Agrobacterium-mediated transformation of the medicinal plant Podophyllum hexandrum Royle (syn. P. emodi Wall. ex Hook.f. & Thomas)
Agrobacterium-mediated transformation of the medicinal plant Podophyllum hexandrum Royle (syn. P. emodi Wall. ex Hook.f. & Thomas)
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DOI：
10.1007/s11240-013-0307-1
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发表时间：
2013-07-01
影响因子：
3
通讯作者：
Ganapathi, Andy
中科院分区：
文献类型：
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作者：
Rajesh, Manoharan;Jeyaraj, Murugaraj;Ganapathi, Andy
以抗癌药物鬼臼毒素的重要来源桃儿七为材料，建立了农杆菌介导的遗传转化方法。用携带pCAMBIA 2301的根癌农杆菌感染高度增殖的胚性细胞，pCAMBIA 2301分别含有npt II和gusA作为选择标记和报告基因。转化体细胞胚和植株分别在含卡那霉素的MS基本培养基和发芽培养基上筛选。GUS组织化学分析、聚合酶链反应和Southern杂交结果表明，gusA基因已成功整合到六雄蕊青霉基因组中并得到表达。与头孢噻肟相比，200毫克升（-1）特美汀完全逮捕农杆菌的生长和有利于体细胞胚发育的胚性细胞。在不同的农杆菌菌株、乙酰丁香酮浓度和共培养时间中，以乙酰丁香酮为外植体的胚性愈伤组织最易发生胚性愈伤组织。在含100 μ M乙酰丁香酮的MS基本培养基上共培养3天，转化率为29.64%。本研究建立的农杆菌介导的遗传转化方法有利于将目的基因导入到六蕊杨中，以提高桃的鬼臼毒素含量，并提高其他有用性状。
An efficient Agrobacterium-mediated genetic transformation method has been developed for the medicinal plant Podophyllum hexandrum Royle, an important source of the anticancer agent podophyllotoxin. Highly proliferating embryogenic cells were infected with Agrobacterium tumefaciens harbouring pCAMBIA 2301, which contains npt II and gusA as selection marker and reporter genes, respectively. The transformed somatic embryos and plantlets were selected on Murashige and Skoog (MS) basal medium containing kanamycin and germination medium, respectively. GUS histochemical analysis, polymerase chain reaction and Southern blot hybridisation confirmed that gusA was successfully integrated and expressed in the P. hexandrum genome. Compared with cefotaxime, 200 mg l(-1) timentin completely arrested Agrobacterium growth and favoured somatic embryo development from embryogenic cells. Among the different Agrobacterium strains, acetosyringone concentrations and co-cultivation durations tested, embryogenic callus infected with A. tumefaciens EHA 105 and co-cultivated for 3 days on MS basal medium containing 100 mu M acetosyringone proved to be optimal and produced a transformation efficiency of 29.64 % with respect to germinated GUS-positive plantlets. The Agrobacterium-mediated genetic transformation method developed in the present study facilitates the transference of desirable genes into P. hexandrum to improve the podophyllotoxin content and to enhance other useful traits.