Cryopreservation of Shoot Tips of Rh.sachalinensis by Encapsulation-vitrification

Shoot tips excised from two resources of Rh.sachalinensis had been cryopreserved by encapsulation-vitrification.In the experiment,effects of sucrose preculture duration,osmoprotection time during encapsulation and exposure time to PVS2 on survival rate of cryopreserved shoot tips of Rh.sachalinensis were discussed.Mature seeds of Rh.sachalinensis were cultured on hormone-free MS medium,and plantlets formed from germinated seeds in 3 weeks.Excised shoot tips were precultured with increasing sucrose concentrations of 0.3,0.5,0.75 and 1 M for 4 days,then transferred to 1 M sucrose for 4 days,and precultured shoot tips were encapsulated and simultaneously osmoprotected with a loading solution of 2 M glycerol and 1 M sucrose for 60 min.Osmoprotected and encapsulated shoot tips were dehydrated with a highly concentrated vitrification solution for 180 to 210 min at 4 ℃ prior to direct immersion in liquid nitrogen for 48 h.After rapid warming in water bath for 3 min at 40 ℃,the shoot tips were rinsed with the modified MS medium containing 1 M sucrose for 20 min,and then cultured on the MS medium supplemented with 6-BA 4.44 μM and NAA 0.54 μM in dark for 1 week prior to exposure to the light at 22 ℃.The best survival rate of encapsulation vitrified shoot tips amounted to nearly 100% and no plantlet abnormality was observed.