Tropane alkaloid production inDatura stramonium root cultures

Summary Tropane alkaloid production was studied in different root cultures ofDatura stramonium. Cultured roots were obtained with 10⁻⁶ M of indolbutyric acid. Their doubling times were from 6 to 19 days. Hyoscyamine content varied from 0.17 to 0.62% dry weight, and scopolamine content from 0.08 to 0.33% dry weight, depending on the lines. A comparison of the bioproductivity of these compounds in the pot-grown plant roots showed that it was two to three orders lower than cultured roots, and it increased one order of magnitude considering the productivity on the whole plant. Bioproductivity, growth capacity and alkaloid production stability during subsequent transfers (more than 2 yr) are reported. Only one root line (N5) showed excretion of the alkaloids to the culture medium. Characterization of three selected lines (N1, N5, and N9) showed that the highest alkaloid production is reached at the stationary phase of growth, with the exception of line N9.     

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.     

Indole alkaloid production by hairy root cultures of Catharanthus roseus

Hairy root cultures of Catharanthus roseus were established by infection with six different Agrobacterium rhizogenes strains. Two plant varieties were used and found to exhibit significantly different responses to infection. Forty-seven hairy root clones derived from normal plants and two derived from the flowerless variety were screened for their growth and indole alkaloid production. The growth rate and morphological appearance showed wide variations between the clones. The alkaloid spectra observed were qualitatively but not quantitatively very similar to that of the corresponding normal plant roots. No vindoline or deacetyltransferase activity could be detected in any of the cultures studied. O-acetylval-lesamine, an alkaloid which has not been previously observed in C. roseus was identified from extracts of hairy root clone No. 8. Two root clones were examined for their growth and alkaloid accumulation during a 26-day culture period. Alkaloid accumulation parallelled growth in both clones with ca. 2 mg ajmalicine and catharanthine per g dry weight being observed.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

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.     

Characterization of transgenic plants derived from hairy roots ofHyoscyamus muticus

Mature plants were regenerated via protoplasts fromAgrobacterium rhizogenes-transformed root cultures ofHyoscyamus muticus L., and chemical analyses were performed on 34 individual plants. The regenerated plants showed strong phenotypic differences from clone to clone as well as from the control plants. Polymerase chain reaction studies revealed that the plants exhibiting the strongest phenotypic alterations contained therol (A, B and C) genes, whereas the plants with fewer alterations had lost them. The plants produced hyoscyamine, scopolamine and a range of different calystegins, and considerable somaclonal variation was observed. Alkaloid production in the plants transgenic for therol genes was clearly reduced. The pattern of calystegins was similar within all the regenerated plants lackingrol genes. Among the plants withrol genes, the calystegin B₁ was not detectable. It seems clear that the presence ofrol genes is detrimental to the alkaloid accumulation in the transgenic plants in contrast to hairy root cultures.Abbreviation PCR Polymerase chain reaction     

Correlation between the nicotine content of tobacco plants and callus cultures

Callus cultures of two low-alkaloid lines of Nicotiana tabacum L. had considerably lower nicotine contents than cultures from the respective highalkaloid cultivars which were isogenic except for the two loci for alkaloid accumulation. Thus, there was a strong correlation between the nicotine content of callus cultures and the plants from which they were derived.     

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.     

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     

Steroidal glycoalkaloids in cell and shoot teratoma cultures ofSolanum dulcamara

Bittersweet (Solanum dulcamara L., Solanaceae) is of interest as a source of steroidal alkaloids for the commercial production of hormones. Since glycoalkaloid production is positively correlated to differentiation, tumor and teratoma cultures of the soladulcidine chemotype were established by transformation withAgrobacterium tumefaciens. A newly developed HPLC-system, which allowed separation and sensitive quantitation of the glycoalkaloids soladulcidine-tetraoside, solamargine and solasonine, was used to analyse glycoalkaloid profiles in plants and cultures. Tumors and teratoma were charcterized by a shift in their alkaloid pattern from soladulcidine tetraoside to the solasodine glycosides solamargine and solasonine. Shoot teratoma showed a total glycoalkaloid content of 1% dw, which is about fivefold higher than in the source plant. A regenerated plant retained the altered alkaloid spectrum; the levels, however, equalled those of the source plant. From the alteration of alkaloid pattern in the transformed cultures suggestions can be made concerning the biosynthetic pathway. Completion of the biosynthesis of the aglycone is likely to be complete before glycosylation occurs.     

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     

Manipulating indole alkaloid production by Catharanthus roseus cell cultures in bioreactors: from biochemical processing to metabolic engineering

Catharanthus roseus plants produce many pharmaceutically important indole alkaloids, of which the bisindole alkaloids vinblastine and vincristine are antineoplastic medicines and the monoindole alkaloids ajmalicine and serpentine are antihypertension drugs. C. roseus cell cultures have been studied for producing these medicines or precursors catharanthine and vindoline for almost four decades but so far without a commercially successful process due to biological and technological limitations. The research thus focused on the one hand on engineering the bioreactor process on the other engineering the cell factory itself. This review mainly summarizes the progress made on biochemical engineering aspects of C. roseus cell cultures in bioreactors in the past decades and metabolic engineering of indole alkaloid production in recent years. The paper also attempts to highlight new strategies and technologies to improve alkaloid production and bioreactor performance. Perspectives of metabolic engineering to create new cell lines for large-scale production of indole alkaloids in bioreactors and effective combination of these up- and down-stream processing are presented.     

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.     

Variation of alkaloid productivity among several clones of hairy roots and regenerated plants ofAtropa belladonna transformed withAgrobacterium rhizogenes 15834

Hairy root cultures ofAtropabelladonna were established by transformation withAgrobacterium rhizogenes 15834. Five clones of them were employed to study the production of hyoscyamine, the main constituent of the plant, together with other tropane alkaloids. The growth and alkaloid production of each clone were differently affected by basal liquid culture media tested. The transgenic plants regenerated from each clone of the hairy roots had different phenotypes and diverse alkaloid productivity both in the cultured condition and in productivitiy both in the cultured condition and in hydroponics.Abbreviations ANOVA analysis of variance - B5 medium Gamborg B5 medium - BA N⁶-benzyladenine - B.S. Balanced Solution - dw dry weight - EC electric conductivity - fw fresh weight - GC/MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - MS medium Murashige and Skoog medium - NAA naphthalene-l-acetic acid - PCR polymerase chain reaction - SDS sodium dodecyl sulfate - TMS trimethylsilyl - WP medium Woody Plant medium     

Regulation of product synthesis in cell cultures of Catharanthus roseus. V. Long-term maintenance of cells on a production medium

Three unselected cell lines of C. roseus maintained on a growth-associated alkaloid production medium were studied over a period of 2 to 5.5 years for the stability of alkaloid production (serpentine and ajmalicine). Large fluctuations in the total alkaloid content of 20-day-old cells were found for all three cell lines at each subculture over a two-year period. Growth rates increased during prolonged subculture and one cell line became unproductive after five years culture. By selection of small autofluorescent aggregates, high alkaloid production was restored in this cell line, while the parent line was found to be unresponsive to alkaloid induction treatments. The instability in both alkaloid production and spectrum and the loss of alkaloid productivity are discussed in relation to the selection pressures present during long-term maintenance of cell suspension cultures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - nHS n-heptane sulphonate     

Genetic stability of hairy root cultures of Datura stramonium

The karyotypic status of three root lines, transformed with Agrobacterium rhizogenes, versus a non-transformed root line of Datura stramonium, as well as grown plants (control), was investigated. In the transformed cultures, the karyotype as well as the number of chromosomes (2n = 24) remained the same as in the control plants. In the normal root line there were clear differences at a morphological level despite showing the same chromosomic number (2n = 24) at a high frequency. The existence of aneuploids (2n = 2n ± x) and tetraploids (2n = 4x = 48) was also observed in the latter. The frequency of chromosomic alterations was very low in all the cultures. Cytogenetic analysis of the transformed root cultures of D. stramonium reported here shows a great chromosomic stability, which is not the case with normal root cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Establishment and characterization of photosynthetic hairy root cultures of Datura stramonium

Twelve different lines of Datura stramonium (normal and hairy) root cultures were subjected to conditions which induce photoautotrophy. Two of the hairy root lines responded to induction, showing clearly a diminished growth rate when compared to heterotrophic cultures, an increase in chlorophyll, a net O₂ evolution, CO₂ fixation, and de novo synthesis of the ribulose 1,5 biphosphate carboxylase enzyme. A time course of growth and tropane alkaloid levels in the tissue and medium, revealed a correlation between the development of the photosynthetic apparatus and the increase in scopolamine. Although normal cultures did not grow photosynthetically, they showed some greening response under the first step of the induction. The correlation between development of photosynthesis and increase in scopolamine synthesis were corroborated with normal root cultures. This experimental model is used for the basic study of the regulatory enzymes involved in the biosynthesis of tropane alkaloids, as well as for the study of their mechanisms of transport.     

Production of higher levels of trigonelline by cell cultures of Trigonella foenum-graecum than by the differentiated plant

Callus cultures of Trigonella foenum-graecum contained 3 to 4 times more trigonelline than the seeds of this plant and 12 to 13 times more than the roots and shoots. Even higher levels of this alkaloid were produced by suspension cultures. This high productivity was maintained during successive subculturing of calli and cell suspensions for eight months. Thus, trigonelline is to be added to the group of the few metabolites whose synthesis in cell cultures exceeds its production in the differentiated plants. Media that had supported the growth of suspension cultures contained one third or more of the total alkaloid, whereas media of callus cultures contained about one tenth of this substance. Trigonelline accumulated in callus and suspension cultures with aging. Raising the level of nicotinic acid in the nutrient medium resulted in some increase of trigonelline production by the culture.Abbreviations 2.4 D 2.4-dichlorophenoxyacetic acid - IAA indoleacetic acid - IPA indolepropionic acid - NAA -naphthaleneacetic acid - GA Gibberellic acid - K kinetin     

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.     

Recovery of phenotypically normal transgenic plants of Brassica oleracea upon Agrobacterium rhizogenes-mediated co-transformation and selection of transformed hairy roots by GUS assay

In this paper we describe the production of transgenic broccoli and cauliflower with normal phenotype using an Agrobacterium rhizogenes-mediated transformation system with efficient selection for transgenic hairy-roots. Hypocotyls were inoculated with Agrobacterium strain A4T harbouring the bacterial plasmid pRiA4 and a binary vector pMaspro::GUS whose T-DNA region carried the gus reporter gene. pRiA4 transfers T_L sequences carrying the rol genes that induce hairy root formation. Transgenic hairy-root production was increased in a difficult-to-transform cultivar by inclusion of 2,4-D in the medium used to resuspend the Agrobacterium prior to inoculation. Transgenic hairy roots could be selected from inoculated explants by screening root sections for GUS activity; this method eliminated the use of antibiotic resistance marker genes for selection. Transgenic hairy roots were produced from two cauliflower and four broccoli culivars. Shoots were regenerated from transgenic hairy root cultures of all four cultivars tested and successfully acclimatized to glasshouse conditions, although some plants had higher than diploid ploidy levels. Southern analysis confirmed the transgenic nature of these plants. T₀ plants from seven transgenic lines were crossed or selfed to produce viable seed. Genetic analysis of T₁ progeny confirmed the transmission of traits and revealed both independent and co-segregation of Ri T_L-DNA and vector T-DNA. GUS-positive phenotypically normal progeny free of T_L-DNA were identified in three transgenic lines out of the six tested representing all the cultivars regenerated including both cauliflower and broccoli.     

Stability of CaMV 35S-gus gene expression in (bird's foot trefoil) hairy root cultures under different growth conditions

Lotus corniculatus is an agronomically important forage legume. Genetic engineering offers opportunities both to improveL. corniculatus as a crop and to increase basic understanding of plant biochemistry and metabolism. Biosynthesis of secondary products and nitrogen fixation are two areas in which gene expression has been studied using hairy root cultures ofL. corniculatus. The stability of foreign gene expression in these cultures is critically important. TwoL. corniculatus root culture lines containing a reporter gene (CaMV 35S-gus) were used to investigate the stability of expression of a foreign gene under a range of conditions likely to be encountered in experiments. The hairy root culture lines were grown under varying conditions of light, temperature, nutrient supply, and in the presence of the auxin 2,4-D, or the elicitor glutathione. Expression of thegus gene, detected by measuring GUS activity, was found to be relatively stable under all of the conditions investigated.