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Title: Use of Agrobacterium rhizogenes strain 18r12v and paromomycin selection for transformation of Brachypodium distachyon and Brachypodium sylvaticum

item Collier, Ray
item BRAGG, JENNIFER - University Of California
item HERNANDEZ, BRYAN - University Of California
item VOGEL, JOHN - Joint Genome Institute
item Thilmony, Roger

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/9/2016
Publication Date: 5/24/2016
Citation: Collier, R.A., Bragg, J., Hernandez, B., Vogel, J., Thilmony, R.L. 2016. Use of Agrobacterium rhizogenes strain 18r12v and paromomycin selection for transformation of Brachypodium distachyon and Brachypodium sylvaticum. Frontiers in Plant Science. doi: 10.3389/fpls.2016.00716.

Interpretive Summary: Brachypodium distachyon and B. sylvaticum are useful model grass species for economically important crops like wheat, barley and switchgrass. The ability to genetically modify these model species using biotechnology is critical for their use in scientific research. Research reported here demonstrates that a novel strain of plant transformation bacteria, carrying newly designed genetic engineering components, can be used to efficiently generate transformed Brachypodium plants that are resistant to paromomycin, an antibiotic toxic to unmodified plants. In addition, the transformed plants were of high quality and typically contained only the intended DNA sequences. This new method complements existing protocols, enables the efficient generation of transgenic Brachypodium plants, and will facilitate the implementation of novel more sophisticated biotechnology approaches in the future.

Technical Abstract: The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum, but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This study demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticum. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. tumefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation.