SUBSTRATE-DEPENDENT NEGATIVE SELECTION IN PLANTS USING A BACTERIAL CYTOSINE DEAMINASE GENE

The inability of many higher eukaryotes to convert 5-fluorocytosine to cytotoxic 5-fluorouracil presents the possibility of using the bacter ial cytosine deaminase codA gene for negative selection. In transforme d plant callus, expression of codA results in cell death on 5-fluorocy tosine. In transgenic tobacco and Lotus japonicus plants the substrate -dependent negative marker segregates as a single dominant gene, and o n 5-fluorocytosine CodA+ seedlings stop growing at the early seedling stage. Positive selection of CodA+ tobacco on the pyrimidine biosynthe tic inhibitor N-(phosphonacetyl)-L-aspartate was obtained, by pyrimidi ne salvage from external cytosine. Activity of cytosine deaminase was determined by conversion of labelled cytosine to uracil followed by se paration in thin layer chromatography. The codA marker therefore provi des substrate-dependent negative and positive selection, together with cytosine deaminase reporter activity.