*ssakuma@tottori-u.ac.jp

Global warming significantly threatens wheat yield production. The viability of pollen and pistil is pivotal for successful grain sets. However, the methods for observing pollen tube elongation, a key process in fertilization, are yet to be optimized. In the present study, we developed an improved simple method. An intergenic cross, rather than self-pollination, was performed to discriminate the obvious pollen tube signals from other factors in the pollen tube pathway. A bread wheat cultivar was utilized as a female parent and a rye cultivar as a pollen parent.

Two days after emasculating bread wheat, rye pollen was applied on wheat pistils by hand. Pistils were collected two hours after pollination. Pollen tubes were visualized by conventional aniline blue staining. The stained pistil was observed using a fluorescence microscope. Due to the thickness of the ovary of bread wheat (ca. 189 µm), the fluorescence of pollen tubes could not be detected by observation of intact ovary.

When the ovary and pistil were cut in half with a razor before staining, fluorescence from pollen tubes was detected in 74.7% of ovary examined. Chemical clearing of the ovary before staining did not provide any improvement. “Focus stacking” greatly improved the clarity of an image.

The proportion of rye pollen tubes reaching the style base was 97.1 ± 9.7%, and the proportion reaching the inside of the ovary from the base of the pistil was 90.6 ± 11.1%. The seed setting rate in the intergenic cross was 76.6 ± 15%.

This method allows observing pollen tube germination and elongation in the style and ovary, which is useful for analyzing the viability of pollen and pistils.