Cell growth kinetics and accumulation of secondary metabolite of Bletilla striata Rchb.f. using cell suspension culture

Abstract

14 15 [Abstract] Bletilla striata (Orchidaceae) is a well-recognized endangered medicinal 16 plant due to inadequate natural reproduction with high market worth. To evaluate the 17 cell growth kinetics and accumulation of secondary metabolites (SMs), the cell 18 suspension culture is proved to be a valuable approach for acquiring the high yield of 19 medicinal parts. An effective cell suspension culture for obtaining B. striata cell 20 growth and its SMs was in vitro induction of callus from B. striata seeds. The cell 21 growth kinetics and accumulation of SMs were analyzed using the mathematical 22 model. Results cell growth kinetic model revealed that the growth curve of B. striata 23 suspension cells was curved as sigmoid shape, indicating the changes of the growth 24 curve of suspension cells. Improved Murashige and Skoog cell growth medium was 25 the utmost favorable medium for B. striata callus formation with the highest cell 26 growth during the stationary phase of cultivation period, the cell growth acceleration 27was started after 7 days and thereafter gradually decrease at 24 day and then reached 28 to highest at 36 day of cultivation period in both dry weight and fresh weight. The 29 coelonin concentration was peak during exponential growth stage and decreased 30 afterward at the stationary phase in the cell suspension culture. The maximum content 31 of coelonin (about 0.3323 mg/g cell dry weight) was observed on the 18th day of the 32 cultivation cycle while the dactylorhin A and militarine reached highest at 24 day, and 33 p-hydroxybenzyl alcohol at 39 day. This investigation also laid a foundation for 34 multi-mathematical model to better describe the accumulation variation of SMs. The 35 production of SMs had shown great specificity during cells growth and development. 36 This research provided a well-organized way to more accumulation and production of 37 SMs, on scale-up biosynthesis in B. striata cell suspension culture. 38[Key words] Bletilla striata; suspension culture; growth curve; secondary 39 metabolites; kinetics; mathematical model 40 41 Introduction 42Bletilla striata is a perennial herb of Orchidaceae, which is an important traditional 43 Chinese herbal medicine recorded in the pharmacopoeia of all previous dynasties. It is 44 sweet, and slightly cold with prominent effects of healing muscles and stopping 45 bleeding. Hence is ideal for treating traumatic bleeding [1] . At present, due to the 46 limitation of the traditional artificial breeding methods, the production of B. striata 47 tuber and its medicinal active ingredients is difficult to meet the market demand [2] . B. 48 striata has turn out to be an endangered class with the falling down wild plant 49 properties due to over utilization in current periods. For the sustainable progress and 50 comprehensive consumption of B. striata, it is essential to recognize its cell growth 51 dynamics with the accumulation of SMs. However, until now, limited approaches 52 have been developed on these perspectives. In addition, the quali...