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Planta

, Volume 241, Issue 4, pp 941–951 | Cite as

Targeted mutagenesis using zinc-finger nucleases in perennial fruit trees

  • Reut Peer
  • Gil Rivlin
  • Sara Golobovitch
  • Moshe Lapidot
  • Amit Gal-On
  • Alexander Vainstein
  • Tzvi Tzfira
  • Moshe A. Flaishman
Original Article

Abstract

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.

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.

Keywords

Targeted genome editing Zinc-finger nuclease (ZFN) Perennial fruit trees Apple (Malus domesticaFig (Ficus carica

Notes

Acknowledgments

This study was supported by the Ministry of Agriculture, Bet Dagan, Israel.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Reut Peer
    • 1
  • Gil Rivlin
    • 1
  • Sara Golobovitch
    • 1
  • Moshe Lapidot
    • 1
  • Amit Gal-On
    • 2
  • Alexander Vainstein
    • 3
  • Tzvi Tzfira
    • 4
  • Moshe A. Flaishman
    • 1
  1. 1.Institute of Plant SciencesAgricultural Research OrganizationBet-DaganIsrael
  2. 2.Department of Plant Pathology and Weed ScienceAgricultural Research OrganizationBet-DaganIsrael
  3. 3.Institute of Plant Sciences and Genetics in AgricultureThe Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of JerusalemRehovotIsrael
  4. 4.Department of Life SciencesBen-Gurion University of the NegevBeershebaIsrael

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