Zaragozá C, Muñoz-Bertomeu J, Arrillaga I
Dpt. Biología Vegetal, Facultad de Farmacia, Universidad de Valencia, 46100 Burjassot, Valencia, Spain.
Plant Cell Rep. 2004 Jun;22(11):832-8. doi: 10.1007/s00299-004-0766-2. Epub 2004 Feb 6.
A protocol based on SAAT (sonication-assisted Agrobacterium-mediated transformation) has been developed to obtain herbicide-resistant transgenic black locust (Robinia pseudoacacia L.) plants. Cotyledon explants were co-cultivated with Agrobacterium AGL1 strain carrying the pTAB16 plasmid (bar and gusA genes). The effects of bacterial concentration (OD550 of 0.3, 0.6, 0.8) and method of infection (sonication vs immersion) on bacterial delivery were determined by assaying cotyledons for transient beta-glucuronidase expression 3 days after infection. SAAT increases transient expression efficiency especially at an OD550 of 0.6. After determining bacterial concentration and infection method, other factors affecting transformation efficiency, such as explant preconditioning and period of time before applying selection, were tested. From these experiments, the preferred protocol for black locust cotyledon transformation should include sonication of preconditioned cotyledons in AGL1 suspension, coculture for 3 days with 100 microM acetosyringone and transfer to selection medium with 4 mg/l phosphinothricin and 150 mg/l timentin. Of the initial explants, 2% produced at least one transgenic shoot. Genetic transformation was confirmed by Southern hybridization, chlorophenol red assay and herbicide tolerance of the regenerated plants.
已开发出一种基于超声辅助农杆菌介导转化（SAAT）的方案，用于获得抗除草剂转基因刺槐（Robinia pseudoacacia L.）植株。子叶外植体与携带pTAB16质粒（bar和gusA基因）的农杆菌AGL1菌株共培养。通过在感染后3天检测子叶中瞬时β-葡萄糖醛酸酶的表达，确定细菌浓度（OD550为0.3、0.6、0.8）和感染方法（超声处理与浸泡）对细菌导入的影响。SAAT可提高瞬时表达效率，尤其是在OD550为0.6时。在确定细菌浓度和感染方法后，测试了其他影响转化效率的因素，如外植体预处理和施加选择前的时间。从这些实验中，刺槐子叶转化的首选方案应包括将预处理的子叶在AGL1悬浮液中进行超声处理，与100 microM乙酰丁香酮共培养3天，然后转移到含有4 mg/l草丁膦和150 mg/l替门汀的选择培养基上。在最初的外植体中，2%产生了至少一个转基因芽。通过Southern杂交、氯酚红测定和再生植株对除草剂的耐受性证实了遗传转化。
