found unchanged or somewhat the production reduced in comparison to nontransgenic plants. Similarly, the production of calystegines was enhanced by treating the hairy roots with 5% sucrose. When Murashige and Skoog (1962) medium was supplemented with 1.0 mM of auxin, the concentration reduced the accumulation of calystegine, but the production of tropane alkaloids remain unchanged in transgenic hairy roots (Rothe et al. 2003). On estimation it was observed that the in vitro hyoscyamine alkaloid production in differentiated leaves was more in yield in comparison to the original plant (Al-Ashaal et al. 2013; Khater et al. 2013).
Scopolamine and hyoscyamine like alkaloids, considered as anesthetic and antispasmodic drugs, are produced commercially by applying hairy root culture technology in A. belladonna. The well-regenerated hairy roots accumulate more scopolamine, hyoscyamine, and/or nicotine in A. belladonna (Endo and Yamada 1985; Yang et al. 2011). By HPLC determination, it was concluded that the accumulation of tropane alkaloids was higher in in vitro regenerated leaves but lower in the roots and shoots. Similarly, the atropine accumulation was higher, but scopolamine accumulated in lower concentration in the leaves of A. acuminata (Banerjee et al. 2008; Ashtiania and Sefidkonb 2011). The transgenic cells (containing H6H gene/s) of A. baetica were elicited with salicylic acid, acetyl salicylic acid, and methyl jasmonate for the induction of synthesis of tropane alkaloids. The synthesis of scopolamine was increased by the acetyl salicylic acid and methyl jasmonate, while salicylic acid did not affect the synthesis. This happened due to the expression of engineered h6h genes and other genes involved in the biosynthesis of alkaloids. The synthesis of scopolamine was increased by 25-fold higher than in control (Jaber-Vazdekis et al. 2008).
The biotransformation of betuligenol into an oxidized product betuloside was performed in the hairy roots of A. belladonna. The yield of biotransformed products increased twofold higher than the control root mass. On the 5th and 10th days of incubation, the highest biotransformation of betuligenol into raspberry ketone and betuligenol into betuloside was reported in hairy roots. The application of hairy roots for biotransformation of raspberry ketone and betuloside opens up new opportunities in the production of medicinally significant secondary metabolites (Srivastava et al. 2013).
The pharmaceutically important tropane alkaloids were isolated from the A. belladonna. The putrescine N-methyltransferase from N. tabacum and hyoscyamine 6β-hydroxylase from Hyoscyamus niger were overexpressed in transgenic A. belladonna for investigation of biosynthesis of tropane alkaloids in stem roots and fruits. The expression of putrescine N-methyltransferase and hyoscyamine 6β-hydroxylase was not reported in wild plant species but were found transgenic plants. The maximum conversion of hyoscyamine was observed in aerial parts, not in underground parts. In aerial parts, the hyoscyamine was fully transformed into scopolamine. The synthesis of hyoscyamine, anisodamine, and scopolamine was reported to have higher yield in underground parts than in aerial parts (Xia et al. 2016).
The effects of KCr(SO4)2 on the accumulation of tropane alkaloids as well as the expression of hyoscyamine 6β-hydroxylase gene was investigated in in vitro regenerated plantlets of A. belladonna. The chromium treatment to plantlets decreased the weights, lengths of the plantlets, and chlorophyll contents but enhanced the levels of proline contents. The chromium treatment also increased the concentration of hyoscyamine and scopolamine. The levels of scopolamine can be correlated with the expression levels of h6h gene with several concentrations of chromium (Jaber-Vazdekis et al. 2009; Vakili et al. 2012).
References
1 Al-Ashaal, H.A., Aboutabl, M.E., Maklad, Y.A., and El-Beih, A.A. (2013). Tropane alkaloids of Atropa belladonna L.: in vitro production and pharmacological profile. Egypt. Pharm. J. 12: 130–135.
2 Arráez-Román, D., Zurek, G., Bässmann, C. et al. (2008). Characterization of Atropa belladonna L. compounds by capillary electrophoresis-electrospray ionization-time of flight-mass spectrometry and capillary electrophoresis-electrospray ionization-ion trap-mass spectrometry. Electrophoresis 29: 2112–2116.
3 Asha Rani, N.S. and Prasad, M.P. (2014). Studies on the optimization conditions of root and callus initiation in Atropa belladonna in in vitro conditions. Int. J. Sci. Res. 3: 25–29.
4 Ashtiania, F. and Sefidkonb, F. (2011). Tropane alkaloids of Atropa belladonna L. and Atropa acuminata Royle ex Miers plants. J. Med. Plants Res. 5: 6515–6522.
5 Banerjee, S., Madhusudanan, K.P., Chattopadhyay, S.K. et al. (2008). Expression of tropane alkaloids in the hairy root culture of Atropa acuminata substantiated by DART mass spectrometric technique. Biomed. Chromatogr. 22: 830–834.
6 Berdai, M.A., Labib, S., Chetouani, K., and Harandou, M. (2012). Atropa belladonna intoxication: a case report. Pan. Afr. Med. J. 11: 72.
7 Bousta, D., Soulimani, R., Jarmouni, I. et al. (2001). Neurotropic, immunological and gastric effects of low doses of Atropa belladonna L., Gelsemium sempervirens L. and Poumon histamine in stressed mice. J. Ethnopharmacol. 74: 205–215.
8 Chadha, S.Y.R. (1985). The Wealth of India, vol. IA, revised edition. New Delhi: CSIR.
9 Chopra, R.N., Nayar, S.L., and Chopra, I.C. (1986). Glossary of Indian Medicinal Plants (Including the Supplement). New Delhi: CSIR.
10 Cikla, U., Turkmen, S., Karaca, Y. et al. (2011). An Atropa belladonna L. poisoning with acute subdural hematoma. Hum. Exp. Toxicol. 30: 1998–2001.
11 Endo, T. and Yamada, Y. (1985). Alkaloid production in cultured roots of three species of Duboisia. Phytochemistry 24: 1233–1236.
12 Gadzikowska, M. and Grynkiewicz, G. (2002). Tropane alkaloids in pharmaceutical and phytochemical analysis. Acta Pol. Pharm. 59: 149–160.
13 Harborne, J.B. and Khan, M.B. (1993). Variations in the alkaloidal and phenolic profiles in the genus Atropa (Solanaceae). Bot. J. Linn. Soc. 111: 47–53.
14 Jaber-Vazdekis, N., Barres, M.L., Ravelo, A.G., and Zárate, R. (2008). Effects of elicitors on tropane alkaloids and gene expression in Atropa baetica transgenic hairy roots. J. Nat. Prod. 71: 2026–2031.
15 Jaber-Vazdekis, N., González, G., Ravelo, A.G., and Zárate, R. (2009). Cloning, characterization and analysis of expression profiles of a cDNA encoding a hyoscyamine 6β-hydroxylase (H6H) from Atropa baetica Willk. Plant Physiol. Biochem. 47: 20–25.
16 Joshi, P., Wicks, A.C., and Munshi, S.K. (2003). Recurrent autumnal psychosis. Postgrad. Med. J. 79: 239–240.
17 Kamada, H., Okamura, N., Satake, M. et al. (1986). Alkaloid production by hairy root cultures in Atropa belladonna. Plant Cell Rep. 5: 239–242.
18 Khater, M.A., Soliman, S.S., Abdel-Hady, M.S., and Fayed, A.H. (2013). Tropane alkaloid production via new promising Atropa belladonna lines by in vivo and in vitro. Nat. Sci. 11: 32–40.
19 Long, S.P., Lu, Y., Wang, Y.X. et al. (2013). Enhancement of tropane alkaloids production in transgenic hair roots of Atropa belladonna by overexpressing endogenous genes AbPMT and AbH6H. Yao Xue Xue Bao 48: 243–249.
20 Maqbool, F., Singh, S., Kaloo, Z.A., and Jan, M. (2014). Medicinal importance of genus Atropa Royle – a review. Int. J. Adv. Res. 2: 48–54.
21 Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15: 473–497.
22 Paul, R. and Datta, K.A. (2011). An updated overview on Atropa belladonna L. Int. Res. J. Pharm. 2: 11–17.
23 Qiang, W., Wang, Y.X., Zhang, Q.Z. et al. (2014). Expression pattern of genes involved in tropane alkaloids
