The embryo sac of Vanilla imperialis (Orchidaceae) is six-nucleate, and double fertilization and formation of endosperm are not observed

Double fertilization and subsequent endosperm formation are two of the most important synapomorphies of the angiosperms. Endosperm is generally lacking in Orchidaceae, but Swamy reported the formation of up to ten endosperm nuclei in Vanilla planifolia in 1947. The observation was documented only by line drawings and has not been confirmed; however, assumptions about endosperm formation occurring in Orchidaceae are primarily founded on Swamy's study. The current study provides the first detailed description of embryo sac formation and early embryogeny in Vanilla since Swamy, and is the first using modern imaging techniques. Flowers of Vanilla imperialis were artificially pollinated at 1-week intervals and the resulting fruits were fixed, embedded and sectioned for light microscopy of embryo sac formation, fertilization and early development of the embryo. Three-dimensional reconstructions of embryo sacs were obtained using confocal laser scanning microscopy on fixed ovules. The mature embryo sac contains only six nuclei as a result of the arrested development of the chalazal nuclei prior to the formation of three antipodals and a polar nucleus. Fertilization was observed 7weeks after pollination, but double fertilization and the formation of endosperm did not occur.