Journal
PLANT JOURNAL
Volume 81, Issue 1, Pages 160-168Publisher
WILEY-BLACKWELL
DOI: 10.1111/tpj.12693
Keywords
gene targeting; marker excision; piggyBac transposon; acetolactate synthase; cleistogamy 1; Oryza sativa; technical advance
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Funding
- Ministry of Agriculture, Forestry and Fisheries of Japan [PGE1001, 23658012, 23310142]
- Grants-in-Aid for Scientific Research [23310142, 23658012] Funding Source: KAKEN
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Precise genome engineering via homologous recombination (HR)-mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR-mediated GT is an extremely rare event, positive-negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re-integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)-tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.
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