Genetic transformation of <Emphasis Type="Italic">Ruta graveolens</Emphasis> L. by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>: hairy root cultures a promising approach for production of coumarins and furanocoumarins

Ruta graveolens L. is a source of pharmacologically active compounds such as coumarins, furanocoumarins and furoquinolone alkaloids. Hypocotyls, callus and shoots of R. graveolens were inoculated with bacteria from two Agrobacterium rhizogenes strains. Hairy root cultures were established after inoculation of hypocotyls with wild A. rhizogenes strain LBA 9402. The transgenic nature of the regenerated tissue was confirmed by PCR amplification. Coumarins, furanocoumarins and alkaloids present in the hairy root tissue were identified by GC and GC-MS and compared with those present in in vitro shoot cultures. The level of pinnarin and rutacultin, bergapten, isopimpinelin and xanthotoxin was approximately twofold higher in hairy root than in shoot cultures. Two additional coumarins: osthole and osthenol, never been found in R. graveolens, were identified in hairy root tissue. Besides coumarins, alkaloids were identified: dictamnine, skimmianine, kokusaginine, rybalinine and an isomer of rybalinine. The levels of nearly all coumarins and alkaloids in hairy roots cultured in the darkness were higher than those accumulated under a photoperiod mode.     

Induction of tropane alkaloid formation in transformed root cultures of Brugmansia suaveolens (Solanaceae)

Hairy root cultures of Brugmansia suaveolens were set up by infection of root tips with Agrobacterium rhizogenes. The successful transformation was confirmed by analysing rolC and virC genes using polymerase chain reaction (PCR). Hairy root cultures were employed to study the formation of tropane alkaloids, such as hyoscyamine. The transformed cultures were incubated with potential elicitors, such as methyljasmonate, quercetin and salicylic acid in order to stimulate the biosynthesis of tropane alkaloids. Profile and amounts of tropane alkaloids were analysed using capillary GLC-MS. At least 18 different tropane alkaloids could be identified. Treatment of the cultures with 200 microM methyljasmonate increased the alkaloid accumulation 25-fold up to a level of 1 mg/g fresh weight as compared to untreated controls. Quercetin enhanced the alkaloid production 10 fold (0.4 mg/g fresh weight) within 24 h. In contrast 100 microM salicylic acid decreased alkaloids to a level of 1 microg/g fresh weight.     

Biotechnology for the production of plant secondary metabolites

The production of plant secondary metabolites by means of large-scale culture of plant cells in bioreactors is technically feasible. The economy of such a production is the major bottleneck. For some costly products it is feasible, but unfortunately some of the most interesting products are only in very small amounts or not all produced in plant cell cultures. Screening, selection and medium optimization may lead to 20- to 30-fold increase in case one has producing cultures. In case of phytoalexins, elicitation will lead to high production. But for many of the compounds of interest the production is not inducible by elicitors. The culture of differentiated cells, such as (hairy) root or shoot cultures, is an alternative, but is hampered by problems in scaling up of such cultures. Metabolic engineering offers new perspectives for improving the production of compounds of interest. This approach can be used to improve production in the cell culture, in the plant itself or even production in other plant species or organisms. Studies on the production of terpenoid indole alkaloids have shown that the overexpression of single genes of the pathway may lead for some enzymes to an increased production of the direct product, but not necessarily to an increased alkaloid production. On the other hand feeding of such transgenic cultures with early precursors showed an enormous capacity for producing alkaloids, which is not utilized without feeding precursors. Overexpression of regulatory genes results in the upregulation of a series of enzymes in the alkaloid pathway, but not to an improved flux through the pathway, but feeding loganin does result in increased alkaloid production if compared with wild-type cells. Indole alkaloids could be produced in hairy root cultures of Weigelia by overexpression of tryptophan decarboxylase and strictosidine synthase. Alkaloids could be produced in transgenic yeast overexpressing strictosidine synthase and strictosidine glucosidase growing on medium made out the juice of Symphoricarpus albus berries to which tryptamine is added. Metabolic engineering thus seems a promising approach to improve the production of a cell factory.     

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.     

Effects of beta-carboline alkaloids on the object recognition task in mice

beta-carboline alkaloids are found in several medicinal plants and display a variety of actions on the central nervous, muscular and cardiovascular systems. The aim of the present study was to evaluate the effects of systemic administration of beta-carboline alkaloids on object recognition in mice. Adult Swiss mice received an intra-peritoneal injection (i.p.) of alkaloids (1.0, 2.5 or 5.0 mg/kg) 30 min before training in an object recognition task. The fully aromatic beta-carbolines, harmine and harmol, induced an enhancement of short-term memory (STM) at all doses tested when compared to controls. Harmaline, a dihydro beta-carboline and inverse agonist of the MK-801 binding site on the N-methyl-d-aspartate (NMDA) receptor, also induced an enhancement of both short-term memory (STM) and long-term memory (LTM). These results demonstrate that systemic administration of beta-carboline alkaloids can improve object recognition memory in mice.     

Canthin-6-one and beta-carboline alkaloids from Eurycoma harmandiana

Two alkaloids, canthin-6-one 9-O-beta-glucopyranoside and 7-hydroxy-beta-carboline 1-propionic acid, were isolated from the roots of Eurcoma harmandiana together with the five known canthin-6-one alkaloids, 9-hydroxycanthin-6-one, 9-methoxycanthin-6-one, 9,10-dimethoxycanthin-6-one, canthin-6-one and canthin-6-one N-oxide, and the two known beta-carboline alkaloids, beta-carboline 1-propionic acid and 7-methoxy-beta-carboline 1-propionic acid. Their structures were based on analyses of spectroscopic data.     

Plantlet regeneration enhances solasodine productivity in hairy root cultures of <Emphasis Type="Italic">Solanum khasianum</Emphasis> Clarke

Summary Shoot regeneration in hairy root cultures of Solanum khasianum Clarke influences root growth, solasodine production. and permeabilization of solasodine into the medium. These parameters are dependent on exogenously supplied auxin and cytokinin: the effect being both concentration-and clone-dependent. Hairy root cultures with no shoot regeneration showed high permeabilization of solasodine into the medium by the sixth week of incubation, suggesting the medium acts as a sink for the solasodine synthesized by the roots. Solasodine in the culture medium was toxic to the transformed roots and caused browning of root tips. In a separate set of experiments, the hairy root cultures showed regeneration of approximately 50–70 mm long shoots after treatment with indole-3-acetic acid and kinetin. These hairy root cultures had inereased levels of solasodine production, compared to cultures without shoot regeneration. The plantlets formed in the hairy root cultures accumulated some of the solasodine, thereby reducing its permcabilization into the medium. Transport of solasodine from root to shoot reduced the toxic effect of solasodine in the root zone and extended the exponential growth phase by 8-10d.     

Occurrence of iridoid glycosides in in vitro cultures and intact plants of <Emphasis Type="Italic">Scrophularia nodosa</Emphasis> L.

Shoot, root, and callus cultures of Scrophularia nodosa L. (Scrophulariaceae) were established and cultivated in vitro. Iridoid glycosides, such as harpagoside, aucubin, and catalpol were identified by LC-ESI-MS and their contents determined by HPLC. For comparison intact plants of S. nodosa were analysed. In shoot cultures slightly lower amounts of detectable iridoid glycosides (4.36% dry weight) were determined than in the field grown plants (4.88%). Concentration of harpagoside was highest in leaves of field plants (1.05%) and in flowers of in vitro plantlets (1.10%). For aucubin the highest amount was found in the leaves of in vitro plantlets (1.67%) whereas the levels of aucubin in the leaves of field plants were remarkably lower. Catalpol was produced as a trace compound in intact plants and shoot cultures. Callus and root cultures were apparently not able to synthesise iridoid glycosides.     

Specific accumulation and revised structures of acridone alkaloid glucosides in the tips of transformed roots of Ruta graveolens

The root tips of Ruta graveolens (common rue) show strong autofluorescence of acridone alkaloids, which are characteristic secondary metabolites of this plant. To study the specific distribution and accumulation of acridone alkaloids in various root segments of Ruta graveolens, root material was harvested from genetically transformed root cultures and extracts were investigated by chromatographic techniques and HPLC-(1)H NMR spectroscopy. The cells of the elongation and differentiation zones contained acridone glucosides and large amounts of acridone alkaloids, mainly rutacridone. Gravacridondiol glucoside was identified as the dominant secondary compound of the root tips and its structure revised by means of spectroscopic methods. In addition, minor acridones, including the structurally revised gravacridontriol glucoside and unknown natural products, were found in the root tip.     

Quantitative determination of secoiridoid and xanthone glycosides of Gentiana dinarica Beck cultured in vitro

Gentiana dinarica Beck, native to the Balkan Dinaric Mountains, was established in vitro from axillary shoot buds. It was maintained in the form of shoot cultures on MS medium supplemented with 1.0 mg l^?1 6-benzyladenine (BA) and 0.1 mg l^?1 α-naphthaleneacetic acid and excised root cultures were maintained on ½ MS medium with 0.5 mg l^?1 indole-3-butyric acid (IBA). Shoot cultures, adventitious roots and excised root cultures were analysed by HPLC techniques for the presence of secoiridoids and xanthones. Gentiopicrin and swertiamarin, the dominant components of shoot cultures, could not be detected in root cultures. Xanthones were present in both shoot and root cultures with norswertianin-1-O-primeveroside as the dominant metabolite. The secoiridoid and xanthone content, although characteristic for certain plant organs, was dependent on the concentration of plant growth regulators (BA and IBA) added to the medium. BA in the shoot multiplication stage strongly increased the secondary metabolite (SEM) content of shoot cultures. IBA had little effect on SEM accumulation in shoots during rooting, while it moderately stimulated SEM accumulation in excised root cultures.     

Effects of natural and synthetic neuroactive substances on the growth and feeding of cabbage looper, Trichoplusia ni

In this study we investigated the effects of two naturally occurring beta-carboline alkaloids and two synthetic tricyclic antidepressants on the growth and food consumption of fifth instar larvae of the cabbage looper, Trichoplusia ni Hübner (Lepidoptera: Noctuidae). In artificial diets at high concentrations (3,000 ppm), harmane, amitriptyline, and imipramine reduce growth and feeding; harmane reduced feeding consistently at a lower concentration (200 ppm). In animals other than insects, beta-carboline alkaloids inhibit monoamine oxidase (MAO) activity and thus affect rates of disposition of serotonin and other monoamine neurotransmitters. Because brain serotonin levels are associated with variation in rates of carbohydrate and protein intake in insects, the effects of beta-carboline alkaloid ingestion on dietary self-selection behavior were examined. Choosing between diets lacking carbohydrate but containing protein and diets lacking protein but containing carbohydrate, larvae consumed a greater proportion of diet containing protein but lacking carbohydrate in the presence of harmane than in its absence. These results are consistent with beta-carboline alkaloid-mediated persistence of serotonin in the brain due to MAO inhibition. Alternatively, these results could reflect alkaloid-mediated peripheral inhibition of sucrose taste receptors influencing ingestive behaviors. That beta-carboline alkaloid ingestion is associated with changes in feeding behavior is consistent with a possible defensive role for these compounds in plant foliage.     

Condensed Tannin Formation by Agrobacterium rhizogenes Transformed Root and Shoot Organ Cultures of Lotus corniculatus

Root cultures of Lotus corniculatus L. cv. Leo transformed withAgrobacterium rhizogenes (C58Cl-pRi15834) grew rapidly in liquidmedium when cultured in the dark and produced large numbersof shoots when illuminated. The shoots, which could be regeneratedto produce fertile plants, were maintained in liquid mediumas shoot-organ cultures The accumulation and cellular distribution of condensed tanninswas determined during the growth of these root and shoot organcultures and in primary callus from non-transformed explants.Root and shoot cultures predominantly accumulated insolublepolymeric tannins which yielded both cyanidin and delphinidinon hydrolysis at ratios equivalent to control plants. Methanol-solublevanillin-positive compounds were isolated but no free oligomericproanthocyanidins, monomeric flavans or dihydroflavonols weredetected in these extracts. Condensed tannin accumulation waslinearly related to root growth and had a similar spatial distributionin ‘tannin’ cells in roots and leaves as comparedto control plants. Tannin-containing cells were absent frommeristematic cells of the root tip and root/shoot interface.Primary callus cultures failed to accumulate condensed tanninson media containing auxins, and exogenously supplied auxinswere found to inhibit tannin accumulation by transformed rootand shoot cultures Key words: Lotus corniculatus, Agrobacterium rhizogenes, hairy roots, condensed tannins, shoot and root cultures.     

Quantification of metabolites in the indole alkaloid pathways of catharanthus roseus: implications for metabolic engineering

In this article, we present a review of the current state of metabolic engineering in Catharanthus roseus. A significant amount of research has contributed to characterization of several individual steps in the biosynthetic pathway of medicinally valuable alkaloids. However, knowledge of the regulation of these pathways is still sparse. Using hairy root cultures, we studied the responses of alkaloid metabolism to environmental stimulation such as light and elicitation. Through precursor feeding studies, the putative rate-limiting steps of the terpenoid pathway in hairy root cultures also have been examined. Relating this knowledge to specific events at the molecular level, and the cloning of corresponding genes are the next key steps in metabolic engineering of the C. roseus alkaloids. Copyright 1998 John Wiley & Sons, Inc.     

Ara-Rhizotron: An Effective Culture System to Study Simultaneously Root and Shoot Development of Arabidopsis

Studying Arabidopsis thaliana (L.) Heynh. root development in situ at the whole plant level without affecting shoot development has always been a challenge. Such studies are usually carried out on individual plants, neglecting competition of a plant population, using hydroponic systems or Agar-filled Petri dishes. Those both systems, however, present some limitations, such as difficulty to study precisely root morphogenesis or time-limited culture period, respectively. In this paper, we present a method of Arabidopsis thaliana (L.) Heynh. cultivation in soil medium, named “Ara-rhizotron”. It allows the non-destructive study of shoot and root development simultaneously during the entire period of vegetative growth. In this system, roots are grown in 2D conditions, comparable to other soil cultures. Moreover, grouping several Ara-rhizotrons in a box enables the establishment of 3D shoot competition as for plants grown in a population. In comparison to a control culture grown in pots in the same environmental conditions, the Ara-rhizotron resulted in comparable shoot development in terms of dry mass, leaf area, number of leaves and nitrogen content. We used this new culture system to study the effect of irrigation modalities on plant development. We found that irrigation frequency only affected root partitioning in the soil and shoot nitrogen content, but not shoot or root growth. These effects appeared at the end of the vegetative growth period. This experiment highlights the opportunity offered by the Ara-rhizotron to point out tardy effects, affecting simultaneously shoot development and root architecture of plants grown in a population. We discuss its advantages in relation to root development and physiology, as well as its possible applications.     

Lysine decarboxylase activity and alkaloid production in Heimia salicifolia cultures

Levels of lysine decarboxylase, thought to exercise control over the biosynthesis of alkaloids derived from the amino acid, have been determined in alkaloid-producing and non-producing cell and organ cultures of Heimia salicifolia. The level of this enzyme has also been measured in cultures grown in the presence and absence of light. In chlorophyllous cell cultures enzyme activity correlates positively with chlorophyll; in shoot cultures the activity also parallels alkaloid production.     

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.