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首页> 外文期刊>Plant Cell Reports >Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium x formolongi
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Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium x formolongi

机译：共培养培养基pH对农杆菌介导的大果百合转化成功的重要性

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An efficient system for Agrobacterium-mediated transformation of Lilium x formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing beta-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified MS medium containing 100 muM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis.
机译：通过防止与MES共培养的培养基中pH的急剧下降，建立了农杆菌介导的大果百合转化的有效系统。用含有质粒pIG121-Hm的根癌农杆菌菌株EHA101的过夜培养物接种分生结节性愈伤组织，所述质粒带有含内含子的β-葡糖醛酸糖苷酶（GUS），潮霉素磷酸转移酶（HPT）和新霉素磷酸转移酶II（NPTII）基因。在含有100μM乙酰丁香酮，30 g / l蔗糖，1 mg / l吡咯烷和不同浓度MES的2 g / l吉兰糖胶固化的MS培养基上共培养三天后，将它们在含有12.5 mg的相同培养基上培养/ l美罗培南消除土壤杆菌2周，然后转移到含有相同浓度美罗培南和25 mg / l潮霉素的培养基中，以选择推定的转基因愈伤组织。仅通过将MES添加至共培养培养基中观察到瞬时GUS表达。仅当将MES添加到共培养培养基中时，尤其是在10mM时，才获得抗潮霉素的转基因愈伤组织。耐潮霉素的愈伤组织在转移到不含吡美仑的培养基上后，成功地再生为小植株。通过组织化学GUS测定，PCR分析和Southern印迹分析确认了植物的转化。

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