1	Cryopreservation using Vitrification with PVS2 Barbara M. Reed USDA-ARS National Clonal Germplasm Repository Corvallis, Oregon CRIS 5358-21000-033-00D
2	PVS2 Vitrification There are many variations of the PVS2 vitrification technique developed by A. Sakai, et al., 1990 This technique requires more careful timing than some others but is faster Only a few samples can be processed at one time Recipes are given at the end of the presentation
3	Basic Steps In-vitro cultured plants (3 weeks from last transfer) Cold-acclimate plantlets for 2-4 weeks with alternating temperatures (8 hr 22°C and 16 hr -1°C) Dissect 1 mm shoot tips and pregrow for 16-48 hr Pretreat in solutions Add PVS2 cryoprotectant to shoot tips Hold in PVS2 Plunge in LN Thaw 1 min in 45 °C water and 2 min in 25°C water Rinse with 1.2 M sucrose liquid MS medium and plate on recovery medium
4	In-vitro cultured plants (3 weeks from last transfer) are placed in cold acclimation for 2-4 weeks
5	Dissect 0.8 mm shoot tips and culture on medium with 5% DMSO and 1 g/L more agar than normal medium
6	Pretreat before PVS2 addition Pretreat in Presoak in 1% Bovine Serum albumen or 10% proline for 2 hour (each in O.4 M sucrose MS medium) Or Presoak in 2M glycerol and 0.4 M sucrose in MS for 20 min.
7	Place cryotubes in a tray and add PVS2 cryoprotectant Add shoot tips and hold for 15-60 min. Close caps and plunge in liquid nitrogen.
8	Thaw quickly in 45°C water for 1 min then move to 25°C water for 2 min
9	Drain off PVS2 and add 1.2M sucrose liquid MS medium. Drain on filter paper and plate on recovery medium in multi-cell plates or petri dishes
10	Conclusions This method is easy to do and successful for many types of plants Be sure to include controls for the dissection and cryoprotectant steps PVS2 is very phytotoxic so cryoprotectant must be carefully timed and controls must be rinsed immediately and plated. Shoots will begin to develop into plantlets in 2-3 weeks
11	Basic Steps for NCGR Procedure (Luo and Reed, 1997, Modified from Yamada et al., 1991) In-vitro cultured plants (3 weeks from last transfer) Cold-acclimate plantlets for 2-4 weeks with alternating temperatures (8 hr 22°C and 16 hr -1°C) Dissect 1 mm shoot tips and grow for 48 hr on 5% DMSO plates in cold acclimation conditions Pretreat in A) Presoak in 1% Bovine Serum albumen or 10% proline for 2 hour in O.4 M sucrose MS medium Or B) presoak in 2M glycerol and 0.4 M sucrose in MS for 20 min. Add PVS2 cryoprotectant to shoot tips Hold in PVS2 for 20 min Plunge in LN Thaw 1 min in 45 °C water and 2 min in 25°C water Rinse with 1.2 M sucrose liquid MS medium and plate on recovery medium
12	Basic Steps for Matsumoto Procedure Vitrification technique for tropical meristems (Matsumoto et al., 1998) In-vitro cultured plants (3 weeks from last transfer) Preculture meristems on ½ MS medium with 0.3 M. sucrose for 16 hr at 20 °C. Loading solution (2 M glycerol + 0.4 M sucrose) for 20 min at 25 °C Add PVS2 for 10 min at 25 °C. LN Rapid warming in 40 °C water Loading solution (2 M glycerol + 0.4 M sucrose) for 20 min at 25 °C. Plate on ½ MS medium with 0.1 mgL-1 BA. Use a filter paper between the meristem and the medium and transfer to new medium after one day by moving the filter paper.
13	Two and Three Step Vitrification T. Matsumoto and A. Sakai. 2003 In-vitro cultured plants (3 weeks from last transfer) Preculture meristems on ½ MS medium with 0.3 M. sucrose for 3 days at 25 °C. Loading solution (2 M glycerol + 0.4 M sucrose) for 20 min at 25 °C. PVS2 for 80 min at 0 °C. (2 step procedure) or 50% PVS2 for 30 min at 0 °C then full strength PVS2 for 50 min (3 step procedure) LN Rapid warming in 40 °C water 1.2 M sucrose MS for 20 min at 25 °C. Plate on ½ MS medium with 1 mgL-1 BA and 3% sucrose. Use a filter paper between the meristem and the medium and transfer to new medium after one day by moving the filter paper.
14	Alternate Method Combining Alginate and Vitrification From Sakai et al 1998 Make beads and do sucrose preculture as in encapsulation-dehydration procedure (Chapter 4) Place beads in loading solution for 1 hr at 25 °C (2 M glycerol + 0.4 M sucrose). Remove loading solution and add PVS2 for 2 hr at 0 °C Immerse in LN Thaw at room temperature Rehydrate in 2 M glycerol + 0.4 M sucrose and plate.
15	Appendix A DMSO Pretreatment Medium
16	Appendix B PVS2 Cryoprotectant