The development of powerful "omics" technologies has enabled researchers to identify many genes of interest for which comprehensive functional analyses are highly desirable. However, the production of lines which ectopically express recombinant genes, or those in which endogenous genes are knocked down via stable transformation, remains a major bottleneck for the association between genetics and gene function in monocotyledonous crops. Methods of effective DNA transfer into regenerable cells of immature embryos from cereals by means of Agrobacterium tumefaciens have been modified in a stepwise manner. The effect of particular improvement measures has often not been significantly evident, whereas their combined implementation has resulted in meaningful advances. Here, we provide updated protocols for the Agrobacterium-mediated generation of stably transgenic barley, wheat, triticale and maize. Based upon these methods, several hundred independent transgenic lines have been delivered, with efficiencies of inoculated embryos leading to stably transgenic plants reaching 86% in barley, 10% in wheat, 4% in triticale, and 24% in maize.