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

采用共培养的方法将细菌Ri(根诱导)质粒导入人参愈伤组织形成的原生质体中。感染后约6周，从多个点开始生根。将根分离并转移到无激素的液体MS培养基中。毛状根在液体培养基中生长旺盛，侧枝较多。激素控制诱导的普通根生长旺盛，无分枝，有时形成愈伤组织状聚集体和/或芽。毛状根与普通根最大的不同之处在于，毛状根可以在无激素的培养基中生长。发根农杆菌Ri载体转化人参毛状根，在无激素条件下培养3周后，毛状根增长3.07倍。毛状根的这种生长速度远远低于许多其他植物。因此，人参海参…在附加IBA和激动素的基础上培养出更多的红根。人参毛状根的活跃生长需要IBA。随后，对瑞香素的生产进行了考察。结果表明，在无激素培养条件下，毛状根产生的皂苷含量和组成与原始愈伤组织几乎相同。此外，毛状根在附加IBA的培养基上培养时，其皂苷产量高于原始愈伤组织和普通培养根，分别是B2K0.1培养基中皂甙的2.85倍和1.98倍。薄层层析和高效液相色谱分析结果表明，毛状根产生的皂苷与普通栽培根和田间生长的天然根相似。人参毛状根在皂素生产中具有一定的应用价值。最后，采用气升式生物反应器对人参毛状根进行渗透处理，使人参毛状根在10%的二甲基亚砜中渗透1h，实现了人参毛状根的连续生产。结果表明，用生物反应器每周投加10%的二甲基亚砜，连续55天成功地生产出皂苷。较少

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