Production of tropane alkaloids in cultured cells of Hyoscyamus niger

Tests for calluses rich in tropane alkaloids were made with newly induced calluses of Atropa belladonna, Datura stramonium and Hyoscyamus niger. Only calluses of H. niger gave an alkaloid-positive test.A Hyoscyamus cell line had the highest total alkaloid content of all the calluses screened by the cell-squash alkaloid assay. Both hyoscyamine and scopolamine were identified in the cultured cells of this line by TLC, GLC and GC-MS.Abbreviations NAA 1-Naphthaleneacetic acid - BA Benzyladenine - BSA N,O-Bis(trimethylsilyl) acetamide     

Alkaloid production in cultured roots of three species of Duboisia

Cultured roots were obtained from calluses of Duboisia leichhardtii, D. myoporoides and D. hopwoodii. Cultured roots of all these species produced both tropane and pyridine-type alkaloids. The selected cultured roots of D. leichhardtii showed high contents of tropane alkaloids (hyoscyamine 0.53%, scopolamine 1.16%, on a dry weight basis).     

High-yield production of tropane alkaloids by hairy-root cultures of aDatura candida hybrid

Hairy root cultures were obtained following inoculation of the stems of sterile plantlets of aDatura candida hybrid withAgrobacterium rhizogenes. The scopolamine and hyoscyamine content was quantified by HPLC and compared with the non-transformed plants. The alkaloid yield (0.68% dry weight) obtained with the hairy roots was 1.6 and 2.6 times the amount found in the aerial parts and in the roots of the parent plants, respectively. Only a small proportion of alkaloids was released into the growth medium. Scopclamine was the principal alkaloid and the scopolamine/hyoscyamine ratio of ca. 5:1 makes these hairy roct cultures worthy of consideration as a source of scopolamine.     

Coculture of genetically transformed roots and shoots for synthesis, translocation, and biotransformation of secondary metabolites

Genetically transformed shooty teratomas of Atropa belladonna and a Duboisia leichhardtii x D. myoporoides hybrid were studied for biotransformation of tropane alkaloids in shake flasks and bioreactors. Although de novo synthesis of hyoscyamine and scopolamine was limited, shoots of both species were able to translocate and accumulate significant quantities of exogenous alkaloid. The maximum yield of scopolamine from hyoscyamine fed to the Duboisia hybrid shoots was 35% w/w; the yield of the scopolamine precursor, 6beta-hydroxyhyoscyamine, was 37% w/w. Biotransformation activity was poor in A. belladonna shooty teratomas provided with exogenous hyoscyamine; however, scopolamine levels comparable with those in leaves of the whole plant accumulated in shoots fed with hairy root extract. Coculture of A. belladonna shooty teratomas and hairy roots in the same hormone-free medium was investigated as a means of providing a continuous source of hyoscyamine for conversion to scopolamine. Of the biotransformation systems tested with A. belladonna, coculture produced the highest levels of scopolamine and the highest scopolamine: hyoscyamine ratios. Cocultured shoots accumulated up to 0.84 mg g(-1) dry weight scopolamine, or 3-11 times the average concentrations found in leaves of the whole plant. The scopolamine: hyoscyamine ratio in coculture ranged from 0.07 to 1.9, a significant improvement over levels of 0-0.03 normally found in A. belladonna hairy roots. Addition of Pluronic F-68 or copper sulfate to the medium and variation in initial medium pH did not improve hyoscyamine release from hairy roots. Scopolamine levels were increased using 1 muM copper sulfate or initial medium pH between 6.0 and 8.0; however, results from elicitation of hairy roots could not match the beneficial effect on scopolamine synthesis of root-shoot coculture. Addition of 0.001-1.0% (w/v) Pluronic F-68 to the roots reduced hyoscyamine release but postponed necrosis in the root tissue for up to 60 d. (c) 1996 John Wiley & Sons, Inc.     

In vitro organogenesis and alkaloid accumulation in Datura innoxia

The kinetics of tropane alkaloids accumulation in different organs such as roots, leaves, stems, flowers and seeds of Datura innoxia was investigated by GC-MS. Twenty-six tropane alkaloids were detected. The ester derivatives of tropine (3alpha-tigloyloxytropine and 3-tigloyloxy-6-hydroxytropine) are the major compounds. Undifferentiated callus were established from the stem explants of Datura innoxia using Murashige and Skoog (MS) medium supplied with 6-benzylaminopurine (BA, 1 mg l(-10) and indole-3-acetic acid (IAA, 0.5 mg l(-1)) in combination for 6 weeks. Callus differentiation was initiated by subculture onto solid MS medium, free from hormones, for more than 10 months. Initially, shoots were formed after four weeks from subculture. Further subculturing in basal MS medium without growth regulators initiated the rooting of a shooty callus after 6 weeks. Investigation of the alkaloid content of the unorganized and organized callus revealed that callus (either green or brown) yielded only trace amounts of alkaloids. On the other hand, re-differentiated shoots contained mainly scopolamine while re-differentiated roots biosynthesized hyoscyamine as the main alkaloid.     

Tailoring Tropane Alkaloid Accumulation in Transgenic Hairy Roots of Atropa baetica by Over-expressing the Gene Encoding Hyoscyamine 6β-hydroxylase

Atropa baetica hairy roots, over-expressing cDNA from Hyoscyamus niger encoding the gene for hyoscyamine 6β-hydroxylase (H6H), were produced by Agrobacterium rhizogenes infection. The transgenic roots over-expressing h6h had an altered alkaloid profile in which hyoscyamine was entirely converted into scopolamine. In the best h6h clone, scopolamine accumulation increased 9-fold compared to plants, amounting to 5.6 mg g dry wt⁻¹, some of which was released into the liquid medium. Only negligible amounts of hyoscyamine were detected. In contrast, the gus control culture contained a much higher amount of hyoscyamine than scopolamine, mimicking the situation in the plant. At the molecular level, a higher conversion of hyoscyamine into scopolamine was related to a higher level of h6h mRNA; in some instances this was 5–10-fold higherThis article is dedicated to the memory of Professor Antonio G. González     

Production of tropane alkaloids by small-scale bubble column bioreactor cultures of Scopolia parviflora adventitious roots

The mass production of tropane alkaloids from adventitious root cultures of Scopolia parviflora, in small-scale bubble column bioreactor (BCB) was attempted. Adventitious roots of S. parviflora produced relatively enhanced levels of scopolamine and hyoscyamine in bioreactor compared to flask type cultures, and rapidly produced root clumps, with continuously increasing biomass throughout the culture period. The production of scopolamine and hyoscyamine in the top and bottom regions of root clumps were higher than in the core region. The adventitious root cultures of S. parviflora in the BCB required a relatively high level of aeration. The optimized conditions for the bioreactor culture growth and alkaloid production were found to be 3g of inoculum, on a fresh weight basis, a 15-day culture period and 0.4vvm of airflow. The elicitation by Staphylococus aureus increased the specific compound of scopolamine, while the production of hyoscyamine was slightly inhibited in BCB cultures.     

Influence of chitosan,acetic acid and citric acid on growth and tropane alkaloid production in transformed roots of Brugmansia candida Effect of medium pH and growth phase

The effects of chitosan, acetic acid and citric acid on production and release of hyoscyamine and scopolamine in hairy root cultures of Brugmansia candida were studied. Chitosan and acetic acid were tested at different concentrations and also at different media pH values. At pH 5.5, and at certain concentrations, acetic acid and chitosan increased the content of root scopolamine and hyoscyamine, and promoted the release of both alkaloids. Lowering the pH to 3.5 and 4.5 reduced the accumulation of both alkaloids in the roots, but at a pH of 4.5, their release increased significantly. Acetic and citric acid stimulated the release of scopolamine and hyoscyamine. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biotransformation of hyoscyamine into scopolamine in transgenic tobacco cell cultures

Hyoscyamine-6beta-hydroxylase (H6H) catalyses the conversion of hyoscyamine into its epoxide scopolamine, a compound with a higher added value in the pharmaceutical market than hyoscyamine. We report the establishment of tobacco cell cultures carrying the Hyoscyamus muticus h6h gene under the control of the promoter CAMV 35S. The cell cultures were derived from hairy roots obtained via genetically modified Agrobacterium rhizogenes carrying the pRi and pLAL21 plasmids. The cultures were fed with hyoscyamine, and 4 weeks later the amount of scopolamine produced was quantified by HPLC. The transgenic cell suspension cultures showed a considerable capacity for the bioconversion of hyoscyamine into scopolamine, and released it to the culture medium. Although the scale-up from shake-flask to bioreactor culture usually results in reduced productivities, our transgenic cells grown in a 5-L turbine stirred tank reactor in a batch mode significantly increased the scopolamine accumulation.     

Somatic embryogenesis and bud formation from immature embryos of buckwheat (Fagopyrum esculentum Moench.)

Immature embryos of Fagopyrum esculentum cv. Pennquad were isolated from field-grown plants and cultured on media containing a high benzylaminopurine to indole-3-acetic acid ratio. Part of the embryos were grown in the presence of 2,4-dichlorophenoxyacetic acid and kinetin for the first 5 days, and then transferred to benzylaminopurine + indole-3-acetic acid medium. From callus tissues developed on hypocotyls and cotyledons, 3 types of tissue were selected in later subcultures: (a) callus tissue strains that produced buds, (b) embryogenic tissue, and (c) unorganized callus tissue, lacking any organogenic capacity. Pretreatment with 2,4-dichlorophenoxyacetic acid increased the number of explants which gave rise to bud forming and embryogenic tissue, but was not essential for morphogenesis. Somatic embryogenesis was confirmed by histological observation. Plantlets could be easily obtained by inducing adventitious roots on shoots, but spontaneous root development in somatic embryos was infrequent.Abbreviations BAP benzylaminopurine - IAA indole-3-acetic acid - 2,4-D dichlorophenoxyacetic acid - IBA indole-3-butyric acid     

Tropane alkaloid production by shoot culture of Duboisia myoporoides R. Br

This work demonstrates the presence of hyoscyamine and scopolamine at different stages of shoot regeneration from non-organogenic and organogenic calli. The 11-week-old non-organogenic calli contained 0.41+/-0.03 and 0.23+/-0.02 microg g(-1) dry wt hyoscyamine and scopolamine respectively. However, no root meristem was found in the calli. The alkaloids were absent in 2-week-old organogenic calli. The shoot-buds induced on the non-organogenic and organogenic calli did not contain these alkaloids. Hyoscyamine and scopolamine contents of the 6-week-old non-rooted shoots regenerated from non-organogenic calli were 7.8+/-0.1 and 6.5+/-0.4 microg g(-1) dry wt respectively and those in the 9-week-old non-rooted shoot regenerated from organogenic calli were 38.5+/-0.4 and 3.6+/-0.1 microg g(-1) dry wt respectively. Hyoscyamine and scopolamine contents of the 4-week-old roots regenerated from non-organogenic and organogenic calli were higher than those in the non-rooted shoots. Since the presence of hyoscyamine and scopolamine in the non-rooted shoot depends on the stage of differentiation, manipulation of culture environment may improve hyoscyamine and scopolamine contents of the non-rooted shoots.     

Hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, in alkaloid-producing root cultures

Root cultures of various solanaceous plants grow well in vitro and produce large amounts of tropane alkaloids. Enzyme activity that converts hyoscyamine to 6β-hydroxyhyoscyamine is present in cell-free extracts from cultured roots of Hyoscyamus niger L. The enzyme hyoscyamine 6β-hydroxylase was purified 3.3-fold and characterized. The hydroxylation reaction has absolute requirements for hyoscyamine, 2-oxoglutarate, Fe²⁺ ions and molecular oxygen, and ascorbate stimulates this reaction. Only the l-isomer of hyoscyamine serves as a substrate; d-hyoscyamine is nearly inactive. Comparisons were made with a number of root, shoot, and callus cultures of the Atropa, Datura, Duboisia, Hyoscyamus, and Nicotiana species for the presence of the hydroxylase activity. Decarboxylation of 2-oxoglutarate during the conversion reaction was studied using [1-¹⁴C]-2-oxoglutarate. A 1:1 stoichiometry was shown between the hyoscyamine-dependent formation of CO₂ from 2-oxoglutarate and the hydroxylation of hyoscyamine. Therefore, the enzyme can be classified as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-). Both the supply of hyoscyamine and the hydroxylase activity determine the amounts of 6β-hydroxyhyoscyamine and scopolamine produced in alkaloid-producing cultures.     

Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus

In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the first committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites.     

Production of emetic alkaloid by in vitro culture of cephaelis ipecacuanha A. Richard

Callus and adventitious roots were induced on leaf segments from shoot culture of Cephaelis ipecacuanha A. Richard on Murashige-Skoog medium containing 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid, 1-naphthaleneacetic acid and kinetin. The contents of emetic alkaloids in calli, roots and root suspension cultures were quantified by HPLC. Roots cultured in solid and liquid Murashige-Skoog media yielded emetine and cephaeline. The amount of the two alkaloids in the root suspension culture was very similar to that of roots from ipecac mother plant grown in a greenhouse. In contrast, calli subcultured on Murashige-Skoog media containing combinations of 2,4-dichlorophenoxyacetic acid and kinetin produced only trace amounts of emetic alkaloids.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA l-naphthaleneacetic acid - Kin kinetin - MS Murashige-Skoog - EM emetine - CP cephaeline - DW dry weight.     

The effect of nitrate and ammonium concentrations on growth and alkaloid accumulation of Atropa belladonna hairy roots

The growth, the alkaloid production, as well as the scopolamine/hyoscyamine ratio of two clones of belladonna hairy roots were studied. The effects of nitrate and ammonium concentrations on these cultures were investigated. A rise in ammonium concentration caused the decline of the hairy roots, while nitrate had a marked effect on the alkaloid content. The alkaloid production obtained with 15.8 mM of NO3- and 20.5 mM of NH4+ was 1.2-1.4 times higher than that obtained when the roots were grown in the standard Murashige and Skoog medium (MS medium, 39.5 mM of NO3- and 20.5 mM of NH4+). The nitrate and ammonium concentrations in the culture medium also had a strong influence on the scopolamine/hyoscyamine ratio. When nitrate or ammonium concentrations were raised, that ratio also was increased 2-3-fold. The hairy root clones originating from transformations with two distinct strains of Agrobacterium had similar responses.     

Alkaloid production by hairy root cultures in Atropa belladonna

Hairy roots were induced by inoculation of stems of sterile plants of Atropa belladonna with Agrobacterium rhizogenes. The axenic culture of the hairy roots isolated from the stems proliferated 60 fold as based on the initial fresh weight after one month of culture. The presence of atropine and scopolamine in hairy roots were examined by TLC and HPLC. Their amounts were analyzed by GLC. The results show that the amount of the two alkaloids in the axenic cultures was the same as or even higher than those of normal plants grown in the field.     

Simultaneous determination of scopolamine, hyoscyamine and littorine in plants and different hairy root clones of Hyoscyamus muticus by micellar electrokinetic chromatography

Hyoscyamus muticus hairy root clones were established following infection with Agrobacterium rhizogenes strains A4, LBA-9402 and 15834 and with A. tumefaciens strain C58C1pRTGus104. The accumulation of tropane alkaloids hyoscyamine, littorine and scopolamine was evaluated by micellar electrokinetic capillary electrophoresis. Littorine was reported for the first time in these clones as well as in the roots of the intact plant and confirmed by collision induced dissociation-mass spectrometry. Tropane alkaloid content in hairy roots was compared with leaves and roots of normal plants at two vegetative stages. Significant differences appeared between the alkaloid contents of the different clones. In particular, all the hairy root clones and the roots of the intact plant produced 1.5-3 and 4.5-9 times more littorine than scopolamine, respectively. The only exception was clone KB7, carrying the h6h gene, which overproduced scopolamine. The aerial parts of H. muticus plants did not contain any littorine, thus indicating different transportation or translocation mechanisms of the various tropane alkaloids.