Location: Crop Improvement and Genetics ResearchTitle: The codA gene as a negative selection marker in Citrus
Submitted to: BioMed Central (BMC)Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/11/2015
Publication Date: 6/17/2015
Citation: Thomson, J.G., Stover, E.W., Peixoto De Olivei, M.L. 2015. The codA gene as a negative selection marker in Citrus. BioMed Central (BMC)Biotechnology. 4:264.
Interpretive Summary: Development of gene targeting systems for plants has been facilitated by the use of appropriate negative selectable marker genes to find cells that have undergone site-specific deletions of DNA no longer needed after transformation. The expression of negative selection genes causes either immediate or conditional cell death in transformed cells and therefore allows selection of cells that have lost these marker genes. In many plant species, the codA gene from E.coli, encoding cytosine deaminase, has been successfully used as a negative selection gene in Agrobacterium-mediated transformation protocols. The gene product of codA deaminates cytosine to uracil, and also converts the uracil analog 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). The latter substance is metabolized to compounds that inhibit both RNA and DNA synthesis, resulting in cell death. The 5-FC analog is not toxic when used in media for wild type cells of many plant species. However, cells of those species that are transformed to express the codA gene are sensitive to 5-FC because they convert it to the toxin 5-FU. This paper describes the first use of the codA positive-negative selection in Agrobacterium-mediated transformation of Citrus cells. It is demonstrated that 5-FC does not affect the growth of untransformed plants, whereas including 100 ug/ml of 5-FC in the media is sufficient to inhibit shoot regeneration of internodes from transgenic plants expressing the introduced codA gene. This research provides an important new tool in the use of site-specific recombination to incorporate new genes with precision into the chromosomes of citrus varieties.
Technical Abstract: Background The use of selectable marker genes is widespread in plant genetic transformation allowing transgenic tissue to grow, but not nontransgenic tissues. The selective agent can confer positive or negative selection according to their capacity to promote the growth or death of transformed cells, respectively. The codA gene of E. coli encodes cytosine deaminase that hydrolyzes 5-flurocytosine into the cytotoxic compound 5-fluorouracil. We tested the transgenic expression of bacterial codA gene in citrus as a conditional negative selection marker, with the goal of selecting against plant tissues in which a transgenic cassette has not been successfully removed. Results We produced transgenic plant lines containing the selection cassette, codA::nptII, driven by d35S promoter, and verified gene integration and expression by southern blot analysis, RT-PCR, and DsRed expression. We then subjected internodes of these transgenic lines to a 5-FC shoot regeneration sensitivity assay. We found that, while wild-type plants were unaffected by the presence of 5-FC, all of the transformed lines displayed symptoms of toxicity, indicating that the codA gene could potentially be used as a negative selectable marker for genome modification in citrus. Conclusion The utility of the codA as a negative selectable marker has been successfully established in Citrus, where it can now be employed for precise genomic modifications.