Arabidopsis protoplast isolation and transformation

The isolation and transfection of Arabidopsis protoplasts were performed as previously described¹⁹ and used for the various assays as indicated. Briefly, 2 g of 14 days old seedlings were sliced and mixed with 15 ml TVL Solution (0.3 M sorbitol and 50 mM CaCl₂). In all, 20 ml of Enzyme Solution was then added containing 0.5 M sucrose, 10 mM MES-KOH pH 5.7, 20 mM CaCl₂, 40 mM KCl, 1% Cellulase (Onozuka R-10), and 1% Macerozyme (R10). This enzyme solution containing protoplasts were filtered through a 75-mm nylon mesh after a gentle swirling motion at room temperature for 15–18 h. Next, the protoplasts were gently covered with 10 ml W5 Solution (2 mM MES pH 5.7, 154 mM NaCl, 125 mM CaCl₂, and 5 mM KCl) without disturbing the sugar content gradient, following centrifugation at 100×g for 7 min. About 10 ml of protoplasts were collected at the interface of Enzyme Solution and W5 Solution were transferred to a new tube. The protoplasts were then washed twice with 15 ml of W5 Solution and centrifuged for 5 min at 60 g. The pelleted protoplasts were resuspended in 1–3 ml MMG Solution containing 4 mM MES pH 5.7, 0.4 M mannitol and 15 mM MgCl₂. For DNA-PEG–calcium transfection, 10–20 μg (about 10 μl) of plasmid was added to 100 μl protoplasts and mixed gently. An aliquot of 110 μl of PEG solution was mixed with this DNA-protoplasts by gently tapping the tube, and then incubated at room temperature for 15 min. The transfection mixture was mixed with 450 μl W5 solution to stop the transfection process, and centrifugated at 100×g for 1–2 min. The pelleted protoplasts were resuspended with 1 ml W5 solution. After incubation overnight in the dark at 23 °C, the protoplast solution was used for the various assays.