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

Abstract

In this study, 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. sylvaticurn. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. turnefaciens 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 genomemore » manipulation strategies which require multiple rounds of transformation.« less

Authors:

Collier, Ray ^[1]; Bragg, Jennifer ^[1]; Hernandez, Bryan T. ^[2]; Vogel, John P. ^[3]; Thilmony, Roger ^[1]

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+ Show Author Affiliations

1. United States Dept. of Agriculture-Agricultural Research Service, Albany, CA, (United States). Crop Improvement and Genetics Research Unit, Western Regional Research Center
2. Univ. of California, Davis, Davis, CA (United States). Dept. of Plant Sciences
3. United States Dept. of Agriculture-Agricultural Research Service, Albany, CA, (United States). Crop Improvement and Genetics Research Unit, Western Regional Research Center; Dept. of Energy, Joint Genome Institute, Walnut Creek, CA (United States)

Publication Date:

Tue May 24 00:00:00 EDT 2016

Research Org.:

United States Dept. of Agriculture-Agricultural Research Service, Albany, CA, (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Science (SC), Joint Genome Institute (JGI)

OSTI Identifier:

1282007

Grant/Contract Number:  

SC0001526; AC02-05CH11231

Resource Type:

Accepted Manuscript

Journal Name:

Frontiers in Plant Science

Additional Journal Information:

Journal Volume: 7; Journal ID: ISSN 1664-462X

Publisher:

Frontiers Research Foundation

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; transformation; Brachypodium; Agrobacterium rhizogenes; paromomycin; tissue culture; callus; selectable marker genes; mediated transformation; plant transformation; particle bombardment; polyubiquitin genes; promoter activity; expression; l.; electroporation; protoplasts