Establishment Of Culture System Of Echinacean Purpurea Hairy Roots And Construction Of Bioreactor For Hairy Root Culture

Echinacea purpurea, famous for its distinct pharmacological efficacy and commercial value, has been using to diminish inflammation, kill virus and bacterium, as well as to enhance people's immunocompetence as a kind of herbal medicine. Hairy roots induced by Agrobacterium rhizogenes have the ability to grow rapidly without phytohormone, and it is easy for hairy roots to control and operate. They also have physiologic, biochemical and genetic stability, showing unique superiorities and large potential for production of secondary metabolites. In this paper, a culture system of E. purpurea hairy roots was established, effects of sound stimulation on growth of the induced hairy roots and accumulation of their secondary metabolites were studied, and a type of sound loading bioreactor for hairy root culture was constructed.At first, leaves of E. purpurea were infected by A. rhizogenes strain A4, at the same time, single-factor experiments and orthogonal experiment for several influential factors were carried out to find the best condition for inducement of E. purpurea hairy roots. As a result, the most appropriate infecting time was about 8min, the most appropriate OD600 of A4 suspension was about 0.8, the most appropriate time of sonication treatment to assist transformation of A4 was about 1.5min, the most appropriate co-culture temperature was about 22℃and the most appropriate pH of culture medium was about 6.0, under which the average inducing rate reached 67%. The induced roots were identified and confirmed to be transformed hairy roots by PCR analysis. It was also obtained that 1/2 MS culture medium was the most favorable one for growth of E. purpurea hairy roots and accumulation of their secondary metabolites among MS, 1/2MS, White, B5 and SH basic culture medium, in which the average growth fold of fresh weight was 38.4, the contents of polysaccharides and phenolic compounds were 17.97% and 2.32%, respectively.Secondly, the effects of sound waves with different intensity and frequency on average growth fold of fresh weight, contents of polysaccharides and phenolic compounds of E. purpurea hairy roots were studied. Furthermore, growth of E. purpurea hairy roots under sound stimulation with certain intensity and frequency was also studied. In general, 108dB and 800Hz sound stimulation had more favorable stimulative performance, under which hairy roots reached their logarithmic growth period and maximum growth 3 days earlier than those of the hairy roots in control group. The maximum average growth fold of fresh weight increased to 48.1, 9.5 times more than that of the control group, while the contents of polysaccharides and phenolic compounds were 18.84% and 2.49% after culture for 27 days, 1.05% and 0.11% higher than those of the hairy roots in control group, respectively.At last, a new type of sound loading bioreactor was designed based on various types of bioreactors in existence for hairy root culture in existence and the study of above-mentioned sound loading experiments. Additionally, technological process design of hairy root culture was finished and the sound field in the bioreactor was analyzed according to spreading character of sound waves. In this sound loading bioreactor, nutriment was transferred by ultrasonic atomization and sound waves with different intensity and frequency could be loaded on the culture. Simultaneously, dissolved oxygen, pH, temperature and conductivity in it could be monitored and determined. When used for hairy root culture, this bioreactor was able to facilitate growth of hairy roots and accumulation of their secondary metabolites by sound stimulation. Thus, it could be a technological innovation for hairy root culture and a contribution to industrialized production of hairy roots of herbal medicine.