Effectiveness of the bacterial gene codA encoding cytosine deaminase as a negative selectable marker in Agrobacterium-mediated plant transformation

Abstract

as a conditional toxic gene was explored during various stages of plant development and in different Agrobacterium, mediated transformation protocols. To this end, several independent tobacco lines transgenic for coda were isolated and these were tested for their sensitivity to 5-fluorocytosine (5-FC) at different developmental stages. On media supplemented with 5-FC, seedling proliferation was inhibited. Leaves failed completely to regenerate sprouts on 5-FC-containing medium. However, 40% of the shoots regenerated on non-selective medium still formed roots on rooting medium with 5-FC. In all these assays, control plants were unaffected by up to 1 mg ml-1 5-FC. Transformation of a coda and nptll-harbouring T-DNA to tobacco leaf discs did not result in any regenerant using a combined 5-FC and kanamycin selection, indicating that coda does not behave as a cell-autonomous marker here. Nevertheless, transformation of the same T-DNA to tobacco protoplasts resulted in some enrichment of codA-nptll+ calluses using the proper combination of 5-FC and kanamycin for selection. Mixing of codA-containing and codA-lacking tobacco protoplasts revealed that the coda gene may behave as a cell autonomous marker under certain, appropriately chosen conditions, which seems to be in paradox with the total absence of escapes in tissue explant transformation. In all these experiments, 250 μg 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 marker in Agrobacterium-mediated gene targeting protocols of tobacco whereby selection at the shoot regeneration level is the most effective.