二乔刺槐叶片在添加5 mg·L~(-1)6-BA的MS培养基上诱导出愈伤组织.将愈伤组织在冻存液(MS+10%DMSO+0.5 mol·L~(-1)蔗糖)中于4℃预冷2 h,置人程序降温仪以1℃·min~(-1)的速率降温至-7℃停留1 h,再以0.1℃·min~(-1)的冷却速率将样品冷却至-20℃,平衡1 h,接下来以0.3℃·min~(-1)的冷却速率将样品冷却至-40℃投入液氮保存,是适宜的降温方法.液氮保存1天后以38℃温水浴解冻.解冻后将愈伤组织用液体培养基(MS+6·BA 5 mg·L~(-1)+30 g·L~(-1)蔗糖)洗涤后转入恢复培养基暗培养,14天后放于光照下培养.光培养3天后冻后愈伤组织上开始长出新生愈伤组织颗粒,成活率最高可达52%,新生愈伤组织可诱导成苗.%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 X.min-1.Being incubated for another hour at -20 ℃ ,the calli were cooled down to - 40 t 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 t 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.
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