Effect of Brassinolide on Growth and Shikonin Formation in Cultured Onosma paniculatum Cells

Abstract

The effect of brassinolide (BR) on cell growth and shikonin and its derivative formation in Onosma paniculatum cell culture was studied. BR addition with IAA and BAP (+BR/+IAA/+BAP) in B(5) medium slightly increased the cell growth at 0.01-0.1 ppb concentration compared with a growth control (-BR/+IAA/+BAP). Only BR addition (+BR/-IAA/-BAP) at 0.001-100 ppb in B(5) medium significantly increased the cell fresh weight compared with a growth control (-BR/-IAA/-BAP). The same concentration of BR tested at 0-1000 ppb increased 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) medium increased shikonin and its derivative content markedly by 31-87%, compared with its control (-BR/+IAA/+BAP). BR at 0.001-1000 ppb without IAA and BAP added to M(9) medium (+BR/-IAA/-BAP) also increased shikonin and its derivative content compared with its control (-BR/-IAA/-BAP). However, the amount 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 results show that BR at 0.01 ppb with IAA and BAP added was the best for cell growth and shikonin formation. Formation of shikonin and its derivative by 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 not as effective as +BR/+IAA/+BAP vs. -BR/+IAA/+BAP for the shikonin formation. 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. Similarly, soluble protein content in the cells treated by BR at 0.01 ppb with 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 soluble protein content over that of -BR/-IAA/-BAP.

Expression analysis of shikonin-biosynthetic genes in response to M9 medium and light in Lithospermum erythrorhizon cell cultures

“…Shikonin and its derivatives, red naphthoquinone secondary metabolites, are produced and accumulated in the roots of members of Boraginaceae plants, such as Lithospermum erythrorhizon (Tabata et al 1974) and Onosma paniculatum (Yang et al 1999). These metabolites are synthesized through the isoprenoid (Heide et al 1989;Yamaga et al 1993;Gaisser and Heide 1996;Wang and Wu 2004) and the phenylpropanoid pathways (Hahlbrock and Scheel 1989).…”

“…1). Moreover, various biotic and abiotic factors, such as fungal elicitors (Tani et al 1992), plant hormones (Yazaki et al 1997b;Yang et al 1999;Touno et al 2005), and light signals (Tabata et al 1974;Heide et al 1989;Gaisser and Heide 1996;Liu et al 2006), have been investigated to study the molecular mechanism of biosynthesis of shikonin and its derivatives. Previously, methyl jasmonate (MeJA), a plant growth regulator known to stimulate the formation of shikonin and its derivatives, has been reported to strongly up-regulate the expression of PGT and CYP98A6 (Matsuno et al 2002;Yazaki et al 2002).…”

Difference in in vitro response and esculin content in two populations of Taraxacum officinale Weber

“…Indeed in Cichorium intybus, according to Rehman et al (2003) esculin content increased under in vitro conditions with increasing age and differentiation. An enhancement in shikonin content was observed in cultured Onosma paniculatum cells when brassinolide, IAA and BA were added (Yang et al 1999). Pectin administered to in vitro cultures of Uncaria tomentosa enhanced the production of triterpene acids (Ursolic and oleanic acid) without affecting growth and sterol accumulation (Flores-Sanchez et al 2002).…”

Effect of Light on Gene Expression and Shikonin Formation in Cultured Onosma Paniculatum Cells

“…The two-stage culture system was used, including a growth stage for cell proliferation, in which the callus was maintained in a B 5 medium at 25°C in light (80 lmol m )2 s )2 8 h/ day) and the subculture was carried out every 16-18 days, and a production stage for the formation of shikonin and its derivatives in M 9 liquid medium (Fujita et al, 1981;Ning and Cao, 1995). IAA at 0.05 mg l )1 and BA at 1 mg l )1 were added to B5 medium as a basic growth regulator combination, while IAA at 0.1 mg l )1 and BA at 1 mg l )1 were added to M 9 production medium (Ning and Cao, 1995;Yang et al, 1999Yang et al, , 2003. To produce shikonin, about 2 g of callus produced during the growth stage in B 5 medium was inoculated in a 250 ml Erlenmayer flask containing 50 ml of M 9 medium and cultured on a rotary shaker at 120 rpm at 25±1°C in the dark.…”

“…To produce shikonin, about 2 g of callus produced during the growth stage in B 5 medium was inoculated in a 250 ml Erlenmayer flask containing 50 ml of M 9 medium and cultured on a rotary shaker at 120 rpm at 25±1°C in the dark. The calluses were harvested after 18-20 days to measure shikonin production (Yang et al, 1999).…”