Cryopreservation of highbush blueberry, strawberry, and saskatoon using V and D cryo-plate methods and monitoring of multiplication ability of regenerated shoots

The purpose of this work was to apply V and D cryo-plate methods for cryopreservation of Vaccinium corymbosum ?Toro?, Fragaria?×?ananassa ?Clery?, and Amelanchier alnifolia (Nutt.)M. Roem. and to monitor the multiplication capacity of shoots regenerated from cryopreserved explants. Shoot tips pre-cultured for 1 d at 23°C in the dark on medium containing 0.3 M sucrose were used as explants. Loading was performed in a solution containing 2 M glycerol and 0.8 M sucrose (30 min at room temperature). In the V cryo-plate, dehydration was carried out at room temperature (20 to 50 min)using the following plant vitrification solutions: original PVS2, 90 percent PVS2 solution, and PVS3. Regarding the D cryo-plate, dehydration was performed in closed glass containers over silica gel for 2, 2.5, or 3 h. In both protocols, rewarming was carried out in a 1.0 M sucrose solution (15 min at 25°C). Regenerated shoots were multiplied and multiplication parameters were monitored after the second subculture. Using the V cryo-plate method, the highest regrowth in highbush blueberry was obtained following 50-min treatment with all three VSs (61.7 to 80.9 percent). The D cryo-plate method was even more suitable with maximum regrowth of 89.4 percent achieved after 2.5 h of desiccation. For strawberry, 62.5 percent was the highest regrowth recorded using PVS3-based V cryo-plate method while 83.3 percent of regrowth was observed using D cryo-plate protocol. Regrowth of saskatoon reached a maximum of 50 percent after 50-min treatment with PVS3 while it did not exceed 40 percent in other treatments. By the second subculture, shoots regenerated from cryopreserved explants regained and even exceeded the multiplication capacity of shoots regenerated from non-cryopreserved explants. This study is the first to present the successful application of the V cryo-plate method in highbush blueberry, as well as the utilization of both V and D cryo-plate methods in saskatoon.