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The resulting protoplasts were layered onto a discontinuous Percoll gradient containing (from bottom to top) 70% (v/v, 3 ml), 40% (v/v, 5 ml), 30% (v/v, 3 ml), 15% (v/v, 3 ml) and 0% (3 ml) Percoll (GE Healthcare, Munich, Germany) in 0.7 M mannitol, 20 mM MgCl 2 , and 5 mM PIPES-KOH (pH 7.0)