Rosemary Callus Induction, Suspension Cell Culture And Determination Of Physical And Chemical Indicators

Rosemary(Rosemarinus officinalis L.)belongs to Labiatae.It is a perennial evergreen sub-shrub,native to Europe and the Mediterranean coast.It is common in France,Spain,Italy,Yugoslavia,Tunisia,Morocco and other countries,and also distributed in the United States,and China.Rosemary's antioxidative components mainly consist of phenolic diterpenes(such as carnosic acid,carnosol,rosmanol,and ursolic acid),as well as phenolic acid(like rosmarinic acid,ferulic acid and others).Rosemary's antioxidants have anti-inflammatory,antibacterial and antiviral,anti-tumor,immunomodulatory effects,as well as the suppression of uric acid and many other pharmacological effects.Its main application areas are food preservation techniques,and cosmetic and pharmaceutical applications.Rosemary's main components will change dramatically depending on regional,environmental,agronomic conditions,the time of harvest,the stage of development of plants,the method of extraction.Using plant suspension cell culture techniques to obtain a stable high-yielding active product's cell line is an ideal way to solve this problem.This paper mainly focus on the cultivation of high-yielding callus of rosemary,the establishment of suspension cell lines,and the law of the physiological and biochemical changes in the process of cell cultivation,as well as the measurement of rosemary culture's antioxidative phenolic diterpenes and rosmarinic acid.The main results of this research are as follows:1.Rosemary calluses were induced by different sterilization methods,with different explants material,basic medium,hormone's concentrations and ratio.The following results are shown:Treating rosemary leaves with 0.1%mercury bichloride solution for 4 minutes,then cutting the leaves into small pieces about 0.5cmx0.5cm,using the inoculation method that the back of leaves contacts with the medium,on MS medium with NAA at 5.0mg/L and 6-BA at 2.0 mg/L,it is possible to induce callus,and the callus are crunchy,green and gritty.2.The main components of rosemary calluses are preliminarily analysied and optimized culture condition to promote the proliferation,the contents of antioxidative phenolic diterpenes and rosmarinic acid of callus lines by using different basic medium,plant growth regulators,the sucrose content in medium and the addition of browning inhibitors and organic coconut milk.The results showed that:Rosemary callus contains a large amount of antioxidative phenolic diterpenes substances and rosmarinic acid.Calli were stabilized through subculturing for several times on the same medium conditions with 2,4-D at 0.8mg/L,KT at 0.2mg/L,sucrose at 30g/L,VC at 20mg/L,10%coconut milk in MS medium,in this way it is possible to derive high-yielding active products'callis of rosemary.3.A stable,active rosemary suspension cell culture system were established through the basic medium selection,the amount of inoculums,pH,and hormone concentrations and ratios.The results show that:after suspension culture for several generations in the MS medium supplemented with 2,4-D at 0.8mg/L,KT at 0.2mg/L,sucrose at 30g/L,VC at 20mg/L,10%coconut milk,it is possible to derive rosemary suspension cell lines with high cell viability and stable growth.4.To determinate the best time to extract the products and subculture the rosemary cell lines by measuring cell growth,the content of antioxidative phenolic diterpenes and rosmarinic acid in cells,medium's pH,conductivity curve and sucrose's content.The results show that;the 18th day in the growth cycle is the best time to extract the product or to subculture the cell lines.