Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis.
Brachypodium distachyon is a promising model system for the structural and functional genomics of temperate grasses because of its physical, genetic and genome attributes. The sequencing of the inbred line Bd21 (http://www.brachypodium.org) started in 2007. However, a transformation method remains to be developed for the community standard line Bd21. In this article, a facile, efficient and rapid transformation system for Bd21 is described using Agrobacterium-mediated transformation of compact embryogenic calli (CEC) derived from immature embryos. Key features of this system include: (i) the use of the green fluorescent protein (GFP) associated with hygromycin selection for rapid identification of transgenic calli and plants; (ii) the desiccation of CEC after inoculation with Agrobacterium; (iii) the utilization of Bd21 plants regenerated from tissue culture as a source of immature embryos; (iv) the control of the duration of the selection process; and (v) the supplementation of culture media with CuSO4 prior to and during the regeneration of transgenic plants. Approximately 17% of CEC produced transgenic plants, enabling the generation of hundreds of T-DNA insertion lines per experiment. GFP expression was observed in primary transformed Bd21 plants (T0) and their progeny (T1). The Mendelian inheritance of the transgenes was confirmed. An adaptor-anchor strategy was developed for efficient retrieval of flanking sequence tags (FSTs) of T-DNA inserts, and the resulting sequences are available in public databases. The production of T-DNA insertion lines and the retrieval of associated FSTs reported here for the reference inbred line Bd21 will facilitate large-scale functional genomics research in this model system.
Vain
Philippe
P
Department of Crop Genetics, John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK. philippe.vain@bbsrc.ac.uk
Worland
Barbara
B
Thole
Vera
V
McKenzie
Neil
N
Alves
Silvia C
SC
Opanowicz
Magdalena
M
Fish
Lesley J
LJ
Bevan
Michael W
MW
Snape
John W
JW
D018797:Cell Cycle Proteins; D004269:DNA, Bacterial / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D005838:Genotype; D016254:Mutagenesis, Insertional / Q000379:methods*; D006109:Poaceae / Q000145:classification / Q000235:genetics* / Q000382:microbiology; D011110:Polymorphism, Genetic; D012231:Rhizobium / Q000235:genetics* / Q000502:physiology; D020079:Terminal Repeat Sequences / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics*
D018797:Cell Cycle Proteins; C121983:DBF4 protein, mouse; D004269:DNA, Bacterial; C036483:T-DNA