Effect of buffering of the medium pH by MES on the development of leaf protoplast-derived cells cultured in the presence of 10 μm 2,4-D. A, Concentration-dependent effect of MES on delaying the acidification of the culture medium caused by the cells. B, Inhibition of medium acidification allows the cells to elongate in the presence of the otherwise inhibitory 10 μm 2,4-D concentration as shown by cell size. C and D, Cell division is delayed parallel with the delay in medium acidification as determined by BrdU incorporation (C) and cell division index (D). E, The appearance of cell activation/division-related proteins like a mitogen-activated protein kinase (MMK1), cyclin-dependent kinase (Cdc2MsA), and A and B type mitotic cyclins (Medsa;CycA2.1 and Medsa;CycB2.2) is also delayed due to medium buffering. F, Cdc2Ms AB-related histone phosphorylation activity is decreased in the cells cultured at buffered medium pH by MES. C, Freshly isolated protoplasts as control.