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Dendrobium cariniferum Rchb. f. is an important crossing parent that is primarily used to produce aromatic Dendrobium orchid cultivars due to its beautiful flowers and pleasant orange fragrance. However, there are no offspring within the limited progenies that fully inherit this odor trait. Thus, converting diploid D. cariniferum into tetraploids for interploid hybridization is another promising approach to creating a new orange aroma germplasm. This study reports a novel in vitro colchicine-induced chromosome doubling protocol that would effectively convert diploid D. cariniferum protocorms into tetraploids. A total of 120 tetraploid genotypes were identified by flow cytometry. The tetraploid induction rate was significantly affected by interaction between the colchicine concentration and exposure time. Treatment with 0.05% colchicine for 24 h produced the optimal results, yielding 54% surviving regenerated plantlets with a 33% tetraploid induction rate. The tetraploid plantlets displayed obvious morphological variations (broader, thicker leaves and greater stem and root diameters) than the diploid plantlets. Tetraploidization also caused a significant change in the leaf anatomical structure, including larger stomata with lower densities, more chloroplasts per stomata, thicker spongy tissues, larger leaf veins and decreased adaxial epidermal cell and trichome densities. We were the first to observe that there are one to five branched trichomes in the leaf sheath in both diploids and tetraploids, and we propose that the leaf adaxial epidermal cell density can be used to identify tetraploids. Moreover, tetraploids exhibited a 1.75-fold greater genome size than diploids. Our study lays the foundation for breeding triploid aromatic Dendrobium orchids.Key message Autotetraploidy causes morphological, anatomical and genome size variations. This is the first report of trichome types in the leaf sheath. Leaf adaxial epidermal cell density is useful for identifying tetraploids.

Disentangling community assembly processes is crucial for fully understanding the function of microbiota in agricultural ecosystems. However, numerous plant microbiome surveys have gradually revealed that stochastic processes dominate the assembly of the endophytic root microbiota in conflict with strong host filtering effects, which is an important issue. Resolving such conflicts or inconsistencies will not only help accurately predict the composition and structure of the root endophytic microbiota and its driving mechanisms, but also provide important guidance on the correlation between the relative importance of deterministic and stochastic processes in the assembly of the root endophytic microbiota, and crop productivity and nutritional quality. Here, we propose that the inappropriate division of dispersal limitation may be the main reason for such inconsistency, which can be resolved after the proportion of dispersal limitation is incorporated into the deterministic processes. The rationality of this adjustment under the framework of the formation of a holobiont between the microbiome and the plant host is herein explained, and a potential theoretical framework for dynamic assembly patterns of endophytic microbiota along the soil–plant continuum is proposed. Considering that the assembly of root endophytic microbiota is complicated, we suggest caution and level-by-level verification from deterministic processes to neutral components to stochastic processes when deciding on the attribution of dispersal limitation in the future to promote the expansion and application of microbiome engineering in sustainable agricultural development based on community assembly patterns.

In vitro mitotic polyploidization using anti-microtubule agents has been commonly used for polyploid production. The present study was an attempt to develop an in vitro oryzalin chromosome doubling and stable tetraploid regenerating protocol for Dendrobium officinale. In this study, we successfully induced tetraploids by treating protocorms developed from the seeds of multiple genotypes. The highest frequency of polyploidy was 37.40%, achieved with 14.4 µM oryzalin treatment for 24 h. Among the obtained polyploid seedlings, 72 solid tetraploid, 33 mixoploid and 3 octoploid genotypes were identified via screening using flow cytometry (FCM). Three peaks were observed in the histograms of the diploid, tetraploid, and octoploid leaves, but four peaks were observed only in mixoploid leaves with FCM, indicating the existence of endopolyploid cells and the occurrence of conventional endoreduplication in the leaves of all ploidy levels. Recurrent ploidy identification of various tetraploid genotype regenerated plantlets obtained via protocorm-like bodies (PLBs) derived from axenic stem-node segments maintained stable tetraploid levels. Comparisons of phenotypic characteristics revealed that relative to the diploid plantlets, the tetraploid plantlets exhibited increased stem diameter, root diameter, labellum width and gynostemium width. Furthermore, the tetraploid plantlets showed lower plant height, leaf length and root length than the diploid plantlets. This efficient polyploid induction and ploidy-stable regeneration protocol can be used for the mass production of tetraploid D. officinale. The tetraploid genotypes regenerated in this study might be useful for nobile Dendrobium breeding in the future.Key messageIn vitro induction of various tetraploid genotypes in D. officinale and subsequent rapid micropropagation through induction of PLBs from axenic nodal segments were performed to obtain ploidy-stable regenerated plantlets

Background Most orchid species have been shown to be severely pollination limited, and the factors affecting reproductive success have been widely studied. However, the factors determining the reproductive success vary from species to species. Habenaria species typically produce nectar but exhibit variable fruit set and reproductive success among species. Here, we investigated the influence of the flowering plant density, inflorescence size, breeding system, and pollinator behaviour on the reproductive success of two rewarding Habenaria species. Results Our observations indicated that Habenaria limprichtii and H. petelotii co-occur in roadside verge habitats and present overlapping flowering periods. Both species were pollination limited, although H. limprichtii produced more fruits than H. petelotii under natural conditions during the 3-year investigation. H. petelotii individuals formed distinct patches along roadsides, while nearly all H. limprichtii individuals clustered together. The bigger floral display and higher nectar sugar concentration in H. limprichtii resulted in increased attraction and visits from pollinators. Three species of effective moths pollinated for H. limprichtii , while Thinopteryx delectans (Geometridae) was the exclusive pollinator of H. petelotii . The percentage of viable seeds was significantly lower for hand geitonogamy than for hand cross-pollination in both species. However, H. limprichtii may often be geitonogamously pollinated based on the behaviours of the pollinators and viable embryo assessment. Conclusions In anthropogenic interference habitats, the behaviours and abundance of pollinators influence the fruit set of the two studied species. The different pollinator assemblages in H. limprichtii can alleviate pollinator specificity and ensure reproductive success, whereas the more viable embryos of natural fruit seeds in H. petelotii suggested reducing geitonogamy by pollinators in the field. Our results indicate that a quantity-quality trade-off must occur between species with different breeding strategies so that they can fully exploit the existing given resources.

Reproductive isolation is a key feature that forms barriers to gene flow between distinct plants. In orchids, prezygotic reproductive isolation has been considered to be strong, because their associations with highly specific pollinators. In this study, the reproductive ecology and reproductive isolation of two sympatric Habenaria species, H. davidii and H. fordii, was investigated by floral phenology and morphology, hand-pollination experiments and visitor observation in southwest China. The two species were dependent on insects for pollination and completely self-compatible. A number of factors have been identified to limit gene flow between the two species and achieved full reproductive isolation. Ecogeographic isolation was a weak barrier. H. fordii and H. davidii had completely overlapped flowering periods, and floral morphology plays an important role in floral isolation. The two species shared the same hawkmoth pollinator, Cechenena lineosa, but the pollinaria of the two orchids were attached on different body parts of pollinators. Prezygotic isolation was not complete, but the interspecific pollination treatments of each species resulted in no seed sets, indicating that unlike many other orchid species, in which the postzygotic reproductive isolation is very weak or complete absence, the post-zygotic isolation strongly acted in the stage of seed production between two species. The results illustrate the reproductive isolation between two species involves multiple plant life-history stages and a variety of reproductive barriers can contribute to overall isolation.

Orchid distribution and population dynamics are influenced by a variety of ecological factors and the formation of holobionts, which play key roles in colonization and ecological community construction. Seed germination, seedling establishment, reproduction, and survival of orchid species are strongly dependent on orchid mycorrhizal fungi (OMF), with mycorrhizal cheating increasingly observed in photosynthetic orchids. Therefore, changes in the composition and abundance of OMF can have profound effects on orchid distribution and fitness. Network analysis is an important tool for the study of interactions between plants, microbes, and the environment, because of the insights that it can provide into the interactions and coexistence patterns among species. Here, we provide a comprehensive overview, systematically describing the current research status of the effects of OMF on orchid distribution and dynamics, phylogenetic signals in orchid–OMF interactions, and OMF networks. We argue that orchid–OMF associations exhibit complementary and specific effects that are highly adapted to their environment. Such specificity of associations may affect the niche breadth of orchid species and act as a stabilizing force in plant–microbe coevolution. We postulate that network analysis is required to elucidate the functions of fungal partners beyond their effects on germination and growth. Such studies may lend insight into the microbial ecology of orchids and provide a scientific basis for the protection of orchids under natural conditions in an efficient and cost-effective manner.

Orchids form mycorrhizal symbioses with fungi in natural habitats that affect their seed germination, protocorm growth, and adult nutrition. An increasing number of studies indicates how orchids gain mineral nutrients and sometime even organic compounds from interactions with orchid mycorrhizal fungi (OMF). Thus, OMF exhibit a high diversity and play a key role in the life cycle of orchids. In recent years, the high-throughput molecular identification of fungi has broadly extended our understanding of OMF diversity, revealing it to be a dynamic outcome co-regulated by environmental filtering, dispersal restrictions, spatiotemporal scales, biogeographic history, as well as the distribution, selection, and phylogenetic spectrum width of host orchids. Most of the results show congruent emerging patterns. Although it is still difficult to extend them to all orchid species or geographical areas, to a certain extent they follow the “everything is everywhere, but the environment selects” rule. This review provides an extensive understanding of the diversity and ecological dynamics of orchid-fungal association. Moreover, it promotes the conservation of resources and the regeneration of rare or endangered orchids. We provide a comprehensive overview, systematically describing six fields of research on orchid-fungal diversity: the research methods of orchid-fungal interactions, the primer selection in high-throughput sequencing, the fungal diversity and specificity in orchids, the difference and adaptability of OMF in different habitats, the comparison of OMF in orchid roots and soil, and the spatiotemporal variation patterns of OMF. Further, we highlight certain shortcomings of current research methodologies and propose perspectives for future studies. This review emphasizes the need for more information on the four main ecological processes: dispersal, selection, ecological drift, and diversification, as well as their interactions, in the study of orchid-fungal interactions and OMF community structure.

BackgroundSeed viability testing is essential in plant conservation and research. Seed viability testing determines the success of ex-situ conservation efforts, such as seed banking but commonly testing protocols of orchids lack consistency and accuracy, therefore, there is a need to select an appropriate and reliable viability test, especially when conducting comparative studies. Here, we evaluated the suitability of three seed viability tests, Evans blue test (EB), Fluorescein diacetate test (FDA) and Tetrazolium test (TTC), with and without sterilization, on seeds of 20 orchid species, which included five epiphytes and fifteen terrestrials, using both fresh seeds and seeds stored at − 18 ºC for 6 to 8 years.ResultsWe found that sterilization and lifeform of seeds affected seed viability across all tests but the storage time was not an influential factor. Sterilization negatively affected seed viability under EB and FDA test conditions but increased the detection of viable seeds in the TTC test in both epiphytic and terrestrial species. The EB test, when administered without sterilization provided the highest viability results. Being non-enzymatic unlike TTC and FDA tests, as expected, the EB test was the most reliable with similar results between sterilized and not sterilized seeds for most epiphytic and terrestrial species as well as when compared between groups.ConclusionsThe lifeform of the species and seed sterilization prior to testing are important influential factors in orchid seed viability testing. Since EB test was found to be reliable we recommend the EB test for seed viability assessment in orchids rather than the less reliable but commonly used TTC test, or the FDA test, which require more expensive and sophisticated instrumentation. Since storage time was not an influential factor in orchid seed viability testing, the recommendations of this study can be used for both fresh as well as long-term stored orchid seeds. This is helpful for research and especially for conservation measures such as seed banking. However, due to the species specificity of the bio-physiology of orchids, we call for comprehensive viability test assessment in the hyper diverse orchid family to be extended to a greater number of species to facilitate efficient conservation and research.

Symbiotic seed germination serves as a preferred method for orchid multiplication related to conservation and reintroduction programs, which involves isolation, identification, and germination-enhancing testing of symbiotic fungi. This study uses seeds of Papilionantheteres, a locally endangered and medicinally valuable epiphytic orchid, to attract germination-enhancing fungi on its four host plants. Only one common and highly compatible fungus (Epa-01 strain, Epulorhiza sp.), isolated from seed baiting near three host plants (Averrhoacarambola, Lagerstroemiavillosa, Callistemonrigidus), enhanced seed germination by more than 80 % and promoted protocorm development to reach stage 5 (with two leaves). Seeds cocultured with the Epa-01 strain and oat meal agar medium significantly outperformed in germination and growth speed compared with those cocultured with asymbiotic germination medium only, indicating that symbiotic seed germination is an effective method for P. teres seedling production. Bark substrate types have profound effects on symbiotic seed germination and protocorm development possibly due to different abundance and growth vitality of the Epa-01 strain on the four host plants. A significant difference was found in the developmental speed of symbiotic seeds between A. carambola and the other three host plants under ex situ and ex vitro seed germination treatments (all P < 0.05). The results suggest that in situ seed baiting may be used to effectively capture germination-enhancing fungi in epiphytic orchids, and testing the effects of bark substrate types on seed germination and protocorm development contributes to selecting appropriate host plants for its reintroduction into natural habitats.

The use of mycorrhizal fungi to enhance orchid seed germination and seedling growth is a promising approach for orchid propagation and conservation, but practical applications remain limited. In this study, we developed a direct seeding technique based on mycorrhizal symbiosis using Dendrobium officinale. Seeds were inoculated with two fungal strains, Serendipita officinale (SO) and Serendipita indica (SI), individually or in combination, and cultivated on three substrates to identify optimal fungus–substrate combinations. SO achieved the highest germination rate (52.9 ± 5.6%) at 30 days on substrate 1 but declined at later stages, while SI performed best on substrate 3 at 30 days (72.3 ± 6.7%) but was less effective after 90 days. The SO and SI mixture showed strong synergistic effects on substrate 1, with peak germination (48.7 ± 5.9%) and seedling formation (45.6 ± 5.1%) at 120 days. Substrates 1 and 3 significantly outperformed 2 (p < 0.05), with 1 promoting rapid early germination and 3 favoring long-term seedling establishment. In contrast, controls without fungi showed less than 5% for all indices, confirming the necessity of symbiotic fungi. Microscopic observations revealed typical orchid mycorrhizal structures and dynamic hyphal turnover, providing histological evidence of the symbiotic mechanism. These findings establish a practical framework for mycorrhizal-assisted propagation and contribute to the ecological cultivation and conservation of D. officinale.