Role of 2,4-dichlorophenoxyacetic acid (2,4-D) in somatic embryogenesis on cultured zygotic embryos of Arabidopsis: cell expansion, cell cycling, and morphogenesis during continuous exposure of embryos to 2,4-D

The relationship between cell expansion and cell cycling during somatic embryogenesis was studied in cultured bent-cotyledon-stage zygotic embryos of a transgenic stock of Arabidopsis thaliana harboring a cyclin 1 At:β-glucuronidase (GUS) reporter gene construct. In embryos cultured in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), following a brief period of growth by cell expansion, divisions were initiated in the procambial cells facing the adaxial side at the base of the cotyledons. Cell division activity later spread to almost the entire length of the cotyledons to form a callus on which globular and heart-shaped embryos appeared in about 10 d after culture. Anatomical and morphogenetic changes observed in cultured embryos were correlated with patterns of cell cycling by histochemical detection of GUS-expressing cells. Although early-stage somatic embryos did not develop further during their continued growth in the auxin-containing medium, maturation of embryos ensued upon their transfer to an auxin-free medium. In a small number of cultured zygotic embryos the shoot apical meristem was found to differentiate a leaf, a green tubular structure, or a somatic embryo. Contrary to the results from previous investigations, which have assigned a major role for the shoot apical meristem and cells in the axils of cotyledons in the development of somatic embryos on cultured zygotic embryos of A. thaliana, the present work shows that somatic embryos originate almost exclusively on the callus formed on the cotyledons. Other observations such as the induction of somatic embryos on cultured cotyledons and the inability of the embryo axis (consisting of the root, hypocotyl, and shoot apical meristem without the cotyledons) to form somatic embryos, reaffirm the important role of the cotyledons in somatic embryogenesis in this plant.