RNA‐guided endonuclease‐driven mutagenesis in tobacco followed by efficient fixation of mutated sequences in doubled haploid plants

Background: Customizable endonucleases are providing an effective tool for genome engineering. The resulting primary transgenic individuals 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 depends not only on meiotic segregation but also on the frequency of mutated germline cells in the chimeric mother plant. Results: RNA-guided endonuclease-mediated mutagenesis was targeted to the green fluorescent protein gene (gfp) harboured by a transgenic tobacco line. Upon retransformation using agfp-specific endonuclease 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. The majority of these mutations proved heritable by each of the three propagation systems used. Regeneration fromin vitrocultured embryogenic pollen allowed for homozygous mutants to be produced more efficiently than via sexual reproduction. The recovery of mutations that were not found among sexually produced progeny was shown to be achievable through vegetative plant propagationin vitro. In addition, a number of mutations not detected in the primary gRNA/Cas9-expressing plants were uncovered in the progeny, irrespective of the mode of propagation. Conclusion: Regeneration from embryogenic pollen culture provides a convenient method to rapidly generate a variety of genetically fixed mutants following site-directed mutagenesis. Induced mutations that are not sexually transmitted can be recovered through vegetative plant regeneration from somatic tissue.