Cryopreservation of Blueberry Dormant Shoot Tips Using V Cryo-plate Method

Cryopreservation of dormant buds of blueberry (Vaccinium virgatum) at -196^oC using V cryo-plate method was investigated. Blueberry was mainly used as the experimental material. Dormant twigs of blueberry were collected from Shimane University orchard in winter and stored at 2°C. After the surface sterilization, shoot tips about 1 mm size were dissected from the dormant buds and precultured overnight at 25°C on solidified 1/4 MS medium containing 0.3 M sucrose. Precultured shoot tips were embedded in calcium alginate gel on aluminium cryo-plates. Osmoprotection treatment for the shoot tips was performed by immersing the cryo-plates for 30 min at 25°C in a LS solution (2 M glycerol plus 0.6 M sucrose in 1/2MS). Then the shoot tips on aluminium cryo-plates were treated with PVS2 solution for different duration 25^oC, cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, cryo-plates were immersed in 1/2MS with 1.0 M sucrose for 20 min at room temperature. The highest survival (84%) after cryopreservation was obtained in shoot tips dehydrated for 30 min by PVS2. The V cryo-plate procedure was applied to 10 additional cultivars of blueberry.  The V cryo-plate procedure may facilitate cryostorage of blueberry germplasm.

This work was supported by a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics-based Technology for Agricultural Improvement, CRS-1001).