Protoplasts were isolated from the leaves of 5-week-old petunia plants using the valosin-containing protein (VCP) enzyme. Briefly, the leaves were chopped and digested in20 mL of enzyme solution at room temperature under gentle shaking (40 rpm) in the dark for 3 h. Following this, the mixture was diluted with an equal volume of W5 solution and filtered through a 100-lm nylon mesh (Falcon, Capitol Scientific, Inc, TX, Austin ) to remove undigested leaf tissue. Protoplasts were then purified and collected using sucrose gradient centrifugation at 70 g for 7 min before being transferred to W5 solution .
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