Optimization of Agrobacterium-mediated transformation and regeneration for CRISPR/Cas9 genome editing of commercial tomato cultivars

Tomato (Solanum lycopersicum) is the second most important horticultural crop worldwide that is widely used as a model plant in genetic manipulation of Solanaceae. CRISPR/Cas9 system has been successfully utilized in several studies for genome edition of model tomato cultivars. However, these genome editing systems should be also optimized for commercial tomato cultivar for direct application of genome editing in field conditions. In this study, we have optimized an Agrobacterium-mediated gene transfer and regeneration system for CRISPR/Cas9 genome editing in two commercial tomato cultivars for the first time. The effect of explant type, genotype, pre-transformation time, Agrobacterium concentration, infection time, and different co-culture periods of bacteria were evaluated to optimize the regeneration and transformation parameters. The highest regeneration capacity of 83% was obtained from cotyledons of Crocker incubated in a medium supplemented with BA (3 mg/L) and IAA (0.1 mg/L). The maximum transformation frequency was obtained by using the following parameters: cotyledon explants of commercial Crocker cultivar that were left for 2 days of pre-transformation incubation, infected with Agrobacterium for 10 min at a concentration of OD600 of 0.6 and co-cultivated with Agrobacterium cells for 48 h. CRISPR/Cas9 system was tested with two gRNAs targeting the phytoene desaturase gene. Fully albino and chimeric plants were successfully produced with optimized transformation and culture conditions in up to 71% of all regenerated plants. In the current study, we optimized the implementation of the CRISPR/Cas9 technique in a commercial tomato cultivar and our method will enable breeders to make necessary changes in traits of interest to improve tomato crops for commercial applications.