Cryopreservation of persimmon (Diospyros kaki Thunb.) by vitrification of dormant shoot tips

Abstract

Shoot tips excised from dormant axillary buds of persimmon (Diospyros kaki Thunb.) were cryopreserved by vitrification. These excised shoot tips were dehydrated in a highly concentrated vitrification solution for 20min at 25°C and then plunged directly into liquid nitrogen. After rapid warming in water at 40°C, the shoot tips were rinsed in a 1.2M sucrose solution for 20min and then plated on a solidified culture medium. Successfully vitrified shoot tips resumed growth within 10days of plating and developed shoots within 3weeks without intermediary callus formation. This simple protocol was successfully applied to the 16 cultivars found in the temperate zone. The average rate of shoot formation was 89%. Even the subtropical species of Diospyros demonstrated a very high recovery growth when the shoot tips had been previously osmoprotected with a mixture of 2M glycerol plus 0.4M sucrose for 20min following sucrose preculture. Little or no contamination occurred in the cryopreserved shoot tips excised from sterilized winter axillary buds. Thus, this simple and reliable vitrification protocol using dormant shoot tips appears to be promising as a routine method for the long-term conservation of Diospyros germplasm of both temperate and subtropical origins.