Elicitation Of Secondary Metabolites In Changium Smyrnioides Suspension Cell Cultures

Changium smyrnioides Wolff is a medicinal herb offamily Apiaceae endemic to China.Its root is used as Chinese medicine "ming dang shen"(Changii Radix).The herb has been regarded as an endangered plant due to human activities and its own biological characteristics.The whole plant and its in vitro cultures contain various furanocoumarins with promising pharmaceutical activities.With systematic study on the establishment of furanocoumarin-oriented suspension culture system and effects of several elicitors,the present research provided a sustainable new resource for natural furanocoumarins.1.Previous studies on the biological characteristics of Changium smyrnioides,main secondary metabolites and their pharmaceutical activities as well as the plant tissue culture practices on Changium smyrnioides were reviewed.The elicitation practices on plant cell cultures aiming for secondary metabolites in recent years were also introduced.2.A furanocoumarin-oriented Changium smyrnioides cell culture system was established with systematic study on the cell lines and the culture conditions.Callus was induced from leaf and petiole.Callus with rapid growth and high contents of furanocoumarins were selected for suspension cultures.The cell lines were selected again and P717 derived from petiole was chosen.The optimal initial inoculums was 2 g/L and the cell cultures were incubated in light at 25℃ in medium with 30 g/L sucrose at pH 5.8.The growth cycle of the established culture was about 15 days.3.The effects of single elicitors and their combinations on Changium smyrnioides cell culture system were investigated.The 10th day of growth cycle was the proper time point for treatment.Methyl jasmonate(MJ),salicylic acid(SA),calcium chloride(CaCl2)and their combined treatments induced the accumulations of 3 furanocoumarins.MJ+CaCl2 enhanced the highest concentrations of furanocoumarins but this treatment suppressed the growth seriously.The highest productions of xanthotoxin(2.08 times of control)and bergapten(40.10 times of control)were induced by MJ while the top production of bergaptol was enhanced by CaCl2(79.40 times of control).4.The changes in biomass,viability of cells,furanocoumarins accumulations and stress-related compounds or enzymes in suspension cells were investigated after elicitation.The effective elicitors or combined treatments aroused oxidative stresses,triggered stress defence in cells and then led to the accumulations of defence-related secondary metabolites such as furanocoumarins.5.The contents of polysaccharide,nucleosides,amino acids and furanocoumarins were quauntified and analyzed.The result showed that the original and regenerated plant contained higher contents of polysaccharide,amino acids and furanocoumarins than samples of callus and cell cultures did.Moreover,original and regenerated plant accumulated more kinds of nucleosides and amino acids than callus and cell cultures did.The cell culture was more sensitive to elicitations than the plant,which resulted higher and more sustaining enhancement of furanocoumarins in suspension cells.