| Licorice has been used as a traditional Chinese medicine extensively for over 2000 years. It not only has antiinflammatory, antibacterial and antiviral activities, but also has immunomodulating, antioxidant and free radical scavenging activities. Glycyrrhiza inflata Batal, an important specie of licorice, has been frequently used to treat phthisis, contagious hepatitis, ague and gastric diseases. One of the major compounds, flavonoids has been demonstrated to carry significant biological or anti-oxidation activities. Thus, the plant of G. inflata has fallen short of supply with an increasing demand for flavonoids. Plant cell culture is a useful method for the production of valuable secondary metabolites. The cell growth and the flavonoids biosynthesis are sensitive to intracellular and environmental conditions. Thus, the present study mainly focused on the establishment of cell suspension culture and of the method for flavonoids determination, and the study of regulation of flavonoids accumulation. The main results were shown as follows:(1) The culture conditions of Glycyrrhiza inflata Bat. were studied, and the cell was obtained with high flavonoids and no glycyrrhizic acid detected. The results showed that the germinations of G. inflata were the highest when treated by H2SO4 with 40min. When the hypocotyl and cotyledon were cultured on MS medium with added different kinds of hormones, the best medium to induce callus was MS+2,4-D (1.0 mg L-1)+6-BA (1.0 mg L-1)+NAA (1.0 mg L-1) with 90% induction rate. Similarly, the callus was cultured well in the medium of MS+NAA (0.5 mg L-1)+2,4-D (0.5 mg L-1)+6-BA (0.5 mg L-1). Biomass accumulation of G. inflata exhibited"S"curve in one culture cycle when the cells were cultured with solid medium, with the greatest values obtained on day 26.(2) The component of the flavonoids in cell suspension culture was analyzed, and a method for flavonoids determination was established. The results showed that the most component of the flavonoids in plant almost detected in cell suspension culture. The molecular mass of 593, 559, 270, 309, 487, 589 and 309 were ensured in the component of the flavonoids by LC-MS. Moreover, characteristic reaction indicated that the flavonoids were composed of flavone, flavonol and flavonone.(3) The stable cell suspension culture was established. When the cells were cultured in the optimized MS medium with the 4%-6% inoculum, the biomass of the cells and the flavonoids production were higher. In one culture cycle,the biomass accumulation exhibited"S"curve, with the greatest values obtained on day 21 (16.4 and 232.4 g L-1 based on dry mass and fresh mass, respectively). Similarly, flavonoids production also got to a peak of 95.7 mg L-1 on day 21.(4) The cell biomass, flavonoids production and nutrient consumption including nitrate, ammonium and phosphate during cultivation were investigated in progressive scale-up shake flasks to understand the characteristic of cell suspension culture of G. inflata during the scaling up process. The maximum biomass, flavonoids production and the maximum specific growth rate all decreased with progressive scale-up flasks. With regard to nutrient consumption, the trends of phosphate, nitrate and ammonium consumption of cells grown in progressive scale-up flasks were very similar. The phosphate concentration in the three levels of flasks was almost exhausted at the logarithm phase. Additionally, in any one shake flask, nitrate and ammonium both decreased. With regard to the carbon source, sucrose was hydrolyzed to glucose and fructose directly absorbed by cells, which was exhausted on day 22. Consequently, a conclusion could be drawn that the maximum biomass and flavonoids production decreased to some extent with the progressive scale-up flasks, and the main culture conditions such as the KLa needed to be taken into account and further optimized during the further development of G. inflata culture process for efficient and higher flavonoids production on a bioreactor scale.(5) The effects of different concentration of methyl jasmonate, salicylic acid and fungal polysaccharide on the cell biomass and flavonoids production in cell suspension cultures of G. inflata were studied. The results showed that the addition of appropriate concentration of the three kinds of elicitors resulted in the defense response,which increased the expression of the antioxidant system such as catalases and peroxidases, and enhanced the flavonoids accumulation finally.(6) The effect of four precursors on flavonoids biosynthesis in cell suspension cultures of G. inflata has been studied. The results showed that all these precursors could promote the flavonoids biosynthesis. Phenylalanine, tyrosine, cinnamic acid and NaAc have the most significant promoting effect on flavonoids synthesis at the concentration of 20, 5, 5 and 5 mg L-1, respectively. Moreover, the optimum time for tyrosine addition is the 5th day during the cell culture, while the optimum time for the other three kinds of precursors is the 10th day during the cell culture. Of the four precursors, tyrosine is the most effective. The maximum flavonoids production reached 113.04 mg L-1, 1.43 times as that of the control. The addition of the two kinds of precursors on the cell suspension culture promoted the flavonoids accumulation more, which suggested that the interaction effect exist.(7) The effect of water deficit on flavonoid production and physiological parameters for oxidative stress were studied in cell suspension culture of G. inflata to investigate its drought tolerance. The result indicated that appropriate water deficit enhanced flavonoid production. Drought stress led to hydrogen peroxide accumulation more than the control. Moreover, under drought conditions, malondialdehyde content, the activities of catalase and peroxidase increased to a greater extent than the control. All above suggested that appropriate water deficit could activate the antioxidative defense enzymes system to maintain stability in plants subjected to drought stress.(8) The effects of different kinds of osmotic regulator on the cell growth and flavonoids accumulation were studied. The results showed that the appropriate concentration of mannitol, KCl and sucrose addition improved the flavonoids production. During the cultivation, both mannitol and KCl could regulate the osmotic to promote the flavonoids accumulation. As to sucrose, it not only could be hydrolyzed to the fructose and glucose used directly by cell, but also might be considered as the osmotic regulator to improve the flavonoids production. |
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