Cell Suspension Culture Of Cornus Officinalis And Analysis Of Its Content Of Ursolic Acid

As traditional Chinese medicine, dogwood is effectively used in treating prostration, astringent refined gas and nourishing liver and kiney. By cell culture, we can effectively use and develope the dogwood resources. In this experiment, we use the different parts of dogwood as experimental material, and then use the plant tissue and cell culture techniques for the purpose of inducing the callus and dogwood callus suspension culture system. We study on several sides systematically such as inducing the callus of dogwood, the method of regulating the loose state, the characteristics of cell suspension culture system and the regularity of its growth. Using spectrophotometry to measure the changes of the ursolic acid in dogwood to provide the theoretical basis for the industrial production of ursolic acid.In the stage of dogwood callus induction, we compare the impacts of different type of explants, different condition of sterilization, different lighting conditions, different hormone types and levels on the callus induction rate, browning and growth state of dogwood. The results show that:KT (3mg/L),+2,4-D (1mg/L) has the best induction result; Under the condition of light intensity between1500and2000lx,3%sucrose, pH value about5.8,the increasing rate of dogwood cell biomass reaches the maximum.During the process of dogwood callus subculture, if the callus is too loose or too tight, they are not both conducive to the suspension culture, so obtaining the relatively loose, and vigorous growth of callus cells is the premise for the cell suspension culture. The results show that the best subculture culture formula:MS+KT (3mg/L),+2,4-D (1mg/L) on the subculture, the optimum pH is from5.6to6.0. We compared the impact of light conditions on the callus growth, and drew a dynamic growth curve of the dogwood callus in35days. We get that callus in this stage presents "S" type growth cycle, in the10th day it entered the logarithmic phase point, in the28th day the cell biomass reached the maximum.We establish the cell suspension culture system and then study the impact of different inoculum size, sucrose concentration, liquid volume, shaking speed, hormone formulations, and the pH changes on the cell suspension culture system. We also measure the Cell dynamic growth curve during the suspension culture and the study the accumulation of the ursolic acid.This article obtains the best suspension culture conditions are as follows:prepare liquid medium, the sucrose concentration is3%, pH is5.6,2g callus in each bottle, the best hormone formula is as follow:the MS+4.0mg/L KT+2.0mg/L2,4-D. Under the above conditions, during the first8th day cell growth growth was in lag phase,and then entered into the logarithmic growth phase after the8th day, the cell biomass reached the maximum in the22th day and its growth rate is more than200%. During suspension culture, the pH value firstly decreased and then increased. Using the spectrophotometry to measure the content of ursolic acid of the cell during the suspension culture, we get that in the26th day it reached the maximum of3.48mg/g, while when dogwood fruit is ripe, the average content of ursolic acid is2.70mg/g. So Ursolic acid in suspension cultured cells is30%much more than ripe fruit...