In flowering plants, each pollen grain forms two highly reduced, non-motile sperm cells, either at anthesis (tricellular pollen) or later, during pollen tube growth (bicellular pollen). Sperm cells are isolated from the tricellular pollen of Plumbago zeylanica by bursting the grains using 10mM MOPS with 0.8M mannitol (pH 4.6) and collected using centrifugation or a micropipette. Sperm cells of Nicotiana tabacum are formed only after 8 or more hours of pollen tube growth; therefore, styles were hand pollinated, tubes were allowed to grow several hours, and then cut styles were immersed in a liquid pollen tube growth medium until tubes grew out of the cut end. Osmotic release of sperm cells was conducted in 10mM MOPS 0.5M mannitol (pH 5.4). During passage in the tube, non-motile sperm cells are conveyed within a surrounding pollen plasma membrane, principally through actin-myosin interactions. Using immunogold electron microscopy, anti-myosin label is found on the cytoplasmic face of the surrounding pollen plasma membrane, but does not appear to occur on the sperm cell surface in either Plumbago or tobacco. Newly-formed, isolated sperm cells of tobacco infrequently undergo non-specific, spontaneous fusion. Sperm cells isolated later lose this ability through apparent surface changes, though fusion may still be induced through use of cellulase and pectinase, calcium or polyethylene glycol. Late in tobacco pollen tube growth, the normally isomorphic sperm cells diverge in size. The sperm cell associated with the vegetative nucleus is consistently larger in tobacco, but smaller in Plumbago. Tobacco sperm cells appear to be clearly dimorphic at the completion of pollen tube growth despite their prior isomorphic appearance. The presence of sperm dimorphism in this well-described model for sperm isomorphism suggests that sperm dimorphism may be more prevalent in angiosperms than was previously thought.

Key words: cell fusion, Nicotiana, Plumbago, sperm cell isolation, tobacco