Root cultures, alkaloid production

In Table XIV through XVIII the occurrence of alkaloids in various types of cell and tissue cultures of Atropa, Datura, Duboisia, Hyoscyamus, and various other species in the family Solanaceae is summarized. From these data it is clear that the production of tropane alkaloids in cell suspension cultures is rather low. Only in root cultures has production similar to, or even higher than, the original plant been obtained. For this reason an extensive discussion on efforts to improve production in cell suspension cultures is not useful instead, we briefly deal with the application of plant biotechnology for the improvement of the tropane alkaloid-producing plants. Finally, we discuss the bioconversion of added precursors. 

Hashimoto T, Yun D-J, Yamada Y. (1993) Production of tropane alkaloids in genetically engineered root cultures. Phytochemistry 32 713-718. 

Goodbody and co-workers (7/9) have examined the production of alkaloids in root and shoot cultures induced from seedlings of C. roseus. The pattern of alkaloids in the root cultures was similar to that of the roots from intact plants. Thus ajmalicine (39) and catharanthine (4) were produced, but no vindoline (3), a major leaf alkaloid, and no bisindole alkaloids. Similarly, the pattern of the alkaloid content of the shoot cultures was like that of the leaves of the intact plant, showing the presence of vindoline (3), catharanthine (4), and ajmalicine (39), with 3 predominating. A search for the bisindole alkaloids in the cultures indicated the presence of anhydrovinblastine (8) and leurosine (11) in the shoot cultures (2.6 and 0.3 xg/g fresh weight, respectively), but no vinblastine (1) or vincristine (2). 

Key words agriculture, alkaloids, biofertilizers, biotechnology, channels, dmgs, food, ions, medicine, plant protection, production, receptors, root cultures... 

In vitro production of alkaloids is possible also with the use of another in vitro technique organ culture. Root cultures are the most common concerning alkaloids, as this part of plants is of great importance for alkaloid synthesis. Root cultures that produce alkaloids have been studied as far back... 

Robins, R. J., Parr, A. J., Payne, J., Walton, N. J. and Rhodes, M. J. C. 1990. Factors regulating tropane alkaloid production in a transformed root culture of a Datura Candida x Datura aurea hybrid. Planta, 181 414-422. 

Hartmann, T. and Toppel. G. 1987. Senecionine A-oxide, the primary product of pyrrolizidine alkaloid biosynthesis in root cultures of Senecio vulgaris. Phytochemistry, 26 1639-1643. 

Hashimoto, T., Yukimune, Y. and Yamada, Y. 1986. Tropane alkaloid production of Hyoscyamus root cultures. Journal of Plant Physiology, 124 61-75. 

Extensive studies to quantitate the production of indole alkaloids in Catharanthus roseus hairy root cultures have revealed that they accumulate several compounds including ajmalicine, serpentine, catharanthine, tabersonine, horhammericine, and lochnericine.27, 28 The presence of tabersonine in hairy roots has raised speculations that this intermediate in vindoline biosynthesis, together with catharanthine, is transported from this potential site of biosynthesis through the vasculature to the stem and to the leaves where tabersonine is further elaborated into vindoline within laticifers and/or idioblasts.26 However, oxidized derivatives of tabersonine, such as horhammericine and lochnericine, are present at 5 to 15 times the levels of tabersonine in hairy roots,27 and presumably this prevents their transport and/or use for vindoline biosynthesis. In this context, it would be interesting to... 

Important for the production of scopolamine, hyoscyamine 6p-hydroxylase was the first enzyme of tropane alkaloid metabolism to be purified and remains the most rigorously studied. It was obtained in pure form from H. niger root cultures and the preparation showed that it is a bifunctional enzyme with activity both as the 7(3-hydroxylase and as the 6,7 3-epoxidase. A clone for H6H was obtained following the purification of enzyme activity. The gene shows some similarity to other hydroxylases, including those involved in oxidative reactions in the formation of ethylene and anthocyanins (Hashimoto and Yamada, 1994). 

Walton, N.J., Robins, R.J. and Rhodes, M.J.C. (1988) Perturbation of alkaloid production by cadaverine in hairy root cultures of Nicotiana rustica. Plant Sci., 54, 125-31. 

Atropa belladonna plants have been transformed with an H6H clone from H. niger. A. belladonna normally produces high levels of hyoscyamine, the precursor for the more pharmaceutically valuable alkaloid scopolamine (Fig. 3b). However, after transformation with the H6H gene, transgenic A. belladonna plants were shown to accumulate scopolamine almost exclusively (164). Additionally, the levels of tropane alkaloid production in a variety of hairy root cultures were altered by overexpression of methyltransferase putrescine-N-methyltransferase and H6H. Overexpression of both of these enzymes in a hairy root cell culture resulted in significant increases in scopolamine production (164, 165). Fluorinated phenyllactic acid substrates... 

There are considerable literatures on the production of tropane alkaloids in tissue and cell cultures derived firom various parts of intact plants [4]. In a number of cases, root differentiation is required for enhanced tropane alkaloid biosynthesis [3, 5]. The production of the economically valuable tropane alkaloids, scopolamine and hyoscyamine, by these cultures has not been commercially successful, however, root cultures are so far the best system to investigate the production and biosynthesis of tropane alkaloids. 

In this article, we demonstrate the establishment of the adventitious and the hairy root cultures of several solanaceous plant species including Datura, Duboisia, Hyoscyamus, and Scopolia etc. for the production and biosynthetic studies of tropane alkaloids. In addition, the isolation and structural elucidation of the new tropane alkaloid 7P-hydroxyhyoscyamine and the piperidone alkaloid hyalbidone from them is also presented. 

Alkaloid production by adventitious root culture of Duboisia hybrid... 

The genus Duboisia consists of three species, D. leichhardtii F. Muell, D. myoporoides R. Br. and D. hopwoodii F. Muell. The leaves of the former two species are a major source of the tropane alkaloids, scopolamine and hyoscyamine [1]. The alkaloid content in Duboisia can be affected by the environment, and a seasonal decline was noted in the scopolamine content between May and September in Australia [18]. Callus and root cultures of Duboisia have been studied with a view to developing a new method to obtain tropane alkaloids more efficiently and the results obtained supported the view that root organ culture is so far the best system for the production of tropane alkaloids [5]. Therefore suitable culture conditions for growth and alkaloid production must be established. 

Fig. 3. Effects of lAA, NAA and 2,4-D on growth and alkaloid production. Roots were cultured in the dark for four weeks. The numbers in brackets represent growth index on a dry weight basis. Ban represent standard deviation of the mean, n=3.

The time course of alkaloid production were also examined with different concentrations of lAA (0.5 mg/1), 4-Cl-IAA (0.1 mg/1) and 5,6-Cl2-IAA (0.01 mg/1) (Fig. 4). The alkaloid yield in roots cultured with 4-Cl-IAA and 5,6-Cl2-IAA increased more rapidly than those with lAA after two weeks of culture. Scopolamine was also detected in the culture medium, especially after four to five weeks, and its yield increased significantly in the presence of 4-Cl-IAA and 5,6-Cl2-IAA as compared to lAA. A continual recovery of useful secondary metabolites from a culture medium might be important for the industrial production. These results indicate that lAA derivatives (4-Cl-lAA and 5,6-Cl2-IAA) may be applied to the industrial production of scopolamine in cultures. 

Alkaloid production in hairy root cultures of Hyoscyamus albus... 

The hairy root cultures of Hyoscyamus species have been studied for alkaloid production by several researchers [3, 23, 24]. However, the influence of phytohormones and light on the production of tropane alkaloids in transformed roots of this genus has not been reported in detail. In this section, we describe the alkaloid production by H. albus hairy roots cultured under different conditions. 

We compared the time course of tropane alkaloid production in the adventitious and two types of hairy roots of H. albus, transformed with the different strains of A. rhizogenes [15]. In the adventitious roots of H. albus, hyoscyamine was the main alkaloid during the first 6 weeks of culture in phytohormone-free MS liquid medium. At the seventh week of culture, with the beginning of the stationary growth, scopolamine became the major constituent (Fig. 7). 

The influence of phytohormones and light on the production of tropane alkaloids in transformed roots of Hyoscyamus albus was investigated [25]. In adventitious roots cultured in the dark, the addition of lAA (up to 4 mg/1) to the culture medium only slightly affected alkaloid production (Fig. 8a). On the other hand, when the adventitious roots were cultured under light, the alkaloid content decreased with the addition of lAA. Any combination of lAA with kinetin rapidly... 

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