DNA-free genome editing in tomato protoplasts using CRISPR/Cas9 ribonucleoprotein delivery

CRISPR/Cas9 ribonucleoproteins enable DNA-free genome editing; thus, improving protoplast culture is essential for the efficient development of mutant plants. However, the use of protoplast cultures is limited because a universal method cannot be applied to diverse plants. Solanum lycopersicum ‘Hein...

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Published inHorticulture, environment and biotechnology Vol. 65; no. 1; pp. 131 - 142
Main Authors Kang, Ga Hui, Kang, Beum-Chang, Han, Jeung-Sul, Lee, Je Min
Format Journal Article
LanguageEnglish
Published Singapore Springer Nature Singapore 01.02.2024
Springer Nature B.V
한국원예학회
Subjects
2,4-D
Agriculture
Benzyladenine
Biomedical and Life Sciences
Callus
Cell culture
Cell division
CRISPR
Cultivars
Deoxyribonucleic acid
Dichlorophenoxyacetic acid
Disease resistance
DNA
Editing
Genome editing
Genomes
Growth regulators
Leaf-curl
Life Sciences
Mutagenesis
Naphthaleneacetic acid
Plant breeding
Plant Breeding/Biotechnology
Plant diseases
Plant Ecology
Plant growth
Plant Physiology
Polyethylene glycol
Protoplasts
Research Report
Ribonucleoproteins
Solanum lycopersicum
Tomatoes
Transfection
Yellow leaf
농학
CRISPR-Cas9 RNPs
DNA-free
Transfection
Tomato
Protoplast culture
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Abstract CRISPR/Cas9 ribonucleoproteins enable DNA-free genome editing; thus, improving protoplast culture is essential for the efficient development of mutant plants. However, the use of protoplast cultures is limited because a universal method cannot be applied to diverse plants. Solanum lycopersicum ‘Heinz 1706,’ a model cultivar for tomato genome analysis, has not yet been studied for DNA-free genome editing. We optimized the protoplast culture method for the tomato model cultivar ‘Heinz 1706’ using combinations of plant growth regulators (PGRs) and basal media. Isolated protoplasts were cultured in R-Ini medium for cell division and micro-calli proliferation, and then the medium was changed to G-207.3 medium for callus formation. Among different concentrations of 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP), the combination of 0.05 mg/L NAA and 0.5 mg/L BAP in the R-Ini medium was observed to be highly efficient for micro-calli development. G-207.3 liquid medium was more efficient for mini-calli formation than the R-Ini liquid medium. For calli formation from the mini-calli, 0.1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 mg/L NAA, and 0.5 mg/L BAP were used in G-207.3 solid medium. In addition, five single guide RNAs (sgRNAs) were designed to target SlPelo using polyethylene glycol-mediated transfection, thereby developing a tool for tomato yellow leaf curl virus (TYLCV) resistance breeding. sgRNAs and Cas9 complexes were delivered into protoplasts using PEG-mediated transfection, and sgRNA2 resulted in a high mutagenesis efficiency. The results will be valuable for DNA-free genome editing in tomato for the development of new breeding materials.
AbstractList CRISPR/Cas9 ribonucleoproteins enable DNA-free genome editing; thus, improving protoplast culture is essential for the efficient development of mutant plants. However, the use of protoplast cultures is limited because a universal method cannot be applied to diverse plants. Solanum lycopersicum ‘Heinz 1706,’ a model cultivar for tomato genome analysis, has not yet been studied for DNA-free genome editing. We optimized the protoplast culture method for the tomato model cultivar ‘Heinz 1706’ using combinations of plant growth regulators (PGRs) and basal media. Isolated protoplasts were cultured in R-Ini medium for cell division and micro-calli proliferation, and then the medium was changed to G-207.3 medium for callus formation. Among different concentrations of 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP), the combination of 0.05 mg/L NAA and 0.5 mg/L BAP in the R-Ini medium was observed to be highly efficient for micro-calli development. G-207.3 liquid medium was more efficient for mini-calli formation than the R-Ini liquid medium. For calli formation from the mini-calli, 0.1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 mg/L NAA, and 0.5 mg/L BAP were used in G-207.3 solid medium. In addition, five single guide RNAs (sgRNAs) were designed to target SlPelo using polyethylene glycol-mediated transfection, thereby developing a tool for tomato yellow leaf curl virus (TYLCV) resistance breeding. sgRNAs and Cas9 complexes were delivered into protoplasts using PEG-mediated transfection, and sgRNA2 resulted in a high mutagenesis efficiency. The results will be valuable for DNA-free genome editing in tomato for the development of new breeding materials.
CRISPR/Cas9 ribonucleoproteins enable DNA-free genome editing; thus, improving protoplast culture is essential for the effi cient development of mutant plants. However, the use of protoplast cultures is limited because a universal method can not be applied to diverse plants. Solanum lycopersicum ‘Heinz 1706,’ a model cultivar for tomato genome analysis, has not yet been studied for DNA-free genome editing. We optimized the protoplast culture method for the tomato model cultivar ‘Heinz 1706’ using combinations of plant growth regulators (PGRs) and basal media. Isolated protoplasts were cultured in R-Ini medium for cell division and micro-calli proliferation, and then the medium was changed to G-207.3 medium for callus formation. Among diff erent concentrations of 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP), the combination of 0.05 mg/L NAA and 0.5 mg/L BAP in the R-Ini medium was observed to be highly effi cient for micro calli development. G-207.3 liquid medium was more effi cient for mini-calli formation than the R-Ini liquid medium. For calli formation from the mini-calli, 0.1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 mg/L NAA, and 0.5 mg/L BAP were used in G-207.3 solid medium. In addition, fi ve single guide RNAs (sgRNAs) were designed to target SlPelo using polyethylene glycol-mediated transfection, thereby developing a tool for tomato yellow leaf curl virus (TYLCV) resistance breeding. sgRNAs and Cas9 complexes were delivered into protoplasts using PEG-mediated transfection, and sgRNA2 resulted in a high mutagenesis effi ciency. The results will be valuable for DNA-free genome editing in tomato for the development of new breeding materials. KCI Citation Count: 0
CRISPR/Cas9 ribonucleoproteins enable DNA-free genome editing; thus, improving protoplast culture is essential for the efficient development of mutant plants. However, the use of protoplast cultures is limited because a universal method cannot be applied to diverse plants. Solanum lycopersicum ‘Heinz 1706,’ a model cultivar for tomato genome analysis, has not yet been studied for DNA-free genome editing. We optimized the protoplast culture method for the tomato model cultivar ‘Heinz 1706’ using combinations of plant growth regulators (PGRs) and basal media. Isolated protoplasts were cultured in R-Ini medium for cell division and micro-calli proliferation, and then the medium was changed to G-207.3 medium for callus formation. Among different concentrations of 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP), the combination of 0.05 mg/L NAA and 0.5 mg/L BAP in the R-Ini medium was observed to be highly efficient for micro-calli development. G-207.3 liquid medium was more efficient for mini-calli formation than the R-Ini liquid medium. For calli formation from the mini-calli, 0.1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 mg/L NAA, and 0.5 mg/L BAP were used in G-207.3 solid medium. In addition, five single guide RNAs (sgRNAs) were designed to target SlPelo using polyethylene glycol-mediated transfection, thereby developing a tool for tomato yellow leaf curl virus (TYLCV) resistance breeding. sgRNAs and Cas9 complexes were delivered into protoplasts using PEG-mediated transfection, and sgRNA2 resulted in a high mutagenesis efficiency. The results will be valuable for DNA-free genome editing in tomato for the development of new breeding materials.
Author Han, Jeung-Sul
Lee, Je Min
Kang, Beum-Chang
Kang, Ga Hui
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  givenname: Jeung-Sul
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  organization: Department of Horticultural Science, Kyungpook National University
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  surname: Lee
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Keywords CRISPR-Cas9 RNPs
DNA-free
Transfection
Tomato
Protoplast culture
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Snippet CRISPR/Cas9 ribonucleoproteins enable DNA-free genome editing; thus, improving protoplast culture is essential for the efficient development of mutant plants....
CRISPR/Cas9 ribonucleoproteins enable DNA-free genome editing; thus, improving protoplast culture is essential for the effi cient development of mutant plants....
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SubjectTerms 2,4-D
Agriculture
Benzyladenine
Biomedical and Life Sciences
Callus
Cell culture
Cell division
CRISPR
Cultivars
Deoxyribonucleic acid
Dichlorophenoxyacetic acid
Disease resistance
DNA
Editing
Genome editing
Genomes
Growth regulators
Leaf-curl
Life Sciences
Mutagenesis
Naphthaleneacetic acid
Plant breeding
Plant Breeding/Biotechnology
Plant diseases
Plant Ecology
Plant growth
Plant Physiology
Polyethylene glycol
Protoplasts
Research Report
Ribonucleoproteins
Solanum lycopersicum
Tomatoes
Transfection
Yellow leaf
농학
Title DNA-free genome editing in tomato protoplasts using CRISPR/Cas9 ribonucleoprotein delivery
URI https://link.springer.com/article/10.1007/s13580-023-00549-4
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