• 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).

• Korean Journal of Plant Tissue Culture
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• v.28 no.4
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• pp.209-213
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• 2001

Spathiphyllum floribundum cv. Cupid was micropropagated on LS medium containing various growth regulators. Shoot tips were cultured on media with different concentrations of thidiazuron (TDZ) or banzyladenine (BA). The medium containing 2.0 mg/L BA was most effective on the shoot multiplication and the formation of adventitious multi-shoot cluster from shoot tips among the different concentrations of TDZ and BA. The combined treatments of BA and paclobutrazol (PBZ) were stimulated the shoot multiplication and the formation of adventitious mu]ti-shoot clusters from shoot tips compared to the ones of BA alone. The adventitious multi-shoot clusters were culled with 5∼7 mm in size and cultured on media with BA or thidiazuron (TDZ). Shoot multiplication and shoot cluster proliferation from the shoot clusters were very favorable on media supplemented with 2.0∼3.0 mg/L BA. The shoots grew and rooted out vigorously on media with 2.0∼3.0 mg/L IBA. The rooted plantlets were acclimatized in the soil mixed with a rate of vermiculite 1 and perlite 1, and survived more than 95% in greenhouse.

• Korean Journal of Plant Resources
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• v.33 no.6
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• pp.675-681
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• 2020

This study describes an efficient and stable droplet vitrification following cryopreservation of strawberry shoot tip (Fragaria × ananassa Duch.) accessions 'Massey' and 'MDUS3816'. The shoot tips were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.7M). Precultured explants were osmoprotected with loading solution (LS, C4) containing 17.5% glycerol and 17.5% sucrose for 40 min and exposed to dehydration solution (B1) containing 50% glycerol and 50% sucrose for 40 min at 25oC. Subsequently, the explants were transferred onto droplets containing 2.5 µL PVS3 on sterilized aluminum foils (4 cm× 0.5 cm) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regrowth rate (%) in both the cultivars was obtained when the shoot tips were precultured with 0.3M sucrose for 30 h + 0.5M sucrose for 16 h at 25oC. The cryopreserved shoots tips exhibited 57.8 % recovery rate by culturing in NH₄NO₃-free MS medium supplemented with 3% sucrose, 1.0 g/L casein, 1.0mg/L GA₃, and 0.5 mg/L BA for 5 weeks and in MS medium supplemented with 0.5 mg/L GA₃ for 8 weeks. Variation was not observed in both of ploidy analysis and morphological investigation on plantlets of two accessions cryopreserved under variable preculture conditions.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.04a
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• pp.80-80
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• 2019

This study describes an efficient and widely applicable droplet-vitrification following cryopreservation for shoot tips of (Fragaria x ananassa DUCH. cvs. 'Derunoka' and 'Jumbo pure berry'. The shoot tips of strawberry were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.7M). Precultured explants were treated with loading solution (LS, C4) containing glycerol 17.5% and sucrose 17.5% for 40 min and exposed to dehydration solution (B1) containing 50% of glycerol and 50% of sucrose for 60 min at $25^{\circ}C$, and then transferred onto droplets containing $2.5{\mu}l$ PVS3 on sterilized aluminum foils ($4cm{\times}0.5cm$) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regeneration rate (%) was obtained when shoot tips were precultured with treatment-2 (exposing of shoot tips to MS + 0.3M Sucrose for 30 h and then treated with MS+0.5 M sucrose for 16 h) at $25^{\circ}C$ in both the cultivars. The viability of cooled samples, followed by culturing on MS medium for 4 weeks was 77.8% and 60.0% for 'Derunoka' and 'Jumbo pure berry', respectively. This result shows droplet-vitrification would be a promising method for cryobanking strawberry germplasm.

• Korean Journal of Plant Resources
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• v.31 no.6
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• pp.675-683
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• 2018

This study describes an efficient and widely applicable droplet-vitrification following cryopreservation for shoot tips of Chrysanthemum morifolium (Ramat.) cvs. 'Borami' and 'Yes morning'. The shoot tips of Chrysanthemum were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.7 M). Precultured explants were treated with loading solution (LS, C6) containing glycerol 20% and sucrose 20% for 30 min and exposed to dehydration solution (B5) containing 40% of glycerol and 40% of sucrose for 60 min at $25^{\circ}C$, and then transferred onto droplets containing $2.5{\mu}l$ PVS3 on sterilized aluminum foils ($4cm{\times}0.5cm$) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regeneration rate (%) was obtained when shoot tips were precultured with treatment-2 (exposing of shoot tips to MS + 0.3M sucrose for 30 h and then treated with MS+0.5 M sucrose for 16 h) at $25^{\circ}C$ in both the cultivars. The viability of cooled samples, followed by culturing on $NH_4NO_3$-free MS medium for first 5 days was increased to two-fold (80.7%) regrowth rate over those cultured on normal MS medium or MS medium containing plant growth regulators. This result shows droplet-vitrification would be a promising method for cryobanking chrysanthemum germplasm.

• 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.

• Journal of Plant Biotechnology
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• v.31 no.1
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• pp.61-65
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• 2004

In order to micropropagate uniform plantlets of Alocasia cadieri Chantrier in vitro, the shoot tips were cultured on media containing various concentrations of BA and thidiazuron (TDZ). Multiple shoot formation from shoot tips was very effective on medium containing 0.1mg/L TDZ. The formed shoots from shoot tips were separated into a shoot, and cultured on media with BA, TDZ, and NM combination for proliferation. The shoots were multiplied very vigorously on medium with 0.5mg/L TDZ and 0.5mg/L NAA. The rooting and growth of multiplied shoots were more effective on medium with 2.0g/L activated charcoal, rather than those with IBA and NAA. Rooted plantlets show high survival in soil mixed with perlite 1: vermiculite 1 or vermiculite alone.

• 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%.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2002.11b
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• pp.53-53
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• 2002

Shoot tips of in vitro propagated plantlets were cryopreserved using encapsulation/dehydration procedures. Shoot tips were excised under filter sterilized antioxidants solution (0.2M phosphate buffer, pH 5.7 supplemented with 5g/1 ascorbic acid and 15g/1 sodium borate). They were drawn up into a sterile 10 $\textrm{cm}^3$disposable pipette and were dropped into the culture medium with 2.5w/v Na-alginate, then into 100mM CaCl$_2$.2$H_2O$. Encapsulated shoot tips were transferred into 10㎤ of liquid culture medium with a range of sucrose concentrations (0.25-1.0M) and were incubated in dark for 24 hours in 18C at 40rpm. Beads were then dehydrated in silica gel for different time intervals (1-24 hours). Then they were freeze dried either rapidly (plunge directly into liquid N2 or in two stages (samples were kept at 20C for 10 minutes, then reduced to 35C at 1C per minute. Then, plunge into liquid $N_2$). The influence of sucrose and silica gel pre-treatment on pre- and post-freeze shoot growth was examined.(중략)