• Journal of Embryo Transfer
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• v.9 no.2
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• pp.173-180
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• 1994

The present experirnents on cryopreservation were carried out to investigate effect of solution toxicity, equilibration time and cell stages on the post-thaw survival of mouse morulae and blastocyst embryos cryopreserved by vitrification in EFS solution. The mouse embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen and thawed rapidly. After the mouse morulae embryos were exposed to EFS solution for 2 and 5 ruin. at room temperature and then they were washed in 0.5 M sucrose solution and basal mediurn(D-PBS + 10% FCS), they were cultured to examined cryoprotectant toxicity induced injury during exposure, most of embryos developed to expanded blastocysts(100 and 90.0%). However, when the exposure time was extended to 10 and 20 min, these development rates dropped dramatically in 10 ruin. (75.0%) and 20 ruin. (4.5%), respectively. When the compacted morulae were vitrified in EFS solution after equilibration for 2 and 5 min, the embryos have developed to normal blastocyst following thawing, washing and culture processes was 89.3 and 89.6%. However, when the exposure time was expanded to 10 ruin, this survival rate dropped to 68.8%. When the blastocyst were vitrified in EFS solution after equilibration for 2, 5 and 10 minutes, the survival rate of embryos which developed to normal blastocyst following thawing and culture processing were 58.5, 46.7 and 22.4%, respectively. The optimal time of equilibration of mouse morula and blastocysts in EFS solution seemed o be 2 and 5 ruin.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2021.04a
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• pp.36-36
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• 2021

Cryopreservation has been broadly used as an efficient method for a long-term conservation for many types of plants especially vegetatively propagated plants. Among several cryopreservation methods, a droplet-vitrification was the most widely applicable and efficient method. Studies have developed protocols for strawberry using droplet-vitrification method and suggested the practical use of the protocol for large number of germplasm with a little modification. In this study, the droplet vitrification method of shoot tip has been tested on 31 accessions provided around the world. Shoot tips were precultured on Murashige and Skoog (MS) liquid medium supplemented with 0.3~0.5M sucrose. Precultured explants were osmoprotected with loading solution, 35% of PVS3 (C4, 17.5% glycerol and 17.5% sucrose) for 40 min and exposed to dehydration solution, PVS3 (B1, 50% glycerol and 50% sucrose) for 60 min. Then, the explants were transferred onto droplets containing 2.5 uL PVS3 on sterilized aluminum foils prior to direct immersion in liquid nitrogen (LN) for 1hr. The cryopreserved shoot tips were rapidly warmed in a water bath at 40C and then unloaded in MS with 0.8M sucrose for 40 min. The shoot tips were cultured in NH₄NO₃-free MS post culture medium for 2 weeks. Subsequently, the explants were moved to the MS medium for 6 weeks and evaluated the regrowth rate. By this droplet-vitrification protocol, twenty-four accessions showed at least 40% regrowth rate. Out of 24 accessions, 'Nonsan1ho' had the highest regeneration rate of 85.8% and 'Jumbo pureberry' had the lowest with 42.1%.

• Korean Journal of Plant Resources
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• v.36 no.6
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• pp.571-579
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• 2023

In this study, cryopreservation by droplet-vitrification was applied to pear (Pyrus spp.) germplasm. We focused on the development and assessment of various strategies for the selection of suitable tissue, osmoprotection, and dehydration. We also evaluated post-thaw recovery of cryopreserved explants by droplet-vitrification. Preferentially, we tested the effects of preculture and loading treatments to determine which tissues were more suitable, either the apical shoot tips or the axillary buds. Apical shoot tips showed the better regrowth rate than in vitro axillary buds. The most effective techniques for cryopreservation were as follows. Shoots from in vitro seedlings which had been cultured for about 5-6 weeks were cold-hardened at 4℃ for one week, excised shoot tips were precultured on liquid MS medium including 0.3 M sucrose for 31 hours and 0.7 M sucrose for 17 hours, osmoprotected in loading solution (LS) for 40 min, and then cryoprotected in dehydration solution (PVS3) for 90 min. In addition, we found that regrowth rates of explants on regrowth medium after exposure to liquid nitrogen (LN) were higher than those on MS medium. Results indicated that the highest regrowth percentage was 95.6% for 'Bartlett' cultivar and 68.9% for 'BaeYun No.3' cultivar. Consequently, apical shoot tips of two pear cultivars, 'Bartlett' (P. communis) and 'BaeYun No.3' (P. pyrifolia), were successfully cryopreserved by droplet-vitrification. Results of this study show that the enhanced droplet-vitrification method described in the present study could be used as an effective means for long-term storage of pear genetic resources.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.29-29
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• 2003

The objective of this study is to examine an effective cryopreservation method and various vitrification containers on the survival vates of embroys. For the vitrification, in vitro produced embryos at blastocyst stage were exposed to ethylene glycol 5.5 M freezing solution (EG5.5) for 20 sec, loaded on containers such as grid, straw and paper and then immediately plunged into - 196$^{\circ}C$ L$N_2$. The blastocysts were thawed serially in 0.5, 0.25 and 0.125%; P < 0.05). Therefore, this study suggests that bovine embryos can be easily, effectively and successfully cryopreserved by grid, straw, and paper in the presence of freezing solution. Furthermore, vitrification using paper may be used as a no M sucrose in CR1aa, each for 1 min, and cultured in CR1 an medium supplemented with 10% FBS. After thawing, there were not significant differences in recovery rates of EM grid, straw and paper as 84.6, 88.3, and 93.7%, respectively (Table 1). However, survival rates of EM grid (78.1%) and paper (77.1%) showed significantly higher than straw (52. 1w method for bovine embryos.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2023.04a
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• pp.33-33
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• 2023

Potatoes are the world's 4th major food crop after maize, rice, and wheat and also are a staple food for 1.3 billion people. Due to their wide adaptability to various environmental conditions, their yeild capacity, and high commercial value, potatoes have contributed to global food security. Many potato germplasms are commonly preserved as whole plants in fields or in storage to maintain their particular genetic combinations. However, field maintenance is expensive and has the risk of potential losses from diseases, pests, plant ageing and climate change. Over the past four decades, meaningful efforts have been made toward the safe long-term conservation of potatoes through cryopreservation methods such as droplet-vitrification. In this study, we tested 4 Korean potato varieties('Golden Egg', 'Golden Ball', 'Ja-Young' and 'Ha-Ryeong') with the modified potato droplet -vitrification protocol. Potato shoot tips are precultured in a sucrose-enriched medium(0.3 and 0.7M for 7 and 17hrs, respectively) and submitted to a loading step with C4 solution for osmoprotection. The treated explants were dehydrated with Plant Vitrification Solution(PVS)2 which is 80% A3 solution in ice for 30 minutes. Thawing and unloading steps were performed with 0.8M sucrose solution for 30 sec(40℃) followed by 30min(25℃, room temperature). In a potato post-culture medium(MS+0.1 mg·L^-1 GA₃+0.1 mg·L^-1 kinetin), we obtained a survival rates of post-thawed explants ranging 16.1-82.2%. The results suggest that modified and optimized protocols are required dependinig on every cultivar, genetic and ecological types. To achieve higher survival and regeneration rates, each step within the cryoprocedure must be carefully optimized.

• Clinical and Experimental Reproductive Medicine
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• v.34 no.2
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• pp.117-124
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• 2007

Objective: The aim of this study was to compare the efficacy of slow freezing with vitrification method for cryopreservation of mouse pronuclear stage embryos. Methods: Mouse pronuclear embryos obtained from superovulated mice and classified into 2 groups of slow freezing and vitrification. Slow freezing solution consisted of 1.5 M PROH, 0.1 M sucrose, while vitrification solution consisted of 40% ethylene glycol, 18% Ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline supplemented with 10% SSS. Recovery and survival rates after thawing and development rates to hatching balstocyst stage were compared between two groups. Results: After freezing and thawing, recovery rate of slow freezing group was 93.8%, whereas vitrification group was 66.5% (p<0.01). Survival rate of recovered embryos were similar between two groups as 83.2% in slow freezing and 87.6% in vitrification. Embryo development rates to 2-cell stage after 24 hrs (77.0% vs 59.1%), 4-cell after 48 hrs (72.6% vs 53.3%), blastocyst after 96 hrs (53.1% vs 40.1%) of thawing were significantly higher in vitrification group than those of slow freezing group, respectively. Conclusion: The vitrification method may provide better developmental competence of frozen-thawed embryos than that of slow freezing method for cryopreservation of mouse pronuclear stage embryos.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2010.05a
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• pp.12-12
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• 2010

We developed droplet-vitrification protocol, a combination of droplet-freezing and solution-based vitrification, and applied to germplasm collections of garlic, potato, lily as well as cell lines, including hairy roots, somatic embryos. To establish a garlic cryobank, four Korean garlic field collections at Danyang, Suwon, Mokpo and Namhae were cryopreserved last five years. The protocol applied consisted of preculture for 3-4 days at $10^{\circ}C$ on solid MS medium with 0.3M sucrose, loading for 40 min in liquid medium with 35% PVS3, dehydration with PVS3 for 150 min, cooling in $5{\mu}l$ droplets of PVS3 placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into pre-heated($40^{\circ}C$) 0.8M sucrose solution for 30s. A total of over 900 accessions of garlic were stored in liquid nitrogen for long-term conservation using unripe inflorescences, cloves or bulbils. Twelve alternative plant vitrification solutions were designed by modifying cryoprotectant concentrations from the original PVS2 and PVS3. The results suggest that PVS2-based vitrification solutions with increased glycerol and sucrose and/or decreased DMSO and EG concentrations can be applied for medium size explants which are tolerant to chemical toxicity and moderately sensitive to osmotic stress. PVS3 and variants can be used widely when samples are heterogeneous, of large size and/or very sensitive to chemical toxicity and tolerant to osmotic stress.

• 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.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2021.04a
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• pp.35-35
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• 2021

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. 'MilkyWay'. 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 ㎕ 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.

• Korean Journal of Agricultural Science
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• v.43 no.4
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• pp.575-580
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• 2016

This study was performed to determine whether supplementation of tauroursodeoxycholic acid (TUDCA), an endoplasmic reticulum (ER) stress inhibitor, during vitrified cryopreservation enhances the development of frozen mouse embryos. Mouse 8-cell stage embryos were collected and exposed to a cryoprotectant solution containing TUDCA or TM (tunicamycin, an ER stress inhibitor) at room temperature and stored in liquid nitrogen following vitrification. The final concentration of TUDCA or TM was $50{\mu}M$. The survival and development rates of mouse 8-cell stage embryos exposed to TUDCA- or TM-containing solutions at room temperature or stored in liquid nitrogen following vitrification were measured. There were no significant differences in survival rate and blastocyst formation rate among control, TUDCA, and TM groups after embryos were exposed to vitrification solutions at RT. When mouse 8-cell stage embryos were treated with TUDCA or TM and then stored in liquid nitrogen, the survival rates of control and TUDCA groups were significantly higher than for the TM group. Blastocyst formation rate of the TUDCA group following in vitro culture was significantly higher than that in control or TM groups. The TM group showed a lower (p < 0.05) blastocyst formation rate than the other two groups. Our results indicate that TUDCA supplementation during cryopreservation of mouse embryos could enhance their development capacity.