期刊
PLANT CELL REPORTS
卷 41, 期 9, 页码 1843-1852出版社
SPRINGER
DOI: 10.1007/s00299-022-02893-8
关键词
Solanum lycopersicum; Mesophyll protoplast regeneration; CRISPR; Cas9; Ribonucleoprotein; SP and SP5G genes
资金
- Swedish University of Agricultural Sciences
- Nilsson-Ehle-donationerna
In this study, a transgene-free breeding method for cultivated tomato was established using the CRISPR/Cas9 technology. The optimization of protoplast isolation and overcoming the obstacle in shoot regeneration from transfected protoplasts resulted in the successful generation of regenerated plants with a high mutation rate.
Key message 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. The application of genome editing as a research and breeding method has provided many possibilities to improve traits in many crops in recent years. In cultivated tomato (Solanum lycopersicum), so far only stable Agrobacterium-mediated transformation carrying CRISPR/Cas9 reagents has been established. 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 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. 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%. We have successfully obtained regenerated plants with a high mutation rate four months after protoplast isolation and transfection. 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.
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