Agrobacterium Protocols

Jennifer N Bragg, Amy Anderton, Rita Nieu, John P Vogel, Jennifer N. Bragg, John P. Vogel, Bragg, Jennifer N., Anderton, Amy, Nieu, Rita, Vogel, John P.

The small grass Brachypodium distachyon has attributes that make it an excellent model for the development and improvement of cereal crops and bioenergy feedstocks. To realize the potential of this system, many tools have been developed (e.g., the complete genome sequence, a large collection of natural accessions, a high density genetic map, BAC libraries, EST sequences, microarrays, etc.). In this chapter, we describe a high-efficiency transformation system, an essential tool for a modern model system. Our method utilizes the natural ability of Agrobacterium tumefaciens to transfer a well-defined region of DNA from its tumor-inducing (Ti) plasmid DNA into the genome of a host plant cell. Immature embryos dissected out of developing B. distachyon seeds generate an embryogenic callus that serves as the source material for transformation and regeneration of transgenic plants. Embryogenic callus is cocultivated with A. tumefaciens carrying a recombinant plasmid containing the desired transformation sequence. Following cocultivation, callus is transferred to selective media to identify and amplify the transgenic tissue. After 2-5 weeks on selection media, transgenic callus is moved onto regeneration media for 2-4 weeks until plantlets emerge. Plantlets are grown in tissue culture until they develop roots and are transplanted into soil. Transgenic plants can be transferred to soil 6-10 weeks after cocultivation. Using this method with hygromycin selection, transformation efficiencies average 42 %, and it is routinely observed that 50-75 % of cocultivated calluses produce transgenic plants. The time from dissecting out embryos to having the first transgenic plants in soil is 14-18 weeks, and the time to harvesting transgenic seeds is 20-31 weeks.