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Cryopreservation of Limonium serotinum apical meristems from in vitro plantlets using droplet-vitrification

Barraco, Giuseppe, Sylvestre, Isabelle, Iapichino, Giovanni, Engelmann, Florent
Scientia horticulturae 2011 v.130 no.1 pp. 309-313
Limonium, apical meristems, callus formation, cooling, cryopreservation, droplets, ethylene glycol, genotype, glycerol, in vitro studies, plantlets, regrowth, shoots, sucrose
In this study in vitro shoot tips of a Sicilian genotype of Limonium serotinum were successfully cryopreserved using the droplet-vitrification technique. Growth recovery of cryopreserved shoot tips was possible only when samples were pretreated for 16h in liquid medium with 0.3M sucrose, then for 5h in liquid medium with 0.7M sucrose before performing the cryopreservation protocol. Optimal conditions included treatment for 20min in a loading solution containing 1.9M glycerol+0.5M sucrose, treatment with vitrification solution B5 (glycerol 40.0%, sucrose 40.0%, w/v) for 60 and 90min or vitrification solution A9 (glycerol 30.0%, dimethylsulfoxide 20.0%, ethylene glycol 20.0%, sucrose 15.0%) for 20min, rapid cooling in minute droplets of vitrification solution, rapid rewarming by immersion for 20min in unloading solution containing 1.2M sucrose. Under these conditions, 37% recovery of cryopreserved shoot tips was achieved. Regrowth of cryopreserved samples was slow but always direct, without callus formation.