Transformation of gentian with Agrobacterium rhizogenes and gentiopicroside contents in the hairy roots.

龙胆(Gentiana scabra Bunge var.)Buergeri Maxim.)接种生根农杆菌野生型菌株MAFF03-01724进行诱导。这些毛状根，在去除细菌后，在无激素的1/2MS液体培养基(Murashige and Skoog(Murashige and Skoog，1962)的一半强度，1.5%蔗糖)中，在黑暗中25ﾟC(100rpm)中旺盛生长。毛状根在含2~3 mg/L 6-苄基氨基嘌呤+0.1~0.2 mg/L 1-萘乙酸的1/2MS固体培养基上，25ﾟ℃光照16h，分化出不定芽。然后将整株植株转移到无激素的MS固体培养基上进行再生。通过检测毛状根中mikimopine(Opinc)的合成，证实了重组质粒T-DNA的整合。转化植株在无激素MS固体外植体上培养时，表现为节间丛生，茎基侧枝旺盛，而未转化植株表现为莲座型。此外，与未转化植株相比，转化植株表现出叶片皱缩、顶端优势降低、根生长增加、根向斜生等形态变化。转移到盆栽土壤中约30d后，转化植株形成花芽，而未转化植株仍保持莲座型。在1/2的Murasige和Skoog培养基上，比较了龙胆苦苷(I)的浓度与野生根的浓度。高效液相色谱法测定龙胆苦苷含量，毛状根中龙胆苦苷(I)的含量为野生龙胆苦苷的1/3。

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.