• Korean Journal of Plant Resources
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• v.34 no.6
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• pp.600-607
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• 2021

Cryopreservation method using a droplet vitrification was applied to the thirty-one strawberry accessions of in vitro grown shoot tips. A protocol with 0.3 - 0.5 M preculture followed by C4 loading and B1 dehydration solutions efficiently implemented cryopreservation of twenty-six strawberry accessions. The highest regrowth rate was 85.8% for PHS0007 and others were ranged between 85.8% and 21.0%. A slightly modified protocol was applied to five accessions. With these two protocols, twenty-eight accessions obtained more than 40% regrowth rate. This study showed that the droplet vitrification method was able to practically implement cryopreservation of in vitro grown shoot tips of broad range of strawberry germplasm (105).

• 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.

• 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.

• Korean Journal of Plant Resources
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• v.36 no.6
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• pp.562-570
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• 2023

The droplet-vitrification technique for cryopreservation has proven successful across a diverse range of germplasm, ensuring safe and effective long term preservation. In this study, we investigate an effective cryopreservation protocol using the droplet-vitrification technique for shoot tips of Freesia hybrida cultivars 'Sunny Gold' and 'Sweet Lemon'. To determine optimal conditions for Freesia cryopreservation, we employed a carefully selected standard procedure along with additional treatments and alternative solutions. For 'Sunny Gold', the highest regrowth rate of 24% was achieved when shoot tips underwent dehydration with PVS3 solution for 120 minutes before direct immersion in liquid nitrogen (LN) for 1 hour, coupled with a standard protocol involving a two-step preculture with 0.3 M - 0.5 M sucrose, loading with C4 for 40 minutes, and unloading with 0.8 M sucrose for 40 minutes. In the case of 'Sweet Lemon,' regrowth of cryopreserved shoot tips was observed with dehydration treatments, including PVS2 (A3) for 60 minutes and PVS3 (B1) for 60 minutes, as well as longer exposure. The results reflect the distinct sensitivity of shoot tips to chemical toxicity and osmotic stress in these two genotypes. This study provides valuable evidence to consistently enhance the effectiveness of cryopreservation methods for the long-term conservation of Freesia germplasm.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2004.10a
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• pp.114-114
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• 2004

• 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.

• Reproductive and Developmental Biology
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• v.30 no.4
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• pp.279-285
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• 2006

Vitrification has been suggested to be an effective method for the cryopreservation of human ES cells. However, the efficiency of vitrification with different vehicles remains a matter of ongoing controversy. The objective of this study was to assess the efficiency of cryopreservation in human ES cells by vitrification using different vehicles. A human ES cell line and a variety of vehicles, including micro-droplet (MD), open-pulled straw (OPS) and electron microscopic grid (EM-grid), were employed in an attempt to assess vitrification efficiency. In order to evaluate the survivability and the undifferentiated state of the post-vitrified human ES cells, we conducted alkaline phosphatase staining and characterization via both RT-PCR and immunofluorescence assays. The survival rates of the post-vitrified human ES cells using MD, OPS and EM-grid were determined to be 61.5%, 66.6% and 53.8%, respectively. There also exist significant differences between slow-freezing and vitrification (p<0.01). However, no significant differences were detected between the vehicle types. Finally, the pluripotency of human ES cells after thawing was verified by teratoma formation. Cryopreservation using vitrification is more effective than slow-freezing, and the efficiency of vehicles proved effective with regard to the preservation of human ES cells.

• 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.