• Plant Biotechnology Reports
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• v.3 no.1
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• pp.1-56
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• 2009

Orchid seeds are nearly microscopic in size. Because of that, many fanciful theories were proposed for the origin of orchids. Almost 400 years separate the time when orchid seeds were seen for the first time and the development of a practical asymbiotic method for their germination. The seeds were first observed and drawn during the sixteenth century. Seedlings were first described and illustrated in 1804. The association between orchid and fungi was observed as early as 1824, while the requirement for mycorrhiza for seed germination was established in 1899. An asymbiotic method for orchid seed germination was developed in 1921. After Knudson's media B and C were formulated, orchids growing and hybridization became widespread. Hybrids which early growers may not have even imagined became possible.

• 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.

• Plant Biotechnology Reports
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• v.3 no.1
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• pp.103-109
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• 2009

In this study we established reliable methods for conservation of seeds of Phaius tankervilleae as an orchid genetic resource. The seeds, which were dehydrated to 5% water content and preserved at $4^{\circ}C$, showed no decrease in viability and germinability after three months. After storage for six months, however, the seeds showed a drastic decrease in germinability, even though survival rate was high. For long-term preservation of seeds of P. tankervilleae, cryopreservation is applied to the freshly harvested seeds. When the seeds were cryopreserved by the vitrification method for up to 12 months there was no apparent deterioration effect of storage time. These results indicate that cryopreservation by the vitrification method is useful for long-term conservation of P. tankervilleae seeds, which are difficult to preserve for more than three months under dry and low-temperature conditions.

• Journal of Plant Biology
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• v.33 no.4
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• pp.271-276
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• 1990

Our study was carried out for plant regeneration via somatic embryogenesis from immature seeds of Bletilla striata. The highest frequency of embryogenic callus formation was obtained from the immature seeds (at 150 days after pollination) cultured on Hyponex and VW medium supplemented with 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg/l kinetin under the dark condition. Multiple somatic embryos were induced when embryogenic callus was transferred to VW medium without growth regulators under continued illumination. Somatic embryos were observed histologically with scanning electron microscopy. Regeneration of Bletilla striata was obtained from somatic embryos with a well-defined scutellum and coleoptile as well as with one or more shoot primordia and root primordia. We think that these methods for orchid multiplication must be useful to access clonal propagation of orchids.

• 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.

• FLOWER RESEARCH JOURNAL
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• v.18 no.4
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• pp.271-277
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• 2010

The study is aimed to obtain the basic data for developing new variations of wild spring orchid. The results was investigated the capsules' formational characteristics and the germination ratio after having been self-pollinated by dividing the flowering period into the 5 stages into budding time, semi-flowering, full-flowering, 10 days after flowering, and 20 days after flowering. The fruit setting ratio was the highest as 100% in the plant which had been pollinated 20 days after the flowering, while the weight of the capsule was heaviest in the orchid which had been pollinated in semi-flowering period. As the result of investigating the germination ratio by dividing the period into 5 stages, it was the highest in the plant which had been pollinated during the semi-flowering period, and in the result of investigating the germination ratio by dividing the seeds harvesting days into the 3 stages, such as, 150 days, 165 days and 180 days after the pollination, it was highest as 5% in the orchid whose seeds had been harvested 150 days after the pollination. In the result of examining the germination ratio of the seeds treated with NaOCl, the those treated with 2% of NaOCl showed the highest as 67% in the germination ratio.

• 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.

• 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 Resources
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• v.19 no.2
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• pp.355-359
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• 2006

This study was carried out to investigate the factors affecting seed germination of Korean native orchids, such as Habenaria radiata, Bletilla striate and Cypripedium macranthum. In general, seeds of H. radiata was effectively germinated on solid medium and the highest germination (29.6%) was achieved on Hyponex solid medium. But shoot growth was better in MS liquid medium containing $100mL\;L^{-1}$ coconut water (MSC medium). Seed germination of B. striata was effectively induced on MS solid medium. However, no seed germination was observed in all liguid medium tested. Seed germination experiments of C. macranthum were performed using 6 kinds of media and seeds harvested 90 days after cross- and self-pollination. Cross-pollinated seeds were germinated on all the media with the best germination ratio of 4.45% on MSC medium. But no germination was observed from self-pollinated seeds.

• Journal of Plant Biotechnology
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• v.41 no.1
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• pp.44-49
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• 2014

In this study, we investigated the effects of sodium hypochlorite (NaOCl) and red or blue light-emitting diode (LED) light on embryo swelling and germination of Oreorchis patens (Lindl.) Lindl. A method for determining the swelling and protocorm formation in O. patens seeds through in vitro examination of immature seeds was established. NaOCl treatment of immature seeds was found to significantly enhance the extent of embryo swelling and protocorm formation in immature zygote embryos compared to those in the untreated controls. Additionally, the effects of white fluorescent light, and red and blue LED lights on embryo swelling and protocorm formation in in vitro cultured seeds were examined and compared to the conditions with or without NaOCl treatment. The most suitable light for embryo swelling and protocorm formation was the red LED light.