【摘要】 以花椒试管苗叶片、愈伤组织和悬浮细胞为试验材料,通过单因素试验研究酶液浓度、渗透压调节剂浓度对花椒原生质体分离的影响,并以花椒试管苗叶片分离出的原生质体为试验材料,研究培养基种类、培养密度、不同激素配比对花椒原生质体培养的影响。结果表明,酶液浓度、渗透压调节剂浓度对花椒原生质体分离有显著影响。在适宜条件下,用甘露醇作花椒原生质体分离的渗透压调节剂,浓度在0.6～0.7mol·L^-1时分离效果最佳。以花椒叶片为原生质体分离材料的最佳酶液组成为CPW-0.7mol·L^-1甘露醇+1.0%（w/v）纤维素酶R-10+1.5%（w/v）果胶酶,酶解时间为10h,纯化后的原生质体产量和活力分别可达82.36×105个·g^-1和72.74%。以愈伤组织和悬浮细胞为分离材料的最佳酶液组成均为CPW-0.6mol·L^-1甘露醇+2.0%（w/v）纤维素酶R-10+0.5%（w/v）果胶酶,酶解12～14h,纯化后的原生质体产量及活力分别为31.26×105个·g^-1、59.15%和53.87×105个·g^-1、63.92%。以花椒试管苗叶片为原生质体分离材料,分离纯化后的花椒原生质体在无激素的WPM培养基中,培养密度为1×105个·mL^-1,培养第3天原生质体第1次分裂,2周分裂3～5次,原生质体28d后形成微细胞团,培养60d后可形成2mm左右的愈伤组织。更多还原

【Abstract】 Leaves,calli and suspension cells of Zanthoxylum bungeanum were used as experimental materials to study the effects of enzyme concentration,enzymolysis time,osmoticum concentration and sucrose concentration on protoplast separating and purifying through single factor experiment.The obtained protoplasts with high yield and vigour derived from the leaves of Z.bungeanum were used to study the effects of culture media,protoplasts density and exogenous hormones on protoplast culture.The results showed that protoplast isolation and purification of Z.bungeanum were significantly affected by enzyme concentration and osmoticum concentration.Using mannitol as osmotic regulator,the suitable concentration was 0.6-0.7 mol·L^-1.The best enzyme combinations for protoplast isolation from the leaves of Z.bungeanum were CPW-0.7 mol·L^-1 mannitol+1.0%（w/v）cellulase onozuka R-10+1.5%（w/v）pectinase,the enzymolysis time was 10 h.Under these conditions,protoplast yield was 82.36×105·g^-1 and the protoplast viability was 72.74%.The suitable enzyme combinations for protoplast isolation from calli and suspension cells were CPW-0.6 mol·L^-1 mannitol+2.0%（w/v）cellulase onozuka R-10+0.5%（w/v）pectinase,the enzymolysis time was about 12-14 h.The yields and viabilities of protoplasts were 31.26×105·g^-1,59.15% and 53.87×105·g^-1,63.92%,respectively.The protoplasts were cultured on the medium of modified WPM.Cell division was found in 3-4 days.Cell division took place 3-5 times after 2 weeks culture.Cell colony was found in 28 days and microcalli in 60 days.更多还原