The CRISPR/Cas9 technology represents enormous potential for plant breeding and plant biotechnology, as it allows precise modification of the nucleotide sequence of endogenous genes and the regulation of their expression. The technology is most commonly used for inducing mutations due to its simplicity, versatility, efficiency, and specificity. In our research, we utilized microspores and protoplasts of five species from the Brassica genus to develop a method for genome editing using plasmids and ribonucleoprotein complexes (RNPs) composed of the Cas9 enzyme and a single guide RNA molecule, excluding the step of stable plant transformation. Experiments were conducted on microspore embryogenesis and plant regeneration by treating cells with abscisic acid and buthionine sulfoximine to enhance regenerative ability. Colchicine was used to double the chromosomes and obtain doubled haploid plants, whose ploidy was determined by flow cytometry. The regeneration ability of the shoots was checked from the isolated protoplasts. Plasmids, both with and without CRISPR/Cas9 genes, and RNPs containing the FRIGIDA gene sequence were introduced into microspores and protoplasts using polyethylene glycol (PEG) and electroporation. The efficiency of introducing plasmid vectors into protoplasts was determined by flow cytometry. Next-generation sequencing was used to detect indel mutations. The percentage of induced indel mutations in microspore transformation with plasmids or RNPs ranged up to 0.03%, reaching up to 6.00% after protoplast transformation with RNP, and up to 2.41% with plasmid vector uptake.
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