Efficient Protoplast Transfection Protocol

Protoplasts were transfected by a modified TEAMP method [4 (link)]. Approximately 5 × 10⁴ protoplasts (2 × 10⁴ to 1 × 10⁵) in 0.2 mL of MMg solution were mixed with approximately 30 (20 to 40) μg of plasmid DNA at room temperature. An equal volume of a freshly-prepared solution of 40% (w/v) PEG (MW 4000; Fluka) with 0.1 M CaCl₂ and 0.2 M mannitol was added, and the mixture was incubated at room temperature for 5 min. After incubation, 3 mL of W5 solution was added slowly, the solution was mixed, and protoplasts were pelleted by centrifugation at 100 × g for 1 min (Eppendorff A-4-44 rotor). This protoplast W5 wash step was repeated twice. The protoplasts were resuspended gently in 1 mL of W5 and were incubated in 6-well plates coated with 1% BSA at room temperature for 16 hr in light.