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

用发根农杆菌野生型菌株MAFF03-01724接种龙胆，诱导龙胆（Gentiana scabra Bunge var. buergeri Maxim.）产生毛状根。这些毛状根在去除细菌后，在无激素的1/2 MS液体培养基（Murashige和Skoog （Murashige和Skoog， 1962）培养基的一半强度，1.5%蔗糖）中，在25‰的温度下（100 rpm）黑暗中茁壮生长。在含2-3 mg/l 6-苄基氨基嘌呤（BAP）和0.1-0.2 mg/l 1-萘乙酸（NAA）的1/ 2ms固体培养基上，在25‰/天光照16 h/d条件下，从毛状根分化出不定芽。然后将整株植株转移到无激素的MS固体培养基上再生。通过检测毛状根中米基平的合成，证实了利质粒T-DNA的整合。再生植株在无激素MS基质上培养时，转化植株节间伸长，茎基部侧枝旺盛，未转化植株呈莲座表型。此外，与未转化植株相比，转化植株表现出叶片皱褶、顶端优势度降低、根系生长加快、根系倾斜等形态变化。转化植株在移入花盆土壤约30天后形成花蕾，而未转化植株仍保持莲座表型。龙胆的毛状根。在1/2 Murasige和Skoog培养基上培养龙胆苦苷(I)，并与野生根进行浓度比较。高效液相色谱法定量分析龙胆苦苷，毛状根龙胆苦苷(I)含量为野生根的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.