The Influence Of Multiple Factors On Grape Cells Culture And Secondary Metabolic Products

As a kind of phytoalexin, resveratrol has multiple functions such as anti-oxidation, anti-free radicals, lowering blood lipid, anti-tumor, anti-mutagenesis, anti-aging and so on. In this thesis, a clone of grape cell line with high yielding of resveratrol has been obtained for the first time, by using plant tissue culture and cell suspension culture techniques. Furthermore, the effects of nutritional and environmental factors on this cell line have been investigated systematically, and comparison has been performed between the cell growth and the accumulation of resveratrol during the whole culture process. And it has also investigated the relationships between the cell amount and factors such as inoculation, dissolved oxygen and the shear stress under the large-scale culture conditions.To improve the survival rate of culture materials and the callus formation, with the grape skin as the explant, NAA 6.0mg/L+6-BA 0.6mg/L have been added into the MS culture medium. A new generation was formed every 21 days, and callus grew well. Callus from a 7-10d secondary culture was adopted to perform the liquid culture. A new generation was formed every 15 days, and the suspended cells proliferated rapidly.The results showed that, in the process of suspension cell and large-scale culture, the grape cell line Jf01 maintained the high growth rate and the good dispersal of the mother plant, and its synthesis capability of target product was significantly improved. Thus, it's a high-yielding cell lines.The grape cell line Jf01 maintained features such as fast growth and good dispersibility in both shake-flask culture and bioreactor culture. And obvious improvement was observed in the synthesis of resveratrol. Thus, it was a high-yielding cell line.To some extent, this cell line is not sensitive to environmental factors and has relatively strong resistance. The optimal culture condition is as follows: 30g/L sucrose, pH5.8 (before sterilization), 150 rpm (stirring velocity) and 100-150g/L (FW, inoculum). The results also showed that cells had some requirements for the concentration of dissolved oxygen in the proliferation stage, with the oxygen-consuming peak at the initial culture stage. With the increase in stirring rate, the shear stress increased and the quantity of cells assembled in cell mass decreased. As a result, cells were deformed and disrupted, the accumulation of biomass was inhibited and even decreased, which eventually caused the cell lysis and the decrease in the synthesis of resveratrol.No obvious changes in the growth state and the synthesis of resveratrol in both shake-flask culture and bioreactor culture of the cell line Jf01. Thus, it possesses some advantages in the large-scale culture.