Purification and immunohistochemical detection of an embryogenic cell protein in carrot

An embryogenic cell protein from carrot (Daucus carota L.), designated ECP31 for embryogenic cell protein and with a relative mass of 31,000, was purified by sequential column chromatographies. Its apparent relative mass was estimated to be 120,000 by gel filtration. Immunoblotting and immunohistochemical studies showed that ECP31 was preferentially localized in the peripheral cells of clusters of embryogenic cells in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) and disappeared during the course of somatic embryogenesis in the absence of 2,4-D. ECP31 began to accumulate on the 33rd day after initiation of cultures of hypocotyl segments on Murashige-Skoog medium with 2,4-D, when callus began to appear on the segments. In dry seeds, lower amounts of ECP31 were located throughout the entire zygotic embryos but not in endosperm. ECP31 was also detected in provascular tissue of malformed somatic embryos.