| Low production of bioactive compounds in cultured plant cells is one of the main limitations for commercial application of plant cell culture technology. The key for producing bioactive compounds in large-scale using plant cell culture technology is to improve the production of the desired metabolites. Plant secondary metabolism is influenced by the surroundings around them. It has been well reported that environmental stresses can stimulate the synthesis of secondary metabolites in plants. High salt is one of the common stresses that plants encounter during their grwoth. In order to investigate if high salt can stimulate the production of secondary metabolites of plant cells, we use Carduus crispus L. cells as a model to check the effects of high salt on flavonoids contents and the growth of Carduus crispus L. cells. Moreover, the mechanism of the salt-induced flavonoids production is explored. The main results are belows:1. Optimizing the extraction condition of total flavonoids in Carduus crispus L. cells The extraction condition of total flavonoids was optimized in this paper. Primary and secondary factors affecting total flavonoids extraction were:Ethanol concentration＞Solid-liquid ratio＞Extraction temperature＞Extraction time.The best combination of all these factors is A2B3C1D3.In other words,60% of Ethanol concentration,1:20 of Solid-liquid ratio,60℃of Extraction temperature and 30 minites of Extraction time.2. Establishing a stable suspension line of Carduus crispus L. cellsIn order to abtain the Carduus crispus L. suspension cell line which is quick in growth,high in secondary metabolites,the effects of different ratio of Hormone concentrations, sucrose concentration, shaking speed and inoculum on cell's growth and metabolism were studied. Flavonoids production as the indicator,the best culture condition is MS+1×10-5mol/L NAA+2×10-6mol/L BA+3% Sucrose, pH5.8,25℃of culture temperature, 110 rpm of Shaking Speed,3 g/50mL medium of inoculum.3. Salt stress can induce cell death and improve flavonoids content in Carduus crispus L. cellsAfter Salt treatment,under fluorescence microscope,the cells were dyed green by Nucleic acid-specific dye Styox,while the control cells can't be imaged. Agarose gel electrophoresis analysis showed that partial nucleic acid were degradated, Showing significant tailing phenomenon while the control group showed a single band telling us the nucleic acid is complete in nomal growth cells.All these are similar to some fetures of PCD,indicating that cells were undergoing programmed cell death under Salt stress.At the same time,we found that flavonoids content was improved apparently after cells were treated with Salt.To obtain the best inducing effect of Salt stress on the flavonoids,we investigated the effects of different Salt concentration and different time treated with Salt on dry weight and flavonoids content.when cells were treated with 40mmol/L Salt solution after six days following subculture,flavonoids content in cells is the highest.Under such treatment,dry weight is 0.83 fold,flavonoids content of per-gram dry weight is 2.015 fold and the total flavonoids production is 1.67 fold as much as the control.4. NO is the essential signaling molecules of mediating Salt stress to induce cell death and stimulate flavonoid synthesisAfter Salt was added,NO production sharply increased in a short time.when cPTIO,a kind of NO-Specific scavenger,was added to eliminate the increased NO,we found that both cell death rate and flavonoid synthesis were inhibited Significantly,which told us Salt stress must dely on NO busting to induce cell death and improve flavonoid synthesis.In other words,NO is the essential signaling molecules of mediating Salt stress to induce cell death and stimulate flavonoid synthesis.5. BI-1 expresion can inhibite cell death induced by Salt stress,but have no influence on the increasement of flavonoid content induced by Salt stressTransgenic BI-1 Carduus crispus L.suspension cells were constructed using Agrobacterium-mediated method.BI-1 expression induced by Est can decrease cell death ratio, but had no influence on the improvement of flavonoid synthesis stimulated by Salt stress, so compared to cells treated only with salt,the flavonoids in the cells treated with both BI-1 and salt is higher. Under this treatment,dry weight is 0.91 fold, the total flavonoids production is 1.86 fold as much as the control. All these implied the Signal pathways mediating cell death and flavonoids synthesis were different.BI-1 expression also didn't decrease NO content induced by salt stress,showing that BI-1 inhibited cell death independent of NO Signaling molecule.Summerized the above mentioned,NO can mediate Salt stress to induce cell death and flavonoid synthesis in Carduus crispus L.cells,and BI-1 independent of NO Signaling molecule inhibited cell death induced by Salt stress but had no influence on flavonoid content stimulated by Salt stress.All these told us Salt stress induced cell death and stimulated flavonoids synthesis by sharing NO and some other Signaling molecules in the upstream but departing into two independent branch in the downstream, and the action site of BI-1 was on the cell death signal transduction Branch.
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