Approaches to minimize variation of transgene expression in plants. Molecular Breeding 16: 79 – 91

The development of genetic transformation methods has led to numerous applications of transgenic plants, including the improvement of certain crop traits, the high-level production of valuable proteins, and the use of transgenic plants as a tool to elucidate gene function. However, considerable variation of transgene expression is often observed within populations of transgenic plants transformed with the same transgene construct and under identical transformation conditions. This inter-transformant variation of transgene expression highly complicates phenotype analyses and the production of commercial crops with stable and predictable transgene performance. To reduce cost, labor, and interpretational flaws, multiple efforts are being directed toward achieving stable expression of transgenes with an expected level of expression. Various factors are thought to influence this variation of transgene expression in plants, including inconsistency of transgene copy numbers, the transgene insertion site, and RNA silencing in the host plant. We recently reviewed the current methodologies to minimize inter-individual variation of transgene expression in nuclear transformed plants.1 This review expounds on different transformation methods, recombinase-mediated approaches, gene targeting, optimization of transgene cassettes, viral suppressor genes, and RNA-silencing mutants. We describe how a widely-used strategy to obtain predictable transgene expression levels is the generation of single-copy transformants, as it is generally believed that the copy number of the transgene influences the level of transgene expression. Several reports suggest that the transgene copy number locus may depend on the transformation method used (Agrobacteriu