Plant breeding is a multidisciplinary science of changing the genetic makeup of plants in order to generate desired traits or characteristics, and thus it can be accomplished through many different techniques ranging from simply selecting plants with desirable traits for propagation to more complex molecular techniques. Both conventional and genetically modified (GM) plant breeding alter or modify the genes of a plant so that a better variety is developed. Breeding using GM tools is achieved for the same reasons as conventional breeding. One prominent distinction is that instead of randomly mixing genes in conventional breeding, which occurs as a result of a sexual cross, a specific gene is directly transferred or selectively inactivated in the new plant variety through GM plant breeding. Site-specific mutagenesis and selection of gene knockout mutants are readily carried out in model plant species, such as Arabidopsis. However, targeted mutation of a specific gene is technically impractical, if not impossible, in most cases. As an alternative approach, RNA interference (RNAi), which is mediated by small interfering RNA (siRNA) and microRNA (miRNA), is routinely employed for targeted silencing of genes in academic and biotechnological studies. Recently, engineered nuclease-based genome editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have been developed to induce site-specific genome modifications in both animals and plants. ZFNs are chimeric DNA restriction enzymes that consist of the nuclease domain of the Fok1 restriction enzyme, which triggers double strand breaks in genomic DNAs, and a custom-designed ZF DNA-binding domain, which guides the ZFNs to specific sequences within genomic DNAs. The double-strand breaks are rejoined by cellular DNA repair machinery, resulting in targeted mutagenesis or targeted gene replacement.
In this work, we employed the ZFN tool to specifically inactivate two flowering genes, such as FCA and GI that also mediate high temperature responses and clock output signaling, respectively, in a bioenergy grass crop, Brachypodium distachyon. We designed extensive sets of ZF recognition sequences that recognize target sequences within the FCA and GI genes. The potential ZFN cassettes were transformed into Brachypodium ecotype Bd21-3. The transformants will be screened to identify those carrying targeted gene mutations. We will also discuss the extension of the ZFN tool to other plant species, including crops.

• Sangmin Lee(Department of Chemistry, Seoul National University)
• Pil Joon Seo(Department of Chemistry, Seoul National University)
• Chung-Mo Park(Department of Chemistry, Seoul National University) Corresponding Author