3.2.2. Quantifying pollen deposited on a receptive stigma per visit or unit time
Usually this parameter is measured by counting the number of pollen grains deposited on the stigma per visit or unit time, regardless of fertilization success. This method implies the microscopic examination of pollen germination and tube growth in the stigma and style. Different stains that stain pollen grains differentially from the surrounding stigmatic tissues can be used. Usually the stigma is gently squashed under a coverslip after staining to better visualize the pollen grains. If needed, stigmas can be fixed in FAA (formaldehyde - acetic acid - 70% ethanol [1:1:18]), 4% paraformaldehyde, glutaraldehyde (2.5% glutaraldehyde in 0.03 mol/L phosphate buffer), 3:1 (v/v) ethanol – acetic acid, or just 70% ethanol and stored at 4ºC for later examination. However, it should be taken into account that before germination, pollen needs to adhere to the stigma and hydrate. Fixing can remove non-adhered pollen grains and consequently the estimate of pollen load may be lower than if fresh stigmas were analysed. In some cases, softening the fixed stigmas should be performed before staining and squashing; this can be done by autoclaving the samples at 1 Kg / cm-2 for 10 to 20 min in 5% (w/v) sodium sulphite and rinsing in distilled water or, alternatively, 1M NaOH can be used for 1 h following a rinse in distilled water. Each of the following methods is acceptable for determining number of pollen grains and extent of their germination.
- Epifluorescence microscopy. This is the most widely used method for visualizing pollen tubes. Stain the pollen grains and pollen tubes with aniline blue (specific for callose, a polysaccharide present in pollen tube walls and plugs produced in pollen tubes of most Angiosperms) (Fig. 7) and observe under ﬂuorescence microscopy. The usual mix is 0.1% (v/v) aniline blue in 0.3 M K3PO4 (Linskens and Esser, 1957). The observer can directly determine the percentage pollen germination on the stigma.
- Light microscopy. Different
methods are available that do not require epifluorescence:
a. Methyl green and Phloxine B (Dafni et al., 2005). Non-germinating grains stain dark brown-red, whereas in germinating pollen grains, the empty grains stain green and the pollen tubes red.
b. Stain with 1% basic fuchsin: 1% fast green (4:1) (Kearns and Inouye, 1993). De-stain and soften the tissue in lactic acid for 12 hours and then squash the tissue under a coverslip. Pollen tubes stain maroon and the background remains white.
c. Acetocarmine/basic fuchsin (Kearns and Inouye, 1993). Add a drop of acetocarmine, followed by a drop of 3% aqueous basic fuchsin and de-stain with a drop of absolute ethanol. Pollen cytoplasm stains red.
- Scanning electron microscopy
can be used to visualize the whole stigmatic surface, but this is a more
time-consuming and complicated than light microscopy.
Fig. 7. Pollen germination in vitro. Left (A): pollen from Japanese plum stained with aniline blue. Right (B): pollen from sweet cherry, unstained.