Efficient cryopreservation by droplet vitrification of pentaploid roses and the phenotype of regenerated plants

Shoot tips from in vitro plants of four rose species were cryopreserved by the droplet vitrification method. Optimized conditions involved exposure to loading solution for 20 min, then treatment with plant vitrification solution (PVS2) for 20 min (Rosa agrestis, R. canina and R. dumalis) or 30 min (R. rubiginosa) followed by freezing in liquid nitrogen. Survival rate ranged from 78.3 to 95.1%, depending on the species. Regrowth rate of shoot tips was 50.5% for R. agrestis, 63.2% for R. rubiginosa, 71.4% for R. dumalis and 78% for R. canina. The preculture of donor plants in a medium with 0.25 µM sucrose facilitated the isolation of shoot tips and increased regrowth rate after cryopreservation. Plant regeneration was carried out in Murashige and Skoog medium with 1 µM 6-benzylaminopurine, 1.5 µM gibberellic acid and 0.087 M sucrose. Plants regenerated from cryopreserved shoot tips did not display morphological alterations in comparison with non-cryopreserved shoot tip – derived plants