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

Liu, Ying and Andersson, Mariette and Granell, Antonio and Cardi, Teodoro and Hofvander, Per and Nicolia, Alessandro (2022). Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum). Plant Cell Reports
[Research article]

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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.

Authors/Creators:Liu, Ying and Andersson, Mariette and Granell, Antonio and Cardi, Teodoro and Hofvander, Per and Nicolia, Alessandro
Title:Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum)
Series Name/Journal:Plant Cell Reports
Year of publishing :2022
Number of Pages:10
Publisher:SPRINGER
ISSN:0721-7714
Language:English
Publication Type:Research article
Article category:Scientific peer reviewed
Version:Published version
Copyright:Creative Commons: Attribution 4.0
Full Text Status:Public
Subjects:(A) Swedish standard research categories 2011 > 4 Agricultural Sciences > 404 Agricultural Biotechnology > Plant Biotechnology
Keywords:Solanum lycopersicum, Mesophyll protoplast regeneration, CRISPR, Cas9, Ribonucleoprotein, SP and SP5G genes
URN:NBN:urn:nbn:se:slu:epsilon-p-118275
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-p-118275
Additional ID:
Type of IDID
DOI10.1007/s00299-022-02893-8
Web of Science (WoS)000819345600001
ID Code:28659
Faculty:LTV - Fakulteten för landskapsarkitektur, trädgårds- och växtproduktionsvetenskap
Department:(LTJ, LTV) > Department of Plant Breeding (from 130101)
Deposited By: SLUpub Connector
Deposited On:30 Aug 2022 13:56
Metadata Last Modified:30 Aug 2022 14:01

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