Efficient Arabidopsis Protoplast Isolation

Leaves (width: 2 cm, length: 5 cm in optimal light condition; width: 0.5 cm; length: 2.5 cm in low light conditions) were collected from 3 to 5-week-old plants grown under optimal light (ca. 150 μE·m^-2·s^-1) or low light (ca. 50·μE m^-2·s^-1) conditions. Arabidopsis protoplasts were isolated in two ways. First, to recreate the current technique, protoplasts were made according to the procedure of Yoo et al. [4 (link)]. Second, in a new technique, selected leaves were used in a 'Tape-Arabidopsis Sandwich' experiment. The upper epidermal surface was stabilized by affixing a strip of Time tape (Time Med, Burr Ridge, IL) while the lower epidermal surface was affixed to a strip of Magic tape (3 M, St. Paul, MN). The Magic tape was then carefully pulled away from the Time tape, peeling away the lower epidermal surface cell layer. The peeled leaves (7 to 10 optimal-light-growth leaves, about 1-2 g, up to 5 g), still adhering to the Time tape, were transferred to a Petri dish containing 10 mL of enzyme solution [1% cellulase 'Onozuka' R10 (Yakult, Tokyo, Japan), 0.25% macerozyme 'Onozuka' R10 (Yakult), 0.4 M mannitol, 10 mM CaCl₂, 20 mM KCl, 0.1% BSA and 20 mM MES, pH 5.7]. The leaves were gently shaken (40 rpm on a platform shaker) in light for 20 to 60 min until the protoplasts were released into the solution. The protoplasts were centrifuged at 100 × g for 3 min in an Eppendorff A-4-44 rotor (Hamburg, Germany), washed twice with 25 mL of pre-chilled modified W5 solution (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 5 mM glucose, and 2 mM MES, pH 5.7) and incubated on ice for 30 min. During the incubation period, protoplasts were counted using a hemocytometer under a light microscope. The protoplasts were then centrifuged and resuspended in modified MMg solution (0.4 M mannitol, 15 mM MgCl2, and 4 mM MES, pH 5.7) to a final concentration of 2 to 5 × 10⁵ cells/mL.