Modest Frequency and Pleiotropic Impacts of Zinc-Finger Mutagenesis in Poplar

Gene flow from recombinant-DNA modified (GMO) trees is major barrier to their public acceptance and regulatory approval. Because many intensively grown trees are vegetatively propagated, complete sexual sterility could be a powerful means to mitigate or prevent gene flow. We tested four pairs of zinc-finger nucleases (ZFNs) as mutagenic agents against the LEAFY and AGAMOUS orthologs in poplar that are expected to be required for sexual fertility. Each of the four ZFN genes was cloned behind a heat-shock promoter for inducible expression and reduced pleiotropic effects. Using Agrobacterium tumefaciens, we transformed more than 21,000 explants. The rate of transformation was substantially reduced by the ZFN constructs; only 391 transgenic shoots were produced (1.8 %).  After heat shock and subsequent development of the transgenic plants, only two events were found to contain mutations; both were 7-bp deletions in one allele at the PtAG2 locus. No mutations were observed at the PtAG1 or PtLFY loci. Our results indicate a mutation rate of zero to 0.3% per explant per allele, among the lowest reported for ZFN mutagenesis in plants. The combined effects of reduced recovery of transgenic plants, and a modest mutation frequency, suggests that ZFNs may be a poor choice for mutagenesis of poplar genes.