Using Tape- Arabidopsis Sandwich for Arabidopsis protoplast isolation. (A) The upper epidermis was stuck onto Time tape and the lower epidermis was stuck to clear 3 M tape. The lower epidermal layer was removed by peeling away the 3 M tape; the blue circle indicates cells from the lower epidermis on the 3 M tape after peeling. (B) The leaves with Time tape still adhering to the top surface (with blue backing) were incubated in enzyme solution. (C) After 1 hr, the cell walls were digested and the protoplasts were released into the bottom of the dish. (D) The enzymes only digested the cell wall of those mesophyll cells that were not protected by lower epidermal cells. Left: intact; right: lower epidermal cells removed. (E) Protoplasts derived from mature Arabidopsis leaves. (F) After incubation in the enzyme solution, all mesophyll cells became protoplasts: microscopy showed that only upper epidermal cells remained. Yellow arrow: Time tape; red line: the relative position of 3 M Magic tape where the lower epidermal cell layer (purple circle) was removed; green arrow; the related position of mesophyll. M: midrib; U: upper epidermis; L: lower epidermis. Bar = 100 μm.