ABSTRACT
Low transformation efficiency and high background of non-targeted events are major constraints to gene targeting in plants. We demonstrate here applicability in maize of a system that reduces the constraint from transformation efficiency. The system requires regenerable transformants in which all of the following elements are stably integrated in the genome: (i) donor DNA with the gene of interest adjacent to sequence for repair of a defective selectable marker, (ii) sequence encoding a rare-cutting endonuclease such as I-SceI, (iii) a target locus (TL) comprising the defective selectable marker and I-SceI cleavage site. Typically, this requires additional markers for the integration of the donor and target sequences, which may be assembled through cross-pollination of separate transformants. Inducible expression of I-SceI then cleaves the TL and facilitates homologous recombination, which is assayed by selection for the repaired marker. We used bar and gfp markers to identify assembled transformants, a dexamethasone-inducible I-SceI::GR protein, and selection for recombination events that restored an intact nptII. Applying this strategy to callus permitted the selection of recombination into the TL at a frequency of 0.085% per extracted immature embryo (29% of recombinants). Our results also indicate that excision of the donor locus (DL) through the use of flanking I-SceI cleavage sites may be unnecessary, and a source of unwanted repair events at the DL. The system allows production, from each assembled transformant, of many cells that subsequently can be treated to induce gene targeting. This may facilitate gene targeting in plant species for which transformation efficiencies are otherwise limiting.
