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Arabinogalactan proteins (AGPs) are extracellular proteoglycans involved in plant growth and development. The addition of β-D-glucosyl Yariv reagent (βGlcY), a synthetic phenylglycoside that specifically reacts with AGPs, to the culture medium notably disturbed microspore embryogenesis in a concentration-dependent manner. The initiation of microspore embryogenesis was clearly inhibited by 30 μM βGlcY and completely inhibited by 50 μM βGlcY. The transfer of microspore-derived embryos at different developmental stages into NLN6 medium containing 50 μM βGlcY prohibited their normal development, as ∼21.24, 43.99, and 59.73%, respectively, of the treated globular-, heart-, and torpedo-stage embryos exhibited numerous root hair-like structures. Both heart-stage and torpedo-stage embryos showed a rapid growth of roots with a large number of clustered root hairs. Some root hair-like structures were also observed on the apical portions of embryos. Microscopy of the treated embryos revealed that the basic patterns of cells at both the radial and apical–basal axes were greatly altered, such that the cells lost their ability to carry out programmed embryogenesis. These results show that the βGlcY–AGP interaction modulates the developmental fate of embryonic cells, especially epidermal cells, and thereby strongly affects root generation and development. Immunofluorescence microscopy revealed that both JIM8 and JIM13 binding to AGP co-localize with βGlcY-binding sites. Thus, AGPs binding to βGlcY, co-localized with Jim8- and Jim13-binding protein, appear to play a crucial role in the initiation of Brassica microspore embryogenesis and the maintenance of cell differentiation during embryonic development. In addition, these proteins may also be involved in the regulation of root generation.