A simple and efficient cryopreservation of in vitro-grown shoot tips of `Troyer' citrange [Poncirus trifoliata (L.) Raf. × Citrus sinensis (L.) Osbeck.] by encapsulation-vitrification

A simple and efficient method was developed for cryopreservation ofin vitro-grown shoot tips of `Troyer' citrange byencapsulation-vitrification. Excised shoot tips were precultured withincreasing sucrose concentrations of 0.3, 0.5, 0.75 and 1 M for 4 days.Precultured shoot tips were encapsulated and simultaneously osmoprotectedwith a loading solution of 2 M glycerol and 1 M sucrose.Osmoprotected and encapsulated shoot tips were dehydrated with a highlyconcentrated vitrification solution prior to direct immersion in LN for 1 h.Optimal survival of cryopreserved shoot tips was obtained when preculturedshoot tips were osmoprotected for 60 min during encapsulation.Dehydration by exposure to modified PVS2 solution for 90 min at24 °C and for 180 to 210 min at 0 °C was found optimalfor survival of cryopreserved shoot tips. Preculture duration largelyinfluenced survival of cryopreserved shoot tips, with the best result obtainedafter 2 to 5 days of preculture with stable 1 M sucrose. With theoptimized parameters, 100% survival of cryopreserved shoot tips wasachieved. Morphologies of plants regenerated from cryopreserved shoottips were similar to those of the seedlings.