Cryopreservation of Dormant Shoot Tips of Non-Astringent Types of Chinese Persimmon by Encapsulation-Vitrification and Plantlets Regeneration

Shoot tips excised from dormant axillary buds of non-astringent types of Chinese persimmon(Diospyros kaki Thunb.), which is originated from China, had been cryopreserved by encapsulation-vitrification for the first time. In the experiment, three factors, namely sucrose concentration, loading time and exposure time to PVS2,were considered and a good protocol was achieved. Excised shoot tips were encapsulated into alginated-gel beads. Then alginate-coated shoot tips were precultured on a modified MS basal medium supplemented with sucrose at 0.3, 0.5, 0.7, 1.0 molL-1 for one day. After preculture, encapsulated shoot tips were loaded with a mixture of 2 mol稬-1 glycerol plus 0.4 mol稬-1sucrose for 20 min. Following loading, encapsulated shoot tips were dehydrated with a highly concentrated vitrification solution (PVS2 solution)for 2 h at 25℃, and then plunged directly into liquid nitrogen. After rapid warming in water for about one to two min at 40℃, the shoot tips were rinsed with the modified MS medium containing 1.2 molL-1 sucrose for 20 min, and then cultured on the modified medium with 2.2 mg稬-1ZT and 600 mg稬-1 PVP in dark prior to exposure to the light. The best survival rate of encapsulation vitrified shoot tips amounted to nearly 100% and no abnormality was observed. Thus the protocol using dormant shoot tips appeared to be promising as a routine method for the long-term conservation of Diospyros spp. germplasm.