Changes in cellular structures and enzymatic activities during browning ofScots 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 ca llus to regenerate is low, we determined the effect of browning on growth a nd changes in cellular structure during culture. Striking alterations in ce llular structure were detected by LM (light microscopy), EM (electron micro scopy) and SEM (scanning electron microscopy). Accumulation of phenolic sub stances 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 act ivity increased rapidly soon after the start of the culture period, but esp ecially between the second and third weeks of culture. At this time, the de gradation of cell membranes and browning began coincident with the loss of chlorophyll. We conclude that browning is associated with cell disorganizat ion and eventual cell death, making tissue culture of mature pine especiall y difficult.