Somatic embryogenesis from cultured carrot cells progresses through successive morphogenetic stages termed globular, heart, and torpedo. To understand the molecular mechanisms underlying plant embryogenesis, we isolated two genes differentially expressed during embryo development. The expression of these two genes is associated with heart-stage embryogenesis. By altering the culture conditions and examining their expressions in a developmental variant cell line, we found that these genes were controlled by the developmental program of embryogenesis and were not directly regulated by 2,4-dichlorophenoxyacetic acid, the growth regulator that promotes unorganized growth of cultured cells and suppresses embryo morphogenesis. These genes are also expressed in carrot zygotic embryos but not in seedlings or mature plants.Embryogenesis involves the commitment of cells to an embryogenic fate and the expression of the embryogenic program to produce multicellular forms. It is presumed that morphogenesis is specified by the selective expression of gene sets in a sequential manner (1). As a model system to identify genes participating in plant embryogenesis and to study their mechanisms of gene regulation, we chose carrot somatic embryogenesis. Cultured carrot cells proliferate as unorganized cell clusters in a chemically defined medium supplemented with an auxin, 2,4-dichlorophenoxyacetic acid. Upon removal of 2,4-dichlorophenoxyacetic acid, these cell clusters readily form embryos that progress from globular to heart and to torpedo stages (2, 3). It is generally believed that the cultured carrot cells possess embryogenic potential, but the execution of embryo morphogenesis is suppressed by 2,4-dichlorophenoxyacetic acid (4).The onset of embryogenesis involves new protein synthesis (5-7) that is followed by an organized pattern of rapid cell division (8), resulting in a globular-shaped structure. The development of the heart stage involves axis formation and polarized growth resulting in a structure with bilateral symmetry. The heart-stage embryos elongate to produce torpedostage embryos that consist of initial cells for shoot and root meristems (9, 10) and three tissue layers, the protoderm, the cortex, and the procambium.Because early plant embryogenesis does not involve changes in the levels of abundant gene products (5), it has been difficult to isolate differentially expressed genes by conventional methods (11). We, therefore, used a combined immunoadsorption and epitope selection method to isolate cDNA clones corresponding to genes preferentially expressed during embryogenesis (12). This paper describes the temporal expression of these genes at the mRNA and protein levels. (17) with 2,4-dichlorophenoxyacetic acid at 0.1 mg/liter. WOiC and HA were subcultured at a cell density of 8 x 105 cells per ml, and 10-day-old cultures were used to produce somatic embryos as follows. Cultures of WOO1C and HA were filtered through a 500-,um mesh nylon screen, and the filtrates were pelleted by low-speed centrifugation and...

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