Optimizing Visualization of Pollen Tubes in Wheat Pistils

Successful pollination and fertilization are crucial for grain setting in cereals. Wheat is an allohexaploid autogamous species. Due to its evolutionary history, the genetic diversity of current bread wheat (<i>Triticum aestivum</i>) cultivars is limited. Introducing favorable alleles from related wild and cultivated wheat species is a promising breeding strategy for resolving this issue. However, wide hybridization between bread wheat and its relatives is hampered by the presence of suppressor genes and difficulties in crossing. Optimized methods for observing pollen tubes are essential for understanding the mechanism of crossability between wheat and its relatives. Here, we improved the crossing procedure between bread wheat and rye (<i>Secale cereale</i>) and established an optimized protocol for visualizing pollen tube behavior. Crossing via detached spike culture significantly enhanced crossing efficiency and phenotypic stability. A combination of canonical aniline blue staining and optimized clearing and sectioning allowed us to visualize pollen tube behavior. The proportion of rye pollen tubes reaching the micropyle was lower than that for pollen tubes germinated on the stigmatic hair, explaining why the hybrid seed-setting rate was approximately 75% instead of 100%. This method sheds light on wide hybridization through deeper visualization of the insides of pistils.