Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum)

Abstract

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. Abstract 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 M0 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.

Genome editing approaches using reproductive cells/tissues in flowering plants

“…In addition to Agrobacterium -mediated methods, genome editing via direct delivery of CRISPR/Cas9 components into plant cells or tissues has been developed. Notably, DNA-free genome editing, which can avoid the introduction of foreign DNA sequences into genomic DNA, has been achieved by the direct delivery of Cas9-gRNA RNP into somatic protoplasts via PEG-Ca 2+ -mediated transfection, such as in tobacco, Arabidopsis , lettuce, rice ( Woo et al, 2015 ), Petunia ( Subburaj et al, 2016 ), grapevine, apple ( Malnoy et al, 2016 ), potato ( Andersson et al, 2018 ), and tomato ( Liu et al, 2022 ). Although a somatic protoplast-based genome editing can use abundant isolated cells for transfection, it remains a major challenge to apply it generally in a wide range of plant species due to difficulties in plant regeneration and obtaining a low frequency of genome-edited plants.…”

Recent Advances in Tomato Gene Editing

“…This approach relies on the establishment of successful protoplast isolation and regeneration procedures. PEG-mediated transfection has been widely used to deliver vector DNA or RNPs into protoplasts of many plant species, including various tomato cultivars [ 78 , 79 , 80 , 81 ] and the wild tomato S. peruvianum [ 82 ].…”

Plant biomacromolecule delivery methods in the 21st century

“…Protoplast isolation and transformation with PEG were performed in many crop plants (Bilang et al, 1994) including but not limited to wheat (Brandt et al, 2020), rice (Lin et al, 2020;Bes et al, 2021), maize (Svitashev et al, 2016), potato (Carlsen et al, 2022), and soybean (Patil et al, 2022). Additionally, in recent years, the PEG-mediated protoplast transfection of Cas9 protein and Cas9 complexed with in vitro synthesized guide RNA (ribonucleoprotein, RNP) was successfully used in major row crop and horticultural crops in a quest to establish a DNA-free genome editing platform (Sant'Ana et al, 2020;Subburaj et al, 2022;Liu et al, 2022).…”