Background: The application of a vacuum during the incubation in cryoprotective agents such as PVS2 allows for increased penetration, reducing total incubation times required before vitrification and post-cryopreservation regeneration is achieved.
Objective: This study compared a conventional droplet-vitrification protocol to the new vacuum infiltration vitrification protocol in four Australian plant species.
Materials and methods: The new vacuum infiltration vitrification applied an 80 kPa vacuum during incubations in loading solution and PVS2. Infiltration of the cryoprotective agents into shoot tips was determined by differential scanning calorimetry measuring ice formation in the thermographs comparing a range of loading solution and PVS2 incubation times.
Results and conclusion: The application of the vacuum infiltration vitrification technique resulted in a significantly reduced PVS2 incubation time for cryogenic survival and regeneration for all four species, reducing the time needed to adequately protect shoot tips by half to a quarter when compared to a conventional droplet-vitrification technique.