title: A bacterial gene codA encoding cytosine deaminase is an effective conditional negative selectable marker in Glycine max.
abstract: Research describes the practical application of the codA negative selection marker in Soybean. Conditions are given for codA selection at both the shooting and rooting stages of regeneration. Conditional negative selection is a powerful technique whereby the absence of a gene product allows survival in otherwise lethal conditions. In plants, the Escherichia coli gene codA has been employed as a negative selection marker. Our research demonstrates that codA can be used as a negative selection marker in soybean, Glycine max. Like most plants, soybean does not contain cytosine deaminase activity and we show here that wild-type seedlings are not affected by inclusion of 5-FC in growth media. In contrast, transgenic G. max plants expressing codA and grown in the presence of more than 200 μg/mL 5-FC exhibit reductions in hypocotyl and taproot lengths, and severe suppression of lateral root development. We also demonstrate a novel negative selection-rooting assay in which codA-expressing aerial tissues or shoot cuttings are inhibited for root formation in media containing 5-FC. Taken together these techniques allow screening during either the regeneration or rooting phase of tissue culture.
authors.affiliation: Department of Plant Sciences, University of California-Davis, Davis, CA, 95616, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Shao
authors.affiliation: Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN, 55108, USA.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Michno
authors.affiliation: Department of Plant Sciences, University of California-Davis, Davis, CA, 95616, USA.
authors.forename: Sara K
authors.initials: SK
authors.lastname: Hotton
authors.affiliation: Western Regional Research Center, USDA-ARS Crop Improvement and Genetics, Albany, CA, 94710, USA.
authors.forename: Ann
authors.initials: A
authors.lastname: Blechl
authors.affiliation: Western Regional Research Center, USDA-ARS Crop Improvement and Genetics, Albany, CA, 94710, USA. James.Thomson@ars.usda.gov.
authors.forename: James
authors.initials: J
authors.lastname: Thomson
mesh_terms: D043525:Cytosine Deaminase / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D005819:Genetic Markers; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics*; D012641:Selection, Genetic; D013025:Glycine max / Q000201:enzymology / Q000235:genetics*
chemical_list: D029968:Escherichia coli Proteins; D005819:Genetic Markers; D043525:Cytosine Deaminase
keywords: 5-Fluorocytosine; Conditional negative selection; Cytosine deaminase; Glycine max; Soybean; codA gene