TY - JOUR
AU - White, C.I.
AB - We have analysed the application of positive-negative selection for the selection 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 gene targeting events. One was the chalcone synthase (CHS) gene present as a single copy and the second an hemizygous chromosomally inserted T-DNA containing the hpt gene, conferring resistance to hygromycin, flanked by CHS sequences. The target lines were transformed with replacement-type T-DNA vectors which contained a positive selectable marker flanked by the regions of the CHS gene and a negative selectable marker to counter-select random insertions. As negative marker we used the Escherichia coli codA gene encoding cytosine deaminase, conferring upon the cells sensitivity to 5-flourocytosine (5-FC). Doubly selected transformants represent 1–4% of the primary transformed cells. Targeting events were not found at the chalcone synthase locus nor at the artificial hpt 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 these cells is associated with substantial deletions of the T-DNA molecule from the right-border end.
TI - Positive-negative selection and T-DNA stability in Arabidopsis transformation
JF - Plant Molecular Biology
DO - 10.1023/A:1006192225464
DA - 2004-09-29
UR - https://www.deepdyve.com/lp/springer-journals/positive-negative-selection-and-t-dna-stability-in-arabidopsis-T619oHMP9c
SP - 83
EP - 93
VL - 39
IS - 1
DP - DeepDyve
ER -