Transformation and Saponin Production by Introduction of Ri Plasmid into Ginseng Cell

采用共培养法将细菌诱导根质粒引入人参愈伤组织原生质体中。感染后约6周，从几个点出现根。将根分离并转移到不含激素的MS液体培养基中。毛状根在液体培养基中生长旺盛，有许多侧枝。激素调控诱导的普通根生长旺盛，无分枝，有时形成愈伤组织样聚集体和（或）芽。与普通根最重要的区别是，毛状根可以在无激素的培养基中生长。结果表明，发根农杆菌的Ri质粒对毛状根进行了转化。在无激素培养基中培养3周后，人参毛状根增加了3.07倍。这种毛状根生长的增长远低于许多其他植物物种。因此，在添加了IBA和激动素的培养基中培养人参根茎。人参毛状根的活性生长需要IBA。随后，研究了大黄皂苷的生产。结果表明，在无激素培养基中培养的毛状根产生的皂苷含量和成分与原始愈伤组织几乎相同。毛状根在添加IBA的培养基中培养时，产生的皂苷比原始愈伤组织和普通培养的根多，分别是B2K0.1培养基中皂苷的2.85倍和1.98倍。薄层色谱（TLC）和高效液相色谱（HPLC）分析结果表明，毛状根产生的皂苷与田间栽培的普通根和本地根相似。由此可见，人参毛状根在皂苷的生产中是有用的。最后，使用由反烧瓶型改进的气升式生物反应器连续生产皂苷，人参毛状根经10% DMSO渗透1小时。结果表明，采用每周一次的10% DMSO处理，连续55天成功生产皂苷。少

Bacterial Ri (root inducing) plasmid was introduced into the protoplast formed from ginseng callus by a co-culture method. Approximately 6 weeks after infection, roots appeared from several points. The roots were isolated and transferred to a liquid hormone-free MS medium. The hairy roots grew vigorously in the liquid medium with numerous lateral branching. The ordinary roots, which were induced by hormonal control, grew vigorously without branching, and sometimes formed Callus-like aggregates and/or shoot. The most important difference from the ordinary roots is the fact that the hairy roots could grow in hormone-free medium. Moreover, it was shown from the detection of opines, that the hairy roots were transformed with Ri plasmid of Agrobacterium rhizogenes.The ginseng hairy roots increased 3.07 times after 3 weeks of the culture in the hormone-free medium. This increase in the growth of the hairy roots was much lower than those in many other plant species. Therefore, the ginseng hai … More ry roots were cultured in the medium supplemented with IBA and kinetin. The ginseng hairy roots required IBA for the active growth. Subsequently, the production of daponins was examined. As a result, it was demonstrated that the hairy roots, cultured in hormone-free medium, produced almost the same amount and constituent of saponins as the original callus. Moreover, when the hairy roots were cultured in the medium supplemented with IBA, they produced a larger amount of saponins than the original callus and the ordinary cultured roots, that is 2.85 and 1.98 times as much in B2K0.1 medium. The results of TLC and HPLC analysis indicated that the hairy roots produced saponins resembling those of the ordinary cultured roots and the native roots grown in the field. Thus, it was apparently shown that the ginseng hairy roots are useful in the saponin production.Finally, the continuous production of saponin using an airlift type bioreactor revised from a reversed flask type, with ginseng hairy root was carried out by permeabilization with 10% DMSO for 1 hr. As a result, the saponin continued to be successfully produced for 55 days by the treatment of 10% DMSO every one week using the bioreactor. Less