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首页> 外文期刊>Plant Cell Reports >Sexually mature transgenic American chestnut trees via embryogenic suspension-based transformation
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Sexually mature transgenic American chestnut trees via embryogenic suspension-based transformation

机译：性成熟的转基因美洲栗树通过基于胚悬浮的转化

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摘要

The availability of a system for direct transfer of anti-fungal candidate genes into American chestnut (Castanea dentata), devastated by a fungal blight in the last century, would offer an alternative or supplemental approach to conventional breeding for production of chestnut trees resistant to the blight fungus and other pathogens. By taking advantage of the strong ability of embryogenic American chestnut cultures to proliferate in suspension, a high-throughput Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into the tree was established. Proembryogenic masses (PEMs) were co-cultivated with A. tumefaciens strain AGL1 harboring the plasmid pCAMBIA 2301, followed by stringent selection with 50 or 100 mg/l Geneticin. A protocol employing size-fractionation to enrich for small PEMs to use as target material and selection in suspension culture was applied to rapidly produce transgenic events with an average efficiency of four independent transformation events per 50 mg of target tissue and minimal escapes. Mature somatic embryos, representing 18 transgenic events and derived from multiple American chestnut target genotypes, were germinated and over 100 transgenic somatic seedlings were produced and acclimatized to greenhouse conditions. Multiple vigorous transgenic somatic seedlings produced functional staminate flowers within 3 years following regeneration.
机译：将抗真菌候选基因直接转移到美洲栗（Castanea dentata）中的系统的可用性，该系统在上个世纪受到霉菌病的破坏，将为常规育种提供一种替代或补充方法，以生产抗该病的栗树。叶枯病真菌和其他病原体。通过利用具有胚性的美国板栗培养物在悬浮液中增殖的强大能力，建立了高通量根癌农杆菌介导的转化方案，用于将外源基因稳定整合到树中。将胚发生质量（PEM）与带有质粒pCAMBIA 2301的根癌农杆菌AGL1共培养，然后用50或100 mg / l遗传霉素严格选择。协议采用大小分级富集小PEM用作目标材料并在悬浮培养中进行选择的方案可快速产生转基因事件，每50 mg靶组织平均发生四个独立转化事件，且逃逸最少。代表18种转基因事件并衍生自多种美国板栗靶基因型的成熟体细胞胚被发芽，并生产了100多种转基因体细胞幼苗并使其适应温室条件。再生后三年内，多个有活力的转基因体细胞幼苗产生了功能稳定的花朵。

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