Research On The Optimzation Culture System Of Hairy Roots Of Scutellaria Baicalensis Georgi-Ⅲ

Baicalin, a kind of flavonoids compounds, distributes in the roots ofScutellaria baicalensis Georgi (S. baicalensis). Baicalin has the effects ofscanvenging oxygen free radicals, anti-oxidation, immune adjustment,anti-inflammatory, antivirus (such as HIV) and antitumor. In recent years, withthe affirmation of baicalin’s medicinal value, the production of baicalin medicinegrows increasingly. In recent years, with the development of medical technology,the development of medicine made from baicalin and S. baicalensis is increasingwhile the cultivated area is declining day by day. Because of the abuse ofpesticide and pollution of herb production areas, the quality and security of crudedrugs are also increasingly serious. So the exploration of new resources tosubstitute the S. baicalensis will be meaningful to the development of Chinesemedicine.In this research, the optimized hairy roots were chose as the researchmaterials. The species of hairy roots induced by our research group and thesynthetic regulation of flavonoids ingredients of hairy roots were investigated aswell. Furthermore, the culture conditions of hairy roots for baicalin synthesiswere studied. In addition, the preliminary research on genetic modification ofhairy roots of S. baicalensis was discussed. The results were as follows:1. The Internal Transcribed Spacer (ITS) of hairy roots of S. baicalensiswas amplified by double primers. The ITS of hairy roots of S. baicalensis were589bp. Then the ITS sequence was blasted using Basic Local Alignment SearchTool with gene sequences and the results showed that the similarity of ITSsequence of hairy roots and Scutellaria baicalensis Georgi (Accession:AB557593.1) was90%. The ITS sequence of hairy roots of S. baicalensis wassubmit to Gen Bank and the accession number was JF824665. It suggested thatthe hairy roots belonging to Scutellaria baicalensis Georgi form molecular level.2. HPLC fingerprint showed that the hairy roots of S. baicalensis had abilities of synthesizing flavonoids ingredients including baicalin, wogonoside,baicalein and wogonin and so on. Baicalin was the major ingredient in hairyroots. The similarity of hairy roots cultured by fructose for30-50d rose from0.660to0.987gradually. The hairy roots cultured by sucrose and glucose for30-50d, the similarity of hairy roots and radix scutellariae (1.000) was higherthan0.9. The similarity of hairy roots and radix scutellariae (1.000) afterenzymolysis is0.969. It suggested that the hairy roots had the hydrolysisenzyme.3. The three-stage culture system of hairy roots of S. baicalensis was set upin this study. The inoculation of the first stage of culturing seeds was5g/L, theculture medium was optimized MS liquid medium with50g/L glucose and thehairy roots were cultured by28℃for18d under110rpm shaker revolutions.The inoculation of the second stage of culturing seeds was also5g/L, the culturemedium was optimized MS liquid medium contending0.2mmol/L Phe withmixed carbon of40g/L sucrose and10g/L glucose. And the hairy roots werecultured by28℃for20d under110rpm shaker revolutions. The third stage ofculturing hairy roots was established by adding0.4mmol/L Phenylalanine and60g/L sucrose to the culture medium, and adopting two-stage culture methodwhich was maintained at28℃for the first20d, and then20℃till40d under110rpm shaker revolutions. Cultured by this method, the biomass and baicalinaccumulations of hairy roots were11.52g/L and20.99%.4. To detect the biomass of hairy roots on line, the relationship of biomass y(g/L)and conductivity x (mS/cm) of the liquid medium was studied. The equation:y=7.090x3－30.886x+24.914(R2=0.998) could be used to calculate thebiomass of hairy roots on line.5. To enhance the hairy roots’ abilities of anti-hypoxia, pBI121-VHbplasmid was transformed to Agrobaterium rhizogenes15834and R1000. Itprovided an effective tool for the transformation of VHb gene to hairy roots.