Cryopreservation of Strawberry Meristems and Mass Propagation of Plantlets1

Abstract

Meristems isolated from strawberry (Fragaria X ananassa Duch. cv. Redcoat) runner-tips were cultured on a Murashige and Skoog (MS) medium supplemented with 6-benzylamino purine (BA), indolebutyric acid (IBA) and gibberellic acid (GA₃) at concentrations of 1, 1 and 0.1 μm, respectively. The meristems regenerated shoots within 3 to 4 weeks when incubated at 26°, 16 hour photoperiods and 4000 lux intensity. On medium supplemented with BA at a concentration of 10 μm these shoots proliferated into “clumps,” each consisting of 150 to 200 shoots. Meristems isolated from these in vitro shoots were precultured on medium supplemented with dimethylsulfoxide (DMSO) or glycerol as cryoprotectants, frozen either slowly at cooling velocities of 0.5° to 1.0°C/minute to −40° or rapidly and stored in liquid nitrogen (−196°). Maximum viability and plant regeneration (95%) was obtained when the meristems were precultured on medium supplemented with 5% DMSO, and then frozen at a cooling velocity of 0.84°/min. A viability of 35% was observed when meristems precultured on 5% glycerol-supplemented medium for 3 days were frozen at a cooling velocity of 0.94°/min. Rapid freezing and rapid dry freezing using either DMSO or glycerol resulted in reduced viability. A viability and plant regeneration frequency of 95% was obtained after the meristems were in storage for one week in liquid nitrogen. From samples assayed after 8 weeks of storage in liquid nitrogen, more than 55% of the meristems regenerated into plantlets.