Establishment Of Stevia Rebaudiana Hairy Root Cultures System And The Studies Of Chlorogenic Acids Aecumulation

Stevia rebaudianum Bertoni is a commercial crop which was introduced into China in the early 80 s. This crop drew researchers’ attention for its high content of diterpenes(Stevia glycosides) in the leaves, which show high sweetness, low calorie and various biological activities, and therefore has been widely cultivated in China. Beside of Stevia glycosides, there are also several other bioactive components in the leaves, such as polyphenols, flavonoids, tirterpenes, alkaloids, and sterols. Although these compounds have been widely used in food, medicine and cosmetic industry, but haven’t caused people’s attention because of its low content and the difficulty to be extracted and purified with stevia glycosides at the same time. In the present study, Stevia rebaudianum hairy rootes were induced by by co-culture of Stevia rebaudiana leaves and Agrobacterium rhizogenes(C58C1) after infection, which were verified by PCR detection of rol B and rolC genes. The major compounds(chlorogenic acids) in the hairy roots were identified by HPLC-MS and HPLC. To obtain a higher production of chlorogenic acids, the critical culture conditions were optimized. This paper provides references for the production of chlorogenic acids by hairy root culture. The main research content is as follows:(1) The inducing and confirmation of hairy root. Stevia rebaudianum hairy rootes were induced by co-culture of Stevia rebaudiana leaves and Agrobacterium rhizogenes(C58C1) after infection. White adventitious roots appeared from the infected leaves in the third week after infection. When the adventitious roots were cultured in exogenous phytohormone free 1/2 MS solid medium, it showed a obvious hairy-root phenotype: vigorous growth, highly branching and Geotropism growth, which indicated that these adventitious roots might be hairy roots.The following PCR determinations showed that the rolB and rol C gene of Agrobacterium rhizogenes Ri plasmid had been successfully transferred into Stevia rebaudianum genome, consequently, the hairy roots were definitely induced by Agrobacterium rhizogenes. The vigorous hairy roots were excised and cultured in 1/2MS liquid medium for subculture. The primary hairy root culture technology had been established after several sub culturing.(2) Qualitative and quantitative analysis of major secondary metabolites in hairy roots. Hairy roots cultured in 1/2 MS liquid medium were extracted by 70% ethanol in an ultrasonic extractor, and the extracts were subjected to determination by HPLC- MS and HPLC. According to the HPLC chromatogram, there are three main peaks in the hairy root extracts. The following HPLC-MS analysis showed that the m/z of molecular ion of these three peaks were 353, 515 and 515, respectively, which indicated that its molecular formula might be C16H18O9, C25H24O12, and C25H24O12. Based on the abovementioned experiments and the analysis of some related literatures, we presumed that the three compounds were chlorogenic acid and dicaffeoylquinic acid, respectively. To further validate these compounds, the authentic compounds named chlorogenic acid(CGA), 3,5-dicaffeoylquinic acid(3,5-CQA), and 4,5-dicaffeoylquinic acid(4,5-CQA) were added into the hairy root extracts respectively, and then the mixtures were determined by HPLC. The results showed that no new peaks appeared in the HPLC chromatogram, and the area and height of corresponding peak increased. Therefore, we deduced that the three compounds were chlorogenic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid, respectively. In addition, a HPLC detection method was established, which could determine those three compounds simultaneously.(3) The screening of high-producing hairy root lines. Eight lines with typical hairy root phenotype and high growth rate were selected for sub-culturing and screening. These hairy root lines were further confirmed by PCR detection of rol gene. Line T3 exhibited the highest growth rate and highest content of chlorogenic acids after 24 days of culture. The fresh weight and dry weight of T3 were 15.23 and 1.54 g/100 mL, and the content of chlorogenic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid were 39.41、48.10 and 3.51 mg/g, respectively. The total production of chlorogenic acids was 140.36 mg/100 mL. Therefore, T3 was selected for further research.(4) Optimization of key culture conditions. To obtain a higher production of chlorogenic acids, the effects of carbon source, medium and sucrose concentration on biomass and chlorogenic acids accumulation were investigated. The results showed that B5+40 g/L sucrose was more suitable for the production of chlorogenic acids. Under the optimized conditions, the fresh weight and dry weight were 17.93 and 2.34 mg/100 mL, and the content of chlorogenic acid, 3,5-dicaffeoylquinic acid, and 4,5- dicaffeoylquinic acid were 39.44、57.72 and 2.48 mg/g, respectively, and the total production of chlorogenic acids was 233.54 mg/100 mL, which was 66.39 percent higher than that before optimization.(5) Establishment of hairy root culture technology for production of chlorogenic acids. The growth curve and chlorogenic acids accumulation curve of line T3 were established, under the optimized culture conditions, and the profile of fresh weight, dry weight, and chlorogenic acids content was studied during a growth cycle. The results suggested that the best subculture time was the 10 th day, and the best harvest time was the 27 th day. Under the optimized conditions, the total production of chlorogenic acids was 234.40 mg/100 mL.In conclusion, Stevia rebaudianum hairy root culture was established by co-culture, and screening of high-producing hairy root line and optimization of culture conditions significantly improved the production of chlorogenic acids in hairy root. Production of chlorogenic acids by hairy root culture of Stevia rebaudiana is promising, and our study provided useful reference for the related researches.