|
In this thesis Yunnan large leaf species spring shoots of leaves as explant material, B5 0.5mg / L 2,4-D 0.1mg / L KT medium induced callus. After several subculture taken loose callus suspension culture, optimization of culture conditions and the eventual establishment of suspension cell lines. Explore the physical and chemical factors and elicitors on cell growth and catechin content and components. The main findings are as follows: 1. Study the impact of different factors on callus induction of tea: MS and B5 medium only callus formation early differences, B5 medium induced better, but after a week no significant difference; seeds explants induced callus induction rate, up to 80% or more; spring callus induction rate compared with the dark culture in autumn; 12 hours, light can significantly reduce the browning , but the long-term light inhibition of callus induction and growth of callus denser, looser callus under dark conditions, suitable for cell suspension culture. 2. Orthogonal experimental design, different shaking speed, liquid volume, inoculum size tea Suspension Cell Growth and catechin yield, the results show that the 80rpm shaking speed, 30ml liquid filling amount, 1g inoculum size The optimum conditions for tea cell suspension culture. Measured under optimum tea cell suspension culture conditions, the cell growth rate, catechin accumulation and substrate consumption, drawing a growth curve. The highest cell dry weight was 3.77 times proliferation, the suspension cells catechin content of 57.83mg/gDCW cultured system catechin total content for 501.39mg / L, but only catechin components EGC, EC and ECG. 3. Study the light and sucrose physicochemical factors on the growth of cells in the tea plant cell suspension cultures, the cumulative effect of the catechins. The results show: the suspended cells in the logarithmic growth phase after the light treatment, the cell fresh weight and dry weight, respectively, than those of CK decreased by 32.73% and 25.61%, respectively, while catechin accumulated on than 61.45% of CK HPLC analysis indicated that the illumination significantly stimulate the C, EC, EGC synthesis, wherein the C content of the fresh leaves of 176.91%, while the EC and EGC 120.98 percent and 359.04 percent of the control, respectively. The sucrose content of 8%, the cumulative cell growth and catechin significantly enhance the role of cell fresh weight, dry weight, and catechin content than CK 29.31%, 19.38%, 19.80%. HPLC analysis showed that the C content is in the sucrose treatment 106.33% of the fresh leaves, EC and EGC 101.25 percent and 307.93 percent of the control, respectively. Two treatments was not detected in the ester catechins ECG of EGCG. . Elicitor salicylic acid (SA), methyl jasmonate (MJ), and yeast extract (YE) growth of cells in the cell suspension cultures of tea catechins cumulative impacts. 1mg / L MJ 5mg / L YE cell growth slightly affected, but to promote the accumulation of catechin their catechin content of 78.02% and 71.10%, respectively, compared with CK. 1mg / L MJ processing of the suspended cells, the C content is fresh leaves of 203%, while the EC only control 44.41%, and its accompanying the increase in the ECG, the control 289 of%, speculated that may exist of EC no food sub-technology process; Further , to the EGC content of 12.75 times higher than that of the control has reached 81.75% of the fresh leaves, but it is still not detected of EGCG, which the mechanism is not clear. 5mg / L YE treated suspension cells, the C content is 256% of the fresh leaves, EC and EGC is not detected, the ECG substantially increased to 753.76%, 48.08% higher than the fresh leaves, also exists in the EC gallic process. Salicylic cumulative effects on cell growth and catechin.
|