Fig. 2.

SR2200 staining to study PT guidance and fertilization. SR2200 can be used for PT staining (a–f). a PTs growing on the placenta surface; micropylar guidance of wild-type PT (b) and pod1-2 mutant PT (c); failed PT reception in fer-5 mutant ovules (d; inset shows overview) in comparison with wild type (e). Arrow head indicates intact tip of PT in d. f Rare case of polytubey in a wild-type ovule (fer-5 ± parent plant) with two PTs targeting one ovule (indicated by arrow heads). Burst of PTs (white) can be seen by release of CFP-ER from the vegetative cell (cyan). Double fertilization has occurred as judged by the 15 dsRed-labeled centromers in each endosperm nucleus (magnification shown in inset; labeled by pHTR12::HTR12-mCherry depicted in orange). The second sperm pair from the other PT failed to fuse with female gametes (indicated by arrows; labeled by pMGH3::MGH3-2xVenus-N7 and shown in green). Auto-fluorescence is shown in yellow. g Overview of unfertilized ovule expressing synergid-specific pNTA≫NLS-tdtomato; h magnification of g; i fertilized ovule of the same transgenic line shown in g. The nuclear-localized tdtomato signal is now visible in the developing endosperm. Developing embryo is indicated by bracket; endosperm nuclei are labeled by asterisks. SR2200 staining is shown in gray scale (a–c, g–i) and in cyan as overlay with DIC image in gray scale (d, e). Scale bar 20 µm