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.     

Seasonal variation in the secondary chemistry of foliar and reproductive tissues of Delphinium nuttallianum

Plant secondary compounds are critical in affecting interactions between plants and their herbivores. The norditerpene alkaloids are secondary compounds in Delphinium (larkspur) species which are divided into two classes: the N-(methylsuccinimido) anthranoyllycoctonine (MSAL-type) and non MSAL-type, and are known to be toxic to herbivorous insects and livestock. Alkaloid concentrations were measured in a whole plant context in vegetative and reproductive tissues in Delphinium nuttallianum at different stages of plant maturity at two locations to explore how plant maturity affected alkaloid concentrations within a growing season. Alkaloid concentrations differed between vegetative and reproductive tissues, with vegetative tissues having significantly lower alkaloid concentrations than reproductive tissues. However, no systematic differences in alkaloid concentrations were observed at different plant maturity stages across the growing season. Based on the data we suggest that alkaloid allocation in different plant parts of D. nuttallianum is influenced by life history of the plant, consistent with plant defense theory. At one location, as pods mature the qualitative alkaloid composition changed through structural diversification of the alkaloids present. The ecological significance of this structural diversification awaits further exploration.     

Stepharine production in morphogenic cell cultures of <Emphasis Type="Italic">Stephania glabra</Emphasis> (ROXB.) Miers

Young leaves of Stephania glabra (ROXB.) Miers. (Menispermaceae) were used to establish callus cultures with the goal of studying their ability to produce the valuable alkaloid stepharine. The establishment of callus cultures from this plant is extremely difficult because primary calli are not viable and must be transferred to a liquid culture medium for subsequent subculturing. All obtained cultures possessed high morphogenic or rhizogenic potential. The methanolic extracts of two examined cell lines were analyzed by HPLC with high-resolution mass spectrometry to determine the alkaloid composition. Eleven alkaloids were detected in both cultures, including stepharine, magnoflorine, menisperine, roemerine, palmatine, corydalmine, N-methylcorydalmine, columbamine, tetrahydropalmatine, jatrorrhizine and tetrandrine. The predominant alkaloid was stepharine, the structure of which was identified through ¹H and ¹³C NMR, UV, HRMS and tandem MS. Quantitative HPLC-UV analysis showed that stepharine was produced at high levels (0.9?% dry cell weight) in the morphogenic callus culture S2-L cultivated in liquid media. To our knowledge, this is the highest level of stepharine production identified in plant cell cultures. The overall production parameters of the culture have remained stable over a long-term cultivation period for 3 years.     

Variability in tissue cultures of Choisya ternata. III comparing alkaloid production in cell lines obtained by various strategies

Callus cultures of Choisya ternata have been prepared by different strategies: aggregate clones, subclones and protoclones obtained from well-established strains; protoclones obtained from mesophyll tissue; cultures transformed by Agrobacterium tumefaciens. All of them show high variability in their dihydrofuroquinoline alkaloid production. As compared to the alkaloid content of the whole plant, one alkaloid (platydesminium) could be obtained in higher amounts in some lines, but it was impossible to get high-balfourodinium accumulating lines. Moreover balfourodinium-producing capacities were lower in transformed cells as compared to those of normal cell lines.Abbreviations MS Murashige and Skoog (1962) - B5 Gamborg B5 (1968) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphtaleneacetic acid - BA N⁶-benzyladenine - EtOH ethanol - d.w. dry weight - sd standard deviation     

Norditerpene alkaloid concentrations in tissues and floral rewards of larkspurs and impacts on pollinators

Plant secondary compounds mediate interactions with insects and other animals. The norditerpene alkaloids are significant secondary compounds in Delphinium (larkspur) species which are divided into two classes: the 7, 8-methylenedioxylycoctonine (MDL-type) and N-(methylsuccinimido) anthranoyllycoctonine (MSAL-type), and are known to be toxic to herbivorous insects and livestock. Alkaloid concentrations were measured in a whole plant context in vegetative and floral tissues as well as rewards (pollen and nectar) in Delphinium barbeyi and Delphinium nuttallianum. Alkaloid concentrations differed between vegetative tissues, floral tissues and floral rewards. Alkaloid concentrations in floral parts were consistent with optimal defense theory, with tissues more closely tied to plant fitness, such as fruits, being more heavily defended than foliage. However, alkaloid concentrations were significantly lower in nectar compared to other tissues. The norditerpene alkaloids influenced the activity of bumble bees, the dominant pollinator of larkspur, but the effects were concentration dependent. Alkaloids in nectar are found at concentrations that have no effect on bee activity; however, if alkaloid concentrations in nectar were similar to those in foliage bee activity would be reduced significantly. These results suggest that nectar with low alkaloid concentrations may be beneficial to plant fitness by limiting adverse effects on pollinator activity.     

Characterization of a novel N-methyltransferase (NMT) from Catharanthus roseus plants

Young leaves from Catharanthus roseus plants contain a novel N-methyltransferase which transfers the methyl group from S-adenosyl-L-methionine specifically to position 1 of (2R, 3R)-2,3-dihydro-3-hydroxytabersonine, producing the N-methylated product. The enzyme shows a high degree of specificity toward substrates containing a reduced double bond at position 2,3 of tabersonine derivatives but the more substituted N-desmethyldeacetylvindoline did not act as a substrate. The enzyme catalyses the third last step in vindorosine and vindoline biosynthesis, and is associated with chlorophyll-containing fractions in partially purified enzyme preparations. The lack of vindoline accumulation in cell suspension cultures is correlated with the lack of expression of this enzyme activity as well as that of an acetyltransferase which catalyses the last step in vindoline biosynthesis. Neither fungal elicitor treatment of cell line #615 nor transfer to alkaloid production medium resulted in expression of these two enzyme activities, nor was either enzyme activity detected in photoautotrophic or hormone autotrophic cultures. Cell lines #200, 615–767 and 916 could not be induced to produce DAT or NMT enzyme activities.     

The development of a single-stage growth and indole alkaloid production medium for Catharanthus roseus (L.) G. Don suspension cultures

By modification of a standard Murashige and Skoog plant tissue culture maintenance medium, a system has been developed for Catharanthus roseus cell suspension cultures in which both growth and indole alkaloid accumulation can occur in a single-stage culture of 14–21 days. Precise optimization of the medium depends upon the cell line under investigation, but the inclusion of lactose as the carbohydrate source, and NAA and kinetin as growth regulators, will generally increase yields of the indole alkaloid catharanthine. Treatment of cells growing in this optimized medium with agents that stimulate the accumulation of secondary metabolites both increases the yield of catharanthine and reduces the time required for production. We believe that this process could be useful for the commercial production of plant secondary metabolites.     

In vitro plantlet regeneration and assessment of alkaloid contents from callus cultures of Ephedra foliata (Unth phog), a source of anti-asthmatic drugs

Ephedra foliata, (Gymnosperm) is a pharmaceutically important plant known for the last 5,000 years and has a number of medicinal properties. We describe here for the first time, a method for plant regeneration from callus established from axillary buds as explant, with the aim of optimizing alkaloids production in vitro. The tissue cultures initiated are being maintained for the last 3 years on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 0.5 mg l^?1 each of 2, 4-D and Kin. Maintained callus cultures exhibited regeneration potential and maximum number (23.5 ± 0.44 shoots per culture vessel) of shoots with an average height (4.94 ± 0.23 cm) was achieved on MS medium containing combination of 0.25 mg l^?1 each of Kin, BA and 0.1 mg l^?1 of NAA. About 84.9 % regenerated shoots were rooted under ex vitro conditions on Soilrite^®, if their base was treated with 500 mg l^?1 of IBA for 5 min. The rooted plantlets were successfully acclimatized under greenhouse conditions with ≈80 % survival rate. We analyzed alkaloid contents of tissue culture raised plants/callus as affected by the different concentrations and combination of two additives, i.e., l-phenylalanine and IBA. The alkaloid production was higher in the in vitro grown cultures than field-grown plants. Highest alkaloid content was recorded in callus culture on M₅ medium having 0.5 mg l^?1 each of 2, 4-D and Kin, 100 mg l^?1 l-phenylalanine and 5 mg l^?1 IBA. The present protocol may be applicable for the large-scale cultivation of E. foliata and selection of cell line having higher secondary metabolite contents of this pharmaceutically important threatened plant species.     

Stimulation of Sanguinarine Production by Combined Fungal Elicitation and Hormonal Deprivation in Cell Suspension Cultures of Papaver bracteatum

Fungal elicitor preparations from either homogenized mycelia of Dendryphion penicillatum (Cda.) Fr., a specific pathogen of Papaver species, or conidia of Verticillium dahliae Kleb., a general pathogen, were added to 14-day-old suspension cultures of Papaver bracteatum. Plant tissue cultures were grown either in the presence or absence of 0.1 milligram of 2,4-dichlorophenoxyacetic acid per liter and 0.5 milligram of 6-benzylam-inopurine per liter. Dendryphion extracts elicited an accumulation of the benzophenanthridine alkaloid, sanguinarine, which was not greatly influenced by hormone deprivation. Millimolar concentrations of dopamine were detected under all conditions. Thebaine was found when cells were cultured in hormone-free media, but it was not elicitor dose dependent. Verticillium-elicited cultures accumulated sanguinarine in an elicitor-dose-dependent manner only under conditions of hormonal deprivation, resulting in an elevation of sanguinarine levels 5- to 500-fold greater than controls (2-10% dry weight). Most of the sanguinarine accumulated in the medium (23 milligrams per liter), with 85% of the alkaloid associated with a 100g sedimenting fraction that, upon light microscopic inspection, proved to be devoid of cells. In bioassays, sanguinarine showed significant biological activity at concentrations as low as 5 to 10 micrograms per milliliter against three general plant pathogens, Verticillium dahliae, Botrytis cinerea Pers. ex Fr., and Rhizoctonia solani Kuehn. Dendryphion was less affected by sanguinarine addition and displayed an ability to metabolize the alkaloid as evidenced by its loss from the media, subsequent accumulation in the mycelia, and ultimate disappearance over a 48-hour period. By comparison, dopamine and thebaine were less toxic to the general plant pathogens.     

Selective uptake of pyrrolizidine N-oxides by cell suspension cultures from pyrrolizidine alkaloid producing plants

The N-oxides of pyrrolizidine alkaloids such as senecionine or monocrotaline are rapidly taken up and accumulated by cell suspension cultures obtained from plants known to produce pyrrolizidines, i.e. Senecio vernalis, vulgaris, viscosus (Asteraceae) and Symphytum officinale (Boraginaceae). The transport of the N-oxides into the cells is a specific and selective process. Other alkaloid N-oxides such as sparteine N-oxide are not taken up. Cell cultures from plant species which do not synthesize pyrrolizidine alkaloids are unable to accumulate pyrrolizidine N-oxides. The suitability of the pyrrolizidine N-oxides in alkaloid storage and accumulation is emphasized.     

Alkaloid production in Vernonia cinerea: Callus, cell suspension and root cultures

Fast-growing callus, cell suspension and root cultures of Vernonia cinerea, a medicinal plant, were analyzed for the presence of alkaloids. Callus and root cultures were established from young leaf explants in Murashige and Skoog (MS) basal media supplemented with combinations of auxins and cytokinins, whereas cell suspension cultures were established from callus cultures. Maximum biomass of callus, cell suspension and root cultures were obtained in the medium supplemented with 1 mg/L alpha-naphthaleneacetic acid (NAA) and 5 mg/L benzylaminopurine (BA), 1.0 mg/L NAA and 0.1 mg/L BA and 1.5 mg/L NAA, respectively. The 5-week-old callus cultures resulted in maximum biomass and alkaloid contents (750 microg/g). Cell suspension growth and alkaloid contents were maximal in 20-day-old cultures and alkaloid contents were 1.15 mg/g. A 0.2-g sample of root tissue regenerated in semi-solid medium upon transfer to liquid MS medium containing 1.5 mg/L NAA regenerated a maximum increase in biomass of 6.3-fold over a period of 5 weeks. The highest root growth and alkaloid contents of 2 mg/g dry weight were obtained in 5-week-old cultures. Maximum alkaloid contents were obtained in root cultures in vitro compared to all others including the alkaloid content of in vivo obtained with aerial parts and roots (800 microg/g and 1.2 mg/g dry weight, respectively) of V. cinerea.     

Fusarium oxysporum homogenates and jasmonate induce limited sanguinarine accumulation in Argemone mexicana cell cultures

In vitro cultures of Argemone mexicana (Papaveraceae) were induced from leaves of mature plants. Sanguinarine, a benzophenanthridine, was the main alkaloid in the cultures, even in the absence of inducers of secondary metabolism. The accumulation of this metabolite was increased by adding methyl jasmonate and fungal elicitors, although in a limited fashion in comparison to other sanguinarine-producing species. Evidence of a transport mechanism, which may be related to the magnitude of the response, was obtained based on the fluorescent properties of bezophenathridines in the elicited cultures.     

Concomitant occurrence of pyrrolizidine and quinolizidine alkaloids in the hemiparasite Osyris alba L. (Santalaceae)

The investigation of the alkaloid extracts of the hemiparasitic plant Osyris alba, collected from three different localities in southern France, revealed the concomitant presence of both pyrrolizidine (PA) and quinolizidine (QA) alkaloids in the samples from two of these localities. The sample from the third locality contained only PAs. The eight QAs identified were sparteine, N-methylcytisine, cytisine, methyl-12-cytisine acetate, hydroxy-N-methylcytisine, N-acetylcytisine, lupanine, and anagyrine. Of the eleven detected PAs, eight were identified as chysin A, chysin B, 1-carboxypyrrolizidine-7-olide, senecionine, integerrimine, retrorsine, senecivernine and a new alkaloid janfestine (7R-hydroxychysin A or 1R-carbomethoxy-7R-hydroxypyrrolizidine). PAs were mainly present as their N-oxides This is, to our knowledge, the first report demonstrating the simultaneous presence of two classes of alkaloids, quinolizidine and pyrrolizidine alkaloids, in a single parasitic plant. As these alkaloids do not occur in the same host plant, the results indicate that Osyris must have tapped more than one host plant concomitantly. Since both quinolizidine and pyrrolizidine alkaloids serve as defence compounds against herbivores, affecting different molecular targets, the simultaneous acquisition of the two types of alkaloids by a single plant could provide a novel mode of defence of hemiparasites against herbivores.     

Alkaloid production by plants regenerated from cultured cells of Datura innoxia

Cellular aggregates in Datura innoxia suspension cultures give rise to large numbers of shoots when such aggregates are cultured in the light on an auxin-free agar medium supplemented with kinetin. These shoots form roots on a kinetin-free medium to develop into complete plants. Most of the regenerated plants are diploid, and the frequencies of ancuploid or polyploid plants are much lower than might be expected from the distribution of chromosome number in the cultured cells. During root ditterentiation and plant development, scopolamine synthesis is initiated and there is a progressive increase in the alkaloid content. Consequently, the general pattern of alkaloid composition is restored to a normal state in the majority of the regenerated plants including aneuploid or polyploids. Nevertheless, some of the plants show an abnormal expression in alkaloid metabolism, such as the complete hydrolysis of scopolamine in the dried leaves.     

Detection of Specific Strains and Variants of Streptococcus cremoris in Mixed Cultures by Immunofluorescence

Antisera against four different strains of Streptococcus cremoris were raised by injecting rabbits with washed suspensions of whole cells. These antisera interacted specifically with the corresponding strain in a mixture of up to nine different S. cremoris strains. The antisera could be used for analyzing the composition of mixed cultures containing these strains by immunofluorescence. Competition experiments were performed in batch and continuous cultures under amino acid limitation. A bacteriophage-sensitive variant of S. cremoris SK11 (SK1128) could be distinguished from a bacteriophage-resistant variant (SK1143) by the same immunofluorescence technique. The competition between the two variants and the stability of both variants in pure cultures were followed with the specific antibodies. Antibodies against the purified proteolytic system of S. cremoris Wg2 were used to determine the presence of proteases by immunofluorescence in several S. cremoris strains under different culture conditions. The described immunofluorescence methods can be used to analyze complex mixed starter cultures common in the dairy industry as the strains and variants present in these mixtures can be recognized microscopically.     

Plant Virology and Next Generation Sequencing: Experiences with a Potyvirus

Next generation sequencing is quickly emerging as the go-to tool for plant virologists when sequencing whole virus genomes, and undertaking plant metagenomic studies for new virus discoveries. This study aims to compare the genomic and biological properties of Bean yellow mosaic virus (BYMV) (genus Potyvirus), isolates from Lupinus angustifolius plants with black pod syndrome (BPS), systemic necrosis or non-necrotic symptoms, and from two other plant species. When one Clover yellow vein virus (ClYVV) (genus Potyvirus) and 22 BYMV isolates were sequenced on the Illumina HiSeq2000, one new ClYVV and 23 new BYMV sequences were obtained. When the 23 new BYMV genomes were compared with 17 other BYMV genomes available on Genbank, phylogenetic analysis provided strong support for existence of nine phylogenetic groupings. Biological studies involving seven isolates of BYMV and one of ClYVV gave no symptoms or reactions that could be used to distinguish BYMV isolates from L. angustifolius plants with black pod syndrome from other isolates. Here, we propose that the current system of nomenclature based on biological properties be replaced by numbered groups (I–IX). This is because use of whole genomes revealed that the previous phylogenetic grouping system based on partial sequences of virus genomes and original isolation hosts was unsustainable. This study also demonstrated that, where next generation sequencing is used to obtain complete plant virus genomes, consideration needs to be given to issues regarding sample preparation, adequate levels of coverage across a genome and methods of assembly. It also provided important lessons that will be helpful to other plant virologists using next generation sequencing in the future.     

Enhanced Alkaloid Production in Callus Cultures of Heliotropium indicum L and Regeneration of Plantlets

Heliotropium indicum L (Boraginaceae) contains the anticancer pyrrolizidine alkaloid, indicine-N-oxide (INO). To study the yield of INO as a function of plant development, plantlets were regenerated in vitro from nodal and hypocotyl explants and also from hypocotyl callus, on Murashige and Skoog’s (MS) medium supplemented with 1-naphthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), asparagine (Asp) and glutamine (Glu). The regenerated plantlets were rooted on MS supplemented with Glu or gibberellic acid (GA₃). While 5-week-old seedlings showed a high amount of INO (0.12% dry wt) that depleted gradually as the plants attained maturity and flowered, fast growing callus produced a much higher yield of INO (0.32% dry wt). As the callus cultures differentiated into shoots and subsequently into plantlets, the INO content decreased to about 0.2% dry wt. It appears that INO is the primary product of pyrrolizidine alkaloid biosynthetic pathway in rapidly growing meristematic tissue of Heliotropium indicum, later reduced to its alkaloidal base and reallocated to other tissues. The article includes an efficient micropropagation method for Heliotropium indicum.     

Accumulation of Ergot Alkaloids During Conidiophore Development in Aspergillus fumigatus

Production of ergot alkaloids in the opportunistic fungal pathogen Aspergillus fumigatus is restricted to conidiating cultures. These cultures typically accumulate several pathway intermediates at concentrations comparable to that of the pathway end product. We investigated the contribution of different cell types that constitute the multicellular conidiophore of A. fumigatus to the production of ergot alkaloid pathway intermediates versus the pathway end product, fumigaclavine C. A relatively minor share (11 %) of the ergot alkaloid yield on a molar basis was secreted into the medium, whereas the remainder was associated with the conidiating colonies. Entire conidiating cultures (containing hyphae, vesicle of conidiophore, phialides of conidiophore, and conidia) accumulated higher levels of the pathway intermediate festuclavine and lower levels of the pathway end product fumigaclavine C than did isolated, abscised conidia, indicating that conidiophores and/or hyphae have a quantitatively different ergot alkaloid profile compared to that of conidia. Differences in alkaloid accumulation among cell types also were indicated by studies with conidiophore development mutants. A ?medA mutant, in which conidiophores are numerous but develop poorly, accumulated higher levels of pathway intermediates than did the wildtype or a complemented ?medA mutant. A ?stuA mutant, which grows mainly as hyphae and produces very few, abnormal conidiophores, produced no detectable ergot alkaloids. The data indicated heterogeneous spatial distribution of ergot alkaloid pathway intermediates versus pathway end product in conidiating cultures of A. fumigatus. This skewed distribution may reflect differences in abundance or activity of pathway enzymes among cell types of those conidiating cultures.     

Accumulation of beta-Carboline Alkaloids and Serotonin by Cell Cultures of Peganum harmala L: I. CORRELATION BETWEEN PLANTS AND CELL CULTURES AND INFLUENCE OF MEDIUM CONSTITUENTS

A number of cell cultures of Peganum harmala were initiated to check for a correlation between the harman alkaloid content of seedlings and cell lines derived therefrom. Despite a poor correlation between callus or suspension culture lines and parent plants, the mean alkaloid contents of strains derived from seedlings with higher alkaloid yields were nevertheless higher than the mean contents of strains derived from low yield plants. Generally, alkaloid accumulation decreased with the numbers of transfers. By permanent visual selection for fluorescent areas of the calluses, however, a mean content of 0.1% harman alkaloids and 0.1% serotonin could be maintained, which was 10 times higher than in unselected callus cultures.     

Morphological and molecular characterization of somaclonal variations in tissue culture-derived banana plants

In this study, 40 000 tissue culture-derived banana plants (vitroplants) at different growth stages, i.e. acclimatization, nursery and open field of banana (Musa spp.) cultivar ‘Grand Naine’ were screened for somaclonal variations using morphological investigations and molecular characterization. The total detected variants were grouped into 25 off-types (two of them died) in addition to the normal plant. Random Amplified Polymorphic DNA (RAPD) was carried out to study the differences among the normal cultivar ‘Grand Naine’ and its 23 variants using 17 arbitrary primers. Cluster analysis results revealed that ‘winged petiole’ and ‘deformed lamina’ were more related to the normal plant. However, ‘Giant plant’ and ‘weak plant’ related to each other and clustered with normal plant. According to principal coordinate analysis, most of the variants were aggregated nearly, whereas ‘variegated plant’ was separated apart from the other variants. This may reflect the genetic difference between ‘variegated plant’ and the other variants. The results obtained from both molecular and morphological analyses were in contiguous with better resolution when using the PCOORDA analysis than cluster analysis. Thus, it can be said that molecular markers can be used to eliminate the undesirable somaclonal variants from the lab without additional culture of the vitroplants in the field in order to save time and efforts.