Targeted mutagenesis using zinc-finger nucleases in perennial fruit trees

Planta. 2015 Apr;241(4):941-51. doi: 10.1007/s00425-014-2224-x. Epub 2014 Dec 21.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Endonucleases / genetics*
  • Ficus / enzymology
  • Ficus / genetics*
  • Fruit / enzymology
  • Fruit / genetics
  • Gene Expression
  • Genes, Reporter
  • Genome, Plant / genetics*
  • Genomics
  • Malus / enzymology
  • Malus / genetics*
  • Mutagenesis, Site-Directed / methods*
  • Mutation
  • Plant Proteins / genetics
  • Plants, Genetically Modified
  • Sequence Alignment
  • Zinc Fingers / genetics

Substances

  • Plant Proteins
  • Endonucleases