Enrichments of gene replacement events by Agrobacterium-mediated recombinase-mediated cassette exchange

We report recombinase-mediated cassette exchange (RMCE), which can permit integration of transgenes into pre-defined chromosomal loci with no co-expressed marker gene by using Agrobacterium-mediated transformation. Transgenic tobacco plants which have a single copy of negative marker genes (codA) at target loci in heterozygous and homozygous conditions were used for gene exchange by the RMCE method. By negative selection, we were able to obtain five heterozygous and four homozygous transgenic plants in which the genes were exchanged from 64 leaf segments of heterozygous and homozygous target plants, respectively. Except for one transgenic plant with an extra copy, the other eight plants had only a single copy of marker-free transgenes, and no footprint of random integrated copies was detected in half of the eight plants. The RMCE re-transformation frequencies were calculated as 6.25 % per explant and were approximately the same as the average percentage of intact single-copy transformation events for standard tobacco Agrobacterium-mediated transformation.