非洲紫罗兰原生质体的制备及不同组分培养基对细胞增殖的影响

目的 探讨非洲紫罗兰（Saintpaulia ionantha）原生质体的制备方法，并比较不同组分培养基对原生质体再生及增殖的影响。方法 以非洲紫罗兰愈伤组织制备悬浮细胞，以细胞生长曲线确定最佳培养时间，并将纤维素酶（cellulase R-10）和果胶酶（pectolyase Y-23）按2︰1（质量比）混合分别酶解悬浮细胞18、20、24 h，比较原生质体得率和细胞活力以确定最佳酶解时间。分别以原生质体基础培养基提取愈伤组织提取物、根尖提取物及培养土浸出物配制培养基培养原生质体16 d，观察原生质体细胞壁再生并比较细胞增殖率。结果 悬浮细胞培养8天细胞增殖率最大，细胞活力93.8%； 4%（质量分数）纤维素酶和2%（质量分数）果胶酶混合酶解21 h原生质体得率达到峰值，细胞活力为75.2%，延长至24 h后得率与21 h相比，差异无统计学意义，但细胞活力降至69.5%。以不同组分培养基培养原生质体，最早于第6 d在愈伤组织提取物培养基中观察到再生细胞壁及细胞分裂；第16天，愈伤组织和根尖提取物培养基细胞增殖率显著高于原生质体基础培养基和培养土浸出物培养基，并以愈伤组织为佳，而培养土浸出物无促进作用。结论 以非洲紫罗兰愈伤组织悬浮细胞培养8 d并以4%纤维素酶和2%果胶酶混合酶解21 h能获得较多高活力的原生质体；愈伤组织提取物培养基能有效促进原生质体再生及细胞增殖。

Preparation of Saintpaulia ionantha protoplast and effect of different cultures on cell growth

Objective To investigate the preparation of Saintpaulia ionantha protoplast and effect of different cultures on its regeneration and growth.Methods Suspended cells of Saintpaulia ionantha were made by the loose callus and cultured to determine the optimal cultivating time by means of its growth curve.Protoplasts were prepared with cellulase and pectinase mixed at a ratio of 2 to 1,incubating for 18,21 and 24 h,respectively,to obtain the best enzymolysis time by comparing the protoplast yields.Basic mediums were added with callus extracts,roots extracts and soil lixivium,respectively,to nurture Saintpaulia ionantha′s protoplasts for 16 days.Results The rate of cell growth reached the highest,and the cell activity was 93.8 % when suspension cells were cultured for 8 days;the maximum yield of protoplast was in 21h under enzymolysising mixed with 4 % cellulase and 2 % pectinase and its activity was 75.2 %.Cell walls and cell divisions were observed at the 6th day in culture medium added with callus extracts.The cell growths in callus extracts medium and roots extracts medium were significantly higher than in basic medium and soil lixivium,of which the best was callus extracts.And soil extracts couldn′t promote the protoplast cell growth.Conclusion Highly active protoplasts of Saintpaulia ionantha can be produced when the suspended cells gained by the loose callus were cultured for 8 days,and then were enzymolysed by 4% cellulase and 2% pectinase for 21 h.Callus extracts medium could promote protoplasts′ regeneration and growth.