Study On Secondary Metabolites Production And Metabolism Regulation Of Tripterygium Wilfordii Hook F. Tissue Culture

Tripterygium wilfordii Hook f. is a traditional Chinese herbal medicines as well as an important resource to product botanical pesticides. Its demand is increasingly in medicine, the development of new pesticides, and other aspects in recent years. However, the resources of wild T. wilfordii are sharply declining because of the blind exploitation. It is an important value through tissue culture methods to produce useful metabolites. The tissue culture was studied in this purpose for protecting natural resources, preserving the ecological environment and laying the foundation for developing sustainable use of resources. The callus induction was studed using the root, stem and leaves of one-year-old T. wilfordii branch cuttings as explants. The suspension cell lines and the adventitious root lines were established using the loose callus after repeated subculture. The triptolide-and alkaloid content of the cell suspension, adventitious root were explored the influence of the explants, medium components, culture conditions, precursor substances, elicitors. The main results and conclusions were given as follows:1.T. wilfordii was used to study the callus induction and to optimize the culture condition.The callus induction was significantly impacted by the explant type, medium type, concentration and composition of the hormone. Three kinds of explants of T. wilfordii, which can be used to induct callus, are roots, stems and leaves. MS medium was suitable for callus induction of T. wilfordii in 6 kinds of basic medium. Callus was induced 47.6% when explants were cultured on MS medium supplemented with 1.0 mg/L 2,4-D and callus was induced 36.32% when explants were cultured on MS medium supplemented with 2.0 mg/LNAA. The callus induction rate were increased when auxin and cytokinin KT or 6-BA were used in conjunction. MS+1.0 mg/L 2,4-D +0.5¹.0 mg/LKT was best for callus induction of T. wilfordii. The callus was induced more than 90%.The callus growth and the accumulation of triptolide and alkaloids were studied by 3 different sources of the callus subculture. The callus growth and the accumulation of triptolide and alkaloids were discussed by the influence of the basic medium, concentration and composition of hormones, prevention of browning and so on. Root, stem and leaf explants of three kinds established callus, in the same medium for more than substituting subculture, the root callus became loose, granular, and the stems, leaves dense callus , a massive, serious browning. The root callus growth and triptolide content was significantly higher than that of the stem, leaf callus, and leaf callus of the alkaloid content was significantly higher than that of the root, stem callus content. 6,7-V was suitable for the callus subculture. White medium was suitable for accumulation of triptolide and alkaloid but the callue growth was low. The medium supplemented with 1.0 mg/L2,4-D and 0.5 mg/LKT increased the callus growth and alkaloid content. The medium supplemented with 4.0 mg/L NAA and 0.5 mg/L6-BA increased the triptolide content. The medium supplemented with 5¹0 mg/L AgNO3 not only inhibited browning callus but also increased triptolide content significantly. The callus growth and the content of triptolide and alkaloid are all highest than others in dark culture, and the callus browning is lesser. The callus growth reached the maximum at the first 50 days, and triptolide reached the maximum level at the first 55 days, alkaloid content is increasing with the extension of time for culturing.2.The establishmeng and optimization of T. wilfordii suspension line and the impact of triptolide and alkaloid content.The suspension line of T. wilfordii was established to studied the cell growth and the accumulation of triptolide and alkaloids through basic medium, subculture time, inoculation density, pH and add seven kinds of amino acids, 10 kinds of non-biological elicitor and seven kinds of precursors. NT medium was favourable to the cell growth. MS medium was favourable to subculture. White medium was favourable to accumulate of triptolide and alkaloid. Inoculating quantity controled in 8¹2 FW g/L was appropriate for cell growth. The suitable time for subculture was 30³5 d. The suitable time for harvest was 55 d. The best pH for the cell growth was 5.8. The best pH for the accumulation of triptolide was 6.4. The best pH for the accumulation of triptolide was 5.2.The cell growth was significantly inhibited by adding aspartic acid, tyrosine and arginine and serine, and the stronger the concentration the greater its inhibition; Joinning phenylalanine, methionine and cysteine at low concentrations, can promote cell growth as well. Seven kinds of amino acids can promote triptolide synthesis at different levels. The contents of triptolide were improved 3.2 times and 2.8 times over control respectively when arginine was 1mmol/L and homocysteine was 2 mmol/L. The synthesis of alkaloids was promoted when other amino acid was joined except of homocysteine. The content of alkaloids was improved 1.5 times over control,and the content of triptolide was improved 2.3 times over control,when methionine was 0.5 mmol/L . The cell growth was significantly inhibited by adding non-biological elicitor, soy protein, salicylic acid, hydrolyzed casein protein, glutamine, acid hydrolysis casein and formaldehyde, while CCC, sodium acetate, yeast extract and sodium benzoate can promote cell growth at low concentrations. The synthesis of triptolide was promoted when others was joined except of hydrolysis casein.The content of triptolide were improved 2.4 times and 2.9 times over control respectively when glutamine was 2 mmol/L and soybean protein was 1 g/ L. The synthesis of alkaloid was promoted when other elicitors were joined except of salicylic acid. The content of alkaloids was improved 1.3 times over control the when yeast extract was 2 g/L. The cell growth was only about 77% of control when formaldehyde concentration was 1.0 mL/L, but the triptolide content was 2 times of control, the total alkaloid content was 1.2 times of control.The cell growth was improved by adding precursor substances at low concentrations except of cinnamic acid. The synthetic triptolide was promoted significantly by adding precursor substances except of malonic acid. The content of triptolide was improved 2 times over control when cinnamic acid was 10 mg/L. Not only the triptolide content was 2.5 times than the control, but also the alkaloid content was 1.5 times than the control when the medium supplemented with 10 ml/L isoprene. And the decline in the growth of cells is not obvious, for the control of 96%. Citric acid, cinnamic acid and pyruvate were inhibited synthesis of alkaloids. Malonic acid, malic acid and tartaric acid can promote the synthesis of the alkaloids. The content of alkaloids was improved 2 times over control when malonic acid was 2.0 mmol/L.3.The establishmeng and optimization of T. wilfordii adventitious roots line and the impact of triptolide and alkaloid content.The adventitious roots line of T. wilfordii was induced and established to study the disciplinarian of medium nutrient consumption. Iarge number of elements, trace elements, carbon and organic and biological elicitor, which can influence the growth of adventitious roots and the accumulation of triptolide and alkaloids is studied. The adventitious roots began to grow by adding 4.0 mg/L NAA in the cells logarithmic phase in cell suspension cultures. After the acclimation training, the stable culture adventitious root line was formed. The triptolide of the adventitious root was about 2.1 times as cell and 4.5 times as root bark respectively.The adventitious root growth was in stagnation because nitrogen, phosphorus, and potassium content were obviously insufficient, through the study of nutrient consumption of NT medium. When NO3-/NH4+consistented with the medium of NT and unchanged, the adventitious root growth increased sharply with the increase of the concentration of nitrogen in the medium. When the nitrogen concentration was 50 mmol/L, the growth of adventitious root reached the maximum. The content of triptolide and alkaloids reached the maximum when the nitrogen concentration was 30 mmol/L. The growth of adventitious root reached the maximum when NO3-/NH4+ was 9︰1, triptolide content reached the highest when nitrogen content is the same as NT medium; Only in ammonium circumstances, the total alkaloids content was the highest. The growth of adventitious root increased with the increase of the concentration of PO43-, K+, Ca²⁺ and Mg²⁺; The content of triptolide was to the maximum when K+ concentration in the medium is 1.3 times of NT medium and ,the Ca²⁺ concentration is 2/3 times of NT medium. The content of triptolide increased with the increase of PO43- concentration. The content of triptolide was to the maximum when Mg²⁺ concentration was content of NT. The content of alkaloids was to the maximum when PO43-,K+,Ca²⁺ and Mg²⁺ concentration were content of NT.Trace elements, Fe²⁺,Zn²⁺ and Cu²⁺ can meet adventitious root growth in NT medium. The adventitious root reached the maximum when 2 times Mn²⁺ concentration of NT. The adventitious root growth was increased with the concentration of Na2MoO4 increase in the pilot area. Medium which was lack of H3BO3, CoCI2 and CoCI2 did not affect adventitious root growth; The triptolide and alkaloids accumulation reached the maximum when Fe²⁺ concentration was 1/3 of normal standards . The content of triptolide and alkaloids were increased with Mn²⁺ concentration increase. The triptolide accumulation reached the maximum when Zn²⁺ concentration was 2 times, and the accumulation of alkaloids was increased with the increase of Zn²⁺ exponentially in the pilot area; The content of triptolide and alkaloids were reached the maximum when Cu²⁺ concentration was 2 times of NT. The synthesis of alkaloids was significantly inhibited by adding H3BO3 and the higher of its concentration the stronger of the inhibition. The lack of KI did not affect the synthesis of alkaloids in medium. The triptolide content increased with the concentration of KI. The lack of CoCI2 did not affect the synthesis of triptolide in medium. and alkaloids content reached the maximum when CoCI2 concentration was 2 times of NT. The synthesis of triptolide and alkaloids were favourable when Na2MoO4 was lack.Sucrose was carbon source which was best for adventitious root growth and triptolide accumulation. Glucose was best for alkaloid accumulation. The adventitious root increment was highest when the sucrose concentration was 45 g/L. The triptolide and alkaloid contents were highest when the sucrose concentration was 20 g/L. NT medium in the normal standard of inositol and VB1 can make adventitious root growth, triptolide and alkaloid content reached the maximum at 7 kinds of organic compounds. The triptolide content reached the maximum by adding 0.5 mg/L nicotinic acid. However, the synthesis of alkaloids was significantly inhibited and and the higher of its concentration the stronger of the inhibition.The adventitious root growth, triptolide and alkaloid content reached the highest when VB6 concentration was 1mg/L . The adventitious root growth, triptolide content were increased with the concentration of glycine in the pilot area and the alkaloid content reached the maximum by adding 1 mg/L glycine. Folic acid and biotin accession did not affect the growth of adventitious roots, but when folic acid concentration was 1 mg/L, triptolide and alkaloid content reached the maximum. Triptolide content reached the maximum when biotin was 0.5 mg/L and the alkaloid content reached the maximum when biotin was 1 mg/L.The triptolide and alkaloid biosynthesis in the adventitious root cultures of T. wilfordii was enhanced via a treatment of seven elicitors separately, streptomycin elicitor and G. cingulata elicitor were the best, but neither of them manifests any noticeable effects on the cell growth of the adventitious root cultures. The triptolide content of the adventitious root cultures treated with G. cingulata elicitor was 88.65μg/gDW, which was 2.2 times than the control (39.62μg/gDW). The total alkaloids content of the adventitious root cultures treated with G. cingulata elicitor was 6.09 mg/g DW, which was 2 times than the control (3.02 mg/gDW). The results showed that elicitation was dependent on the elicitor concentration, the incubation period and the physiological stage at which the adventitious root cultures were treated. In addition, we found that for V G. cingulata, the optimum concentration was 100μg carbohydrate per milliliter medium, the strongest response of T. wilfordii adventitious root cultures to the elicitation was at the late exponential growth stage, and the highest triptolide content of the adventitious root cultures was on the 15th day post treatment.The alkaloid content raised with the extension of time the elicitor.In different size of flash, according to 2/5 volume of flash to join the medium. With the increase of the volume of flash, adventitious root growth of T. wilfordii declined slightly, dropped 2.92% and 6.57% respectively in 1 L and 5 L flash. The adventitious root growth ,triptolide and the total alkaloids content in the medium were almost not affected. The triptolide and the total alkaloids are secreted. The triptolide yield was 56% and the total alkaloids was 65% after filtering adventitious roots in the culture medium.Different extracts of culture production have obvious inhibition and toxicity effect on P. xylostella larvae.. Treated with the extraction of adventitious root cultures, the P. xylostella has a negative amount of weight. After 72 h, about 70 percent was died, the weight of survival insects dropped by 18.33 percent before test, LC50 was 6.72 mg/mL. Toxicity effects was 2 times of the root bark.4.The insecticidal activity and inhibition of the growth and development of Plutella xylostella was studed through tissue culture products extract of T. wilfordii. It was established in the same extract of HPLC method for triptolide and UV method for total alkaloids using the root bark, leaves, the cultures and medium of T. wilfordii Hook. f.The linear relationship of the triptolide concentrations and peak areas was good in range of 1¹00μg/ mL (R2=0.999 9);The linear relationship of the total alkaloids concentrations and absorbance was good in range of 5-100μg/ mL (R2=0.999 8). In the same extract the average recovery of triptolide was 100.72%(RSD=0.975%)and the average recovery of total alkaloids was 100.33%(RSD=2.56%). Toxicity activity of different cultures extracts on P. xylostella larvae was an obviously evident. The adventitious root extract was the strongest activity on P. xylostella larvae. LC50 was 6.72 mg/mL. Toxicity effects was 2 times of the root bark. Effect different cultures extracts on the growth and development of P. xylostella larvae was inhibited significantly. Extraction of adventitious root cultures of the P. xylostella a negative amount of weight, 72 h after about 70 percent was died, the survival of insects weight dropped 18.33 percent before test.5.The establishment of regeneration system and the study of rapid propagation of T. wilfordiiThe embryogenic callus were used to study the influence factors of the occurrence of somatic embryogenesis and plant regeneration process, the proliferation of shoots. Histological observation has indicated that T. wilfordii somatic embryogenesis callus originated from a internal single cell, and the development process of somatic cell embrvos of T. wilfordii is similar to that of zygotic embryos. NAA is better than 2,4-D; 6-BA is better than KT, callus tissue regeneration rate is improved by the use of NAA and 6-BA. For the test, such as six kinds of medium, callus plant regeneration rate of MS and B5 reached 100 percent; Not only callus tissue regeneration rate reached 100 percent, but also the average callus formation bud of each callus will be more on MS medium supplemented with 0.1 mg/L NAA and 1.0 mg/L 6-BA.; The embryogenic callus of T. wilfordii ,which was in the process of subculture plant , regeneration rate and the number of small seedlings formation by callus differentiation, gone from a low to high and then to a low process. The regenerative capacity of embryogenic callus regeneratived between 18 months and a year was strongest, and up to 100%. The embryogenic callus of T. wilfordii was loss capacity of differentiation after 20 months. Stem proliferation was better on 1/2 MS +2.0 mg/L IBA.Plantlets in 1/2 MS +2.0 mg/L IBA +0.5 g/L of activate carbon was suitable to the rooting medium culture, rooting rate of 100%. The survival rate up to 96%, when transplanted to mixture, which was composed by 0% perlite and 50% humus soi.