|Budget Amount *help
¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1997 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1996 : ¥700,000 (Direct Cost : ¥700,000)
Hairy roots of gentian (Gentiana scabra Bunge var. buergeri Maxim.) were induced following the inoculation with wild type of Agrobacterium rhizogenes strain MAFF03-01724. These hairy roots, after the elimination of bacteria, vigorously grew in hormone free 1/2 MS liquid medium (half strength of Murashige and Skoog (Murashige and Skoog, 1962) medium, 1.5% sucrose), at 25ﾟC in the dark (at 100 rpm). Adventitious shoots were differentiated from the hairy roots by culturing on 1/2 MS solid medium containing 2-3 mg/l 6-benzylaminopurine (BAP) and 0.1-0.2 mg/l 1-naphthaleneacetic acid (NAA) at 25ﾟC under 16 h/day light. Then the whole plants were regenerated by transfer to hormone free MS solid medium. The integration of Riplasmid T-DNA was confirmed by detection of mikimopine (opinc) synthesis in the hairy roots. The transformed plants, when the regenerated plants were cultured on hormone free MS solid modium, showed clongation of internodes and vigorous emergence of lateral branches at the base of the stems, while the non-transformed plants showed rosette phenotype. Furthermore, in comparison with the non-transformed plants, the transformed plants showed several morphologic alterations such as wrinkled leaves, reduced apical dominance, increased root growth, and plagiotropic roots. About 30 days after the transfer to the soil in the pots, the transformed plants formed flower buds, while the non-transformed plants still kept showing rosette phenotype.
The hairy roots of Gentiana scabra BUNGE var. Buergeri MAXIM.were cultured on 1/2 Murasige and Skoog medium and the concentration of gentiopicroside (I) was compared with that of the wild root. Quantitative analysis of gentiopicroside by high performance liquid chromatography showed that the content of gentiopicroside (I) of the hairy roots was 1/3 of the wild roots.