Instituto de Investigaciones Agrobiológicas de Galicia, Consejo Superior de Investigaciones Cientificas, Avda. de Vigo s/n, Santiago de Compostela, Spain.
Plant Cell Rep. 2010 Dec;29(12):1411-22. doi: 10.1007/s00299-010-0931-8. Epub 2010 Oct 24.
A protocol was developed for genetic transformation of somatic embryos derived from juvenile and mature Quercus robur trees. Optimal transformation conditions were evaluated on the basis of the results of transient GUS expression assays with five oak embryogenic lines and a strain of Agrobacterium tumefaciens (EHA105) harbouring a p35SGUSINT plasmid containing a nptII and a uidA (GUS) genes. For stable transformation, embryo clumps at globular/torpedo stages (4-10 mg) were inoculated with EHA105:p35SGUSINT bacterial cultures, cocultivated for 4 days and selected in proliferation medium with 75 mg/l of kanamycin. Putatively transformed masses appeared after 20-30 weeks of serial transfers to selective medium. Histochemical and molecular analysis (PCR and Southern blot) confirmed the presence of nptII and uidA genes in the plant genomes. Transformation efficiencies ranged from up to 2% in an embryogenic line derived from a 300-year-old tree, to 6% in a juvenile genotype. Twelve independent transgenic lines were obtained from these oak genotypes, and transgenic plantlets were recovered and acclimatized into the soil. This is the first demonstration of the production of transformed somatic embryos and regenerated plants from juvenile and mature trees of Q. robur and suggests the possibility of introducing other genetic constructions to develop trees that are tolerant/resistant to pathogens and/or biotic stresses.
建立了一个从幼龄和成熟的欧洲栎树体胚中进行遗传转化的方案。通过对五个欧洲栎胚性系和一株携带 p35SGUSINT 质粒的根癌农杆菌（EHA105）的瞬时 GUS 表达分析，评估了最佳的转化条件，该质粒含有一个 nptII 和一个 uidA（GUS）基因。对于稳定转化，将处于球形/鱼雷形阶段（4-10mg）的胚簇接种到 EHA105:p35SGUSINT 细菌培养物中，共培养 4 天，然后在含有 75mg/L 卡那霉素的增殖培养基中进行选择。在选择性培养基上进行 20-30 周的连续传代后，出现了疑似转化的团块。组织化学和分子分析（PCR 和 Southern blot）证实了植物基因组中存在 nptII 和 uidA 基因。转化效率在一个来自 300 年树龄的胚性系中高达 2%，在一个幼龄基因型中高达 6%。从这些欧洲栎基因型中获得了 12 个独立的转基因系，并且已经回收了转基因苗并将其驯化到土壤中。这是首次从幼龄和成熟的欧洲栎树体胚和再生植株中获得转化体的证明，表明有可能引入其他遗传构建体来培育对病原体和/或生物胁迫具有耐受性/抗性的树木。
