Rapid delivery of Cas9 gene into the tomato cv.‘Heinz 1706’through an optimized Agrobacterium-mediated transformation procedure

Solanum lycopersicum‘Heinz 1706’is a pioneer model cultivar for tomato research,whose whole genome sequence valuable for genomics studies is available.Nevertheless,a genetic transformation procedure for this cultivar has not yet been reported.Meanwhile,various genome editing technologies such as transfection of clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)ribonucleoprotein complexes into cells are in the limelight.Utilizing the Cas9-expressing genotype possessing a reference genome can simplify the verification of an off-target effect,resolve the economic cost of Cas9 endonuclease preparation,and avoid the complex assembly process together with single-guide RNA(sgRNA)in the transfection approach.Thus,this study was designed to generate Cas9-expressing‘Heinz 1706’lines by establishing an Agrobacterium tumefaciens-mediated transformation(ATMT)procedure.Here,we report a rapid and reproducible transformation procedure for‘Heinz 1706’by finetuning various factors:A.tumefaciens strain,pre-culture and co-culture durations,a proper combination of phytohormones at each step,supplementation of acetosyringone,and shooting/rooting method.Particularly,through eluding subculture and simultaneously inducing shoot elongation and rooting from leaf cluster,we achieved a short duration of three months for recovering the transgenic plants expressing Cas9.The presence of the Cas9 gene and its stable expression were confirmed by PCR and qRT-PCR analyses,and the Cas9 gene integrated into the T_(0) plant genome was stably transmitted to T_(1) progeny.Therefore,we anticipate that our procedure appears to ease the conventional ATMT in‘Heinz 1706’,and the created Cas9-expressing‘Heinz 1706’lines are ultimately useful in gene editing via unilateral transfection of sgRNA into the protoplasts.