Preparation of guard cell protoplasts from Medicago sativa

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%.