Methyl-β-cyclodextrin and coronatine as new elicitors of tropane alkaloid biosynthesis in Atropa acuminata and Atropa belladonna hairy root cultures.

Abstract

Hyoscyamine (HYO) and scopolamine (SCO) are tropane alkaloids acting as anticholinergic factors on the parasympathetic nervous system in humans and are produced by Solanaceous plants. Two strains of Agrobacterium rhizogenes, A4 and LBA9402, were used to infect Atropa acuminata Royle ex Miers and Atropa belladonna L. leaf explants. A. acuminata was inoculated either by direct infection or sonicated-assisted A. rhizogenes-mediated transformation (SAAT) was performed. A. belladonna was inoculated with the A4 strain using a direct method. The selected hairy root lines of both species were elicited with 50 mM methyl-β-cyclodextrin (β-CD), 0.5μM coronatine (Cor) or 50 mM β-CD +0.5μM Cor on Day 14 of culture. The elicitor effect on growth and HYO and SCO content was analyzed after one (T1) and two (T2) weeks of treatment. In A. acuminata explants, the highest transformation percentage (T%) was obtained with strain A4 and the SAAT method (T%: 96.43). Cor significantly reduced the growth of A. acuminata hairy roots (fresh weight and dry weight [DW]: 2.52 and 0.3g, respectively), whereas β-CD increased their DW (0.4g). Also, the combined β-CD + Cor treatment had a positive significant effect on the DW of A. belladonna hairy roots (0.41 g). In A. acuminata hairy roots, the HYO level was lower under Cor treatment than in the control at both sampling times. In contrast, the SCO content was increased 10-fold by Cor elicitation at T1 compared to the control (10.95 mg g-1 DW) and was also positively affected by β-CD + Cor. In A. belladonna hairy roots, all the elicitors had a negative effect on both HYO and SCO production. This report is the first assessment of the effect of β-CD and Cor elicitors on tropane alkaloid production.