The present invention provides assays and methods for efficiently testing a polynucleotide of interest for a phenotype in a root. In some embodiments, the assays and methods include regenerating green tissue that is transgenic for at least one polynucleotide of interest into one or more transgenic plantlets that have at least one transgenic root. Further provided are methods of making a root assay by contacting green tissue with a first rooting medium to produce a plantlet and a plurality of roots. Additionally provided are methods of assaying for insecticidal activity on a live root. Accordingly provided herein is a substantially contamination-free, root bioassay. Further provided are methods of identifying a promoter having activity in a root.