FIGURE 4.

Immunofluorescence of H3K9me2 during pollen development and microspore embryogenesis of Hordeum vulgare. Confocal laser scanning microscopy analysis of vacuolated microspores, starting point of the two developmental pathways (A–A”), tricellular pollen, advanced stage of gametophytic development (B–B”), proembryos, early stage after reprogramming (C–C”) and coleoptilar embryo, advanced embryogenesis stage (D–D”). (A–D): Nomarsky’s differential interference contrast (DIC) images showing the cellular organization of the different structures. (A’–D’): DAPI staining of nuclei (blue). (A”–D”): H3K9me2 immunofluorescence signal over nuclei (green). The same structures are visualized under different microscopy modes in (A–A”), (B–B”), (C–C”) and (D–D”). Inset shows a detail of (D”) at higher magnification. The exine showed unspecific autofluorescence in some images (A’,A”,B”,C”). Bars represent: (A–A”), (B–B”): 10 μm, (C–C”): 20 μm, (D–D”): 75 μm.