Changes in cellular structures and enzymatic activities during browning of Scots pine callus derived from mature buds

Visible browning is a typical feature of callus cultures derived from shoot tips of mature Scots pine (Pinus sylvestris L.). Because the ability of callus to regenerate is low, we determined the effect of browning on growth and changes in cellular structure during culture. Striking alterations in cellular structure were detected by LM (light microscopy), EM (electron microscopy) and SEM (scanning electron microscopy). Accumulation of phenolic substances was shown by histochemical staining. Staining for beta-glucosidase activity of soluble proteins that had been subjected to polyacrylamide gel electrophoresis indicated lignification of cells. The measured growth rate of callus was low compared with a hypothetical growth curve. Peroxidase activity increased rapidly soon after the start of the culture period, but especially between the second and third weeks of culture. At this time, the degradation of cell membranes and browning began coincident with the loss of chlorophyll. We conclude that browning is associated with cell disorganization and eventual cell death, making tissue culture of mature pine especially difficult.