20.500.12556/RUL-151619 Optimization of cabbage (Brassica oleracea var. capitata L.) protoplast transformation for genome editing using CRISPR/Cas9 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 µg 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. CRISPR/Cas9 genome editing protoplasts PEG-mediated transformation Brassica oleracea var. capitata B. oleracea zelje urejanje genoma genetika protoplasti genetske transformacije true false true Angleški jezik Slovenski jezik Članek v reviji 2023-10-12 14:19:59 2023-10-12 14:20:03 2024-12-06 11:41:54 0000-00-00 00:00:00 2023 0 0 9 str. art. 1245433 Vol. 14 2023 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 575 1664-462X 10.3389/fpls.2023.1245433 168169731 fpls-14-1245433.pdf fpls-14-1245433.pdf 1 28674B3C2101555CE093788132D17857 20fe6919957e6769a8e0afd2d149d67bee1051fec4dacc3ddcd7f5743a7558f8 ca91d6dc-68f9-11ee-a464-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=176634 https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1245433/full 0 6c2753ee-b3bc-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=196572 Biotehniška fakulteta 0 0 0