prized medicinal orchids with large-scale application in various nutraceutical and herbal preparations. However, due to paucity in the availability of plant materials, its usage is often restricted. In order to ensure continuous supply of raw material for the pharmaceutical and herbal industries, plant tissue culture plays a crucial role. In the present research, transverse thin cell layer (t -TCL) derived in vitro cultures of M. acuminata were initiated and maintained in Murashige and Skoog (MS) medium supplemented with 1.5 mg/l meta -topolin (m T), 5.0 mg/l chitosan, 1.5 mg/l indole-3- butyric acid (IBA) and 5.0 mg/l phloroglucinol (PG) for three years time period under culture room conditions by regular intermittent sub-culturing. However, the success of in vitro propagated plant bio-resources are often adversely impacted due to the occurrence of clonal variations which is considered as a serious limitation for the commercial utilization. In order to ascertain the clonal fidelity of the long term - propagated in vitro lines of M. acuminata , genetic homogeneity amongst the regenerated plantlets was accessed using two advanced gene targeted molecular markers namely start codon targeted (SCoT) and CAAT box derived polymorphism (CBDP). The plants were found to exhibit 93.34% clonal fidelity in comparison to their mother plant. Keeping in consideration the therapeutic attributes of M. acuminata , the phytochemical makeup of the wild and in vitro - derived plant parts was compared using high performance thin layer chromatography (HPTLC) technique. Furthermore, M. acuminata is considered to be one the most precious medicinal orchids having rich reserves of biomolecules which can be used against various pathogenic bacterial strains. The antibacterial activities of various tissue extracts of M. acuminata were also evaluated against the opportunistic bacterial strains viz. Escherichia coli (MTCC43), Salmonella typhimurium (MTCC733), Staphylococcus aureus (MTCC96) and Bacillus subtilis (MTCC121). The results of antibacterial assays demonstrated the therapeutic efficacy of the in vitro - derived plant parts at identical magnitude with that of their wild counterparts. The present research provides a holistic insight into the clonal stability of the long term - propagated in vitro plants of M. acuminata with promising antibacterial activity. This study can also serve as a model approach for the maintenance of genetically stable in vitro lines of therapeutically important endangered orchid species over a period of time. [ABSTRACT FROM AUTHOR]

• Epidendrum secundum Jacq is a terrestrial orchid that grows in Ecuadorian southern highlands. • Enzymatic and non-enzymatic antioxidant assays were performed in E. secundum. • E. secundum organic extracts displayed antibacterial activity against Staphylococcus aureus. • Catalase activity was significantly higher in E. secundum in vitro seedling leaves. A comparison of antioxidant and antibacterial activities among in vitro cultured seedlings and Orchidarium plant specimens of Epidendrum secundum Jacq , a terrestrial orchid from southern Ecuadorian highlands, was performed. The aim of this work was to evaluate the enzymatic and non-enzymatic antioxidant compounds production from in vitro cultured seedlings and Orchidarium plants from the orchid species under study. For this purpose, a group of spectrophotometric assays was set up, comparing the free radical scavenging capacity, phenolic and flavonoids content from seedling leaves and plant leaves organic extracts, as non-enzymatic antioxidant indicators. In addition, the antibacterial activity of the organic plant extracts was evaluated versus Escherichia coli and Staphylococcus aureus. Catalase and ascorbate peroxidase activities were assessed as enzymatic antioxidant systems in aqueous extracts. The results showed higher non-enzymatic antioxidant activity for leaves from Epidendrum secundum Orchidarium plants compared with leaves from in vitro seedling cultures. The organic extracts from the orchid under study displayed moderate antibacterial activity against Staphylococcus aureus. In the enzymatic antioxidant evaluation, catalase activity was significantly higher at in vitro conditions. The results from this work allow us to highlight the potential of Epidendrum secundum Jacq as a source of bioactive metabolites with promising antioxidant and antibacterial activities. [ABSTRACT FROM AUTHOR]

• Members of Genus Paphiopedilum are categorised under rare and endangered of IUCN and CITES. • Conventional method of propagation is a slow process while the population of this species is reducing at an alarming rate in the wild. • Emphasis needs to be given on developing efficient protocols for in vitro mass multiplication to meet commercial and conservation strategies. • Significant observations on the response of the species on four types of cytokinins and auxins. • First report on the effect of Meta-topolin, IPA as phytohormones on growth and development of Paphiopedilum villosum. Paphiopedilum villosum Lindl. Stein , is a horticulturally important orchid as it is widely traded for its cut-flower and ornamental value, which is mainly attributed to long shelf-life of the flowers. High commercial demands of this genus have led to its extensive collection leading to the population diminution from the wild habitats. In vitro propagation technology is a prominent tool for multiplication of orchids for commercial and conservational purposes. In the present study, the effects of nutrient media composition and phytohormones in enhancing the shoot multiplication and seedling proliferation of P. villosum using in vitro technique was assessed. The optimal medium for shoot proliferation and root development was found to be Murashige and Skoog (MS, 1962) with the highest response of 70.8% in seedling development. Amongst the cytokinins, 10 µM meta -topolin (m T) supplemented in MS medium was found to be the most conducive for developing longest shoots (5.8 cm) and roots (4.5 cm). The auxin, indole-3-propionic acid (IPA) at 5 μM in MS medium resulted in 4.7 cm long shoots and 4.1 cm long roots. During acclimatization, the in vitro -raised seedlings showed 63.1% survival in compost mix substratum. The protocol described in the present study would be of immense importance in capping the over-exploitation of P. villosum from its natural habitat and to further enhance its large-scale propagation its large-scale propagation and conservation. [ABSTRACT FROM AUTHOR]

Dactylorhiza hatagirea , a terrestrial orchid endemic to North-Western Himalayas has immense medicinal importance attributed to dactylorhin. Inspite of therapeutic relevance, quantification of dactylorhin from this orchid has not been reported so far. Also, there is tremendous need to conserve D. hatagirea through in vitro approaches to harness its various bio-constituents. In the current study, it was found that flowers/inflorescences were enriched with dactylorhin along with tubers in D. hatagirea. Other aerial parts such as stem and leaves were not contributing towards the accumulation of active secondary metabolites collectively in whole plant. Also, the protocol for regeneration of multiple shoots with tubers/roots and callus initiation in D. hatagirea was optimized. BM2 terrestrial orchid media supplemented with 1 mg/L indole-3-butyric acid (IBA) and 6 mg/L 6-Benzylaminopurine (BAP) promoted shoot and root regeneration rapidly and efficiently. Moreover, Murashige Skoog (MS) media containing 0.5 mg/L 1-Naphthaleneacetic acid (NAA) and 1 mg/L BAP was suitable with a high frequency rate and a shorter duration for callus initiation. Further, it was observed that there was less enrichment of dactylorhins in in vitro cultures compared to naturally grown plant parts, but can be exploited alternatively if needed. [Display omitted] • In vitro protocol for shoot/root regeneration and callus initiation was standardized in D. hatagirea. • Detailed quantification of dactylorhins was performed in D. hatagirea. • Dactylorhins were more accumulated in tubers along with floral parts. • Alternative production of dactylorhins in vitro cultures was reported. [ABSTRACT FROM AUTHOR]

• PCR-based techniques are the most widely used diagnostic tools for virus indexing. • Out of 681 total genera, only 49 genera were reported for virus infection. • Cypripedioideae is the most affected among the sub-family in Orchidaceae. • Production of Virus-free orchids using in vitro techniques is a desirable approach. The maximum number of genera in the family Orchidaceae are well represented by their floricultural importance and are exported globally for trade exchanges. However, economic loss due to viral diseases has a profound impact on the orchid trade industry worldwide. Further, enduring phytosanitary certificates and undergoing stringent quarantine measures for virus-free orchids pose a major setback during the exchange of breeding materials between regions or countries. Fortunately, the spread of orchid viruses can be effectively controlled if proper diagnosis of viral diseases is made at an early stage and strict application of appropriate eradication methods are deliberated in a holistic approach. This bibliographic review highlights the substantial advancements in the diagnostic and eradication techniques of orchid viruses during the past few decades. The review shows that PCR-based techniques, which are represented as the "gold standard" method, are the most widely used diagnostic tools because of their accuracy, simplicity, and sensitivity. Considering the large diversity of orchids, only 7.2% of the genera belonging to the sub-families Epidendroideae, Orchidoideae, Cypripedioideae, and Vanilloideae have been detected for virus infection. The tribes Podochileae, Thaieae, Nervilieae, Gastrodieae, Xerorchideae, Triphoreae, Wullschlaegelieae, Tropidieae, Sobralieae, and Neottieae of the family Orchidaceae are under-represented. In vitro techniques, including meristem culture, thermotherapy, chemotherapy, and their combinations, represent the most desirable and effective approaches for orchid viral elimination. The use of cryotherapy, a simple technique based on cryopreservation, should have a high priority for viral elimination from infected orchids. This review could pave the way forward for comprehensive research on the production of virus-free orchids. [ABSTRACT FROM AUTHOR]

• High frequency of somatic embryo regeneration protocol for medicinal orchids within a short span using thidiazuron. • Triphenyl tetrazolium chloride test for viability check of somatic embryos. • Higher germination frequency of encapsulated somatic embryos through synthetic seed technology. Flickingeria nodosa (Dalz.) Seidenf. is a vulnerable epiphytic orchid bearing short lived flowers. Due to the unavailability of the specific pollinators the fruit setting is difficult. So the alternative method is through regeneration by using the vegetative propagules. The aim of the present study was to standardize the protocol for direct regeneration of somatic embryos from the leaf explants of Flickingera nodosa and future development of complete plantlets through synthetic seed technology. The young leaves were treated with lower concentration of 0.5 mg/L thidiazuron (TDZ) and inoculated on Knudson C basal medium (KCBM) prepared with diverse concentrations and combinations of auxin and cytokinin to standardize the optimum growth regulator combination. Development of somatic embryos was observed with only adenine sulphate and indole acetic acid (IAA) combination of growth regulator's. The highest number of somatic embryo development was observed in KCBM fortified with 4 mg/L adenine sulphate and 2 mg/L IAA. Triphenyl tetrazolium chloride test confirmed the viability of developed globular and heart shaped somatic embryos through microscopic observation. The developed somatic embryos were encapsulated with sodium alginate (2%) dipped in CaCl 2 (100 mM) solution and were incubated for 30 min to make firm, transparent and consistent synthetic seeds. Formed seeds were inoculated on BN3FBM with 2 mg/L adenine sulphate and 1 mg/L IAA for germination. The fully developed plantlets were effectively established with 82% survival frequency after acclimatization and hardening. [ABSTRACT FROM AUTHOR]

• An efficient model seed germination protocol for Vanda. • Addition of KN in the growth medium enhanced the frequency of PLBs proliferation and in vitro seedling multiplication. • Genetic fidelity and phytochemical analysis between the mother and in vitro regenerated plants. • A GC–MS method for simultaneous analysis of bioactive compounds. In the present investigation, an attempt was made to study in vitro propagation of Vanda cristata Wall. ex Lindl., an endangered and medicinally important epiphytic orchid, using seed germination which may reduce the pressures on its heedless collections from natural habitats. In vitro cultures were raised via asymbiotic seed germination technique on MS (Murashige and Skoog, 1962) medium with and without growth additives. When medium was supplemented with Kinetin, 99.75±0.25% seeds successfully germinated in 5.50±0.28 days followed by early protocorm formation (32.25±0.47 days) and complete seedling development (99.25±0.47 days). Indole-3-acetic acid+6-benzyl aminopurine in the medium showed synergistic effect during initial stages of seed germination and seedling development. Comparative phytochemical analysis, however, revealed that mother plant had a higher yield of secondary metabolites than in vitro regenerated plants. GC–MS analysis of the leaf methanolic extracts revealed 54 different phytochemicals. The outcome of the present study may be highly beneficial for the germplasm conservation and commercial cultivation of V. cristata for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Mulberry is an economically important crop for sericulture in China. Mulberry plantations are shifting inland, where they face high salinity. Arbuscular mycorrhizal fungi (AMF) reportedly enhance mulberry's tolerance to salinity. Here, we assessed if additional adaptive advantages against salinity are provided by sex differences beyond those provided by mycorrhizal symbiosis. In a pot experiment, male and monoecious plants were exposed to three salinity regimes (0, 50, and 200 mM NaCl) and two mycorrhiza-suppressed conditions (with or without benomyl application) for more than 16 months. We noticed that salinity alone significantly decreased the mycorrhizal colonization rate, salinity tolerance, K+ concentrations, and the ionic ratios of all plants. Mycorrhizal association mildly ameliorated the salt-induced detrimental effects, especially for monoecious plants, and sex-specific responses were observed. Meanwhile, both sexes had adopted different strategies to enhance their salinity resistance. Briefly, mycorrhizal monoecious plants exhibited a higher net photosynthetic rate and lower translocation of Na+ from root to shoot compared with mycorrhizal males under saline conditions. Their salt tolerance was probably due to the Ca2+/Na+ in roots. In comparison, male plants exhibited lower Na+ acquisition, more Na+ translocated from root to shoot, higher root biomass allocation, and higher N concentrations under harsh saline conditions, and their salt tolerance was mainly related to the K+/Na+ in their shoots. In conclusion, our results highlight that AMF could be a promising candidate for improving plant performance under highest salinity, especially for monoecious plants. Cultivators must be mindful of applying fungicides, such as benomyl, in saline areas. • There were sex-specific differences between mycorrhizal male and monoecious mulberry plants. • Both sexes of mulberry plants adopt different strategies to enhance salinity tolerance. • Monoecious plants benefited more from fungal symbiosis than males. [ABSTRACT FROM AUTHOR]

• Hawaiian orchid seed morphometrics differ from those seen in mainland orchids. • Terrestrial orchid seeds are larger, embryos have the highest mean area. • Epiphytic orchid seeds are smaller and the least buoyant. • Testa morphometrics may be useful in developing in vitro seed germination protocols. Ultrastructure and dimensions of whole seed, embryo, and testa cells of 10 orchid species native to North America and Hawaii are reported. Seeds of seven terrestrials (Cleistes bifaria, Isotria medeoloides, Liparis elata, L. hawaiensis, Platanthera holochila, P. integrilabia , and P. leucophaea), two epiphytes (Encyclia tampensis, Epidendrum nocturnum), and one semi-aquatic (Habenaria repens) orchid were studied by light and scanning electron microscopy. In contrast to the epiphytes and the semi-aquatic orchid, seeds of terrestrial species were larger in size evidenced by a small length/width ratio, had greater seed volume, a larger number of testa cells, and embryos with the highest mean area, supporting the existing literature. Interestingly, seeds of both Hawaiian species differed noticeably from their mainland counterparts (L. hawaiensis vs. L. elata; P. holochila vs. P. integrilabia and P. leucophaea). Our study lends support for differences between genera, but also within at least two genera (Liparis, Platanthera), for orchids occupying different habitats separated by thousands of km (Hawaii, Florida). For orchids threatened with immediate extinction in North America and worldwide, a better understanding of seed morphometrics, and especially the structure and function of the testa, may be useful in developing more effective protocols aimed at in vitro seed germination. [ABSTRACT FROM AUTHOR]

• In vitro propagation of medicinally exploited orchid Pholidota articulata has been accomplished. • Protocol can be exploited for a regular supply of metabolites from the in vitro -raised plantlets of P. articulata. • Study useful for reducing pressures exerted on natural populations for medicinal purposes. The present study enumerates an efficient and genetically stable mass multiplication protocol through in vitro culture technology with enhanced production of secondary metabolites for an endangered orchid Pholidota articulata Lindl. Higher frequency plantlet regeneration of P. articulata was accomplished in Gamborg B 5 medium supplemented with chitosan as compared to the other conventional plant growth hormones used. Chitosan-treated seeds showed the highest germination (97.06%) and the developing plantlets cultured in medium supplemented with 4 mg/l of chitosan showed the best rooting (37.33/ plantlet), shooting (8.67/ plantlet), and high acclimatizing ability (83.33%). Mass scale propagation of Pholidota articulata was obtained in B 5 medium supplemented with chitosan using axillary buds as secondary explants from the in vitro – raised plantlets. These micropropagated plantlets were assessed for clonal fidelity using inter-specific sequence repeats (ISSR) and start codon targeted polymorphism marker (SCoT) and were found to be genetically uniform wherein 91.8% of monomorphism was recorded. A comparative analysis of secondary metabolites and antioxidant potential among the in vivo and in vitro plants revealed a higher accumulation capacity in chitosan-treated plantlets. An interrelationship between phytochemicals' accumulation and chitosan concentration in the medium was deduced using the matrix plot which showed that 4 mg/l of chitosan was optimum for the accumulation of phytochemicals. Also, a correlation between the accumulated phytochemicals and the antioxidant activity in P. articulata could be shown using the Pearson correlation and Principal Component Analysis. The comprehensive in vitro regeneration protocol developed in this study can be of enormous importance for both ex-situ conservation and harvest of various bioactive compounds from P. articulata thereby reducing the pressure on its natural populations. [ABSTRACT FROM AUTHOR]

Habenaria intermedia (D. Don) Szlach is a rare orchid species of broad spectrum medicinal importance. It is commonly known as 'Riddhi' in the ayurvedic system of medicine. Pseudobulbs of this species are used in the ayurvedic formulations such as ' Chyawanprash '. The edible tubers are used for the treatment of several chronic diseases such as asthma, leprosy, skin diseases, memory loss, and as an aphrodisiac, depurative, anthelmintic, rejuvenating, and also as a general tonic. In the present work, the pharmacognostic, physiochemical, phytochemical, nutraceutical and antioxidant potencies of H. intermedia have been presented. In pharmacognostic evaluation, we studied sensory, macroscopic, and histochemical parameters. The physicochemical parameters evaluated were total ash (2.5 ± 0.2%), acid insoluble ash (1 ± 0.1%) , water-soluble ash (2 ± 0.2%), foreign matter (nil), loss on drying (0.4 ± 0.2 %), foaming index (less than 100), swelling index (20 ml ± 2%), pH, and crude fibre content (1 ± 0.01%.). In phytochemical evaluation, qualitative and quantitative analyses were performed. For qualitative phytochemical analysis, benzene, chloroform, ethyl acetate, ethanol and aqueous extracts were used. The phytochemical & nutraceutical analysis showed the presence of carbohydrates, amino acids, gums, glycosides, flavonoids, tannins, proteins, saponins, phenols, resins, and alkaloids. In quantitative analysis, total phenolic content (TPC) was found 8.91 ± 1.13 µg/ml (Gallic acid equivalents/gm extract). Total flavonoids content (TFC) was 16.03 ± 2.04 µg/ml against the standard quercetin. The total sugar content was 34.3 ± 4.27 µg/ml against the standard of glucose, and total resin content was 4.5 ± 0.21%. Tannin content was 0.03 ± 0.1% in pseudobulbs whereas fats content was 1 ± 0.12%. In antioxidant assays, aqueous extract showed higher antioxidant potency in terms of % radical scavenging activity (RSA) as compared to hydroalcoholic extracts. The above parameters provide critical information for setting quality parameters of crude drugs used in various formulations and will contribute significantly to orchid research. These findings encourage the potential use of this plant in disease management & treatments, which justify its usage in traditional systems of medicines. Research is required in the isolation of potent compounds and their clinical trials. [Display omitted] • Habenaria intermedia (D. Don) Szlach is a rare medicinal orchid, the edible tubers are used in various ayurvedic formulations. • Pseudobulbs are used to treat asthma, leprosy, skin diseases, memory and as aphrodisiac, depurative, anthelmintic, rejuvenating and general tonic. • Pharmacognostic, physiochemical, phytochemical and nutraceutical potency have been studied quantitatively and qualitatively. • Antioxidant assays (Total antioxidant activity and FRAP) were performed and showed promising results. [ABSTRACT FROM AUTHOR]

• Cymbidium and Dendrobium orchids harbor diverse endophytic fungi. • These endophytic fungi have antimicrobial activities. • Fusarium was the dominant genus of endophytes from orchids. • Novel discovery of Buergenerula spartinae and Exserohilum rostratum from orchids. This study reports the isolation of fungal endophytes from common orchids in Malaysia (Cymbidium and Dendrobium sp.), their antimicrobial activities and phytochemical constituents. Culturable fungal endophytes were sampled from surface-sterilized root tissues of Cymbidium and Dendrobium orchids, then identified via molecular approaches. To establish their antibacterial and antifungal activities, the identified fungal endophytes were tested by the agar well diffusion assay and dual culture technique, respectively. Results revealed a total of 59 fungal endophytes were isolated, and they were represented in eight different genera (Fusarium, Nigrospora, Lasiodiplodia, Exserohilum, Curvularia, Buergenerula, Trichoderma and Daldinia). Fusarium was the most dominant genus among the pool of isolates, with high isolation frequencies of 62.7% (37 isolates). Rare genera such as Buergenerula and Exserohilum were also discovered at lower frequencies (3.33%, 1 isolate each). Among the 59 isolates, 8 isolates (13.56%) demonstrated antibacterial activities (5.67 ± 0.58 to 25 ± 0.00 mm) against all 4 pathogens tested (Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa). All endophytic isolates were, however, capable of inhibiting the growth of at least one pathogenic fungus (Ganoderma boninense, Pythium ultimum, Fusarium solani, Candida albicans) (1.16 to 91.86% inhibition). Prominent antimicrobial activities were displayed by Fusarium incarnatum (C4) and Trichoderma asperellum (D7). Phytochemical analysis of extracts from these endophytes revealed that antimicrobial activities may be attributed to constituents like alkaloids, flavonoids, phenols, saponins, tannins and terpenoids. These results highlighted the potential of fungal endophytes from Orchidaceae as an alternative source for antimicrobial agents. [ABSTRACT FROM AUTHOR]

• Highly efficient leaf regeneration protocol reported for Vanda cristata. • KC medium found best for regeneration response. • Regeneration response via callusing, protocorm like bodies and shoot bud formation. • The best regeneration was obtained in NAA(10.6 µM)+BAP(8.8 µM). • Genetic fidelity has been assessed using ISSR molecular markers. In the present paper, a protocol for plant regeneration was developed for micropropagation of an epiphytic orchid, Vanda cristata Wall. ex Lindl., by using whole leaf explants procured from five months old in vitro grown cultures. The regeneration competence of leaf explants was tested on two different nutrient media (M and KC) supplemented with auxins (IAA, IBA, NAA) and cytokinins (BAP, KN, TDZ) at different concentrations. The regeneration response was invariably initiated mostly at the bases of the leaf explants via shoot bud, protocorm like bodies (PLBs) and callus formation whereas leaf tips (apical side) showed very less response. KC medium supplemented with NAA(10.6 µM)+BAP(8.8 µM), acted synergistically; cent per cent explants initiated regeneration response via formation of callus, PLBs and shoot buds at the basal part of leaf explant and complete plantlets were obtained. Highest number of regenerants (6) was observed in this combination as compared to the control. The genetic stability analysis of in vitro regenerated plantlets of V. cristata was performed by using inter-simple sequence repeats (ISSR) primers; 6.67% polymorphism and 93.33% of monomorphism was calculated using five ISSR primers, that showed a high degree of genetic stability to obtain true-to-type plantlets. The protocol developed in this study may be useful for conserving V. cristata including other orchid species using different biotechnological approaches. [ABSTRACT FROM AUTHOR]

• ORSV was found to be more prevalent than CymMV with the incidence percentages of 53.5% and 46.4% respectively. • Mixed infection of ORSV and CymMV was found to be 35.7%. • The Nucleotide sequences of ORSV and CymMV CP genes from Meghalaya showed high sequence identities of 96.90–99.56% and 96.91–99.66% respectively with other available sequences in Genbank. Odontoglossum ringspot virus (ORSV) and cymbidium mosaic virus (CymMV) are the most prevalent viruses infecting orchids all around the world. These viruses cause a serious threat to the orchid industry as they can spread from infected plants to healthy plants through vegetative propagation, contaminated tools, and human hands. This result in negative vigour of infected plants, showing stunted growth, producing fewer flowers, and decreasing flower quality. In this study, 84 leaf samples of orchids were collected from nine horti-hubs and orchid nurseries in Meghalaya. The orchid leaf samples were subjected to multiplex RT-PCR amplification using gene-specific primers targeting the coat protein (CP) genes of CymMV and ORSV. The most prevalent virus detected was ORSV with a 53.5% incidence while CymMV showed a 46.4% incidence. Mixed infections of ORSV and CymMV were detected in 30 samples (35.7%). The nucleotide sequences of ORSV and CymMV CP genes from Meghalaya showed high sequence identities of 96.90–99.56% and 96.91–99.66%, respectively when compared to other sequences in GenBank. The phylogenetic relationship among ORSV isolates and CymMV, based on infected host or geographical origin, showed no clear-cut clustering of isolates. [ABSTRACT FROM AUTHOR]

• 100% germination of E. cordigera was achieved in MS ½-strenght + 0,15 g L-1 Ac. • Efficient protocols for in vitro propagation and acclimatization of E. cordigera were established. Encyclia cordigera is a Mexican native orchid, of major ornamental importance due to its beautiful and fragrant flowers. The said characteristics also create a risk for the extinction of the species, thus, the urgent necessity to stablish propagation strategies to prevent its loss. The objective of this study was to establish a protocol for asymbiotic seed germination and acclimatization of E. cordigera. For asymbiotic germination, different concentrations (¼ and ½ and full-strength) of MS medium were evaluated, adding 0.15% of activated charcoal (AC). MS medium without AC was used as a control treatment. The germination percentage was evaluated after 16 weeks of culture. The treatments used for the germination stage were also used for the development of seedlings which lasted 20 weeks, then plant length, number of roots, root length, number of shoots, and number of leaves per shoot were measured. Finally, the plants obtained were acclimatized. The ½-strength MS + 0.15% AC resulted in 100% seed germination, while ¼-strength MS + 0.15% AC yielded longer seedlings (1.53 cm long). As for the number and length of roots, MS + 0.15% AC proved to be the best medium, with 2 roots and 2 cm respectively. No significant differences between culture media were found for the number of shoots and number of leaves per shoot. The plants obtained were transferred to an acclimatization process for eight weeks in the greenhouse, 209 plants (97% survival rate) were obtained during the acclimatization process. The presented protocols will contribute to the propagation and conservation of E. cordigera , an orchid species with ornamental and economic importance. [ABSTRACT FROM AUTHOR]

• A total of 25 putative PLD proteins from the genomes of Phalaenopsis equestris (7) , Dendrobium catenatum (9) and Apostasia shenzhenica (9) were identified. • These proteins are comprised of two HKD domains and two additional domains; C2 and PXPH domains at N-terminal region. • The multiple sequence alignment, domain and motifs analysis revealed their conserved nature and homology structure simulation and Ramachadran plot analysis revealed the abundance of beta sheets in tertiary structures. • The presence of Root-specific element, DOF proteins binding domain, scaffold or matrix attachment region, pollen-specific, dehydration-responsive, light-responsive, gibberellin-responsive, ABA-responsive elements and Meja-responsive upstream cis- regulatory promoter elements indicated their role in various physiological processes. Phospholipase D (PLD) plays a fundamental role in various aspects of plant growth, development and stress responses. In present study, 25 putative PLD proteins from the genomes of Phalaenopsis equestris (7) , Dendrobium catenatum (9) and Apostasia shenzhenica (9) were identified on the basis of presence of two HKD domains and two additional domains; C2 and PXPH domains at N-terminal region. These proteins were clustered into five sub-groups (α, β, δ, ζ and φ) along with their orthologs in Arabidopsis thaliana and Oryza sativa upon phylogenetic analysis. Gene characterization divulged that most of the PePLD, DcPLD and AsPLD genes within the same sub-group exhibited similar exon-intron organization and highly conserved motifs. RNA-seq based spatio-temporal studies indicated differential expression in various vegetative and reproductive tissues. PePLDα 1 , DcPLDα 1 and AsPLDα 1 were showing significant expression in all the tissues, while for other genes the expression was limited either in vegetative or reproductive tissues. Homology structure simulation revealed the abundance of random coils in secondary structures and beta sheets in tertiary structures. The presence of specific upstream cis- regulatory promoter elements emphasized the role of PLD s in various physiological processes. This report is the first genome-wide characterization of the PLD gene family in orchids and shall provide new insight into the functional characterization of these genes. [ABSTRACT FROM AUTHOR]

• An efficient protocol for the in-vitro conservation of S. tigrina is established. • ABA are necessary to in-vitro conservation S. tigrina for 180 days without affecting the survival of the shoots. • This study may contribute to the rescue and conservation programs of this valuable orchid. Stanhopea tigrina is a plant endemic to Mexico, listed as threatened in national and international standards because of the fragmentation of its habitat and the overexploitation of its natural populations. Therefore, programs for the conservation and propagation of this genetic resource should be implemented. This study aimed to establish an efficient protocol for the in vitro conservation of S. tigrina. To this end, three plant growth inhibitors were evaluated: abscisic acid (ABA), paclobutrazol (PBZ), and ancymidol (ACD), at concentrations of 0, 0.5, 1.0, and 2.0 mg L−1. In addition, an osmoregulator, polyethylene glycol (PEG), at 0, 10, 20, and 30 g L−1, was evaluated. Subsequently, the conserved material was regenerated using 2 mg L−1 of 6-benzylaminopurine (BAP). After 180 days of in vitro conservation, growth and development variables (survival rate, number of shoots and roots, shoot length) were evaluated. For in vitro conservation 2 mg L−1 ABA generated, the lowest shoot height (1.6 cm), the lowest number of shoots (4.3), and the lowest number of roots (3.7). In contrast, with the control treatment, in which shoots of 7.2 cm height, 4.5 shoots per explant, and 6.95 roots were obtained. Regeneration of 3.2 shoots per explant was achieved with 2 mg L−1 BAP, in addition to root growth during the multiplication phase. During the acclimatization process, 80% plant survival was observed. That according to the results obtained, this study contributes to conservation studies on this species, which is currently threatened with extinction. [ABSTRACT FROM AUTHOR]

• This review states the isolation, culture, characterization and application of OMF. • Major bottlenecks of traditional identification of OMF and its elucidation are discussed. • States the application OMF for bio-hardening and growth promotion of seedlings. • Provides insights for mass cultivation and conservation of orchids. In nature all orchids develop a mutualistic association with specialized group of fungi called orchid mycorrhiza. In natural environment, orchids are exclusively reliant on mycorrhizal fungi for seed germination, establishment, growth and development. In the early developmental stages, all orchids are mycoheterotrophic, and in most cases the association subsists throughout their lifespan. The significance of orchid mycorrhizal fungi (OMF) for seed germination, nourishment, growth promotion, and survival has been established more than a century ago and tremendous advancements were made in recent decades. Till date, understanding the mode of interaction between orchids and mycorrhizal fungi and determining the architecture of interaction networks are major challenges in ecology and evolutionary biology. The development of molecular techniques has empowered us to unscramble the fungal diversity comprised in the symbiosis and has facilitated us to overcome the difficulties associated with the conventional identification of OMF and orchid-fungal interactions. In addition, the application of modern biotechnological approaches resolved many controversial issues of OMF. Recently, OMF are used for biological hardening and growth promotion of in vitro raised seedlings, early flowering and quality improvement of flowers, and diseases and pests management. The aim of this communication is to update and summarize orchid mycorrhizal research including isolation, culture, and characterization of OMF, understanding the mechanisms of orchid-fungus symbiosis, and application of OMF in seed germination, growth promotion, and survival of asymbiotically raised seedlings and conservation of orchids. This appraisal will convey valuable insights to orchidologists engaged in biology, propagation, and conservation of orchids. [ABSTRACT FROM AUTHOR]

• Seed germination and plantlet growth were impacted by organic constituents and nitrogen sources. • Individual application of organic nitrogen compounds led to higher germination efficiencies. • The present protocol could be used for large-scale in vitro micropropagation of terrestrial orchids. 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. [ABSTRACT FROM AUTHOR]

• We reveal the floral trends and reproductive biology of two Mediterranean Clematis species. • Volatile compounds were sampled using a SPME-HS followed by GC–MS. • 90% of the volatile compounds were monoterpenes in both species. • Pollen supply and nectar production are important for pollinators during critical periods. • Hedges plant formations play an essential role in the conservation of pollinators and act as a linking semi-natural environment and farmland. Clematis cirrhosa L. and C. flammula L. (Ranunculaceae) are perennial climbing species, that are sympatric and that bloom in two different seasons: winter and summer. Both have colourful flowers representing the two types of Clematis flowers present in the Western Mediterranean basin: bell-shaped and disc-shaped flowers, respectively. This morphological and positional diversity encourages the comparative analysis of their reproductive biology, as well as the profiles of their floral volatiles. Morphological floral traits and the shape of flowers are different and are highly correlated with the rainy season pollinator's availability. Both species exhibit facultative autogamy. Volatile compounds were extracted using a standardised solid-phase microextraction of headspace (SPME-HS) followed by gas chromatography and mass spectrometry (GC-–MS). The spectrum of flower scents highlights the importance of monoterpenes (93% in C. cirrhosa and 99% in C. flammula) as the major components identified in both species. The study helps to explain the relationship between the floral shape, the pollination system, and the reproductive characteristics of Clematis. In addition, these two species act as food reservoirs for pollinators supplying pollen and nectar in critical periods of the year when resources are scarce. The two species play an important role in maintaining the populations of Apis mellifera, Bombus terrestris , and other seasonal pollinators. [ABSTRACT FROM AUTHOR]

• Recent medicinal importance of Arundina graminifolia are described. • This review brings the current status of phytochemicals status of Arundina graminifolia. • Pharmacological studies and in vitro conservation strategies of Arundina graminifolia are also discussed. Traditional medicines play an important role in protecting the health of all ethnic groups around the world, and their importance in the development of medical and health services cannot be overstated. The Orchidaceae family includes some of the most beautiful flowers in the world, each with its own unique shape, color, and form. Potted and cut flower orchids are big business because the bioactive components in them are great for your health. Arundima graminifolia (Orchidaceae) has been extensively used for wide range of ethnopharmacological activities, including rheumatism, food poisoning, snake bites, traumatic injuries, detoxification and heat clearance. It is also utilized to treat pneumonia, tuberculosis and bronchitis by the ethnic people. The recent two decades of fruitful achievements in its chemical constituent investigations have laid a foundation for the study of its therapeutically material basis and action mechanism, which will help clarify its broad-spectrum detoxification mechanism and develop into a modern pharmaceutical preparation. This suggests that the whole plant of A. graminifolia is more important than other orchid species for extracting bioactive constituents such as flavonoids, phenylpropanoid, phenanthrene, glycosides, bibenzyl and phenolic compounds for medicinal purposes. The bamboo orchid is a promising species for studying the efficacy to obtain pharmaceutical products due to its short life cycle and accumulation of bioactive ingredients in multiple plant parts. This review summarizes the current medicinal usage, phytochemistry and pharmacological activities of this high value species. [ABSTRACT FROM AUTHOR]

• We identify and characterize 18 FAD genes in the Vanilla planifolia genome. • FAD expression differs between different plant tissues and development stages. • Some VpFAD genes were found to participate in the interaction between Vanilla planifolia and Fusarium oxysporium. Fatty acid desaturase (FAD) plays an essential role in plants; however, FAD genes have not previously been characterized in orchids. Vanilla planifolia is a valuable natural spice in the family Orchidaceae. In this study, we identified 18 FAD genes in this species and analyzed their protein characteristics, chromosome distribution, phylogeny, gene structure, and conserved motifs. Transcriptome data analysis showed that the 18 genes showed different expression patterns in six tissues (seed, placental laminae, mesocarp, hair, stem, and leaf) and various stages of seed development (6 weeks, 8 weeks, 10 weeks, 3 months, 5 months, and 6 months). In addition, the expression profile of infected Fusarium oxysporum indicated that the FAD genes play an essential role in dealing with pathogen invasion. This study provides a comprehensive understanding of the FAD family in Vanilla planifolia as well as information on pathogen immunity, which can provide a reference for further functional research. [ABSTRACT FROM AUTHOR]

• Positive influence of MM medium on germination of A. pyramidalis and G. conopsea was determined. • Germination was influenced by full darkness and nutrient medium enriched with organic aditives. • In seedling growth, 2iP initiated the formation of the aboveground part of G. conopsea. • In A. pyramidalis, Kin initiated the leaf formation, while Ba promote root formation. Protocols for asymbiotic seed germination and seedling development of two terrestrial orchid species – Gimnadenia conopsea and Anacamptis pyramidalis are presented in this paper. Detail examination covered nutrient media: Knudson C (KC) and Malmgren (MM); organic supplements: coconut water (CW), pineapple juice (PJ), peptone (PE), casein hydrolysate (CA) and glutamine (A); different lighting conditions – 0/24 Light / Dark (L / D) and 16/8 (L / D) as well as cytokinins: 6-benzyladenine (BA), kinetin (Kin), and 6- (γ, γ-dimethylallylamino) purines (2 -iP) aiming to assess asymbiotic seed germination in controlled in vitro conditions, to define the optimal protocol for their mass propagation. The obtained results indicate that the nutrient medium MM and conditions of complete darkness encourage seed germination in both investigated species, while the organic supplements amendment resulted in a shortened period necessary for seed germination as well as a significantly higher percentage of germination. In Gymnadenia conopsea , the highest percentage of germination (73,79%) accompanied by the highest embryo/protocorm morphometric characteristics (1.40 mm average height, 1.21 mm average width, and 0.30 mm average shoot height) was achieved on MM-CW nutrient medium. A similar result was observed for Anacamptis pyramidalis with 69,88% germinated seeds on MM-CW nutrient medium, while the highest morphometric values were 1.10 mm (on MM-CW) for height, 1.00 mm (on MM-PE) for width and 0.57 mm (on MM-PE) for bud height. On the contrary, the results obtained on nutrient medium KC and under the 16/8 L / D lighting conditions indicate the inhibitory effect of these conditions on the seeds germination in examined species. A experiment with chitokinin treatments indicates that the best result in G. conopsea was achieved on the medium with the addition of 0.3 mg L−1 2iP with 5.33 mm for plant height, 1,13 root numbers and 12.26 mm root lenght, while in A. pyramidalis 0.3 mg L−1 Kin emerged as the most suitable for the development of the upper part of the plant (3.33 mm), while the medium enriched with 0.3 mg L−1 BA had a positive effect on the formation of the root system. [ABSTRACT FROM AUTHOR]

Vanda bicolor Griff. is a vulnerable orchid species having immense horticultural importance. This communication reports the outcome of a successful attempt towards the formulation of immature seed culture protocol of Vanda bicolor , factors affecting effective germination, plant regeneration. This study also endeavored to formulation of a simple and effective hardening process which supports direct transfer of plantlets from the culture condition to the field. Immature seeds from green pods of 7 months old registered 88.2% germination on MS nutrient medium supplemented with 3% sucrose, 3 μM each of NAA and BA under normal culture conditions. The germinating embryos converted to protocorms and protocorm-like bodies (PLBs) on the same germination medium within 55 days of culture and subsequently converted to plantlets and supported culture proliferation and simultaneous healthy roots development on MS medium fortified with sucrose (3%), NAA and BA (3 + 6 μM respectively) and activated charcoal (0.6%) where as many as 6.2 shoots developed per subculture per PLBs and 3.3 roots per plant. A new hardening technique was developed with fortified pieces of dead wood and burnt wood. It was found that after 5–6 wk of transfer to the hardening condition, velamenous roots started adhering to the wood pieces. These plants could be transferred to polyhouse as well as in the nature directly where 98.25 and 97.75% survival rate registered respectively after 8 wk of transfer. [ABSTRACT FROM AUTHOR]

• Low cost and eco-friendly substrata as agar alternative in tissue culture media. • Foam, betel nut coir, coconut coir, paddy straw, forest leaf litter, sugarcane bagasse and saw dust as alternative for agar. • Low cost protocols for in vitro propagation of orchids. • Potential commercial scale propagation of orchid through low cost tissue culture techniques. Agar has been the popular choice of the masses as tissue culture gelling matrix. But, in culture medium one of the costlier components is agar which accounts for ∼38% of the media cost and puts a limitation for commercial exploitation of the technique. Different low cost substrata such as polyurethane foam, betel nut coir, coconut husk, paddy straw, forest leaf litter, sugarcane bagasse, moist cotton pad, saw dust etc. have been screened as they are cost effective and most of them are eco-friendly which could be used as complete agar alternative in orchid in vitro propagation. This communication reviews the possibilities of using different low cost substrata as agar alternative in orchid tissue culture to make the planting materials production platform more attractive for horticulturists. Further, these substrata can also be used with suitable modification for in vitro culture of non-orchid species which will help conserve the species economically. [ABSTRACT FROM AUTHOR]

Cancer is one of the deadly diseases all over the world which is defined as unregulated growth and division of the cells. Natural remedies, particularly derived from plants, were used to treat a range of illnesses for thousands of years, including cancer. Several orchid species have been reported as a cure to treat various types of cancers. Orchids are huge family of flower bearing plants. They are traditionally used in folk medicine for the cure of various infectious diseases and tumour. Many of bioactive compounds isolated from orchid plants such as Dendrobium longicornu Lindl., Dendrobium transparens, Rhyncostylish retusa , Vanda cristata, Anoectochilus formosanus etc. possess anticancer activities via modifying the biotransformation of potential carcinogens by xenobiotic-metabolizing enzymes, alteration of hormone synthesis, inhibition of cancer cell proliferation, suppression of protein expression, impede of cell cycle and impairment of cell growth. In actual circumstances active constituents present in orchidaceae family are not so explored. So, in this review we covered the article published till 2022 using different electronic databases PubMed/ MEDLINE, Scopus, Web of Science, and Google Scholar. The present review highlights the orchid plant family and its bioactive constituents with anticancer activity via various signalling pathways, as well as their possible mechanisms of action. [Display omitted] • Cancer is a spectrum of disorders thats can leads to death. • Several orchid plants have been reported to cure various types of cancers. • Many bioactive compounds isolated from orchid plants such as Dendrobium longicornu, Dendrobium transparens, Rhyncostylish retusa, Vanda cristata, Anoectochilus formosanus etc. possess anticancer activities. • They showed anticancer activity against various types of cancer cell line. • In this study, we reviewed several anticancer properties identified in orchid plants via various signalling pathways, as well as their possible mechanisms of action. [ABSTRACT FROM AUTHOR]

• The first effective micropropagation protocol for the rare orchid Vanda brunnea. • ORCHIMAX multiplication medium has a subsequent effect on the acclimatization stage. • Brassinolide (BL) can relieve the damaging effects of stress during acclimatization. Orchids are one of the most popular groups of ornamental plants, and growing demand for them forces the horticultural industry to increase their production. This can be effectively done by in vitro propagation. This study focued on the development of an efficient protocol for in vitro propagation of a rare orchid species, Vanda brunnea (syn. Vanda henryi Schltr.). Two media, ORCHIMAX and Murashige and Skoog (MS) were tested, with or without 0.5 mg·L−1 benzyladenine (BA). Acclimatization of microplants to ex vitro conditions was done using water, 0.05% brassinolide (BL), or 1% abscisic acid (ABA). The best regeneration (100%, 6 microplants/explant) was observed on the ORCHIMAX medium (regardless of the BA presence). At the acclimatization stage, a lower stomatal conductance was observed in microplants regenerating on ORCHIMAX medium and treated with 0.05% BL. Plants regenerating on ORCHIMAX had a lower hydrogen peroxide content and a higher catalase activity than regenerants from the MS. In general, the lowest peroxide level and the highest catalase activity were found in microplants treated with BL which evidently contributed to the reduction of the oxidative stress at the acclimation stage. This resulted in 100% survival rate and a highlly efficient method for micropagation of this species. [ABSTRACT FROM AUTHOR]

Orchidaceae is one of the largest and most diversified group of flowering plants within the families of angiosperm. Medicinal properties of orchids are less explored, but it is drawing significant attention of the scientific communities recently. The north eastern Himalayan region of India is well-known orchid hotspot with prevalence of two-third of total orchid species of the country. In the present study, an attempt was made to investigate the phytochemical composition of six native, endangered medicinal orchid species of this region namely Dendrobium nobile, Dendrobium moschatum, Dendrobium densiflorum, Acampe papillosa, Coelogyne nitida and Arundina graminifolia. The nutritional as well as phytochemical composition and antioxidant activity of the extracts were analyzed by spectrophotometric methods. Minerals were estimated by atomic absorption spectroscopy (AAS) and flame photometer. Quantification of phenolics and bioactive metabolites from methanol extracts of the selected orchid species were carried out by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GCMS) methods. Results revealed that Dendrobium moschatum contained highest total carbohydrate (6.49 g 100g−1) and protein (11.30 g 100g−1). Dendrobium densiflorum was rich in total flavonoid content (35.54 mg quercetin equivalent QE 100g−1) and total alkaloid content (11.70 g 100g−1). It also showed highest 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activity (64.4 µg ml−1). Acampe papillosa exhibited maximum total phenol content (4.09 g gallic acid equivalent GAE 100g−1), tannin (3.17 g tannic acid equivalent TAE 100g−1) and saponin (7.20 g 100g−1) content whereas Coelogyne nitida showed 281.7 µg ml−1 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) scavenging activity. Iron, zinc, copper, manganese, sodium, potassium and calcium contents were higher in the Dendrobium species compared to other medicinal orchids under study. Quercetin (4518.3 mg kg−1 in Dendrobium moschatum) and catechin (1211.1 mg kg−1 in Dendrobium densiflorum) were the major phenolic compounds present abundantly in these orchid species. Metabolite analysis by GCMS showed presence of total twenty-six major bio-active compounds (like dendroban-12-one, embramine, scoparone etc.) in these six orchid species. The current study presents scientific evidence in support of the traditional therapeutic application of the studied orchid species and proves that these are potential sources of biologically and pharmacologically active ingredients. [Display omitted] • Phytochemical composition analysis and bio prospecting of six medicinal orchids were carried out. • Dendrobium leaves are rich in carbohydrate and minerals but stems are rich in protein content. • Dendrobium densiflorum is rich in flavonoid, alkaloid, zinc, bio active compounds and antioxidant activity. • Acampe papillosa is rich in total phenol, total tannin and total saponin content. • Coelogyne nitida exhibits higher antioxidant activity due to higher quercetin content. [ABSTRACT FROM AUTHOR]

• Embryonic grafting of monocots used to successfully produce two photosynthetically different plants- Orchid (CAM) and Maize(C4). • The grafts developed with various morphological features corresponding to the grafted region. • Embryonic grafting in monocots eliminates the need for vascular cambium. Traditional grafting practices exclude monocots due to their physical incompatibilities. Their distinct anatomical features, such as scattered vascular bundles and parallel venation and lack of vascular cambium, don't allow for graft healing. The recent development of embryonic grafting has disapproved the requirement of vascular cambium for grafting, instead relying on undifferentiated tissue. We grafted the embryos of two photosynthetically different plants- Zea mays (C4) and Dendrobium ovatum (CAM). We successfully generated grafts through a 'whole embryo-protocorm' method. The grafts showed multiple morphological characteristics, which included curling of leaves, stunted growth, and callus-like structure. We did an anatomical analysis to confirm graft union and vascular connectivity. The grafts showed over an 80 % survival rate across all morphology 30 days after grafting. The embryonic meristematic cells proliferated and healed the graft junction, thus allowing the grafted embryo to grow as an individual plant. This technique opens avenues for trait transfer between monocot crops. [Display omitted] [ABSTRACT FROM AUTHOR]

Human beings have disturbed the original characters of plants through interspecies hybridization, especially for economically producing cultivars with enhanced traits. Phalaenopsis is remoulded for exquisite flowers with huge shape and popular colors. However, the current challenge in Phalaenopsis breeding is that existing varieties tend to lose their original floral scent during the complex process of super-paraent hybridization. To address this, we collected a wide range of native species and hybrids to form the composition background and correlated the dynamics of Phalaenopsis fragrance for the futher breeding. A total of 208 volatile organic compounds (VOCs), with 83 prominent compounds constituting about 40 % of the identified substances. The primary species included all three major classes of floral fragrance compounds, with native species accounting for 83.33 % of volatile compounds released at levels exceeding 100 ng g−1 h−1. Both primary species and cultivars exhibited distinct aroma profiles, characterized by ten specific substances. Phalaenopsis stobartiana , originating from China, shows potential for benzenoid scents in fragrant flower breeding. On the other hand, hybrids primarily consisted of terpenoids and contained a higher abundance of low-threshold floral fragrance compounds. We also observed significant correlations between terpenoid and benzenoid scents with respect to color and flower shape. The height and width of the plants also showed a significant negative correlation with phenylpropanoid/ benzenoids and nitrogen-containing scent compounds. By conducting an extensive investigation into floral fragrance detection, we were able to concisely present the interrelationships among various characteristics of Phalaenopsis. This endeavor lays a primary foundation for exploring polymorphism in floral scent, providing guidance in Phalaenopsis breeding and facilitating ongoing genome-based molecular design for cultivating high-quality and fragrant orchids. [Display omitted] • Primary species and cultivars exhibited distinct aroma profiles separated by ten main characteristic substances. • Metabolic shunting and hybridization origin are possible reasons for the loss of floral fragrances in Phalaenopsis cultivars. • Phalaenopsis stobartiana can be prospective parent to make up for the benzenoid floral substances of Phalaenopsis. [ABSTRACT FROM AUTHOR]

• Ethylene affected anthocyanin degradation of cut Vand a 'Sansai Blue' orchid flowers in the transcription level of genes encoding for anthocyanin biosynthesis enzymes. • Inhibitor of ethylene action, 1-methylcyclopropene (1-MCP), effectively prevented the ethylene effects on discoloration of 'Sansai Blue' Vand a orchid. • Sucrose could not overcome the effect of ethylene in enhancing anthocyanin degradation. • Sucrose alone improved flower quality, such as RFW, water uptake and floret senescence, and extended the vase life of 'Sansai Blue' Vand a orchid. The 'Sansai Blue' Vand a orchid flower is very sensitive to exogenous ethylene although the flowers produce very low amounts of endogenous ethylene. We reported previously that there is a rapid ethylene-induced anthocyanin breakdown, which occurs before the senescence processes could be detected (Khunmuang et al. 2019b). In addition, there is a possibility that at the same time, ethylene may inhibit the anthocyanin biosynthesis, which also continues during vase life. Therefore, the purpose of the present study was to understand how ethylene, the ethylene inhibitor, 1-MCP, and sucrose affect anthocyanin content and flower quality parameters during vase life. For this purpose, the cut inflorescences were exposed to air (control), ethylene, 1-MCP or sucrose, and the effects of these treatments was examined on four genes encoding for anthocyanin biosynthesis enzymes, and on various quality parameters in cut Vanda 'Sansai Blue' orchid flowers at the bloom stage during vase life. The results revealed that exogenous ethylene caused petal discoloration in cut 'Sansai Blue' Vanda orchid flowers in accordance with their anthocyanin degradation, as well as the down-regulation of expression of genes encoding for the four anthocyanin biosynthesis enzymes. Treatments of 1-MCP alone or 1-MCP before ethylene treatment up-regulated the expression of the four genes, as compared to the control two days after treatments. This suggests that 1-MCP can prevent the effects of both endogenous and exogenous ethylene on anthocyanin biosynthesis. 1-MCP was also very effective in preventing ethylene responses in 'Sansai Blue' Vanda orchids by delaying the decrease in anthocyanin and sugar contents, and maintaining color appearance of flowers. Treatment with sucrose alone significantly inhibited floret discoloration, induced higher accumulation of anthocyanin and soluble sugar contents during vase life, and delayed quality parameters, as well as floret senescence throughout the vase period. However, sucrose could not overcome the effect of ethylene in enhancing anthocyanin degradation. It seems therefore, that these positive effects of sucrose may stem from its senescence-delaying effects, rather than being an anti-ethylene agent. Our findings indicate that the 1-MCP and sucrose treatments enhanced anthocyanin accumulation in the petals of cut 'Sansai Blue' Vanda orchid flowers at the bloom stage, but in different mechanisms. The present study also provides further support to the conclusion that the two processes of ethylene-induced discoloration and senescence proceed separately and independently during vase life of cut 'Sansai Blue' Vanda flowers. [ABSTRACT FROM AUTHOR]

• -Orchidaceae from subtropical Brazilian Atlantic Forest are, in their majority, epiphytes. • -The two functional groups were identified: xerophytes and meso /hygrophytes. • -Strategies for water and nutrient storage predominate in the xerophyte group. • -Meso/hygrophytes species require greater attention in conservation regarding climate change. Ecological anatomy studies seek to link structural aspects to environmental pressures, improving knowledge about the occurrence and distribution of species. Orchidaceae is a group with high species richness and ecological diversity, well represented in the Brazilian Atlantic Forest, a biodiversity hotspot. The Iguaçu National Park (ParNa Iguaçu) is an important remnant of the southern Brazilian Atlantic Forest with a high richness of Orchidaceae. Given the family's diversity and the relevance of ecological anatomy studies for interpreting traits associated with the survival of plants in certain environments, we studied 38 species of Orchidaceae from ParNa Iguaçu, seeking to identify possible functional groups. For this, we surveyed 57 leaf morpho-anatomical characters and conducted histochemical and statistical tests. The morpho-anatomical traits group the species into two functional groups, with Cyclopogon congestus emerging isolated. In group A, xeromorphic morphoanatomical characters predominate, and group B combines members with meso /hygromorphic characteristics. We advocate the importance of understanding the ecological behavior of species, especially Orchidaceae, due to their important ecological role, in conservation actions in protected areas. Furthermore, our results highlight the morphological and anatomical diversity of the Orchidaceae found in ParNa Iguaçu, as well contributing to the development of better management practices for these plants, considering the threats posed by the opening of clearings and climate change. [ABSTRACT FROM AUTHOR]

• Density of leafless vanilla vines and phorophytes increase in degraded forest. • Morphological plasticity of attachment roots is to cope up with habitat degradation. • Anatomical traits of underground roots are modulated to withstand water scarcity. • Changes in root traits ensure safety when facing forest degradation and drought. Human activities and climate crises have resulted in severe disturbances affecting tropical forests. Here, we address the responses of leafless Vanilla spp. to open habitats, drought, and scarcity of large trees as phorophytes, with a focus on the root system. First, we compared the abundance of vanilla plants and root morphology in conserved and degraded forests. Next, we analysed the variations in root anatomical traits along a rainfall gradient. Our results showed contrasting responses depending on the ecoregion. In the northern humid forest, both the density of phorophytes and the number of vanillas per phorophyte were higher in the degraded forest than in the conserved forest. No such effects were observed in the southern dry forest. Furthermore, vanilla vines in the degraded humid forest developed more filiform roots for anchorage to smaller supports. Vanilla species occurring in semiarid climates, such as V. bosseri and V. decaryana , have larger underground roots with a thicker cortex to compensate for water rarity, as well as more vascular bundles to promote hydraulic conductivity, and more aerenchyma to decrease radial transport. Xylem vessel diameter is a plastic trait that increases with drought severity, as observed in one V. madagascariensis ecotype. These findings underline the high plasticity of the root system in leafless Vanilla spp. when facing habitat degradation and drought, and provide insights into their possible use in conservation and crop breeding. [ABSTRACT FROM AUTHOR]

• Association of endophytes ensure survival of orchids in nature. • Non-mycorrhizal endophytes are predominantly ascomycetes. • Orchid-endophyte interaction is not clade specific. • The interaction trends from specialised in basal to generalised in nested clades. • The endophytes do not appear to have co-evolved epiphytic orchid hosts. Fungal endophytes are known to play a vital role in perpetuation and survival of orchids. While orchid–mycorrhizal relationship has been studied in detail, dynamics of orchid–non-mycorrhizal associations are insufficiently explored. There is paucity of empirical evidence for host specificity, phylogenetic preference, co-evolution, and habitat selection amongst orchids and their non-mycorrhizal endophytes. We explored these aspects by studying the root associated non-mycorrhizal fungal assemblages from 11 epiphytic orchid species in two tropical communities located at Indo-Burma biodiversity hotspot. A total of 94 non-mycorrhizal fungal isolates were mined. Of these 55 isolates could be identified at species level, while 39 isolates were circumscribed at generic level using the ITS region. The core group fungal assemblages predominantly belonged to Ascomycota. The extent of mutualistic reliance was inferred by mapping the bipartite interaction in a phylogenetic framework. The analysis shows that the orchid–non-mycorrhizal associations are neither clade specific nor nested, rather they depend on the availability of suitable fungal taxon at the sites. The study also indicates the absence of phylogenetic preference between the partners. However, topology of the bipartite interaction network suggests that there has been a shift from specialised interaction in the basal clades, to generalised one in the later evolved clades. [ABSTRACT FROM AUTHOR]

Crepidium acuminatum (D. Don) Szlach (Syn. Malaxis acuminata , Trade Name: Jeevak) is an amazing nutraceutical orchid. In the ayurveda sector, it is a well-known herb because of its immense therapeutic potential. Pseudobulbs are the most in-demand component in the industry, out of all the segments of the plant. The activity is connected to the presence of several active metabolites. There are only a few research papers on C. acuminatum secondary metabolite extraction and characterization. We extracted and characterized β-sitosterol, Stigmasterol, and Eugenol from the pseudobulbs of this plant by High Performance Thin Layer Chromatography (HPTLC), Column Chromatography and High Performance Liquid Chromatography (HPLC). Nuclear Magnetic Resonance (NMR), Mass Spectroscopy (MS), Ultra Violet (UV), and Fourier Transform-Infrared (FT-IR) spectroscopy were utilised to characterise compounds. HPLC analysis revealed 99.98 μg/ml of β-sitosterol in the sample. Eugenol, Stigmasterol, and β-sitosterol, metabolites have a high therapeutic potential, indicating that they will be important medications in the future. Clinical trials are needed to confirm their effects on life-threatening disorders. The project's purpose is to inspire other scientists to launch a series of scientific inquiries into Crepidium acuminatum (Malaxis acuminata), and other orchids to fill in the gaps in the knowledge. [Display omitted] • Crepidium acuminatum (Malaxis acuminata) is a miraculous orchid, traditionally used as nutraceutical and in various Ayurveda formulations. • Pseudobulbs are extensively used by herbal industry. • Eugenol and stigmasterol has been isolated from pseudobulbs.by HPTLC method. • Characterization has been done by UV, FT-IR, NMR, and MS • β-sitosterol has been isolated, identified quantified from pseudobulbs. • Optimized conditions for quantification of β-sitosterol by HPLC method has been reported in this paper. • HPLC-PDA method was used for quantification. Reported method can be used for quantification in other orchids also. • Eugenol, stigmatserol and β-sitosterol are notable drugs of the future and clinical studies are needed to prove its implications on life threatening diseases. [ABSTRACT FROM AUTHOR]

• Auxin-cytokinin interaction allows asymbiotic germination of white variant of L. anceps. • Organic compounds help in vitro rooting of white variant of L. anceps. • Propagules of white variant of L. anceps were increased. Laelia anceps ssp. anceps is a species endemic to Mexico. The aim of this study was the establishment of a protocol of asymbiotic germination of L. anceps ssp. anceps , white variant. For asimbiotic germination, we evaluated different treatments: (a) 2 mg L−1 of 6-benzylaminopurine (BAP) + 2 mg L−1 of indoleacetic acid (IAA), (b) 2 mg L− BAP + 2 mg L−1 IAA + 2 mg L−1 naphthalenacetic acid (NAA) and (c) 2 mg L−1 Kinetin (KIN) + 2 mg L−1 BAP + 2 mg L−1 NAA. Included a control treatment with no PGR supplementation. The percentage of germinated seeds was assessed. For in-vitro rooting, different organic compounds were evaluated: (a) 1 mg L−1 IAA + 150 mL L−1coconut water, (b) 1 mg L−1 IAA + 80 g L−1banana pulp, and (c) 2 mg L−1 BAP + 2 mg L−1 IAA + 2 mg L−1 NAA. We also included a control treatment with no supplementation of PGRs or organic compounds. The percentage of rooted plants was evaluated and transferred to acclimatization process. 2 mg L−1 BAP + 2 mg L−1 IAA+ 2 mg L−1 NAA produced 82.20% seed germination; seedlings showed an average length of 1.11 cm and 1.64 leaves per explant. The treatment with 1 mg L−1 IAA + 150 ml L−1coconut water yielded a 78.20% rooting. 85% survival rate of plants was observed during the acclimatization process. This protocol will favor the propagation of the valuable germplasm of this orchid species that is endangered of extinction. [ABSTRACT FROM AUTHOR]

• TDZ was effective for inducing PLBs from leaf explants of C. tigrina. • GA 3 significantly increases shoot regeneration from PLBs. • Cattleya tigrina leaves are endopolyploid, with 2C, 4C and 8C DNA content cytotypes. • 26.5% of the plantlets regenerated from PLB presented duplicated ploidy levels. • The ploidy level of the regenerants changes stomatal density and morphology. The induction and subsequent regeneration of protocorm-like bodies (PLB) is the most common morphogenetic pathway for orchid micropropagation. The regeneration of plants from PLB can be a bottleneck for some species due to asynchronicity. Genetic fidelity is another issue, and the cytogenetic stability of regenerated plantlets deserves special attention due to the common occurrence of endopolyploidy in orchids. In the present work, we defined optimal conditions for PLB induction and plant regeneration for Cattleya tigrina , a threatened orchid from the Brazilian Atlantic Rain Forest. We estimated the optimum thidiazuron (TDZ) concentration for PLB induction from leaf explants, while also comparing whole leaves vs thin cell layer (TCL) as explants. For plantlet regeneration from PLB clusters, we investigated the effects of different concentrations of GA 3 and sucrose, as well as the dehydration of PLBs and the use of temporary vs continuous immersion systems. Furthermore, the cytogenetic stability of regenerated plantlets was analyzed using flow cytometry, and the differences in stomatal morphology were studied. Exogenous TDZ (25 µM) was the most effective concentration for PLB induction, with 17.5% induction rate. Leaf basal regions, either in whole leaves or in TCL, were the most responsive for PLB induction. The dehydration of PLBs for 120 min increased the fresh weight increment (FWI) in plantlet regeneration, but at the expense of decreased shoot number and increased mortality. Temporary-immersion bioreactors were found to be the most suitable system for PLB multiplication compared to continuous immersion system. Shoot development was challenging due to previously acquired habituation to TDZ. Sucrose and GA 3 showed an interactive effect on shoot growth. GA 3 improved shoot formation, and maximum shoot production was estimated to occur at 10.3 and 9.7 µM GA 3 on media containing 1.5% and 2% sucrose, respectively, resulting in larger number of developed plantlets (∼220) per culture flask. The flow cytometry analysis showed that 26.5% of plants regenerated from PLBs had doubled ploidy levels. The ploidy alterations were reflected in changes on leaf epidermis, such as increased stomatal density and number. [ABSTRACT FROM AUTHOR]