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.

The same anthocyanins served four different ways: Insights into anthocyanin structure-function relationships from the wintergreen orchid, Tipularia discolor.

Over 500 unique anthocyanins have been described to date, which vary in color, antioxidant, light-attenuating, and antimicrobial properties. Identification of anthocyanin chemical structure may therefore serve as an important clue to their in situ function in plants. We characterized the histological and chemical structures of anthocyanins associated with diverse leaf color patterns in the terrestrial orchid, Tipularia discolor, as a step towards understanding their ultimate function. Tipularia discolor produces a single wintergreen leaf in autumn, which is drab brown in color during expansion. Upper (adaxial) surfaces of fully-expanded leaves may be green, purple-spotted, or solid purple, while lower (abaxial) surfaces are bright magenta. Our results showed that the same three cyanidin 3,7,3'-triglucosides, in similar concentrations and proportions, accounted for coloration in each of these cases, and that different colors result from differences in histological location of anthocyanins (i.e. abaxial/adaxial epidermis, mesophyll). Anthocyanins with 3,7,3' linkage positions are rare in plants, occurring only within the orchid subfamily Epidendroideae, to which Tipularia belongs. These results are important to the discussion of anthocyanin structure-function because they serve as a reminder that 1) plants may employ the same anthocyanins in different anatomical locations to achieve a broad range of colors (and potentially adaptive functions), and 2) anthocyanin chemical structure and anatomical location are influenced by phylogenetic inertia, as well as natural selection.

Labellum structure of Bulbophyllum echinolabium J.J. Sm. (section Lepidorhiza Schltr., Bulbophyllinae Schltr., Orchidaceae Juss.).

This micromorphological, chemical and ultrastructural study is a continuation of research conducted on the section Lepidorhiza. The Bulbophyllum echinolabium flowers comprised features that characterize a sapromyophilous syndrome, having large, glistening parts that emit an intense scent of rotten meat. The secretory activity was described in the hypochile (nectary in longitudinal groove and in the prickles) and the epichile (putative osmophore). The ultrastructural studies revealed a dense cytoplasm in the epidermis and subepidermal tissue with large nuclei and numerous mitochondria, the profiles of SER and RER, and dictyosomes. Large amounts of heterogeneous residues of secreted material (possibly phenolic) were present on the cuticle surface, similar to the unusual prominent periplasmic space with flocculent secretory material. The chemical analysis (GC/MS) of the scent profile of lips comprised carbohydrates and their derivatives (29.55% of all compounds), amino acids (1.66%), lipids (8.04%) and other organic compounds (60.73%). A great number of identified compounds are Diptera attractants (mainly Milichiidae, Tephritidae, Drosophilidae, Muscidae, Sarcophagidae, Tachinidae). The examination of visual and olfactory features indicates correlation between colour of flowers and the type of olfactory mimicry, where a dark colour labellum emits strong smell of rotten waste.

Evolution of anatomical characters in Acianthera section Pleurobotryae (Orchidaceae: Pleurothallidinae).

Acianthera section Pleurobotryae is one of ten sections of the genus Acianthera and include four species endemic to the Atlantic Forest. The objective of this study was to describe comparatively the anatomy of vegetative organs and floral micromorphology of all species of Acianthera section Pleurobotryae in order to identify diagnostic characters between them and synapomorphies for the section in relation of other sections of the genus. We analyzed roots, ramicauls, leaves and flowers of 15 species, covering eight of the nine sections of Acianthera, using light microscopy and scanning electron microscopy. Acianthera section Pleurobotryae is a monophyletic group and the cladistic analyses of anatomical and flower micromorphology data, combined with molecular data, support internal relationship hypotheses among the representatives of this section. The synapomorphies identified for A. sect. Pleurobotryae are based on leaf anatomy: unifacial leaves, round or elliptical in cross-section, round leaves with vascular bundles organized in concentric circles, and mesophyll with 28 to 30 cell layers. Within the section, the clade (A. crepiniana + A. mantiquyrana) presented more differences in vegetative organ morphology and higher support values in combined analyses when compared to the second clade, (A. atropurpurea + A. hatschbachii). For each of these clades an exclusive set of homoplasies of vegetative and floral organs were also identified. The results support the argument that vegetative organs are more evolutionarily stable in comparison to reproductive organs and thus helpful for inference of internal phylogenetic relationships in Acianthera, while flowers are highly variable, perhaps due to the diversity of pollinator attraction mechanisms. The analyses indicate that the elliptical leaves observed in A. crepiniana have originated from round leaves observed in the other species of this section, suggesting an adaptation to increase the area of exposure of the leaf and better the efficiency of capture of sunlight in shaded environments such as the Atlantic Forest. The presence of papillose regions in both vegetative and floral organs indicated that micromorphological characters are also useful for the delimitation of species and sections within the genus.

Comparative survey of secretory structures and floral anatomy of Cohniella cepula and Cohniella jonesiana (Orchidaceae: Oncidiinae). New evidences of nectaries and osmophores in the genus.

The morpho-anatomical structure of nectaries, osmophores, and elaiophores, and the anatomical and micromorphological features of floral pieces of Cohniella cepula Hoffmans. and Cohniella jonesiana Rchb.f. were comparatively analyzed. In both species, bracteal and sepal nectaries are structured, i.e., they present a secretory epidermis, secretory parenchyma, and vascular bundles. Nectar secretion is released through stomata. The anatomical and micromorphological traits are similar in both nectaries, which can be detected only if the nectar drops are secreted. Considering the location of these nectaries, the secreted nectar would not be a reward to pollinators. Osmophores are located at the base of both callus and laterals lobes, and consist of a layer of secretory epidermis composed of quadrangular cells and papillae. Elaiophores are found on the callus of the labellum and are of the epithelial type. The anatomical features of floral pieces are similar in both species. The anatomical analysis of sepals and petals showed a few differences, which could be of potential taxonomic value. Our results contribute valuable and novel information for the knowledge of these species and the genus, which will be useful in future taxonomic evaluations.

Comparative anatomical properties of some Epidendroideae and Orchidoideae species distributed in NE Turkey.

In this research, anatomical, leaf micromorphological features of the samples belonging to 25 taxa (Anacamptis Rich., Cephalanthera Rich., Dactylorhiza Necker ex Nevski, Gymnadenia R.Br., Himantoglossum Spreng., Limodorum Boehm., Ophrys L., Orchis L., Platanthera Rich., Serapias L., Spiranthes Rich. and Steveniella Schltr.) spread in the Karadeniz Region have been evaluated comparatively. In anatomical studies, the transverse section from root, stem and leaf, and surface section from leaves of plants were examined. In addition, micromorphological properties of leaf were determined by electron microscopy. Morphometrical analyses were carried out using the anatomical and leaf micromophological characters of each taxa. The data matrices were obtained by examining the results of at least three samples collected from different localities. The data were evaluated using Statistical Package for the Social Sciences (SPSS) and PAleontological STatistics (PAST) statistical programs with PCA, linear discriminant analysis (LDA), and unweighted pair group method with arithmetic mean analysis. Anatomical characteristics of plants such as root epidermis cell length, cortex diameter and pith cell diameter, leaf upper epidermis length-width and bulliform cell length-width were determined to be important characteristics. It was concluded that these characters are especially important in grouping at the genus level.

Ultrastructural studies and molecular characterization of root-associated fungi, of Crepidium acuminatum (D.Don) Szlach.: a threatened and medicinally important taxon.

Crepidium acuminatum (Orchidaceae) is a threatened medicinal orchid that grows under shady and moist forest floor where light remains for a very short period of time. Mycorrhizal association is known to be essential for seed germination and seedling establishment in amajority of orchids. Identification of fungi that form mycorrhizae with orchids is of crucial importance for orchid conservation. We used both morphological as well as molecular approaches to study this plant-fungal interaction. Scanning electron microscopy showed that fungi grow and proliferate in the middle layers of the cortex. Also, spiral-root hairs were foundalong with root hairs, which is an unusual observation. Spiral-root hairs provide more surface area for fluid absorption and entrance of colonizers. Further, total root genomic DNA was isolated and fungal internal-transcribed spacer (ITS) regions were polymerase chain reaction (PCR)-amplified using specific primer combinations ITS1F/ITS4 and ITS1/ITS4tul. ITS sequences were obtainedand analysed to know the closest sequence matche in the GenBank using BLASTn hosted by NLM-NCBI. Subject sequences were identified to be belonging to three main genera, namely, Tulasnella, Aspergillus and Penicillium. Results indicate that mycorrhizal association is necessary for the growth and development of the plant. In addition, this symbiosis influences the distribution and rarity of this medicinally valuable taxon. Specific fungal partners may lead to an enhanced seed germination rate and increased efficiency of nutrient exchange between both the partners. Hence, knowledge of mycorrhizal fungi is essential for future in vitro germination and seedling establishment programmes, because they rely on fungi for germination. Identification of mycorrhizal fungi can be usedfor orchid propagation and conservation programmes.

Floral nectary and osmophore of Epipactis helleborine (L.) Crantz (Orchidaceae).

The analysis of flowers collected at different stages of anthesis provides strong evidence to conclude that the shell-shaped hypochile and the knobs of epichile form a nectary. The scent comes from the aromatic constituents of nectar and the epichile tissue and the apices of all tepals (osmophores). The comparison between pollinated and unpollinated flowers revealed that the anthesis of unpollinated flowers lasted up to the 16th day. The nectariferous secretory cells formed single-layered epidermis and several layers of underlying parenchyma built by small, isodiametric cells with thin walls and dense cytoplasm, relatively large nuclei, supplied by collateral vascular bundles. During the floral lifespan, the residues of secreted material were higher on the hypochile cells. The lipoid-carbohydrate material and lipid globules in the cell walls and in the cytoplasm were localised. The abundance of starch grains was observed at the beginning of anthesis and their gradual reduction during the flower lifespan. At the end of anthesis in unpollinated flowers, the lipoid-carbohydrate-phenolic materials have been demonstrated. The phenolic material was the same as in plastoglobuli. The features such as irregular plasmalemma, the secretory vesicles that fuse with it, fully developed dictyosomes, numerous profiles of ER indicate vesicle-mediated process of secretion. The substances could be transported by vesicles to the periplasmic space via granulocrine secretion and then to the external surface. Both micro-channels and slightly developed periplasmic space were visible in the hypochile epidermis. This is the first time for anatomical survey of secretory tissue in pollinated and unpollinated flowers of E. helleborine.

Evolutionary and functional potential of ploidy increase within individual plants: somatic ploidy mapping of the complex labellum of sexually deceptive bee orchids.

Background and Aims: Recent tissue-level observations made indirectly via flow cytometry suggest that endoreplication (duplication of the nuclear genome within the nuclear envelope in the absence of subsequent cell division) is widespread within the plant kingdom. Here, we also directly observe ploidy variation among cells within individual petals, relating size of nucleus to cell micromorphology and (more speculatively) to function. Methods: We compared the labella (specialized pollinator-attracting petals) of two European orchid genera: Dactylorhiza has a known predisposition to organismal polyploidy, whereas Ophrys exhibits exceptionally complex epidermal patterning that aids pseudocopulatory pollination. Confocal microscopy using multiple staining techniques allowed us to observe directly both the sizes and the internal structures of individual nuclei across each labellum, while flow cytometry was used to test for progressively partial endoreplication. Key Results: In Dactylorhiza, endoreplication was comparatively infrequent, reached only low levels, and appeared randomly located across the labellum, whereas in Ophrys endoreplication was commonplace, being most frequent in large peripheral trichomes. Endoreplicated nuclei reflected both endomitosis and endocycling, the latter reaching the third round of genome doubling (16C) to generate polytene nuclei. All Ophrys individuals studied exhibited progressively partial endoreplication. Conclusions: Comparison of the two genera failed to demonstrate the hypothesized pattern of frequent polyploid speciation in genera showing extensive endoreplication. Endoreplication in Ophrys appears more strongly positively correlated with cell size/complexity than with cell location or secretory role. Epigenetic control of gene overexpression by localized induction of endoreplication within individual plant organs may represent a significant component of a plant's developmental programme, contributing substantially to organ plasticity.

Floral features of two species of Bulbophyllum section Lepidorhiza Schltr.: B. levanae Ames and B. nymphopolitanum Kraenzl. (Bulbophyllinae Schltr., Orchidaceae).

Two representatives of section Lepidorhiza, previously sometimes considered conspecific, Bulbophyllum levanae and Bulbophyllum nymphopolitanum, demonstrated both similarities and differences in floral features. There were significant differences in the length of sepals and micromorphological features of the labellum. In both species, osmophores are located on the extended apices of sepals and possibly on petals. An abundance of proteins in tepals is probably associated with the unpleasant scent of the flowers, whereas the thin wax layers on the epidermis are probably involved in the maintenance of the brilliance of floral tepals, which strongly attracts flies. In all tepals of both species, we noted the presence of dihydroxyphenolic globules in the cytoplasm after staining with FeCl3. Comparison with ultrastructure results revealed that they were associated with plastids containing plastoglobuli. The most remarkable feature was the presence of a prominent periplasmic space in the epidermal cells of both investigated species. Furthermore, in the labellum of B. levanae, the cuticle contained microchannels. The combination of periplasmic space and microchannels has not previously been recorded.

Nectar and oleiferous trichomes as floral attractants in Bulbophyllum saltatorium Lindl. (Orchidaceae).

Although many Orchidaceae have deceit flowers that produce no reward, the most common reward, when present, is nectar. Bulbophyllum, however, is unusual in that the labellar secretions of most species investigated to date lack sugars, and, therefore, cannot be considered true nectar. The African species Bulbophyllum saltatorium is an exception in that it produces not only nectar but also possesses specialized, capitate oleiferous trichomes. The nectary of B. saltatorium is borne on the labellum and is represented by a deep, narrow, median longitudinal groove, having a small aperture, and flanked by trichomes. Isodiametric epidermal cells lining this groove secrete nectar which collects both in the groove and on the surface of the labellum. As well as a nectary, the labellum of B. saltatorium also bears three types of unicellular trichomes: the longest trichomes are borne distally and abaxially; the marginal ones form a rim around the entire labellum, and finally, massive, capitate trichomes occur proximally and adaxially. These are oleiferous, containing large quantities of oil which might function as precursors of volatile components of fragrance or provide a food-reward. To the best of our knowledge, this is the first time for such oleiferous trichomes to be described for Bulbophyllum. Therefore, apart from their color and markings, flowers of this species are able to attract pollinators in at least two, possibly three ways: food-reward in the form of nectar; fragrance; and possibly food-rewards in the form of food-hairs.

Floral micromorphology and transcriptome analyses of a fragrant Vandaceous Orchid, Vanda Mimi Palmer, for its fragrance production sites.

BACKGROUND: Vanda Mimi Palmer (VMP) is commercially valuable for its strong fragrance but little is known regarding the fragrance production and emission sites on the flowers. RESULTS: Olfactory perception detected fragrance only from the petals and sepals. Light and Environmental Scanning Electron microscopy analyses on fresh tissues showed distributions of stomata and trichomes concentrated mostly around the edges. These results paralleled the rich starch deposits and intense neutral red stain, indicating strong fragrance and trichomes as potential main fragrance release sites. Next Generation Sequencing (NGS) transcriptomic data of adaxial and abaxial layers of the tissues showed monoterpene synthase transcripts specifically linalool and ocimene synthases distributed throughout the tissues. qPCR analyses taken at different time points revealed high levels of linalool and ocimene synthases transcripts in the early morning with maximal level at 4.00 am but remained low throughout daylight hours. CONCLUSIONS: Knowledge of the VMP floral anatomy and its fragrance production characteristics, which complemented our previous molecular and biochemical data on VMP, provided additional knowledge on how fragrance and flower morphology are closely intertwined. Further investigation on the mechanisms of fragrance biosynthesis and interaction of potential pollinators would elucidate the evolution of the flower morphology to maximize the reproduction success of this plant.

Hormonal and morphological changes during seed development of Cypripedium japonicum.

With the goal of evaluating the factors that may be influencing the reproductive success of an endangered, terrestrial orchid Cypripedium japonicum Thunb., the anatomical and morphological characteristics of seed development were assessed, as were the levels of endogenous hormones to investigate the possible relationship between hormones and seed development. The results showed that morphological characteristics of seeds were changing from milky white and filiform to brown and fusiform. The development of ovule that initiated on the fertile valves only began after pollination, and a mature embryo sac was produced at 40 days after pollination (DAP). Fertilization occurred 50 to 60 DAP, and the newly formed zygote divided into two unequal cells that became the embryo proper and the suspensor. Seeds reached maturity at about 115 DAP, at which point they consisted only of a globular embryo and two layers of seed coat, without endosperm. The suspensor was lost, and only lipid particles and a large number of starch grains distributed in the cytoplasm. The peak of gibberellic acid (GA3), zeatin riboside (ZR), and indole acetic acid (IAA) occurred in a time sequence, as 20, 30, and 40 DAP. There was a highly significant positive correlation among them (P < 0.01). Abscisic acid (ABA) content increased at 60 DAP and reached the maximum at 115 DAP. ABA contents were significantly negatively correlated with the three hormones above (P < 0.05). It indicated that the lack of endosperm and suspensor tissues, delayed differentiation of embryos, and high levels of ABA may lead to the difficulty of germination.

The long pollen tube journey and in vitro pollen germination of Phalaenopsis orchids.

KEY MESSAGE: Pollen biology in P. aphrodite. Orchids have a distinct reproductive program. Pollination triggers ovule development and differentiation within flowers, and fertilization occurs days to months after pollination. It is unclear how pollen tubes travel through the developing ovaries during ovule development and when pollen tubes arrive at the mature embryo sac to achieve fertilization. Here, we report a robust staining protocol to image and record the timing of pollen germination, progressive growth of pollen tubes in ovaries, and arrival of pollen tubes at embryo sacs in Phalaenopsis aphrodite. The pollen germinated and pollen tubes entered the ovary 3 days after pollination. Pollen tubes continued to grow and filled the entire cavity of the ovary as the ovary elongated and ovules developed. Pollen tubes were found to enter the matured embryo sacs at approximately 60-65 days after pollination in an acropetal manner. Moreover, these temporal changes in developmental events such as growth of pollen tubes and fertilization were associated with expression of molecular markers. In addition, we developed an in vitro pollen germination protocol, which is valuable to enable studies on pollen tube guidance and tip growth regulation in Phalaenopsis orchids and possibly in other orchid species.

Proteomic and morphometric study of the in vitro interaction between Oncidium sphacelatum Lindl. (Orchidaceae) and Thanatephorus sp. RG26 (Ceratobasidiaceae).

Orchidaceae establish symbiotic relationships with fungi in the Rhizoctonia group, resulting in interactions beneficial to both organisms or in cell destruction in one of them (pathogenicity). Previous studies have focused mostly on terrestrial species with a few, preliminary studies, on epiphytes. To further our understanding of the molecular mechanisms involved in these symbioses, we evaluated the interaction between Oncidium sphacelatum Lindl. and the mycorrhizal fungus Thanatephorus sp. strain RG26 (isolated from a different orchid species) in vitro using morphometric and proteomic analyses. Evidence from the morphometric and microscopic analysis showed that the fungus promoted linear growth and differentiation of orchid protocorms during 98 days interaction. On day 63, protocorm development was evident, so we analyzed the physiological response of both organisms at that moment. Proteome results suggest that orchid development stimulated by the fungus apparently involves cell cycle proteins, purine recycling, ribosome biogenesis, energy metabolism, and secretion that were up-regulated in the orchid; whereas in the fungus, a high expression of proteins implicated in stress response, protein-protein interaction, and saccharides and protein biosynthesis were found in the symbiotic interaction. This is the first work reporting proteins differentially expressed in the epiphytic orchid-fungus interaction and will contribute to the search for molecular markers that will facilitate the study of this symbiosis in both wild orchids and those in danger of extinction.

Floral nectary anatomy and ultrastructure in mycoheterotrophic plant, Epipogium aphyllum Sw. (Orchidaceae).

Epipogium aphyllum is a European-Asian obligatory mycoheterotrophic orchid containing no chlorophyll. Flowers are not resupinate with a sack-shape spur and cordate lip, which is divided into two parts: the basal (hypochile) and distal one (epichile). The floral analysis provides strong evidence to conclude that nectar is secreted on the upper surface of pink-coloured papillate ridges and epidermal (adaxial) cells at different place in spur, especially at the apex. The exudation on papillae has been observed through the entire anthesis and it has been stained on polysaccharides, proteins, and lipids. The dense cytoplasm of papillae contains profuse endoplasmic reticulum, plentiful vesicles (bigger ones with tannin-like materials), numerous mitochondria, sometimes dictyosomes, starch grains, and plastids with tubular structures. The large electron-dense bodies in cell walls are structurally the same as tannin-like materials from vesicles that are in contact with plasmalemma. The rupture of thin layer of swelled cuticle is caused by pressure of gathered substances exuded due to granulocrine secretion. The idioblasts with raphides occur mainly in tepals tissue. The dynamic changes of the nectar exudation, released through endocrine secretion, have been noticeable during the anthesis: both on the lip and inside the spur. The nectar secretion is not dependent on the colour form of E. aphyllum blooming shoots. The floral biology and ultrastructure differ from mycoheterotrophic plants known up to date.

Ultramicroscopic examination of mature massulae of Habenaria arinaria (Orchidaceae).

The mature massula of H. arinaria was examined by means of transmission electron microscopy, with the aim to understand the nature of cohesion between grains, the accumulation of pollen storage reserves, and the behavior of the nucleus of the vegetative cell in this composite type of pollen. The massula was a union of a large number of polygonal pollen grains that were tightly linked together. The exine within the massula were highly simplified, consisting of a single layer of nexine-2, lacking tectum, bacula, and nexine-1, while all the four layers comprised the exine on the massula surface. The two layers of nexine-2 of adjacent grains fused into a seamless whole. Undoubtedly the fusion of the nexine-2 was the mechanism by which the grains of the massula were linked together. No starch grains, lipid bodies, or storage proteins were present in the mature massula, and so the composite pollen of this species belonged to a novel type with regard to storage reserves. The vegetative nucleus was not lobed and revealed a huge amount of highly condensed chromatin, indicating a quiescent status. The condensed status of the vegetative nuclei in this composite type of pollen system is in striking contrast to the highly decondensed status reported in the free type of pollen grains.

Effect of cryopreservation on in vitro seed germination and protocorm growth of Mediterranean orchids.

BACKGROUND: Cryopreservation is an effective method for the long-term conservation of plant germplasm. OBJECTIVE: In the present study the effect of liquid nitrogen on seed structure, germination and protocorm development of eight Mediterranean orchid species was evaluated. MATERIALS AND METHODS: A scanning electron microscopy analysis was conducted to investigate the effect of the immersion in liquid nitrogen on seed integuments. Germination and protocorms growth were obtained applying in vitro cultures techniques. RESULTS: No micro-morphological differences emerged between cryopreserved and untreated seeds. The effect of the treatment on the seed germination varied on the basis of the species. For Ophrys sphegodes ssp. passionis and Orchis mascula, freezing resulted in a significant increase of germination percentages. Protocorms of all species showed a positive growth trend although some significant differences of size occurred among the protocorms derived from treated and untreated seeds. CONCLUSION: Cryogenic techniques seem to have great potential in orchid germplasm conservation.

Labellar anatomy and secretion in Bulbophyllum Thouars (Orchidaceae: Bulbophyllinae) sect. Racemosae Benth. & Hook. f.

BACKGROUND AND AIMS: Floral secretions are common in Bulbophyllum Thouars, and the labella of a number of Asian species are said to produce secretions rich in lipids that act as food rewards for insect pollinators. Although some of these reports are based on simple histochemical tests, a much greater number are anecdotal and, hitherto, neither the ultrastructure of the labellum nor the secretory process has been investigated in detail. Furthermore, sophisticated histochemical approaches have generally not been applied. Here, both the labellar structure and the secretory process are investigated for four species of Asian Bulbophyllum sect. Racemosae Benth. & Hook. f., namely Bulbophyllum careyanum (Hook.) Spreng., B. morphologorum Kraenzl., B. orientale Seidenf. and B. wangkaense Seidenf., and compared with those of unequivocal lipid-secreting orchids. METHODS: Labellar, secretory tissue was investigated using light microscopy, scanning electron microscopy, transmission electron microscopy and histochemistry. KEY RESULTS: The adaxial median longitudinal groove of the labellum contained secretory tissue comprising palisade-like epidermal cells, similar to those of certain lipid-secreting Oncidiinae Benth. However, these cells and their secretions gave positive results mainly for protein and mucilage, and their organelle complement was consistent with that of cells involved in protein and mucilage synthesis. Sub-cuticular accumulation of secretion resulted in cuticular distension and blistering. The sub-epidermal layer of isodiametric parenchyma contained starch and, like the epidermal cells, ultrastructure consistent with mucilage synthesis. Lipids were mainly confined to the cuticle, and hardly any intracellular lipid droplets were observed. CONCLUSIONS: It is proposed that mucilage is produced by dictyosomes present in the palisade-like epidermal cells. Mucilage precursors may also be produced by these same organelles in sub-epidermal cells and are thought to pass along the symplast via plasmodesmata into the adjoining palisade-like secretory cells, which contain abundant arrays of rough endoplasmic reticulum. Here, they become chemically modified and form a protein-rich, mucilaginous secretion that, following vesicle-mediated transport across the cytoplasm, traverses the cell wall and accumulates in blisters formed from the distended cuticle. Rupture of these blisters releases the secretion onto the labellar surface. However, in certain species, there is some evidence that the secretion may traverse the cuticle via cuticular pores, and micro-channels may permit the passage of fragrance. Hydrolysis of sub-epidermal starch probably generates the carbohydrate and, together with mitochondria, much of the energy required for the secretory process. This anatomical organization resembles that found in certain lipid-secreting, Neotropical species of Bulbophyllum and Oncidiinae, but since the chemical composition of their secretions is different, and these taxa occur on a separate continent and have different insect pollinators, parallelism of floral anatomy is likely.

Comparative anatomy of floral elaiophores in Vitekorchis Romowicz & Szlach., Cyrtochilum Kunth and a florally dimorphic species of Oncidium Sw. (Orchidaceae: Oncidiinae).

BACKGROUND AND AIMS: Recently, molecular approaches have been used to investigate the phylogeny of subtribe Oncidiinae, resulting in the re-alignment of several of its genera. Here, a description is given of the structure of the floral elaiophores (oil glands) of four species formerly assigned to Oncidium Sw. Those of Vitekorchis excavata (Lindl.) Romowicz & Szlach., Cyrtochilum meirax (Rchb.f.) Dalström and a species of Oncidium displaying floral dimorphism, namely O. heteranthum Poepp. & Endl. var. album, are compared with that of Gomesa longipes (Lindl.) M.W. Chase & N.H. Williams, whose epithelial elaiophores are typical of many Oncidiinae, in order to extend our understanding of elaiophore diversity within this subtribe. METHODS: Floral elaiophore structure was examined and compared at anthesis for all four species using light microscopy, scanning electron microscopy, transmission electron microscopy and histochemistry. KEY RESULTS: In all species investigated, with the exception of C. meirax, the floral elaiophore occurs on the labellar callus and is of the intermediate type, possessing both glabrous and trichomatous regions. By contrast, although all four species produce lipid secretions, C. meirax lacks an obvious elaiophore. In each case, the secretory tissue is represented by a single-layered epidermis of cuboidal cells (trichomatous and/or atrichomatous). Palisade cells are absent. The secretion may be wax- or oil-like and is usually produced by smooth endoplasmic reticulum (SER). However, in C. meirax, where rough endoplasmic reticulum (RER) predominates, oil accumulates as plastoglobuli within elaioplasts. These plastoglobuli are then discharged into the cytoplasm, forming oil bodies. In some species, oil usually accumulates within vesicles at the plasmalemma or in the periplasmic space before traversing the cell wall and accumulating beneath the cuticle, sometimes with distension of the latter. Gomesa longipes is unusual in its production of a heterogeneous secretion, whereas Vitekorchis excavata is equally remarkable for the protuberances found on the walls of its secretory cells. CONCLUSIONS: Anatomically, the secretory tissues of all four species, despite currently being assigned to four different genera, are remarkably similar and indicative of homoplasy. This supports previous investigations of the floral elaiophore in Oncidiinae, which showed that the same elaiophore characters may be shared by different clades, but not always by species of the same genus. Consequently, elaiophores are considered to be of limited value in investigating the phylogeny of this subtribe. Furthermore, floral dimorphism does not greatly modify elaiophore structure in the fertile flowers of Oncidium heteranthum var. album. Based on the presence or absence of well-defined elaiophores, the nature of the secretion and the cell ultrastructure, it is likely that floral oil may be produced in Oncidiinae in one of two ways: by the ER (mainly SER) or by plastids, most notably elaioplasts. Once the oil is discharged into the cytoplasm as oil bodies or oil droplets, there is little difference between the subsequent stages of oil secretion; the oil traversing the cytoplasm (often vesicle-mediated) and cell wall before accumulating beneath the cuticle.