Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a Root-inducing (Ri) plasmid induces the formation of hairy roots at the wounding site after the transfer of T-DNA from the Ri plasmid into the plant genome. The protocol describes in detail the procedure of the injection-based hairy root induction in Brassica napus DH12075 and Arabidopsis thaliana Col-0. The hairy roots may be used to analyze a transgene of interest or processed for the generation of transgenic plants. Regeneration medium containing cytokinin 6-benzylaminopurine (5 mg/L) and auxin 1-naphthaleneacetic acid (8 mg/L) successfully elicits shoot formation in both species. The protocol covers the genotyping and selection of regenerants and T1 plants to obtain plants carrying a transgene of interest and free of T-DNA from the Ri plasmid. An alternative process leading to the formation of a composite plant is also depicted. In this case, hairy roots are kept on the shoot (instead of the natural roots), which enables the study of a transgene in hairy root cultures in the context of the whole plant.