Optimization of tissue culture and Cas9 transgene expression in tomato: A step towards CRISPR/Cas9-based genetic improvement.

Tomato (Solanum lycopersicum L.) is an essential source of antioxidants and a prime candidate for bioengineering experiments. Many studies have aimed to improve tomatoes using CRISPR/Cas9 technology; however, the success rate is limited due to the lack of efficient regeneration and genetic modification techniques. Here, we report an efficient regeneration and transformation procedure focused on developing efficient Cas9 gene transgenic tomato plants using the Agrobacterium tumefaciens strain LBA4404 harbouring pCRISPR/Cas9TK2-NIC binary vector. We optimized the concentrations and combinations of growth hormones to promote direct shoot and root regeneration via hypocotyl explants. We found that MS medium 2.0 mg/l Zeatin (Zn) + 1.5 mg/l Indole -3- acetic acid (IAA) + 0.3 mg/l Benzyl amino purine (BAP) was preeminent for shoot regeneration medium, and 0.5 mg/l BAP+ 0.1 mg/l IAA was appropriate for root regeneration. Cas9 transgenes in the tomato genome of putative tomato plants were validated using various methods, including polymerase chain reaction (PCR), and confirmed via Southern blotting. The developed protocol showed improved regeneration and transformation efficiencies in tomatoes of 88 % and 54 %, respectively. In this study, we successfully established a gene delivery platform for tomatoes using the CRISPR/Cas9 system.