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作 者 ：王鹏凯,吕伟光,胡雪怡,陈金慧,施季森*

期 刊 ：西北植物学报 2013年 2期 页码：254~260

关键词：北美鹅掌楸；原生质体；酶解条件；愈伤组织；

Keywords:Liriodendron tulipifera, protoplasts, enzymolysis conditions, callus,

摘 要 ：以鹅掌楸属植物北美鹅掌楸的悬浮细胞和组培苗叶片为材料,对北美鹅掌楸原生质体分离、纯化与培养条件进行研究。结果表明:叶片和悬浮细胞用含有0.1% 2-吗啉乙磺酸（MES）和0.6 mol/L甘露醇的Cell Protoplast Wash（60M-CPW）溶液25 ℃预处理1 h效果最好;悬浮细胞最佳酶解液为60M-CPW+1%纤维素酶+1%半纤维素酶+0.2%果胶酶Y-23+0.1% MES,每克材料25 ℃酶解6 h有效原生质体产量可以达到3×10⁶个;叶片最佳酶解液为60M-CPW+2%纤维素酶+1%半纤维素酶+0.2%果胶酶Y-23+0.1% MES,每克材料25 ℃酶解10 h有效原生质体产量可以达到11×10⁶个;悬浮细胞原生质体易于培养,在KM8p+1.0 mg/L 2,4-D+0.5 mg/L 6-BA培养基中培养25 d可形成肉眼可见的愈伤组织。

Abstract:The suspension cells and leaves of Liriodendron tulipifera were used as experiment materials to isolate protoplasts.The influences of different factors including pretreatment osmotic pressure,the kind and combination of enzymes,digestion time,culture medium were compared.The result showed that the pretreatment of leaves and suspension callus cells in the Cell Protoplast Wash containing 0.1% 2-(4-Morpholino) ethanesulfonic acid (MES) and 0.6mol/L mannitol (60M-CPW) for 1 h at 25 ℃ and light-resistant has the best effect in the protoplasts isolation.The best enzyme solution of suspension cells contained 60M-CPW+1% cellulase+1% hemicellulase+0.2% pectinase Y-23+0.1% MES,incubated at 25 ℃ for 6 h and the protoplasts yield was about 3×10⁶ per gram.The best enzyme solution of leaves contained 60M-CPW+2% cellulase+1% hemicellulase+0.2% pectinase Y-23+0.1% MES,incubated at 25 ℃ for 10 h and the protoplasts yield was about 11×10⁶ per gram.It was easy to culture the protoplasts of suspension callus cells and the culture KM8p was better than the modified MS.In the culture medium of KM8p containing 1.0 mg/L 2,4-D+0.5 mg/L 6-BA,the protoplasts of suspension cells will be formed visible callus in 25 days.

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