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，置入程序降温仪以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 ℃·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.
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