Effect of brassinolide on growth and shikonin formation in cultured Onosmapaniculatum cells

The effect of brassinolide (BR) on cell growth and shikonin and its derivat ive formation in Onosma paniculatum cell culture was studied. BR addition w ith IAA and BAP (+BR/+IAA/+BAP) in B-5 medium slightly increased the cell g rowth at 0.01-0.1 ppb concentration compared with a growth control (-BR/+IA A/+BAP). Only BR addition (+BR/-IAA/-BAP) at 0.001-100 ppb in B-5 medium si gnificantly increased the cell fresh weight compared with a growth control (-BR/-IAA/-BAP). The same concentration of BR tested at 0-1000 ppb increase d the cell fresh weight of +IAA/+BAP significantly more than that of -IAA/- BAP. BR at 0.001-0.1 ppb with IAA and BAP added (+BR/+IAA/+BAP) in M-9 medi um increased shikonin and its derivative content markedly by 31-87%, compar ed with its control (-BR/+IAA/+BAP). BR at 0.001-1000 ppb without IAA and B AP added to M-9 medium (+BR/-IAA/-BAP) also increased shikonin and its deri vative content compared with its control (-BR/-IAA/-BAP). However, the amou nt of shikonin and derivative formed of +IAA/+BAP was greater than that of -IAA/-BAP only at the same concentration of BR at 0-1 ppb. These combined r esults show that BR at 0.01 ppb with IAA and BAP added was the best for cel l growth and shikonin formation. Formation of shikonin and its derivative b y adding BR at 0.01 ppb with IAA and BAP (+BR/+IAA/+BAP) in M-9 medium was significantly enhanced 4 days after BR addition compared with a production control (-BR/+IAA/+BAP). In contrast, +BR/-IAA/-BAP vs. -BR/-IAA/-BAP was n ot as effective as +BR/+IAA/+BAP vs. -BR/+IAA/+BAP for the shikonin formati on. The time course study for shikonin formation also showed that +BR/+IAA/ +BAP and -BP/+IAA/+BAP only slightly increased cell growth in M-9 medium. S imilarly, soluble protein content in the cells treated by BR at 0.01 ppb wi th IAA and BAP (+BR/+IAA/+BAP) exceeded that of the control (-BR/+IAA/+BAP) 4 days after BR addition. And +BR/-IAA/-BAP only slightly increased the so luble protein content over that of -BR/-IAA/-BAP.