title: True-breeding targeted gene knock-out in barley using designer TALE-nuclease in haploid cells.
abstract: Transcription activator-like effector nucleases (TALENs) are customizable fusion proteins able to cleave virtually any genomic DNA sequence of choice, and thereby to generate site-directed genetic modifications in a wide range of cells and organisms. In the present study, we expressed TALENs in pollen-derived, regenerable cells to establish the generation of instantly true-breeding mutant plants. A gfp-specific TALEN pair was expressed via Agrobacterium-mediated transformation in embryogenic pollen of transgenic barley harboring a functional copy of gfp. Thanks to the haploid nature of the target cells, knock-out mutations were readily detected, and homozygous primary mutant plants obtained following genome duplication. In all, 22% of the TALEN transgenics proved knocked out with respect to gfp, and the loss of function could be ascribed to the deletions of between four and 36 nucleotides in length. The altered gfp alleles were transmitted normally through meiosis, and the knock-out phenotype was consistently shown by the offspring of two independent mutants. Thus, here we describe the efficient production of TALEN-mediated gene knock-outs in barley that are instantaneously homozygous and non-chimeric in regard to the site-directed mutations induced. This TALEN approach has broad applicability for both elucidating gene function and tailoring the phenotype of barley and other crop species.
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, Gatersleben, Germany.
authors.forename: Maia
authors.initials: M
authors.lastname: Gurushidze
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, Gatersleben, Germany.
authors.forename: Goetz
authors.initials: G
authors.lastname: Hensel
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, Gatersleben, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Hiekel
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, Gatersleben, Germany.
authors.forename: Sindy
authors.initials: S
authors.lastname: Schedel
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, Gatersleben, Germany; Institute of Genetics and Biophysics, Naples, Italy.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Valkov
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, Gatersleben, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Kumlehn
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D001947:Breeding; D004720:Endonucleases / Q000235:genetics*; D055786:Gene Knockout Techniques; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006238:Haploidy; D006720:Homozygote; D001467:Hordeum / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D004720:Endonucleases