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

Autor: Pavese V; Dipartimento di Scienze Agrarie, Forestali e Alimentari-Department of Agricultural, Forest and Food Sciences (DISAFA), Università degli Studi di Torino, Torino, Italy., Moglia A; Dipartimento di Scienze Agrarie, Forestali e Alimentari-Department of Agricultural, Forest and Food Sciences (DISAFA), Università degli Studi di Torino, Torino, Italy., Milani AM; Dipartimento di Scienze Agrarie, Forestali e Alimentari-Department of Agricultural, Forest and Food Sciences (DISAFA), Università degli Studi di Torino, Torino, Italy., Marino LA; Dipartimento di Scienze Agrarie, Forestali e Alimentari-Department of Agricultural, Forest and Food Sciences (DISAFA), Università degli Studi di Torino, Torino, Italy., Martinez MT; Mision Biologica de Galicia, Sede de Santiago, Consejo Superior de Investigaciones Cientificas, Santiago de Compostela, Spain., Torello Marinoni D; Dipartimento di Scienze Agrarie, Forestali e Alimentari-Department of Agricultural, Forest and Food Sciences (DISAFA), Università degli Studi di Torino, Torino, Italy., Botta R; Dipartimento di Scienze Agrarie, Forestali e Alimentari-Department of Agricultural, Forest and Food Sciences (DISAFA), Università degli Studi di Torino, Torino, Italy., Corredoira E; Mision Biologica de Galicia, Sede de Santiago, Consejo Superior de Investigaciones Cientificas, Santiago de Compostela, Spain.
Jazyk: angličtina
Zdroj: Frontiers in plant science [Front Plant Sci] 2024 Feb 19; Vol. 15, pp. 1323390. Date of Electronic Publication: 2024 Feb 19 (Print Publication: 2024).
DOI: 10.3389/fpls.2024.1323390
Abstrakt: The CRISPR/Cas9 ribonucleoprotein (RNP)-mediated technology represents a fascinating tool for modifying gene expression or mutagenesis as this system allows for obtaining transgene-free plants, avoiding exogenous DNA integration. Holm oak ( Quercus ilex ) 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 in vitro leaves and proembryogenic masses. Proembryogenic masses represented the best material to get high protoplast yield (11 x 10 6 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 phytoene desaturase ( pds ) 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 4 NO 3 and KNO 3 supplemented with 0.1 mg/L benzylaminopurine and 0.1 mg/L 2,4-dichlorophenoxyacetic acid.
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision
(Copyright © 2024 Pavese, Moglia, Milani, Marino, Martinez, Torello Marinoni, Botta and Corredoira.)
Databáze: MEDLINE
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