Production Of Paeonol From Paeonia Suffruticosa Andr. Callus Tissue: Induction And Growth Optimization

Paeonol,one of important bioactive substances in the root bark of Paeonia suffrutiscosa Andrew,has comprehensive range of benefits,such as antisepsis,protecting kidney and liver,anti-tumor and treating diabetes,cardiovascular and cerebrovascular diseases and so on.A series of studies in this paper were conducted to overcome the disadvantages in the traditional paeonol preparation process and further improve its comprehensive utilization value and preparation efficiency of paeonol.P.suffruticosa cv.‘ Luoyang Red ',an ornamental peony,and P.ostii T.Hong et J.X.Zhang,a medical peony as experimental materials,the content of paeonol in different parts of peony was compared.The technique of medicinal plant tissue culture was adopted to establish aseptic culture system of different explants,investigate the effects of different factors on inducing the callus and the content of paeonol in callus,and optimize the optimum culture program by response surface methodology.Under the guidance of the optimal formulation,the growth and content of paeonol of callus was facilitated.The results of this study preliminarily demonstrate that there are feasibility and superiority of plant cell engineering technology applied to large-scale prepare paeonol.1.The establishment of culture systemAsepsis system of embryo,bulbil,petiole and young stem segment was established,respectively,and browning phenomenon among these process was treatment study and browning of young stem was reduced effectively.The callus of embryo,bulbil,petiole and young stem was induced successfully by optimizing culture conditions.Comparing the effect of different explants,basic media and hormone on inducing their callus,the embryo's rate of callus and growth vigour was higher than others,thus it was assumed the best explant material to secondary culture,and the optimum nutrition formulas of two kinds of peony were determined,respectively.For P.ostii,its optimum nutrition formula is MS+ 6-BA0.5mg/L+2,4-D 0.5 mg/L,and the optimum nutrition formulaof P.suffruticosa cv.‘Luoyang Red' is MS+ 6-BA 0.5mg/L+2,4-D 1.0 mg/L.2.The determination of paeonolThe content of paeonol in different explants and their callus was determined by UV-spectrophotometry and the effect of different induction conditions on paeonol accumulation of embryo's callus was inspected.The results showed that paeonol widely exists in different parts of peony,non-traditional medicinal parts also have medicinal value,and the content of the paeonol in different parts' callus was also different.The effects of different factors on the content of paeonol in callus of embryo were inspected by single factor.The results showed that the content of paeonol in 30-days' callus was the highest.The content of paeonol in the fourth-generation embryo's callus was the highest,thus the third-generation callus was further studied.The conditions,such as MS basic culture medium,0.5mg/L6-BA,4.0mg/L 2,4-D,Cu0.125 mg/L,Ca1760 mg/L,phe 60mg/L,sucrose as carbon source,pH 5.8,culture 30 days by ordinary fluorescent lamp and conserved under8?,could contribute to increase the content of paeonol in callus.3.Determination of optimal culture programThe main factors influencing on the content of paeonol in embryo's callus among single factor experiments were investigated,the important factors were filtrated firstly by Plackett-Burman experiments and the specific level of these factors was determined by the steepest climbing experiment and Central Composite Design.Finally,the optimum culture programs suitable for the accumulation of paeonol in embryo's callus were optimized preliminary respectively,which were MS+6-BA 0.5mg/L+2,4-D 4.0mg/L +Ca 2771.38mg/L+Cu 0.11mg/L+phe 120mg/L+agar 7.5g/L+sucrose 30g/L for P.ostii and MS+ 6-BA 0.26mg/L+2,4-D 4.0mg/L+Ca 3117.62mg/L+Cu 0.11mg/L+ phe 75.23mg/L for P.suffruticosa cv.‘Luoyang Red',30-day culture,normal light,saved in 8?.With culturing by guidance of the optimum culture programs,the content of paeonol in these plants' callus was 10.21 and 5.86 times than the original level,showing a good optimization effectiveness and these conclusions have some certain reference value.