The codA gene as a negative selection marker in Citrus

Abstract

The use of positive selectable marker genes is widespread in plant genetic transformation allowing transgenic cells to grow while repressing non-transgenic cells. Negative selectable markers, on the contrary, allow the repression or ablation of transgenic cells. The codA gene of Escherichia coli encodes cytosine deaminase that hydrolyzes 5-fluorocytosine (5-FC) into the cytotoxic compound 5 fluorouracil. We tested the transgenic expression of the 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. We developed transgenic citrus lines containing the selection cassette, codA::nptII, driven by double enhanced CaMV35S promoter, verified by Southern blot analysis, RT-PCR, DsRed expression and subjected these transgenic lines to a 5-FC sensitivity assay. We found that, while non-transgenic citrus were unaffected by the presence of 5-FC, all of the transformed lines displayed symptoms of toxicity, indicating that the codA gene could be used as a negative selectable marker in Citrus, for post-transformation detection of the removal of undesired sequences.