Isolation of sperms from the pollen tubes of flowering plants during fertilization

Sperm cells have been isolated from pollen tubes growing in style segments of the dicotlyledon Rhododendron macgregoriae and the monocotyledon Gladiolus gandavensis by the in vivo/in vitro method at various stages of fertilization. Pollen tubes emerged from the cut end of the style into agar medium, and more than 95% contained sperm cells. Sperm cells were released from the pollen tubes by osmotic shock or by placing styles in wall-degrading enzymes: 0.5% macerozyme and 1% cellulase. The isolated sperms were ellipsoidal protoplasts of diameter about 2 × 3 micrometers in Gladiolus and about 3 × 4 micrometers in Rhododendron. After isolation, a proportion of the sperm cells occurred in pairs linked at one end by finger-like connections. The pairs of isolated sperms were dimorphic in terms of surface area and volume. By cutting the styles at various positions and times after pollination, the potential exists to detect changes in sperm gene expression associated with fertilization.