Biochemical and morphological stratification in Acer saccharum root callus

Root callus of sugar maple (Acer saccharum, Marsh.) exhibits differential metabolic activities and cellular morphologies at different depths. A thin layer of acid phosphatase-active cells encloses a second layer of cells that reduce nitroblue tetrazolium (NBT). Mitosis occurs within the cells of this NBT reducing layer (which is one-third of the total depth of the callus), and the cells beneath it show acid phosphatase activity. Morphologically the cells at the outer surface appear tubular and loosely attached, with intact nuclei and cytoplasm. The cells of the NBT region are isodiametric and possess a large vacuole. The cells below the NBT region show progressive degeneration with depth by loss of nuclei, cytoplasm, and cell wall integrity. There is new cell growth only from the NBT region when aseptically cut callus sections are placed on new medium, regardless of whether or not this region is in direct contact with the agar. Several hypotheses to explain sugar maple callus organization are discussed.