Confocal scanning laser microscopy, a new technique used in an embryological study of Dactylorhiza maculata (Orchidaceae)

Ovules of Dactylorhiza maculata, fixed in FPA50 and made transparent in Herr's clearing fluid, were investigated with confocal scanning laser microscopy. This new technique makes it possible to obtain thin optical serial sections in perfect alignment without damaging the material. It made possible an interpretation of the development of the embryo sac differing from that based on previous investigations with traditional technique. Using the new technique we found that the young embryo sac generally contains seven nuclei, two of which fuse, and the mature sac is 6-nucleate. The pollen tube does not penetrate any of the synergids when entering the embryo sac. Double fertilization takes place, and all nuclei are still alive at that moment. Four to six endosperm nuclei are formed, but later they degenerate and the growing embryo fills the entire embryo sac.