Although gene targeting (
GT
) is a useful technology for precise mutagenesis of target sequences, its frequency is quite low. Establishing experimental procedures using a model system will enable us to improve this frequency and apply to
GT
as a universal system. Here, we propose a convenient system with which to evaluate the frequency of site-directed mutagenesis via
GT
using a positive-negative selection method. We constructed a
GT
vector harboring a partial rice
acetolactate synthase gene with mutations conferring bispyribac sodium (BS) tolerance and a gene conferring blasticidin-S tolerance as a positive selection marker. In addition,
diphtheria toxin A subunit gene was used as a negative selection marker to enrich
GT
cells. We regenerated
GT
candidate plants successfully at a frequency of 2.1 putative
GT
events/gram
Agrobacterium-infected callus following dual selection on BS and blasticidin-S. Moreover, molecular analyses confirmed that
GT
events occurred in >80% of regenerated plants. Existing
GT
methods using positive-negative selection require that true putative
GT
events be verified by molecular analysis because of the growth of large numbers of cells in which partial
GT
vectors containing positive selection marker cassettes, but lacking the negative selection marker, have inserted at random loci. In contrast, the present method with dual selection on both BS and blasticidin-S allows direct enrichment of
GT
cells at high frequency without the need for further extensive molecular screening.
抄録全体を表示