【摘要】 分离制备高活力的植物原生质体是从细胞水平研究植物生理、生化和分子生物学的基础。由于植物原生质体外包裹着细胞壁，其分离和培养尚存在诸多难点。以全球性恶性杂草牛筋草(Eleusine indica)的叶片和叶鞘为材料，采用单因素试验方法优化牛筋草原生质体制备条件，包括材料用量、酶液组分、酶解时间和离心速率等，获得了高活力的牛筋草原生质体。具体方法：取5～6叶期牛筋草叶片2 g,在含有1.0%纤维素酶、0.5%离析酶、0.5%果胶酶的10 mL酶解体系中酶解3 h, 2 500 r/min离心收集，原生质体产量最高可达2.075×10~7个/g,有活力的细胞可达90%以上，而且利用叶片制备的原生质体的得率比叶鞘高。本研究建立了一个优化的牛筋草叶片原生质体制备方法，为从单细胞水平研究牛筋草的抗药性机制奠定了基础，也可以为其他禾本科杂草原生质体的制备提供参考。更多还原

【Abstract】 The isolation and cultivation of high-viability plant protoplasts serve as the foundation for studying plant physiology, biochemistry, and molecular biology at the cellular level. However, due to the presence of a cell wall surrounding plant protoplasts, there are numerous challenges in their isolation and culture. In this study, using leaves and sheaths of the globally invasive weed Eleusine indica as materials, single-factor experiments were conducted to optimize the conditions for protoplast preparation from E. indica,including material quantity, enzyme solution composition, enzymatic hydrolysis time, and centrifugation speed. High-activity protoplasts of E. indica were successfully obtained. Specifically, 2 g of leaves from E. indica plants at 5-6 leaf-stage were enzymatically hydrolyzed for 3 hours in a 10 mL enzyme solution containing 1.0% cellulase, 0.5% macerozyme, and 0.5% pectinase. The protoplasts were then collected by centrifugation at 2 500 r/min. The maximum protoplast yield reached 2. 075 ×107protoplasts/g,with over 90% viability. Furthermore,the yield of protoplasts prepared from leaves was higher than that from sheaths. This study established an optimized method for preparing protoplasts from E. indica leaves,laying the groundwork for studying the herbicide resistance mechanisms of E. indica at the single-cell level and providing a reference for the preparation of protoplasts from other grass weeds.更多还原