title: RNA-Guided Cas9-Induced Mutagenesis in Tobacco Followed by Efficient Genetic Fixation in Doubled Haploid Plants.
abstract: Customizable endonucleases are providing an effective tool for genome engineering. The resulting primary transgenic individuals (T0) are typically heterozygous and/or chimeric with respect to any mutations induced. To generate genetically fixed mutants, they are conventionally allowed to self-pollinate, a procedure which segregates individuals into mutant heterozygotes/homozygotes and wild types. The chances of recovering homozygous mutants among the progeny depend not only on meiotic segregation but also on the frequency of mutated germline cells in the chimeric mother plant. InNicotianaspecies, the heritability of Cas9-induced mutations has not been demonstrated yet. RNA-guided Cas9 endonuclease-mediated mutagenesis was targeted to thegreen fluorescent protein(GFP) gene harbored by a transgenic tobacco line. Upon retransformation using aGFP-specific guide RNA/Cas9 construct, the T0plants were allowed to either self-pollinate, or were propagated via regeneration fromin vitrocultured embryogenic pollen which give rise to haploid/doubled haploid plants or from leaf explants that form plants vegetatively. Single or multiple mutations were detected in 80% of the T0plants. About half of these mutations proved heritable via selfing. Regeneration fromin vitrocultured embryogenic pollen allowed for homozygous mutants to be produced more efficiently than via sexual reproduction. Consequently, embryogenic pollen culture provides a convenient method to rapidly generate a variety of genetically fixed mutants following site-directed mutagenesis. The recovery of a mutation not found among sexually produced and analyzed progeny was shown to be achievable through vegetative plant propagationin vitro, which eventually resulted in heritability when the somatic clones were selfed. In addition, some in-frame mutations were associated with functional attenuation of the target gene rather than its full knock-out. The generation of mutants with compromised rather than abolished gene functionality holds promise for future approaches to the conclusive functional validation of genes which are indispensible for the plant.
authors.affiliation: Plant Reproductive Biology, Physiology and Cell Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Germany.
authors.forename: Sindy
authors.initials: S
authors.lastname: Schedel
authors.affiliation: Plant Reproductive Biology, Physiology and Cell Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Pencs
authors.affiliation: Plant Reproductive Biology, Physiology and Cell Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Germany.
authors.forename: Götz
authors.initials: G
authors.lastname: Hensel
authors.affiliation: Plant Reproductive Biology, Physiology and Cell Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Müller
authors.affiliation: Structural Cell Biology, Physiology and Cell Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Germany.
authors.forename: Twan
authors.initials: T
authors.lastname: Rutten
authors.affiliation: Plant Reproductive Biology, Physiology and Cell Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Kumlehn
keywords: genome engineering; haploid technology; mutant fixation; pollen embryogenesis; site-directed mutagenesis