马铃薯‘春薯4号’原生质体的分离纯化

Isolation and Purification of Protoplasts from Potato Chunshu 4

植物原生质体是去掉细胞壁的由细胞膜包被的有活力的裸细胞,它具有活细胞的一切特征,是基础生命科学研究及作物育种改良的理想材料。本研究以马铃薯‘春薯4号’组培苗叶片为外植体,对‘春薯4号’原生质体的分离和纯化条件进行了探索和优化。结果表明,含3 mg/L硝酸银的MS培养基培养21 d可显著增大叶面积,为原生质体制备提供充足的材料;0.4%纤维素酶+0.5%果胶酶+0.1%离析酶,在转速为35 r/min、25℃、黑暗条件下酶解叶片14 h,原生质体产量较高,质量最好;界面法纯化原生质体,得到的原生质体大小一致,破碎细胞较少;用上述方法制备的原生质体活力达90%以上,可用于原生质体培养。

Plant protoplasts are viable naked cells without cell wall and coated by cell membranes.They have all the characteristics of living cells and are ideal materials for basic life science research and crop breeding improvement.In this study,the leaves of potato Chunshu 4 tissue culture seedlings were used as explants,the isolation and purifi⁃cation conditions of Chunshu 4 protoplasts were explored and optimized.The results showed that MS medium con⁃taining 3 mg/L silver nitrate for 21 days could significantly increase leaf area and provide sufficient materials for pro⁃toplast preparation.When 0.4%cellulase+0.5%pectinase+0.1%isolating enzyme were used to hydrolyze leaves for 14 h under the condition of rotating speed of 35 rpm,25℃and darkness,the yield of protoplast was higher and the quality was the best.The protoplast was purified by interface method,and the protoplast size was the same,and there were few broken cells.The activity of protoplast prepared by the above method is over 90%,which can be used for protoplast culture.