• The Korean Journal of Mycology
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• v.43 no.2
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• pp.129-132
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• 2015

In this study, orchid mycorrhizal fungi (OMF) were isolated from four terrestrial orchids on Mt. Hambeak, Platnathera chlorantha, Platnathera mandarinorum, Cephalanthera falcate, and Cephalanthera longibracteata. OMF were identified using morphological and sequences analysis of fungal internal transcribed spacer (ITS) regions by specific primer of basidiomycetous orchid mycorrhizas; ITS1-OF and ITS4-OF. Four species of orchid mycorrhizal fungi were identified as Ceratobasidium sp, Epulorhiza anaticula, Tulasnella calospora and Tulasnella sp.

• The Korean Journal of Mycology
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• v.40 no.3
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• pp.132-135
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• 2012

This study was performed to isolate and identify the orchid mycorrhizal fungi (OMF) from roots of five species of terrestrial orchids in Korea; Cymbidium goeringii, Spiranthes sinensis, Calanthe discolor, Bletilla striata, Pogonia minor. DNA was extracted from isolated OMF and ITS region was amplified using primers, ITS1-OF and ITS4-OF. Four species of OMF belong to Tulasnellaceae and Sebacinaceae were identified; Tulasnella calospora, Tulasnella irregularis, Tulasnella sp., Sebacina vermifera.

• The Korean Journal of Mycology
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• v.39 no.1
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• pp.7-10
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• 2011

Orchid mycorrhizal fungi (OMF) were examined in roots of the six terrestrial species of orchids collected in Ulleung Islands. Seven OMF isolates from the roots of orchids were identified based on morphological and molecular characters. Internal transcribed spacer region of OMF DNA was amplified using basidiomycete-specific ITS primers, ITS1-OF and ITS4-OF. OMF beloning to Tulasnellaceae and Ceratobasidaceae was identified through molecular analysis.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.04a
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• pp.53-53
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• 2019

Cypripediums, popularly called lady's slippers or moccasin flowers, are the showiest and most sought after hardy terrestrial orchids, collected and grown by orchid and alpine plant enthusiasts alike. In Korea, 4 species of cypripedium are reported as Cypripedium japonicum, C. macranthos, C. guttatum, and C. calceolus. We had already reported the feasibilities of C. macranthos and C. guttatum with in vitro germination methods from immature seeds. The seeds of white lady's slipper orchid (Cypripedium macranthos Sw. alba) were collected 65 days after pollination in 2018. The green pods were sterilized with flame and sowed immediately on the POM(Phytomax orchid maintenance media(R), Sigma) supplemented with BAP 0, 0.5, 1.0 mg/L and NAA 0, 1, 2mg/L. The germination of seed was observed 90 days after sowing, and the plantlets were subcultured to the same media according to the size of the protocorm with 1~2, 2~3, 3~4, 5~6, 7~8mm. The time of the subculture to the new media seems to be critical factors of forming rhizoids which is the hairy root of the cypripediums. As a results, the protorms of the white lady's slipper orchid was successfully germinated in the POM media supplemented BAP 0.5 and NAA 1.0 mg/L. The roots and rhizoids were formed in 5~6mm protocorms subculture over 95% survival ratio. We also tried to subculture to liquid medium without activated charcoal, however the browning or malformation of the roots was observed in the root. The formation of shoots from the protocorm was effectively enhanced in the POM media with non-additives of plant growth regulators. These results indicate the possibility of high and stable production and practical industrialization of endangered white lady's slipper orchids.

• The Korean Journal of Mycology
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• v.43 no.1
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• pp.20-25
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• 2015

This study was conducted to investigate diversity of orchid endophytic fungi from roots of two terrestrial orchids, Cypripedium japonicum and Cypripedium macranthum in Korea. The endophytic fungi were identified using morphological and DNA sequences isolated from the roots. Totally, 11 taxa of the endophytic fungi were identified from the roots of C. japonicum and 15 taxa from C. macranthum. Three species of the fungi were common in both host species; Leptodontidium orchidicola, Humicola fuscoatra var. fuscoatra, Umbelopsis dimorpha. Six species of the fungal isolates were the first report in Korea; Oidiodendron echinulatum, Pseudogymnoascus roseus, Geomyces vinaceus, Cryptosporiopsis ericae, U. dimorpha, Chaetomium cupreum.

• Journal of Plant Biotechnology
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• v.40 no.1
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• pp.43-48
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• 2013

Oreorchis coreana Finet is threatened globally by over-collection from its natural habitats for horticultural purposes. Its rarity in nature makes this plant one of the most endangered species in Korea. In this study, we investigated the effects of sodium hypochlorite (NaOCl) on orchid seed viability and seed germination. An in vitro bioassay swelling test using immature seeds was compared with a standard chemical procedure using triphenyl tetrazolium chloride (TTC) to test seed viability. In general, the bioassay was more appropriate for estimating embryo viability after a prolonged pre-treatment (more than 1 h) in 1% NaOCl, a surface sterilant often used to enhance germination of seeds of terrestrial plants. Therefore, an efficient method for investigating in vitro swelling of immature seeds is urgently needed. We established a method for determining the viability and swelling of O. coreana seeds via in vitro examination of immature seeds. Treatment of immature seeds with 1% NaOCl for 10 min greatly enhanced the extent of swelling of immature zygote embryos when compared to untreated seeds. These data obtained here appear to be comparable to viability and swelling that occurs in O. coreana seeds via asymbiotic germination.

• Journal of Species Research
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• v.6 no.1
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• pp.68-75
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• 2017

We investigated the effects of sodium hypochlorite (NaOCl) and culture medium on embryo swelling and germination of Calanthe discolor Lindl., and established a method for determining the swelling and protocorm formation of C. discolor seeds via in vitro examination of immature seeds. Treatment of immature seeds with NaOCl greatly enhanced the extent of embryo swelling and protocorm formation of immature zygote embryos compared to seeds without NaOCl treatment. The effects of the culture media were also evaluated with regard to embryo swelling and protocorm formation of in vitro cultured seeds with and without NaOCl treatment. Additionally, the effects of white fluorescent light and red and blue LEDs lights on seedling growth in in vitro culture were examined. The most suitable condition for seedling growth after 12 weeks of culture was the red LEDs light with POM medium. These results show effective asymbiotic germination and growth of C. discolor.

• Korean Journal of Plant Taxonomy
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• v.45 no.4
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• pp.353-361
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• 2015

Spatio-temporal variation in fruit set in orchids would affect long-term population viability and will influence genetic diversity over many generations. The aim of this study was to examine the breeding system of the nectariferous terrestrial orchid Epipactis thunbergii, to specifically determine levels of fruit set in terms of time and space under natural conditions. We examined pollination under natural conditions and conducted hand pollination experiments during a 2-year survey in four populations located along 1.5 km of coastal line in Jinguiri (rual village) [Jeollanam-do (province), southern Korea]. We found that, over a 2-year period, levels of percentage of fruit set were similar within patches of the four populations. By contrast, we detected significant differences in the percentage of fruit set among patches. We also found that plants with larger inflorescence size produced significantly more fruits than plants with fewer flowers. Over a 2-year period, the percentage of fruit set for E. thunbergii was similar but low (14.1%) compared to that averaged for eighty-four rewarding species (37.1%). However, an increase in fruit set was achieved by hand-pollinations: artificial self-pollination (90.5-95.2%), artificial geitonogamy (94.7-95.0%), and cross-pollination (artificial xenogamy, 91.3-91.4%). No emasculated flowers produced fruits and no automatic pollination was found in E. thunbergii. Our findings suggest that E. thunbergii is a self-compatible terrestrial orchid that depends on pollinators (insects) to achieve fruit set in natural habitats, and that local environmental conditions were similar over a period of 2 years in the study area. Our results also highlight the cryptic variation of fruit production in time, but more pronounced variability in space.

• Korean Journal of Plant Taxonomy
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• v.49 no.2
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• pp.118-126
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• 2019

The taxonomic rank of the tiny-leaved terrestrial orchid Cephalanthera subaphylla Miyabe & $Kud{\hat{o}}$ has been somewhat controversial, as it has been treated as a species or as an infraspecific taxon, under C. erecta (Thunb.) Blume [C. erecta var. subaphylla (Miyabe & $Kud{\hat{o}}$) Ohwi and C. erecta f. subaphylla (Miyabe & $Kud{\hat{o}}$) M. Hiro]. Allozyme markers, traditionally employed for delimiting species boundaries, are used here to gain information for determining the taxonomic status of C. subaphylla. To do this, we sampled three populations of five taxa (a total of 15 populations) of Cephalanthera native to the Korean Peninsula [C. erecta, C. falcata (Thunb.) Blume, C. longibracteata Blume, C. longifolia (L.) Fritsch, and C. subaphylla]. Among 20 putative loci resolved, three were monomorphic (Dia-2, Pgi-1, and Tpi-1) across the five species. Apart from C. longibracteata, there was no allozyme variation within the remaining four species. Of the 51 alleles harbored by these 17 polymorphic loci, each of the 27 alleles at 14 loci was unique to a single species. Accordingly, we found low average values of Nei's genetic identities (I) between ten species pairs (from I = 0.250 for C. erecta versus C. longifolia to I = 0.603 for C. falcata vs. C. longibracteata), with C. subaphylla being genetically clearly differentiated from the other species (from I = 0.349 for C. subaphylla vs. C. longifolia to 0.400 for C. subaphylla vs. C. falcata). These results clearly indicate that C. subaphylla is not genetically related to any of the other taxa of Cephalanthera that are native to the Korean Peninsula, including C. erecta. In a principal coordinate analysis (PCoA), C. subaphylla was positioned distant not only from C. falcata, C. longibracteata, and C. longifolia, but also from C. erecta. Finally, K = 5 was the best clustering scheme using a Bayesian approach, with five clusters precisely corresponding to the five taxa. Thus, our allozyme results strongly suggest that C. subaphylla merits the rank of species.

• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.239-244
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• 2015

Calanthe striata f. sieboldii Decne. ex Regel is a terrestrial orchid with beautiful flowers arranged in racemose inflorescences. This species is threatened due to over-collection and loss of suitable habitats. Asymbiotic germination is useful in the conservation efforts to re-establish plants in the wild, and for commercial propagation. In this study, we investigate the effects of sodium hypochlorite (NaOCl), three types of culture media (Phytomax Orchid Maintenance - POM, Seed Germination Maintenance - SGM, and Murashige and Skoog 1962 - MS), and plant growth regulators on embryo swelling, protocorm formation, and embryo diameter of C. striata f. sieboldii. Treatment with 1% NaOCl for 30 min greatly enhanced embryo swelling (28.3%), embryo diameter ($205.8{\mu}m$), and embryo protocorm formation (54.8%) compared to seeds without NaOCl treatment (embryo swelling 8.5%, embryo diameter $14.6{\mu}m$, and protocorm formation 13.4%) on POM medium. Protocorm formation on POM medium supplemented with 1.0 mg/L N6-benzyladenine (BA) (95.6%) was better than the control (54.5%). Additionally, the effects of activated charcoal (AC) and sucrose on seedling growth in in vitro culture were examined. The protocorm converted into healthy plants with well-developed shoot primordia on the POM medium with AC and sucrose. The most suitable conditions for seedling growth after 10 weeks of culture were the POM medium with AC or sucrose. These results show effective asymbiotic seed germination and in vitro seedling growth of C. striata f. sieboldii.