A novel method to produce massive seedlings via symbiotic seed germination in orchids.

Orchids produce large numbers of dust-like seeds that rely heavily on orchid mycorrhizal fungi (OMFs) for germination. Using OMFs to facilitate orchid proliferation is considered an effective method for orchid conservation but still presents challenges in practice. In this study, orchid seed-fungus complexes, in which orchid seeds and fungal mycelia were embedded together to form granules, were developed as platforms to facilitate seed germination and seedling production. Overall, seedlings were produced by seed-fungus complexes for five orchid species with large variations in the percentages of seedlings produced among species/treatments. For the different fungal treatments in Dendrobium officinale, Sebacinales LQ performed much better than the other fungal strains. At 90 days after sowing, 75.8±2.6% seedlings were produced in the LQ treatment, which was significantly higher than in the Tulasnella sp. JM (22.0±3.0%) and Tulasnella sp. TPYD-2 (5.3±1.0%) treatments, as well as in the LQ and TPYD-2 cocultured treatment (40.4±3.2%), while no seedlings were formed in the Tulasnella sp. SSCDO-5 or control treatments. For the other four orchid species, only one compatible fungus for each species was used, and the percentages of seedlings in epiphytic Dendrobium devonianum (67.2±2.9%) and D. nobile (38.9±2.8%) were much higher than those in terrestrial Paphiopedilum spicerianum (2.9±1.1%) and Arundina graminifolia (6.7±2.1%) at 90 days after sowing. Adding 1% polymer water-absorbent resin to the seed-fungus complexes of D. officinale seeds with fungal strain Sebacinales LQ significantly increased seedling formation, while other additional substances showed negative effects on seedling formation. For the storage of seed-fungus complexes, it is recommended to store the seed-fungus complexes in valve bags at room temperature for a short time and at a low temperature of 4°C for no more than 30 days. As a platform for symbiotic seed germination, the seed-fungus complex can facilitate seed germination, produce seedlings and support subsequent seedling growth, and its seedling productivity depends on seed germination characteristics, seed viability, and the efficiency of fungi. Seed-fungus complexes have great potential to be used as propagules in orchid conservation.

What role does the seed coat play during symbiotic seed germination in orchids: an experimental approach with Dendrobium officinale.

BACKGROUND: Orchids require specific mycorrhizal associations for seed germination. During symbiotic germination, the seed coat is the first point of fungal attachment, and whether the seed coat plays a role in the identification of compatible and incompatible fungi is unclear. Here, we compared the effects of compatible and incompatible fungi on seed germination, protocorm formation, seedling development, and colonization patterns in Dendrobium officinale; additionally, two experimental approaches, seeds pretreated with NaClO to change the permeability of the seed coat and fungi incubated with in vitro-produced protocorms, were used to assess the role of seed coat played during symbiotic seed germination. RESULTS: The two compatible fungi, Tulasnella sp. TPYD-2 and Serendipita indica PI could quickly promote D. officinale seed germination to the seedling stage. Sixty-two days after incubation, 67.8 ± 5.23% of seeds developed into seedlings with two leaves in the PI treatment, which was significantly higher than that in the TPYD-2 treatment (37.1 ± 3.55%), and massive pelotons formed inside the basal cells of the protocorm or seedlings in both compatible fungi treatments. In contrast, the incompatible fungus Tulasnella sp. FDd1 did not promote seed germination up to seedlings at 62 days after incubation, and only a few pelotons were occasionally observed inside the protocorms. NaClO seed pretreatment improved seed germination under all three fungal treatments but did not improve seed colonization or promote seedling formation by incompatible fungi. Without the seed coat barrier, the colonization of in vitro-produced protocorms by TPYD-2 and PI was slowed, postponing protocorm development and seedling formation compared to those in intact seeds incubated with the same fungi. Moreover, the incompatible fungus FDd1 was still unable to colonize in vitro-produced protocorms and promote seedling formation. CONCLUSIONS: Compatible fungi could quickly promote seed germination up to the seedling stage accompanied by hyphal colonization of seeds and formation of many pelotons inside cells, while incompatible fungi could not continuously colonize seeds and form enough protocorms to support D. officinale seedling development. The improvement of seed germination by seed pretreatment may result from improving the seed coat hydrophilicity and permeability, but seed pretreatment cannot change the compatibility of a fungus with an orchid. Without a seed coat, the incompatible fungus FDd1 still cannot colonize in vitro-produced protocorms or support seedling development. These results suggest that seed coats are not involved in symbiotic germination in D. officinale.

Fatty acid composition in seeds of holoparasitic Orobanchaceae from the Caucasus region: Relation to species, climatic conditions and nutritional value.

The specialization of parasitic plants from the Orobanchaceae family to the heterotrophic lifestyle caused several morphological, physiological and molecular changes. One of the adaptations to the parasitic lifestyle is the production of a large number of the smallest seeds in world flora, also called "dust-seeds". Seeds of 34 holoparasitic species from the Cistanche, Orobanche, Phelipanche, and Phelypaea genera were collected in the Caucasus region (54 samples) and their fatty acid content and compositions analysed. Of these seeds, 28 were investigated for the first time, and 12 are endemic to the Caucasus (one of the most important biodiversity hotspots in the world). The influence of different hosts, populations, habitats, and climatic conditions on the fatty acid content and composition, as well as some connections of taxonomic classification are discussed. The fatty acid content in the species varied between 0.9 and 42.5%, and showed quantitative differences at generic and infrageneric levels, while displaying uniform fatty acid composition. Thirteen fatty acids were identified, of which nine were undescribed for Orobanchaceae. The fatty acid composition of the Orobanchaceae seeds represented a mixture of saturated fatty acids (SFAs) (average 7.8%) and unsaturated fatty acids (UFAs) (average 92.2%). The fatty acid content in the Orobanchaceae seeds was directly unrelated to taxonomy, while the n-6/n-3 fatty acid ratio supported the clear separation of the Orobanche and Phelipanche genera. Orobanchaceae seeds contained mainly linoleic and oleic acids, thus they could be a potential nutritional source of the unsaturated fatty acids. Additionally, the studies confirmed the hypothesis that the degree of seed oil fatty acid unsaturation increased in colder climatic conditions, especially for the Orobanche genus.

Transitions between the Terrestrial and Epiphytic Habit Drove the Evolution of Seed-Aerodynamic Traits in Orchids.

Orchids are globally distributed, a feature often attributed to their tiny dustlike seeds. They were ancestrally terrestrial but in the Eocene expanded into tree canopies, with some lineages later returning to the ground, providing an evolutionarily replicated system. Because seeds are released closer to the ground in terrestrial species than in epiphytic ones, seed traits in terrestrials may have been under selective pressure to increase seed dispersal efficiency. In this study, we test the expectations that seed airspace-a trait known to increase seed flotation time in the air-is (i) larger in terrestrial lineages and (ii) has increased following secondary returns to a terrestrial habit. We quantified and scored 20 seed traits in 121 species and carried out phylogenetically informed analyses. Results strongly support both expectations, suggesting that aerodynamic traits even in dust seeds are under selection to increase dispersal ability, following shifts in average release heights correlated with changes in habit.

Mycorrhizal divergence and selection against immigrant seeds in forest and dune populations of the partially mycoheterotrophic Pyrola rotundifolia.

Plant populations occupying different habitats may diverge from each other over time and gradually accumulate genetic and morphological differences, ultimately resulting in ecotype or even species formation. In plant species that critically rely on mycorrhizal fungi, differences in mycorrhizal communities can contribute to ecological isolation by reducing or even inhibiting germination of immigrant seeds. In this study, we investigated whether the mycorrhizal communities available in the soil and associating with the roots of seedlings and adult plants of the partially mycoheterotrophic Pyrola rotundifolia differed between populations growing in sand dunes and forests. In addition, reciprocal germination experiments were performed to test whether native seeds showed higher germination than immigrant seeds. Our results showed that the mycorrhizal communities differed significantly between forest and dune populations, and that within populations seedlings and adults also associated with different mycorrhizal communities. In both forest and dune populations, mycorrhizal communities were dominated by members of the Thelephoraceae, but dune populations showed a higher incidence of members of the Inocybaceae, whereas forest populations showed a high abundance of members of the Russulaceae. Reciprocal germination experiments showed that native seeds showed a higher germination success than immigrant seeds and this effect was most pronounced in dune populations. Overall, these results demonstrate that plants of P. rotundifolia growing in dune and forest habitats associate with different mycorrhizal communities and that reduced germination of non-native seeds may contribute to reproductive isolation. We conclude that selection against immigrants may constitute an important reproductive barrier at early stages of the speciation process.

An annotated translation of Noël Bernard's 1899 article 'On the germination of Neottia nidus-avis'.

We translate Noël Bernard's discovery of orchid symbiotic germination discovered on Neottia nidus-avis, as published in the May 1899 issue of the Comptes rendus hebdomadaires des séances de l'Académie des sciences. In his note, Bernard (1874-1911) establishes the need for a fungus, which is also forming mycorrhizae in adults, for seeds germination. We provide illustrations reproduced from his later works, and summaries of the French text he cited. In our annotations, we show how early this discovery was done in Bernard's career, and insist on the scientific framework at the end of the nineteenth century, where orchid germination was mysterious and the need for vicinity of parents was not fully understood. We comment the text of Bernard on the basis of the most recent knowledge on Neottia nidus-avis and on orchid mycorrhizal fungi. Introducing his following papers, we finally discuss the emergence of the concept of peloton digestion, and how Bernard's work quickly paved the way to a general understanding of mycoheterotrophic germination in orchids and beyond.

Specificity of fungal associations of Pyroleae and Monotropa hypopitys during germination and seedling development.

Mycoheterotrophic plants obtain organic carbon from associated mycorrhizal fungi, fully or partially. Angiosperms with this form of nutrition possess exceptionally small 'dust seeds' which after germination develop 'seedlings' that remain subterranean for several years, fully dependent on fungi for supply of carbon. Mycoheterotrophs which as adults have photosynthesis thus develop from full to partial mycoheterotrophy, or autotrophy, during ontogeny. Mycoheterotrophic plants may represent a gradient of variation in a parasitism-mutualism continuum, both among and within species. Previous studies on plant-fungal associations in mycoheterotrophs have focused on either germination or the adult life stages of the plant. Much less is known about the fungal associations during development of the subterranean seedlings. We investigated germination and seedling development and the diversity of fungi associated with germinating seeds and subterranean seedlings (juveniles) in five Monotropoideae (Ericaceae) species, the full mycoheterotroph Monotropa hypopitys and the putatively partial mycoheterotrophs Pyrola chlorantha, P. rotundifolia, Moneses uniflora and Chimaphila umbellata. Seedlings retrieved from seed sowing experiments in the field were used to examine diversity of fungal associates, using pyrosequencing analysis of ITS2 region for fungal identification. The investigated species varied with regard to germination, seedling development and diversity of associated fungi during juvenile ontogeny. Results suggest that fungal host specificity increases during juvenile ontogeny, most pronounced in the fully mycoheterotrophic species, but a narrowing of fungal associates was found also in two partially mycoheterotrophic species. We suggest that variation in specificity of associated fungi during seedling ontogeny in mycoheterotrophs represents ongoing evolution along a parasitism-mutualism continuum.

Tansley Review No. 110.: Numerical and physical properties of orchid seeds and their biological implications.

Orchid seeds are very small, extremely light and produced in great numbers. Most range in length from c. 0.05 to 6.0 mm, with the difference between the longest and shortest known seeds in the family being 120-fold. The 'widest' seed at 0.9 mm is 90-fold wider than the 'thinnest' one, which measures 0.01 mm (because orchid seeds are tubular or balloon-like, 'wide' and 'thin' actually refer to diameter). Known seed weights extend from 0.31 lg to 24 µg (a 78-fold difference). Recorded numbers of seeds per fruit are as high as 4000000 and as low as 20-50 (80000-200000-fold difference). Testae are usually transparent, with outer cell walls that may be smooth or reticulated. Ultrasonic treatments enhance germination, which suggests that the testae can be restrictive. Embryos are even smaller: their volume is substantially smaller than that of the testa. As a result, orchid seeds have large internal air spaces that render them balloon-like. They can float in the air for long periods, a property that facilitates long-distance dispersal. The difficult-to-wet outer surfaces of the testa and large internal air spaces enable the seeds to float on water for prolonged periods. This facilitates distribution through tree effluates and/or small run-off rivulets that may follow rains. Due to their size and characteristics, orchid seeds may also be transported in and on land animals and birds (in fur, feathers or hair, mud on feet, and perhaps also following ingestion). contents Summary 367 I. Introduction 367 II. Number 368 III. Size 379 IV. Air space in the seeds 381 V. Floatation and dispersal 383 VI. Conclusions 417 Acknowledgements 417 References 418.