Studies On Tissue Culture And Metabolic Regulation Of Saussurea Involucrate Kar.et Kir.

Saussurea involucrate Kar. et Kir. is a perennial herbaceous plant belonging to the family of Compositae. Pharmacological studies have demonstrated that S. involucrate and its constitutes have anti-inflammatory, cardiotonic, terminating the gestation, anti-cancer and anti-fatigue actions. S. involucrate is indispensably rare and valuable medical herb in our ethnic drug. S. involucrate plant were formally recorded in the new edition of Chinese Pharmacopoeia (2005).We induced the plant from the ripe seed, from which callus was induced. The most suitable medium of callus induction was MS+NAA 0.5 mg·L-1+6-BA 2.0 mg·L-1 +sucrose 3% +agar0.45%. During subculturing, high-yielding cell line was screened, and the growth medium and production medium were screened preliminary. The subculture medium of callus was MS+NAA 5.0 mg·L-1+6-BA 0.5 mg·L-1+sucrose 3% +agar 0.45%; the bud differentiation medium was MS+NAA 0.2 mg·L-1+6-BA 2.0 mg·L-1+sucrose 3% +agar 0.45%; the bud subculture medium was MS+NAA 1.0 mg·L-1+6-BA2.0 mg·L-1+sucrose 3% +agar 0.45%; the root differentiation medium was MS+NAA 0.2 mg·L-1+IAA 0.2 mg·L-1+sucrose 3% +agar 0.45% +0.2% active carbon. Synchronized cell was obtained by cell size screening and starvation culture, with which the suspension culture system was established. The most suitable inoculation concentration was 2.53 g·L-1。The cell suspension culture condition was: MS+NAA 5.0 mg·L-1+6BA 0.5 mg·L-1 +sucrose3%; pH5.8; temperature:24±1°C; light period:12 h·d-1; light strength:85μmol·m-2s-1; rotary speed:120±5rpm.S. involucrate cell suspension culture growth cycle was 16 d. The lag phase was 0-2 d, early log phase was 2-6 d, middle log phase was 6-14 d, and stable phase was 14-16 d. We studied of kinetics of cell growth, product accumulation, and substrate consumption in suspension culture system. It provided theoretical basis for regulating the path of secondary metabolism.Three abiotic elicitors were added into suspension culture system.50μmol·L-1 NO were added into the system on the 14th day, and the content and yield of flavonoids reached 46.15 mg·g-1 and 830.7 mg·L-1. As the same,50μmol·L-1 SA were added into the system on the 14th day, and the content and yield of flavonoids reached 46.81 mg·g-1 and 701.79 mg·L-1. We added 100μmol·L-1 MJ on the 6th day, and the content and yield of flavonoids reached 52.33 mg·g-1 and 924.67 mg·L-1. The three abiotic elicitors could enhance the content of flavonoids and the activity of PAL, and could enhance the biomass except SA. We added four fungal elicitors (F1817、F1832、F1780、F1953、F2563), the content of flavonoids were all enhanced, the content of flavonoids and syringin reached 46.56 mg·g-1 and 3.03 mg·g-1. The best fungal elicitor was F1953.The content of active component of S. involucrate was increased to a great extent by induced with biotic and abiotic elicitors. The content of flavonoids in induced suspension culture reached 60.41 mg·g-1,3 times higher than each parts of wild material. The content of syringin in induced suspension culture reached 3.10 mg·g-1, 11.48 times higher than each parts of wild material.