Development of Alternative Plant Vitrification Solutions to Be Used in Droplet-Vitrification Procedures

Cryopreservation of Coffea arabica L. Zygotic Embryos by Vitrification

“…At higher temperatures, the vitrifying solution (PVS2) has a higher penetration capacity in tissues (Yi et al, 2012). Conversely, at lower temperatures, solution mobility is reduced, decreasing the penetration rate through the plasma membrane, preventing the toxic effect and excessive explant dehydration (Kim et al, 2009).…”

Effect of the Duration of Liquid Nitrogen Storage on the Regrowth of Blackberry Cryopreserved by Droplet Vitrification

“…The regrowth rates of cryopreserved shoot tips achieved after the PVS A3 dehydration for 40 min on ice did not notably differ from the previous results (61.8%). Although the most commonly employed vitrification solution for cryopreservation of plant tissues is PVS2, it is very toxic for most plant species, thus the dehydration duration has to be determined very precisely (Kim et al 2009b). Sample dehydration at 0 °C mitigates the cytotoxicity of this vitrification solution.…”

“…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.…”

Cryopreservation of cherry rootstock Gisela 5 using vitrification procedure

“…After the loading treatment, the loading solution was replaced with 2 ml of filter sterilised vitrification solution. Vitrification (dehydration) was performed on ice (0 o C) using 2 types of vitrification solutions: PVS2 vitrification solution (Sakai et al 1990) (liquid, hormone-free MS medium containing in w/v, 13.7% sucrose, 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide -DMSO) and modified PVS2 vitrification solution -PVS A3 solution (Kim et al 2009) (liquid, hormone-free MS medium containing in w/v: 22.5% sucrose, 37.5% glycerol, 15% ethylene glycol and 15% DMSO). The following durations of dehydration were used for both types of vitrification solutions: 30, 40 and 50 minutes.…”