New insights to a MYB candidate gene for the induction of male sterility in Petunia x hybrida through a CRISPR-edited DNA-free by transient transformation system

The inability of a plant to produce pollen, male gametes or functional anthers is known as male sterility (MS). The introduction of the MS trait into parental lines in plant breeding programs is an important task in many horticultural crops, to reduce the cost of hybrid seed production and to ensure varietal purity for the generation of F1 hybrids. In ornamentals, the introgression of MS traits for the production of F1 hybrids has not received much attention because propagation is usually done by agamic methods. However, vegetative propagation allows the direct propagation and commercialization of male-sterile ornamentals, which could be very beneficial for several reasons, such as for example the removing pollen allergens. Recent advances in genome editing (GE) technology, based on CRISPR/Cas-system, have ushered in a new era, also linked to progress in the acquisition of first-generation non-transgenic modified plants and the development of foreign DNA-free editing techniques. Based on these assumptions, we describe the setting of the initial stages for the development of a CRISPR/Cas9-based breeding strategy for the implementation of the MS trait in Petunia x hybrida hort. ex E. Vilm, a model system of the Solanaceae family and always a point of reference in ornamental research. The general objective is to create a temporary protoplast transfection system mediated by CRISPR/Cas-edited DNA-free plant material. This will be achieved by direct delivery of a pre-assembled ribonucleoprotein (RNP) complex consisting of pure Cas9 protein and in vitro synthesized single guide RNA (sgRNA) molecules. In particular, we report the results and ongoing work on targeting a Petunia MYB candidate gene: several sgRNAs were then identified and selected based on their potential ability to target the investigated sequence at the CDS level and the predicted TSS region. In addition, an expression kinetic analysis of target gene in vegetative tissues and flower buds at different phenological stages has suggested its potential importance in the formation and development of relative anther tissues and pollen.