A review on endophytic bacteria of orchids: functional roles toward synthesis of bioactive metabolites for plant growth promotion and disease biocontrol.

MAIN CONCLUSION: In this review, we have discussed the untapped potential of orchid endophytic bacteria as a valuable reservoir of bioactive metabolites, offering significant contributions to plant growth promotion and disease protection in the context of sustainable agriculture. Orchidaceae is one of the broadest and most diverse flowering plant families on Earth. Although the relationship between orchids and fungi is well documented, bacterial endophytes have recently gained attention for their roles in host development, vigor, and as sources of novel bioactive compounds. These endophytes establish mutualistic relationships with orchids, influencing plant growth, mineral solubilization, nitrogen fixation, and protection from environmental stress and phytopathogens. Current research on orchid-associated bacterial endophytes is limited, presenting significant opportunities to discover new species or genetic variants that improve host fitness and stress tolerance. The potential for extracting bioactive compounds from these bacteria is considerable, and optimization strategies for their sustainable production could significantly enhance their commercial utility. This review discusses the methods used in isolating and identifying endophytic bacteria from orchids, their diversity and significance in promoting orchid growth, and the production of bioactive compounds, with an emphasis on their potential applications in sustainable agriculture and other sectors.

Biosolids Treated as a Nutritional Alternative for in vitro Culture of Bowdichia Virgilioides Kunth.

The safe management of sewage waste is a current concern due to population growth and waste production. Biosolids, derived from sewage sludge treatment, are globally used as organic fertilizers, aligning with Sustainable Development Goal 6 for resource recycling. However, biosafety concerns arise due to the presence of metals and microplastics in biosolids, potentially impacting soil and water. This study investigated biosolids' use for in vitro cultivation of Bowdichia virgilioides Kunth. Results indicate that while biosolids can replace traditional nutritional media, balancing their concentration is crucial for optimizing plant growth. The WPM (Wood Plat Medium) remains essential for in vitro cultivation, but substituting it with biosolids at concentrations of up to 2 g L- 1 is feasible, providing similar plant development compared to the WPM medium. However, when combined, there is a complex and challenging interaction between biosolids and the culture medium.

Orchid species diversity across a forest disturbance gradient in west Mau forest, Kenya.

Orchidaceae is one of the most diverse and widespread groups of flowering plants. Despite their immense ecological and socio-economic value, their spatial distribution across forest disturbance gradient is not well understood, particularly in tropical montane forests. This study assessed the influence of forest degradation on orchid species richness and abundance in West Mau Forest, Kenya. Stratified systematic sampling was adopted across three different disturbance regimes consisting of relatively intact forest, moderately disturbed forest and highly degraded forest. A total of five orchid species were recorded from nine host-tree species. The intact forest had seven host tree species with five orchid species. The moderately degraded forest had four host-tree species with two orchid species, while the highly degraded forest that had no orchids. Polystachya confusa was the most abundant orchid species (600.0±227.9 clumps ha-1) followed by Bulbophyllum sp (340.0±112.2 clumps ha-1), Chamaeangis sp (300.0±115.5 clumps ha-1), Aerangis sp (200.0±57.7 clumps ha-1) and Tridactyle sp (100.0±0.0 clumps ha-1). The results of this study indicate that forest degradation reduces orchid species diversity in tropical montane forests. They also show that orchids are bioindicators of forest degradation status.

Plastome Evolution, Phylogenomics, and DNA Barcoding Investigation of Gastrochilus (Aeridinae, Orchidaceae), with a Focus on the Systematic Position of Haraella retrocalla.

Gastrochilus is an orchid genus containing about 70 species in tropical and subtropical Asia with high morphological diversity. The phylogenetic relationships among this genus have not been fully resolved, and the plastome evolution has not been investigated either. In this study, five plastomes of Gastrochilus were newly reported, and sixteen plastomes of Gastrochilus were used to conduct comparative and phylogenetic analyses. Our results showed that the Gastrochilus plastomes ranged from 146,183 to 148,666 bp, with a GC content of 36.7-36.9%. There were 120 genes annotated, consisting of 74 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. No contraction and expansion of IR borders, gene rearrangements, or inversions were detected. Additionally, the repeat sequences and codon usage bias of Gastrochilus plastomes were highly conserved. Twenty hypervariable regions were selected as potential DNA barcodes. The phylogenetic relationships within Gastrochilus were well resolved based on the whole plastome, especially among main clades. Furthermore, both molecular and morphological data strongly supported Haraella retrocalla as a member of Gastrochilus (G. retrocallus).

The Complete Mitogenome of Apostasia fujianica Y.Li & S.Lan and Comparative Analysis of Mitogenomes across Orchidaceae.

Apostasia fujianica belongs to the genus Apostasia and is part of the basal lineage in the phylogenetic tree of the Orchidaceae. Currently, there are only ten reported complete mitochondrial genomes in orchids, which greatly hinders the understanding of mitochondrial evolution in Orchidaceae. Therefore, we assembled and annotated the mitochondrial genome of A. fujianica, which has a length of 573,612 bp and a GC content of 44.5%. We annotated a total of 44 genes, including 30 protein-coding genes, 12 tRNA genes, and two rRNA genes. We also performed relative synonymous codon usage (RSCU) analysis, repeat sequence analysis, intergenomic transfer (IGT) analysis, and Ka/Ks analysis for A. fujianica and conducted RNA editing site analysis on the mitochondrial genomes of eight orchid species. We found that most protein-coding genes are under purifying selection, but nad6 is under positive selection, with a Ka/Ks value of 1.35. During the IGT event in A. fujianica's mitogenome, the trnN-GUU, trnD-GUC, trnW-CCA, trnP-UGG, and psaJ genes were identified as having transferred from the plastid to the mitochondrion. Compared to other monocots, the family Orchidaceae appears to have lost the rpl10, rpl14, sdh3, and sdh4 genes. Additionally, to further elucidate the evolutionary relationships among monocots, we constructed a phylogenetic tree based on the complete mitogenomes of monocots. Our study results provide valuable data on the mitogenome of A. fujianica and lay the groundwork for future research on genetic variation, evolutionary relationships, and breeding of Orchidaceae.

Synthetic Tetraploid of Oncidium crispum Lodd. (Orchidaceae).

In ornamental plants, artificial polyploidization has enabled the creation of new cultivars. Due to their high commercial value in the international flower market and their ornamental characteristics, such as the shape, size, color, and durability of their flower, orchids have received great attention in studies of artificial polyploidization. Here we described the protocol used for polyploid induction in Oncidium crispum, an epiphyte species native of southeastern Brazil, of great ornamental interest and widely sold in flower shops. The species stands out for having inflorescence with large flowers, brown in color with yellow spots. In addition, O. crispum has great potential for use in genetic improvement programs since the species is widely used in interspecific crosses. Closed capsules containing mature O. crispum seeds were subjected to running sterilized water for 10 min and then to a 1.5% sodium hypochlorite solution for 10 min. Small portions of seeds were introduced into 50 mL of water-soluble fertilizer with macro- and micronutrients (B>M) plus 0.7% agar. Explants originating from seeds previously in vitro germinated were submitted to 0.05% and 0.1% of colchicine for 4 days and 8 days. Flow cytometry and chromosome counts confirmed that the protocol successfully produced synthetic polyploid plants.

Perturbation of periodic spot-generation balance leads to diversified pigmentation patterning of harlequin Phalaenopsis orchids: in silico prediction.

BACKGROUND: A retrotransposon HORT1 in the promoter of the anthocyanin activator gene PeMYB11, microRNA858 (miR858) that targets PeMYB11, and a repressor PeMYBx have been implicated in pigmentation patterning diversity of harlequin Phalaenopsis orchids. However, the interrelationship among them remains to be elucidated. RESULTS: To understand how these factors interact to generate anthocyanin spots in Phalaenopsis, we successfully developed a mathematical model based on the known reaction-diffusion system to simulate their interplay and refined the conceptual biological model. Intriguingly, the expression of both PeMYBx and PeMYB11 were in phase for purple spot formation, even though they showed adverse effects on anthocyanin accumulations. An increase in the self-activation rate of PeMYB11 resulted in the increased size of purple spots, but no effects on spot fusion. Decreased degradation rate of miR858 in the purple regions, led to disruption of the formation of spotted pigmentation patterning and a full-red pigmentation pattern. Significantly, the reduced miR858 level promotes the fusion of large dark purple dots induced by the solo-LTR of HORT1, eventually generating the purple patches. In addition, the spatially heterogeneous insertion of HORT1 caused by the remnant solo-LTR of HORT1 derived from random homologous unequal recombination of HORT1 in individual cells of floral organs could explain the diverse pigmentation patterning of harlequin Phalaenopsis. CONCLUSIONS: This devised model explains how HORT1 and miR858 regulate the formation of the pigmentation patterning and holds great promise for developing efficient and innovative approaches to breeding harlequin Phalaenopsis orchids.

Genome-Wide Identification and Expression Pattern Analysis of GATA Gene Family in Orchidaceae.

The GATA transcription factors play crucial roles in plant growth, development, and responses to environmental stress. Despite extensive studies of GATA genes in many plants, their specific functions and mechanisms in orchids remain unexplored. In our study, a total of 149 GATA genes were identified in the genomes of seven sequenced orchid species (20 PeqGATAs, 23 CgGATAs, 24 CeGATAs, 23 DcaGATAs, 20 DchGATAs, 27 DnoGATAs, and 12 GelGATAs), classified into four subfamilies. Subfamily I typically contains genes with two exons, while subfamily II contains genes with two or three exons. Most members of subfamilies III and IV have seven or eight exons, with longer introns compared to subfamilies I and II. In total, 24 pairs (CgGATAs-DchGATAs), 27 pairs (DchGATAs-DnoGATAs), and 14 pairs (DnoGATAs-GelGATAs) of collinear relationships were identified. Cis-acting elements in GATA promoters were mainly enriched in abscisic acid (ABA) response elements and methyl jasmonate (MeJA) elements. Expression patterns and RT-qPCR analysis revealed that GATAs are involved in the regulation of floral development in orchids. Furthermore, under high-temperature treatment, GL17420 showed an initial increase followed by a decrease, GL18180 and GL17341 exhibited a downregulation followed by upregulation and then a decrease, while GL30286 and GL20810 displayed an initial increase followed by slight inhibition and then another increase, indicating diverse regulatory mechanisms of different GATA genes under heat stress. This study explores the function of GATA genes in orchids, providing a theoretical basis and potential genetic resources for orchid breeding and stress resistance improvement.

Genome-Based Identification of the Dof Gene Family in Three Cymbidium Species and Their Responses to Heat Stress in Cymbidium goeringii.

As an important genus in Orchidaceae, Cymbidium has rich ecological diversity and significant economic value. DNA binding with one zinc finger (Dof) proteins are pivotal plant-specific transcription factors that play crucial roles in the growth, development, and stress response of plants. Although the Dof genes have been identified and functionally analyzed in numerous plants, exploration in Orchidaceae remains limited. We conducted a thorough analysis of the Dof gene family in Cymbidium goeringii, C. ensifolium, and C. sinensis. In total, 91 Dof genes (27 CgDofs, 34 CeDofs, 30 CsDofs) were identified, and Dof genes were divided into five groups (I-V) based on phylogenetic analysis. All Dof proteins have motif 1 and motif 2 conserved domains and over half of the genes contained introns. Chromosomal localization and collinearity analysis of Dof genes revealed their evolutionary relationships and potential gene duplication events. Analysis of cis-elements in CgDofs, CeDofs, and CsDofs promoters showed that light-responsive cis-elements were the most common, followed by hormone-responsive elements, plant growth-related elements, and abiotic stress response elements. Dof proteins in three Cymbidium species primarily exhibit a random coil structure, while homology modeling exhibited significant similarity. In addition, RT-qPCR analysis showed that the expression levels of nine CgDofs changed greatly under heat stress. CgDof03, CgDof22, CgDof27, CgDof08, and CgDof23 showed varying degrees of upregulation. Most upregulated genes under heat stress belong to group I, indicating that the Dof genes in group I have great potential for high-temperature resistance. In conclusion, our study systematically demonstrated the molecular characteristics of Dof genes in different Cymbidium species, preliminarily revealed the patterns of heat stress, and provided a reference for further exploration of stress breeding in orchids.

Plastid genome evolution in leafless members of the orchid subfamily Orchidoideae, with a focus on Degranvillea dermaptera.

PREMISE: Leafless, heterotrophic plants are prime examples of organismal modification, the genomic consequences of which have received considerable interest. In particular, plastid genomes (plastomes) are being sequenced at a high rate, allowing continual refinement of conceptual models of reductive evolution in heterotrophs. However, numerous sampling gaps exist, hindering the ability to conduct comprehensive phylogenomic analyses in these plants. METHODS: Using floral tissue from an herbarium specimen, we sequenced and analyzed the plastome of Degranvillea dermaptera, a rarely collected, leafless orchid species from South America about which little is known, including its phylogenetic affinities. RESULTS: The plastome is the most reduced of those sequenced among the orchid subfamily Orchidoideae. In Degranvillea, it has lost the majority of genes found in leafy autotrophic species, is structurally rearranged, and has similar gene content to the most reduced plastomes among the orchids. We found strong evidence for the placement of Degranvillea within the subtribe Spiranthinae using models that explicitly account for heterotachy, or lineage-specific evolutionary rate variation over time. We further found evidence of relaxed selection on several genes and of correlations among substitution rates and several other "traits" of the plastome among leafless members of orchid subfamily Orchidoideae. CONCLUSIONS: Our findings advance knowledge on the phylogenetic relationships and paths of plastid genome evolution among the orchids, which have experienced more independent transitions to heterotrophy than any other plant family. This study demonstrates the importance of herbarium collections in comparative genomics of poorly known species of conservation concern.

Salt tolerance in a neotropical orchid in the absence of local adaptation to salt spray.

PREMISE: Salt tolerance has rarely been investigated regionally in the neotropics and even more rarely in Orchidaceae, one of the largest families. Therefore, investigating local adaptation to salt spray and its physiological basis in Epidendrum fulgens, a neotropical orchid species, brings important new insights. METHODS: We assessed the degree of salt tolerance in E. fulgens by testing whether coastal populations are more tolerant to salt, which could point to local adaptation. To understand the physiological basis of such salt tolerance, we exposed wild-collected individuals to salt spray for 60 days, then measured leaf expansion, osmotic potential, sodium leaf concentration, chlorophyll leaf index, chlorophyll fluorescence, relative growth rate, and pressure-volume curves. RESULTS: There is no local adaptation to salt spray since both inland and coastal plants have a high tolerance to salt stress. This tolerance is explained by the ability to tolerate high concentrations of salt in leaf tissues, which is related to the high succulence displayed by this species. CONCLUSIONS: We showed an unprecedented salt tolerance level for an orchid species, highlighting our limited knowledge of that trait beyond the traditional studied groups. Another interesting finding is that salt tolerance in E. fulgens is linked to succulence, is widespread, and is not the result of local adaptation. We suggest that E. fulgens and its allied species could be an interesting group to explore the evolution of important traits related to tolerance to salt stress, like succulence.

[Material basis and mechanism of Bletillae Rhizoma for melasma, gastrointestinal hemorrhage, lung cancer and bronchoplumonary inflammation as "homotherapy for heteropathy" based on UHPLC-Q-Exactive Orbitrap HRMS coupled with network pharmacology and molecular docking].

Based on UHPLC-Q-Exactive Orbitrap HRMS coupled with the network pharmacology and molecular docking, the common material basis and molecular mechanisms of Bletillae Rhizoma for melasma, gastrointestinal hemorrhage, lung cancer and bronchoplumonary inflammation as "homotherapy for heteropathy" were explored. The fingerprint of 17 batches of Bletillae Rhizoma from different areas was established using HPLC, and the similarity analysis was carried out. The common chemical components of the 17 batches of Bletillae Rhizoma were identified using UHPLC-Q-Exactive Orbitrap HRMS. Depending on the bioavailability and drug-like properties of the common components, the active chemical components were screened, and then their protein targets were collected using the Traditional Chinese Medicine Database and Analysis Platform(TCMSP) and SwissTargetPrediction database. The protein targets related to diseases were retrieved from the databases DrugBank, TTD and GeneCards to produce a Venn diagram. The shared targets were obtained between drugs and diseases as "homotherapy for heteropathy" targets. The protein-protein interaction(PPI) was analyzed with the STRING database, and KEGG and GO analyses of the "homotherapy for heteropathy" targets were performed using the Bioconductor database. Cytoscape 3.7.2 software was employed to construct the "chemical components of Bletillae Rhizoma-homotherapy for heteropathy targets" network and PPI network, and topological analysis was conducted to screen out the key active chemical components and core targets. Finally, the affinity between the active components and core targets was evaluated using the molecular docking by AutoDock Vina 4.2.6, which verified the interaction between them. Thirteen common peaks were identified by fingerprint chromatography, and the similarity between different batches was 0.941-0.998. Fifty-three chemical components were identified by mass spectrometry in Bletillae Rhizoma, and 18 common chemical constituents were obtained in the 17 batches of Bletillae Rhizoma. Network pharmacologic screening showed that the pharmacodynamic substances of Bletillae Rhizoma for melasma, gastrointestinal hemo-rrhage, lung cancer and bronchoplumonary inflammation with "homotherapy for heteropathy" were 11 compounds, such as polysaccharides, biphenanthrenes, dihydrophenanthrenes and bibenzyls. There were 42 common targets identified for the treatment of different diseases. These targets were involved in biological processes such as cell response to chemical stress, reactive oxygen species and positive regulation of protein kinase B signal transduction. They were also involved in 121 signaling pathways, encompassing vital pathways such as PI3K-Akt, ErbB, Rap1, FoxO, MAPK and estrogen. Molecular docking results showed a strong affinity between the key active components and the core targets. This study provides a preliminary explanation of how Bletillae Rhizoma exerts its therapeutic effect on chloasma, gastrointestinal hemorrhage, lung cancer, and bronchopneumonic lesions as "homotherapy for heteropathy" through a combined action involving multiple components, targets, and pathways. These findings offer a certain theoretical basis for the further deve-lopment and application of Bletillae Rhizoma.

Phylogenomic insights into the historical biogeography, character-state evolution, and species diversification rates of Cypripedioideae (Orchidaceae).

Cypripedioideae (slipper orchids; Orchidaceae) currently consist of âˆ¼200 herbaceous species with a strikingly disjunctive distribution in tropical and temperate regions of both hemispheres. In this study, an updated phylogeny with representatives from all five cypripedioid genera was presented based on maximum likelihood and Bayesian inference of plastome and low-copy nuclear genes. Phylogenomic analyses indicated that each genus is monophyletic, but some relationships (e.g., those among Cypripedium sects. Acaulia, Arietinum, Bifolia, Flabellinervia, Obtusipetala and Palangshanensia) conflict with those in previous studies based on Sanger data. Cypripedioideae appeared to have arisen in South America and/or the adjacent Qinghai-Tibet Plateau and Hengduan Mountains âˆ¼35 Mya. We inferred multiple dispersal events between East Asia and North America in Cypripedium, and between mainland Southeast Asia and the Malay Archipelago in Paphiopedilum. In the Americas, divergences among four genera (except Cypripedium) occurred around 31-20 Mya, long before the closure of the Isthmus of Panama, indicating the importance of long-distance dispersal. Evolutionary patterns between morphological and plastome character evolution suggested several traits, genome size and NDH genes, which are likely to have contributed to the success of slipper orchids in alpine floras and low-elevation forests. Species diversification rates were notably higher in epiphytic clades of Paphiopedilum than in other, terrestrial cypripedioids, paralleling similar accelerations associated with epiphytism in other groups. This study also suggested that sea-level fluctuations and mountain-building processes promoted the diversification of the largest genera, Paphiopedilum and Cypripedium.

Separation and purification of benzylester glucosides and derivatives from tubers of Gymnadenia conopsea (L.) R. Br. by linear gradient counter-current chromatography combined with elution-extrusion mode.

Tubers of Gymnadenia conopsea (L.) R. Br. (Orchidaceae), a traditional medicine and food homologous plant, has a broad application and development prospect in the food and drug industries. Benzylester glucosides, the main effective active components in this plant, are difficult to separate due to their similar structures and high polarity. In this study, linear gradient counter-current chromatography was used to separate benzylester glucosides and derivatives, combined with elution-extrusion mode. The main separation parameters were optimized, including the ratio of mobile phase and sample loading. Finally, seven compounds were successfully separated, including 4-hydroxybenzyl alcohol (1), 4-hydroxybenzaldehyde (2), dactylorhin B (3), loroglossin (4), dactylorhin A (5), 4-(ethoxymethyl) phenol (6), and militarine (7). The structures were analyzed by mass spectrometry and nuclear magnetic resonance spectrometry. According to our findings, the established method was an efficient approach to separate benzylester glucosides and derivatives from tubers of G. conopsea. The established strategy could be applied to purify other similar high-polarity compounds from complex natural products.

Integrated multi-omic analysis reveals the carbon metabolism-mediated regulation of polysaccharide biosynthesis by suitable light intensity in Bletilla striata leaves.

Bletilla striata, valued for its medicinal and ornamental properties, remains largely unexplored in terms of how light intensity affects its physiology, biochemistry, and polysaccharide formation. In this 5-month study, B. striata plants were exposed to three different light intensities: low light (LL) (5-20 µmol m-2·s-1), middle light (ML) (200 µmol m-2·s-1), and high light (HL) (400 µmol m-2·s-1). The comprehensive assessment included growth, photosynthetic apparatus, chlorophyll fluorescence electron transport, and analysis of differential metabolites based on the transcriptome and metabolome data. The results indicated that ML resulted in the highest plant height and total polysaccharide content, enhanced photosynthetic apparatus performance and light energy utilization, and stimulated carbon metabolism and carbohydrate accumulation. HL reduced Chl content and photosynthetic apparatus functionality, disrupted OEC activity and electron transfer, stimulated carbon metabolism and starch and glucose accumulation, and hindered energy metabolism related to carbohydrate degradation and oxidation. In contrast, LL facilitated leaf growth and increased chlorophyll content but decreased plant height and total polysaccharide content, compromised the photosynthetic apparatus, hampered light energy utilization, stimulated energy metabolism related to carbohydrate degradation and oxidation, and inhibited carbon metabolism and carbohydrate synthesis. Numerous genes in carbon metabolism were strongly related to polysaccharide metabolites. The katE and cysK genes in carbon metabolism were strongly related not only to polysaccharide metabolites, but also to genes involved in polysaccharide biosynthesis. Our results highlight that light intensity plays a crucial role in affecting polysaccharide biosynthesis in B. striata, with carbon metabolism acting as a mediator under suitable light intensity conditions.

Genome of the early spider-orchid Ophrys sphegodes provides insights into sexual deception and pollinator adaptation.

Pollinator-driven evolution of floral traits is thought to be a major driver of angiosperm speciation and diversification. Ophrys orchids mimic female insects to lure male pollinators into pseudocopulation. This strategy, called sexual deception, is species-specific, thereby providing strong premating reproductive isolation. Identifying the genomic architecture underlying pollinator adaptation and speciation may shed light on the mechanisms of angiosperm diversification. Here, we report the 5.2 Gb chromosome-scale genome sequence of Ophrys sphegodes. We find evidence for transposable element expansion that preceded the radiation of the O. sphegodes group, and for gene duplication having contributed to the evolution of chemical mimicry. We report a highly differentiated genomic candidate region for pollinator-mediated evolution on chromosome 2. The Ophrys genome will prove useful for investigations into the repeated evolution of sexual deception, pollinator adaptation and the genomic architectures that facilitate evolutionary radiations.

Cattleya walkeriana Gardner (Orchidaceae) propagation: culture medium, sealing system and irradiance.

This study aimed to evaluate the influence of culture media, irradiance, and sealing system on the in vitro and ex vitro growth of Cattleya walkeriana Gardner. We used MS medium as culture medium, supplemented with 30 g L-1 of sucrose and solidified with 7.0 g L-1 of bacteriological agar. This medium served as a control, while for the other treatments we supplemented the media as follows: 2) MS with 150 g L-1 of banana pulp = P150; 3) MS with 300 g L-1 of banana pulp = P300; 4) MS with 150 g L-1 of banana peel = PE150; and 5) MS with 300 g L-1 of banana peel = PE300. The irradiances were provided by 3000K LED lamps: 86 µmol m-2 s-1 (Irradiance-1) and 128 µmol m-2 s-1 (Irradiance-2) and the conventional sealing (CSS) and sealing systems that allow gas exchange (GESS). After 120 (in vitro) and 180 days (ex vitro) of cultivation, we evaluated them for pseudobulb (PN), leaf (LN) and root number (RN), plant height (PH), pseudobulb diameter (PD), longest leaf (LL) and root length (RL), fresh mass (TFM) and survival (%SURV). There was a significant interaction for all the variables analyzed. The CM x SS double interaction was significant for PH, LL, and RL. The CM x I x SS interaction was significant for PN, LN, RN, PD, TFM, and %SURV traits of C. walkeriana grown in vitro. There was a significant interaction between CM x I x SS for all C. walkeriana traits evaluated in ex vitro culture. Using the medium with up to 150 g L-1 of banana pulp combined with Irradiance-2 and CSS provided the highest values for in vitro plant growth. However, prior cultivation in MS medium, Irradiance-1, and CSS provided the greatest survival and establishment of this species plants in ex vitro culture.

Combined metabolomics and transcriptomics reveal the secondary metabolite networks in different growth stages of Bletilla striata (Thunb.) Reichb.f.

BACKGROUND: Bletilla striata (Thunb.) Reichb.f. (B. striata) is a traditional Chinese medicinal herb. B. striata polysaccharides (BSP), stilbenes and 2-isobutyl malic acid glucosoxy-benzyl ester compounds are the main active ingredients in B. striata. However, there is limited report on the changes of medicinal components and their biosynthesis regulation mechanisms in the tubers of B. striata at different stages. METHOD: The tubers of B. striata were collected during the flowering period, fruiting period, and harvest period to determine the total polysaccharide content using the phenol sulfuric acid method. The changes in secondary metabolites in the tubers at these stages were analyzed by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS), and transcriptomics was conducted for further exploration of their biosynthetic pathways. RESULT: The BSP content gradually increases from the flowering period to the fruiting period as the tubers develop, reaching its peak, but subsequently decreases at harvest time, which may be associated with the germination of B. striata buds in later stage. A total of 294 compounds were identified in this study. Among them, a majority of the compounds, such as 2-isobutyl malate gluconoxy-benzyl ester, exhibited high content during the fruit stage, while stilbenes like coelonin, 3'-O-methylbatatasin III, and blestriarene A accumulated during the harvesting period. The transcriptome data also revealed a substantial number of differentially expressed genes at various stages, providing a partial explanation for the complex changes in metabolites. We observed a correspondence between the expression pattern of GDP-Man biosynthesis-related enzyme genes and cumulative changes in BSP. And identified a positive correlation between 9 transcription factors and genes associated with polysaccharide biosynthesis, while 5 transcription factors were positively correlated with accumulation of 2-isobutyl malate gluconoxy-benzyl ester compounds and 5 transcription factors exhibited negative correlated with stilbene accumulation. CONCLUSION: It is imperative to determine the appropriate harvesting period based on the specific requirements of different active ingredients and the accumulation patterns of their metabolites. Considering the involvement of multiple transcription factors in the biosynthesis and accumulation of its active ingredients, a comprehensive investigation into the specific regulatory mechanisms that facilitate high-quality cultivation of B. striata is imperative.

Complete chloroplast genome structural characterization of two Aerides (Orchidaceae) species with a focus on phylogenetic position of Aerides flabellata.

BACKGROUND: The disputed phylogenetic position of Aerides flabellata Rolfe ex Downie, due to morphological overlaps with related species, was investigated based on evidence of complete chloroplast (cp) genomes. The structural characterization of complete cp genomes of A. flabellata and A. rosea Lodd. ex Lindl. & Paxton were analyzed and compared with those of six related species in "Vanda-Aerides alliance" to provide genomic information on taxonomy and phylogeny. RESULTS: The cp genomes of A. flabellata and A. rosea exhibited conserved quadripartite structures, 148,145 bp and 147,925 bp in length, with similar GC content (36.7 ~ 36.8%). Gene annotations revealed 110 single-copy genes, 18 duplicated in inverted regions, and ten with introns. Comparative analysis across related species confirmed stable sequence identity and higher variation in single-copy regions. However, there are notable differences in the IR regions between two Aerides Lour. species and the other six related species. The phylogenetic analysis based on CDS from complete cp genomes indicated that Aerides species except A. flabellata formed a monophyletic clade nested in the subtribe Aeridinae, being a sister group to Renanthera Lour., consistent with previous studies. Meanwhile, a separate clade consisted of A. flabellata and six Vanda R. Br. species was formed, as a sister taxon to Holcoglossum Schltr. CONCLUSIONS: This research was the first report on the complete cp genomes of A. flabellata. The results provided insights into understanding of plastome evolution and phylogenetic relationships of Aerides. The phylogenetic analysis based on complete cp genomes showed that A. flabellata should be placed in Vanda rather than in Aerides.

Evidence of introduced honeybees (Apis mellifera) as pollen wasters in orchid pollination.

Biological invasions threaten global biodiversity, altering landscapes, ecosystems, and mutualistic relationships like pollination. Orchids are one of the most threatened plant families, yet the impact of invasive bees on their reproduction remains poorly understood. We conduct a global literature survey on the incidence of invasive honeybees (Apis mellifera) on orchid pollination, followed by a study case on Australian orchids. Our literature survey shows that Apis mellifera is the primary alien bee visiting orchids worldwide. However, in most cases, introduced honeybees do not deposit orchid pollen. We also test the extent to which introduced honeybees affect orchid pollination using Diuris brumalis and D. magnifica. Diuris brumalis shows higher fruit set and pollination in habitats with both native and invasive bees compared to habitats with only introduced bees. Male and female reproductive success in D. magnifica increases with native bee abundance, while conversely pollinator efficiency decreases with honeybee abundance and rises with habitat size. Our results suggest that introduced honeybees are likely involved in pollen removal but do not effectively deposit orchid pollen, acting as pollen wasters. However, Apis mellifera may still contribute to pollination of Diuris where native bees no longer exist. Given the global occurrence of introduced honeybees, we warn that certain orchids may suffer from pollen depletion by these invaders, especially in altered habitats with compromised pollination communities.