Asymbiotic Germination Research Articles

In Vitro Micropropagation of Eria Lasiopetala (Willd.) Ormerod- A Native Medicinal Orchid in Bangladesh

The present study focuses on the asymbiotic germination of seeds through in vitro culture of Eria lasiopetala, a native medicinal orchid in Bangladesh. Out of the four basal media, MS was found to be the most effective medium, where 93.58±0.374% seed germinations were recorded. Different plant growth regulators (PGRs), e.g. BAP, Kin, NAA and were used for secondary protocorm proliferation, plantlet development, and multiplication of shoot buds through in vitro conditions. The maximum number of secondary protocorms (12.80±0.115) were developed that were derived from primary protocorms on MS medium fortified with 0.5 mg/l BAP and 1.0 mg/l NAA. Data revealed that 0.5 mg/l BAP + 0.25 mg/l NAA showed the best combination for height elongation of plantlets (3.12±0.012 cm) in MS medium. While for multiple shoot buds (MSBs) 1.0 mg/l BAP + 0.5 mg/l NAA showed better (6.8±0.127) performance than other combinations of PGRs. The single shoots were isolated from the multiple shoots and sub-cultured on ½MS medium supplemented with NAA (0.5 - 2.0 mg/l), IAA (0.5 - 2.0 mg/l) and IBA (0.5 - 2.0 mg/l) for root induction. The maximum number of roots (7.53±0.176) and its growth (4.61±0.01 cm) were found in ½MS medium supplemented with 1.0 mg/l IAA. The well rooted plantlets were hardened and successfully transferred after acclimatization in pots containing the mixture of coconut husk, charcoal and brick pieces in the ratio of 2:1:1 and eventually established under natural condition. J. Bio-Sci. 32(1): 45-60, 2024

Effect of zeatin and casein hydrolysate on in vitro asymbiotic germination of immature seeds of Vanilla planifolia Jacks ex Andrews (Orchidaceae)

The germination of Vanilla planifolia seeds under natural environmental conditions is very low (0.1 %) because of specific mycorrhizal requirements. Therefore, the species is typically propagated spending vegetative using stem cuttings. Thus, all cultivated plants are genetically identical and susceptible to multiple abiotic and biotic stressors. Artificial propagation from seeds is difficult for most terrestrial orchids native to temperate regions, and V. planifolia is no exception. As propagation from the seeds of this species has low success rates, in this study, an effective reproducible protocol for the in vitro asymbiotic germination of Vanilla planifolia seeds was established. For this purpose, the effects of three asymbiotic culture media on seed germination and seedling development of Vanilla planifolia [Murashige and Skoog (MS), Vacin and Went (VW), Knudson ‘C’ (KC)] were tested, two zeatin concentrations (Z = 0.25 and 0.50 mg L−1), two concentrations of casein hydrolysate (CH=50 and 500 mg L−1), and two combinations of these (Z = 0.25 mg L−1 + CH=250 mg L−1) and (Z = 0.50 mg L−1 + CH=500 mg L−1). The highest germination rate (74.5%) was recorded for the treatment with MS salts at a quarter strength supplemented with 500 mg L−1 of CH. All the seeds that germinated under this treatment showed high survival and vigorous seedling growth. In contrast, in the treatment with MS salts at a quarter strength supplemented with combination of CH and Z (CH=500 mg L−1 + Z = 0.50 mg L−1) resulted in the production of calluses (53 %).

The patterns of inbreeding depression in food-deceptive Dactylorhiza orchids.

Inbreeding depression (ID) in food-deceptive plants have been reported previously, however, it has not been often proven that selfed seeds germinate better than outbred ones or that selfing affects ID. To resolved these issues, food-deceptive related Dactylorhiza majalis, D. incarnata var. incarnata and D. fuchsii orchids were investigated. Hand pollination treatments and control pollination were conducted. Fruit set, number of seeds per fruit, seed length, number of well-developed seeds per fruit, and proportion of in vitro asymbiotic germination seeds, were analyzed in relation to inflorescence levels and used as fitness indicators for these orchids. The ID and pollen limitation were measured. The lowest ID (δ = -1.000) was in D. majalis, and present in combination with a high pollen limitation in its populations. D. fuchsii showed higher ID (δ = 0.366), and D. incarnata var. incarnata weak ID (δ = 0.065), although ID varied between its fitness components. The seed number per fruit differed significantly between the treatments and the inflorescence levels in treatments. This study emphasizes that the breeding system rather than the flower position on the inflorescence shaped the quality and quantity of reproductive output. The ID and its effect on germination of food-deceptive orchid seeds undoubtedly played an important role.

Asymbiotic germination, seedling establishment and fungal uptake of Pterostylis montana and P. paludosa, two orchid species endemic to New Zealand

ABSTRACT Asymbiotic germination of orchid seeds has been successful for many species, and this could be an important method to generate seedlings to restore threatened populations. However, successful reintroduction of orchids into wild habitats depends on establishment of mycorrhizal symbioses. This communication reports on asymbiotic methods used to propagate two greenhood species endemic to New Zealand, Pterostylis montana (Not Threatened) and P. paludosa (At Risk – Declining). We furthermore investigated whether asymbiotically generated seedlings were colonised with orchid mycorrhizal fungi (OMF) when introduced to soil collected from natural forest areas where these species are not known to occur. We found that seedlings transferred to natural soil hosted at least seven different endophytic fungi, but no known OMF. We caution against the use of asymbiotically germinated seeds for reintroductions before getting a better understanding of the dynamics of mycorrhizal infections and their distribution in the soil.

Gibberellic Acid Inhibits Dendrobium nobile-Piriformospora Symbiosis by Regulating the Expression of Cell Wall Metabolism Genes.

Orchid seeds lack endosperms and depend on mycorrhizal fungi for germination and nutrition acquisition under natural conditions. Piriformospora indica is a mycorrhizal fungus that promotes seed germination and seedling development in epiphytic orchids, such as Dendrobium nobile. To understand the impact of P. indica on D. nobile seed germination, we examined endogenous hormone levels by using liquid chromatography-mass spectrometry. We performed transcriptomic analysis of D. nobile protocorm at two developmental stages under asymbiotic germination (AG) and symbiotic germination (SG) conditions. The result showed that the level of endogenous IAA in the SG protocorm treatments was significantly higher than that in the AG protocorm treatments. Meanwhile, GA3 was only detected in the SG protocorm stages. IAA and GA synthesis and signaling genes were upregulated in the SG protocorm stages. Exogenous GA3 application inhibited fungal colonization inside the protocorm, and a GA biosynthesis inhibitor (PAC) promoted fungal colonization. Furthermore, we found that PAC prevented fungal hyphae collapse and degeneration in the protocorm, and differentially expressed genes related to cell wall metabolism were identified between the SG and AG protocorm stages. Exogenous GA3 upregulated SRC2 and LRX4 expression, leading to decreased fungal colonization. Meanwhile, GA inhibitors upregulated EXP6, EXB16, and EXP10-2 expression, leading to increased fungal colonization. Our findings suggest that GA regulates the expression of cell wall metabolism genes in D. nobile, thereby inhibiting the establishment of mycorrhizal symbiosis.

Asymbiotic germination of Prasophyllum (Orchidaceae) requires low mineral concentration

Context Asymbiotic germination is used for propagation of orchids and to assess seed viability. However, asymbiotic germination media remain undeveloped for Prasophyllum (Orchidaceae), a genus with 39 threatened species in southern Australia. Aims To develop asymbiotic germination and replate media for Prasophyllum, and determine whether survival of asymbiotically grown seedlings under nursery conditions is enhanced through the addition of a mycorrhizal fungus to potting media. Methods Using Prasophyllum frenchii as a model, we tested 81 asymbiotic germination medium combinations. The three best-performing media were then compared against four untested commercial media, with and without added coconut water and/or banana. The best-performing medium was trialled on 18 additional Prasophyllum species. Twenty-four replate media were then tested for their ability to support seedling development. We measured the survival of seedlings under nursery conditions with and without the addition of mycorrhizal Ceratobasidium fungus to potting media. Key results Low concentrations of minerals, auxins and cytokinins were necessary for asymbiotic germination of P. frenchii, although germination was highest overall on the commercial medium W9.5 with 5% coconut water. The most effective replate medium was W9 with 5% banana. However, survival of seedlings was 1.7%, 2 years after transferral to potting media in the nursery, regardless of the addition or absence of a mycorrhizal fungus. Conclusions Asymbiotic media can effectively germinate Prasophyllum seed, although deflasked seedlings have low survival rates. Implications Our media are broadly applicable to assess seed viability in Prasophyllum. However, poor survival of asymbiotically grown seedlings contrasts with recently published evidence of high seedling survival using symbiotic propagation methods, indicating that plants should be propagated symbiotically for conservation programs.

The influence of pre-treatment on germination of three species of Catasetum (Orchidaceae)

Abstract: Asymbiotic germination techniques are successful in species of the Catasetum genus. The diverse composition of the culture media can be critical; however, viable seeds are required for asymbiotic germination. The reliable method for viability assessment is the tetrazolium test. A decisive step in the efficiency of the tetrazolium test is the seeds preconditioning with sucrose solution, which has the potential to improve germination. Therefore, this work aimed to evaluate the seed germination of three species of the genus Catasetum in culture media, preconditioned or not with sucrose solution. Seeds of the species Catasetum osculatum, Ctsm. galeritum and Ctsm. complanatum were assessed by tetrazolium test and germinated in two different media, pretreated or not with 10% sucrose solution. The statistical analysis showed that the use of sucrose pretreatment significantly increased the germination rates of the species. We conclude that the pretreatment with 10% sucrose for 24 hours, regardless of the species and the culture medium, significantly increases the germination of Ctsm. complanatum, Ctsm. galeritum and Ctsm. osculatum seeds.

Clonal Micropropagation of Cymbidium erythrostylum Rolfe

Cymbidium erythrostylum Rolfe is one of the most beautiful species of the genus cymbidium which is used as a cut flower or indoor plant. However, it was registered as a rare species even in its original habitat. This study was carried out to develop a protocol for in vitro propagation of C. erythrostilum. We used protosomes obtained by the asymbiotic germination of seed on 1/2 of Murashige and Skoog nutrient medium (MS) supplemented with 1 mg/L 2-Isopentenyl adenine (2iP) as objects of study. During the multiplication stage, the number of formed protosomes on a culture medium containing 0.8 mg/L 6-Benzylaminopurine (6-BAP) was higher (3.8 ± 0.2 protosomes). During the subsequent cultivation on ½ MS medium supplemented with 2 mg/L 6-BAP and 0.5 mg/L a-naphthaleneacetic acid (NAA), the highest numbers of shoots (4.00 ± 0.19 shoots/plantlet) and leaves (4.50 ± 0.14 leaves/plantlet) were obtained. At the rooting stage under in vitro conditions, the most effective was the use of ½ MS nutrient medium with the addition of 0.5 mg/L of indolyl-3-butyric acid IBA, 1 g/l charcoal, and 50 g/l banana puree. The obtained plants were successfully adapted to a substrate consisting of bark, perlite, and peat in a ratio of 1:1:1.

In vitro seed germination and acclimatization of Encyclia cordigera (Kunth) Dressler

• 100% germination of E. cordigera was achieved in MS ½-strenght + 0,15 g L-1 Ac. • Efficient protocols for in vitro propagation and acclimatization of E. cordigera were established. Encyclia cordigera is a Mexican native orchid, of major ornamental importance due to its beautiful and fragrant flowers. The said characteristics also create a risk for the extinction of the species, thus, the urgent necessity to stablish propagation strategies to prevent its loss. The objective of this study was to establish a protocol for asymbiotic seed germination and acclimatization of E. cordigera . For asymbiotic germination, different concentrations (¼ and ½ and full-strength) of MS medium were evaluated, adding 0.15% of activated charcoal (AC). MS medium without AC was used as a control treatment. The germination percentage was evaluated after 16 weeks of culture. The treatments used for the germination stage were also used for the development of seedlings which lasted 20 weeks, then plant length, number of roots, root length, number of shoots, and number of leaves per shoot were measured. Finally, the plants obtained were acclimatized. The ½-strength MS + 0.15% AC resulted in 100% seed germination, while ¼-strength MS + 0.15% AC yielded longer seedlings (1.53 cm long). As for the number and length of roots, MS + 0.15% AC proved to be the best medium, with 2 roots and 2 cm respectively. No significant differences between culture media were found for the number of shoots and number of leaves per shoot. The plants obtained were transferred to an acclimatization process for eight weeks in the greenhouse, 209 plants (97% survival rate) were obtained during the acclimatization process. The presented protocols will contribute to the propagation and conservation of E. cordigera , an orchid species with ornamental and economic importance.

In vitro asymbiotic germination, seedling formation and tuberization of Orchis sancta

Plant tissue culture techniques have recently become attractive tools for propagating rare and endangered species and producing their economically important natural products. Here, we introduce a protocol for an effective, fast and reliable production route of plantlets and tubers from salep orchid, Orchis sancta, a native orchid species growing wild in Turkey. In the course of this study, the efficacy of basal media and plant growth regulators on asymbiotic germination of mature seed, seedling formation, tuberization and glucomannan contents of tubers were assessed. Amongst all basal media evaluated, Orchimax including activated charcoal gave the best germination rate (62.46%). Supplementing this medium with 1.0 mg/L zeatin resulted in the highest germination rate (81.18%). Lowest thidiazuron concentration (0.25 mg/L) was very effective on shoot elongation while kinetin (0.5 and 1.0 mg/L) promoted leaf formation. The aforesaid basal medium with IBA favored root extension (31.16 mm) and root formation (4.5 roots per plantlet) at 1.0 mg/L and 2.0 mg/L IBA, respectively. Cytokinins, zeatin (2.0 mg/L) in particular, promoted tuber formation and glucomannan accumulation. Plantlets were successfully adapted to soil step by step.

Cypripedium subtropicum embryo development and cytokinin requirements for asymbiotic germination

BackgroundCypripedium subtropicum is a unique, endangered lady’s slipper orchid with evergreen leaves on non-dormant shoots that is native to southwestern China. This study documents the major developmental events in C. subtropicum seed development from fertilization to seed maturity, determines the optimum period for seed collection, and examines the cytokinin requirements for asymbiotic germination and protocorm survival.ResultsStructural studies revealed that embryo development proceeded after successful fertilization at 60 days after pollination (DAP). At 105 DAP, a globular embryo with the shrinking inner seed coat was observed, and seeds collected at this time point exhibited optimal germination. After 120 DAP, most seeds had a mature embryo within the capsule, and within the cells of the embryo proper, numerous proteins/lipid bodies were present as the main storage products. In addition, the inner seed coat had compressed into a thin layer that tightly enclosed the embryo, while the outer seed coat had progressively elongated, resulting in a hair-like appearance of the mature seed. Histochemical staining using Nile red and toluidine blue O (TBO) indicated that the lignified inner and outer seed coats may lead to coat-imposed dormancy. Seeds collected at this stage germinated poorly. Analyses of cytokinin preferences and optimal concentrations for germination and protocorm survival showed that both 6-(γ,γ-dimethylallylamino) purine (2iP) and 6-benzylaminopurine (BA) enhanced germination compared with the control, although higher concentrations of BA (4 and 8 μM) suppressed germination. The protocorm survival rate improved with increasing 2iP concentration.ConclusionsThis study provides a reproducible procedure for culturing immature seeds of C. subtropicum based on a defined time schedule of seed development. In addition, the cytokinin 2iP was shown to improve germination and protocorm survival. This study provides a scientific basis for seedling establishment through asymbiotic seed culture for further reintroduction efforts.

The challenges of growing orchids from seeds for conservation: An assessment of asymbiotic techniques.

Lewis Knudson first successfully germinated orchid seeds asymbiotically on artificial medium in 1922. While many orchid species have since been grown asymbiotically, the tremendous variation in how species respond to artificial medium and growth conditions ex situ has also become apparent in the past century. In this study, we reviewed published journal articles on asymbiotic orchid seed germination to provide a summary of techniques used and to evaluate if these differ between terrestrial and epiphytic species, to identify areas where additional research is needed, and to evaluate whether asymbiotic germination could be used more often in ex situ conservation. We found articles reporting successful asymbiotic germination of 270 species and 20 cultivars across Orchidaceae. Researchers often used different techniques with epiphytic versus terrestrial species, but species‐specific responses to growth media and conditions were common, indicating that individualized protocols will be necessary for most species. The widespread success in generating seedlings on artificial media suggests that asymbiotic techniques should be another tool for the conservation of rare orchid species. Further advances are needed in understanding how to introduce mycorrhizae to axenically grown orchids and to maximize the viability of seedlings reintroduced into natural habitats to fully utilize these methods for conservation.

Efficient protocol for in vitro propagation of Laelia anceps ssp. anceps white variant from asymbiotic seed germination

Laelia anceps ssp. anceps is a species endemic to Mexico. The aim of this study was the establishment of a protocol of asymbiotic germination of L. anceps ssp. anceps, white variant. For asimbiotic germination, we evaluated different treatments: (a) 2 mg L−1 of 6-benzylaminopurine (BAP) + 2 mg L−1 of indoleacetic acid (IAA), (b) 2 mg L− BAP + 2 mg L−1 IAA + 2 mg L−1 naphthalenacetic acid (NAA) and (c) 2 mg L−1 Kinetin (KIN) + 2 mg L−1 BAP + 2 mg L−1 NAA. Included a control treatment with no PGR supplementation. The percentage of germinated seeds was assessed. For in-vitro rooting, different organic compounds were evaluated: (a) 1 mg L−1 IAA + 150 mL L−1coconut water, (b) 1 mg L−1 IAA + 80 g L−1banana pulp, and (c) 2 mg L−1 BAP + 2 mg L−1 IAA + 2 mg L−1 NAA. We also included a control treatment with no supplementation of PGRs or organic compounds. The percentage of rooted plants was evaluated and transferred to acclimatization process. 2 mg L−1 BAP + 2 mg L−1 IAA+ 2 mg L−1 NAA produced 82.20% seed germination; seedlings showed an average length of 1.11 cm and 1.64 leaves per explant. The treatment with 1 mg L−1 IAA + 150 ml L−1coconut water yielded a 78.20% rooting. 85% survival rate of plants was observed during the acclimatization process. This protocol will favor the propagation of the valuable germplasm of this orchid species that is endangered of extinction.

Perkecambahan Asimbiotik Biji Anggrek Dendrobium bicaudatum pada Media dengan Penambahan Ekstrak Tomat secara In Vitro

Asymbiotic Germination of Dendrobium bicaudatum Orchid Seeds on Media with In Vitro Addition of Tomato Extract. Dendrobium bicaudatum is a natural orchid that needs to be preserved. Orchid seeds propagation naturally (symbiotic) has a low success rate. Orchid seeds do not have an endosperm. Asymbiotic germination with in vitro culture techniques can be an alternative solution for the propagation of orchid seeds. This study aimed to determine the effect of giving tomato extract on modified media on the asymbiotic germination of Dendrobium bicaudatum orchid seeds. This research used the Completely Randomized Design with five treatments of tomato extract concentration, namely 0%, 5%, 10%, 15%, 20%, with 4 replications. The variables observed in this study were the time and percentage of phase 1; time and percentage of phase 2; time and percentage of phase 3; time and percentage of phase 4; time and percentage of phase 5; and time and percentage of phase 6. The results showed that the tomato extract treatment had a significant or very significant effect on the germination of Dendrobium bicaudatum orchid seeds and the best treatment for the fastest and the highest percentage of germination from protocorm to First Leaf Primodium was the addition of tomato extract with a concentration of 15% or 20%, and a concentration of 0% tomato extract for the early stages of swelling of the embryo.

Comparative Transcriptomics Analysis of the Symbiotic Germination of D. officinale (Orchidaceae) With Emphasis on Plant Cell Wall Modification and Cell Wall-Degrading Enzymes.

Orchid seed germination in nature is an extremely complex physiological and ecological process involving seed development and mutualistic interactions with a restricted range of compatible mycorrhizal fungi. The impact of the fungal species' partner on the orchids' transcriptomic and metabolic response is still unknown. In this study, we performed a comparative transcriptomic analysis between symbiotic and asymbiotic germination at three developmental stages based on two distinct fungi (Tulasnella sp. and Serendipita sp.) inoculated to the same host plant, Dendrobium officinale. Differentially expressed genes (DEGs) encoding important structural proteins of the host plant cell wall were identified, such as epidermis-specific secreted glycoprotein, proline-rich receptor-like protein, and leucine-rich repeat (LRR) extensin-like protein. These DEGs were significantly upregulated in the symbiotic germination stages and especially in the protocorm stage (stage 3) and seedling stage (stage 4). Differentially expressed carbohydrate-active enzymes (CAZymes) in symbiotic fungal mycelium were observed, they represented 66 out of the 266 and 99 out of the 270 CAZymes annotated in Tulasnella sp. and Serendipita sp., respectively. These genes were speculated to be involved in the reduction of plant immune response, successful colonization by fungi, or recognition of mycorrhizal fungi during symbiotic germination of orchid seed. Our study provides important data to further explore the molecular mechanism of symbiotic germination and orchid mycorrhiza and contribute to a better understanding of orchid seed biology.

Advances in Breeding, Bioprospecting, and In Vitro Culture of Laelia Orchid Species

Orchids (Orchidaceae) are plants that are highly appreciated by their beautiful flowers worldwide. Moreover, they represent a source of metabolites with applications in medicine and biotechnology. Within the Orchidaceae family, the Laelia genus is a group of orchid species from the Neotropics and is probably one of the most representative genera of America. Laelia orchids are cultivated by their splendid flowers and are widely used in orchid breeding. Here, we revise the use of the Laelia genus in orchid breeding and metabolite bioprospecting. We also analyze the use of plant tissue culture (PTC) as an alternative to conventional propagation and as a strategy for the recovery of those Laelia species threatened with extinction. We summarize and discuss the recent advances in the application of different PTC techniques for mass multiplication based on asymbiotic germination, organogenesis, protocorm-like bodies development, and somatic embryogenesis, and the advances of in vitro conservation by cryoconservation and the use of slow-growth promoting hormones. Finally, we suggest future directions and venues in research for Laelia species.

Asymbiotic germination, initial development in vitro and acclimatization of Cyrtopodium paludicolum Hoehne, a Brazilian Savanna orchid species

Abstract Cyrtopodium paludicolum is a terrestrial orchid species, native to Brazil, whose natural propagation is jeopardized by the intensive collection from the wild and is threatened by agricultural expansion in Cerrado areas. In light of that, this investigation aimed at studying the in vitro germination and early development of C. paludicolum as influenced by culture medium, sucrose and growth regulators as well as its micropropagation by using dark-grown stem segments. A protocol for its acclimatization is also detailed. The effects of Murashige & Skoog (MS), Knudson C (KC) and Vacin & Went (VW) media on the in vitro germination and initial development were tested. The influence of different concentrations of BA, NAA, and of sucrose on plant multiplication and growth were evaluated. The possibility of using etiolated stem segments for micropropagation was also assessed. Acclimatization was accomplished in two phases by using three different substrates. The results showed that VW was the best medium for germination whereas for seedling formation KC was the most advantageous since they were healthy and vigorous. Sucrose at 2% favored the greatest seedling growth and development. Shoot and root proliferation and development were best promoted in the presence of 2.28/2.28 and 0.57/0.57 µM BA/NAA, respectively. The use of etiolated stem segments for micropropagation was effective. Successful acclimatization was accomplished by initially growing plants in community pots containing a 3:1 (v/v) mix of Bioplant and dried Sphagnum moss followed by their transfer to individual pots containing a 2:1 (v/v) mix of Bioplant and Ouro Negro substrates.

Influence of Culture Conditions on In Vitro Asymbiotic Germination of Anacamptis longicornu and Ophrys panormitana (Orchidaceae).

This study is the first approach to in vitro asymbiotic germination of two species of Sicilian threatened terrestrial orchids, Anacamptis longicornu and Ophrys panormitana. Seeds were collected in the wild and cultured in two different media—Orchimax medium (OM) and Murashige and Skoog (MS)—and exposed to different photoperiods and temperatures to evaluate the best conditions for the specific stages of development. The germination of A. longicornu was very high on OM (95.5%) and lower on MS medium (21.4%), whereas O. panormitana germinated only on OM medium, with significantly lower percentages (12.0%), compared with A. longicornu. This difference is caused by variation in quality and quantity of nutrients used, primarily by nitrogen source. The results show that temperature and photoperiod widely affect seed germination and development. Although further investigations on asymbiotic and symbiotic germination are needed for the improvement of conservation of Mediterranean terrestrial orchids, our results contribute to the conservation of this group of plants.

Characterization of embryo and protocorm development of Paphiopedilum spicerianum

Paphiopedilum spicerianum (P. spicerianum) is a rare orchid species with high ornamental value. Asymbiotic germination is the most efficient propagation method for conservation and commercial purposes because clonal propagation is very difficult and the separation of native species of Paphiopedilum through aseptic seeding is uncommon owing to their conservatism. However, a high protocorm developmental arresting rate during the asymbiotic germination is the major obstacle for seedling establishment. The fundamental understanding of embryo and protocorm developmental mechanisms will guide the development of an effective propagation method. The morphological and physiological characterization of the key developmental process of embryos and protocorms shows that the mature seeds of P. spicerianum consist of a spherical embryo without an endosperm. Seed coats become heavily lignified once the embryo is mature. Embryo cell size is relatively uniform, and significant structure polarity and cell size gradients occur at the early protocorm stage. The high level of auxin and cytokinin accumulation at the early stage of embryo development and protocorm stage may help to facilitate cell division. The transcriptome profiles of protocorms at three different developmental stages were compared to explore the regulatory mechanism of protocorm development. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that differentially expressed genes were implicated in secondary metabolite metabolism, plant hormone signal transduction and photosynthesis. The temporal expression patterns of candidate genes related to embryo and shoot development were analyzed to reveal their roles in protocorm development: in the early stage of protocorm development, embryonic development related genes such as SERKs and BBM1 were active, while in the late stage of protocorm, shoot apical meristem related genes such as WOX8, CLAVATA2, CUC2, and SCR were active.

Клональное микроразмножение представителей рода Dactylorhiza Neck. ex Nevski

Abstract The aim of this study is to develop and improve methods of in vitro propagation of representatives of Dactylorhiza: D.baltica , D. fuchsii. For the study, we used protocorms obtained by the asymbiotic germination of seed during 90 days. It has been established that half-strength of Murashige and Skoog (1962) medium (½ MS) supplemented with 1-2 mg/l 6-Benzylaminopurine(6-BAP), potato puree (20g/l), and charcoal (1g/l) effectively influenced the development of protocorms, and seedlings formation in the studied species. The result of the study showed that the survival rate of protocorms was high in all experimental culture media, but in D. fuchsii it was better at a concentration 2mg/l of 6-BAP (95.4%), while in D. baltica it was high at 1mg/l (87.0%). The highest percentage of multiple protocorms (68%) and the formation of new secondary protocorms in D. fuchsii (5,5±0,3 units) were observed on a culture medium containing 2 mg/l 6-BAP. The highest percent of rooting of D. fuchsii protosoms (78%) and length of roots (0.9cm) observed in ½ MS medium without growth regulators. During the development of D. baltica protosoms, the culture medium of ½ MS containing 1 mg/l 6-BAP had the best effect on the number of roots (1.8±0.1root/protosom), while the medium supplemented with 2mg/l of 6-BAP contributed to the formation of a larger number of new secondary protocorms (3,2±0,1protocorm/unit). During the subsequent cultivation of protosoms of D. baltica on a culture medium containing 1 mg/l it was observed an increase in the height of shoots (4,8±0,3 см), and the length of roots (2,2±0,1 см), wherein the number of newly formed protocorms was higher by 30% on the medium supplemented with 2 mg/l 6-BAP. Keywords: DACTYLORHIZA BALTICA, DACTYLORHIZA FUCHSII, IN VITRO, PROTOCORMS, ORGANIC ADDITIVES