Exogenous inorganic ions, partial dehydration, and high rewarming temperatures improve peach palm (Bactris gasipaes Kunth) embryogenic cluster post-vitrification regrowth

Previous studies on the vitrification of peach palm (Bactris gasipaes Kunth) embryogenic cluster highlighted PVS3 as essential for survival, but toxic nonetheless. The objective of this work was to identify methods to improve embryogenic cluster survival through either modification to PVS3 or other changes to the established protocol. PVS3 reduced to 80% strength was less toxic to non-vitrified controls and showed no significant difference in vitrified embryogenic cluster at any incubation time (60, 120, 180, or 240 min) compared to full strength PVS3. The addition of 0.1 M KCl, MgCl2, MgSO4, or K2H(PO4) to 80% PVS3 increased vitrified embryogenic cluster regrowth. D2O had no significant effect on embryogenic cluster regrowth when used as a solvent for PVS3 compared to H2O. Larger embryogenic cluster showed greater regrowth than smaller ones. Regrowth increased with rising rewarming temperature up to 70 °C, the highest tested temperature. The duration of rewarming between 1, 2, or 3 min had no overall significant effect, suggesting that the most critical events in rewarming occur within the first minute. The most effective strips used for droplet vitrification were made from aluminum or silver, which were significantly more effective than polystyrene and expanded polystyrene. Droplet vitrification was initially more effective than conventional cryovial immersion vitrification at 60 min incubation in PVS3, but not significantly different at 120, 180 or 240 min incubations. Partial dehydration for 1 h in a laminar flow hood significantly increased regrowth, but longer dehydration times decreased regrowth when combines with longer PVS3 incubation times.