Isolation and identification of littorine from hairy roots of Atropa belladonna

A hairy root clone (M8) of Atropa belladonna, producing high levels of tropane alkaloids, was established by transformation with Agrobacterium rhizogenes (MAFF 03-01724). Littorine, an intermediate of tropane alkaloids, was detected by high-performance liquid chromatography and gas chromatography-mass spectrometry in the alkaloid fraction of the hairy roots and identified by nuclear magnetic resonance analysis. Littorine was also detected in the non-transformed root culture of A. belladonna. Received: 18 March 1998 / Revision received: 15 June 1998 / Accepted: 3 August 1998     

Production of Pharmaceuticals from Papaver Cultivars in Vitro

Iranian (Papaver bracteatum Lindl.) and opium poppy (P. somniferum L.) plantlets obtained from germinated seeds grown on a Murashige and Skoog basal medium (BM) readily manifest alkaloids. Temperature had a profound effect on growth and alkaloid production after 8 weeks in culture. Plantlets of poppy cultivars (cvs.) grew best at 18.5 and 20°C compared to 15 or 25°C. An alkaloid survey study with 24 Iranian and 21 opium poppy cvs. revealed that total morphinan alkaloids ranged from 0 to 6.55 mg/g dw. Prolific axillary branching was achieved from poppy cvs. by maintaining shoots on BM containing 1.0 mg/L N⁶‐benzyladenine and 0.01 mg/L α‐naphthalene acetic acid for an additional 16 weeks. The influence of vessel size on the growth response of established shoot clumps was determined by subculture in a variety of culture vessels for 8 weeks. The tested culture vessels included culture tubes (55 mm³ capacity (cap.)), babyfood jars (143 mm³ cap.), Magenta GA‐7 containers (365 mm³ cap.), and polycarbonate jars (1890 mm³ cap.) employing an in vitro hydroponics system (i.e. an automated plant culture system (APCS)). Highest growth rates occurred employing the APCS. The culture vessel capacity had a significant positive correlation on shoot length, fresh weight, number of leaves, and number of shoots. Shoot length, fresh weight, leaves, and shoots grown in the APCS exhibited increases of 1‐, 21.5‐, 7.8‐, and 8.3‐fold, respectively, compared to shoots grown in culture tubes. Higher culture growth rates that occurred in the larger‐size vessels were correlated with lower alkaloid production (mg alkaloids/g dw). However, the overall total alkaloids/vessel [(mg alkaloid/g dw)×g culture dw] increased because of greater biomass production per vessel. The alkaloid content was found to remain stable for shoots grown over a 6–month evaluation period.     

Traits of transgenic Atropa belladonna doubly transformed with different Agrobacterium rhizogenes strains

Hairy root cultures of Atropa belladonna L. were established by infection either with Agrobacterium rhizogenes ATCC 15834 or MAFF 03-01724, and transgenic plants were obtained from both hairy root cultures. Doubly transformed roots were induced by re-infection of the leaf segments of transgenic Atropa belladonna plants (A. rhizogenes 15834) with MAFF 03-01724. Shoots and viviparous leaves were regenerated from the doubly transformed roots. The genetic transformation was determined by the opine assay (agropine, mannopine and/or mikimopine) and polymerase chain reaction. Physiological changes and tropane alkaloid biosynthesis in the hairy roots (singly and doubly transformed) were investigated. The alkaloid content in the doubly transformed root strain was intermediate as compared to the root strains which were singly transformed. On the other hand endogenous IAA levels in doubly transformed roots were significantly decreased compared to both singly transformed roots.Abbreviations BA benzyladenine - IAA indoleacetic acid - NAA naphthaleneacetic acid - PCR polymerase chain reaction - t-ZR trans-zeatin     

Root, Callus, and Cell Suspension Cultures, from Atropa belladonna, L. and Atropa belladonna, Cultivar lutea Doll

Root, callus, and cell suspension cultures have been establishedfrom seedlings of Atropa belladonna, L. and Atropa belladonna,cultivar lutea Döll. The growth of these cultures is described.Callus cultures transferred to auxin (-naphthaleneacetic acid)-freemedium initiated roots and shoots. Excised root cultures havebeen established from such roots and plants from such shoots.Extracts of the cultures have been submitted to the Vitali—Morinreaction and following chromatography, to the Dragendorff reaction.Cultured excised roots and plants raised from shoots initiatedon cultured callus were shown to contain atropine (hyoscyamine)and reactive substances corresponding in Rf to hyoscine andcuscohygrine. These alkaloids were absent from cultured callusand cultured cell suspensions and from leaves when initiatedwithout roots on callus. The cultured calluses and cell suspensionscontained choline (0.022–0.027 g per 100 g dry weightof root callus). The growth of cell suspension cultures wasnot inhibited by incorporating atropine sulphate, L-hyoscyamine,L-hyoscine hydrobromide, or DL-scopoline nitrate in the culturemedium at 250 mg/I. These alkaloids were absorbed by the cells,a high proportion of the added alkaloid could be recovered fromthe cultures even after 4 weeks' growth and no evidence wasobtained of the presence of degradation products of the alkaloids.The suppression of alkaloid formation in actively growing callusand cell suspension cultures is discussed.     

Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids

California poppy (Eschscholzia californica Cham.) root cultures produce a variety of benzophenanthridine alkaloids, such as sanguinarine, chelirubine and macarpine, with potent biological activity. Sense and antisense constructs of genes encoding the berberine bridge enzyme (BBE) were introduced into California poppy root cultures. Transgenic roots expressing BBE from opium poppy (Papaver somniferum L.) displayed higher levels of BBE mRNA, protein and enzyme activity, and increased accumulation of benzophenanthridine alkaloids compared to control roots transformed with a -glucuronidase gene. In contrast, roots transformed with an antisense-BBE construct from California poppy had lower levels of BBE mRNA and enzyme activity, and reduced benzophenanthridine alkaloid accumulation, relative to controls. Pathway intermediates were not detected in any transgenic root lines. Suppression of benzophenanthridine alkaloid biosynthesis using antisense-BBE also reduced the growth rate of the root cultures. Two-dimensional ¹H-NMR spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic roots lines. However, transformed roots with low levels of benzophenanthridine alkaloids contained larger cellular pools of certain amino acids compared to controls. In contrast, cellular pools of several amino acids were reduced in transgenic roots with elevated benzophenanthridine alkaloid levels relative to controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was only marginally altered in all transgenic root lines; thus, altering metabolic flux through benzophenanthridine alkaloid pathways can affect cellular pools of specific amino acids. Consideration of such interactions is important for the design of metabolic engineering strategies that target benzophenanthridine alkaloid biosynthesis.     

Effect of differentiation on the regulation of indole alkaloid production in Catharanthus roseus hairy roots

In vitro cultures of hairy root derived from Catharanthus roseus accumulate higher levels of indole alkaloids than cell suspension cultures. Hairy roots were interconverted to undifferentiated cells by manipulation of the culture medium. When the concentration of micronutrients in the culture medium was five times that of Phillips and Collins (1979) medium, cell suspensions formed from the hairy roots. The alkaloid content was five times lower in the cell suspensions than in the control, but upon regeneration of the roots the alkaloid content regained its original level. The formation of cell suspensions from hairy roots was also accompanied by a reduction in tryptophan decarboxylase and the strictosidine synthase activity to less than 5% and 30%, respectively. 3-Hydroxymethylglutaryl coenzyme A reductase activity was the same in the cell suspension and in the regenerated line. Received: 12 February 1998 / Revision received: 21 May 1998 / Accepted: 5 June 1998     

The effect of Tiron, a water soluble radical scavenger, on growth, morphology and alkaloid content of adventitious roots in Atropa belladonna

 The effect of Tiron (disodium 1,2-dihydroxybenzene-3,5-disulfonate) on the growth, morphology and alkaloid content of adventitious roots in Atropa belladonna was investigated. High concentrations of Tiron had an inhibitory effect on growth of the root. The appearance of cultured roots was significantly changed from rough roots accompanied with callus-like tissue in control cultures to fine roots without callus formation. Alkaloid content was drastically increased by the addition of 1 mM Tiron to the medium. The influence of NAA, which has an inhibitory effect on alkaloid production, was partially restored by Tiron treatment, indicating that this radical scavenger may affect the production of alkaloids through modulation of the mode of action of auxin. Glutathione content of the root was not influenced by Tiron. Received: 3 June 1999 / Revision received: 28 September 1999 / Accepted: 30 September 1999     

Alkaloid patterns and biosynthetic capacity of root cultures from some pyrrolizidine alkaloid producing Senecio species

Root cultures of Senecio vulgaris, S. vernalis, S. erucifolius and S. squalidus were established. The patterns of pyrrolizidine alkaloids found in these root cultures were analyzed by high-resolution GC and GC-MS and compared with the alkaloids present in the respective plants. In vitro cultured roots produce alkaloid patterns and accumulate quantities which are comparable to those found in soil grown plants. With the exception of the otonecine derivative senkirkine all pyrrolizidines accumulate as N-oxides. Only senkirkine is partially released into the medium. The cultures incorporate biosynthetic precursors, e.g. ¹⁴C-labelled putrescine or spermidine with high efficiency into the alkaloids. Senecionine N-oxide was found to be the main product of biosynthesis. Evidence is presented that senecionine N-oxide is directly transformed into senkirkine, the main alkaloid of S. vernalis root cultures.Abbreviations GC Gas chromatography - MS Mass spectroscopy - PND Phosphorous-Nitrogen-Detector - FID Flame Ionization Detector - fr.wt Fresh weight     

Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.)

In this study, the effects of ploidy level and culture medium were studied on the production of tropane alkaloids. We have successfully produced stable tetraploid hairy root lines of Hyoscyamus muticus and their ploidy stability was confirmed 30?months after transformation. Tetraploidy affected the growth rate and alkaloid accumulation in plants and transformed root cultures of Egyptian henbane. Although tetraploid plants could produce 200% higher scopolamine than their diploid counterparts, this result was not observed for corresponding induced hairy root cultures. Culture conditions did not only play an important role for biomass production, but also significantly affected tropane alkaloid accumulation in hairy root cultures. In spite of its lower biomass production, tetraploid clone could produce more scopolamine than the diploid counterpart under similar growth conditions. The highest yields of scopolamine (13.87?mg?l^?1) and hyoscyamine (107.7?mg 1^?1) were obtained when diploid clones were grown on medium consisting of either Murashige and Skoog with 60?g/l sucrose or Gamborg??s B5 with 40?g/l sucrose, respectively. Although the hyoscyamine is the main alkaloid in the H. muticus plants, manipulation of ploidy level and culture conditions successfully changed the scopolamine/hyoscyamine ratio towards scopolamine. The fact that hyoscyamine is converted to scopolamine is very important due to the higher market value of scopolamine.     

Optimization of induction and culture conditions and tropane alkaloid production in hairy roots of <Emphasis Type="Italic">Anisodus acutangulus</Emphasis>

In this study, an efficient transformation system for the medicinal plant Anisodus acutangulus was successfully developed and optimized using Agrobacterium rhizogenes. Three bacterial strains, A4, R1601, and modified C58C1 and three explant types, leaf blade, petiole, and stem, were examined. The highest transformation efficiency of 94.44% was achieved using strain C58C1 with stem explants. Over 20 independent hairy root lines were successfully established with strain C58C1 using stem explants, all of which contained the ro/B and ro/C genes as confirmed by polymerase chain reaction (PCR). Out of four media compositions, the liquid 1/2 MS medium was found the most suitable for hairy root growth. The maximum biomass of one hairy root line increased up to 80 times in liquid 1/2 MS medium after a 30 day culture period. Different hairy root lines displayed a varied capacity for tropane alkaloid production and the best hairy root line (T4) from the C58C1-stem combination produced up to 10.21 mg/g (dw) of hyoscyamine, which was about 1.5-fold higher than in the wild type plants. To our knowledge, this is the first report to demonstrate the production of tropane alkaloids in hairy roots of A. acutangulus.     

Alternative metabolic fates of hygrine in transformed root cultures of Nicandra physaloides

Alkaloid production by several Agrobacterium rhizogenes-transformed root lines of Nicandra physaloides was studied. Early in the culture cycle all lines contained predominantly hygrine, which has previously been reported to be the major alkaloid in roots of this plant. A number of other hygrine-derived alkaloids were identified, and these generally increased in importance as the cultures matured. Some lines were found to differ quite markedly in the relative importance of the different hygrine-derived alkaloids. The possibility is raised that alkaloid biosynthesis in N. physaloides root cultures may be a good model system in which to study metabolite partitioning in branched metabolic pathways.Abbreviations FW fresh weight - GC gas chromatography - MS mass spectrometry - MW molecular weight     

Catharanthus roseus: micropropagation and in vitro techniques

Different methods of in vitro culture of Catharanthus roseus provide new sources of plant material for the production of secondary metabolites such as indole alkaloids. Callus, cell suspension, plantlets, and transgenic roots cultured in the bioreactor are used in those experiments. The most promising outcomes include the production of the following indole alkaloids: ajmalicine in unorganised tissue, catharanthine in the leaf and cell culture in the shake flask and airlift bioreactor, and vinblastine in shoots and transformed roots. What is very important, enzymatic coupling of monomeric indole alkaloids, vindoline and catharanthine, is possible to form vinblastine in cell cultures. The method of catharanthine and ajmalicine production in the suspension culture in bioreactors has been successful. In this method, elicitation may be used acting on different metabolic pathways. Also of interest is the method of obtaining arbutin from the callus culture of C. roseus conducted with hydroquinone. The transformed root culture seems to be the most promising for alkaloid production. The genetically transformed roots, obtained by the infection with Agrobacterium rhizogenes, produce higher levels of secondary metabolites than intact plants. Also, whole plants can be regenerated from hairy roots. The content of indole alkaloids in the transformed roots was similar or even higher when compared to the amounts measured in studies of natural roots. The predominant alkaloids in transformed roots are ajmalicine, serpentine, vindoline and catharanthine, found in higher amounts than in untransformed roots. Transformed hairy roots have been also used for encapsulation in calcium alginate to form artificial seeds.     

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.     

Production of steroidal alkaloids by hairy roots of Solanum aviculare and the effect of gibberellic acid

Cultures of Solanum aviculare hairy roots were established after transformation with Agrobacterium rhizogenes A4. High levels of steroidal alkaloids measured as solasodine equivalents were produced in shake-flasks and bioreactor, even though relatively low concentrations are found in roots in vivo. In shake flasks the maximum alkaloid yield was 32 mg g^-1 dry weight; in a 3-1 air-driven bioreactor the yield was 29 mg g^-1. These yields represent a 5-fold increase over previous reports for in vitro production, and are comparable with levels found in the aerial parts of intact S. aviculare plants. Production of steroidal alkaloids was growth-associated. High sugar levels at stationary phase and insensitivity to increased levels of medium components suggest that root cultures were limited by oxygen mass-transfer. In Petri-dish culture with and without exogenous gibberellic acid, root length and number of root tips increased exponentially; growth proceeded with a constant length per root tip of about 35 mm. Addition of gibberellic acid enhanced growth but reduced the specific steroidal-alkaloid level. Taking into account both growth and alkaloid yield, accumulation of steroidal alkaloids was improved by about 40% at gibberellic-acid concentrations of 10 and 100 g l^-1.     

Effects of phytosulfokine-α on growth and tropane alkaloid production in transformed roots of Atropa belladonna

Phytosulfokine (PSK)- is a sulphated pentapeptide, isolated fromthe medium of cultured Asparagus officinalis mesophyllcells, that promotes cell proliferation. It is a putative key factor inconditioned medium required for the growth of low-density plant cell cultures.The present study investigates the effect of PSK- on growth and tropanealkaloid production in Atropa belladonna hairy rootstransformed with Agrobacterium rhizogenes (MAFF 03-01724). Although the growth rates ofhairy roots cultured in medium with orwithout PSK- for 4 weeks did not show any differences, the productivityof tropane alkaloids, especially of hyoscyamine, was enhanced by10^–7 or 10^–8 M PSK-. Inaddition, the content of tropane alkaloids in transformed roots treated withPSK- was 1.4 times higher than that of untreated roots after 4 weeks ofculture. The time course of growth and tropane alkaloid production inAtropa belladonna transformed roots suggested thatPSK- influenced the growth of transformed roots during the activegrowingphase, but not tropane alkaloid production.     

Incorporation of arginine, ornithine and phenylalanine into tropane alkaloids in suspension-cultured cells and aseptic roots of intact plants oi Atropa belladonna

Label from U-¹⁴C-arginine (Arg), -ornithine (Orn) and -phenylalanine(Phe) was incorporated into hyoscyam-ine and scopolamine byboth dissected roots of intact plants and homogeneous or aggregatingsuspension cultures of Atropa belladonna L. The early biosyntheticcompounds (hygrine, tropinone, tropanol) were found only inthe roots, and alkaloid synthesis proceeded as far as to scopolaminethere. In the synthesis of the tropane skeleton, Orn was usedmore efficiently than Arg. Phe was quickly metabolized bothin roots and suspension cultures, and the label was incorporatedinto both ethanol-insoluble compounds, particularly proteinsand different ethanol-soluble compounds, especially phenolics. When the callus, used to initiate suspension cultures, was repeatedlysubcultured, the degree of organization of the suspension-culturedcells (second suspension passage) grown in the presence of 2.5µM 2-naphthaleneacetic acid (NAA), changed first fromhomogeneous to aggregating and later to heavily rooty. Simultaneouslywith an increasing degree of organization, alkaloid synthesisdecreased. At first, the rate of incorporation of Arg and Phe,and later Orn into alkaloids decreased. In heavily rooty suspensionsneither hyoscyamine nor scopolamine were found, but traces oftropanol were detected. Hence, the synthesis of tropane alkaloidsseems to be regulated at the later biosynthetic steps. Key words: Arginine, Atropa, hyoscyamine, omithine, phenylalanine, roots, scopolamine, suspension culture, tropane alkaloids     

Calystegines as a new group of tropane alkaloids in Solanaceae

Calystegines are a new group of polyhydroxy alkaloids with a nortropane skeleton. They were detected in Atropa belladonna root cultures by chromatographic methods (TLC, GC) and identified by NMR and mass spectroscopy. Their occurrence was examined in several species of the Solanaceae. The biosynthesis of these compounds is suggested to proceed via the tropane alkaloid pathway, the first metabolite being pseudotropine. A pseudotropine-forming tropinone reductase was isolated and characterized from Atropa belladonna root cultures. Further evidence is given for the significance of tropinone and pseudotropine in calystegine formation by feeding experiments that increased calystegine formation. ¹⁵N-tropinone was shown to be incorporated into calystegines.Abbreviations GC gas chromatography - TBON 8-thiabicyclo[3.2.1]octan-3-one - TLC thin-layer chromatography     

Organogenesis in Cell Suspension Cultures of Atropa belladonna L. and Atropa belladonna Cultivar lutea Doll

Callus cultures have been initiated from excised cultured rootsof Atropa belladonna and A. belladonna cultivar lutea and usedto establish suspension cultures in a synthetic culture medium(referred to as SSM) containing 2.0 mg/1 -naphthaleneaceticacid (NAA). Visible cellular aggregates develop in these suspensioncultures and when such aggregates are cultured in an NAA-omittedmedium, large numbers of roots arise superficially on the aggregates.Root development is enhanced by incorporating into the auxin-freemedium either tropic acid or I-naphthoxyacetic acid and boththese substances can promote root initiation in the presenceof levels of NAA which alone suppress organogenesis. The aggregatesalso give rise under appropriate conditions of culture to shootsand to embryo-like structures. The nature and frequency of suchmorphogenesis in the aggregates is dependent not only upon thecomposition of the culture medium but upon the number and thelength of previous culture passages through which the callusand suspension cultures have been propagated in the SSM. Theultimate loss of morphogenetic potential in serially propagatedcallus is discussed. The structure of the aggregates in SSM and in the media promotingroot initiation is described. Examination of the aggregatesfor their content of alkaloids indicates that the major belladonnaalkaloids can only be detected in cultures forming roots.     

Indole alkaloid production by transformed and non-transformed root cultures ofCatharanthus roseus

Summary Ten transformed and two non-transformed root lines ofCatharanthus roseus were established. A systematic study of the growth kinetics and alkaloid content was performed over a culture cycle and showed significant differences between transformed and non-transformed cultures. Mean doubling times for transformed and normal root lines were 2.8 and 19.5 days, respectively. Alkaloid content in hairy roots was from two- to threefold higher than in the non-transformed tissues. The established transformed root lines produced a wide variety of indole alkaloids as can be observed from their complex thin layer chromatography patterns. A large quantity of serpentine was determined in two of the transformed root cultures. Alkaloid content, both quantitatively and qualitatively, has been stable in the hairy root cultures for more than 2 yr of subculturing.     

Influence of exogenous hormones on the growth and secondary metabolite formation in transformed root cultures

Transformed organ cultures formed following transformation of plant tissues with Agrobacterium species owe their phenotypes to alterations in hormone metabolism. Exogenously supplied hormones have been used to probe the relationship between the growth and morphology of transformed root cultures of a number of species and their ability to accumulate secondary products. Auxins in the presence of low levels of kinetin induce the rapid disorganisation of transformed roots of Nicotiana rustica ultimately toform suspension cultures of transformed cells and this process is associated with a decrease in nicotine content of the cells. This is related to cells in the culture losing competence in alkaloid biosynthesis. In contrast, exogenously supplied GA₃ enhanced branching in two transformed root clones of the tropane-alkaloid producing species, Brugmansia candida and so enhanced their typical hairy root phenotype. This growth substance had the effect of reducing the overall alkaloid accumulation but in one case significantly altered the relative concentrations of different tropine esters.In transformed roots of Cucumis sativus, the phenotype of the roots is influenced by the expression of auxin synthesis genes on T_R-DNA resulting in roots with two distinct morphologies. The pattern of expression of the enzyme ascorbate oxidase in populations of control roots of different morphologies is described. The significance of these phenotypic variations on the utility of transformed root cultures for the study of secondary metabolic pathways will be discussed.Abbreviations AO ascorbate oxidase - DW dry weight - FW fresh weight - GA₃ gibberellic acid