58. Development of alternative plant vitrification solutions and loading solutions in droplet-vitrification procedures

Kim, Haeng-Hoon; No, Na-Young; Shin, Donghyun; Baek, Hyung-Jin; Kang, Jung-Hoon; Kim, Jung-Gon; Cho, Eun-Gi

doi:10.1016/j.cryobiol.2009.10.072

Cited by 30 publications

(38 citation statements)

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“…Recovery was significantly higher with DV, in comparison with the ED technique. The current results might be improved by optimizing various steps of the DV protocol, including conditioning in vitro plants or microcuttings on medium enriched with growth regulators such as BAP or zeatine riboside [27], or using alternative loading and vitrification solutions [28,29]. Before its large scale application can be envisaged, the optimized protocol should be validated through its application to a range of grapevine cultivars.…”

Section: Discussionmentioning

confidence: 99%

Cryopreservation of grapevine (Vitis vinifera L.) in vitro shoot tips

Marković¹,

Chatelet

Sylvestre

et al. 2013

Open Life Sciences

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In this work, we compared the efficiency of encapsulation-dehydration and droplet-vitrification techniques for cryopreserving grapevine (Vitis vinifera L.) cv. Portan shoot tips. Recovery of cryopreserved samples was achieved with both techniques; however, droplet-vitrification, which was used for the first time with grapevine shoot tips, produced higher regrowth. With encapsulationdehydration, encapsulated shoot tips were precultured in liquid medium with progressively increasing sucrose concentrations over a 2-day period (12 h in medium with 0.25, 0.5, 0.75 and 1.0 M sucrose), then dehydrated to 22.28% moisture content (fresh weight). After liquid nitrogen exposure 37.1% regrowth was achieved using 1 mm-long shoot tips and only 16.0% with 2 mm-long shoot tips. With droplet-vitrification, 50% regrowth was obtained following treatment of shoot tips with a loading solution containing 2 M glycerol + 0.4 M sucrose for 20 min, dehydration with half-strength PVS2 vitrification solution (30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% dimethylsulfoxide and 0.4 M sucrose in basal medium) at room temperature, then with full strength PVS2 solution at 0°C for 50 min before direct immersion in liquid nitrogen. No regrowth was achieved after cryopreservation when shoot tips were dehydrated with PVS3 vitrification solution (50% (w/v) glycerol and 50% (w/v) sucrose in basal medium).

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Section: Discussionmentioning

confidence: 99%

Cryopreservation of grapevine (Vitis vinifera L.) in vitro shoot tips

Marković¹,

Chatelet

Sylvestre

et al. 2013

Open Life Sciences

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“…Also, Hirai and Sakai (2003) showed that in sweet potato cryopreservation, both a higher concentration of sucrose (1.6 M) in the LS and a longer period of osmoprotection (3 h at 25°C) were necessary to increase the osmotolerance. Kim et al (2009) indicated for developing a new LS solution for the droplet vitrification procedure that the loading treatment may act as an osmotic stress neutralizer and/or induce a physiological adaptation of tissues and cells prior to both dehydration and vitrification. Also, they pointed out that appropriate LS should be selected for plant species which are highly sensitive to the cryotoxicity of the PVS solutions.…”

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confidence: 99%

“…transferred onto aluminum strips with a droplet of PVS2 or PVS3 just before immersion into LN, which is cumbersome (Kim et al 2009). The V-Cryo-plate method can overcome these disadvantages since all treatments can be carried out only by moving and transferring the cryo-plate (with attached shoot tips) from one solution to another.…”

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confidence: 99%

Cryopreservation of in vitro-grown shoot tips of carnation (Dianthus caryophyllus L.) by vitrification method using aluminium cryo-plates

Sekizawa

Yamamoto

Rafique

et al. 2011

Plant Biotechnology

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Cryopreservation using an aluminum cryo-plate was successfully applied to in vitro-grown carnation (Dianthus caryophyllus L.) shoot tips. The shoot tips (1-1.5 mmϫ1 mm) were dissected from the shoot and precultured at 25°C for 2 days on MS medium containing 0.3 M sucrose. The precultured shoot tips were placed on the aluminum cryo-plate containing ten wells embedded with alginate gel. Osmoprotection was performed by immersing the cryo-plates in loading solution (2 M glycerol and 1.4 M sucrose) for 90 min at 25°C. Then, dehydration was performed by immersing the cryoplates in PVS2 for 25 min at 25°C. After storage in liquid nitrogen, shoot tips attached to cryoplate, were directly immersed into 2 ml 1 M sucrose solution for regeneration. Using this procedure, the average regrowth level of vitrified shoot tips of 4 carnation cultivars reached 95%. This new method has many advantages and will facilitate the cryostorage of reference cultivars of carnation. (LS) and PVS2 without floating and/or clinging of shoot tips and also with no or low level of injury to those shoot tips. In this paper, this V-Cryo-plate procedure was applied for the cryopreservation of carnation.The carnation plants used in this study were obtained from the Nishinihon Station, NCSS, sub-bank of NIAS Genebank Project, in Japan in April 2010. Four cultivars of carnation, 'Melissa', 'Grana', 'Terra Cotta' and 'Yell', were tested. Apical shoots, excised from in vivo explants, were surface-sterilized successively with 70% ethanol for 1 min, and 0.5% sodium hypochlorite for 10 min, and then rinsed twice with sterilized distilled water. After surface sterilization, shoot tips with basal plates (1 mm longϫ1 mm wide) were excised from the shoots and cultured on 70 ml of 0.4% (w/v) gellan gum-solidified MS medium (Murashige, Skoog 1962) in a culture flask (80 mmϫ100 mm) containing 1 mg l Ϫ1 thidiazuron, 1 mg l Ϫ1 naphthalene acetic acid and 3% (w/v) sucrose (Jose et al. 2010). After 50 days of culture, shoots were transferred onto gellan gum-solidified MS medium devoid of plant growth regulators. Then stock plants were subcultured every two months on MS medium. Cultures were incubated at 25°C with a 16 h photoperiod under white fluorescent light (52 mmol m Ϫ2 s Ϫ1) in the culture flask (standard condition). For cryopreservation of in vitro shoot tips of carnation, the V-Cryo-plate procedure was applied. The size of the aluminum cryoplate used was 7 mmϫ37 mmϫ0.5 mm with ten wells (diameter 1.5 mm, depth 0.75 mm) ( Figure 1B). These plates fit in 2 ml cryotube. The different steps of the VCryo-plate procedure are as follows; 1) Cut shoots (5 mm) with a lateral bud and plate on solidified MS medium and culture for 2 weeks at 25°C in standard conditions ( Figure 1A). Then, dissect shoot tips with basal plate (1-1.5 mm longϫ1 mm wide) from the shoots and preculture for 2 days at 25°C on the MS medium with 0.3 M sucrose. 2) Place an aluminum cryo-plate in Petri-dish and pour 2.0-2.5 ml 2% (w/v) Na-alginate solution with 0.4 M sucrose in calcium-free M...

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“…The cryopreservation experiments were performed using 1-2 mm long apical shoot tips excised from in vitro shoots, which were maintained on the MS multiplication medium for 3 weeks. The explants taken were cryopreserved using the optimized droplet vitrification protocol (Vujović et al 2015): (1) pre-treatment of explants in the liquid MS medium with BA 1, IBA 0,1 and GA 3 0,1 mg l -1 and a progressively increasing sucrose concentration (0.3 M for 15 h and 0.7 M for 5 h), in the dark at 23 °C; (2) loading treatment with the hormone-free liquid MS medium comprising 1.9 M glycerol and 0.5 M sucrose (C4 solution; Kim et al 2009a), 30 min at room temperature; (3) dehydration with PVS3 [50% glycerol (w/v) and 50% (w/v) sucrose; Nishizawa et al 1993] 40 min at room temperature and the modified PVS2 solution [PVS A3 -37.5% (w/v) glycerol, 15% (w/v) dimethylsulfoxide, 15% (w/v) ethylene glycol and 22.5% (w/v) sucrose; Kim et al 2009b] 40 min on ice; (4) transfer of explants in 10 µ droplets of vitrification solution attached to aluminium foil strips; (5) direct plunging of aluminium foil strips in LN where they were kept for 2, 4, 8 and 24 h; (6) rapid warming by direct plunging of aluminium foils in a preheated (37°C) unloading solution (0.8 M sucrose) for 30 s, and further 30 min-incubation in the equal volume of unloading solution at room temperature. The following adequate controls were included for each step of the procedure: (1) pre-growth control refers to shoot tips just pre-treated in the liquid MS medium and directly placed on the growth medium; (2) loading controls were loaded but both non-dehydrated and non-cryopreserved; (3) dehydration controls were treated in the same way as the cryopreserved explants but without the LN immersion.…”

Section: Methodsmentioning

confidence: 99%