Establishment of hairy root cultures ofDatura stramonium. Characterization and stability of tropane alkaloid production during long periods of subculturing

The aim of this paper was the screening of the variability of growth patterns, biomass and tropane alkaloid production of 500 hairy root lines ofDatura stramonium. Data on the long term stability in alkaloid production of these lines for more than 5 years are also provided. In an effort to obtain high alkaloid-producing root clones, it is demonstrated that systematic selection is necessary. Comparisons are made, mainly concerning alkaloid production and its stability, with normal root cultures initiated from the same mother plants when necessary. Hairy root cultures were found to have a hyoscyamine and scopolamine bioproductivity of 2 orders of magnitude higher than mother plants.     

Genetic variation in constitutive and inducible pyrrolizidine alkaloid levels inCynoglossum officinale L.

The constitutive pyrrolizidine alkaloid (PA) concentration of both shoots and roots differed significantly between 17 selfed families. The broad-sense heritability accounted for 33–43% of the variation in PA levels. Families also differed significantly in the amount and the direction of PA induction in both shoots and roots, 24 h after punching 15 holes in the leaves. We found a significantly negative relationship between the changes in PA content of the shoots and changes in PA content of the roots. The total PA content of the plants did not increase. We thus concluded that changes in PA distribution over the plant resulted from transport of PAs within the plant. The direction of transport differed between families: some transported PAs to the shoots, others to the roots. This makes it questionable whether PAs act as damage-induced defences. The effect of damage on the PA concentration is far less than the differences found between families in the constitutive PA concentration. This again strongly suggests that damage-induced defences inCynoglossum officinale do not play an important role. We argue that the general lack of attention that is given to genotype in induction experiments, has led to false conclusions.     

Response of a leaf beetle to two food plants,only one of which provides a sequestrable defensive chemical

Oreina elongata is a chemically defended leaf beetle. If its food plant contains pyrrolizidine alkaloids, all life stages of the beetle sequester them. However, one of the two known host-plant genera does not contain these alkaloids. In this paper we compare the adult feeding preference and larval performance of two populations, one feeding on Adenostyles alliariae (which contains alkaloids) and one on Cirsium spinosissimum (devoid of alkaloids). Adults of the population living on C. spinosissimum preferred the alkaloid-containing A. alliariae, while adults of the population feeding on A. alliariae showed no preference for either plant. On the other hand, larval growth of both populations is better on C. spinosissimum, without alkaloids. This is especially so in the population that never naturally encounters pyrrolizidine alkaloids; the population living on A. alliariae is apparently better adapted to its host's secondary compounds. The data are discussed in terms of cost of defense and trade-offs between growth and defense.     

Cost assessment of the production of pyrrolizidine alkaloids in ragwort (Senecio jacobaea L.)

Costs of pyrrolizidine alkaloid (Pa) production in vegetative ragwort (Senecio jacobaea) were examined under conditions in which plant growth was limited by light, nitrogen and phosphorus. Measurable costs of Pa production were demonstrated under light-limiting conditions. Plants with higher Pa concentrations grew more slowly than those with lower Pa concentration. Under nitrogen- and phosphorus-limited conditions no trade-off between Pa production and growth was observed.Publication of the Meijendel-comité, new series no. 116     

绣线菊碱H,I及O的化学结构

绣线菊碱Ｈ,Ｉ,Ｏ为Ａｔｉｓｉｎｅ型的新二匝生物碱,从毛萼绣线菊根中分离得到。本文根据波谱解析及化学转换,分别测定了它们的化学结构。     

Esterification reactions in the biosynthesis of tropane alkaloids in transformed root cultures

Transformed root cultures of Datura stramonium and of related species contain both aliphatic and aromatic tropane esters. It has been shown that these esters are produced by the action of several acyl transferases that transfer the acidic moiety to tropan-3-ol (tropine) or tropan-3-ol (pseudotropine) from various acyl-coenzyme A thioesters. The presence of these enzymes has been examined in a range of tropane-alkaloid-producing and non-tropane-alkaloid-producing species. Activities that esterify tropine appear to be confined to species that accumulate tropane alkaloids, whereas a number of species that do not accumulate tropane alkaloids possess some ability to esterify pseudotropine.The present state of knowledge of these enzymes is reviewed. One of these activities, tigloyl-Coenzyme A:pseudotropine acyl transferase, has been purified to near homogeneity and the properties of this enzyme are summarized.Abbreviations CoA coenzyme A - gc gas chromatography - ms mass spectrometry     

Catharanthine and ajmalicine synthesis in Catharanthus roseus hairy root cultures

Two year old, transformed root cultures of Catharanthus roseus accumulate ajmalicine and catharanthine (0.57 and 0.36 mg g^-1 DW, or 7.0 and 3.0 mg l^-1, respectively). Changes in the concentration of the medium components, as well as the addition of hydrolytic enzymes and biotic elicitors, were used as strategies to increase these alkaloid yields. Regarding the components of the medium, the results obtained, when sucrose was raised from 3 to 4.5%, are noteworthy. The nitrogen source induced differential responses in the individual alkaloid yields. No net change in the alkaloid content was observed either with changes in the concentration of vitamins or macro-and micronutrients. Though the root culture only shows a limited response to elicitors, Aspergillus treatment and the use of macerozyme increased the accumulation of ajmalicine selectively, while the addition of methyl jasmonate increased the yield of both alkaloids.Abbreviations MeJa methyl jasmonate - mU milliunits     

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.     

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.     

Caffeine biosynthesis in young leaves of Camellia sinensis: In vitro studies on N-methyltransferase activity involved in the conversion of xanthosine to caffeine

The aim of this study was to investigate the S -adenosylmethionine dependent N -methyltransferase(s) (NMT) associated with the three methylation steps in the caffeine biosynthesis pathway in tea ( Camellia sinensis L.). NMT activity in cell-free preparations from young leaves was purified by anion-exchange and gel-filtration column chromatography. In both systems, a single zone of NMT activity, with broad substrate specificity was detected. The N-3 position of dimethylxanthine and monomethylxanthines was methylated more readily than N-1 while comparatively little substitution occurred at the N-7 locus. When xanthosine was used as a substrate only the N-7 position was methylated. These results indicate that a single NMT may participate in the conversion of xanthosine to caffeine. The apparent M_r of the NMT, estimated by gel filtration chromatography, was 61 000. The substrate specificity of the NMT is compatible with the operation of a xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine pathway as the main biosynthetic route to caffeine in young tea leaves. The data also indicate that the conversion of 7-methylxanthine → paraxanthine → caffeine may function as one of a number of minor pathways that also contribute to the production of caffeine.