• 한국작물학회지
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• 제49권4호
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• pp.354-357
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• 2004

A simplified technique which cryoprotects zygotic embryos by encapsulation-dehydration was developed for the germplasm conservation of herbaceous peony (Paeonia lactiflora Pall.). The highest survival rate $(85\%)$ was obtained from embryos treated by encapsulation-dehydration. The zygotic embryos were precultured on MS medium containing 0.3mg/L $GA_3$ for 1 day. The precultured embryos were encapsulated in $3\%$ (w/v) alginate beads and immersed for 1 h in MS medium containing 2 M glycerol and 0.5 M sucrose. The encapsulated embryos were dehydrated for 5h by air drying prior to direct immersion in liquid nitrogen. This encapsulation-dehydration method appears to be a promising technique for germplasm cryopreservation of a herbaceous peony.

• 한국자원식물학회지
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• 제33권6호
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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.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2019년도 추계학술대회
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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.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2021년도 춘계학술대회
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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.

• Journal of Plant Biotechnology
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• 제7권3호
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• pp.183-186
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• 2005

Cryopreservation has been recognized as a practical and efficient tool for the long-term storage of vegetatively propagated plants. This study was conducted to investigate the effects of slow-freezing techniques on the cryopreservation of potato. In vitro plantlets of the potato genotypes of 'Atlantic', 'Superior’, 'Namseo', 'J138', and 'CTO5-5' were cold acclimated, and the excised axillary buds were precultured, osmoprotected, exposed to plant vitrification solution, frozen slowly to $-40^{\circ}C$ and then rapidly plunged into liquid nitrogen, thawed and finally plated on the regeneration medium. It was found that the higher the sucrose concentrations in the subculture medium of donor plantlets, the higher the survival rates of shoot tips after cryopreservation, and the highest survival (20%) was observed in the medium added with 0.25 M sucrose. As for the effect of cooling, $0.3^{\circ}C/min$ cooling speed showed the highest survival (25%). Different varieties showed different responses over different cryopreservation treatments. Survival rate was increased by slow-freezing technique method as compared with that of the basic cryopreservation method of vitrification alone in the diverse potato genotypes. Leaf and tuber morphologies of potatoes regenerated after cryopreservation using slow freezing technique were similar to those derived from the in vitro stock plantlets.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.327-332
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• 2000

Supended cells of Camptotheca acuminata were observed to lose their ability to synthesize camptothecin and its derivatives as a result of repeated cultures. Accordingly, the maintenance of high-yield cells by cryopreservation was sudied to overcome this stability problem, and various factors involved were optimized. Pregrowing the cells in 8% myoinositol for 4 days was found to be the most effective in improving survival. The highest survival was obtained when the pregrown cells were cryoprotected with a mixture of 10% DMSO, 0.6M mannitol, and 10% glycerol. When the cryopreserved cells were maintained in a freezer at $-70^{\circ}C$, 94% survival was obtained after 4 months. The survivals after 5 and 8 months of storage decreased to 52% and 45%, respectively. No loss of biosynthetic capacility of camptothecin was observed after short to medium term cryopreservation of C. acuminata.

• 한국동물생명공학회지
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• 제36권4호
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• pp.299-306
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• 2021

Vitrification methods are commonly used for mammalian reproduction through the long-term storage of blastocyst produced in vitro. However, the survival and quality of embryos following vitrification are significantly low compared with blastocyst from in vitro production (IVP). This study evaluates that the survival of frozen-thawed bovine embryos was relevant to mitochondrial superoxide derived mitochondrial activity. Here we present supplementation of the cryopreservation medium with Mito-TEMPO (0.1 µM) induced a significant (p < 0.001; non-treated group: 56.8 ± 8.7%, reexpanded at 24 h vs Mito-TEMPO treated group: 77.5 ± 8.9%, re-expanded at 24 h) improvement in survival rate of cryopreserved-thawed bovine blastocyst. To confirm the quality of vitrified blastocyst after thawing, DNA fragmentation of survived embryos was examined by TUNEL assay. As a result, TUNEL positive cells rates of frozen-thawed embryos were lower in the Mito-TEMPO treated group (4.2 ± 1.4%) than the non-treated group (7.1 ± 3.5%). In addition, we investigated the intracellular ROS and mitochondrial specific superoxide production using DCF-DA and Mito-SOX staining in survived bovine embryos following vitrification depending on Mito-TEMPO treatment. As expected, intracellular ROS levels and superoxide production of vitrified blastocysts after cryopreservation were significantly reduced (p < 0.05) according to Mito-TEMPO supplement in freezing medium. Also, mitochondrial activity measured by MitoTracker Orange staining increased in the frozen-thawed embryos with Mito-TEMPO compared with non-treated group. These results indicate that the treatment of Mito-TEMPO during cryopreservation might induce reduction in DNA fragmentation and apoptosis-related ROS production, consequently increasing mitochondrial activation for developmental capacity of frozen-thawed embryos.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2021년도 춘계학술대회
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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%.

• 한국자원식물학회지
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• 제36권6호
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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.

• Journal of Microbiology and Biotechnology
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• 제6권3호
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• pp.209-212
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• 1996

A two-step cooling technique has been developed for cryopreservation of suspension cultured Scutellaria baicalensis cells. Efficient regrowth of cryopreserved cells was obtained in cryoprotected cells with a mixture of 1.5 M glycerol and 0.4 M sucrose in Schenk and Hildebrandt medium without pretreatment in high osmotic medium. Optimum freezing conditions were found to be a cooling rate of $0.5^{\circ}C$ min from $4^{\circ}C$ to $-40^{\circ}C$, and then retaining samples at $-40^{\circ}C$ for 30 min prior to plunging into liquid nitrogen. A regrowth rate of approximately 95$%$ was obtained after three month storage in liquid nitrogen. Callus cultures established from the cryopreserved cells were found to produce the same patterns of flavonoid accumulation and retain their baicalin producing activity.