EFFECTIVENESS OF THE BACTERIAL GENE CODA ENCODING CYTOSINE DEAMINASE AS A NEGATIVE SELECTABLE MARKER IN AGROBACTERIUM-MEDIATED PLANT TRANSFORMATION

The usefulness of the E. coli codA gene encoding cytosine deaminase as a conditional toxic gene was explored during various stages of plant development and in different Agrobacterium-mediated transformation pro tocols. To this end, several independent tobacco lines transgenic for codA were isolated and these were tested for their sensitivity to 5-fl uorocytosine (5-FC) at different developmental stages. On media supple mented with 5-FC, seedling proliferation was inhibited. Leaves failed completely to regenerate sprouts on 5-FC-containing medium. However, 4 0% of the shoots regenerated on non-selective medium still formed root s on rooting medium with 5-FC. In all these assays, control plants wer e unaffected by up to 1 mg ml(-1) 5-FC. Transformation of a codA and n ptII-harbouring T-DNA to tobacco leaf discs did not result in any rege nerant using a combined 5-FC and kanamycin selection, indicating that codA does not behave as a cell-autonomous marker here. Nevertheless, t ransformation of the same T-DNA to tobacco protoplasts resulted in som e enrichment of codA(-) npfII(+) calluses using the proper combination of 5-FC and kanamycin for selection. Mixing of codA-containing and co dA-lacking tobacco protoplasts revealed that the codA gene may behave as a cell autonomous marker under certain, appropriately chosen condit ions, which seems to be in paradox with the total absence of escapes i n tissue explant transformation. In all these experiments, 250 mu g ml (-1) 5-FC was found to be the most optimal for selection. Our results suggest that codA can be successfully used as a negative selectable ma rker in Agrobacterium-mediated gene targeting protocols of tobacco whe reby selection at the shoot regeneration level is the most effective.