Protocorm Formation Research Articles

In vitro seed propagation of Dendrobium moschatum as influenced by different plant growth regulators

This study aimed to develop an effective in vitro protocol for germinating and multiplying Dendrobium moschatum seeds by identifying optimal concentrations of plant growth regulators for shoot proliferation and root formation. Five different MS media formulations were utilized, including a control (M?) and those supplemented with varying concentrations of kinetin and NAA (M?, M?, M?) and BAP (M?). Results showed significant variation in seed germination time across media, with the longest duration observed in M? (76 days) and the shortest in M? (55 days). Protocorm formation and plantlet development were quickest in M?, while M? and M? exhibited the longest duration. The initiation of leaves or shoots occurred earliest in M? (103 days) and latest in M? (149 days). Notably, leaf and plantlet growth differed between three- and five-month intervals, with M? demonstrating optimal growth over the longer term. These findings underscore the importance of carefully balanced plant growth regulator concentrations in achieving successful seed germination and subsequent growth in D. moschatum.

Phorophyte preference of an epiphytic orchid (Dendrobium wangliangii) is independent of orchid mycorrhizal fungi that promote seed germination

Abstract Epiphytism is an example of an obligate interspecific interaction between plants, where a plant species grows on another plant species for physical support. In epiphytic orchids that rely on suitable host tree species (phorophytes) and mycorrhizal fungi for germination and growth, a distinct preference has been observed for particular host tree species. However, the factors driving this host preference remain unclear. In this study, we investigated whether mycorrhizal fungi that promote seedling development determine the preference of phorophytes in the epiphytic orchid Dendrobium wangliangii. First, we identified the preferred host tree species for this orchid. Next, in vitro and in situ germination experiments were conducted to identify potential fungi that promote seed germination. Finally, high‐throughput sequencing was used to assess the distribution of these fungi on both colonized host trees and potential host trees, including individual trees of the host species not yet colonized by the orchid and trees of another species growing in the vicinity of a colonized tree. Dendrobium wangliangii exhibited a preference for trees within the Fagaceae family. Two Fusarium species from the bark of host trees were identified as the primary fungi that promote seed germination. Successful protocorm formation and seedling development were observed exclusively on preferred phorophytes. Fungal community analyses further indicated that both Fusarium spp. occurred on all potential host trees, suggesting that the host preference of this epiphytic orchid is not constrained by the distribution of suitable mycorrhizal fungi. Synthesis: Regarding the epiphytes that depend on mycorrhizal fungi for successful seedling recruitment, we conclude that strong epiphyte–phorophyte associations do not arise from the limited distribution of mycorrhizal fungi but are more likely the result of other factors inherent in the phorophyte environment.

Influences of Organic Additives on Asymbiotic Seed Germination of Dendrobium cruentum Rchb. f. for In Vitro Micropropagation

Dendrobium cruentum Rchb. f., a commercial orchid in Thailand, faces the risk of extinction and should be conserved, and propagated for commercial. Therefore, the aim of this research was to identify a suitable VW medium supplemented with organic additives for the micropropagation of D. cruentum through asymbiotic seed germination. The VW media supplemented with separate organic additives (coconut water (CW) (100 mL/L), ‘Hom Thong’ banana homogenate (BH) (1000 mg/L), and potato homogenate (PH) (1000 mg/L) or its combination was determined, and the seed germination rate (%), protocorm formation, and plantlet development were observed. The results showed that a combination of BH and PH resulted in the highest seed germination (94.69 ± 1.55 %) when compared to basal VW (control). Protocorm formation was highest with CW alone (47.38 ± 7.17 %) and a combination of CW and PH (52.13 ± 6.20 %). Additionally, after 10 weeks of culture, supplementation with BH yielded the highest number of plantlets. However, after 24 weeks of culture, each organic additives gave the different developments of healthy plantlets. To investigate suitable potting media for acclimatization of well-grown plantlets in greenhouse conditions, 2 pieces of foam, dried coconut husk, and a combination of peat moss: Sphagnum moss: Pumice: Perlite (3:1:1:1) were investigated. The highest survival rate percentage was found in foam-planted plantlets (80 %). Ultimately, the effectively simple mediums containing organic additives and a one-step protocol were provided for the micropropagation of D. cruentum via seeds, and the derived information could be used for D. cruentum conservation and production. HIGHLIGHTS The effects of organic additives on asymbiotic seed germination for in vitro micropropagation of Dendrobium cruentum Rchb. f. was firstly reported The media supplemented with organic additives yield significantly the highest seed germination, protocorm formation, and plantlet formation and development of D. cruentum when compared to basal media In greenhouse conditions, 2 pieces of foam gave the highest survival rate percentage of plantlets (80 %) The practical and beneficial media supplemented with organic additives for micropropagation of D. cruentum was provided GRAPHICAL ABSTRACT

First evidence for multimodal animal seed dispersal in orchids

Identifying the mechanisms for seed dispersal and persistence of species is a central aim of ecology. Seed dispersal by animals is an essential form of dissemination in many plant communities, including seeds of over 66% of neotropical canopy tree species.1,2 Besides physical dispersal, animals influence seed germination probabilities through scarification, breaking dormancy, and preventing rotting, so plants often invest important resources in attracting them. Orchids are predominantly adapted to wind dispersal, having dust-like seeds that are easily uplifted. Exceptions include bird-,3,4 cricket-,5,6 and mammal-dispersed7 species, featuring fleshy fruits with hard seeds that germinate after passing the animal's digestive system. Given the similarity in fruit and seed morphology, zoochory has also been suggested in Vanilla,8,9,10,11,12,13,14,15 a pantropical genus of 118 species with vine-like growth.16,17,18 We test this prediction through in situ and ex situ experimentation using fruits of Vanilla planifolia, and wild relatives, from which vanillin-a widely used natural aroma and flavoring-is obtained. Seeds from dehiscent fruits are removed by male Euglossini collecting fragrances, a unique case in plants, and female Meliponini bees gathering nest-building materials, a first among monocots. By contrast, mammals, mostly rodents, consume the nutritious indehiscent fruits, passing the seeds up to 18h after consumption. Protocorm formation in digested and undigested seeds proves that scarification in the gut is not strictly required for germination. Multimodal seed dispersal mechanisms are proven for the first time in Orchidaceae, with ectozoochory and endozoochory playing crucial roles in the unusually broad distribution of Vanilla.

In vitro asymbiotic seed germination, protocorm formation, and plantlet development of Orchis simia Lam.: A threatened terrestrial orchid species

• Seed germination and plantlet growth were impacted by organic constituents and nitrogen sources. • Individual application of organic nitrogen compounds led to higher germination efficiencies. • The present protocol could be used for large-scale in vitro micropropagation of terrestrial orchids. The present study examined the seed morphometric and viability, asymbiotic seed germination, protocorm formation, and plantlet growth in Orchis simia Lam., an at-risk Euro-Mediterranean terrestrial orchid species. Furthermore, tissue histology pattern in vitro grown plantlets and field-established was compared. After one month, shoot and root primordia started to grow on developing protocorms and small plantlets became visible after 2–3 subculturing. Asymbiotic in vitro seed germination and plantlet growth attributes were significantly ( P <0.05) impacted by both type of main organic constituent and nitrogen sources. The seeds sown on media supplemented with pineapple juice (PJ) and casein hydrolysate (CH) had the highest germination percentage. In terms of required days to see the first signs of germination, CW+AV treatment brings about the fastest germination (6.8 ± 0.20 days). Protocorms grown on the media comprising PJ in combination with Aminoven (AV) or casein hydrolysate (CH) or Pep showed higher dimensions and therefore higher weight (% 450 increase) compared to protocorms grown on other media. Altogether, treatments with higher germination percentage, faster germination, and bigger protocorms finally resulted in better plantlet growth. Acclimatization of in vitro grown plantlets occurred by converting scattered vascular tubes into fully developed xylem and phloem vessels facilitating sap flow. Taking into account the fragmentation of O. simia populations in the Middle East and the Euro-Mediterranean basin mainly due to tuber overharvesting and climate change, the present procedure could be used for large-scale multiplication of in vitro raised seedlings to re-introduce into endangered regions.

Addition of fungal inoculum increases seed germination and protocorm formation in a terrestrial orchid

Because orchid mycorrhizal fungi are important for orchid seed germination, addition of germination-supporting fungal inoculum to the soil can be an efficient way to improve in situ seed germination of terrestrial orchids, but empirical evidence is still largely lacking. In this study, we used seed inoculation experiments to test the hypothesis that addition of fungal inoculum increases seed germination of the terrestrial orchid Gymnadenia conopsea under natural field conditions. In China, this species has suffered dramatic declines in abundance due to habitat loss, grazing and over-collection, while observational studies have shown very low recruitment of seedlings into natural populations. Previous research has shown that the fungal strain GS2 (Ceratobasidiaceae) was able to support germination under in vitro conditions. Seed packages were used to bury orchid seeds in the soil with and without fungal inoculum and to assess seed germination. In total, 11.96 ± 2.65 % of seeds had the testa ruptured when fungal inoculum was added to the seed packages, 10.63 ± 3.32 % of seeds germinated to the protocorm formation stage, 5.24 ± 2.71 % of the seeds grew to the protocorm differentiation stage, and 3.16 ± 1.63 % of seeds finally formed early seedlings. In contrast, no seed germination was observed in the control groups without fungal inoculum. We isolated the fungi associating with the germinating seeds and confirmed that seed germination was supported by fungal strain GS2. In addition, several other fungi were isolated from germinating seeds, including four other Ceratobasidiaceae strains that are known to support seed germination in vitro. Our results highlight the potential of fungal inoculum to induce seed germination of terrestrial orchids under field conditions and may be useful when developing more efficient symbiotic seed germination protocols for the restoration and conservation of terrestrial orchids, especially in situations where natural germination is severely limited.

Epidendrumradicans Fungal Community during Ex Situ Germination and Isolation of Germination-Enhancing Fungi.

Orchids exhibit varying specificities to fungi in different microbial environments. This pilot study investigated the preference of fungal recruitment during symbiotic germination of Epidendrum radicans Pav. ex Lindl. Two different orchid substrates were used for ex situ seed baiting: pine bark and rotten oak leaf, with Basidiomycota and Ascomycota as the respective dominant groups. Both substrates promoted seed germination, with a higher protocorm formation rate on pine bark (65.75%). High-throughput sequencing characterized the fungal communities of germinated protocorms. Basidiomycota was the dominant group in protocorms that symbiotically germinated on both substrates. The family-level community structures of endophytic fungi in protocorms that symbiotically germinated on both substrates were close to those of protocorms that germinated in vitro on MS1 medium. For protocorms, the dominant fungal groups recruited from substrates differed at the genus level; from pine bark, they were genera belonging to unclassified Sebacinales (41.34%), Thanatephorus (14.48%) and Fusarium (7.35%), while, from rotten oak leaf, they were Rhizoctonia (49.46%), Clitopilus (34.61%), and Oliveonia (7.96%). Four fungal isolates were successfully obtained and identified as belonging to the family Tulasnellaceae, genera Ceratobasidium and Peniophora, which could promote seed germination to the seedling stage. The data indicate that endophytic fungi for E. radicans germination on two different substrates are affected at the genus level by the substrate, with a degree of specificity at the family level.

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

BackgroundOrchids 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.ResultsThe 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.ConclusionsCompatible 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.

Post-embryonic development and seedling morphogenesis of Dendrobium moniliforme (L.) Sw. under asymbiotic culture conditions

Dendrobium moniliforme is an endangered orchid species in China. It is important to understand the seed germination and plant morphogenesis for the protection of D. moniliforme germplasm resources. Using asymbiotic culture, morphological and anatomical method, we found the polar spherical embryo formed, and seed germination was completed after 6-9 days culture. It took about 27 days that the polar spherical embryo continued cell division and tissue differentiation, including the apical meristem formed, the rhizoid on the base of embryo developed. The mature embryo is consisted of cotyledon, the apex growth cone, the axial structure, and the rhizoid, but there is no differentiation of radicle primordia. After 57-60 days culture, the adventitious root primordia could be observed on the adjacent to the apical meristem zone, and also the first true leaf has developed, which meant the mature embryo developed into protocorm. With the development of the second true leaf, the stem axis extended significantly, the adventitious roots were growing out laterally as well, and the seedling morphogenesis was completed after 63-67 days culture. From a seed to the seedling, it should be divided into four stages: seed germination, post-embryonic development, protocorm formation and seedling morphogenesis. Not only the post-embryonic development is the key step in the mature embryo formation, but also the protocorm is a unique structure to be different from the embryo in morphogenesis process of D. moniliforme. Our research work resolves the controversy over the embryogenesis and development of Dendrobium and provides basic data for plant biological theory.

Addition of fungal inoculum increases germination of orchid seeds in restored grasslands

Grasslands restored on arable land often retain high residual nutrients, modified soil biota, and lower plant species diversity. Establishment of rare plant species with complex multitrophic interactions, typical of undisturbed nutrient-poor environments, may be hindered by the absence of interacting organisms. We hypothesised that the addition of a mycorrhizal symbiont improves the seed germination of orchids that crucially depend on fungi. We focused on grasslands restored on arable land 1–15 years ago featuring residual mineral nutrients and low organic matter contents compared to semi-natural grasslands and on four orchid species differing in the level of mycorrhizal specificity: high – Anacamptis pyramidalis and Orchis mascula – and low – Platanthera bifolia and Gymnadenia conopsea. Five fungal isolates obtained from non-green underground mycorrhizal orchid seedlings (protocorms) or adults' roots were tested for orchid-fungus compatibility under conditions in vitro. Orchid seeds inserted in retrievable seed packets were subsequently co-introduced with selected fungal isolates grown either on agar or sterilized hay into the soil of nine restored grasslands and incubated for twelve months. The identity of mycorrhizal fungi in retrieved protocorms was verified by molecular methods. The isolates that supported protocorm establishment in vitro enabled also protocorm formation in situ, but success rates differed among orchid species. While mycorrhizal specialists produced most protocorms after inoculation, the mycorrhizal generalists took advantage of naturally occurring fungi and produced some protocorms both in inoculated and uninoculated treatments. We showed that the addition of mycorrhizal fungi enhanced protocorm formation regardless of the modified soil environment, especially in mycorrhizal specialist orchids. This method may help to restore populations of native orchid species in their former distribution ranges, including farming-altered habitats.

Pollination Compatibility of Dendrobium Polinela Lampung, and The Effect of Media Composition with Coconut Water on Seed Germination In Vitro

Efforts to increase the quality of Dendrobium orchids can be made by increasing the genetic quality through crosses while increasing the quantity can be done by propagation through in vitro culture. Information about the compatibility of an orchid species is very important to maximize the acquisition of seeds as propagation material. The seeds from the cross were propagated by in vitro culture technique. The purpose of this study was to obtain a compatible crossing method between crossing and selfing D.Polinela Lampung orchids with selected parents and to determine the effect of giving a combination of media with coconut water concentration in inducing germination and growth of orchid seeds. This study consisted of two experiments, namely (1) the compatibility test of D.Polinela Lampung orchids with three types of Indonesian orchids (D.discolour, D.leporinum, D.lasientera), and (2) the effect of basic media (MS, VW, Growmore, Gandasil) and water concentration CW (0, 50, 150, and 250 ml/1) on seedling growth of D.Polinela Lampung x D.lasientera orchids. In conclusion, among the sixteen crosses of Dendrobium orchids, ten compatibility crosses were found (pollination that produces fruit). The addition of 15 and 25% CW in the growmore medium (91.37% and 98.03%), MS (90.97% and 96.00 %), and Gandasil (90.67% and 92.67%) gave the highest percentage results. Seed germination and protocorm formation. The protocorm that forms the first leaves produced on Growmore media added with 50-150 ml/1 coconut water gave the most results compared to other treatments. The results of the research are expected as basic information in orchid propagation, especially the study of compatibility and germination of orchid seeds.

Compatible and Incompatible Mycorrhizal Fungi With Seeds of Dendrobium Species: The Colonization Process and Effects of Coculture on Germination and Seedling Development.

Orchids highly rely on mycorrhizal fungi for seed germination, and compatible fungi could effectively promote germination up to seedlings, while incompatible fungi may stimulate germination but do not support subsequent seedling development. In this study, we compared the fungal colonization process among two compatible and two incompatible fungi during seed germination of Dendrobium officinale. The two compatible fungi, i.e., Tulasnella SSCDO-5 and Sebacinales LQ, originally from different habitats, could persistently colonize seeds and form a large number of pelotons continuously in the basal cells, and both fungi promoted seed germination up to seedling with relative effectiveness. In contrast, the two incompatible fungi, i.e., Tulasnella FDd1 and Tulasnella AgP-1, could not persistently colonize seeds. No pelotons in the FDd1 treatment and only a few pelotons in the AgP-1 treatment were observed; moreover, no seedlings were developed at 120 days after incubation in either incompatible fungal treatment. The pattern of fungal hyphae colonizing seeds was well-matched with the morphological differentiation of seed germination and seedling development. In the fungal cocultural experiments, for both orchids of D. officinale and Dendrobium devonianum, cocultures had slightly negative effects on seed germination, protocorm formation, and seedling formation compared with the monocultures with compatible fungus. These results provide us with a better understanding of orchid mycorrhizal interactions; therefore, for orchid conservation based on symbiotic seed germination, it is recommended that a single, compatible, and ecological/habitat-specific fungus can be utilized for seed germination.

Isolating ecological-specific fungi and creating fungus-seed bags for epiphytic orchid conservation

More than 1/3 of Chinese native orchids are used as Traditional Chinese Medicine (TCM) including many species of Dendrobium, and these Dendrobium species have been massively collected. The restoration-friendly cultivation model, i.e. planting targeted species in natural settings, is considered a new conservation tool for these epiphytic medicinal orchids, especially for Dendrobium species. In this study, we set out to develop an easy-to-use solution for practice of restoration-friendly cultivation in D. officinale, an critically endangered orchid with high medicinal value. We isolated fungi both from naturally occurred protocorms and roots of adult plants, and successfully obtained nine orchid mycorrhizal fungi (OMFs). Among them, five OMFs have been experimentally tested to promote seed germination and seedling development, but varied greatly in effectiveness. The most effective fungus, Sebacinales LQ, could quickly promote seedling formation and development in D. officinale. The percentage of protocorm formation could reach 65.90 ± 5.1%, and 4.57 ± 1.3% of seeds developed into seedlings at 30 days after incubation. At 60 days after incubation, the protocorm formation (83.71 ± 2.9%) already reached culmination and the percentage of seedlings (68.19 ± 3.8%) approached to peak level in LQ treatment. The fungus-seed bags, containing mixtures of fungal powders of LQ strains and seeds of D. officinale, were used as propagules and released in the original habitats of D. officinale. After one year, the percentages of seedlings in fungus-seed bags ranged from 13.16 ± 1.1% up to 20.88 ± 2.2% at three sites, and most seedlings appeared seemingly healthy. The fungus-seed bags showed many advantages in practice, e.g., low-cost mass production, long term storage, convenient transportation, controllable seedling quantity and density, ease of use in the field, and environmentally-friendly biodegradable paper bags. This low-cost and easy-to-use method could well achieve the target of restoration-friendly cultivation in D. officinale, and has universal applications for conservation translocations of epiphytic orchid based on symbiotic seeds germination.

Pollination compatibility of Dendrobium spp. orchids from Bali, Indonesia, and the effects of adding organic matters on seed germination under in vitro culture

Abstract. Darmawati IAP, Astarini IA, Yuswanti H, Fitriani Y. 2021. Conservation of Dendrobium spp. Bali orchids (Indonesia) through in vitro seed culture. Biodiversitas 22: 2554-2559. Orchid species, including Dendrobium spp., originated from Bali are threatened to extinction due to habitat destruction, illegal hunting, small population size, and the effects of global environmental change. Conservation strategies are therefore required to preserve the remaining germplasm, one of which is through in vitro seed propagation. This research aimed to investigate the pollination compatibility (i.e., pollination that produce fruits) of Dendrobium orchids originated from Bali, and to determine the effects of adding organic matters (i.e., coconut water, tomato extract, and peptone) to the base media on orchid seed germination under in vitro culture condition. Self-pollination and sibling pollination were implemented to seven orchid species, namely Dendrobium macrophyllum, D. heterocarpum, D. fimbriatum, D. linearifolium, D. spathilingue, D. secundum, and D. plicatile. The results showed that only four species, i.e., D. macrophyllum, D. heterocarpum, D. fimbriatum, and D. linearifolium, that produced fruits after being pollinated. The subsequent in vitro propagation of seeds produced by the four species showed that the addition of organic matters of tomato extract, coconut water, and peptone significantly affected the germination of Dendrobium forma Bali orchid seeds, indicated by different germination rates. The addition of 20% coconut water to Vacin and Went (VW) media gave the highest percentages of seed germination and protocorm formation for D. macrophyllum, D. heterocarpum, and D. fimbriatum at 98.33% and 95.00%; 91.67% and 91.67%; and 95% and 98.33%, respectively. Our findings can serve as baseline information when developing conservation strategies of Dendrobium orchids from Bali, particularly from the aspect of propagation

Hybridization and In Vitro Seed Germination of a Commercial Hybrid Oncidium Orchid in Indonesia

Oncidium is the one of orchid family that is not native Indonesia. In the development of Oncidium breeding are widely encountered difficulties, especially when using advanced commercially hybrid varieties. Hybridization and seed germination of Oncidium is still not established yet. The objectives of this study were to obtain population of hybridization and to identify the best medium culture of seed germination and planlet development of a commercial Oncidium Orchid in Indonesia. The observations were made at a percentage of the success of crosses, seed maturity, the length of the protocorm formation, the effect of media type and sucrose concentration. In this study, the percentage of the cross-success of Oncidium was 15.7% (85 capsules) from 566 crossing and 6.18% (35 capsules) germinated. The fruit harvest age of Oncidium crosses varies between 165 days to 245 days after crossing. The duration of protocorm germination varies between 17-82 days after spreading seed. Protocorms were growth well and fast on medium Tsuchiya supplemented with 1 mg L−1 BAP and 0.25 mg L−1 NAA. and plantlet formation were promoted on the same based media Tsuchiya but supplemented using 0.5 mgL−1 BAP and 0.1 mg L−1 NAA. The optimized protocol required about 24–30 weeks from the spread seed to the plantlet formation. Seedling were grown at size 5-7 cm immediately planted as pot community. The time of acclimatization varies between 6-8 months after the last subculture. while individual time ranges between 4-5 months after acclimatization. Hopefully, the current study will assist with future development of Oncidium Orchid breeding in Indonesia.

Transcriptome sequencing and differential gene expression analysis reveal the mechanisms involved in seed germination and protocorm development of Calanthe tsoongiana.

Calanthe tsoongiana is a rare orchid species native to China. Asymbiotic seed germination is of great importance in the ex situ conservation of this species. Based on morphological characteristics and anatomical structures, the C. tsoongiana developmental process from seeds to seedlings was divided into four stages (SA, PB, PC and PD), and subsequently, changes in endogenous hormone contents and gene expression were assessed using RNA-seq analysis. K-means analysis divided the DEGs into eight clusters. The gene expression decreased markedly between the imbibed seed and globular protocorm stages, with this being the most notably enriched cluster. During the seed germination period, DEGs were dominated by ATP metabolic processes, respiration and photosynthesis. A small change in gene expression was found in the globular protocorm versus the finger-like protocorm stages. During the last developmental stage, DEGs were significantly enriched in lignin catabolic processes and plant-type secondary cell wall biogenesis. DEG homologs, such as TSA1, DAO, NCED1, STM, and CUC2, were related to phytohormones and the morphogenesis of shoots, leaves and roots. Particularly, interactions between CUC2 and STM as well as AS1 and STM were likely involved in protocorm formation and development. Furthermore, TSA1 and DAO were distinctly validated and implicated in the synthesis and metabolism of auxin, which has a pivotal role in plant development. Our study is the first to combine morphological and transcriptome analysis to examine the process of protocorm formation and development. The results provide a foundation for understanding the mechanisms of seed germination and protocorm development of C. tsoongiana.

Asymbiotic germination and morphological studies of seeds of Atlantic Rainforest micro-orchids (Pleurothallidinae).

The morphological and morphometric characters of seeds belonging to 11 species of the subtribe Pleurothallidinae using light and scanning electron microscopy were studied to understand the in vitro germination process. Qualitative data (color, shape, ornamentation) and quantitative ones were also evaluated in seeds and embryos (length, width, volume and air space percentage between the integument and the embryo). The viability of the seeds was evaluated by in vitro germination in woody plant medium (WPM), and by analysis of the developmental stages of protocorms until seedling formation (two to 24 weeks). Morphometric data showed variations within the genus Acianthera and between species of different genera. The best germination and protocorm formation responses occurred with Acianthera prolifera (92%) and Acianthera ochreata (86%), with the formation of seedlings after 12 and 16 weeks of sowing, respectively. The seeds and embryos of A. prolifera and A. ochreata were larger (length, width, and volume) with a structural polarity that may have facilitated their germination comparing to others studied species. Other characteristics of A. prolifera seeds that may have contributed to these results include the presence of a thin testa without ornamentation and a suspensor. The protocorms of Anathalis obovata, Dryadella liliputiana, and Octomeria gracillis developed slowly in the WPM, not reaching the seedling stage in 24 weeks of cultivation. This morphological and morphometric study contributes to the understanding of asymbiotic germination of some micro-orchid species.

Cryopreservation in liquid nitrogen of Brazilian orchid seeds Miltonia flavescens Lindl.

Miltonia flavescens is a species vulnerable to extinction, which justifies research on preservation methods. Cryopreservation in liquid nitrogen (LN) consists of maintaining biological material at a low-temperature (-196 °C). Thus, the aim of the experiment is to evaluate the influence of different cryoprotective solutions on cryopreservation in LN of the Brazilian orchid Miltonia flavescens seeds. The experiment was conducted in a completely randomized design (CRD), with eight treatments and six replications. The treatments were composed as follows: control; immersion in LN, no cryoprotectant adding; and immersion in LN, with the addition of cryoprotectants: sucrose 0,4 mol L-1; glycerol 2 mol L-1; protection by vitrification in solution (PVS)1; PVS2; PVS2 + 1% phloroglucinol, and PVS3. Except for the control treatment, which was kept in a freezer (10±2 °C), the others remained frozen for 15 days. After this period, the viability of the seeds was evaluated. These seeds were sown and, 30 days after germination, then the frequency of protocorm formation was verified. Before the cryopreservation, the seeds showed 75% viability and 9.5% water content. After cryopreservation, the seeds varied between 67 to 75% viability. However, treatment with glycerol 2 mol L-1 exhibited lower performance than the others (58%). The control treatment showed a higher percentage of protocorm formation (71%) followed by treatments PVS1 (63%), PVS2 (64%), and PVS2PHLO (66%). For the purpose of preserving Miltonia flavescens seeds in liquid nitrogen for a prolonged period, the treatments PVS1, PVS2, and PVS2PHLO proved to be viable and promising alternatives.

Co-Cultures of Mycorrhizal Fungi Do Not Increase Germination and Seedling Development in the Epiphytic Orchid Dendrobium nobile.

Orchids are highly dependent on mycorrhizal fungi for seed germination and subsequent growth to a seedling as they provide essential carbon, water, and mineral nutrients to developing seeds. Although there is mounting evidence that orchid seeds are often colonized by multiple fungi simultaneously, most in vitro germination experiments focus on mycorrhizal monocultures and little is known about how mycorrhizal assemblages affect seed germination and growth of seedlings. In this study, we compared the effects of mycorrhizal monocultures and co-cultures on seed germination and seedling growth of the epiphytic orchid Dendrobium nobile. In situ baiting was used to isolate mycorrhizal fungi from protocorms for germination experiments. Germination experiments were conducted under two light regimes for 90 days. In total, five fungal strains were isolated from protocorms of D. nobile, indicating that the species was not highly specific to its fungal partners. Four strains (JC-01, JC-02, JC-04, and JC-05) belonged to the Serendipitaceae and one (JC-03) to the Tulasnellaceae. In vitro germination experiments showed that germination percentages were higher under light-dark conditions than under complete dark conditions, supporting previous findings that light facilitates germination in epiphytic orchids. While all strains were able to induce protocorm formation and growth into the seedling stage, large differences between fungal strains were observed. Co-cultures did not result in significantly higher seed germination percentages and seedling development than monocultures. Taken together, these results demonstrate that effects of fungal assemblages are not predictable from those of component species, and that more work is needed to better understand the role of fungal assemblages determining seed germination and subsequent growth under natural conditions.

In vitro plant regeneration through protocorm formation in orchid Calanthe odora

The regeneration of plants through formation of protocorm and protocorm-like bodies was achieved by in vitro asymbiotic immature seed culture of Calanthe odora. This is the first report on in vitro regeneration of plantlets of Calanthe odora Griff., the IUCN red listed threatened species. The immature seeds as explants were most regenerative than mature seeds. The MS media fortified with different concentrations of Kinetin alone or in combination with NAA were tested to sort out as the best regeneration media. From the experimental results the best medium was sorted as MS medium supplemented with 1.0 mg/l Kinetin and 1.0 mg/l NAA for germination and formation of protocorm-like bodies from immature seed explants followed by transfer to MS media fortified with 1.5 mg/l Kinetin and 1.0 mg/l NAA for development of protocorm to plantlets. The plantlets after hardening in in vitro were transferred to polyhouse where 86.7% survival rate was recorded on vermiculite potting media.