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Research article - Peer-reviewed, 2022

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

Liu, Ying; Andersson, Mariette; Granell, Antonio; Cardi, Teodoro; Hofvander, Per; Nicolia, Alessandro

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. 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.

Keywords

Solanum lycopersicum; Mesophyll protoplast regeneration; CRISPR; Cas9; Ribonucleoprotein; SP and SP5G genes

Published in

Plant Cell Reports
2022,
Publisher: SPRINGER

Authors' information

Swedish University of Agricultural Sciences, Department of Plant Breeding
Swedish University of Agricultural Sciences, Department of Plant Breeding
Granell, Antonio
Consejo Superior de Investigaciones Cientificas (CSIC)
Cardi, Teodoro
Consiglio per la Ricerca in Agricoltura e L'analisi Dell'economia Agraria (CREA)
Cardi, Teodoro
Istituto di Bioscienze e Biorisorse (IBBR-CNR)
Swedish University of Agricultural Sciences, Department of Plant Breeding
Nicolia, Alessandro
Consiglio per la Ricerca in Agricoltura e L'analisi Dell'economia Agraria (CREA)

UKÄ Subject classification

Plant Biotechnology

Publication Identifiers

DOI: https://doi.org/10.1007/s00299-022-02893-8

URI (permanent link to this page)

https://res.slu.se/id/publ/118275