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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 (1.5–2.0 mm in length) including 4–5 leaf primordia were excised from the cold-hardened stock shoots and precultured with 0.3 M sucrose for 1 day. Precultured shoot tips were treated with a loading solution containing 1.0 M sucrose and 2 M glycerol for 30 min, followed by exposure to PVS2 for 40 min at 0 °C. Dehydrated shoot tips were then transferred onto 3.5 μL PVS2 droplets on aluminum foil strips, prior to a direct immersion into liquid nitrogen for 1 h. Frozen foils with shoot tips were re-warmed in 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for a recovery medium containing 0.3 mg L −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. [ABSTRACT FROM AUTHOR]

A droplet-vitrification procedure is described for cryopreservation of Malus shoot tips. Survival patterns, recovery types, histological observations, and genetic integrity were compared for Malus shoot tips cryopreserved using this droplet-vitrification procedure and an encapsulation-dehydration procedure that was previously reported by us. In both procedures, three types of shoot tip recovery were observed following cryopreservation: callus formation without shoot regrowth, leaf formation without shoot regrowth, and shoot regrowth. Three categories of histological observations were also identified in cross-sections of shoot tips recovered after cryopreservation using the two cryogenic procedures. In category 1, almost all of the cells (94–95%) in the apical dome (AD) were damaged or killed and only some cells (30–32%) in the leaf primordia (LPs) survived. In category 2, only a few cells (18–20%) in the AD and some cells (30–31%) in the LPs survived. In category 3, majority of the cells (60–62%) in the AD and some cells (30–33%) in the LPs survived. These data suggest that shoot regrowth is correlated to the presence of a majority of surviving cells in the AD after liquid nitrogen exposure. No polymorphic bands were detected by inter-simple sequence repeats or by random amplified polymorphic DNA assessments, and ploidy levels analyzed by flow cytometry were unchanged when plants recovered after cryoexposure were compared to controls. The droplet-vitrification procedure appears to be robust since seven genotypes representing four Malus species and one hybrid recovered shoots following cryopreservation. Mean shoot regrowth levels of these seven genotypes were 48% in the droplet-vitrification method, which were lower than those (61%) in the encapsulation-dehydration procedure reported in our previous study, suggesting the latter may be preferred for routine cryobanking applications for Malus shoot tips. [ABSTRACT FROM AUTHOR]