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.

Biochemical and transcriptomic analyses of the symbiotic interaction between Cremastra appendiculata and the mycorrhizal fungus Coprinellus disseminatus.

BACKGROUND: Cremastra appendiculata is a rare terrestrial orchid with a high market value as an ornamental and medicinal plant. However, the species depends entirely on fungi for seed germination under natural conditions. In a previous study, we have successfully isolated and identified the mycorrhizal fungus Coprinellus disseminatus which was able to induce the germination of C. appendiculata seeds. We then speculated that C. disseminatus may do so by breaking the testa imposed dormancy of the seeds. In this study, biochemical and transcriptomic analyses were used to characterize the germination of C. appendiculata seeds, collected at different stages of germination, as affected by C. disseminatus. RESULTS: The lignocellulose in the seeds coat of C. appendiculata was degraded by the mycorrhizal fungus resulting in facilitated absorption of water. The rate of decline in lignin content was 67 and 73% at 6 and 12 days after sowing, respectively. The water content increased from 13 to 90% during symbiosis. A total of 15,382 genes showing significantly different levels of expression (log2 FPKM≥2.0, Qvalue≤0.05) were successfully identified among all libraries, where the highest number of DEGs was shared between 6 days versus 0 day after symbiotic germination. Gene annotation results suggested that 15 key genes related water-status, such as DHN gene family and Xero 1 were down-regulated. The genes zeaxanthin epoxidase ZEP, 9-cis-epoxycarotenoid dioxygenase NCED3 and ß-carotene hydroxylase involved in the biosynthesis of abscisic acid (ABA) were significantly down-regulated in 6 days as compared to 0 day after symbiotic germination. CONCLUSIONS: This work demonstrates that mycorrhizal fungus C. disseminatus can stimulate C. appendiculata seeds germination through a mechanism of breaking the testa imposed dormancy and inducing water absorption of the embryo.

FT-like paralogs are repressed by an SVP protein during the floral transition in Phalaenopsis orchid.

KEY MESSAGE: An SVP protein, PhSVP, bound to the CArG-boxes in the promoter regions of FT-like paralogs and repressed their expression, thus affecting the floral transition in Phalaenopsis orchid. Phalaenopsis is an important ornamental flower native to tropical rain forests. It usually reaches vegetative maturity after 4-5 leaves and, after a juvenile stage, forms a flower spike (inflorescence) from the axillary buds. The PEBP gene family encodes a phosphatidyl-ethanolamine-binding protein (PEBP) domain involved in regulating flowering and other aspects of plant development. Here, we identified eight PEBP family genes in Phalaenopsis and detected the expression patterns of seven of them in various organs. Among them, PhFT1 (Phalaenopsis hybrid FLOWERING LOCUS T1), PhFT3, PhFT5, and PhMFT (Phalaenopsis hybrid MOTHER OF FT AND TFL1) promoted flowering in transgenic Arabidopsis, while PhFT6 inhibited flowering. PhSVP (Phalaenopsis hybrid SHORT VEGETATIVE PHASE), an SVP protein that repressed flowering in Arabidopsis, bound to the CArG-boxes in the promoter regions of PhFT3, PhFT6, and PhMFT in a yeast one-hybrid assay. Additionally, dual-luciferase and transient expression assays showed that PhSVP significantly inhibits the expression of both PhFT3 and PhFT6. Together, our work provides a comprehensive understanding of the PhFT-like genes that can promote or repress flowering, and it suggests strategies for regulating the floral transition in Phalaenopsis that exploit the evolutionary versatility of PhFTs to respond to various signals stimuli.

R2R3-MYB genes coordinate conical cell development and cuticular wax biosynthesis in Phalaenopsis aphrodite.

Petals of the monocot Phalaenopsis aphrodite (Orchidaceae) possess conical epidermal cells on their adaxial surfaces, and a large amount of cuticular wax is deposited on them to serve as a primary barrier against biotic and abiotic stresses. It has been widely reported that subgroup 9A members of the R2R3-MYB gene family, MIXTA and MIXTA-like in eudicots, act to regulate the differentiation of conical epidermal cells. However, the molecular pathways underlying conical epidermal cell development and cuticular wax biosynthesis in monocot petals remain unclear. Here, we characterized two subgroup 9A R2R3-MYB genes, PaMYB9A1 and PaMYB9A2 (PaMYB9A1/2), from P. aphrodite through the transient overexpression of their coding sequences and corresponding chimeric repressors in developing petals. We showed that PaMYB9A1/2 function to coordinate conical epidermal cell development and cuticular wax biosynthesis. In addition, we identified putative targets of PaMYB9A1/2 through comparative transcriptome analyses, revealing that PaMYB9A1/2 acts to regulate the expression of cell wall-associated and wax biosynthetic genes. Furthermore, a chemical composition analysis of cuticular wax showed that even-chain n-alkanes and odd-chain primary alcohols are the main chemical constituents of cuticular wax deposited on petals, which is inconsistent with the well-known biosynthetic pathways of cuticular wax, implying a distinct biosynthetic pathway occurring in P. aphrodite flowers. These results reveal that the function of subgroup 9A R2R3-MYB family genes in regulating the differentiation of epidermal cells is largely conserved in monocots and dicots. Furthermore, both PaMYB9A1/2 have evolved additional functions controlling the biosynthesis of cuticular wax.

Genetic insights into the regulatory pathways for continuous flowering in a unique orchid Arundina graminifolia.

BACKGROUND: Manipulation of flowering time and frequency of blooming is key to enhancing the ornamental value of orchids. Arundina graminifolia is a unique orchid that flowers year round, although the molecular basis of this flowering pattern remains poorly understood. RESULTS: We compared the A. graminifolia transcriptome across tissue types and floral developmental stages to elucidate important genetic regulators of flowering and hormones. Clustering analyses identified modules specific to floral transition and floral morphogenesis, providing a set of candidate regulators for the floral initiation and timing. Among candidate floral homeotic genes, the expression of two FT genes was positively correlated with flower development. Assessment of the endogenous hormone levels and qRT-PCR analysis of 32 pathway-responsive genes supported a role for the regulatory networks in floral bud control in A. graminifolia. Moreover, WGCNA showed that flowering control can be delineated by modules of coexpressed genes; especially, MEgreen presented group of genes specific to flowering. CONCLUSIONS: Candidate gene selection coupled with hormonal regulators brings a robust source to understand the intricate molecular regulation of flowering in precious orchids.

Ex vitro symbiotic germination of the seeds of Anacamptis coriophora (L.) R.M. Bateman, Pritgeon & M.W. Chase and Orchis anatolica Boiss.

Rapid destruction of orchid habitats and over-collection of the tubers are the greatest threats to orchid diversity. To counter these threats, it is necessary to grow orchid tubers easily and quickly for economic reasons and to reintroduce populations in the habitats of species that are facing extinction. This study demonstrates a simple viability test for orchid seeds and the ex vitro symbiotic seed germination of temperate orchids. Viability of the seeds of two orchid species, Anacamptis coriophora and Orchis anatolica, was determined without any chemical treatment of the seed coat. Seeds were incubated in packs in moist cocopeats for five days during which seed viability tests being performed daily. The highest viability rate was found in the seeds that were incubated for five days (64.33% for O. coriophora; 67.19% for O. anatolica). The seeds of these orchids were sown non-axenically into a pre-inoculated soil mixture with a compatible fungus, Ceratobasidium sp. AG A. The seeds of both the orchids germinated 18 days after sowing. Leafy and rooted seedlings developed two months after sowing and the first tubers of both the species developed seven months later.

Stage Specificity, the Dynamic Regulators and the Unique Orchid Arundina graminifolia.

Orchids take years to reach flowering, but the unique bamboo orchid (Arundina graminifolia) achieves reproductive maturity in six months and then keeps on year round flowering. Therefore, studying different aspects of its growth, development and flowering is key to boost breeding programs for orchids. This study uses transcriptome tools to discuss genetic regulation in five stages of flower development and four tissue types. Stage specificity was focused to distinguish genes specifically expressed in different stages of flower development and tissue types. The top 10 highly expressed genes suggested unique regulatory patterns for each stage or tissue. The A. graminifolia sequences were blasted in Arabidopsis genome to validate stage specific genes and to predict important hormonal and cell regulators. Moreover, weighted gene co-expression network analysis (WGCNA) modules were ascertained to suggest highly influential hubs for early and late stages of flower development, leaf and root. Hormonal regulators were abundant in all data sets, such as auxin (LAX2, GH3.1 and SAUR41), cytokinin (LOG1), gibberellin (GASA3 and YAB4), abscisic acid (DPBF3) and sucrose (SWEET4 and SWEET13). Findings of this study, thus, give a fine sketch of genetic variability in Orchidaceae and broaden our understanding of orchid flower development and the involvement of multiple pathways.

Evaluation of in vitro cytotoxicity and in vivo potential toxicity of the extract from in vitro cultivated Anoectochilus roxburghii Lindl.

Anoectochilus roxburghii Lind. (A. roxburghii) has promising anti-oxidant, hyperglycemic, hepatoprotective, and immunomodulatory activities as well as anti-tumor effects. However, the pharmacological actions of in vitro cultured plants remain to be determined. Therefore, the objective of the study was to assess in vitro cytotoxicity and in vivo potential toxicity of an extract derived from in vitro cultivated A. roxburghii, termed as iARE. The total flavonoid content and predominant flavonoid compounds of extract were identified and quantitatively analyzed. The in vitro cytotoxicity of iARE was examined using several cancer and normal cell lines. The apoptotic activity and expression of apoptosis-associated genes were also examined in MCF7 cells to determine the underlying mechanisms related to anti-proliferative effects. In vivo potential toxicity of iARE was assessed following acute and subchronic oral administration in Sprague Dawley rats. Quercetin, kaempferol, and isorhamnetin were three flavonoid components identified in iARE. The extract exerted cytotoxic effects on various cancer cells but not normal fibroblasts. Apoptosis in MCF7 cells was induced by iARE in a concentration-dependent manner associated with increased Bax/Bcl-2 ratio and reduced mitochondrial membrane potential ΔΨm, leading to release of cytochrome c, activation of caspase-3/7 and caspase-9, and cleavage of PARP. In the acute oral toxicity study, no mortality or toxicological signs were observed in rats at 1000 or 5000 mg/kg. In a subchronic oral toxicity study, iARE at a dosage of up to 1000 mg/kg produced no mortality or treatment-related adverse effects on general behavior, food intake, body weight, relative organ weights. No apparent marked changes in the histopathology of the liver and kidney were detected. Data demonstrated that iARE induced in vitro cytotoxic effects in cancer cells are associated with lackof invivo toxicity. Thus, iARE was suggested to be considered as apotential therapeutic candidate for cancer treatment.

Reproductive development and genetic structure of the mycoheterotrophic orchid Pogoniopsis schenckii Cogn.

BACKGROUND: Pogoniopsis schenckii Cogn. is a mycoheterotrophic orchid that can be used as a model to understand the influence of mycoheterotrophy at different stages of the reproductive cycle. We aimed to verify the presence of endophytic and epiphytic fungi at each stage of the reproductive process and investigated how the breeding system may relate to genetic structure and diversity of populations. In this study we performed anatomical and ultrastructural analyses of the reproductive organs, field tests to confirm the breeding system, and molecular analysis to assess genetic diversity and structure of populations. RESULTS: During the development of the pollen grain, embryo sac and embryogenesis, no fungal infestation was observed. The presence of endophytic fungal hyphae was observed just within floral stems and indehiscent fruit. Beyond assuring the presence of fungus that promote seed germination, specific fungi hyphae in the fruit may affect other process, such as fruit ripening. As other mycoheterotrophic orchids, P. schenckii is autogamous, which may explain the low genetic diversity and high genetic structure in populations. CONCLUSIONS: We discuss an interesting interaction: fungal hyphae in the indehiscent fruit. These fungal hyphae seem to play different roles inside fruit tissues, such as acting in the fruit maturation process and increasing the proximity between fungi and plant seeds even before dispersion occurs. As other mycoheterotrophic orchids, P. schenckii is autogamous, which may explain the low genetic diversity and high genetic structure in populations. Altogether, our findings provide important novel information about the mechanisms shaping ecology and evolution of fragmented populations of mycoheterotrophic plant.

Vegetative traits can predict flowering quality in Phalaenopsis orchids despite large genotypic variation in response to light and temperature.

Phalaenopsis is an economically important horticultural ornamental, but its growth is slow and costly. The vegetative cultivation phase is long and required to ensure sufficient plant size. This is needed to develop high quality flowering plants. We studied the effects of temperature (27 or 31 °C) and light intensity (60 or 140 µmol m-2 s-1) on plant growth and development during the vegetative cultivation phase in two experiments, with respectively 19 and 14 genotypes. Furthermore, the after-effects of treatments applied during vegetative growth on flowering traits were determined. Increasing light intensity in the vegetative phase accelerated both vegetative plant growth and development. Increasing temperature accelerated vegetative leaf appearance rate, but strongly reduced plant and root biomass accumulation when temperatures were too high. Flowering was greatly affected by treatments applied during vegetative growth, and increased light and temperature increased number of flower spikes, and number of flowers and buds. Genotypic variation was large in Phalaenopsis, especially in traits related to flowering, thus care is needed when generalising results based on a limited number of cultivars. Plant biomass and number of leaves during vegetative growth were positively correlated with flowering quality. These traits can be used as an early predictor for flowering capacity and quality of the final product. Additionally, this knowledge can be used to improve selection of new cultivars.

New intergeneric orchid hybrid found in Romania × Pseudorhiza nieschalkii (Senghas) P.F.Hunt nothosubsp. siculorum H.Kertész & N.Anghelescu, 2020.

We describe the first reported intergeneric, which naturally occurs between two subspecies belonging to different genera, Dactylorhiza fuchsii subsp. sooana (genus Dactylorhiza) and Pseudorchis albida subsp. tricuspis (genus Pseudorchis), as × Pseudorhiza nieschalkii (Senghas) P.F.Hunt nothosubsp. siculorum H.Kertész & N.Anghelescu, 2020. The hybrid was found and digitally photographed for the first time by Hajnalka Kertész in June, 2020, within Terra Siculorum, in one of the Natura 2000 protected areas, known as Harghita Madaraș, ROSCI00090. Following detailed morphometric analysis using 67 characters and molecular karyological analyses, we identified this unique specimen as an intergeneric hybrid, new to science. The hybrid, an F1 generation plant, most likely representing a single intergeneric pollination event, is phenotypically intermediate between its parental species in most of the characters scored, but it significantly closely resembles Pseudorchis albida subsp. tricuspis parent. Since several individuals of the parental species occurred in near proximity, within 1-10 meters distance, we suggest that the production of this hybrid required a minimum travel distance of ca 1-10 meters, by the pollinators and frequent exchange of pollen between the parental species was very likely. The parental species and the hybrid, which display a considerable synchronicity in their flowering time, overlap in the pollinator community, sharing various species of Hymenopterans and Dipterans, very abundant in the heathland. This Terra Siculorum hybrid is thus best described as a rarely occurring intergeneric hybrid that shows strong Pseudorchis albida subsp. tricuspis parental dominance in inheritance patterns.

Comparative transcriptomic analyses of normal and peloric mutant flowers in Cymbidium goeringii Rchb.f identifies differentially expressed genes associated with floral development.

Cymbidium geringii has high ornamental and economic importance. Its traits, including flower shape, size, and color, are highly sought by orchid breeders. Gaining insights into the molecular basis of C. geringi flower development would accelerate genetic improvement of other orchids. Methods and Results: Here, C. goeringii RNA was purified from normal and peloric mutant flowers, and cDNA libraries constructed for Illumina sequencing. We generated 329,156,782 clean reads, integrated them, and then assembled into 236,811 unigenes averaging 595 bp long. A total of 11,992 differentially expressed genes s, of which 6119 were upregulated and 5873 downregulated, were uncovered in peloric mutant flower buds relative to normal flower buds. Kyoto Encyclopedia of Genes and Genomes enrichment assessments posited that these differentially expressed genes are associated with "Photosynthesis", "Linoleic acid metabolism", as well as "Plant hormone signal transduction" cascades. The DEGs were designated to 12 remarkably enriched GO terms, and 16 cell wall associated GO terms. The expression level of 16 determined genes were verified using RT-qPCR. Conclusions: Our gene expression data may be used to study the regulatory mechanism of flower organ development in C. geringi.

Selection of suitable reference genes for quantitative real time PCR in different Tulasnella isolates and orchid-fungus symbiotic germination system.

Under natural conditions, mycorrhizal symbiosis accompanies nearly the entire life cycle of orchids from seed germination through to flowering and fruiting. Tulasnella-like orchid mycorrhizal fungi are the most common mycorrhizal fungi found in association with orchid species. Presently suitable reference genes have not been systematically selected for the quantification of gene expression via Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). We evaluated 12 candidate Tulasnella genes in nine different Tulasnella isolates and in the Dendrobium-fungal symbiotic germination associations followed by statistical analysis using the programs Bestkeeper, geNorm, and Normfinder to analyze the expression stability of the individual genes. The results showed that the EF2, UBC, and PP2A genes had the highest rankings with relatively stable expression levels across the different genotypes and during the symbiotic seed germination process by the three programs, and may be suitable for RT-qPCR normalization. Furthermore, the gene encoding C-5 Sterol desaturase (C5SD) was selected to verify the reliability of EF2, UBC, and PP2A expression during the Tulasnella-Dendrobium symbiotic seed germination process. This study is the first systematic exploration of optimal reference genes for gene expression studies during the colonization of orchid seeds by the mycorrhizal fungus Tulasnella.

Cytological, Biochemical, and Transcriptomic Analyses of a Novel Yellow Leaf Variation in a Paphiopedilum (Orchidaceae) SCBG COP15.

The genus Paphiopedilum, belonging to the Orchidaceae, has high ornamental value. Leaf variations can considerably improve the economic and horticultural value of the orchids. In the study, a yellow leaf mutant of a Paphiopedilum hybrid named P. SCBG COP15 was identified during the in vitro plant culture process; however, little is known about their molecular mechanisms. For this, RNA-seq libraries were created and used for the transcriptomic profiling of P. SCBG COP15 and the yellow mutant. The Chl a, Chl b, and carotenoid contents in the yellow leaves decreased by approximately 75.99%, 76.92%, and 56.83%, respectively, relative to the green leaves. Decreased chloroplasts per cell and abnormal chloroplast ultrastructure were observed by electron microscopic investigation in yellowing leaves; photosynthetic characteristics and Chl fluorescence parameters were also decreased in the mutant. Altogether, 34,492 unigenes were annotated by BLASTX; 1,835 DEGs were identified, consisting of 697 upregulated and 1138 downregulated DEGs. HEMA, CRD, CAO, and CHLE, involved in Chl biosynthesis, were predicted to be key genes responsible for leaf yellow coloration. Our findings provide an essential genetic resource for understanding the molecular mechanism of leaf color variation and breeding new varieties of Paphiopedilum with increased horticultural value.

Transcriptome sequencing and differential gene expression analysis reveal the mechanisms involved in seed germination and protocorm development of Calanthe tsoongiana.

Calanthe tsoongiana is a rare orchid species native to China. Asymbiotic seed germination is of great importance in the ex situ conservation of this species. Based on morphological characteristics and anatomical structures, the C. tsoongiana developmental process from seeds to seedlings was divided into four stages (SA, PB, PC and PD), and subsequently, changes in endogenous hormone contents and gene expression were assessed using RNA-seq analysis. K-means analysis divided the DEGs into eight clusters. The gene expression decreased markedly between the imbibed seed and globular protocorm stages, with this being the most notably enriched cluster. During the seed germination period, DEGs were dominated by ATP metabolic processes, respiration and photosynthesis. A small change in gene expression was found in the globular protocorm versus the finger-like protocorm stages. During the last developmental stage, DEGs were significantly enriched in lignin catabolic processes and plant-type secondary cell wall biogenesis. DEG homologs, such as TSA1, DAO, NCED1, STM, and CUC2, were related to phytohormones and the morphogenesis of shoots, leaves and roots. Particularly, interactions between CUC2 and STM as well as AS1 and STM were likely involved in protocorm formation and development. Furthermore, TSA1 and DAO were distinctly validated and implicated in the synthesis and metabolism of auxin, which has a pivotal role in plant development. Our study is the first to combine morphological and transcriptome analysis to examine the process of protocorm formation and development. The results provide a foundation for understanding the mechanisms of seed germination and protocorm development of C. tsoongiana.

New species of Tulasnella associated with Australian terrestrial orchids in the Cryptostylidinae and Drakaeinae.

Many orchids have an obligate relationship with Tulasnella mycorrhizal fungi for seed germination and support into adulthood. Despite the importance of Tulasnella as mycorrhizal partners, many species remain undescribed. Here, we use multiple sequence locus phylogenetic analyses to delimit and describe six new Tulasnella species associated with Australian terrestrial orchids from the subtribes Cryptostylidinae and Drakaeinae. Five of the new species, Tulasnella australiensis, T. occidentalis, T. punctata, T. densa, and T. concentrica, all associate with Cryptostylis (Cryptostylidinae), whereas T. rosea associates with Spiculaea ciliata (Drakaeinae). Isolates representing T. australiensis were previously also reported in association with Arthrochilus (Drakaeinae). All newly described Tulasnella species were delimited by phylogenetic analyses of four loci (nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 [ITS], C14436 [ATP synthase], C4102 [glutamate synthase], and mt 16S rDNA [mtLSU]). The pairwise sequence divergence between species for the ITS region ranged from 5.6% to 25.2%, and the maximum sequence divergence within the newly described species ranged from 1.64% to 4.97%. There was a gap in the distribution of within- and between-species pairwise divergences in the region of 4-6%, with only one within-species value of 4.97% (for two T. australiensis isolates) and one between-species value of 5.6% (involving an isolate of T. occidentalis) falling within this region. Based on fluorescence staining, all six new Tulasnella species are binucleate and have septate, cylindrical hyphae. There was some subtle variation in culture morphology, but colony diameter as measured on 3MN+vitamin medium after 6 wk of growth did not differ among species. However, T. australiensis grew significantly (P < 0.02) slower than others on ½ FIM and » potato dextrose agar (PDA) media. Formal description of these Tulasnella species contributes significantly to documentation of Tulasnella diversity and provides names and delimitations to underpin further research on the fungi and their relationships with orchids.

Silencing of FOREVER YOUNG FLOWER-Like Genes from Phalaenopsis Orchids Promotes Flower Senescence and Abscission.

Ectopic expression of FOREVER YOUNG FLOWER (FYF) delays floral senescence and abscission in transgenic Arabidopsis. To analyze the FYF function in Phalaenopsis orchids, two FYF-like genes (PaFYF1/2) were identified. PaFYF1/2 were highly expressed in young Phalaenopsis flowers, and their expression decreased significantly afterward until flower senescence. This pattern was strongly correlated with the process of flower senescence and revealed that PaFYF1/2 function to suppress senescence/abscission during early flower development. Interestingly, in flowers, PaFYF1 was consistently expressed less in petals than in lips/sepals, whereas PaFYF2 was expressed relatively evenly in all flower organs. This difference suggests a regulatory modification of the functions of PaFYF1 and PaFYF2 during Phalaenopsis flower evolution. Delayed flower senescence and abscission, which were unaffected by ethylene treatment, were observed in 35S::PaFYF1/2 and 35S::PaFYF1/2 + SRDX transgenic Arabidopsis plants due to the downregulation of the ethylene signaling and abscission-associated genes EDF1-4, IDA and BOP1/2. These results suggest a possible repressor role for Phalaenopsis PaFYF1/2 in controlling floral senescence/abscission by suppressing ethylene signaling and abscission-associated genes. To further validate the function of PaFYF1/2, PaFYF1/2-VIGS (virus-induced gene silencing) Phalaenopsis were generated and analyzed. Promotion of senescence and abscission was observed in PaFYF1/2-VIGS Phalaenopsis flowers by the upregulation of PeEDF1/2, PeSAG39 and PeBOP1/2 expression, the early occurrence of greening according to their increased chlorophyll content and the reduction in water content in flower organs. Our results support that PaFYF1/2 function as transcriptional repressors to prohibit flower senescence and abscission in Phalaenopsis.

Effects of inoculated mycorrhizal fungi and non-mycorrhizal beneficial micro-organisms on plant traits, nutrient uptake and root-associated fungal community composition of the Cymbidium hybridum in greenhouse.

AIMS: The aim of this study was to assess the effects of beneficial micro-organisms on the growth, nutrient accumulation and root-associated fungal species composition of pot orchids grown in the greenhouse. METHODS AND RESULTS: A greenhouse pot experiment was conducted to investigate the beneficial effects of a mycorrhizal fungus, Epulorhiza repens isolate ML01, an endophytic fungus, Umbelopsis nana isolate ZH3A-3 and a mixed commercial inoculum Rem, alone or in combination. Nested PCR assays showed that both isolates ML01 and ZH3A-3 can successfully establish in inoculated soil. All the inoculants significantly increased the plant total dry weight of Cymbidium hybridum 'Golden Boy', whereas only co-inoculation with the endophytic fungus ZH3A-3 and the Rem enhanced the fresh weight and height of host plants. The mycorrhizal fungus positively affected P, K, Ca, Mg content in shoots and Zn content in roots, while the endophytic fungus improved N, P, Ca accumulation in shoots and roots. Co-inoculation with the Rem and ML01 improved root to shoot translocation of Fe and Zn. In addition, inoculation with ZH3A-3, ML01+Rem and ZH3A-3+Rem decreased the relative frequency of Fusarium sp. on orchid roots. Trichoderma sp. were isolated from the roots treated with ML01, ML01+Rem and ZH3A-3+Rem. CONCLUSIONS: Both mycorrhizal and endophytic fungi had the potential to create favourable microflora in the orchid roots and stimulate the growth of transplanted plantlets under greenhouse condition. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly isolated endophytic strain ZH3A-3 showed significant application value in orchid production.

Asymbiotic germination and morphological studies of seeds of Atlantic Rainforest micro-orchids (Pleurothallidinae).

The morphological and morphometric characters of seeds belonging to 11 species of the subtribe Pleurothallidinae using light and scanning electron microscopy were studied to understand the in vitro germination process. Qualitative data (color, shape, ornamentation) and quantitative ones were also evaluated in seeds and embryos (length, width, volume and air space percentage between the integument and the embryo). The viability of the seeds was evaluated by in vitro germination in woody plant medium (WPM), and by analysis of the developmental stages of protocorms until seedling formation (two to 24 weeks). Morphometric data showed variations within the genus Acianthera and between species of different genera. The best germination and protocorm formation responses occurred with Acianthera prolifera (92%) and Acianthera ochreata (86%), with the formation of seedlings after 12 and 16 weeks of sowing, respectively. The seeds and embryos of A. prolifera and A. ochreata were larger (length, width, and volume) with a structural polarity that may have facilitated their germination comparing to others studied species. Other characteristics of A. prolifera seeds that may have contributed to these results include the presence of a thin testa without ornamentation and a suspensor. The protocorms of Anathalis obovata, Dryadella liliputiana, and Octomeria gracillis developed slowly in the WPM, not reaching the seedling stage in 24 weeks of cultivation. This morphological and morphometric study contributes to the understanding of asymbiotic germination of some micro-orchid species.