These methods are largely based on those originally developed by Uragami (9) and Sakai (11) and has since been adapted extensively by Kim et al. (17) and Thin et al. (18) to produce different derivative methodologies. These use different combinations of cryoprotectants applied at higher concentrations than would be the case for standard cryoprotection. They also include protocol permutations that incorporate pregrowth and dehydration treatments that assist the recovery of more sensitive genotypes (11). The basic protocols are presented with some alternative options highlighted.
1. Prepare PVS2 (see Subheading 2.5.; see Note 22).
2. Prepare a range of diluted PVS2 solutions (e.g., 10-80% [v/v] PVS2 solutions in standard liquid medium).
3. Prepare a solution of standard liquid medium containing 1.2 M sucrose (see Note 23).
PVS3 is an alternative solution comprising 50% (w/v) sucrose and 50% (w/v) glycerol prepared in standard liquid culture medium (8).
3.6.2. Basic Vitrification Procedure
Prepare shoot tips (see Subheading 3.3.) and construct a toxicity test (see Notes 23 and 24) for the PVS2 solutions as follows:
1. Add on ice, chilled 1-mL aliquots of PVS2 within the range of 50-80% (v/v) to a specific concentration for 1-10 min and remove almost all the vitrification solution (see Note 25).
2. Replace the intermediate concentrations with a stepwise, higher concentration of PVS2 solution (see Note 6).
3. Gradually increase the concentration of PVS2 until the tissues are in the 100% solution (see Note 24).
4. Remove the 100% PVS2 and replace with "unloading" solution containing 1.2 M sucrose in standard liquid medium. Perform two to three washes in fresh unloading solution and maintain in this solution for up to 30 min.
5. Expel vial contents onto a filter paper; the paper will soak away the liquid excess.
6. Transfer the shoots to recovery medium and select the treatment that permits maximum survival and cryoprotection.
7. After the stepwise delivery of PVS2 has been optimized, transfer the vials directly to liquid nitrogen.
8. For rewarming, place vials in a water bath at 45°C and recover shoot tips as described in steps 4-5 of this protocol.
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