Abstract:

Robinia bella-rosea calli were initiated from the leaves on MS medium supplemented with 5 mg·L^-1 6-BA. The calli were cooled down at 4 ℃ for 2 h in a cryoprotectant （MS+10%DMSO＋0.5 mol·L^-1 Sucrose）, and further refrigerated to -7 ℃ at a rate of 1 ℃·min^-1 by using a programmed temperature-changing freezer. After one hour at -7 ℃ for 1 h, the calli in the cryoprotectant were further refrigerated to -20 ℃ at a rate of 0.1 ℃·min^-1. Being incubated for another hour at -20 ℃, the calli were cooled down to -40 ℃ at a rate of 0.3 ℃·min^-1 and then the calli were transferred and preserved in liquid nitrogen for a day. The low temperature treated calli were thawed at 38 ℃ water bath, and the thawed calli were washed with liquid MS medium with 6-BA 5 mg·L^-1 and 30 g·L^-1 sucrose, then transferred to solid medium and cultured in darkness. Two weeks later, the calli were cultured in light, and showed able to produce fresh and green new callus after 3 d， and the survival rate of the low temperature treated calli reached to 52%. New-produced calli were able to regenerate plantlets.

Key words: Robinia bella-rosea, callus, cryopreservation, freezing method