This technology has surpassed the other genome editing tools, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), as it is more easily and cheaply customized and yields high mutation efficiency in some species (Lowder et al. Variations of CRISPR/Cas9 technology have been applied for genome editing in recent years (Gao 2021). Out of 110 regenerated M 0 plants obtained, 35 (31.8%) were mutated targeting both SP and SP5G genes simultaneously and the editing efficiency was up to 60% in at least one allele in either SP or SP5G genes. We have successfully obtained regenerated plants with a high mutation rate four months after protoplast isolation and transfection. We have identified that the shoot regeneration medium containing 0.1 mg/L IAA and 0.75 mg/L zeatin was the best hormone combination with a regeneration rate of up to 21.3%. In this study, we report the implementation of a transgene-free breeding method for cultivated tomato by CRISPR/Cas9 technology, including the optimization of protoplast isolation and overcoming the obstacle in shoot regeneration from transfected protoplasts.
Shoot regeneration from transfected protoplasts is the major bottleneck in the application of DNA-free genome editing via ribonucleoprotein-based CRISPR/Cas9 method in cultivated tomato. In cultivated tomato ( Solanum lycopersicum), so far only stable Agrobacterium-mediated transformation carrying CRISPR/Cas9 reagents has been established. The application of genome editing as a research and breeding method has provided many possibilities to improve traits in many crops in recent years. We have established a DNA-free genome editing method via ribonucleoprotein-based CRISPR/Cas9 in cultivated tomato and obtained mutant plants regenerated from transfected protoplasts with a high mutation rate.
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