Arabinogalactan-proteins in Cichorium somatic embryogenesis:: effect of β-glucosyl Yariv reagent and epitope localisation during embryo development

\Direct somatic embryogenesis was induced in root tissues of the Cichorium hybrid '474' (C. intybus L. var. sativum x C. endivia L. var. latifolia). Addition of beta-D-glucosyl Yariv reagent (beta GlcY), a synthetic phenyl-glycoside that specifically binds arabinogalactan-proteins (AGPs), to the culture medium blocked somatic embryogenesis in a concentration-dependent manner with complete inhibition of induction occurring at 250 mu M beta GlcY. The AGP-unreactive alpha-D-galactosyl Yariv reagent had no biological activity in this system. Upon transfer of 250 mu M beta GlcY-treated roots to control conditions, somatic embryogenesis was recovered with a time course similar to that of control roots. The beta GlcY penetrated roots and bound abundantly to developing somatic embryos, to the root epidermis and the stele. Immunofluorescence and immunogold labelling using monoclonal antibodies (JIM13, JIM16 and LM2) revealed that AGPs were localised in the outer cell walls peripheral cells of the globular embryo. A spatiotemporal expression of AGPs appeared to be associated with differentiation events in the somatic embryo during the transition from the globular stage to the torpedo stage. To verify beta GlcY specificity, molecules that bound beta GlcY were extracted from treated conditioned medium and identified as AGPs by using the same monoclonal antibodies. In addition, AGPs were found to be abundantly present in the medium during embryogenic culture. All of these results establish the implication of AGPs in embryo development, and their putative role in somatic embryogenesis is discussed.