Cryopreservation of Viola cornuta shoot tips using vitrification procedure

Cryopreservation represents a suitable method for long term storage of different plant genetic resources. The aim of this study was to develop protocol for cryopreservation of Viola cornuta shoot tips using one step freezing method with chemical dehydration of tissue with modified Plant Vitrification Solutions (PVS2 or PVS3). Shoot tips (1-2 mm) of two-week cold acclimated shoots were cultured on ½MS medium with 0.3 M sucrose for one day before treatment with loading solution (2 M glycerol, 0.4 M sucrose) for 30 min. Osmotic dehydration with PVS2 solution (30% glycerol, 15% ethylene glycol and 15% DMSO in liquid ½MS medium with 0.4 M sucrose) were tested at 0 °C or 24 °C. Osmotic dehydration with PVS3 (50% sucrose, 50% glycerol in liquid ½MS medium) were tested at 24 °C for 45 min. After the treatment the explants were directly immersed in liquid nitrogen (LN) for at least one day. Re-warming was performed at 42 °C in water bath for 2 min. After re-warming, the PVS solutions were replaced with unloading solution containing 1.2 M sucrose for 20 min. Re-warmed shoot tips were cultured on ½MS medium with 0.1 mg L-1 BAP. We observed that PVS2 solution is cytotoxic for V. cornuta shoot tips and cannot be used for cryopreservation. However, cryopreservation with PVS3 solution was successful, where 71.9-100% shoot tips survived treatment before immersion to LN and 31-40% survived after re-warming from LN. Regrowth of cryopreserved shoot tips with new well-formed leaves was obtained after four weeks of culture.