FIXATION INDUCES DIFFERENTIAL TIP MORPHOLOGY AND IMMUNOLOCALIZATION OF THE CYTOSKELETON IN POLLEN TUBES

The effects of chemical fixation on tip morphology and immunolocalization of the cytoskeleton in Tradescantia virginiana pollen tubes were evaluated using four different fixation protocols differing in fixative type/concentration, fixation time, buffer system and additives. Apical regions were much more sensitive to fixation manipulations than more basipetal areas. The presence of the calcium chelator EGTA at 5 mM led to tip rupture and/or swelling in over 80% of germinated grains. However, low or no EGTA levels during fixation resulted in poor immunolocalizations, although tips had more normal morphology. Double fixation in which pollen tubes were first treated for a short period with higher fixative and lower EGTA (0.5 mM) concentrations, followed by lower fixative and higher EGTA (5.0 mM) concentrations, resulted in both improved preservation of pollen tube tip morphology and microfilament/microtubule localizations.