AUXIN LEVELS AT DIFFERENT STAGES OF CARROT SOMATIC EMBRYOGENESIS

The role of auxin in somatic embryogenesis was evaluated by characterizing the changes in the concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and their conjugates in callus suspension cells and developing embryos of Daucus carota. Both embryogenic and non-embryogenic lines exhibited similar growth rates and levels of IAA and 2,4-D on 2,4-D-supplemented medium. Total endogenous IAA in both lines exposed to 2,4-D reached high levels greater than 600 ng g-1 fresh weight which suggests that IAA levels in carrot callus are not regulated via auxin feedback mechanisms. After being transferred to 2,4-D-free medium, the embryogenic line exhibited a rapid decline in both free and conjugated 2,4-D metabolites within seven days, while IAA levels remained relatively steady for seven days in the preglobular stage after which the levels declined steadily in all subsequent stages of embryo development. Individual analyses of different embryo fractions collected from asynchronous cultures confirmed that each stage in embryo development had lower IAA levels than the preceding stage. The non-embryogenic line maintained similar 2,4-D levels but higher IAA levels than the embryogenic line throughout the experiment. The present results suggest that high IAA levels may be necessary but are not sufficient for the initial events in plant embryogenesis, whereas low IAA levels are associated with the later stages of embryo development.