Journal
FRONTIERS IN PLANT SCIENCE
Volume 14, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fpls.2023.1245433
Keywords
CRISPR/Cas9; genome editing; protoplasts; PEG-mediated transformation; B. oleracea
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The study evaluated the factors affecting transformation efficiency in cabbage protoplasts for efficient delivery of the CRISPR/Cas9 vector. By optimizing the transformation protocol, a high editing efficiency was achieved. These findings have practical significance for successful genome editing in cabbage and other brassicas.
Genome editing techniques, such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR/Cas9) are undoubtedly becoming an indispensable tool for improving food crops and tackling agricultural challenges. In the present study, key factors affecting transformation efficiency, such as PEG4000 concentration, incubation time, and plasmid amount were evaluated to achieve efficient delivery of CRISPR/Cas9 vector into cabbage protoplasts. Using amplicon sequencing, we confirmed a significant effect of PEG4000 concentration and incubation time on the induced target mutations. By optimizing the transformation protocol, editing efficiency of 26.4% was achieved with 40 mu g of plasmid and 15 minutes incubation with 50% PEG4000. While these factors strongly affected the mutation rate, the viability of the transformed protoplasts remained high. Our findings would be useful for successful genome editing in cabbage and other brassicas, as well as in research areas such as gene function analysis and subcellular localization that rely on transient transformation methods in protoplasts.
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