The rhizosphere microbiome and its influence on the accumulation of metabolites in Bletilla striata (Thunb.) Reichb. f.

BACKGROUND: Bletilla striata (Thunb.) Reichb. f. (B. striata) is a perennial herbaceous plant in the Orchidaceae family known for its diverse pharmacological activities, such as promoting wound healing, hemostasis, anti-inflammatory effects, antioxidant properties, and immune regulation. Nevertheless, the microbe-plant-metabolite regulation patterns for B. striata remain largely undetermined, especially in the field of rhizosphere microbes. To elucidate the interrelationships between soil physics and chemistry and rhizosphere microbes and metabolites, a comprehensive approach combining metagenome analysis and targeted metabolomics was employed to investigate the rhizosphere soil and tubers from four provinces and eight production areas in China. RESULTS: Our study reveals that the core rhizosphere microbiome of B. striata is predominantly comprised of Paraburkholderia, Methylibium, Bradyrhizobium, Chitinophaga, and Mycobacterium. These microbial species are recognized as potentially beneficial for plants health. Comprehensive analysis revealed a significant association between the accumulation of metabolites, such as militarine and polysaccharides in B. striata and the composition of rhizosphere microbes at the genus level. Furthermore, we found that the soil environment indirectly influenced the metabolite profile of B. striata by affecting the composition of rhizosphere microbes. Notably, our research identifies soil organic carbon as a primary driving factor influencing metabolite accumulation in B. striata. CONCLUSION: Our fndings contribute to an enhanced understanding of the comprehensive regulatory mechanism involving microbe-plant-metabolite interactions. This research provides a theoretical basis for the cultivation of high-quality traditional Chinese medicine B. striata.

Assembly and comparative analysis of the complete multichromosomal mitochondrial genome of Cymbidium ensifolium, an orchid of high economic and ornamental value.

BACKGROUND: Orchidaceae is one of the largest groups of angiosperms, and most species have high economic value and scientific research value due to their ornamental and medicinal properties. In China, Chinese Cymbidium is a popular ornamental orchid with high economic value and a long history. However, to date, no detailed information on the mitochondrial genome of any species of Chinese Cymbidium has been published. RESULTS: Here, we present the complete assembly and annotation of the mitochondrial genome of Cymbidium ensifolium (L.) Sw. The mitogenome of C. ensifolium was 560,647 bp in length and consisted of 19 circular subgenomes ranging in size from 21,995 bp to 48,212 bp. The genome encoded 35 protein-coding genes, 36 tRNAs, 3 rRNAs, and 3405 ORFs. Repeat sequence analysis and prediction of RNA editing sites revealed a total of 915 dispersed repeats, 162 simple repeats, 45 tandem repeats, and 530 RNA editing sites. Analysis of codon usage showed a preference for codons ending in A/T. Interorganellar DNA transfer was identified in 13 of the 19 chromosomes, with plastid-derived DNA fragments representing 6.81% of the C. ensifolium mitochondrial genome. The homologous fragments of the mitochondrial genome and nuclear genome were also analysed. Comparative analysis showed that the GC content was conserved, but the size, structure, and gene content of the mitogenomes varied greatly among plants with multichromosomal mitogenome structure. Phylogenetic analysis based on the mitogenomes reflected the evolutionary and taxonomic statuses of C. ensifolium. Interestingly, compared with the mitogenomes of Cymbidium lancifolium Hook. and Cymbidium macrorhizon Lindl., the mitogenome of C. ensifolium lost 8 ribosomal protein-coding genes. CONCLUSION: In this study, we assembled and annotated the mitogenome of C. ensifolium and compared it with the mitogenomes of other Liliidae and plants with multichromosomal mitogenome structures. Our findings enrich the mitochondrial genome database of orchid plants and reveal the rapid structural evolution of Cymbidium mitochondrial genomes, highlighting the potential for mitochondrial genes to help decipher plant evolutionary history.

Development and Application of a Duplex RT-RPA Assay for the Simultaneous Detection of Cymbidium mosaic virus and Odontoglossum ringspot virus.

Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are among the world's most serious and widespread orchid viruses; they often infect orchids, causing devastating losses to the orchid industry. Therefore, it is critical to establish a method that can rapidly and accurately detect viruses in the field using simple instruments, which will largely reduce the further spread of viruses and improve the quality of the orchid industry and is suitable for mass promotion and application at grassroots agrotechnical service points. In this investigation, we established a rapid amplification method for virus detection at 39 °C for 35 min to detect the presence of CymMV and ORSV simultaneously, sensitively, and specifically in orchids. Primers for the capsid protein (CP)-encoding genes of both viruses were designed and screened, and the reaction conditions were optimized. The experimental amplification process was completed in just 35 min at 39 °C. There were no instances of nonspecific amplification observed when nine other viruses were present. The RPA approach had detection limits of 104 and 103 copies for pMD19T-CymMV and pMD19T-ORSV, respectively. Moreover, the duplex RT-RPA investigation confirmed sensitivity and accuracy via a comparison of detection results from 20 field samples with those of a gene chip. This study presents a precise and reliable detection method for CymMV and ORSV using RT-RPA. The results demonstrate the potential of this method for rapid virus detection. It is evident that this method could have practical applications in virus detection processes.

Phylogenomics and biogeographical diversification of Collabieae (Orchidaceae) and its implication in the reconstruction of the dynamic history of Asian evergreen broadleaved forests.

The tribe Collabieae (Epidendroideae, Orchidaceae) comprises approximately 500 species. Generic delimitation within Collabieae are confusing and phylogenetic interrelationships within the Collabieae have not been well resolved. Plastid genomes and nuclear internal transcribed spacer (ITS) sequences were used to estimate the phylogenetic relationships, ancestral ranges, and diversification rates of Collabieae. The results showed that Collabieae was subdivided into nine clades with high support. We proposed to combine Ancistrochilus and Pachystoma into Spathoglottis, merge Collabium and Chrysoglossum into Diglyphosa, and separate Pilophyllum and Hancockia as distinctive genera. The diversification of the nine clades of Collabieae might be associated with the uplift of the Himalayas during the Late Oligocene/Early Miocene. The enhanced East Asian summer monsoon in the Late Miocene may have promoted the rapid diversification of Collabieae at a sustained high diversification rate. The increased size of terrestrial pseudobulbs may be one of the drivers of Collabieae diversification. Our results suggest that the establishment and development of evergreen broadleaved forests facilitated the diversification of Collabieae.

Phenotypic variation seems not to be associated with the genetic profile in Zygopetalum (Orchidaceae): a case study of a high-elevation rocky complex.

BACKGROUND: Hybridization associated with polyploidy studies is rare in the tropics. The genus Zygopetalum (Orchidaceae) was investigated here as a case study of Neotropical plants. In the rocky highlands of the Ibitipoca State Park (ISP), southeast Brazil, individuals with intermediate colors and forms between the species Z. maculatum and Z. triste were commonly identified. METHODS AND RESULTS: Chromosomal analysis and DNA quantity showed a uniform population. Regardless of the aspects related to the color and shape of floral structures, all individuals showed 2n = 96 chromosomes and an average of 14.05 pg of DNA. Irregularities in meiosis associated with chromosome number and C value suggest the occurrence of polyploidy. The genetic distance estimated using ISSR molecular markers revealed the existence of genetic variability not related to morphological clusters. Morphometric measurements of the flower pieces revealed that Z. maculatum shows higher variation than Z. triste although lacking a defined circumscription. CONCLUSION: The observed variation can be explained by the polyploid and phenotypic plasticity resulting from the interaction of the genotypes with the heterogeneous environments observed in this habitat.

Root photosynthesis prevents hypoxia in the epiphytic orchid Phalaenopsis.

Orchids (Phalaenopsis spp.) growing in tropical and subtropical regions are epiphytes. As such, they grow on trees with the root system utilised to anchor themselves to tree branches. These roots are highly specialised, display a large diameter and are often green, suggesting the ability to carry out photosynthesis. However, the role of photosynthesis in orchid roots is controversial. Orchids that are leafless can photosynthesise in their roots, thus indicating that some orchid roots carry out photosynthesis in a similar manner to leaves. However, the primary site of photosynthesis in orchids are in their leaves, and the roots of epiphytic orchids may mostly conduct internal refixation of respiratory CO2 . Besides contributing to the overall carbon metabolism of orchid plants, oxygen produced through root photosynthesis may also be important by alleviating potential root hypoxia. The bulky tissue of most epiphytic orchid roots suggests that oxygen diffusion in these roots can be limited. Here, we demonstrate that the bulky roots of a widely commercially cultivated orchid belonging to the genus Phalaenopsis are hypoxic in the dark. These roots are photosynthetically active and produce oxygen when exposed to light, thus mitigating root hypoxia.

A dual network cross-linked hydrogel with multifunctional Bletilla striata polysaccharide/gelatin/tea polyphenol for wound healing promotion.

Wound healing is a dynamic and complex biological process, and traditional biological excipients cannot meet the needs of the wound healing process, and there is an urgent need for a biological dressing with multifunctionality and the ability to participate in all stages of wound healing. This study developed tea polyphenol (TP) incorporated multifunctional hydrogel based on oxidized Bletilla striata polysaccharide (OBSP) and adipic acid dihydrazide modified gelatin (Gel-ADH) with antimicrobial, antioxidant hemostatic, and anti-inflammatory properties to promote wound healing. The composite OBSP, Gel-ADH, TP (OBGTP) hydrogels prepared by double crosslinking between OBSP, TP and Gel-ADH via Schiff base bonding and hydrogen bonding had good rheological and swelling properties. The introduction of TP provided the composite hydrogel with excellent antioxidant antibacterial activities against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coil). In the rat liver hemorrhage model and skin injury model, the OBGTP composite hydrogel had significant (p < 0.001) hemostatic ability, and had the ability to accelerate collagen deposition, reduce the expression of inflammatory factors, and promote rapid wound healing. In addition, OBGTP hydrogels had adhesive properties and good biocompatibility. In conclusion, OBGTP multifunctional composite hydrogels have great potential for wound healing applications.

Partial mycoheterotrophy in the leafless orchid Eulophia zollingeri specialized on wood-decaying fungi.

Although the absence of normal leaves is often considered a sign of full heterotrophy, some plants remain at least partially autotrophic despite their leafless habit. Leafless orchids with green stems and capsules probably represent a late evolutionary stage toward full mycoheterotrophy and serve as valuable models for understanding the pathways leading to this nutritional strategy. In this study, based on molecular barcoding and isotopic analysis, we explored the physiological ecology of the leafless orchid Eulophia zollingeri, which displays green coloration, particularly during its fruiting phase. Although previous studies had shown that E. zollingeri, in its adult stage, is associated with Psathyrellaceae fungi and exhibits high 13C isotope signatures similar to fully mycoheterotrophic orchids, it remained uncertain whether this symbiotic relationship is consistent throughout the orchid's entire life cycle and whether the orchid relies exclusively on mycoheterotrophy for its nutrition during the fruiting season. Our study has demonstrated that E. zollingeri maintains a specialized symbiotic relationship with Psathyrellaceae fungi throughout all life stages. However, isotopic analysis and chlorophyll data have shown that the orchid also engages in photosynthesis to meet its carbon needs, particularly during the fruiting stage. This research constitutes the first discovery of partial mycoheterotrophy in leafless orchids associated with saprotrophic non-rhizoctonia fungi.

The effect of Bletilla striata polysaccharide on the physical and healing properties of curdlan-based hydrogel for wound healing.

Bletilla striata polysaccharide (BSP) was added to curdlan to form a blend hydrogel through a simple heating-cooling procedure to improve the hydrophilicity and healing efficacy of curdlan-based hydrogel used in wound healing. We explored the interplay between BSP and curdlan, studied how BSP concentration affects the physical properties and microstructures of hydrogels, and examined the biocompatibility and healing properties of the blend hydrogel. It was proved that the hydrogel framework was primarily formed by ordered arranged curdlan molecules, with BSP uniformly dispersed and intertwined with curdlan through hydrogen bonding. This effectively improved its hydrophilicity and strengthened the microstructure. Curdlan was found to be compatible with BSP. The blend hydrogel B3Cd3 (containing 1.5% BSP and 1.5% curdlan, w/v) was identified as the optimal formulation based on its higher water adsorption, water retention, thermal stability and interconnected microstructure, and was thus selected for further research. In vitro experiments revealed the highest cell viability of L929 in B3Cd3 extracts compared to those extracts of single-component curdlan hydrogel (Cd). In vivo, animal studies indicated that the B3Cd3 accelerated wound healing compared to the control group by improving re-epithelialization and blood vessel regeneration. On Days 3 and 11, the therapeutic benefits of B3Cd3 exceeded those of the Cd group, and no significant differences were observed in wound healing rates between the B and B3Cd3 groups from Day 7. The study proves that BSP enhances the physical and healing properties, as well as cell proliferation, of the curdlan-based hydrogel. The blend hydrogel B3Cd3, with its exceptional properties, holds potential for future application as a material for non-infected wound healing.

In sight the behavior of natural Bletilla striata polysaccharide hydrocolloids by molecular dynamics method.

Plant polysaccharides, distinguished by diverse glycosidic bonds and various cyclic sugar units, constitute a subclass of primary metabolites ubiquitously found in nature. Contrary to common understanding, plant polysaccharides typically form hydrocolloids upon dissolution in water, even though both excessively high and low temperatures impede this process. Bletilla striata polysaccharides (BSP), chosen for this kinetic study due to their regular repeating units, help elucidate the relationship between polysaccharide gelation and temperature. It is suggested that elevated temperatures enhance the mobility of BSP molecular chains, resulting in a notable acceleration of hydrogen bond breakage between BSP and water molecules and consequently, compromising the conformational stability of BSPs to some extent. This study unveils the unique relationship between polysaccharide dissolution processes and temperature from a kinetics perspective. Consequently, the conclusion provides a dynamical basis for comprehending the extraction and preparation of natural plant polysaccharide hydrocolloids, pharmaceuticals and related fields.

A review of the Stelidium group of Stelis Panzer, 1806 (Hymenoptera: Megachilidae) with two new species from western North America.

The Stelidium group is readily distinguished from all other members of the subgenus Stelis Panzer, 1806 by the combination of small body size ( 6 mm), pale maculations on the head adjacent to the inner margins of the compound eyes and laterally on the vertex in both sexes, and females with sternum 6 extended beyond tergum 6, the former with the dorsal lip trowel-shaped with the apex broadly rounded or subtruncate to more narrowly pointed. This monophyletic clade, which is endemic to North America, currently consists of members previously placed into two species groups: the permaculata group containing S. anasazi Parker & Griswold, 2013, S. ashmeadiellae Timberlake, 1941, S. permaculata Cockerell, 1898, and S. robertsoni Timberlake, 1941, and the palmarum group containing S. broemelingi Parker & Griswold, 2013, S. elongativentris Parker, 1987, and S. palmarum Timberlake, 1941; two additional species, S. herberti (Cockerell, 1916) from Mexico, and S. nyssonoides (Brues, 1903) from Texas, United States, have not been definitively placed in either species group. Two new species are herein described, one from southcentral British Columbia, Canada, the other from New Mexico, United States. A preliminary molecular phylogeny places both new species in the permaculata species group. In addition, S. herberti is also placed within the permaculata species group based on morphological similarity, sharing the multi-spotted maculation pattern on the terga. Based on molecular affinity, S. broemelingi also belongs to the permaculata species group. Because no type specimen for S. nyssonoides is seemingly available for examination, it is hereby considered nomen dubium until the specimen is found and its taxonomic status clarified in relation to the more recently described species in the permaculata species group. A key to females and diagnoses are provided for all known taxa.

The Complete Chloroplast Genomes of Bulbophyllum (Orchidaceae) Species: Insight into Genome Structure Divergence and Phylogenetic Analysis.

Bulbophyllum is one of the largest genera and presents some of the most intricate taxonomic problems in the family Orchidaceae, including species of ornamental and medical importance. The lack of knowledge regarding the characterization of Bulbophyllum chloroplast (cp) genomes has imposed current limitations on our study. Here, we report the complete cp genomes of seven Bulbophyllum species, including B. ambrosia, B. crassipes, B. farreri, B. hamatum, B. shanicum, B. triste, and B. violaceolabellum, and compared with related taxa to provide a better understanding of their genomic information on taxonomy and phylogeny. A total of 28 Bulbophyllum cp genomes exhibit typical quadripartite structures with lengths ranging from 145,092 bp to 165,812 bp and a GC content of 36.60% to 38.04%. Each genome contained 125-132 genes, encompassing 74-86 protein-coding genes, 38 tRNA genes, and eight rRNA genes. The genome arrangements, gene contents, and length were similar, with differences observed in ndh gene composition. It is worth noting that there were exogenous fragment insertions in the IR regions of B. crassipes. A total of 18-49 long repeats and 38-80 simple sequence repeats (SSRs) were detected and the single nucleotide (A/T) was dominant in Bulbophyllum cp genomes, with an obvious A/T preference. An analysis of relative synonymous codon usage (RSCU) revealed that leucine (Leu) was the most frequently used codon, while cysteine (Cys) was the least used. Six highly variable regions (rpl32-trnLUAG > trnTUGU-trnLUAA > trnFGAA-ndhJ > rps15-ycf1 > rbcL-accD > psbI-trnSGCU) and five coding sequences (ycf1 > rps12 > matK > psbK > rps15) were identified as potential DNA markers based on nucleotide diversity. Additionally, 31,641 molecular diagnostic characters (MDCs) were identified in complete cp genomes. A phylogenetic analysis based on the complete cp genome sequences and 68 protein-coding genes strongly supported that 28 Bulbophyllum species can be divided into four branches, sects. Brachyantha, Cirrhopetalum, and Leopardinae, defined by morphology, were non-monophyly. Our results enriched the genetic resources of Bulbophyllum, providing valuable information to illustrate the complicated taxonomy, phylogeny, and evolution process of the genus.

Identification of Viruses Infecting Phalaenopsis Orchids Using Nanopore Sequencing and Development of an RT-RPA-CRISPR/Cas12a for Rapid Visual Detection of Nerine Latent Virus.

Phalaenopsis orchids are one of the most popular ornamental plants. More than thirty orchid viruses have been reported, and virus-infected Phalaenopsis orchids significantly lose their commercial value. Therefore, the development of improved viral disease detection methods could be useful for quality control in orchid cultivation. In this study, we first utilized the MinION, a portable sequencing device based on Oxford Nanopore Technologies (ONT) to rapidly detect plant viruses in Phalaenopsis orchids. Nanopore sequencing revealed the presence of three plant viruses in Phalaenopsis orchids: odontoglossum ringspot virus, cymbidium mosaic virus, and nerine latent virus (NeLV). Furthermore, for the first time, we detected NeLV infection in Phalaenopsis orchids using nanopore sequencing and developed the reverse transcription-recombinase polymerase amplification (RT-RPA)-CRISPR/Cas12a method for rapid, instrument-flexible, and accurate diagnosis. The developed RT-RPA-CRISPR/Cas12a technique can confirm NeLV infection in less than 20 min and exhibits no cross-reactivity with other viruses. To determine the sensitivity of RT-RPA-CRISPR/Cas12a for NeLV, we compared it with RT-PCR using serially diluted transcripts and found a detection limit of 10 zg/µL, which is approximately 1000-fold more sensitive. Taken together, the ONT platform offers an efficient strategy for monitoring plant viral pathogens, and the RT-RPA-CRISPR/Cas12a method has great potential as a useful tool for the rapid and sensitive diagnosis of NeLV.

Process optimization and character evaluation of Bletilla striata polysaccharide (BSP) and chitosan (CS) composite hemostatic sponge (BSP-CS).

Bletilla striata polysaccharide (BSP) and chitosan (CS) were chemically cross-linked using oxalyl chloride to prepare a composite hemostatic sponge (BSP-CS), and the process parameters were optimized using the Box-Behnken design (BBD) with response surface methodology. To optimize the performance of the hemostatic sponge, we adjusted the ratio of independent variables, the amount of oxalyl chloride added, and the freeze-dried volume. A series of evaluations were conducted on the hemostatic applicability of BSP-CS. The characterization results revealed that BSP-CS had a stable bacteriostatic effect on Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa within 72 h, and the bacteriostatic rate was above 30%. The CCK-8 cytotoxicity test demonstrated that BSP-CS had a certain effect on promoting cell proliferation of L929 cells. In the mouse tail-cutting experiment, the hemostasis time of BSP-CS was 463.0±38.16 s, shortened by 91.3 s on average compared with 554.3±34.67 s of the gauze group. The blood loss of the BSP-CS group was 28.47±3.74 mg, which was 34.7% lower than that of the control gauze group (43.6±3.83 mg). In the in vitro coagulation experiment, the in vitro coagulation index of the BSP-CS group was 97.29%±1.8%, which was reduced to 8.6% of the control group. The CT value of the BSP-CS group was 240±15 s, which was 155 s lower than that of the gauze group (355±31.22 s). All characterization results indicate that BSP-CS is an excellent hemostatic material.

A new orchid species expands Darwin's predicted pollination guild in Madagascar.

The world-renowned pollination system of the long-spurred orchid Angraecum sesquipedale Thouars and the long-tongued hawkmoth Xanthopan praedicta (Rothschild & Jordan, 1903), from Madagascar, is the best-known example of the predictive power of evolutionary ecology1,2, yet its actual degree of specialisation remains poorly described due to the incompleteness of the pollination record of X. praedicta. Here, we describe another species from Madagascar, an angraecoid orchid distantly related to the genus Angraecum Bory, that has evolved these extreme adaptations to a single pollinator after a pollinator shift. It bears the longest spur of any flowering plant, relative to flower diameter, reaching 33 cm. The discovery of a species with such an exceptionally long spur is a rare event, the most recent dating to 19653. This novelty is described here as Solenangis impraedicta (Figure 1A-F) and discussed in a phylogenetic framework. Its conservation status is assessed as Endangered.

Isolation, invitro, invivo anti-inflammatory, analgesic and antioxidant potential of Habenaria plantegania Lindl.

Habenaira plantaginea belong to orchid family which is native to Asia. Members of this family are commonly famous for the cure of pain and inflammation. To date, no research was found on isolation of compounds from this plant for the treatment of inflammation and analgesia nor has been published to our knowledge. The purpose of this study was to evaluate an analgesic, anti-inflammatory and anti-oxidant activity of the isolated compound from the most potent chloroform sub-fraction and the isolated compounds form the habenaria plantaginea. Anti-inflammatory analgesic and antioxidant potential of the various chloroform sub-fractions and isolated compounds from the most potent sub-fraction (HP-1 & HP-1) were screened for their in vitro enzymatic assays. Furthermore, prior to in-vivo investigation, the isolated compounds were subjected for their toxicity study. The potent compound was then examined for acetic acid-induced writhing, hot plate test, carrageenan-induced inflammation assays. Further various phlogistic agents were used for the evaluation of mechanism. In the COX-2 inhibitory assay the chloroform sub fraction Cf-4 demonstrated excellent activity as compared to the other sub-fraction with 92.15% inhibition. The COX-2 enzyme make prostaglandins which are directly involved in inflammation. Likewise against 5-LOX the Cf-4 was the most potent sub-fraction with IC50 3.77 µg/mL. The 5-LOX catalyzes the biosynthesis of leukotrienes which is a group of lipid mediators of inflammation derived from arachidonic acid. Free radicals can induce inflammation through cellular damage while chronic inflammation generates a large number of free radicals, whose eventually lead to inflammation. In antioxidant assays the Cf-4 fraction was displayed excellent results against ABTS, DPPH and H2O2 free radical with 88.88, 77.44, and 65.52% inhibition at highest concentration. Likewise, the compound HP-1 demonstrated 88.81, 89.34 and 80.43% inhibition while compound HP-2 displayed 84.34, 91.52 and 82.34% inhibition against ABTS, DPPH and H2O2 free radical which were comparable to the standard drug ascorbic acid respectively. This study's findings validate the use of this species as traditional use.

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

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

Breeding of ornamental orchids with focus on Phalaenopsis: current approaches, tools, and challenges for this century.

Ornamental orchid breeding programs have been conducted to develop commercially valuable cultivars with improved characteristics of commercial interest, such as size, flower color, pattern, shape, and resistance to pathogens. Conventional breeding, including sexual hybridization followed by selection of desirable characteristics in plants, has so far been the main method for ornamental breeding, but other techniques, including mutation induction by polyploidization and gamma irradiation, and biotechnological techniques, such as genetic transformation, have also been studied and used in ornamental breeding programs. Orchids are one of the most commercially important families in floriculture industry, having very particular reproductive biology characteristics and being a well-studied group of ornamentals in terms of genetic improvement. The present review focuses on the conventional and biotechnological techniques and approaches specially employed in breeding Phalaenopsis orchids, the genus with highest worldwide importance as an ornamental orchid, highlighting the main limitations and strengths of the approaches. Furthermore, new opportunities and future prospects for ornamental breeding in the CRISPR/Cas9 genome editing era are also discussed. We conclude that conventional hybridization remains the most used method to obtain new cultivars in orchids. However, the emergence of the first biotechnology-derived cultivars, as well as the new biotechnological tools available, such as CRISPR-Cas9, rekindled the full potential of biotechnology approaches and their importance for improve ornamental orchid breeding programs.

Mycobiont identity and light conditions affect belowground morphology and physiology of a mixotrophic orchid Cremastra variabilis (Orchidaceae).

We have investigated whether mycobiont identity and environmental conditions affect morphology and physiology of the chlorophyllous orchid: Cremastra variabilis. This species grows in a broad range of environmental conditions and associates with saprotrophic rhizoctonias including Tulasnellaceae and saprotrophic non-rhizoctonian fungi from the family Psathyrellaceae. We cultured the orchid from seeds under aseptic culture conditions and subsequently inoculated the individuals with either a Tulasnellaceae or a Psathyrellaceae isolate. We observed underground organ development of the inoculated C. variabilis plants and estimated their nutritional dependency on fungi using stable isotope abundance. Coralloid rhizome development was observed in all individuals inoculated with the Psathyrellaceae isolate, and 1-5 shoots per seedling grew from the tip of the coralloid rhizome. In contrast, individuals associated with the Tulasnellaceae isolate did not develop coralloid rhizomes, and only one shoot emerged per plantlet. In darkness, δ13C enrichment was significantly higher with both fungal isolates, whereas Î´15N values were only significantly higher in plants associated with the Psathyrellaceae isolate. We conclude that C. variabilis changes its nutritional dependency on fungal symbionts depending on light availability and secondly that the identity of fungal symbiont influences the morphology of underground organs.