Positive-negative selection and T-DNA stability in Arabidopsis transformation

We have analysed the application of positive-negative selection for the sel ection of homologous recombination interactions between the chromosome and a T-DNA molecule after transformation of plant cells. Two different genomic loci in a cell suspension of Arabidopsis thaliana were chosen to study gen e targeting events. One was the chalcone synthase (CHS) gene present as a s ingle copy and the second an hemizygous chromosomally inserted T-DNA contai ning the hpr gene, conferring resistance to hygromycin, flanked by CHS sequ ences. The target lines were transformed with replacement-type T-DNA vector s which contained a positive selectable marker flanked by the regions of th e CHS gene and a negative selectable marker to counter-select random insert ions. As negative marker we used the Escherichia coli codA gene encoding cy tosine deaminase, conferring upon the cells sensitivity to 5-flourocytosine (5-FC). Doubly selected transformants represent 1-4% of the primary transf ormed cells. Targeting events were not found at the chalcone synthase locus nor at the artificial hpr locus in a total of 4379 doubly selected calli, corresponding to at least 109 475 individual primary transformants, We show by PCR and Southern analysis that the 5-FC resistance in the majority of t hese cells is associated with substantial deletions of the T-DNA molecule f rom the right-border end.