FERTILIZATION IN NICOTIANA-TABACUM - ULTRASTRUCTURAL ORGANIZATION OF PROPANE-JET-FROZEN EMBRYO SACS IN-VIVO

Ovules of Nicotiana tabacum L. were cryofixed with a propane-jet freez er and freeze-substituted in acetone to examine technique-dependent ch anges in pre- and post-fertilization embryo sacs using rapidly frozen material. Freezing quality was acceptable in 10% of the embryo sacs in the partially dissected ovules, with ice-crystal damage frequently ev ident in vacuoles and nuclei. One of the two synergids begins to degen erate before pollen-tube arrival in cryofixed material, with breakdown of the plasma membrane and large chalazal vacuole delayed until the p enetration of the pollen tube. Early synergid degeneration involved ch aracteristic increases in cytoplasmic electron density and the generat ion of cytoplasmic bodies to the intercellular space through ''pinchin g-off''. Upon pollen-tube arrival, the male gametes are released throu gh a terminal aperture into the degenerate synergid. Sperm cells under go morphological alteration before gametic fusion: their mitochondrial electron density increases, the endoplasmic reticulum dilates, cytopl asm becomes finely vacuolated and the surrounding pollen plasma membra ne is lost, causing the sperm cells and vegetative nucleus to dissocia te. Discharge of the pollen tube results in the formation of numerous enucleated cytoplasmic bodies which are either stripped or shed from s perm cells and pollen-tube cytoplasm. Two so-called X-bodies are found in the degenerate synergid after pollen-tube penetration: the presume d vegetative nucleus occurs at the chalazal end and the presumed syner gid nucleus near the micropylar end.