Modified droplet-vitrification cryopreservation of arctic bramble (Rubus arcticus) and hybrid arctic bramble

The goal of this study was to test a cryopreservation method for arctic bramble (Rubus arcticus ssp. arcticus) and hybrid arctic bramble (Rubus arcticus ssp. arcticus x Rubus arcticus ssp. stellatus) and to proceed to the long-term cryopreservation of the mandate arctic brambles collections at Natural Resources Institute Finland (Luke). The droplet-vitrification cryopreservation method has been successfully modified and applied at Luke for long-term cryopreservation of in vitro grown shoot tips of strawberries and lateral buds of raspberries. Pretreatments were tested within the in vitro cultures of the Finnish arctic brambles 'Pima' and 'Susanna' and the Swedish hybrid arctic brambles 'Beata' and 'Sofia'. Cold acclimatization pretreatment was implemented, after which shoot tips were excised for long-term cryopreservation using modified droplet-vitrification cryopreservation method. Four different factors including the in vitro growth media of last subculture interval, the duration of the cold pretreatment, the sucrose pretreatment before freezing and the plant vitrification solution 2 (PVS2) dehydration were tested. The highest regeneration rates were obtained by one week cold pretreatment for in vitro cultures grown on fructose-based medium and by increasing sucrose concentrations from 0.25 to 0.5 M for meristems before PVS2 dehydration for 30 min and freezing. As a result, the cultured buds from cryopreserved treatment showed regeneration into well dividing in vitro cultures. As a conclusion, this cryopreservation method was shown applicable for cryopreservation of arctic brambles and long-term cryopreservation for two clonal lines for each cultivar with two repetitions was performed.