1989 Fiscal Year Final Research Report Summary
Transformation and Saponin Production by Introduction of Ri Plasmid into Ginseng Cell
| Project/Area Number |
63870097
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Kitasato University |
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Principal Investigator |
FURUYA Tsutomu Kitasato University Pharmaceutical Sciences Professor, 薬学部, 教授 (10050345)
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| Co-Investigator(Kenkyū-buntansha) |
KAWAGUCHI Kiichiro Kitasato University Pharmaceutical Sciences Assistant Professor, 薬学部, 助手 (10146334)
YOSHIKAWA Takafumi Kitasato University Pharmaceutical Sciences Associate Professor, 薬学部, 助教授 (80050540)
SYONO Kunihiko University of Tokyo Department Applied Sciences of Pure and Professor, 教養学部, 教授 (60050457)
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| Project Period (FY) |
1988 – 1989
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| Keywords | Panax ginseng / Ginseng / Agrobacterium rhizogenes / Ri plasmid / Saponin production / Hairy root / Transformation / Bioreactor |
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| Research Abstract |
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
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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
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