Silene latifolia serves as a model species to study dioecy, the evolution of sex chromosomes, dosage compensation and sex-determination systems in plants. Currently, no protocol for genetic transformation is available for this species, mainly because S. latifolia is considered recalcitrant to in vitro regeneration and infection with Agrobacterium tumefaciens. Using cytokinins and their synthetic derivatives, we markedly improved the efficiency of regeneration. Several agrobacterial strains were tested for their ability to deliver DNA into S. latifolia tissues leading to transient and stable expression of the GUS reporter. The use of Agrobacterium rhizogenes strains resulted in the highest transformation efficiency (up to 4.7% of stable transformants) in hairy root cultures. Phenotypic and genotypic analyses of the T1 generation suggested that the majority of transformation events contain a small number of independent T-DNA insertions and the transgenes are transmitted to the progeny in a Mendelian pattern of inheritance. In short, we report an efficient and reproducible protocol for leaf disc transformation and subsequent plant regeneration in S. latifolia, based on the unique combination of infection with A. rhizogenes and plant regeneration from hairy root cultures using synthetic cytokinins. A protocol for the transient transformation of S.latifolia protoplasts was also developed and applied to demonstrate the possibility of targeted mutagenesis of the sex linked gene SlAP3 by TALENs and CRISPR/Cas9.

Centre of Plant Structural and Functional Genomics Institute of Experimental Botany Academy of Sciences of the Czech Republic 783 71 Olomouc Holice Czech Republic

Department of Genetics Cell Biology and Development Center for Genome Engineering University of Minnesota Minneapolis MN 55455 USA

Department of Plant Developmental Genetics Institute of Biophysics Academy of Sciences of the Czech Republic 61200 Brno Czech Republic

Laboratory of Growth Regulators and Department of Chemical Biology and Genetics Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science Palacký University and Institute of Experimental Botany AS CR Šlechtitelů 11 CZ 783 71 Olomouc Czech Republic