• 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.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.10a
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• pp.37-37
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• 2018

We use two different methods for laboratory propagation from seed of lady's slipper orchid(Cypripedium macranthos Sw.); immature seed which also called green capsule or fully mature seed about 120~130 days from pollination. In green capsule culture, the seed pods should be collected within precisely right time. The right time of seed collection could be diverse under the wether conditions or nutritional factors of the plants. In fully matured seed culture, the more complicated procedures are needed to break the dormancy of the seed; thermal or chemical treatment. The seedlings in this study were easily germinated from immature seeds in Harvais medium; 53 days after pollination(DAP) in Cypripedium pubescens, DAP 65 in C. parviflorum and C. macranthos. The germinated seedlings were transplanted to hormone free media immediately to avoid abnormal growth of seedlings. When the seedlings have roots with a minimum length of around 2-3cm and have visible dormant buds, the seedlings were removed from the flask and stored in refrigerator for vernalization. To examine the correlation of seedlings and maternal plants, the 125 seedlings of C. macranthos were transplanted in the soil bed at a distance of 20-100 cm from mother plants on April 20. The survival rate of seedlings were 92% in 20 cm distance from the ripe plants, and 56 % in 100 cm distance. The seedlings which were transplanted near mother plants showed vigorous growth in plant height, leaf width, and especially dormant buds. Considering the existence of mycorrhiza which is a symbiotic association between a fungus and the roots of a orchid vascular, the various fungus from mother plants could affect the growth of the seedlings. These results indicate the possibility of high and stable production and practical industrialization of endangered lady's slipper orchids.

• Journal of Plant Biotechnology
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• v.43 no.1
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• pp.138-145
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• 2016

To find the optimal propagation condition for endangered Cypripedium macranthos Sw., also known as lady's slipper orchid, the effect of various organic additives on in vitro germination, protocorm formation and seedling growth was investigated during asymbiotic seed culture. When $100ml{\cdot}L^{-1}$ coconut water was added to the basal medium, the highest germination rate and protocorm formation rate were achieved, with 70.8% and 74.2% respectively. Supplementation of phloem sap from birch tree or maple tree also showed a facilitating effect to improve the germination and protocorm development. With $100ml{\cdot}L^{-1}$ birch sap or maple sap, both the germination and protocorm formation rates were roughly more than 65% and 68%. The roots and buds of the seedlings grew vigorously in the medium containing $100ml{\cdot}L^{-1}$ coconut water or phloem sap, in particular, their bud formation rates increased by more than 70%. Addition of banana powder and peptone could not create a more significantly favorable culture condition, and non-addition had the worst results. Our results demonstrated that proper organic amendments such as coconut water and phloem sap might be preferred to in vitro germination and the growth of seedlings developed from the protocorm of C. macranthos Sw. during asymbiotic seed culture.

• Journal of Plant Biotechnology
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• v.43 no.1
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• pp.132-137
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• 2016

The genus Cypripedium, known as lady's slipper orchid, has been considered to have a considerable marketability as a potted and garden plant with beautiful flowers; however, this species is becoming endangered and, in some places extinct, due to habitat destruction and illegal collections. As such, an optimized artificial propagation system is necessary for its conservation and horticultural cultivation. For the establishment of the in vitro proliferation of the endangered Cypripedium macranthos Sw., native to Korea, the effects of medium strength and sucrose concentrations on germination, protocorm formation and seedling growth were investigated through asymbiotic seed culture. The highest germination rate and protocorm formation rate were obtained with a 1/4 MS medium; higher MS medium strengths did not generate the favorable conditions required for germination and protocorm growth. The overall growth characteristics of roots and buds developed from protocorms were best in cultures of 1/4 MS medium. On this medium containing $10g{\cdot}L^{-1}$ sucrose, both the germination rate and protocorm formation rate increased significantly. The general growth properties of seedlings developed on the medium with $10g{\cdot}L^{-1}$ sucrose were best, showing the highest bud formation rate and root number. Our results demonstrate that the 1/4 MS medium, supplemented with $10g{\cdot}L^{-1}$ sucrose, could improve in vitro germination, and facilitate the growth of seedlings developed from the protocorms of C. macranthos Sw.

• Journal of the Korean Society of Environmental Restoration Technology
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• v.23 no.3
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• pp.45-62
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• 2020

This study is to present the basic data for the conservation and management of natural resources of National Park by understanding the ecological characteristics of special protected areas located in Tohamsan District, Gyeongju National Park, South Korea. The numbers of vascular plants were summarized as 106 taxa including 47 families, 79 genera, 96 species, 1 subspecies, 7 varieties and 2 forms. For status by sites, there were 59, 50 and 55 taxa in A area(Pedicularis ishidoyana), B area(Millettia japonica) and C area(Cypripedium macranthos), respectively. In the dominant species, the tree layer was Quercus serrata(A area), Platycarya strobilacea and Pinus koraiensis(B area) and Fraxinus rhynchophylla(C area), the subtree layer was Carpinus cordata and Quercus serrata(A area), Pinus koraiensis and Castanea crenata(B area) and Fraxinus rhynchophylla(C area), the 1^st shrub layer was Toxicodendron trichocarpum and Rhododendron schlippenbachii(A area), Lindera erythrocarpa and Styrax japonicus(B area) and Fraxinus rhynchophylla and Lindera erythrocarpa(C area), 2^nd shrub layer was Styrax obassis and Lespedeza maximowiczii(A area), Lespedeza maximowiczii and Rhododendron mucronulatum(B area) and Lindera erythrocarpa(C area), the herbaceous layer was Athyrium yokoscense, Dryopteris chinensis, Dryopteris lacera and Lindera obtusiloba(A area), Athyrium yokoscense, Millettia japonica, Carex humilis Leyss. var. nana and Carex ciliato-marginata(B area) and Hydrangea macrophylla (Thunb.) Ser. subsp. serrata, Ajuga spectabilis and Oplismenus undulatifolius(C area).

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.04a
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• pp.45-45
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• 2018

We had already reported the successful germination for green pods of purple lady's slipper orchid (Cypripedium macranthos Sw.). The green pod methods is to take immature seeds in green capsules, sterilize the capsule, and take out the sterile seeds. This method, however, needs very critical time of harvest. The critical time of seed harvest changes depending upon the species, condition of the specimen, and climatic influence, and the right time lies between 5 and 12 weeks after fertilization. In this study, the mature seeds were collected after 120-130 days with hand-polination of lady's slipper orchids. Mature seeds are usually dormant and it has to be overcome, either with hormone or storing the seeds near freezing for two or three months to break dormancy. The seeds were first surface sterilized with 70% ethanol and then transferred 1% NaOCl for 10-15 minutes, followed by rinses 3 times with sterilized distilled water. The cypripedium seeds consists of an embryo within a seed coat known as a testa. The testa is water repellent and the seed has a large air space between the embryo and testa so the seed tends to float on water. We had resolved the problems with vacuum pump to soak water into the testa before sterilization. The seeds were placed on liquid or agar solidified germination media. Cultures were incubated at $24{\pm}1^{\circ}C$ in dark. The seeds were germinated in 6-8 weeks in liquid suspension culture (germination percentage over 18%); however, the seeds on agar solidified media took more than 5 months to germinate and the germination percentage less than 5%. The most effective media for liquid culture was 1/4 strength Murashige and Skoog (MS) medium with 50 ml/l coconut water ($4brix^{\circ}$) at pH 5.8.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2020.08a
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• pp.11-11
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• 2020

Background : The genus Cypripediums are typically adapted to the low temperature and cold climates of the high land in Korea. The species which were reported in Korean peninsula are C.japonicum, C.macranthos, C.calceolus and C.guttatum. We had already reported the successful germination of the C.macranthos and C.guttatum. The small spotted lady's slipper orchid(C.guttatum) is most endangered plants to extinction of which population was reported less than 100 in Korea. The objective of the present research is restoration of the C.guttatum in ex situ and adaptation in high land and nursery. Methods and Results : The 350 seedlings of C.guttatum were transplanted in soil at alpine area(altitude 750m) and the nursery of Korea National Arboretum(KNA) in YangPyeong city on May 2019 and 2020 in order to verify the possibility of ex situ restoration. The sprouted ratio of the seedlings were less than 10% in the alpine area and 83% in the nursery of KNA. The reasons of the diverse survival ratio are presumed as the low moisture content in the soil and heavy shade of the alpine area. The 15 cypripedium species and 29 hybrids were also effectively adapted in the KNA rare plant garden. Conclusion : These results demonstrated the difficulties of the restoration for small spotted lady's slipper orchid in the natural habitation. From the above results, we may conclude that the critical factors of the genus Cypripedium are intensive care for the fragile seedlings during the 1^st year acclimatization.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.10a
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• pp.23-23
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• 2019

Cypripedium is an orchid genus of over 50 species widely distributed in the northern temperate zone, with a few taxa extending central America and the Himalayas and the southwesternmost tip of China in Asia. Of the known species approximately two thirds are found in China. In Korea, 4 species were reported to find with rare popularity ; C. macranthos, C. guttatum, C. japonicum, and C. calceolus. The seed has a firm brown fusiform testa with an opening at one end where the micropyle of the ovule was situated. Cypripedium has small, fusiform, wind-dispersed seeds that weigh about $1{\sim}2{\mu}g$ and range from 0.1 to 2 mm long and 0.07 to 0.4 mm across. The germination of Cypripedium was previously thought to be hard to cultivate in one's garden and impossible to propagate artificially, nowadays lab-propagated seedlings are readily available in EU or United States. We had already reported the successful micropropagation of lady's slipper orchids with green pod sowing methods. The collected Cypripedium species and hybrids were planted in Korea National Arboretum (KNA) on Oct. 2018. The 11 species including C. candidum and the 31 hybrids such as C. Ingrid were introduced from Germany with CITES certification. The lady's slipper orchids bloomed on May to June, and the flowers were pollinated in the same species or hybrids to get seeds for in vitro germination on 2~3 days after fully flowering. The green pods were collected after 49~70 days after pollination. The 6 species of Cypripedium seeds were easily germinated on the POM within 70 days after sowing, and the 12 hybrids PLBs were observed with small roots. We had established the scheme of lady's slipper orchids germination in 3 months with green pods, and get the possibility of seedling in 10 months of sowing in vitro.

• Proceedings of the National Institute of Ecology of the Republic of Korea
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• v.2 no.1
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• pp.70-75
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• 2021

The present study aimed to clarify flora living at the area of Restoration Center for Endangered Species in Yeongyang, Gyeongbuk Province. In May, August, and September 2019 and in May and July 2020, all of vascular plants were recorded, and endangered, Korea endemic, and exotic plant species were further identified. The study site contained a total of 418 floral taxa (98 families, 261 genera, 384 species, 4 subspecies, 27 variations, and 3 formations), in which Magnoliophyta accounted for larger proportion (95.2%) than Pteridophyta (3.6%) and Pinophyta (1.2%). In addition, 1 endangered (Cypripedium macranthos Sw.) and 5 Korea endemic species (Aconitum pseudolaeve Nakai, Eleutherococcus divaricatus var. chiisanensis [Nakai] C.H. Kim & B.-Y. Sun, Lonicera subsessilis Rehder, Paulownia coreana Uyeki, and Weigela subsessilis [Nakai] L.H. Bailey) were detected. The number of exotic species was 33, consisting of 4 invasive-exotic, 4 potentially invasive-exotic, and 25 non-invasive species. Compared to a previous assessment before the establishment of the center (in 2014), there were increases in total floral taxa (from 361 to 418), endangered species (from 0 to 1), and exotic species (from 26 to 33). These results possibly reflect temporal changes in floral community, which should be confirmed through subsequent long term monitoring.