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Background and Aims: Although abscisic acid (ABA) is commonly recognized as a primary cause of seed dormancy, there is a lack of information on the role of ABA during orchid seed development. In order to address this issue, the localization and quantification of ABA were determined in developing seeds of Cypripedium formosanum., Methods: The endogenous ABA profile of seeds was measured by enzyme-linked immunosorbent assay (ELISA). Temporal and spatial distributions of ABA in developing seeds were visualized by immunohistochemical staining with monoclonal ABA antibodies. Fluoridone was applied to test the causal relationship between ABA content and seed germinability., Key Results: ABA content was low at the proembryo stage, then increased rapidly from 120 to 150 days after pollination (DAP), accompanied by a progressive decrease in water content and seed germination. Immunofluorescence signals indicated an increase in fluorescence over time from the proembryo stage to seed maturation. From immunogold labelling, gold particles could be seen within the cytoplasm of embryo-proper cells during the early stages of seed development. As seeds approached maturity, increased localization of gold particles was observed in the periplasmic space, the plasmalemma between embryo-proper cells, the surface wall of the embryo proper, and the inner walls of inner seed-coat cells. At maturity, gold particles were found mainly in the apoplast, such as the surface wall of the embryo proper, and the shrivelled inner and outer seed coats. Injection of fluoridone into capsules resulted in enhanced germination of mature seeds., Conclusions: The results indicate that ABA is the key inhibitor of germination in C. formosanum. The distinct accumulation pattern of ABA suggests that it is synthesized in the cytosol of embryo cells during the early stages of seed development, and then exported to the apoplastic region of the cells for subsequent regulatory processes as seeds approach maturity., (© The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company.All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background and Aims: The Orchidaceae have a history of recurring convergent evolution in floral function as nectar production has evolved repeatedly from an ancestral nectarless state. However, orchids exhibit considerable diversity in nectary type, position and morphology, indicating that this convergence arose from alternative adaptive solutions. Using the genus Disa, this study asks whether repeated evolution of floral nectaries involved recapitulation of the same nectary type or diversifying innovation. Epidermis morphology of closely related nectar-producing and nectarless species is also compared in order to identify histological changes that accompanied the gain or loss of nectar production., Methods: The micromorphology of nectaries and positionally equivalent tissues in nectarless species was examined with light and scanning electron microscopy. This information was subjected to phylogenetic analyses to reconstruct nectary evolution and compare characteristics of nectar-producing and nectarless species., Key Results: Two nectary types evolved in Disa. Nectar exudation by modified stomata in floral spurs evolved twice, whereas exudation by a secretory epidermis evolved six times in different perianth segments. The spur epidermis of nectarless species exhibited considerable micromorphological variation, including strongly textured surfaces and non-secreting stomata in some species. Epidermis morphology of nectar-producing species did not differ consistently from that of rewardless species at the magnifications used in this study, suggesting that transitions from rewardlessness to nectar production are not necessarily accompanied by visible morphological changes but only require sub-cellular modification., Conclusions: Independent nectary evolution in Disa involved both repeated recapitulation of secretory epidermis, which is present in the sister genus Brownleea, and innovation of stomatal nectaries. These contrasting nectary types and positional diversity within types imply weak genetic, developmental or physiological constraints in ancestral, nectarless Disa. Such functional convergence generated by morphologically diverse solutions probably also underlies the extensive diversity of nectary types and positions in the Orchidaceae.

Premise of the Study: Protocorm-like bodies (PLBs) of orchids are important in orchid micropropagation and outwardly resemble somatic embryos in form and development. To determine whether PLBs are truly embryogenetic, we compared PLBs with somatic embryos and zygotic embryos to determine whether they had similar surface molecules and whether hydroxyproline-rich glycoprotein (HRGP) inhibitors similarly alter their growth., Methods: Embryogenic calluses (ECs), zygotic embryos, and protocorms were collected for histological and histochemical studies with light microscopy. The presence of JIM11 and JIM20 epitopes was determined using immunodot blots and immunolocalization procedures. The importance of wall proteins in the formation of PLBs was investigated using 3,4-dehydro-l-proline (3,4-DHP), an inhibitor of HRGP biosynthesis., Key Results: At the early stages of PLB formation, the cytoplasm of the globular cell clusters and meristemoids took on a vacuolated appearance. Starch granules and protein bodies appeared, albeit transitory in nature. Positive localizations of JIM11 and JIM20 were noted in the embryogenic culture and developing PLBs similar to zygotic embryos. The inclusion of an inhibitor to HRGPs inhibited PLB formation., Conclusions: This study demonstrates that during the early stages of PLB formation, the cells show cytological characteristics and cell wall markers similar to zygotic embryo development, justifying the statement that PLBs are indeed somatic embryos of orchids. Thus, these results suggest that PLBs could be used as an experimental embryological system for physiological or molecular characterization.

The suspensor plays an active role during the early embryo development of flowering plants. In orchids, the suspensor cells are highly vacuolated without structural specializations, and the possible mechanism(s) that enable the suspensor to serve as the nutrient uptake site is virtually unknown. Here, we used the fluorescent tracer CFDA to characterize the pathway for symplastic transport in the suspensor cells of developing embryos and to provide direct visual evidence that the orchid suspensor has unique physiological properties. The embryo proper uptakes the fluorescent dye through the suspensor. CF could first be detected throughout the suspensor cell and then subsequently in the embryo proper. A plasmolysis experiment clearly indicates that suspensor cells have a more negative osmotic potential than the adjoining testa cells. It is proposed that the preferential entry of CFDA into the suspensor cell of the Nun orchid is aided by the more negative osmotic potential of the suspensor than neighboring cells, providing a driving force for the uptake of water from the apoplast into the symplast.

Background and Aims: Owing to large-scale collecting, the lady's slipper orchid, Paphiopedilum delenatii, is under threat of extinction. Asymbiotic germination provides a useful way to re-establish plants in the wild and for commercial propagation. A detailed study of embryo development would provide information on subsequent germination events and aid in the propagation of the species., Methods: Developing capsules were collected for histochemical and ultrastructural studies by using both light and transmission electron microscopy., Key Results: The suspensor of this species consists of three vacuolated cells. During the early globular stage of embryo development, structural differentiation occurs, revealing an abundance of smooth endoplasmic reticulum cisternae and wall ingrowths within the suspensor cells. These features are not present in cells of the embryo proper. Furthermore, the results of Nile red staining demonstrate that a cuticular layer is present only in the embryo proper, but absent from the suspensor. Cuticular material is also present in the inner walls of the seed coat, and persists through seed maturation., Conclusions: The morphological features of the transfer cell and the absence of cuticular material in the suspensor cell wall corroborate the hypothesis that the suspensor is the major nutrient uptake site for the developing embryo in the lady's slipper orchid. The absence of an endosperm and presence of cuticular material in the inner walls of the seed coat enclosing the embryo proper further support the notion that nutrient uptake by the embryo is confined to the micropylar end of the seed through the suspensor.

Fertilization in flowering plants involves the fusion of one sperm with the egg cell and a second sperm with the central cell. In the Nun orchid, Phaius tankervilliae (Aiton) Bl., the pollen tube deposits two sperms in the "cytoplasmic mass" of the degenerating synergid. Initially both sperms stay close together. Soon afterwards, the two sperms undergo migration. The leading sperm migrates towards the central cell, while the other sperm moves laterally towards the egg cell. The degenerated synergid cytoplasmic content fills in the available space between the central cell and the egg cell, forming the actin coronas. Due to the high actin content, the bright fluorescence initially prevents the visualization of cellular details. With the subsequent reduction in fluorescence, actin staining reveals that the two sperms are pear-shaped with pointed tails. As the sperms approach their respective target cells, cellular extensions form near the point of sperm entry in both the egg cell and the central cell. These structures appear to aid in the cell fusion process. The morphological and structural features observed provide evidence that the process of double fertilization requires the active participation of not only the two sperms but also the egg cell and the central cell.