Cargando…

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

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Liu, Ying, Andersson, Mariette, Granell, Antonio, Cardi, Teodoro, Hofvander, Per, Nicolia, Alessandro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9395478/
https://www.ncbi.nlm.nih.gov/pubmed/35773498
http://dx.doi.org/10.1007/s00299-022-02893-8
_version_ 1784771702400483328
author Liu, Ying
Andersson, Mariette
Granell, Antonio
Cardi, Teodoro
Hofvander, Per
Nicolia, Alessandro
author_facet Liu, Ying
Andersson, Mariette
Granell, Antonio
Cardi, Teodoro
Hofvander, Per
Nicolia, Alessandro
author_sort Liu, Ying
collection PubMed
description 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 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00299-022-02893-8.
format Online
Article
Text
id pubmed-9395478
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Springer Berlin Heidelberg
record_format MEDLINE/PubMed
spelling pubmed-93954782022-08-24 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 Plant Cell Rep Original Article 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 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00299-022-02893-8. Springer Berlin Heidelberg 2022-06-30 2022 /pmc/articles/PMC9395478/ /pubmed/35773498 http://dx.doi.org/10.1007/s00299-022-02893-8 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Original Article
Liu, Ying
Andersson, Mariette
Granell, Antonio
Cardi, Teodoro
Hofvander, Per
Nicolia, Alessandro
Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum)
title Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum)
title_full Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum)
title_fullStr Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum)
title_full_unstemmed Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum)
title_short Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum)
title_sort establishment of a dna-free genome editing and protoplast regeneration method in cultivated tomato (solanum lycopersicum)
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9395478/
https://www.ncbi.nlm.nih.gov/pubmed/35773498
http://dx.doi.org/10.1007/s00299-022-02893-8
work_keys_str_mv AT liuying establishmentofadnafreegenomeeditingandprotoplastregenerationmethodincultivatedtomatosolanumlycopersicum
AT anderssonmariette establishmentofadnafreegenomeeditingandprotoplastregenerationmethodincultivatedtomatosolanumlycopersicum
AT granellantonio establishmentofadnafreegenomeeditingandprotoplastregenerationmethodincultivatedtomatosolanumlycopersicum
AT carditeodoro establishmentofadnafreegenomeeditingandprotoplastregenerationmethodincultivatedtomatosolanumlycopersicum
AT hofvanderper establishmentofadnafreegenomeeditingandprotoplastregenerationmethodincultivatedtomatosolanumlycopersicum
AT nicoliaalessandro establishmentofadnafreegenomeeditingandprotoplastregenerationmethodincultivatedtomatosolanumlycopersicum