Wang, Q., Department of Virology, Agricultural Research Organization, Volcani Center, P.O.B. 6, Bet Dagan 50250, Israel
Batuman, Ö., Department of Virology, Agricultural Research Organization, Volcani Center, P.O.B. 6, Bet Dagan 50250, Israel
Li, P., Department of Virology, Agricultural Research Organization, Volcani Center, P.O.B. 6, Bet Dagan 50250, Israel
Bar-Joseph, M., Department of Virology, Agricultural Research Organization, Volcani Center, P.O.B. 6, Bet Dagan 50250, Israel
Gafny, R., Department of Virology, Agricultural Research Organization, Volcani Center, P.O.B. 6, Bet Dagan 50250, Israel

A simple and efficient method was developed for cryopreservation of in vitro-grown shoot tips of 'Troyer' citrange by encapsulation-vitrification. Excised shoot tips were precultured with increasing sucrose concentrations of 0.3, 0,5, 0.75 and 1 M for 4 days. Precultured shoot tips were encapsulated and simultaneously osmoprotected with a loading solution of 2 M glycerol and 1 M sucrose. Osmoprotected and encapsulated shoot tips were dehydrated with a highly concentrated vitrification solution prior to direct immersion in LN for 1 h. Optimal survival of cryopreserved shoot tips was obtained when precultured shoot tips were osmoprotected for 60 min during encapsulation. Dehydration by exposure to modified PVS2 solution for 90 min at 24°C and for 180 to 210 min at 0°C was found optimal for survival of cryopreserved shoot tips. Preculture duration largely influenced survival of cryopreserved shoot tips, with the best result obtained after 2 to 5 days of preculture with stable 1 M sucrose. With the optimized parameters, 100% survival of cryopreserved shoot tips was achieved. Morphologies of plants regenerated from cryopreserved shoot tips were similar to those of the seedlings.