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Peer, R., Institute of Plant Sciences, Agricultural Research Organization, P.O. Box 6, Bet-Dagan, Israel
Rivlin, G., Institute of Plant Sciences, Agricultural Research Organization, P.O. Box 6, Bet-Dagan, Israel
Golobovitch, S., Institute of Plant Sciences, Agricultural Research Organization, P.O. Box 6, Bet-Dagan, Israel
Lapidot, M., Institute of Plant Sciences, Agricultural Research Organization, P.O. Box 6, Bet-Dagan, Israel
Gal-On, A., Department of Plant Pathology and Weed Science, Agricultural Research Organization, P.O. Box 6, Bet-Dagan, Israel
Vainstein, A., Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel
Tzfira, T., Department of Life Sciences, Ben-Gurion University of the Negev, Beersheba, Israel
Flaishman, M.A., Institute of Plant Sciences, Agricultural Research Organization, P.O. Box 6, Bet-Dagan, Israel
Genome editing is a powerful tool for precise gene manipulation in any organism; it has recently been shown to be of great value for annual plants. Classical breeding strategies using conventional cross-breeding and induced mutations have played an important role in the development of new cultivars in fruit trees. However, fruit-tree breeding is a lengthy process with many limitations. Efficient and widely applied methods for targeted modification of fruit-tree genomes are not yet available. In this study, transgenic apple and fig lines carrying a zinc-finger nuclease (ZFNs) under the control of a heat-shock promoter were developed. Editing of a mutated uidA gene, following expression of the ZFN genes by heat shock, was confirmed by GUS staining and PCR product sequencing. Finally, whole plants with a repaired uidA gene due to deletion of a stop codon were regenerated. The ZFN-mediated gene modifications were stable and passed onto regenerants from ZFN-treated tissue cultures. This is the first demonstration of efficient and precise genome editing, using ZFN at a specific genomic locus, in two different perennial fruit trees—apple and fig. We conclude that targeting a gene in apple or fig with a ZFN introduced by transient or stable transformation should allow knockout of a gene of interest. Using this technology for genome editing allows for marker gene-independent and antibiotic selection-free genome engineering with high precision in fruit trees to advance basic science as well as nontransgenic breeding programs.Main conclusion: Targeting a gene in apple or fig with ZFN, introduced by transient or stable transformation, should allow genome editing with high precision to advance basic science and breeding programs. © 2014, Springer-Verlag Berlin Heidelberg.
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Targeted mutagenesis using zinc-finger nucleases in perennial fruit trees
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Peer, R., Institute of Plant Sciences, Agricultural Research Organization, P.O. Box 6, Bet-Dagan, Israel
Rivlin, G., Institute of Plant Sciences, Agricultural Research Organization, P.O. Box 6, Bet-Dagan, Israel
Golobovitch, S., Institute of Plant Sciences, Agricultural Research Organization, P.O. Box 6, Bet-Dagan, Israel
Lapidot, M., Institute of Plant Sciences, Agricultural Research Organization, P.O. Box 6, Bet-Dagan, Israel
Gal-On, A., Department of Plant Pathology and Weed Science, Agricultural Research Organization, P.O. Box 6, Bet-Dagan, Israel
Vainstein, A., Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel
Tzfira, T., Department of Life Sciences, Ben-Gurion University of the Negev, Beersheba, Israel
Flaishman, M.A., Institute of Plant Sciences, Agricultural Research Organization, P.O. Box 6, Bet-Dagan, Israel
Targeted mutagenesis using zinc-finger nucleases in perennial fruit trees
Genome editing is a powerful tool for precise gene manipulation in any organism; it has recently been shown to be of great value for annual plants. Classical breeding strategies using conventional cross-breeding and induced mutations have played an important role in the development of new cultivars in fruit trees. However, fruit-tree breeding is a lengthy process with many limitations. Efficient and widely applied methods for targeted modification of fruit-tree genomes are not yet available. In this study, transgenic apple and fig lines carrying a zinc-finger nuclease (ZFNs) under the control of a heat-shock promoter were developed. Editing of a mutated uidA gene, following expression of the ZFN genes by heat shock, was confirmed by GUS staining and PCR product sequencing. Finally, whole plants with a repaired uidA gene due to deletion of a stop codon were regenerated. The ZFN-mediated gene modifications were stable and passed onto regenerants from ZFN-treated tissue cultures. This is the first demonstration of efficient and precise genome editing, using ZFN at a specific genomic locus, in two different perennial fruit trees—apple and fig. We conclude that targeting a gene in apple or fig with a ZFN introduced by transient or stable transformation should allow knockout of a gene of interest. Using this technology for genome editing allows for marker gene-independent and antibiotic selection-free genome engineering with high precision in fruit trees to advance basic science as well as nontransgenic breeding programs.Main conclusion: Targeting a gene in apple or fig with ZFN, introduced by transient or stable transformation, should allow genome editing with high precision to advance basic science and breeding programs. © 2014, Springer-Verlag Berlin Heidelberg.
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