• Korean Journal of Agricultural Science
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• v.38 no.2
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• pp.227-233
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• 2011

This study aimed at developing cryopreservation protocol for chrysanthemum (Dendranthema grandiflora Tzelevcv. peak) shoot apices based on droplet-vitrification procedure, which is a combination of droplet-freezing and solution based vitrification. Progressive preculture of shoot apices in liquid MS medium supplemented with 0.3 and 0.7 M sucrose for 31 and 17 hours, respectively, was found optimum among preculture treatments tested. The composition of both loading and vitrification solutions significantly affected recovery growth of shoot tips before and after cryopreservation. Balancing glycerol and sucrose concentrations in the solutions was beneficial for recovery growth. The highest recovery after cryopreservation was observed when apical shoot tips were extracted from 4-week-old in vitro plantlets, progressively precultured with 0.3-0.5-0.7 M sucrose for 32-16-6 hours, respectively, then treated with loading solution comprising of 1.9 M glycerol + 0.5 M sucrose (35% PVS3 solution). Apices were then dehydrated with the vitrification solution consisted of 50% glycerol + 50% sucrose for 90 minutes then directly immersed in liquid nitrogen.

• 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.

• The Korean Journal of Malacology
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• v.26 no.4
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• pp.255-260
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• 2010

This study was conducted to investigate protocol standardization for spermatozoa cryopreservation of the Scapharca broughtonii (Schrenck). Among the freezing rates, freezing at a height of 2 cm above liquid nitrogen surface for 5 minute gave higher activity and survival rate. Among the various diluents, Ringer's solution was the best for S. broughtonii sperm cryopreservation. The suitability of cryoprotectants dimethylsulfoxide (DMSO), dimethylacetamide (DMA), glycerol and methanol were tested against three freezing rates. DMSO gave significantly higher activity and survival rates than others.

• Interdisciplinary Bio Central
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• v.2 no.4
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• pp.16.1-16.4
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• 2010

Cryopreservation of mouse embryos and spermatozoa has become the foremost technique for preserving large numbers of different strains of mice with induced mutations. In 1998, our mouse bank was established in the Center for Animal Resources and Development (CARD), Institute of Resource Development and Analysis, Kumamoto University, Japan, based on the Preservation, supply and development of genetically engineered animals report published by the Ministry of Education, Culture, Sports, Science and Technology. We cryopreserve mouse embryos and sperm, supply these resources, organize training courses to educate people and form part of a domestic and international network of both mutagenesis and resource centers. We currently have over 1,500 mouse strains, 842,000 frozen embryos and 26,000 straws containing frozen sperm. Moreover, we disclose information about 1,300 deposited strains. Furthermore, over 400 strains of frozen embryos or mice produced from frozen embryos and sperm are being supplied to the requesters both domestically and internationally. Additionally we hold training courses on the cryopreservation of mouse germplasm 2~3 times a year, both domestically and internationally. In the course, we teach basic reproductive engineering techniques to trainees on a man-to-man basis. We have already held 28 training courses on the cryopreservation of mouse germplasm at our center and at other institutes.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.5
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• pp.372-378
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• 2000

The cell culture of Angelica gigas Nakai producing decursin derivatives and immunostimulating polysaccharides was preserved in liquid nitrogen after pre-freezing in a deep freezer at -70$^{\circ}C$ for 480 min. The effects of the cryoprotectant and pretreatment before cooling were investigated to obtain the optimal procedure for cyropreservation. When compared to mannitol, sorbitol, or NaCl with a similar osmotic pressure, 0.7 M sucrose was found to be the best osmoticum for the cryopreservation of A. gigas cells. In the pre-culture medium, the cells in the exponential growth phase showed phase showed the best post-freezing survival after cryopreservation. A mixture of sucrose, glycerol, and DMSO was found to be an effective cryoprotectant and a higher concentration of the cryoprotectant provided better cell viability. When compared with the vitrification, the optimum cryopreservation method proposed in this study would seem to be more effective for the long-term storage of suspension cells. The highest relative cell viability established with the procedure was 89%.

• Korean Journal of Plant Resources
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• v.19 no.6
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• pp.665-669
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• 2006

Somatic embryos do not survive at exposure to liquid nitrogen temperatures without cryoprotective treatments. A simplified technique which simultaneously induces and cryoprotects embryogenic calli using plant vitrification solution 2 (PVS2) followed by dehydration was developed for the cryopreservation of Soap berry genetic resources. Vitrification is a way of removing the moisture in vegetation through PVS2. The PVS2 vitrification solution consisted of 30% glycerol (w/v), 15% ethylene glycol (w/v), 15% Dimethylsulfoxide (w/v) in B5 medium containing 0.4M sucrose. Two tests were done. The one was to eliminate moisture at $0^{\circ}C$ and the other at $25^{\circ}C$. In both cases the best results came out at a vitrification time of $10{\sim}20$ minutes. It was also found that the survival rate was higher at $0^{\circ}C$ than at $25^{\circ}C$. In particular, the survival rate reached more than 80%. Water-damaged embryos turned brown and stoped growth, but energetic embryos took on a milky hue and show a very vigorous growth rate. Successful cryopreservation of somatic embryos of soapberry can be used to establish in vitro genebanks for long-term conservation of Soapberry genetic resources to complement field genebanks and other in vitro methods already being used.

• Korean Journal of Plant Resources
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• v.33 no.6
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• pp.689-695
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• 2020

This study was conducted to improve and supplement the system of cryopreservation for adventitious bulbs induced by tissue cultured bulb-scales of lily (Lilium spp.) cvs. 'Milky way'. The explants, bulblets and bulb-scale-bulblets, were treated to low temperature (4℃) for 7 days prior to the pre-culture. The adventitious bulbs were pre-cultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3 and 0.7M). The pre-cultured adventitious bulbs were treated to loading solution (LS1 or LS2, C4 or C6) containing 35% of PVS3 (LS1, C4) or 40% of PVS3 (LS2, C6) for 40 min and exposed to dehydration solution (PVS3, B1) containing 50% glycerol and 50% sucrose for 60 min at 25℃. The adventitious bulbs were moved onto droplets containing 3 µl PVS3 on sterilized aluminum foils, and then soaked into liquid nitrogen (LN) for 60 min. The result of highest regrowth rate as 65.7% was obtained in cold treatment (4℃), osmoprotected with LS1 solution, and cultured in PCM3 medium by using bulb-scale-bulblet for cryopreservation. This result shows that droplet-vitrification could be used as a promising method for long-term storage of lily genetic resource.

• Journal of Plant Biotechnology
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• v.45 no.4
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• pp.333-339
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• 2018

The mature zygotic embryos of the Hevea brasiliensis were cryopreserved through the use of the vitrification and encapsulation/dehydration techniques. In all the experiments, the zygotic embryos were pre-cultured for three days in the MS medium supplemented with 0.3 M sucrose before they were used for the cryopreservation technique. In the vitrification procedure, the effect of the plant vitrification solutions (PVS2 and PVS3) and exposure time were studied. The highest survival rate (88.87%) and regrowth (66.33%) were achieved when the precultured zygotic embryos were incubated in a loading solution for 20 minutes at $0^{\circ}C$. They were subsequently exposed to PVS2 for 120 minutes at $0^{\circ}C$ and plunged directly into liquid nitrogen. Cryopreservation by the encapsulation-dehydration method was successfully done by leaving the encapsulated zygotic embryos in a laminar flow for 4 hours prior to plunging into a LN. The survival rate and regrowth of the encapsulated zygotic embryos were 37.50% and 27.98%, respectively. The cryopreserved zygotic embryos were able to develop into whole plants.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.337-342
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• 2018

Cryopreservation is mainly used for preservation of boar sperm. However, this method stresses the sperm by reactive oxygen species (ROS), and the conception rate and the litter size are not more efficient than the liquid preservation of spermatozoa. Therefore, we use chitosan which is a natural product derived antioxidant compound. We used GnHA (chitosan+hyaluronic acid) and GnHG (chitosan hydrogel) as chitosan complexes to cryopreserve boar sperm for improve sperm metabolism and function. Sperm parameter (sperm motility, progressive motility, path velocity, straight-line velocity, curvilinear velocity) is measured by computer-assisted sperm analysis (CASA) using frozen sperm with GnHA or GnHG (0, 0.25, 0.5, 1 mg/mL), respectively. Also, lipid peroxidation analysis using malondialdehyde (MDA) is performed to confirm the antioxidative effect of chitosan in frozen spermatozoa. CASA analysis showed GnHA and GnHG are effective against cryopreserved boar sperm. And antioxidant effect is measured by lipid peroxidation analysis. GnHA and GnHG, which is chitosan complex are effective for boar sperm cryopreservation by antioxidant effect.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.10a
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• pp.37-37
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• 2019

Cryopreservation is one of the ideal and suitable methods for long-term storage of plant germplasm. The plant contaminated with diseases and pathogens are decreased the multiplication rate, survival rate and high quality of plants after cryopreservation. The aim of this work was to improve a micropropagation method for lily in Korea, which are cultivars and indigenous plant. In the last process of rinsing scales after surface-sterilization, we tried to control the diseases and pathogens lived within the tissue by rinsing in 0.03% sodium hypochlorite (NaClO) instead of sterile distilled water. Bulb scales of Lilium were cultured in vitro on MS medium supplemented with Plant Preservative Mixture (PPM). The newly small bulb formed from bulb-scales was transferred to MS medium. We checked the non-contamination and survival rate after 2 weeks in culture. Non-contamination was shown to be 70 to 90% in formed small bulbs. This study will help to mitigate microbial contamination in Lilium species micropropagation for cryopreservation.