Gametophyte and embryonic ontogeny: understanding the reproductive calendar of Cypripedium japonicum Thunb. (Cypripedoideae, Orchidaceae), a lady's slipper orchid endemic to East Asia.

BACKGROUND: The genus Cypripedium L. is one of the five genera of the subfamily Cypripedioideae, members of which are commonly known as lady's slipper orchids. Cypripedium japonicum is a perennial herb native to East Asia, specifically China, Japan, and Korea. Due to its limited distribution, the species is included in the Endangered category of the IUCN Red List. RESULTS: We investigated gametophyte development, including complete embryogenesis, in C. japonicum. The complete reproductive cycle is presented based on our observations. Anther development begins under the soil, and meiosis of pollen mother cells begins 3 weeks before anthesis, possibly during early April. The megaspore mother cells develop just after pollination in early May and mature in mid-late June. The pattern of embryo sac formation is bisporic, and there are six nuclei: three forming the egg apparatus, two polar nuclei, and an antipodal cell in the mature embryo sac. Triple fertilization results in the endosperm nucleus, which degenerates when the proembryo reaches the eight-to-sixteen-cell stage. CONCLUSION: Our overall comparisons of the features of gametophyte and embryo development in C. japonicum suggest that previous reports on the embryology of Cypripedium are not sufficient for characterization of the entire genus. Based on the available information, a reproductive calendar showing the key reproductive events leading to embryo formation has been prepared.

Development of polyspermic zygote and possible contribution of polyspermy to polyploid formation in angiosperms.

Fertilization is a general feature of eukaryotic uni- and multicellular organisms to restore a diploid genome from female and male gamete haploid genomes. In angiosperms, polyploidization is a common phenomenon, and polyploidy would have played a major role in the long-term diversification and evolutionary success of plants. As for the mechanism of formation of autotetraploid plants, the triploid-bridge pathway, crossing between triploid and diploid plants, is considered as a major pathway. For the emergence of triploid plants, fusion of an unreduced gamete with a reduced gamete is generally accepted. In addition, the possibility of polyspermy has been proposed for maize, wheat and some orchids, although it has been regarded as an uncommon mechanism of triploid formation. One of the reasons why polyspermy is regarded as uncommon is because it is difficult to reproduce the polyspermy situation in zygotes and to analyze the developmental profiles of polyspermic triploid zygotes. Recently, polyspermic rice zygotes were successfully produced by electric fusion of an egg cell with two sperm cells, and their developmental profiles were monitored. Two sperm nuclei and an egg nucleus fused into a zygotic nucleus in the polyspermic zygote, and the triploid zygote divided into a two-celled embryo via mitotic division with a typical bipolar microtubule spindle. The two-celled proembryos further developed and regenerated into triploid plants. These suggest that polyspermic plant zygotes have the potential to form triploid embryos, and that polyspermy in angiosperms might be a pathway for the formation of triploid plants.

FEATURES OF EMBRYONIC DEVELOPMENT OF DIENIA OPHRYDIS (ORCHIDACEAE).

On example of Dienia ophrydis (J. Köenig) Seidenf (Orchidaceae), we have described a new type of embryogenesis of orchids ­ Dienia-type, which is differs from Liparis-type learned earlier in the tribe Malaxideae. Embryogenesis of Dienia-type is characterized by 1) the development of a single-celled suspensor formed by cb-derivative, 2) linear arrangement of germ cells in the tetrad stage, 3) the special structure of the embryo in the stages of tetrads and octants (l, lR, m, ci, cb), and 4) the absence of ci and cb cell division. The convergent similarity of embryogenesis of Dienia- and Caryophyllaceae-types is proposed. A number of specific for D. ophrydis structures of embryo sac and embryo, including «petassum¼, «fitting¼ and «suspensor coat¼ are described for the first time. Petassum represents remains of the cell walls of pollen tube, and perhaps of filamentous apparatus of synergids, plugging the micropyle side of the fertilized embryo sac. The only cell of suspensor has a specific appendix («fitting¼), that connects it with the embryo itself. There is «suspensor coat¼ which surrounds the only suspensor cell, including «fitting¼, but does not extend to the basal cells of the embryo itself.

In vitro propagation of Paphiopedilum orchids.

Paphiopedilum is one of the most popular and rare orchid genera. Members of the genus are sold and exhibited as pot plants and cut flowers. Wild populations of Paphiopedilum are under the threat of extinction due to over-collection and loss of suitable habitats. A reduction in their commercial value through large-scale propagation in vitro is an option to reduce pressure from illegal collection, to attempt to meet commercial needs and to re-establish threatened species back into the wild. Although they are commercially propagated via asymbiotic seed germination, Paphiopedilum are considered to be difficult to propagate in vitro, especially by plant regeneration from tissue culture. This review aims to cover the most important aspects and to provide an up-to-date research progress on in vitro propagation of Paphiopedilum and to emphasize the importance of further improving tissue culture protocols for ex vitro-derived explants.

Biogeography of the Phalaenopsis amabilis species complex inferred from nuclear and plastid DNAs.

BACKGROUND: Phalaenopsis is one of the important commercial orchids in the world. Members of the P. amabilis species complex represent invaluable germplasm for the breeding program. However, the phylogeny of the P. amabilis species complex is still uncertain. The Phalaenopsis amabilis species complex (Orchidaceae) consists of subspecies amabilis, moluccana, and rosenstromii of P. amabilis, as well as P. aphrodite ssp. aphrodite, P. ap. ssp. formosana, and P. sanderiana. The aims of this study were to reconstruct the phylogeny and biogeographcial patterns of the species complex using Neighbor Joining (NJ), Maxinum Parsimony (MP), Bayesian Evolutionary Analysis Sampling Trees (BEAST) and Reconstruct Ancestral State in Phylogenies (RASP) analyses based on sequences of internal transcribed spacers 1 and 2 from the nuclear ribosomal DNA and the trnH-psbA spacer from the plastid DNA. RESULTS: A pattern of vicariance, dispersal, and vicariance + dispersal among disjunctly distributed taxa was uncovered based on RASP analysis. Although two subspecies of P. aphrodite could not be differentiated from each other in dispersal state, they were distinct from P. amabilis and P. sanderiana. Within P. amabilis, three subspecies were separated phylogenetically, in agreement with the vicariance or vicariance + dispersal scenario, with geographic subdivision along Huxley's, Wallace's and Lydekker's Lines. Molecular dating revealed such subdivisions among taxa of P. amabilis complex dating back to the late Pleistocene. Population-dynamic analyses using a Bayesian skyline plot suggested that the species complex experienced an in situ range expansion and population concentration during the late Last Glacial Maximum (LGM). CONCLUSIONS: Taxa of the P. amabilis complex with disjunct distributions were differentiated due to vicariance or vicariance + dispersal, with events likely occurring in the late Pleistocene. Demographic growth associated with the climatic oscillations in the Würm glacial period followed the species splits. Nevertheless, a subsequent population slowdown occurred in the late LGM due to extinction of regional populations. The reduction of suitable habitats resulted in geographic fragmenttation of the remaining taxa.

LONG-TERM CONSERVATION OF PROTOCORMS OF Brassavola nodosa (L) LIND. (ORCHIDACEAE): EFFECT OF ABA AND A RANGE OF CRYOCONSERVATION TECHNIQUES.

BACKGROUND: Populations of Brassavola nodosa have been severely affected by habitat destruction and illegal collecting, and as with the majority of orchid species, it is critical to take action to guarantee their continued survival. OBJECTIVE: The present study aimed to establish protocols for the long-term conservation of protocorms of species. MATERIALS AND METHODS: Four different cryogenic techniques were compared: encapsulation-dehydration (ED), encapsulation-vitrification (EV), encapsulation-dehydration-vitrification (EDV) and vitrification. RESULTS: Preculture of protocorms with ABA was a critical factor in obtaining high percentages of regrowth. With vitrification, 100% regrowth was achieved in five treatments, mainly when protocorms were dehydrated with PVS2 for 120 min. 100% regrowth was also obtained with EDV, where the protocorms were precultured with ABA 5 mg/l for 3 days and incubated with PVS2 for 60 min. With the ED, regrowth of 72% was achieved with the preculture of protocorms with ABA 5 mg/l for the three times of incubation used (3, 6 and 9 days). In the case of EV, 92% regrowth, was recorded when protocorms were precultured for 9 days with ABA 3 mg/l and incubated with PVS2 for 90 min. CONCLUSION: Although regrowth of protocorms was obtained with all the techniques used, the vitrification technique is preferred since it requires less labour and is less costly.

Cold response in Phalaenopsis aphrodite and characterization of PaCBF1 and PaICE1.

Phalaenopsis is a winter-blooming orchid genus commonly cultivated in tropical Asian countries. Because orchids are one of the most economically important flower crops in Taiwan, it is crucial to understand their response to cold and other abiotic stresses. The present study focused on gene regulation of P. aphrodite in response to abiotic stress, mainly cold. Our results demonstrate that P. aphrodite is sensitive to low temperatures, especially in its reproductive stage. We found that after exposure to 4°C, plants in the vegetative stage maintained better membrane integrity and photosynthetic capacity than in the flowering stage. At the molecular level, C-repeat binding factor1 (PaCBF1) and its putative target gene dehydrin1 (PaDHN1) mRNAs were induced by cold, whereas inducer of CBF expression1 (PaICE1) mRNA was constitutively expressed. PaICE1 transactivated MYC motifs in the PaCBF1 promoter, indicating that up-regulation of PaCBF1 may be mediated by the binding of PaICE1 to MYC motifs. Overexpression of PaCBF1 in transgenic Arabidopsis induced AtCOR6.6 and RD29a without cold stimulus and maintained better membrane integrity after cold stress. Herein, we present evidence that cold induction of PaCBF1 transcripts in P. aphrodite may be transactivated by PaICE1 and consequently protect plants from cold damage through up-regulation of cold-regulated (COR) genes, such as DHN. To our knowledge, this study is the first report of the isolation and characterization of CBF, DHN and ICE genes in the Orchidaceae family.

High-resolution secondary ion mass spectrometry analysis of carbon dynamics in mycorrhizas formed by an obligately myco-heterotrophic orchid.

Mycorrhiza formation represents a significant carbon (C) acquisition alternative for orchid species, particularly those that remain achlorophyllous through all life stages. As it is known that orchid mycorrhizas facilitate nutrient transfer (most notably of C), it has not been resolved if C transfer occurs only after lysis of mycorrhizal structures (fungal pelotons) or also across the mycorrhizal interface of pre-lysed pelotons. We used high-resolution secondary ion mass spectrometry (nanoSIMS) and labelling with enriched (13) CO2 to trace C transfers, at subcellular scale, across mycorrhizal interfaces formed by Rhizanthella gardneri, an achlorphyllous orchid. Carbon was successfully traced in to the fungal portion of orchid mycorrhizas. However, we did not detect C movement across intact mycorrhizal interfaces up to 216 h post (13) CO2 labelling. Our findings provide support for the hypothesis that C transfer from the mycorrhizal fungus to orchid, at least for R. gardneri, likely occurs after lysis of the fungal peloton.

Taxonomic placement of Paphiopedilum canhii (Cypripedioideae; Orchidaceae) based on cytological, molecular and micromorphological evidence.

Paphiopedilum canhii was discovered in Northern Vietnam. Since its description in 2010, it has caused a stir among taxonomists due to its interesting mixture of morphological features, i.e. marbled, relatively thick leaves, flowers similar to species classified in the section Barbata, and unique, large staminodial shield. On the basis of these features, it is difficult to classify the species to existing infrageneric units. Using cytological data, phylogenetic analyses based on plastid and nuclear genes and the study of the adaxial epidermis of the leaves and gynostemium structure obtained from Scanning Electron Microscopy (SEM) and Light Microscopy (LM), the taxonomic position of P. canhii was determined. These results suggest that P. canhii forms an independent phylogenetic line within the genus Paphiopedilum deserving subgeneric status, already proposed by Braem and Gruss (2011) as Megastaminodium.

Physical wounding and ethylene stimulated embryogenic stem cell proliferation and plantlet regeneration in protocorm-like bodies of Phalaenopsis orchids.

Phalaenopsis orchids have been regenerated by inducing protocorm-like bodies (PLBs) from etiolated leaf sections. However, the physiological and molecular mechanisms of secondary PLB development and subsequent proliferation have not been explored. Bisectionally cutting primary PLBs resulted in more secondary PLBs at 5 weeks, suggesting an embryogenic stem cell property imposed by wounding of primary PLB tissues. The ethylene precursors ethephon and 1-aminocyclopropanecarboxylic acid and the ethylene perception inhibitor silver nitrate increased PLB formation, while aminoethoxyvinylglycine decreased PLB formation. Ethylene content in wounded PLB explants increased over culture time in media containing ethylene precursors or inhibitors. mRNA levels of PhACS2, PhACS3, and PhACO were increased by ethephon and decreased by ethylene inhibitors. Expression of genes in the ethylene signaling pathway was enhanced following ethylene-precursor treatment and was mitigated by ethylene inhibitors during PLB proliferation. Transcription of PhETR and PhEIN3, as well as PhERS, PhCTR, and PhGTP, was significantly increased 12 h after ethylene treatment. Ethylene and physical wounding stimulated secondary PLB formation in Phalaenopsis, probably through ethylene biosynthesis and signal transduction.

In vitro regeneration of Coelogyne nervosa A.Rich. and Eria pseudoclavicaulis Blatt., threatened orchids of Western Ghats, India.

The seeds of C. nervosa and E. pseudoclavicaulis were germinated asymbiotically on Knudson C (KC) and Schenk and Hildebrandt basal medium (SH). Growth regulators such as 2,4-Dichlorophenoxyacetic acid (2,4-D) individually and in combinations with benzyladenine (BA) and kinetin were used for callus induction from the protocorm like bodies. Coelogyne nervosa showed maximum (90%) callus induction in Knudson C medium supplemented with 2,4-D (2.26 microM) and Eria pseudoclavicaulis showed 60% callus induction in Schenk and Hildebrandt medium supplemented with 2,4-D (2.26 microM). Calli developed a route of production of protocorm-like bodies and eventually developed into plantlets on transfer to growth regulator free half strength basal medium. The well rooted plants were hardened successfully in the potting mixture containing coconut husk, charcoal, and brick pieces in the ratio 2:1:1.

Complete chloroplast genome of the genus Cymbidium: lights into the species identification, phylogenetic implications and population genetic analyses.

BACKGROUND: Cymbidium orchids, including some 50 species, are the famous flowers, and they possess high commercial value in the floricultural industry. Furthermore, the values of different orchids are great differences. However, species identification is very difficult. To a certain degree, chloroplast DNA sequence data are a versatile tool for species identification and phylogenetic implications in plants. Different chloroplast loci have been utilized for evaluating phylogenetic relationships at each classification level among plant species, including at the interspecies and intraspecies levels. However, there is no evidence that a short sequence can distinguish all plant species from each other in order to infer phylogenetic relationships. Molecular markers derived from the complete chloroplast genome can provide effective tools for species identification and phylogenetic resolution. RESULTS: The complete nucleotide sequences of eight individuals from a total of five Cymbidium species' chloroplast (cp) genomes were determined using Illumina sequencing technology of the total DNA via a combination of de novo and reference-guided assembly. The length of the Cymbidium cp genome is about 155 kb. The cp genomes contain 123 unique genes, and the IR regions contain 24 duplicates. Although the genomes, including genome structure, gene order and orientation, are similar to those of other orchids, they are not evolutionarily conservative. The cp genome of Cymbidium evolved moderately with more than 3% sequence divergence, which could provide enough information for phylogeny. Rapidly evolving chloroplast genome regions were identified and 11 new divergence hotspot regions were disclosed for further phylogenetic study and species identification in Orchidaceae. CONCLUSIONS: Phylogenomic analyses were conducted using 10 complete chloroplast genomes from seven orchid species. These data accurately identified the individuals and established the phylogenetic relationships between the species. The results reveal that phylogenomics based on organelle genome sequencing lights the species identification-organelle-scale "barcodes", and is also an effective approach for studying whole populations and phylogenetic characteristics of Cymbidium.

Methylation effect on chalcone synthase gene expression determines anthocyanin pigmentation in floral tissues of two Oncidium orchid cultivars.

The anthocyanin-biosynthetic pathway was studied in flowers of Oncidium Gower Ramsey with yellow floral color and mosaic red anthocyanin in lip crests, sepals and petals, and compared with the anthocyanin biosynthesis in flowers of Oncidium Honey Dollp, a natural somatoclone derived from tissue culture of Gower Ramsey, with a yellow perianth without red anthocyanins in floral tissues. HPLC analysis revealed that the red anthocyanin in lip crests of the Gower Ramsey cultivar comprised peonidin-3-O-glucoside, delphinidin-3-O-glucoside and cyanidin-3-O-glucoside, whereas Honey Dollp was devoid of anthocyanin compounds. Among the five anthocyanin-biosynthetic genes, OgCHS was actively expressed in lip crests of Gower Ramsey flowers, but no transcripts of OgCHS were detected in Honey Dollp floral tissues. Transient expression of OgCHS by bombardment confirmed that recovery of the OgCHS gene expression completed the anthocyanin pathway and produced anthocyanin compounds in lip crests of Honey Dollp flowers. Transcription factor genes regulating anthocyanin biosynthesis showed no distinctive differences in the expression level of OgMYB1, OgbHLH and OgWD40 between the two cultivars. A methylation assay revealed that the promoter of OgCHS was not methylated in Gower Ramsey, while a positive methylation effect was present in the upstream promoter region of OgCHS in Honey Dollp. Overall, our results suggest that the failure of anthocyanin accumulation in Honey Dollp floral tissues may be attributed to inactivation of the OgCHS gene resulting from the epigenetic methylation of 5'-upstream promoter region.

Mixotrophy of Platanthera minor, an orchid associated with ectomycorrhiza-forming Ceratobasidiaceae fungi.

• We investigated the fungal symbionts and carbon nutrition of a Japanese forest photosynthetic orchid, Platanthera minor, whose ecology suggests a mixotrophic syndrome, that is, a mycorrhizal association with ectomycorrhiza (ECM)-forming fungi and partial exploitation of fungal carbon. • We performed molecular identification of symbionts by PCR amplifications of the fungal ribosomal DNA on hyphal coils extracted from P. minor roots. We tested for a (13)C and (15)N enrichment characteristic of mixotrophic plants. We also tested the ectomycorrhizal abilities of orchid symbionts using a new protocol of direct inoculation of hyphal coils onto roots of Pinus densiflora seedlings. • In phylogenetic analyses, most isolated fungi were close to ECM-forming Ceratobasidiaceae clades previously detected from a few fully heterotrophic orchids or environmental ectomycorrhiza surveys. The direct inoculation of fungal coils of these fungi resulted in ectomycorrhiza formation on P. densiflora seedlings. Stable isotope analyses indicated mixotrophic nutrition of P. minor, with fungal carbon contributing from 50% to 65%. • This is the first evidence of photosynthetic orchids associated with ectomycorrhizal Ceratobasidiaceae taxa, confirming the evolution of mixotrophy in the Orchideae orchid tribe, and of ectomycorrhizal abilities in the Ceratobasidiaceae. Our new ectomycorrhiza formation technique may enhance the study of unculturable orchid mycorrhizal fungi.

Minority cytotypes in European populations of the Gymnadenia conopsea complex (Orchidaceae) greatly increase intraspecific and intrapopulation diversity.

BACKGROUND AND AIMS: Patterns of ploidy variation among and within populations can provide valuable insights into the evolutionary mechanisms shaping the dynamics of plant systems showing ploidy diversity. Whereas data on majority ploidies are, by definition, often sufficiently extensive, much less is known about the incidence and evolutionary role of minority cytotypes. METHODS: Ploidy and proportions of endoreplicated genome were determined using DAPI (4',6-diamidino-2-phenylindole) flow cytometry in 6150 Gymnadenia plants (fragrant orchids) collected from 141 populations in 17 European countries. All widely recognized European species, and several taxa of less certain taxonomic status were sampled within Gymnadenia conopsea sensu lato. KEY RESULTS: Most Gymnadenia populations were taxonomically and/or ploidy heterogeneous. Two majority (2x and 4x) and three minority (3x, 5x and 6x) cytotypes were identified. Evolution largely proceeded at the diploid level, whereas tetraploids were much more geographically and taxonomically restricted. Although minority ploidies constituted <2 % of the individuals sampled, they were found in 35 % of populations across the entire area investigated. The amount of nuclear DNA, together with the level of progressively partial endoreplication, separated all Gymnadenia species currently widely recognized in Europe. CONCLUSIONS: Despite their low frequency, minority cytotypes substantially increase intraspecific and intrapopulation ploidy diversity estimates for fragrant orchids. The cytogenetic structure of Gymnadenia populations is remarkably dynamic and shaped by multiple evolutionary mechanisms, including both the ongoing production of unreduced gametes and heteroploid hybridization. Overall, it is likely that the level of ploidy heterogeneity experienced by most plant species/populations is currently underestimated; intensive sampling is necessary to obtain a holistic picture.

Uptake of ant-derived nitrogen in the myrmecophytic orchid Caularthron bilamellatum.

BACKGROUND AND AIMS: Mutualistic ant-plant associations are common in a variety of plant families. Some myrmecophytic plants, such as the epiphytic orchid Caularthron bilamellatum, actively form hollow structures that provide nesting space for ants (myrmecodomatia), despite a substantial loss of water-storage tissue. This study aimed at assessing the ability of the orchid to take up nitrogen from ant-inhabited domatia as possible trade-off for the sacrifice of potential water storage capacity. METHODS: Nitrogen uptake capabilities and uptake kinetics of (15)N-labelled compounds (NH(4)(+), urea and l -glutamine) were studied in field-grown Caularthron bilamellatum plants in a tropical moist forest in Panama. Plants were either labelled directly, by injecting substrates into the hollow pseudobulbs or indirectly, by labelling of the associated ants in situ. KEY RESULTS: Caularthron bilamellatum plants were able to take up all tested inorganic and organic nitrogen forms through the inner surface of the pseudobulbs. Uptake of NH(4)(+) and glutamine followed Michaelis-Menten kinetics, but urea uptake was not saturable up to 2 mm. (15)N-labelled compounds were rapidly translocated and incorporated into vegetative and reproductive structures. By labelling ants with (15)N in situ, we were able to prove that ants transfer N to the plants under field conditions. CONCLUSIONS: Based on (15)N labelling experiments we were able to demonstrate, for the first time, that a myrmecophytic orchid is capable of actively acquiring different forms of nitrogen from its domatia and that nutrient flux from ants to plants does indeed occur under natural conditions. This suggests that beyond anti-herbivore protection host plants benefit from ants by taking up nitrogen derived from ant debris.

Duplicated C-class MADS-box genes reveal distinct roles in gynostemium development in Cymbidium ensifolium (Orchidaceae).

The orchid floral organs represent novel and effective structures for attracting pollination vectors. In addition, to avoid inbreeding, the androecium and gynoecium are united in a single structure termed the gynostemium. Identification of C-class MADS-box genes regulating reproductive organ development could help determine the level of homology with the current ABC model of floral organ identity in orchids. In this study, we isolated and characterized two C-class AGAMOUS-like genes, denoted CeMADS1 and CeMADS2, from Cymbidium ensifolium. These two genes showed distinct spatial and temporal expression profiles, which suggests their functional diversification during gynostemium development. Furthermore, the expression of CeMADS1 but not CeMADS2 was eliminated in the multitepal mutant whose gynostemium is replaced by a newly emerged flower, and this ecotopic flower continues to produce sepals and petals centripetally. Protein interaction relationships among CeMADS1, CeMADS2 and E-class PeMADS8 proteins were assessed by yeast two-hybrid analysis. Both CeMADS1 and CeMADS2 formed homodimers and heterodimers with each other and the E-class PeMADS protein. Furthermore, transgenic Arabidopsis plants overexpressing CeMADS1 or CeMADS2 showed limited growth of primary inflorescence. Thus, CeMADS1 may have a pivotal C function in reproductive organ development in C. ensifolium.

Comparative anatomy of the nectary spur in selected species of Aeridinae (Orchidaceae).

BACKGROUND AND AIMS: To date, the structure of the nectary spur of Aeridinae has not been studied in detail, and data relating to the nectaries of ornithophilous orchids remain scarce. The present paper compares the structural organization of the floral nectary in a range of Aeridinae species, including both entomophilous and ornithophilous taxa. METHODS: Nectary spurs of Ascocentrum ampullaceum (Roxb.) Schltr. var. aurantiacum Pradhan, A. curvifolium (Lindl.) Schltr., A. garayi Christenson, Papilionanthe vandarum (Rchb.f.) Garay, Schoenorchis gemmata (Lindl.) J.J. Sm., Sedirea japonica (Rchb.f.) Garay & H.R. Sweet and Stereochilus dalatensis (Guillaumin) Garay were examined by means of light microscopy, scanning electron microscopy and transmission electron microscopy. KEY RESULTS AND CONCLUSIONS: The diverse anatomy of the nectary is described for a range of Aeridinae species. All species of Ascocentrum investigated displayed features characteristic of ornithophilous taxa. They have weakly zygomorphic, scentless, red or orange flowers, display diurnal anthesis, possess cryptic anther caps and produce nectar that is secluded in a relatively massive nectary spur. Unicellular, secretory hairs line the lumen at the middle part of the spur. Generally, however, with the exception of Papilionanthe vandarum, the nectary spurs of all entomophilous species studied here (Schoenorchis gemmata, Sedirea japonica, Stereochilus dalatensis) lack secretory trichomes. Moreover, collenchymatous secretory tissue, present only in the nectary spur of Asiatic Ascocentrum species, closely resembles that found in nectaries of certain Neotropical species that are hummingbird-pollinated and assigned to subtribes Maxillariinae Benth., Laeliinae Benth. and Oncidiinae Benth. This similarity in anatomical organization of the nectary, regardless of geographical distribution and phylogeny, indicates convergence.

Photosynthetic Mediterranean meadow orchids feature partial mycoheterotrophy and specific mycorrhizal associations.

PREMISE OF THE STUDY: We investigated whether four widespread, photosynthetic Mediterranean meadow orchids (Ophrys fuciflora, Anacamptis laxiflora, Orchis purpurea, and Serapias vomeracea) had either nutritional dependency on mycobionts or mycorrhizal fungal specificity. Nonphotosynthetic orchids generally engage in highly specific interactions with fungal symbionts that provide them with organic carbon. By contrast, fully photosynthetic orchids in sunny, meadow habitats have been considered to lack mycorrhizal specificity. METHODS: We performed both culture-dependent and culture-independent ITS sequence analysis to identify fungi from orchid roots. By analyzing stable isotope ((13)C and (15)N) natural abundances, we also determined the degree of autotrophy and mycoheterotrophy in the four orchid species. KEY RESULTS: Phylogenetic and multivariate comparisons indicated that Or. purpurea and Oph. fuciflora featured lower fungal diversity and more specific mycobiont spectra than A. laxiflora and S. vomeracea. All orchid species were significantly enriched in (15)N compared with neighboring non-orchid plants. Orchis purpurea had the most pronounced N gain from fungi and differed from the other orchids in also obtaining C from fungi. CONCLUSIONS: These results indicated that even in sunny Mediterranean meadows, orchids may be mycoheterotrophic, with correlated mycorrhizal fungal specificity.

Low night temperature acclimation of Phalaenopsis.

The capability of Phalaenopsis to acclimate its photosynthetic capacity and metabolic activity to cool night temperature conditions is crucial for improving orchid production in terms of efficient greenhouse heating. The extent to which Phalaenopsis possesses acclimation potential and the mechanistic background of the metabolic processes involved, have, however, not been studied before. Plants were subjected to a direct and gradual shift from a day to night temperature regime of 28/28-28/16°C, the cold stress and cold acclimation treatment, respectively. In comparison with the cold stress treatment, the cold acclimation treatment led to a higher malate accumulation and a reduction in leaf net CO(2) uptake. Consistently, the contribution of respiratory CO(2) recycling to nocturnal malate synthesis was calculated to be 23.5 and 47.0% for the cold stress and cold acclimation treatment, respectively. Moreover, the lower levels of starch measured in the cold acclimated leaves confirmed the suggested enhanced respiratory CO(2) recycling, implying that Phalaenopsis CAM operation evolved towards CAM idling. It is, however, plausible that this adjustment was not an effect of the low night temperature per se but a consequence of cool-root induced drought stress. Apart from that, at the start of the photoperiod, membrane stability showed a depression which was directly counteracted by an increased generation of glucose, fructose and sucrose. From these observations, it can be concluded that the observed plasticity in CAM operation and metabolic flexibility may be recognized as important steps in the low night temperature acclimation of Phalaenopsis.