Targeted mutagenesis using zinc-finger nucleases in perennial fruit trees
- 793 Downloads
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.
KeywordsTargeted genome editing Zinc-finger nuclease (ZFN) Perennial fruit trees Apple (Malus domestica) Fig (Ficus carica)
This study was supported by the Ministry of Agriculture, Bet Dagan, Israel.
- Brown SK (2012) Apple (Malus × domestica). In: Badnes ML, Byrne D (eds) Fruit breeding. Handbook of plant breeding, vol 8. Springer, US, pp 329–367Google Scholar
- Curtin SJ, Zhang F, Sander JD, Haun WJ, Starker C, Baltes NJ, Reyon D, Dahlborg EJ, Goodwin MJ, Coffman AP, Dobbs D, Joung JK, Voytas DF, Stupar RM (2011) Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases. Plant Physiol 156:466–473CrossRefPubMedCentralPubMedGoogle Scholar
- Flaishman MA, Rodov V, Stover E (2008) The fig: botany, horticulture, and breeding. Hortic Rev 34:113–196Google Scholar
- Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui X, Meng X, Vincent A, Lam S, Michalkiewicz M, Schilling R, Foeckler J, Kalloway S, Weiler H, Ménoret S, Anegon I, Davis GD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jacob HJ, Buelow R (2009) Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325:433CrossRefPubMedCentralPubMedGoogle Scholar
- Hanke MV, Reidel M, Reim S, Flachowsky H (2007) Analysis of tissue uniformity in transgenic apple plants. Acta Hortic 738:301–306Google Scholar
- Janick J, Moore JN (eds) (1996) Fruit breeding, tree and tropical fruits, vol 1. Wiley, New YorkGoogle Scholar
- Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO 6:3901–3907Google Scholar
- Norelli J, Mills J, Aldwinckle H (1996) Leaf wounding increases efficiency of Agrobacterium-mediated transformation of apple. HortScience 31:1026–1027Google Scholar
- Tripathi S, Suzuki J, Gonsalves D (2007) Development of genetically engineered resistant papaya for papaya ringspot virus in a timely manner. In: Ronald PC (ed) Plant-pathogen interactions. Humana Press, Totowa, pp 197–240Google Scholar