滇黄芩毛状根的诱导及其黄芩苷含量测定

本文利用发根农杆菌A grobacterizum rhizogenes1.2556感染滇黄芩再生苗的茎段和叶片,建立了毛状根培养及其植株再生体系。毛状根可直接从受伤的茎、叶外植体表面产生,在无外源激素的MS固体和液体培养基上自主生长,表现出典型的发根特征。毛状根茎段的诱导率较叶片高,最高可达到14.44%;经rolB基因PCR分析和甘露碱纸电泳检测,证明Ri质粒T-DNA已整合到滇黄芩基因组中并表达;毛状根在附加6-BA2mg/L和NAA0.2mg/L的MS固体培养基上直接诱导不定芽,并在MS培养基上生根,形成再生植株。获得的毛状根系经MS液体培养基培养30d后通过HPLC都能检测到黄芩苷,其中1个转化系黄芩苷含量为2.59%,是药材黄芩的0.20倍,而从3年单位时间黄芩苷生成量计算,毛状根是药材黄芩的7.18倍。本研究建立的毛状根培养体系,将对滇黄芩转基因技术的完善和利用毛状根生产黄芩苷的生物转化提供了实验基础。

Agrobacterium rhizogenes Mediated Transformation of Scutellaria amoena and Production of Baicalin in Hairy Roots

The objective of this research was to establish an efficient system of genetic transformation and plant regeneration from hairy roots by infecting the leaf sections and stem segments of in vitro Scutellaria amoena C. H.Wright plantlets. Hairy roots were induced from the wounded surface of the explants on hormone free MS medium after infection by Agrobacterium rhizogenes strain 1.2556 and which showed the representative charac-teristic of hairy root. The frequency of stem segments was higher than that of leaf sections, the highest of which can reach 14.44%. PCR analysis and opine paper electrophoresis confirmed that the T-DNA fragment ofRi plas-mid from A. rhizogenes strain had been inserted into the genome ofScutellaria amoena and expressed. The hairy roots could be directly induced into adventitious buds on the MS solid medium supplemented with 6-BA 2 mg/L and NAA 0.2 mg/L and then took roots on MS solid medium. Finally, formed regeneration plantlets. The con-tents of baicalin in different hairy root clones were determined by HPLC after cultured in liquid MS medium for 30 days. The contents ofhaicalin in one of transformations was 2.59%, which was 0.20 times of medicine Baikal skullcap root, but if we calculated the contents of baicalin from the unit time of 3 years, the baicalin of hairy roo twas 7.18 times than that of medicine Baikal skullcap root. The culture system of hairy roots was established in this research would provide a experiment foundation for perfecting the technique of Scutellaria amoena transgenic and using biotransformation of hairy roots yield to baicalin.