Studies On Polysaccharide Production In Suspension Cell Culture Of Cyclocarya Paliurus

Cyclocarya paliurus belongs to the Cyclocarya Iljinskaja (Juglandaceae),which is one of the endangered plants under state protection. As a good health food resources,Cyclocarya paliurus has many functional compounds, such as flavonoid. polysaccharide.triterpenoids and so on.Due to the scarcity of Cyclocarya paliurus resources.and the sexual and asexual reproduction problems, which seriously restrict the development and utilization of Cyclocarya paliurus,using cell culture technology to produce active ingredient of Cyclocarya paliurus has been considered as a feasiable alternative method.The new approach has important meaning for protecting of Cyclocarya paliurus and its sustainable use.This thesis studied the law of suspension cell growth,polysaccharide synthesis and nutrients consumption.The effect of many physical and chemical factors on suspension cell growth and polysaccharide accumulation were systematically explored.the factors include medium type, carbon sources, macronutrient nutrients, culture condition and elicitor.The results showed as follows:1. Close relations exist between cell growth,polysaccharide synthesis and nutrients consumption. Growth curve basic showed "S" type,0to4days for lag phase,4-10days for exponential growth phase,10-14days for stationary phase,14-18days for decline phase.The content of polysaccharide rapidly increase in the logarithmic period,and reached highest at day10.after that it begin to fall.Suger consumption was slow in delay period,and quickly consumed in logarithmic period.Suger was used up at day12.As to the absorption of nitrogen source,NH4+superior to NO3-. NO3-was almost line dropped. The consumption of PO43-was very fast,80%of PO43-was used at day6.2. Among the seven basic medium of WPM?MS?1/2MS?B5?N6?DKW and White tested, WPM?MS?B5were favourable for the growth of suspension cells,MS was good for polysaccharide accumulation.Among the six carbon sources of sucrose, glucose, fructose, maltose, lactose, starch,use sucrose or fructose as carbon source,the biomass of suspension cells was significantly higher than the other carbon sources.Sucrose the most appropriate carbon source consider of polysaccharide production and cost.The best sucrose concentration was40g/L.Adding15g/L sucrose at the end of exponential growth phase can greatly improve the polysaccharide priduction,which was1.64times that of control.3. Effects of macronutrient on suspension cell growth and polysaccharide content were studied with single factor experiments,based on this,the composition of sucrose,total nitrogen, Mg2+,Ca2+was optimized by L9(34) orthogonal design. The results showed as follows:the optimal level to cell growth was40g/L sucrose+30mmol/L total nitrogen+2 mmol/L Mg2++4mmol/L Ca2+.The optimal level to polysaccharide content was40g/L sucrose+30mmol/L total nitrogen+3mmol/L Mg2++3mmol/L Ca2+4. Effects of four plant hormones on suspension cell growth and polysaccharide content were investigated by L16(45) by orthogonal experiment.The result indicated that2,4-D was the key factor to the growth of suspension cells and had a significant effect on the growth. Suspension cells grew well in MS medium supplemented with0.4mg/L2,4-D,0.4mg/L NAA.0.4mg/L KT and0.4mg/L6-BA.and the polysaccharide content was high in MS medium supplemented with0.8mg/L2,4-D,0.4mg/L NAA,0.4mg/LKT.5. Experimental results indicated that the impact order of four factors on the growth and polysaccharide accumulation was listed as follows:inoculum densities>liquid volume> rotation speed>initial pH,the optimal culture condition conbination was8%of Inoculum densities,40%of liquid medium volume,115r/min of rotation speed and initial pH5.0.6. Effects of salicylic acid and Ce3+on suspension cell growth and polysaccharide accumulation were examined. Adding20?mol/L of SA or Ce3+can significantly improve the yield of polysaccharide,1.44,1.40times of control respectively.