• 한국자원식물학회지
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• 제31권6호
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• pp.684-694
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• 2018

This study describes the successful establishment of a cryopreservation protocol for Citrus limon cultivars: 'Frost Eureka limon' and 'Cook Eureka limon', using a droplet-vitrification method. The shoot tips that were excised from in vitro grown seedlings of the two cultivars were preserved in liquid nitrogen (LN) and successfully regenerated into whole plants. Excised shoot tips were pre-cultured for 1 or 2 days in 0.3 M and 0.5 M sucrose solutions at $25^{\circ}C$ and incubated in a loading solution (LS) composed of 17.5% glycerol + 17.5% sucrose in Murashige and Skoog (MS) medium for 40 min at $25^{\circ}C$. Prior to direct immersion in LN for 1 h, the shoot tips were dehydrated with plant vitrification solution 2 (PVS2) at $0^{\circ}C$ or PVS3 at $25^{\circ}C$. The frozen shoot tips were re-warmed and unloaded with 1.2 M sucrose in $\text\tiny{^1/_2}$ MS for 30 min at $25^{\circ}C$. Shoot tips were post-cultured overnight on survival medium and then micrografted onto 'trifoliate orange' (Poncirus trifoliate (L.) Raf. seedling rootstocks for recovery and to produce whole plants. The highest regrowth rates were 53.5% and 50.3% for cryopreserved shoot tips of 'Frost Eureka limon' and 'Cook Eureka limon', respectively, when pre-cultured in 0.3 M and 0.5 M sucrose concentrations in a sequencing manner, with LS and treated with PVS2 for 60 min at $0^{\circ}C$. We also investigated whether the ammonium ion concentration on post-culture medium affected the viability of the cryopreserved Citrus shoot tips. The viability of cooled samples, following culturing on woody plant media (WPM) containing $\text\tiny{^1/_4}$ ammonium nitrate overnight before micrografting, was the highest (70.3%) in 'Frost Eureka limon'. The study described here is a cost-effective and safe method to conserve Citrus fruit cultivars, for the improvement and large-scale multiplication of fruit plants and for breeding disease resistance.

• 한국자원식물학회지
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• 제30권6호
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• pp.612-617
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• 2017

The preservation of pear germplasm, like that of other clonal germplasms, is difficult because it requires conservation of whole plants or their tissues. Among the currently available methods for long-term conservation of clonal germplasm, cryopreservation of shoot tips is the most reliable and cost- and space-effective option. Alginate-coated axillary shoot tips from in vitro-grown pear were conserved successfully in liquid nitrogen (LN) following dehydration. Shoot recovery from cryopreserved shoot tips was improved greatly after 8 weeks of cold acclimation, but recovery decreased slightly after then. The highest regeneration rate was observed when in vitro shoot tips were preincubated in MS (Murashige and Skoog) medium with 0.3 M sucrose for 48 h, and when alginate-coated shoot tips were precultured in MS medium with increasing sucrose concentrations (0.5 M and 0.7 M) for 8 and 16 h, respectively. When the encapsulated beads were dehydrated for up to 7 h [25% water content (fresh weight basis)] under laminar flow, the highest regeneration rate was observed in "BaeYun No. 3" (55.7%) and "Whanggeum" (43.3%) after warming from LN. This technique is useful as a practical procedure to cryopreserve plant material that is sensitive to freezing of the surrounding cryoprotectant medium. Therefore, this technique appears to be promising for the cryopreservation of shoot tips from in vitro-grown plantlets of pear germplasm.

• 한국자원식물학회지
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• 제29권6호
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• pp.635-641
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• 2016

Sweet potato (Ipomoea batatas L.) shoot tips grown in vitro were successfully cryopreserved by encapsulation-vitrification. Encapsulated explants are very easily manipulated, due to the relatively large size of the alginate beads, and a large number of samples can be treated simultaneously. In this study, the effects of sucrose preculture, cryoprotectant preculture, and post-warm recovery media on regrowth, following liquid nitrogen (LN) exposure, were investigated to establish an efficient encapsulation-vitrification protocol for sweet potato. Shoot tips of plants grown in vitro were precultured in 0.3 M sucrose for 2 d before encapsulation. Encapsulated shoot tips were pre-incubated in liquid MS (Murashige and Skoog) medium containing 0.5 M sucrose for 16 h, before preculturing in sucrose-enriched medium (0.7 M sucrose) for 8 h. Shoot tips were osmoprotected with 35% plant vitrification solution 3 (PVS3) for 3 h, before being dehydrated with PVS3 for 2 h at $25^{\circ}C$. The encapsulated and dehydrated shoot tips were transferred to 2 mL cryotubes, suspended in 0.5 mL PVS3, and plunged directly into liquid N. High levels of shoot formation were obtained for the cv. Yeulmi (65.7%) and Yeonwhangmi (80.3%). The regrowth rates of cryopreserved samples in Yeulmi (78.9%) and Yeonwhangmi (91.3%), following culture on ammonium-free MS medium for 5 d, were much higher than those cultured on standard MS medium (65.7% and 80.3%, respectively). This encapsulation-vitrification is a promising method for the long-term preservation of sweet potato.

• 식물조직배양학회지
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• 제22권2호
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• pp.101-104
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• 1995

작약동아의 정아 및 액아의 기내배양에 의한 유묘의 증식에 필요한 배양조건을 구명코자 실험한 결과는 다음과 같다. 정아배양의 경우, 생장조절재의 조성에 무관하게 두지방종에서 100% 신초가 신장하였으나 생장은 의성지방종 은 NAA 0.1 mg/L 단용배지, 영천지방종은 NAA 0.01 mg/L 단용배지에서 가장 양호하였다. 액아배양의 경우, NAA 0.01 mg/L와 zeatin 5.0 mg/L 혼용배지에서 의성지방종은 100%, 영천지방종은 50%의 가장 높은 신초신장율을 보였고 신초의 생장도 양호한 경향이었다. 정아 및 액아배양으로부터 유도된 신초는 vermiculite를 지지물로 한 NAA 0.1 mg/L 첨가배지에서 30.0%의 발근율을 보였으나 뿌리의 생장은 양호하였다.

• 한국자원식물학회지
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• 제36권6호
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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.

• Journal of Plant Biotechnology
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• 제2권3호
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• pp.151-155
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• 2000

Shoot proliferation was obtained from shoot tips and nodal explants of Vitex negundo L. on MS medium supplemented with either BAP or KIN (0.1-2.0 mg/L) alone or in combination with NAA (0.1 mg/L). The concentrations of cytokinins combined with NAA produced multiple shoots from shoot tips and nodal explants. The highest mean percentage (84.3$\pm$8.0) of shoot multiplication's were observed on nodal explants in the presence of BAP (1.5 mg/L) and NAA (0.1 mg/L) followed by shoot tips (65.0$\pm$5.0). The regenerated shootlets were rooted on MS basal medium IAA, IBA, NAA (0.1-1.5 mg/L). The maximum number of roots (51.0$\pm$2.6) was achieved on the medium containing IBA (1.0 mg/L) followed by other auxins (NAA, IAA). The regenerated plants were successfully transferred to a mixture of vermiculate and soil. About 95% of the plantlets survived when transferred to the field.

• 한국작물학회지
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• 제59권4호
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• pp.498-504
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• 2014

Potato virus Y (PVY) and potato leafroll virus (PLRV) are among the most damaging potato viruses and prevalent in most potato growing areas. In this study, cryopreservation was used to eradicate PVY and PLRV using two cryogenic methods. Potato shoot tips proliferated in vitro were cryopreserved through droplet-vitrification and encapsulation-vitrification using plant vitrification solution 2 (PVS2; 30% glycerol + 15% dimethyl sulfoxide + 15.0% ethylene glycol + 13.7% sucrose) and modified PVS2. Both cryogenic procedures produced similar rates of survival and regrowth, which were lower than those from shoot tip culture alone. The health status of plantlets regenerated from shoot tip culture alone and cryopreservation was checked by reverse transcription-polymerase chain reaction. The frequency of virus-free plants regenerated directly from highly proliferating shoot tips reached 42.3% and 48.6% for PVY and PLRV, respectively. In comparison, the frequency of PVY and PLRV eradication after cryopreservation was 91.3~99.7% following shoot-tip culture. The highest cryopreserved shoot tip regeneration rate was observed when shoot tips were 1.0~1.5 mm in length, but virus eradication rates were very similar (96.4~99.7%), regardless of shoot tip size. This efficient cryotherapy protocol developed to eliminate viruses can also be used to prepare potato material for safe long-term preservation and the production of virus-free plants.

• Journal of Plant Biotechnology
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• 제7권3호
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• pp.175-181
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• 2005

This work described some essential factors necessary for micro-propagation of banana for mass production of synthetic seeds for germ plasm conservation, and how peroxides activity of conserved tissue was influenced. Shoot tips of field grown plants were used to obtain shoot clusters on shoot proliferation medium (MS medium supplemented with 5 mg/l BAP). Using longitudinally-split shoot tip technique, 18720, 8640, 7488, 2016 plantlets were obtained from one shoot tip of Maghraby, Grand Naine, Balady, and Williams, respectively, in six subculture, one month each, on solid medium. Shoot tips excised from in vitro grown plantlets were encapsulated in calcium-alginate beads and stored at $4^{\circ}C$ for one month on half-strength MS basal medium without growth regulators or sugars. After one month all the viable-conserved synseeds formed shoots when they were transferred to MS basal medium, some of them showed synchronous formation of shoot and root systems in one week. Plants retrieved from encapsulated shoot tips were hardened off and transferred to soil.

• 한국자원식물학회지
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• 제32권6호
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• pp.689-697
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• 2019

This study describes an efficient and widely applicable droplet-vitrification following cryopreservation for shoot tips of strawberry (Fragaria × ananassa Duch.) cvs. 'Wonkyo3114' and 'Gurumi40'. The shoot tips were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.5M). Precultured explants were osmoprotected with loading solution (LS, C4) containing 20% glycerol and 20% sucrose for 40 min and exposed to dehydration solution (B5) containing 40% glycerol and 40% sucrose for 40 min at 25℃, 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 MS + 0.3M sucrose for 40 h at 25℃. The cryopreserved shoots tips exhibited 55% regrowth rate by culturing in NH₄NO₃-free MS medium supplemented with 3% sucrose, 1.0 g/L casein, 1.0 mg/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. This result shows that droplet-vitrification could be employed as a promising method for cryostorage of strawberry germplasm.

• 식물조직배양학회지
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• 제21권3호
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• pp.161-166
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• 1994

본 연구는 마늘 무병우량종구의 효율적인 생산, 증식 및 보급체계를 확립하기 위한 기초 자료를 얻기 위하여 한지형 서산종 마늘의 생장점 배양시 기관분화에 미치는 배양 온도, 배양시기, 생장조절물질 및 저온처리 영향을 검토하기 위하여 실시하였다. 동절기의 생장점 배양은 28/2$0^{\circ}C$의 변온 조건에서 배양하는 것보다 24$^{\circ}C$ 항온조건에서 배양하는 것이 생존율 및 shoot 형성과 생장이 양호하였다. 마늘인편 휴면기인 7, 8월에는 kinetin 2 mg/L와 NAA 0.1 mg/L을 첨가한 배지에서 생존율 및 shoot의 생장이 양호한 반면에, GA$_3$ 1 mg/L 첨가한 배지는 억제되었다. 또한 휴면기에는 생장점 배양 전 종구를 15-60일간 4$^{\circ}C$에서 저온처리 함으로서 shoot 생장 및 발근을 촉진시켰으며, 또한 구형성 배지에 계대배양시 저온처리기간이 증가할수록 기내인경구 형성 및 비대를 촉진하였다.