3.2.4. Evaluating pollen germination and pollen tube growth in vivo
The most common procedure is to study pollinated stigmas and styles in squashed preparations stained with aniline blue (specific for callose) and observed under ﬂuorescence microscopy (as described in section 3.1.). The tissues have to be softened, usually in 5% sodium sulfite to allow squashing, and the time of softening is species dependent; it is advisable to start processing the samples overnight and, if needed, the samples can be autoclaved at 1 Kg / cm-2 for 10 min in 5% (w/v) sodium sulphite or placed in 1M NaOH for 1 h. Depending on the species, varying concentration of sodium hydroxide (from 1N to 4N) at different temperatures (ambient temperature overnight or 60ºC for an hour) can also be tried for softening. For staining, 0.1% (v/v) aniline blue in 0.3 M K3PO4 (Linskens and Esser, 1957) can be used and the observations made with a fluorescence microscope. Pollen tube walls and the callose plugs produced by growing pollen tubes show a distinct fluorescence signal (Kearns and Inouye, 1993). This test is also useful to determine the presence of gametophytic self-incompatibility in which incompatible pollen tubes get arrested during pollen tube growth in the style.