| The traditional Chinese medicinal plant Polygonum cuspidatum Sie bet Zucc, a keratin shape shrub of Polygonaceae Polygonum genus, widely distributs in Liaoning , Shanxi, Gansu, Sichuan, Guizhou, Yunnan, the east and mid south of China. Recently, the further research of the active components in Polygonum cuspidatum has suggested that resveratrol exhibits many pharmic functions, such as anti-carcinogenic, anti-cardiovascular disease and female estrogenic balance. It was attached importance to the study and exploitation of resveratrol today. Furthermore experiment showed by content of resveratrol in Polygonum cuspidatum was highest in many plants with analysis of secondary metabolite and active components. To establish the callus suspension culture system in vitro, the rhizome and bud of Polygonum cuspidatum were employed as explants, researches on the biosynthesis, modulation and biotransformation and content difference of resveratrol were also researched in the current paper. The results as follows:1. Species resource research:The resveratrol content of Polygonum cuspidatum plant growing in different districts of Wuling Mountains region is obviously different. The investigation on the growth circumstance in 30 different districts and geographic localities suggested the most desirable growth condition of Polygonum cuspidatum were: slope towards south, slope location upgrade, soil yellow-red earth ,clay, shale soil matrix, scattered illumination. The yield of resveratrol is highest from August to September. Resveratrol is mostly accumulated in the rhizome and hardly existed in other parts, especially in the rhizome buds, so rhizome buds were regarded as the preferred explant for callus induction via and tissue culture in vitro.2. Histochemical localization and quantification of resveratrolA method of resveratrol histochemical localization and determination was established by experiment, providing a theoretic and technique platform to constructing granule callus suspension culture system. Bright-field optical microscope and fluorescence microscope were employed for observation after staining or other pretreatment of Polygonum cuspidatum, such as AlCl3 or H2O treatment. The results showed that resveratrol were distributed in the of fibre tube tissue , namely phloem bast fibre , xylem vessel, xylem fibre cell and periderm phellem cell. Most resveratrol distributed in callus periderm and vessel tissue node and knot. This makes a larger scale tissue culture possible.The quantitative analysis by High Performance Liquid Chromatography andfluorescence photometer verified that resveratrol content in marrow was the lowest , and its content in xylem is relatively lower than in phloem.3. Analysis on factors affecting resveratrol content in Polygonum cuspidatumThe research showed that eight endogenous hormones contents in Polygonum Cuspidatum roots and stems significantly changed from January to December by analysis on factors affecting resveratrol content. The accumulation contribution ratio of the former five principal components was 91.487%, contribution ratio of the first and the second principal component was 39.026% ,17.816% respective .They had the most variances contribution ratio of resveratrol of Polygonum cuspidatum in different season. Z and ABA were the largest variable factors in the first principal component. Both of their was related to the phytorones sinals, So called them "promoting phytormones growth of root and stems and transferring stress signals factors. Their correlation with resveratrol accumulation yield were significant, complex correlation coefficient was 0.589.The results showed that eight inorganic element contents in Polygonum Cuspidatum roots and stems, the three atmosphere factors (month average temperature, month sunshine times and month precipition) changed significantly largely from January to December. The accumulation contribution ratio of the former five principal components reached 94.087%, and contribution ratio of the first and the second principal component was 35.539% and 33.538% respectively. Both of them had them most contribution ratio of resveratrol in Polygonum cuspidatum in different season. Mg, K, and Zn were the largest variable factors in the first principal component. They were called "mineral element promoting enzyme reaction" factors according to their function. Month average temperature, month sunshine time and Cu were the largest variable factors in the second principal component, and they were called "high temperature and strong shines" factors. Analysis of the correlation between the five principal component and resveratrol contents showed that the second principal component, had the significant relation to resveratrol contents among the five principal components, and its complex correlation coefficient was 0.617. The research provides the base to for the resveratrol regulation.4. Suspension culture of granule callusThe bud of the rhizome was the best explants to induce callus on the medium of Murashige and Skoog's (MS) medium with 2 mg-I/'NAA and 0.1 mg-L'1 KT, because the induction ratio of callus is higher and the induction time is shorter. Resveratrol content in rhizome bud callus is highest and the maximum value is 8100u g ? g"1 in Cy8 cell line. Cy8 was used as the explants of suspension culture .Cy8 callus of rhizome bud were subculture for many times. Growing activity, loose and light yellow or pink callus were selected as the materials for callus suspension culture. The optimized suspension culture conditions were: MS culture medium with 2 mg ? L"1 NAA ,0.1 mg ? L'KT, 56 g ? L"1 sucrose and 300 mg ? L"1 casein hydrolyzed(CH), 45g ? L'1 inoculation quantity and circumrotion rate 80r ? min"1. granule callus suspension culture system Stable high resveratrol yield of was established experiments, but the relation between cell growth and accumulation of resveratrol was not positive relation.5. 2L air-lift bio-reactor suspension culture of Polygonum Cuspidatum granule callus in 2L air -lift bio-reactorsuspension cultures of Polygonum Cuspidatum granule callus were cultured in a 2L air-lift bioreactor, its growth periods was about 14-16 days .The cell thickness reached the highest and growth velocity was quick when the cell grew for 12 days. Results showed content of resveratrol content was the highest in cell masses whose diameter were 2-3mm, and content of resveratrol analogue was highest in 3—5mm cell. It makes granule callus suspension enlarging culture by bio-reactor possible.6. Resveratrol biosynthesis pathway in Polygonum cuspidatumPhenylalanine, a precursor of resveratrol, was added in the cell suspension culture system, results showed that resveratrol content was related to PAL enzyme activity and increased dramatically in the culture system adding lmL 1 mmol 'L"1 or 2 mmol 'L^phenylalanine. biosynthesis pathway phenylalanine- flavonoid and stilbenes were presumed to be involved in the resveratrol biosynthesis pathway. The synthesis rate of Polygonum cuspidatum resveratrol can be promoted by adding 40mg ? L"1 resveratrol analog in the suspension culture system , furthermore, cell development in the system were little affected. 20 mg ? L"1 and 40mg ? L"1 resveratrol analogue had no significant effects on cell growth in the system. Resveratrol analogue maybe become thetarget in future culture.7. Regulation of Polygonum Cuspidatum resveratrol production and accumulation by elicitorIt is a beneficial to resveratrol synthesis when the content of Mg2+ in MS culture medium increased 1.5 times, all content of Mg2+ over or under 1.5 times, is unbeneficial. It is advantageous to the growth of Polygonum Cuspidatum callus when Cu2+ was added or decreased properly in MS medium. 1.0 mg ? I/'is secondary, and 3 mg' L'!KT restrained the resveratrol synthesis of Polygonum Cuspidatum in suspension culture system. The phenomenon microbial elicitor restrained cell growth and make resveratrol content increase was not observed. It...
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