A protocol for Agrobacterium-mediated transformation of Brachypodium distachyon community standard line Bd21.
Brachypodium distachyon is a novel model system for structural and functional genomics studies of temperate grasses because of its biological and genetic attributes. Recently, the genome sequence of the community standard line Bd21 has been released and the availability of an efficient transformation system is critical for the discovery and validation of the function of Brachypodium genes. Here, we provide an improved procedure for the facile and efficient Agrobacterium-mediated transformation of line Bd21. The protocol relies on the transformation of compact embryogenic calli derived from immature embryos using visual and chemical screening of transformed tissues and plants. The combination of green fluorescent protein expression and hygromycin resistance enables early identification of transformation events and drastically reduces the quantity of tissue to be handled throughout the selection process. Approximately eight independent fully developed transgenic Bd21 plants can be produced from each immature embryo, enabling the generation of thousands of T-DNA lines. The process--from wild-type seeds to transgenic T(1) seeds--takes approximately 8 months to complete.
Department of Crop Genetics, John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK.
Sílvia C
SC
Alves
Barbara
B
Worland
Vera
V
Thole
John W
JW
Snape
Michael W
MW
Bevan
Philippe
P
Vain
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D030821:Plants, Genetically Modified / Q000196:embryology; D006109:Poaceae / Q000196:embryology / Q000235:genetics*; D012639:Seeds / Q000235:genetics; D014170:Transformation, Genetic