Agrobacterium-Mediated Transformation of Brachypodium distachyon

Abstract

Brachypodium distachyon is an excellent model system for the grasses and has been adopted as a research organism by many laboratories around the world. It has all of the biological traits required for a model system, including small stature, short life cycle, small genome, simple growth requirements, and a close relationship to major crop plants (cereals). In addition, numerous resources have been developed for working with this species, including genome sequences for many lines, sequenced mutant collections, and a large, freely available germplasm collection. Fortunately, among grasses B. distachyon is one of the most easily transformed species, an absolute necessity for a model system. Agrobacterium-mediated transformation is the preferred method to transform plants because it usually results in simple insertions of target DNA. In this article, we describe a method for Agrobacterium-mediated transformation of the inbred B. distachyon lines Bd21 and Bd21-3. Embryogenic callus induced from immature embryos is co-cultivated with Agrobacterium tumefaciens strain AGL1 or Agrobacterium rhizogenes strain 18r12v. Hygromycin and paromomycin are used as selective agents, with comparable transformation efficiencies (defined as the percentage of co-cultivated callus that produce transgenic plants) of 40% to 70%. It takes 20 to 30 weeks to obtain T₁ seeds starting from the initial step of dissecting out immature embryos. This protocol has been shown to be efficient and facile in several studies that resulted in the creation of over 22,000 T-DNA mutants. © 2019 by John Wiley & Sons, Inc.