Soybean pollen-tube pathway transformation Abstract. The interest in developing tissue culture-independent geneStihcoturaentsfaolr. mation methods for plants has been growing. The pollen-tube pathway transformation technique is one method; however, this method is controversial because it is difficult to duplicate and produces insufficient molecular evidence to confirm transformation. Our objective was to evaluate the robustness of the soybean pollen-tube pathway technique (Glycine max L. Merr.). Solutions of purified DNA constructs carrying a bar marker gene and a gus reporter gene or a gene of interest (npk1) were applied to severed styles of flowers 6-8h after self-pollination. The experiment was repeated 3 summers in the field, in which 4 DNA constructs and 7 soybean genotypes were tested. A total of 4793 progeny seeds were harvested from 5590 individually treated soybean flowers. All seeds were germinated and screened for transformants with herbicide spray, histochemical GUS assay, and Southern blot analysis. Although 2% of progenies showed partial resistance to the herbicide, no positive plants were identified from GUS assay and Southern analysis. Our results indicate that soybean pollen-tube pathway transformation is not reproducible.