Droplet vitrification cryopreservation of Rosa canina and Rosa rubiginosa using shoot tips from in situ plants

• Rosa canina and Rosa rubiginosa meristems collected from in situ plants were cryopreserved.
• Droplet vitrification method using LS and PVS2 was applied.
• After rewarming the in vitro cultures were establish successfully for meristems originated from dormant buds.
• Developed protocol provides high survival rate and satisfactory regeneration after cryopreservation.

In this study two wild Rosa species in vivo shoot tips were successfully cryopreserved using the droplet vitrification technique. The growth recovery of cryopreserved shoot tips was satisfactory when the explants were isolated from the lateral buds in situ plants at the end of the winter dormancy period (February) and following the disinfection of the buds surface with 70% ethanol. The optimal condition included treatment for 20 min in the loading solution (2 M glycerol and 0.4 M sucrose), treatment with plant vitrification solution (PVS2) containing 30% glycerol, 15% ethylene glycol, 15% DMSO and 0.4 M sucrose for 20 min (Rosa canina) or 30 min (Rosa rubiginosa). A rapid freezing was conducted on strips of aluminium and, after cryopreservation, followed by a rapid, immersed re-warming for 20 min in the recovery solution, which contained 1.2 M sucrose and, additionally, 0.2% of sodium hypochlorite. Post-freezing regeneration was performed by the in vitro culture on MS media with 5 μM BA, 1.5 μM and 0.087 M sucrose. Under these conditions, 93.5–96% of the shoot tips survived, and over 83.3–86.7% of these regenerated axillary shoots. This procedure facilitates the routine use of cryopreservation of rose in vivo shoot tips and eliminates the maintenance of in vitro cultures as a source of explants for cryopreservation.