| We obtained high quality, high yield of substitute for Scutellaria baicalensis secondary metabolites by using suspension culture that its advantages are high-throughput and strong antioxidant capacities.S. baicalensis is one of the traditional Chinese medicines and its secondary metabolites (baicalin) with good efficiency on clearing damp-drying anti-inflammatory, antiviral, antibacterial and anticancer has been applied to international medical circles wildly. In our study, callus induced from S. baicalensis seedling stems were used to establish a cell suspension culture system. The industrialization theory for using cell suspension culture to produce secondary metabolites was deduced by adding different elicitors, such as methyl jasmonate(MJ) and salicylic acid(SA) to the cell. We have also investigated the hyperhydricity mechanism of S. baicalensis regenerated shoots and established the method of reduced hyperhydricity from S. baicalensis regenerated shoots.The results are as follows:1. The establishment of S.baicalensis cell suspension culture systemCalluses were induced from 2 month old S. baicalensis seedling stems. The stems were cultured on callus induction and regeneration medium. The suitable medium for suspension culture for S. baicalensis cells was 100ml liquid MS medium with 1.5mg/L 6-BA,0.5mg/L NAA,2%(w/v) sucrose and 0.75%(w/v) agar. Then, we measured antioxidant capacity of S. baicalensis cell from different times. A comprehensive subordinate degree represented the antioxidant capacity. The results showed that the comprehensive subordinate degree of antioxidant capacity from S. baicalensis cell reached its maximum(2.69) after 20 days in the suspension culture. The baicalin content reached a high of 78.31mg/g.2. Influence of elicitor on cell suspension culture of S. baicalensis.We established the optimum method of suspension culture for S. baicalensis cells. Adding 150 umol/L MJ took the cell growth rate to reach 967%,and baicalin content reached 131.21 mg/g after suspending culture for 72 hours. As expected, its antioxident capacity improved, FRAP reached 558.46, the clearance rate of DPPH had also reached 101.51%. There was any significant effect of SA on the cell suspension culture. It supplied the industrialization theory for using elicitors on cell suspension culture to produce secondary metabolites.3. The research of decreasing the hyperhydricity and rooting method of S. baicalensis.For reducing hyperhydric rate and improving rooting percentage of in vitro regenerated shoots from S. baicalensis. In the present study, regenerated shoots induced from aseptic seedling stems were used to study the effect of 6-BA, sucrose, agar and paclobotrazol(PP333) on hyperhydricity of regenerated shoots in S. baicalensis. As well, the effects of IBA on rooting of regenerated shoots were studied. The results showed that the hyperhydric rate significantly decreased with lower 6-BA concentration on the medium. The lowest hyperhydric rate and highest multiplication rate were found on medium containing 0.2 mg/L 6-BA. The best multiplication coefficient in vitro shoots without hyperhydricity were found on the medium containing 25g/L sucrose. On the medium containing 7.5 g/L agar, hyperhydric rate of S. baicalensis adventitious shoots was the lowest with higher proliferation coefficient. Simultaneously, we found that the medium containing PP333 positively affected the hyperhydric occurrence of regenerated shoots. With the increase of PP333 concentration on the medium, the hyperhydric rate decreased gradually and in vitro shoots were stronger and healthier. The optimal rooting was achieved from the medium containing 0.1 mg/L IBA. The rooting percentage was 100%with 7 roots per shoot. The survival rate of rooting plantlets was over 95%after transfer to soil.4. Research on the hyperhydricity mechanism of S. baicalensis regenerated shoots.In the present study, morphologic and biochemical changes in hyperhydric shoots compared to normal shoots of S. baicalensis were characterized. In contrast to normal shoots, hyperhydric shoots presented some anatomical abnormalities. In hyperhydric leaves, epidermal cells displayed discontinuity, palisade tissue was absent, and the spongy mesophyll showed larger, unorganized intercellular spaces. The abnormal stomata with deformed guard cells were also found in the hyperhydric leaves. In hyperhydric stems, the epidermal cells were discrete, part of the cortical parenchyma cells and myeloid cells were swollen and burst. In addition, chlorophyll and soluble protein content in hyperhydric shoots decreased significantly. The malondialdehyde(MDA) and the H2O2 level showed marked increase of 43.75%and 46.43% respectively. While, superoxide dismutase (SOD), peroxidase(POD) and catalase(CAT) activities significantly increased in hyperhydric shoots compared to normal shoots. Our results indicated that biochemical changes and abnormal anatomy occurrence in hyperhydric shoots of 5. baicalensis were caused by oxidative stress. |
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