Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene

Transgenic tobacco plants were produced that contained single-copy pART54 T -DNA, with a 35S-uidA gene linked to loxP-flanked kanamycin resistance (npt II) and cytosine deaminase (codA) genes. Retransformation of these plants w ith pCre1 (containing 35S transcribed cre recombinase and hygromycin (hpt) resistance genes) resulted in excision of the loxP-flanked genes from the g enome. Phenotypes of progeny from selfed-retransformed plants confirmed npt II and codA excision and integration of the cre-linked hpt gene. To avoid i ntegration of the hpt gene, and thereby generate plants totally free of mar ker genes, we attempted to transiently express the cre recombinase. Agrobac terium tumefaciens (pCre1) was cocultivated with leaf discs of two pART54-t ransformed lines and shoots were regenerated in the absence of hygromycin s election. Nineteen of 773 (0.25%) shoots showed tolerance to 5-fluorocytosi ne (5-fc) which is converted to the toxic 5-fluorouracil by cytosine deamin ase. 5-fc tolerance in six shoots was found to be due to excision of the lo xP-flanked region of the pART54 T-DNA. In four of these shoots excision cou ld be attributed to cre expression from integrated pCre1 T-DNA, whereas in two shoots excision appeared to be a consequence of transient cre expressio n from pCre1 T-DNA molecules which had been transferred to the plant cells but not integrated into the genome. The absence of selectable marker genes was confirmed by the phenotype of the T-1 progeny. Therefore, through trans ient cre expression, marker-free transgenic plants were produced without se xual crossing. This approach could be applicable to the elimination of mark er genes from transgenic crops which must be vegetatively propagated to mai ntain their elite genotype.