Restricted Pollination for Tracing Individual Pollen Tubes in a Pistil

As one of the essential steps to complete sexual reproduction, a pollen tube is precisely guided to an embryo sac to deliver the sperm cells. This ovule targeting by a pollen tube is one of the essential steps in pollen tube guidance. To assess the ovule targeting ability of the pollen tube from a certain mutant line, comparative analysis of pollen tube behaviors between wild-type and mutant genotypes is important. Here, we provide a protocol that traces all pollen tubes germinated from the quartet tetrad in a pistil by restricted pollination and aniline blue staining. By this analysis, statistical comparison between wild-type and the mutant pollen tube functions under the same in vivo condition is possible.