AUTHOR=Pavese Vera , Moglia Andrea , Milani Anna Maria , Marino Lorenzo Antonio , Martinez Maria Teresa , Torello Marinoni Daniela , Botta Roberto , Corredoira Elena 

TITLE=Advances in Quercus ilex L. breeding: the CRISPR/Cas9 technology via ribonucleoproteins

JOURNAL=Frontiers in Plant Science

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2024.1323390

DOI=10.3389/fpls.2024.1323390

ISSN=1664-462X

ABSTRACT=<p>The CRISPR/Cas9 ribonucleoprotein (RNP)-mediated technology represents a fascinating tool for modifying gene expression or mutagenesis as this system allows for obtaining <italic>transgene-free</italic> plants, avoiding exogenous DNA integration. Holm oak (<italic>Quercus ilex</italic>) has an important social, economic, and ecological role in the Mediterranean climate zones of Western Europe and North Africa and is severely affected by oak decline syndrome. Here we report the first example of the application of the CRISPR/Cas9-RNP technology in holm oak. Firstly, we evaluated the protoplast isolation from both <italic>in vitro</italic> leaves and proembryogenic masses. Proembryogenic masses represented the best material to get high protoplast yield (11 x 10<sup>6</sup> protoplasts/ml) and viability. Secondly, the protoplast transfection ability was evaluated through a vector expressing green fluorescence protein as marker gene of transfection, reaching a transfection percentage of 62% after 24 hours. CRISPR/Cas9 RNPs were successfully delivered into protoplasts resulting in 5.6% ± 0.5% editing efficiency at <italic>phytoene desaturase</italic> (<italic>pds</italic>) target genomic region. Protoplasts were then cultured in semisolid media and, after 45 days in culture, developed embryogenic calli were observed in a Murashige and Skoog media with half concentration of NH<sub>4</sub>NO<sub>3</sub> and KNO<sub>3</sub> supplemented with 0.1 mg/L benzylaminopurine and 0.1 mg/L 2,4-dichlorophenoxyacetic acid.</p>