紫花苜蓿保卫细胞原生质体的制备

摘要

为了获得紫花苜蓿(Medicago sativa)保卫细胞原生质体,并为后续的紫花苜蓿保卫细胞原生质体的相关研究奠定基础,本研究探索了适合紫花苜蓿保卫细胞的提取方法.利用研磨和匀浆机处理的方式获得表皮条,将获得的表皮条再通过两步酶解法去除残留的叶肉细胞和细胞壁,并经多次过滤和离心获得保卫细胞原生质体.最后统计原生质体数目并用FDA染色检测保卫细胞原生质体活性.结果表明,本研究成功分离并获得了高活性的紫花苜蓿保卫细胞原生质体.每5片成熟叶获得1 000个保卫细胞原生质体,经FDA染色后在荧光下显示活性保卫细胞原生质体占90％以上.%To obtain protoplasts of alfalfa (Medicago sativa) guard cells and lay the foundation for further research into guard cell protoplasts,the utility of a method for extracting guard cells from alfalfa leaves was explored.Epidermal peels were obtained by grinding and using a blender.Two-step enzyme digestion was used to remove mesophyll cells from the epidermal peels and guard cell walls;guard cell protoplasts were obtained through repeated filtration and centrifugation.Finally,the number of protoplasts was counted and their viability was assessed by fluorescein diacetate (FDA) staining.The results showed that this method yielded alfalfa guard cell protoplasts in an efficient manner,with 1 000 protoplasts isolated from samples of five leaves and a rate of viable guard cell protoplasts of more than 90 ％.