Standardization Of Tissue Culturing System And Bioreactor Micropropagation Of Pinellia Ternata (thunb.)breit.

Pinellia, Pinellia ternata (Thunb.) Breit., which has been used as a medicinal plant for more than 2000 years, is one of the traditional Chinese medicinal plant species, belonging to family Araceae. In this paper, we have set up a standardized tissue culture system through artificial division of the growth periods and optimized process. A novel temporary immersion bioreactor was designed to overcome the disadvantages for traditional tissue culture, which costs lots of human power and material resources. Then a new way for large-scale automated production of pinellia was tested with some preliminary experiments for micropropagation in this bioreactor.1. Establishment of standardization of tissue culture system. The established procedures of the standardization of tissue culture system are as follows. A petiole was put into Ms+6-BA 1.0 mg/L+NAA 0.05 mg/L+ sucrose 3%+agar 5.0 g/L medium to induce callus and multiple shoot clumps after treated by 75% alcohol 20s, then in mercuric chloride for 8min. When the shoots grow to"petiole elongation stage", the clumps was cut into a single bud to switching into Ms+6-BA 1.0 mg/L+NAA 0.02 mg/L+ sucrose 3%+agar 5.0g/L medium for proliferation. The"Leaf maturity stage"buds were cut into 1/2Ms+IBA 0.03 mg/L+NAA 0.01 mg/L+ sucrose 3%+agar 5.0 g/L+AC 0.3 g/L medium to induce root growth and transplant the plantlet into farm after 30 days growth, when the seedlings are needed for a direct cultivation in greenhouse. When the seedlings are not required for direct planting, the"Leaves proliferating stage"buds were cut into 1/2Ms+IBA 0.03 mg/L+NAA 0.01 mg/L+ sucrose 5% + agar 5.0 g/L + AC 0.3 g/L medium to induce root growth. As the plantlet growing leaves withered and In vitro tubers matured. In vitro tubers can be stored in a culture bottle a long time. In vitro tubers can be cleaned and planted when the weather is suitable for cultivation. 2. Development of temporary immersion bioreactor and its utilization for micropropagation. When the temporary immersion bioreactor was designed and assembled, it was found to work steadily, and the utilization on micropropagation of pinellia was tested. On the one hand, we studied the growth situation of different explants in the bioreactor by comparison of biomass, dry matter rate, multiplication and the state of plantlet. With multiple-buds and petioles as explants, 1957 and 1382 plantlets in each bioreactor were obtained after 60 days culture, and the multiplication ratio were 39.15 and 27.65. With petioles as explants we can get more robust plantlets, so petioles are the ideal explants. On the other hand, with petioles as explants, we compared the situation of plantlets in three kinds of culture methods, including temporary immersion culture, solid culture and Liquid shake flask culture. Bioreactor performed well on Biomass, dry matter rate, multiplication and so on. Such as, the multiplication ratio in bioreactor culture was 24.73, followed by solid culture ratio of 14.75, and it was 12.26 in liquid shake flask culture. Apparently, we can get the most robust plantlets on solid culture. There is no difference between solid culture and bioreactor culture, and they are better than liquid shake flask, judged by the numbers of chloroplast. This shows that bioreactor culture has a high multiplication compared with traditional solid culture. Also the bioreactor have advantages of higher automation, when vaccination, it needs only to replace the liquid medium. In conclusion, the newly developed temporary immersion bioreactor is suitable for culturing pinellia.