以苹果试管苗叶片为原生质体分离材料，对影响原生质体分离和培养的因素进行了研究。结果表明适合叶片酶解的酶液组成是Cellulase-Onzuka R-10 0.8% + Pectinase 0.5% + PVP 1% + 甘露醇 0.65 mol/L + MES0.1%；以改良MT + BA 1.0 mg/L + 2,4-D 0.2 mg/L + 甘露醇0.65 mol/L + Vc 5.0 mg/L + Glu 500 mg/L + CH 100 mg/L + ME 500 mg/L + Arg 50 mg/L为培养基对原生质体进行培养，固液双层培养效果较好，最适培养密度为1×105 (个/ml)，培养1-2 d原生质体变形，3-4 d第一次分裂，2 w分裂3-5次。平邑甜茶原生质体一个月后形成微细胞团，两个半月形成肉眼可见的微愈伤组织。鲁加5号和M7均只形成7-10个细胞的细胞团，嘎拉未见细胞分裂。

The factors effecting isolation and culture of apple protoplast from leaves in vitro were studied. The results indicated that the optimum enzyme solution for apple leaves was Cellulase-Onzuka R-10 0.8% + Pectinase 0.5% + PVP 1% + mannitol 0.65 mol/L + MES 0.1%. The appropriate culture density of protoplasts was 1×105 (No. of protoplasts/ml) and the proper culture method was Liquid-Solid-Double-Layer Culture. The protoplasts were cultured on the medium of modified MT + BA 1.0 mg/L + 2.4-D 0.2 mg/L + mannitol 6.5 mol/L + Vc 5 mg/L + Glu 500 mg/L + CH 100 mg/L + ME 500 mg/L + Arg 50 mg/L. Cell enlargement was found in 1-2 days and cell division was found in 3-4 days. Cell division took place 3-5 times after 2 weeks culture. Cell colony was found in one month and microcalli in two and half months of Malus hupehensis Rehd. The cell number of the cell colony of M7 and Lujia 5 were 7 to 10. And no cell division was found in Gala.