Influence And Preliminary Mechanism Of Elicitors On Growth And Tropane Alkaloids In Hairy Roots Of Atropa Belladonna L.

Atropa belladonna L. is a perennial herbaceous plant and an important medicinal plant specie. Recently, adding elicitor is an effective method for improving the effective component in medicinal plant, although low content of medicinal ingredient in medicinal plants has been a big problem to people. At present, the study of Atropa belladonna L. mainly focuses on the investigation of key enzyme genes in metabolic pathways and has little research about improving the accumulation of secondary metabolites by adding the inducer. In this paper, we take uniform and vigorous hairy roots of Atropa belladonna L. as experiment material, screening the optimal inducer by determining the change of the growth and tropane alkaloids content with adding elicitors（MJ, Ag NO₃, SA, YE） into liquid medium. Furthermore, we also study the preliminary mechanism of elicitors on growth and tropane alkaloids in hairy roots from adversity physiological characteristics, NO and H₂O₂ burst, gene expression of key enzyme in secondary metabolites. The main findings are as follows:1. According to experiments, the growth curve of hairy roots that was cultured in the liquid medium shows S shape, there are four states :delayed periods（0-3 d）,logarithmic periods（3-21 d） and stable periods（21-27 d）. After 27 d, the mass of fresh and dry appear downtrend, meanwhile the contents of tropane alkaloids also reduce.About the change of content of tropane alkaloids, which rapid increases during 6-12 d and in the days until 21 d which followed the contents is in the stabilization process.2. Four kinds of inducer have different influence on growth and tropane alkaloids of hairy roots in Atropa belladonna L.. Results show that MJ restrains the growth and tropane alkaloids accumulation of hairy roots and the production of tropane alkaloids are increased compered with control group under the treatment of 100 μmol·L-1Ag NO₃,though Ag NO₃ inhibits the growth of hairy roots. Meanwhile SA has little influence on growth of hairy roots and stimulates the accumulation of Scopolamine. Moreover, bothhairy roots growth and content of tropane alkaloids show a upward trend with the increasing of YE concentration. To sum up, elicitors have selective influence on growth and trapane alkaloids in hairy roots of Atropa belladonna L., the best elicitor on accumulation of trapane alkaloids is Ag NO₃ and YE could effectively improve of the hairy roots growth and production of tropane alkaloids.3. Studying the change of several physiological indexes during growth of hairy roots that has been added four elicitors into liquid medium find that adding elicitors can lead to the content of MDA increased, especially the SA. Then the treatment groups significantly improve the hairy roots of the content of Pro compared to the control, its content up to 8.16μg·g-1FM, which is 3.92 times of control group. At the same time, the contents of soluble sugar and soluble protein are significantly improved by adding four elicitors, Ag NO₃ and SA on the influence of the soluble sugar contents are more obvious, respectively1.58 and 1.60 times of control. However, YE can obvious improve the contents of soluble protein compared with other elicitors, which up to10.53mg·g-1FM that increased 22.31% compared with CK.4. The elicitor treatment groups significantly increase the content of NO compared with the control group, especially the MJ, which content of NO is 51.76 μmol·L-1·g FM that is 1.61 times of CK and have significantly difference compared with SA, YE treatments. Then four kinds of elicitor treatments also stimulate the burst of H₂O₂, three kinds of treatments are significantly improve content of H₂O₂ compared with the control,increased by 28.25%（MJ）, 72.45%（Ag NO₃） and 17.84%（SA） respectively with the exception of YE treatment that only 4.79% increased over the control.5. Inducers can produce effects on gene expression of key enzymes in the metabolic pathway of hairy roots. First of all, Ag NO₃, SA, YE significantly boost to pmt gene expression and YE that is research 242.22% as CK is more obvious effect on pmt gene expression, while MJ can’t stimulate the expression of pmt gene. Secondly,expression of tr I gene is stimulated by Ag NO₃, SA and YE but inhibited by MJ. lastly,compared to the control group, four kinds of inducer all improve expression of h6 h gene and the results is in the order of MJ, Ag NO₃, SA,YE.