欢迎访问《中国农学通报》，
中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (31): 200-204.

所属专题： 园艺

• 林学 园艺 园林 • 上一篇    下一篇

酿酒葡萄‘桂葡1号’幼叶原生质体分离研究

唐文忠 廖芬 黄茂康 卢塔山 黄伟雄   

  • 收稿日期:2011-05-23 修回日期:2011-07-19 出版日期:2011-12-05 发布日期:2011-12-05
  • 基金资助:

    广西自然科学基金项目

Research on Protoplast Isolation from Young Leaves of Wine Grape ‘GuiPu No.1’

  • Received:2011-05-23 Revised:2011-07-19 Online:2011-12-05 Published:2011-12-05

PDF (PC)

912

摘要：

研究酿酒葡萄‘桂葡1号’幼叶原生质体的最佳分离条件。以‘桂葡1号’20~30日龄无菌苗幼叶为分离原生质体试材，对分离过程中酶液组合、酶解时间、稳定剂浓度、纯化时离心速度及离心时间进行比较分析。酶组合以2%纤维素酶+0.5%果胶酶为宜。随着酶解时间的延长，原生质体的产量逐渐增高，适合‘桂葡1号’幼叶原生质体的酶解时间以8 h为宜。在0.45~0.55 mol/L的甘露醇范围内，随浓度提高，原生质的产率明显上升，0.55 mol/L时达到最大2.48×106个/(g?FW)，活力为79.78%。在悬浮纯化原生质体时，以1000 rpm离心6~8 min的效果较好。分离材料的适宜酶液组合为2% Celluasw Onozuka R-10+0.5% PectolyaseY-23+25 mmol/L MES+0.55 mol/L甘露醇，解离时间以8 h为宜。悬浮纯化时，蔗糖浓度以30%、1000 r/min离心6~8 min效果较好。

关键词: 连作, 连作, 大蒜, 生长, 生理生化

Abstract:

The optimal isolation conditions of protoplasts from young leaves of wine grape ‘GuiPu No.1’ was studied in this paper. The influence of some factors, such as enzyme composition, time of enzymatic hydrolysis, concentration of stabilizer, as well as speed and time of centrifugation, on isolation of protoplasts was comparatively analyzed, using young leaves of ‘GuiPu No.1’ aseptic seedling with 20-30 day as materials. The appropriate enzyme composition was 2%cellulase + 0.5%pectinase. The protoplast yield gradually increased with extension of enzymatic hydrolysis time. The appropriate enzymatic hydrolysis time was 8 hours for protoplast isolation from young leaves of ‘GuiPu No.1’. The protoplast yield significantly increased with rise of mannitol concentration in the range from 0.45~0.55 mol/L and the yield reached highest at mannitol concentration 0.55 mol/L 2.48×106 protoplast/(g?FW) and activity 79.78% The centrifugation speed of 1000 rpm and the centrifugation time of 6～8 min was appropriate for suspension purification of protoplast. The optimal enzyme solution for protoplast isolation was as follows: 2% Celluasw Onozuka R-10+0.5% PectolyaseY-23+25 mmol/L MES+0.55 mol/L Mannitol. Protoplasts with high yield and viability were obtained when incubation for 8 h. The centrifugation at 1000 r/min for 6-8 min in 30% sucrose was good for suspension purification of protoplast.