An efficient droplet-vitrification cryopreservation for valuable blueberry germplasm

Abstract

We report a droplet-vitrification for efficient cryopreservation of valuable germplasm of blueberry. In this protocol, in vitro stock shoots were cold-hardened in the dark at 4°C for 3 weeks. Shoot tips (1.5–2.0mm in length) including 4–5 leaf primordia were excised from the cold-hardened stock shoots and precultured with 0.3M sucrose for 1day. Precultured shoot tips were treated with a loading solution containing 1.0M sucrose and 2M glycerol for 30min, followed by exposure to PVS2 for 40min at 0°C. Dehydrated shoot tips were then transferred onto 3.5μL PVS2 droplets on aluminum foil strips, prior to a direct immersion into liquid nitrogen for 1h. Frozen foils with shoot tips were re-warmed in 1.2M sucrose for 20min at room temperature, followed by post-thaw culture for a recovery medium containing 0.3mgL−1 zeatin for shoot regrowth. With optimized parameters, shoot regrowth levels were 46.7% for ‘O’ Neal’ and 91.7% for ‘Misty’, with a mean shoot regrowth level of 66.5% obtained across the five valuable blueberry genotypes tested. Histological observations showed that many cells in the upper part of apical dome and some in leaf primordia 1–3 survived, while those in lower part of apical dome and leaf primordia 4–5 were seriously damaged, following cryopreservation. No polymorphic bands were detected using inter-simple sequence repeat markers (ISSR) and random amplified polymorphic DNA (RAPD) in regenerants of ‘O’ Neal’ and ‘Misty’ recovered from cryopreservation. To the best our knowledge, this is the first report on blueberry cryopreservation by droplet-vitrification, which would provide a technical platform for establishment of cryo-banks of valuable blueberry germplasm.