A HISTOCHEMICAL STUDY OF CALLUS INITIATION FROM CARROT TAPROOT PHLOEM CULTIVATED IN VITRO

Cylinders of carrot taproot secondary phloem were cultured on one of four media: 1) 2% sucrose + 1% agar (SA); 2) Heller's basal medium (NA); 3) NA + 10^-5 g/liter 2,4-D (H4); and 4) NA + H4 + 15% coconut milk (HW). Samples were taken from the cultured explants at 3-day intervals. A morphological study of the cultured explants revealed no differences between callus-initiating explants (cultured on HW medium) and noncallus-initiating explants (cultured on SA, NA, and H4 media) within the first 3 days of culture. All explants exhibited a typical wound response. Cell division ceased in the NA and SA explants after the sixth day in culture. Extensive cell division occurred in the subsurface layer of dividing cells in the HW explants and resulted in the formation of callus by the ninth day in culture. Histochemical staining revealed that the activity of NAD diaphorase, succinic dehydrogenase, and cytochrome oxidase were closely correlated with the wound response and with callus initiation in the cultured explants. The activity of these enzymes was high in the layer of dividing cells of all explants after 3 days of culture, but with longer periods of culture the activity of these enzymes was closely correlated with the extent of cell division. Acid phosphatase activity was associated with the dividing cell layers of all explants, but comparatively little acid phosphatase activity was observed in the NA, SA, and H4 explants as compared to the HW explants, and acid phosphatase was strongly correlated with callus initiation by the HW explants. Using the nitroso reaction, “catechol tannins” were found in the surface layers of the NA, SA, and H4 explants, while no nitroso-reaction-positive substances were detected in the HW explants during the period of callus initiation.