PDF(Pubmed)
Abstract：
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 106 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 NH4NO3 and KNO3 supplemented with 0.1 mg/L benzylaminopurine and 0.1 mg/L 2,4-dichlorophenoxyacetic acid.
摘要：
CRISPR/Cas9核糖核蛋白（RNP）介导的技术代表了一种用于修饰基因表达或诱变的迷人工具，因为该系统允许获得无转基因植物。避免外源DNA整合。霍尔姆橡树（Quercusilex）具有重要的社交能力，经济,和在西欧和北非的地中海气候区的生态作用，并受到橡树下降综合症的严重影响。在这里，我们报告了CRISPR/Cas9-RNP技术在holmoak中的应用的第一个示例。首先,我们评估了从离体叶片和前胚发生块中分离的原生质体。前胚发生质量代表获得高原生质体产量(11×106原生质体/ml)和活力的最佳材料。其次,通过表达绿色荧光蛋白作为转染标记基因的载体评估原生质体转染能力，在24小时后达到62%的转染百分比。将CRISPR/Cas9RNP成功递送到原生质体中,导致在八氢烯去饱和酶(pds)靶基因组区域处的5.6%±0.5%编辑效率。然后在半固体培养基中培养原生质体，在培养45天后，在Murashige和Skoog培养基中观察到发育的胚性愈伤组织，其中一半浓度的NH4NO3和KNO3补充了0.1mg/L苄基氨基嘌呤和0.1mg/L2,4-二氯苯氧基乙酸。
