Studies On The Cell Suspension Culture And The Regulation Of Secondary Metabolism Of Rhodiola Fastigiata S.H.Fu

As the main functional secondary metabolites contained in roots and rhizomes of Rhodiola fastigiata S.H.Fu that is a medicinal plants, salidroside is a kind of environmental adaptation medicine with the most exploitation perspectives and possessing functions of anti-fatigue, anti-senescence, anti-anoxic, anti-microwave radiation, and anti-tumour. Due to influences from factors such as the abrupt increases of social demands and the gradual degradation of eco-environments, the wild resource of Rhodiola fastigiata has been decreased rapidly, and the species has been facing a severe situation of nearly extinction. In order to find a new way of compensating the wild resources of the species, we at first conducted experiments to screening the high-productive lines of granule calli and the high-productive lines of suspension-cultured cells. Taking the high-productive cell lines as research materials, the morphological characteristics, the growth patterns, the main medicinal compositions and the biosynthesis and accumulation of salidroside were thoroughly studied. Furthermore, effects of the constituents of culture media, temperature, light, pH on growth of the cells and production of the targeted metabolites were observed. Through changing light intensities and duration of irradiation, effects of photons at different wavelengths on bioactivities of phenylalanine ammonia lyase (PAL), the rate-limiting enzyme located at up-stream of the biosynthesis path way of salidroside, and the content changes of main cell inclusions caused by the changes of bioactivities of this enzyme.Expression of some biological characters of the cell lines established in this study, such as the capacities of cell proliferation and secondary metabolism, were much strong than that of original cells. For examples, the average biomass increased 3.23 times, the growth period extended to approximately 24 days. Moreover, the biosynthetic capacity of salidroside was strengthen, the persisting time of biosynthesis extended to the end of growth of the cells, and the accumulation content of salidroside was 2.72 times of that of the original cells. Furthermore, structures of the cell lines became much denser, and the cells became green in color due to occurrence of a small quantity of chlorophylls. These would provide probabilities for further research on the biological, physiological and biochemical characters of the photoautotrophic cells.Culture conditions and the nutrients in the media were optimized using a method of changing individual factors to further characterize the biological properties of the suspension-cultured cell lines of Rhodiola fastigiata. The results show that MS medium could be the most suitable one for obtaining the highest biomass and salidroside productivity. The level of carbon source (sucrose) level in favor of achieving high biomass was 30mg/L, and that in favor of achieving high accumulation rate of salidroside was much higher (50mg/L). The capacities of cell growth and salidroside production was the highest at a ratio of 1:2 between NH₄⁺ and NO₃^- and under the precondition of keeping the total nitrogen at a same level. However, production of salidroside reached to the highest level when the total nitrogen level was 1/2 time of the basic MS medium, indicating that higher ration of C/N favored the biosynthesis of salidroside.The growth of the suspension-cultured cell behaved similar under lower temperature conditions, because wild plants of Rhodiola fastigiata grow well in the conditions of high altitude and cold mountainous regions of south-east Tibet. Productivity of salidroside of the cells under a temperatue of 13℃ increased 39.25%, and that under a temperature of 37℃ decreased 16.21% comparing to that under the room temperature (25℃). Therefore, it was obviously that higher temperature weakened the capacity of salidroside biosynthesis of the cells.It is found by the study of regulation of photons at different wavelengths on biosynthesis of salidroside that photons at the red region of the wavelengths possessed much stronger influences on induction of the activities of PAL, although this influence became saturated as the light intensity was further increased. The effect of photons at a wavelength of 440 nm on the activity of PAL was not obvious comparing to that of photons at the red region, and the content of soluble sugars of the cells under photons of 440 nm was even lower than those of 660 nm and 400 nm.The present dissertation carried out firstly thorough studies on the suspension-cultured cells of Rhodiola fastigiata, a medicinal plant that has grown and survived well in the most rigorous environments of high altitudes. The results obtained could provide solid grounds for researches on the regulation mechanisms of secondary metabolism and the bio-production of targeted metabolites of none-model plants. The experimental systems established in these studies could also provide support for further researches on functional genomics and metabolomics related to the secondary metabolism.