Arabinogalactan-proteins in Cichorium somatic embryogenesis: effect of beta-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). A ddition of beta-D-glucosyl Yariv reagent (beta GlcY), a synthetic phenyl-gl ycoside that specifically binds arabinogalactan-proteins (AGPs), to the cul ture medium blocked somatic embryogenesis in a concentration-dependent mann er with complete inhibition of induction occurring at 250 mu M beta GlcY. T he AGP-unreactive alpha-D-galactosyl Yariv reagent had no biological activi ty in this system. Upon transfer of 250 mu M beta GlcY-treated roots to con trol conditions, somatic embryogenesis was recovered with a time course sim ilar to that of control roots. The beta GlcY penetrated roots and bound abu ndantly to developing somatic embryos, to the root epidermis and the stele. Immunofluorescence and immunogold labelling using monoclonal antibodies (J IM13, JIM16 and LM2) revealed that AGPs were localised in the outer cell wa lls 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 extrac ted from treated conditioned medium and identified as AGPs by using the sam e monoclonal antibodies. In addition, AGPs were found to be abundantly pres ent in the medium during embryogenic culture. All of these results establis h the implication of AGPs in embryo development, and their putative role in somatic embryogenesis is discussed.