Studies On Browning Control Technology And Its Mechanism In Tissue Culture Of Anemone Flaccida Fr. Schmidt

Anemone flaccida Fr. Schmidt is a species of Anemone genus. The dried rhizoma of A. flaccida locally named as "Diwu" is used as a Chinese traditional herb medicine with functions of expelling rheumatism, relieving detumescence and pain, and being conducive to physique. It has magnitude significance of development. The phenomenon of browning is widespread in the rapid propagation in vitro culture of Anemone flaccida Fr. Schmidt, and becoming one of the limiting factors on explant differentiation and proliferation and tissue culture success. In order to find the effective ways to control browning and explain the mechanisms, this thesis focused on the effect of different treatment which affected the explant differentiation and proliferation and browning, such as pre-culturing on blank medium, at low temperature or darkness, antioxidants immersion treatment and adding adsorbents or antioxidants to the culture medium. The relation between the content of total phenols, PPO activity, POD activity and browning was explored in this study. The main results are as follows:1. Pre-culturing on blank medium which contained1g/L active carbon for2days could reduce the browning degree and increase the multiplication coefficient.2. Pre-culturing at low temperature before subculture for7days could reduce the browning degree, but reduce the multiplication coefficient at the same time.3. Pre-culturing at low temperature or darkness after subculture for4days could reduce the browning degree, but also reduce the multiplication coefficient. Pre-culturing at low temperature could effectively control browning in early days, but the browning index rose sharply under normal condition.4.0.5g/L Na2S2O3by immersion treatment could reduce the browning of explants tissue to the greatest extent and increase the multiplication coefficient.5. The order of inhibition effect on browning of different antioxidants was NaiS2O3> ascorbic acid> citric acid> active carbon.0.1g/L (active carbon) and0.2g/L (Na2S2O3) were the best concentrations respectively.6. Halved the content of sucrose or microelement deficiency and doubling the content of inositol could both reduce the degree of browning and increase the multiplication coefficient.7. Explant cultured on medium supplemented with Na2S2O3showed varying degrees of reduction in browning, total phenols, POD activity and PPO activity. Total phenols, POD activity and PPO activity of the explant were all the reasons that lead to browning. The effect of PPO activity was more significant than POD activity on browning.