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为了探讨利用美洲商陆毛状根生产其药用成分的可能性,研究了美洲商陆毛状根诱导及其离体培养的影响因素。结果表明,美洲商陆叶片外植体被发根农杆菌ATCC 15834感染约18 d后,从其叶片外植体形态学下端叶脉切口处产生毛状根,其中以预培养1 d,农杆菌感染20 min,共培养4 d时的毛状根诱导率最高,达到70%。PCR扩增和硅胶薄层层析结果显示,发根农杆菌Ri质粒的rol C基因以及冠瘿碱合成酶基因已在美洲商陆毛状根基因组中整合和表达。所获得的美洲商陆毛状根系都能在无外源激素的MS固体培养基上快速自主生长;其中以毛状根根系2的生长速度最快、分生侧根能力最强和根表面的根毛密度最高;毛状根根表面呈紫红色或呈白色。在供试的MS、1/2MS、B5和6,7-V液体培养基中,以无外源激素的MS培养基最适合美洲商陆毛状根根系生长。与无外源激素的MS培养基相比,6,7-V培养基更有利于毛状根中商陆皂苷甲的合成与积累。本文所建立的美洲商陆毛状根诱导及其离体培养的适宜条件为今后利用其毛状根株系的规模培养来生产其药用有效成分商陆皂苷甲奠定了实验和技术基础。
In order to investigate the possibility of using the hairy roots of the genus Plum to produce their medicinal components, the factors affecting the induction and the in vitro culture of hairy roots of the genus Plum in the Americas were studied. The results showed that the hairy roots were produced from the explant morphology of the leaf explants at the tip of the vein explants after about 18 days of Agrobacterium rhizogenes ATCC 15834 infection. The leaf explants were pre-cultured for 1 day and Agrobacterium tumefaciens Hairy root induction rate reached 70% at 20 min after co-culture for 4 days. PCR amplification and silica gel thin layer chromatography showed that the rol C gene and the opine synthase gene of Agrobacterium rhizogenes Ri plasmid were integrated and expressed in the hairy root genomic genome of P. polyporus. The hairy root system obtained from Populus euphratica could grow autonomously on MS solid medium without exogenous hormones. Among them, root system 2 had the fastest growth rate, the highest lateral root capacity and the highest root hair density on the root surface Hairy root surface was purple or white. In MS, 1 / 2MS, B5 and 6,7-V liquid media, MS medium with no exogenous hormone was the most suitable for root hair growth of Puccinia striiformis. Compared with MS medium without exogenous hormones, 6,7-V medium is more conducive to the synthesis and accumulation of esculentoside A in hairy roots. In this paper, the suitable conditions for the induction of hairy root and the in vitro culture of Psidium chrysogenum have laid the foundation of experiment and technology for the future production of its medicinal effective ingredient, Psidium alpina, on the scale of its hairy root system.