Preculture Condition for Stable Recovery of Strawberry (Fragaria × ananassa Duch.) Shoot Tips after Cryopreservation Using Droplet Vitrification

This study describes an efficient and stable droplet vitrification following cryopreservation of strawberry shoot tip (Fragaria × ananassa Duch.) accessions 'Massey' and 'MDUS3816'. The shoot tips were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.7M). Precultured explants were osmoprotected with loading solution (LS, C4) containing 17.5% glycerol and 17.5% sucrose for 40 min and exposed to dehydration solution (B1) containing 50% glycerol and 50% sucrose for 40 min at 25oC. Subsequently, the explants were transferred onto droplets containing 2.5 µL PVS3 on sterilized aluminum foils (4 cm× 0.5 cm) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regrowth rate (%) in both the cultivars was obtained when the shoot tips were precultured with 0.3M sucrose for 30 h + 0.5M sucrose for 16 h at 25oC. The cryopreserved shoots tips exhibited 57.8 % recovery rate by culturing in NH₄NO₃-free MS medium supplemented with 3% sucrose, 1.0 g/L casein, 1.0mg/L GA₃, and 0.5 mg/L BA for 5 weeks and in MS medium supplemented with 0.5 mg/L GA₃ for 8 weeks. Variation was not observed in both of ploidy analysis and morphological investigation on plantlets of two accessions cryopreserved under variable preculture conditions.