Figure 4. The detection of protoplast membrane permeability.

The images of protoplasts stained with 10 μg mL⁻¹ Evans blue (EB) were observed at different developmental stages under fluorescence microscopy. (A–D) EB red fluorescence remained on the plasma membrane (PM), and the corresponding bright images showed the intact vacuole. (E,F) The vacuole disappeared under bright field. EB red fluorescence penetrated the PM (E, white arrows), and the corresponding bright image showed the vague boundary between the cytoplasm and the PM (E, black arrow). A mass of EB red fluorescence appeared (F), and the protoplast shrunk in the corresponding bright image. Both experiments were repeated, and similar images were obtained. The bar represents 10 μm.