Cytosine deaminase as a substrate-dependent negative selectable marker in Brassica napus

The enzyme cytosine deaminase, encoded by the codA gene, catalyzes the deam ination of the nontoxic compound 5-fluorocytosine (5-FC) to the highly toxi c compound 5-fluorouracil (5-FU). Cytosine deaminase activity is not found in higher plants and Brassica napus seedlings are unaffected by the presenc e of 5-FC in the growth medium. In codA-transformed B. napus seedlings, exp ression of cytosine deaminase results in a reduction of root and hypocotyl lengths, and a severe suppression of true leaf development. This phenotype is dependent on the presence of the 5-FC substrate and no effects an seen i n plants grown in the absence of the substrate or in sibling plants lacking the transgene. The codA transformants have been assessed over three genera tions of growth and in each generation the transgene is stably inherited an d confers the same 5-FC-sensitive phenotype. Transfer of 5-FC-sensitive see dlings to soil results in the restoration of normal growth in up to 100% of the seedlings. These results indicate that codA is a versatile dominant ma rker gene that can be used effectively in B. napus for substrate-dependent negative selection.