Redirecting tropane alkaloid metabolism reveals pyrrolidine alkaloid diversity in Atropa belladonna.

Plant-specialized metabolism is complex, with frequent examples of highly branched biosynthetic pathways, and shared chemical intermediates. As such, many plant-specialized metabolic networks are poorly characterized. The N-methyl Δ1 -pyrrolinium cation is a simple pyrrolidine alkaloid and precursor of pharmacologically important tropane alkaloids. Silencing of pyrrolidine ketide synthase (AbPyKS) in the roots of Atropa belladonna (Deadly Nightshade) reduces tropane alkaloid abundance and causes high N-methyl Δ1 -pyrrolinium cation accumulation. The consequences of this metabolic shift on alkaloid metabolism are unknown. In this study, we utilized discovery metabolomics coupled with AbPyKS silencing to reveal major changes in the root alkaloid metabolome of A. belladonna. We discovered and annotated almost 40 pyrrolidine alkaloids that increase when AbPyKS activity is reduced. Suppression of phenyllactate biosynthesis, combined with metabolic engineering in planta, and chemical synthesis indicates several of these pyrrolidines share a core structure formed through the nonenzymatic Mannich-like decarboxylative condensation of the N-methyl Δ1 -pyrrolinium cation with 2-O-malonylphenyllactate. Decoration of this core scaffold through hydroxylation and glycosylation leads to mono- and dipyrrolidine alkaloid diversity. This study reveals the previously unknown complexity of the A. belladonna root metabolome and creates a foundation for future investigation into the biosynthesis, function, and potential utility of these novel alkaloids.

Engineering tropane alkaloid production and glyphosate resistance by overexpressing AbCaM1 and G2-EPSPS in Atropa belladonna.

Atropa belladonna is an important industrial crop for producing anticholinergic tropane alkaloids (TAs). Using glyphosate as selection pressure, transgenic homozygous plants of A. belladonna are generated, in which a novel calmodulin gene (AbCaM1) and a reported EPSPS gene (G2-EPSPS) are co-overexpressed. AbCaM1 is highly expressed in secondary roots of A. belladonna and has calcium-binding activity. Three transgenic homozygous lines were generated and their glyphosate tolerance and TAs' production were evaluated in the field. Transgenic homozygous lines produced TAs at much higher levels than wild-type plants. In the leaves of T2GC02, T2GC05, and T2GC06, the hyoscyamine content was 8.95-, 10.61-, and 9.96 mg/g DW, the scopolamine content was 1.34-, 1.50- and 0.86 mg/g DW, respectively. Wild-type plants of A. belladonna produced hyoscyamine and scopolamine respectively at the levels of 2.45 mg/g DW and 0.30 mg/g DW in leaves. Gene expression analysis indicated that AbCaM1 significantly up-regulated seven key TA biosynthesis genes. Transgenic homozygous lines could tolerate a commercial recommended dose of glyphosate in the field. In summary, new varieties of A. belladonna not only produce pharmaceutical TAs at high levels but tolerate glyphosate, facilitating industrial production of TAs and weed management at a much lower cost.

Development of Atropa belladonna L. Plants with High-Yield Hyoscyamine and without Its Derivatives Using the CRISPR/Cas9 System.

Atropa belladonna L. is one of the most important herbal plants that produces hyoscyamine or atropine, and it also produces anisodamine and scopolamine. However, the in planta hyoscyamine content is very low, and it is difficult and expensive to independently separate hyoscyamine from the tropane alkaloids in A. belladonna. Therefore, it is vital to develop A. belladonna plants with high yields of hyoscyamine, and without anisodamine and scopolamine. In this study, we generated A. belladonna plants without anisodamine and scopolamine, via the CRISPR/Cas9-based disruption of hyoscyamine 6ß-hydroxylase (AbH6H), for the first time. Hyoscyamine production was significantly elevated, while neither anisodamine nor scopolamine were produced, in the A. belladonna plants with homozygous mutations in AbH6H. In summary, new varieties of A. belladonna with high yields of hyoscyamine and without anisodamine and scopolamine have great potential applicability in producing hyoscyamine at a low cost.

[Nitrogen metabolism and secondary metabolism regulation of Atropa belladonna by exogenous NO under NaCl stress].

Atropa belladonna seedlings were used as experimental materials and cultivated by soil culture method. Different concentrations(0,0.05,0.1,0.2,0.5 mmol·L~(-1))of NO donor sodium nitroprusside(SNP) were sprayed on the leaves. The effects of different concentrations of SNP and different treatment time(4,8,12,16 d) on nitrogen metabolism, secondary metabolite content, precursor content of tropane alkaloid synthesis pathway and expression of key enzyme genes under 100 mmol·L~(-1) NaCl stress were studied. The results showed that with the prolongation of salt stress, the nitrogen metabolism and the accumulation of secondary metabolites of A. belladonna were inhibited to some extent. After treatment with different concentrations of exogenous SNP, the ammonium nitrogen content decreased dramatically, and the contents of nitrate nitrogen, free amino acid, soluble protein and the activities of key enzymes of nitrogen metabolism(NR, GS, GDH) were all greatly improved; the contents of precursor amino acids(ornithine, arginine) and polyamines(Put, Spd, Spm) in the secondary metabolic pathway have increased to varying degrees. The qRT-PCR analysis showed that exogenous SNP treatment can effectively promote the high expression of key enzyme genes PMT, TRⅠ and H6H in the secondary metabolic pathway of A. belladonna, and the production of hyoscyamine and scopolamine were increased notably. In summary, the application of appropriate concentration of SNP can effectively alleviate the inhibition of salt stress on the nitrogen metabolism and secondary metabolism of Atropa belladonna, and enhance its salt tolerance. Overall, 0.1 mmol·L~(-1) and 0.2 mmol·L~(-1) SNP treatment achieved the most remarkable effect.

Homeopathic Treatment of Insomnia and Symptom Clusters Related to Cerebral Chemotoxicity in Oncology.

BACKGROUND: During chemotherapy, the correlation between insomnia and fatigue, anxiety, pain, depressed mood, and cognitive disorders makes these subjective complaints a 'symptom cluster' with common biological mechanisms. The theory of cerebral inflammation following the production of pro-inflammatory cytokines (high level of interleukin 1-ß [IL1-ß], IL6 and tumour necrosis factor-alpha) is currently the most generally accepted. Understanding these mechanisms should allow us to propose a chemoprotective homeopathic treatment of the nervous system. METHODS: By retaining the inflammatory aetiology, we combined the rubrics 'Inflammation of the brain', 'Inflammation of the meninges', 'Inflammation of the nerves' with the symptom cluster: insomnia, fatigue, depressive state and memory disorders. RESULTS: After repertorisation, we propose the following homeopathic protocol: Belladonna 15c, Phosphorus 15c, Cerebral cortex 4c and Nerves 4c, two pills of each medicine to be sucked together before breakfast, lunch and dinner, on each day of chemotherapy and for the following 2 days. CONCLUSION: This selected protocol, derived from a physiopathological knowledge of the symptoms, seems to be well suited to the prevention and treatment of post-chemotherapeutic cerebral inflammation. It is essential to start the homeopathic treatment before the chemotherapy session to anticipate the emergence of the 'chemo-brain' side effects. This proposed prevention protocol must be confirmed and quantified by randomised studies.

[Effects of methyl jasmonate on metabolism of topical alkaloids and expression of relate genes in Atropa belladonna].

Hyoscyamine and scopolamine are important secondary metabolites of tropane alkaloid in Atropa belladonna with pharmacological values in many aspects.In this study, the seedlings of A.belladonna were planted by soil culture and treated with different concentrations of methyl jasmonate (MeJA). The contents of hyoscyamine and scopolamine,the upstream products in alkaloid synthesis,and the expression levels of key enzyme genes PMT, TR Ⅰ and H6H in secondary metabolites of A. belladonna seedlings were measured to clarify the mechanism of MeJA regulating alkaloids synthesis.The results showed that MeJA(200 µmol·L⁻¹) treatment was more favorable for the accumulation of alkaloids.The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine,the both increased first and then decreased with the increased MeJA concentration and the content of putrescine reached the highest at 200 µmol·L⁻¹ MeJA.Further detection of gene expression of PMT, TR Ⅰ and H6H in TAs synthesis pathway showed that no significant trend in PMT gene expression levels.The expression levels of TR Ⅰ and H6H in leaves and roots under 200 µmol·L⁻¹ MeJA were the highest.It can be speculated that the regulation of the formation of hyoscyamine and scopolamine by MeJA mainly through affecting the expression of key enzyme genes.Appropriate concentration of MeJA increased the gene expression of TR Ⅰ in both leaves and roots as well as H6H in roots,promoting the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine.

Evolutional dynamics of 45S and 5S ribosomal DNA in ancient allohexaploid Atropa belladonna.

BACKGROUND: Polyploid hybrids represent a rich natural resource to study molecular evolution of plant genes and genomes. Here, we applied a combination of karyological and molecular methods to investigate chromosomal structure, molecular organization and evolution of ribosomal DNA (rDNA) in nightshade, Atropa belladonna (fam. Solanaceae), one of the oldest known allohexaploids among flowering plants. Because of their abundance and specific molecular organization (evolutionarily conserved coding regions linked to variable intergenic spacers, IGS), 45S and 5S rDNA are widely used in plant taxonomic and evolutionary studies. RESULTS: Molecular cloning and nucleotide sequencing of A. belladonna 45S rDNA repeats revealed a general structure characteristic of other Solanaceae species, and a very high sequence similarity of two length variants, with the only difference in number of short IGS subrepeats. These results combined with the detection of three pairs of 45S rDNA loci on separate chromosomes, presumably inherited from both tetraploid and diploid ancestor species, example intensive sequence homogenization that led to substitution/elimination of rDNA repeats of one parent. Chromosome silver-staining revealed that only four out of six 45S rDNA sites are frequently transcriptionally active, demonstrating nucleolar dominance. For 5S rDNA, three size variants of repeats were detected, with the major class represented by repeats containing all functional IGS elements required for transcription, the intermediate size repeats containing partially deleted IGS sequences, and the short 5S repeats containing severe defects both in the IGS and coding sequences. While shorter variants demonstrate increased rate of based substitution, probably in their transition into pseudogenes, the functional 5S rDNA variants are nearly identical at the sequence level, pointing to their origin from a single parental species. Localization of the 5S rDNA genes on two chromosome pairs further supports uniparental inheritance from the tetraploid progenitor. CONCLUSIONS: The obtained molecular, cytogenetic and phylogenetic data demonstrate complex evolutionary dynamics of rDNA loci in allohexaploid species of Atropa belladonna. The high level of sequence unification revealed in 45S and 5S rDNA loci of this ancient hybrid species have been seemingly achieved by different molecular mechanisms.

Generalisability of prognostic factor research: further analysis of data from the IIPCOS2 study.

Prognostic factor research is important as it helps in refining diagnosis, taking clinical and therapeutic decisions, enhances the design and analysis of intervention trials and helps to identify targets for new interventions that aim to modify the course of a disease. Prognostic factor research in homeopathy can be done by applying Bayes' theorem. This paper considers Bayes' theorem; Likelihood Ratio, conditional probability and research in subpopulations of a condition with examples. We analysed the likelihood ratios for 11 homeopathic medicines for the symptom 'cough' and other upper respiratory tract symptoms, based on data from the IIPCOS2 study. This yielded useful information since several medicines, including Belladonna, had LR >1 for cough, indicating that cough is not an indication for this medicine. The implications for improving homeopathic prescribing are discussed.

Accumulation of mannitol in the cytoplasm and vacuole during the expansion of sepal cells associated with flower opening in Delphinium × belladonna cv. Bellamosum.

MAIN CONCLUSION: The role of mannitol differs from that of glucose, fructose and sucrose in sepal cell expansion associated with flower opening in Delphinium × belladonna. Sepals of Delphinium × belladonna are colored and much larger than the petals. To determine whether the role of mannitol in sepal growth associated with flower opening differs from those of ubiquitous metabolic sugars including glucose, fructose and sucrose, we investigated changes in cell number, subcellular concentrations of soluble carbohydrates, and osmotic potential in sepals during flower opening in Delphinium × belladonna cv. Bellamosum. The number of epidermal cells in the sepals did not increase from the stage when sepal pigmentation started, whereas the cell area increased during flower opening, indicating that petal growth during flower opening depends on cell expansion. Mannitol concentrations in the vacuole at three different stages were approximately 100 mM, which were much higher than the other carbohydrate concentrations, but they decreased slightly at open stage. In contrast, mannitol concentration in the cytoplasm was 56 mM at bud stage, but it increased to 104 mM at open stage. Glucose and fructose concentrations in the vacuole at open stage increased to 45 and 56 mM, respectively. Total osmotic potential in apoplast and symplast, which was partially due to soluble carbohydrates, was almost constant during flower opening. Therefore, mannitol may be acting constitutively as the main osmoticum in the vacuole where it may contribute to the maintenance of the osmotic balance between the cytoplasm and vacuole in open flowers. The role of mannitol differs from those of glucose, fructose, and sucrose in sepal cell expansion in Delphinium × belladonna.

Removal of Phenol by A. belladonna L. Hairy Root.

Phenolic compounds that present in the several industries are harmful and dangerous for human health. In this study we have studied the potential of Atropa belladonna hairy roots in phenol removal of wastewater. The optimal conditions for the removal process were evaluated using different phenol (10-500 mg.1(-1)) and H2O2 (1-15 Mm) concentrations. In the presence of H2O2, Roots were able to remove phenol concentrations up to 500 mg.1(-1). in the wide range of pH (4-9), reaching high removal efficiency. When roots were re-used for five consecutive cycles, phenol removal efficiency decreased from 98-62%, in the last cycle. After the removal process, the solutions were obtained from the experiment were estimated for their toxicity using a test with Lactaca sativa L. seeds. Results showed that the treated solution was less toxic than the parent solution.

[Expression pattern of genes involved in tropane alkaloids biosynthesis and tropane alkaloids accumulation in Atropa belladonna].

Atropa belladonna is a medicinal plant and main commercial source of tropane alkaloids (TAs) including scopolamine and hyoscyamine, which are anticholine drugs widely used clinically. Based on the high throughput transcriptome sequencing results, the digital expression patterns of UniGenes representing 9 structural genes (ODC, ADC, AIH, CPA, SPDS, PMT, CYP80F1, H6H, TRII) involved in TAs biosynthesis were constructed, and simultaneously expression analysis of 4 released genes in NCBI (PMT, CYP80F1, H6H, TRII) for verification was performed using qPCR, as well as the TAs contents detection in 8 different tissues. Digital expression patterns results suggested that the 4 genes including ODC, ADC, AIH and CPA involved in the upstream pathway of TAs, and the 2 branch pathway genes including SPDS and TRII were found to be expressed in all the detected tissues with high expression level in secondary root. While the 3 TAs-pathway-specific genes including PMT, CYP80F1, H6H were only expressed in secondary roots and primary roots, mainly in secondary roots. The qPCR detection results of PMT, CYP80F1 and H6H were consistent with the digital expression patterns, but their expression levels in primary root were too low to be detected. The highest content of hyoscyamine was found in tender stems (3.364 mg x g(-1)), followed by tender leaves (1.526 mg x g(-1)), roots (1.598 mg x g(-1)), young fruits (1.271 mg x g(-1)) and fruit sepals (1.413 mg x g(-1)). The highest content of scopolamine was detected in fruit sepals (1.003 mg x g(-1)), then followed by tender stems (0.600 mg x g(-1)) and tender leaves (0.601 mg x g(-1)). Both old stems and old leaves had the lowest content of hyoscyamine and scopolamine. The gene expression profile and TAs accumulation indicated that TAs in Atropa belladonna were mainly biosynthesized in secondary root, and then transported and deposited in tender aerial parts. Screening Atropa belladonna secondary root transcriptome database will facilitate unveiling the unknown enzymatic reactions and the mechanisms of transcriptional control.

Functional divergence of two arginine decarboxylase genes in tropane alkaloid biosynthesis and root growth in Atropa belladonna.

Putrescine, produced via the arginine decarboxylase (ADC)/ornithine decarboxylase (ODC)-mediated pathway, is an initial precursor for polyamines metabolism and the root-specific biosynthesis of medicinal tropane alkaloids (TAs). These alkaloids are widely used as muscarinic acetylcholine antagonists in clinics. Although the functions of ODC in biosynthesis of polyamines and TAs have been well investigated, the role of ADC is still poorly understood. In this study, enzyme inhibitor treatment showed that ADC was involved in the biosynthesis of putrescine-derived metabolites and root growth in Atropa belladonna. Further analysis found that there were six ADC unigenes in the A. belladonna transcriptome, with two of them, AbADC1 and AbADC2, exhibiting high expression in the roots. To investigate their roles in TAs/polyamines metabolism and root growth, RNA interference (RNAi) was used to suppress either AbADC1 or AbADC2 expression in A. belladonna hairy roots. Suppression of the AbADC1 expression resulted in a significant reduction in the putrescine content and hairy root biomass. However, it had no noticeable effect on the levels of N-methylputrescine and the TAs hyoscyamine, anisodamine, and scopolamine. On the other hand, suppression of AbADC2 expression markedly reduced the levels of putrescine, N-methylputrescine, and TAs, but had no significant effect on hairy root biomass. According to ß-glucuronidase (GUS) staining assays, AbADC1 was mainly expressed in the root elongation and division region while AbADC2 was mainly expressed in the cylinder of the root maturation region. These differences in expression led to functional divergence, with AbADC1 primarily regulating root growth and AbADC2 contributing to TA biosynthesis.

A novel bHLH gene responsive to low nitrogen positively regulates the biosynthesis of medicinal tropane alkaloids in Atropa belladonna.

Medicinal tropane alkaloids (TAs), including hyoscyamine, anisodamine and scopolamine, are essential anticholinergic drugs specifically produced in several solanaceous plants. Atropa belladonna is one of the most important medicinal plants that produces TAs. Therefore, it is necessary to cultivate new A. belladonna germplasm with the high content of TAs. Here, we found that the levels of TAs were elevated under low nitrogen (LN) condition, and identified a LN-responsive bHLH transcription factor (TF) of A. belladonna (named LNIR) regulating the biosynthesis of TAs. The expression level of LNIR was highest in secondary roots where TAs are synthesized specifically, and was significantly induced by LN. Further research revealed that LNIR directly activated the transcription of hyoscyamine 6ß-hydroxylase gene (H6H) by binding to its promoter, which converts hyoscyamine into anisodamine and subsequently epoxidizes anisodamine to form scopolamine. Overexpression of LNIR upregulated the expression levels of TA biosynthesis genes and consequently led to the increased production of TAs. In summary, we functionally identified a LN-responsive bHLH gene that facilitated the development of A. belladonna with high-yield TAs under the decreased usage of nitrogen fertilizer.

[Enhanced biosynthesis of scopolamine in transgenic Atropa belladonna by overexpression of h6h gene].

Transgenic Atropa belladonna with high levels of scopolamine was developed by metabolic engineering. A functional gene involved in the rate limiting enzyme of h6h involved in the biosynthetic pathway of scopolamine was over expressed in A. belladonna via Agrobacterium-mediation. The transgenic plants were culturing till fruiting through micropropogating and acclimating. The integration of the h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction (PCR) analysis. Analysis of the difference of plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight was carried out using SPSS software. The content of hyoscyamine and scopolamine in roots, stems, leaves and fruits was determined by HPLC. The investigation of the expression levels of Hnh6h by qPCR. Both Kan(r) and Hnh6h genes were detected in five transgenic lines of A. belladonna plants (A8, A11, A12, C8 and C19), but were not detected in the controls. The plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight of transgenic plants did not decrease by comparison with the non-transgenic ones, and furthermore some agronomic characters of transgenic plants were better than those of the controls. The highest level of scopolamine was found in leaves of transgenic A. belladonna, and the content of scopolamine was also higher than that of hyoscyamine in leaves. The contents of scopolamine of leaves in different transgenic lines were listed in order: C8 > A12 > C19 > A11 > A8, especially, the content of scopolamine in transgenic line C8 was 2.17 mg x g(-1) DW that was 4.2 folds of the non-transgenic ones (0.42 mg x g(-1) DW). The expression of transgenic Hnh6h was detected in all the transgenic plants but not in the control. The highest level of Hnh6h expression was found in transgenic leaves. Overexpression of Hnh6h is able to break the rate limiting steps involved in the downstream pathway of scopolamine biosynthesis, and thus promotes the metabolic flux flowing toward biosynthesis of scopolamine to improve the capacity of scopolamine biosynthesis in transgenic plants. As a result, transgenic plants of A. belladonna with higher level of scopolamine were developed.

Revealing evolution of tropane alkaloid biosynthesis by analyzing two genomes in the Solanaceae family.

Tropane alkaloids (TAs) are widely distributed in the Solanaceae, while some important medicinal tropane alkaloids (mTAs), such as hyoscyamine and scopolamine, are restricted to certain species/tribes in this family. Little is known about the genomic basis and evolution of TAs biosynthesis and specialization in the Solanaceae. Here, we present chromosome-level genomes of two representative mTAs-producing species: Atropa belladonna and Datura stramonium. Our results reveal that the two species employ a conserved biosynthetic pathway to produce mTAs despite being distantly related within the nightshade family. A conserved gene cluster combined with gene duplication underlies the wide distribution of TAs in this family. We also provide evidence that branching genes leading to mTAs likely have evolved in early ancestral Solanaceae species but have been lost in most of the lineages, with A. belladonna and D. stramonium being exceptions. Furthermore, we identify a cytochrome P450 that modifies hyoscyamine into norhyoscyamine. Our results provide a genomic basis for evolutionary insights into the biosynthesis of TAs in the Solanaceae and will be useful for biotechnological production of mTAs via synthetic biology approaches.

Enhancing the scopolamine production in transgenic plants of Atropa belladonna by overexpressing pmt and h6h genes.

Atropa belladonna is officially deemed as the commercial plant to produce scopolamine in China. In this study we report the simultaneous overexpression of two functional genes involved in biosynthesis of scopolamine, which encode the upstream key enzyme putrescine N-methyltransferase (PMT) and the downstream key enzyme hyoscyamine 6ß-hydroxylase (H6H), respectively, in transgenic herbal plants Atropa belladonna. Analysis of gene expression profile indicated that both pmt and h6h were expressed at a higher level in transgenic lines, which would be favorable for biosynthesis of scopolamine. High-performance liquid chromatography result suggested that transgenic lines could produce higher accumulation of scopolamine at different levels compared with wild-type lines. Scopolamine content increased to 7.3-fold in transgenic line D9 compared with control lines. This study not only confirms that co-overexpression of pmt and h6h is an ideal method to improve the biosynthetic capacity of scopolamine but also successfully cultivates the transgenic line D9, which significantly enhanced the scopolamine accumulation. Our research can serve as an alternative choice to provide scopolamine resources for relative industry, which is more competitive than conventional market.

Modification and translocation of Rac/Rop guanosine 5'-triphosphate-binding proteins of Scoparia dulcis in response to stimulation with methyl jasmonate.

Translocation of two Rac/Rop guanosine 5'-triphosphate-binding proteins from Scoparia dulcis, Sdrac-1 and Sdrac-2, was examined employing transformed belladonna which overproduces these proteins as glutathione-S-transferase-tagged forms. The transferase activities of the fused proteins in microsomal fraction of belladonna markedly increased by the incubation with methyl jasmonate either in Sdrac-1 or Sdrac-2 transformant, while low and constant activities were observed in the untreated control. Recombinant Sdrac-2 protein was found to bind to prenyl chain in the presence of cell extracts prepared from methyl jasmonate-treated S. dulcis, however, Sdrac-1 was palmitoylated by the addition of the cell extracts. These results suggest that both Sdrac-1 and Sdrac-2 translocate to plant membranes by the stimulation with methyl jasmonate, however, targeting of these proteins is triggered by the independent modification mechanisms, palmitoylation for Sdrac-1 and prenylation for Sdrac-2.

Differential production of tropane alkaloids in hairy roots and in vitro cultured two accessions of Atropa belladonna L. under nitrate treatments.

Plants are a potential source of a large number of valuable secondary metabolites. In vitro cultures are being considered as an alternative to agricultural processes for studying valuable secondary metabolites. In this way, nutritive factors are important parameters influencing the production of these compounds in plants. Effects of nitrate concentrations (KNO3) on the production of two tropane alkaloids, hyoscyamine and scopolamine, and the growth of aerial parts and roots of two in vitro propagated accessions of Atropa belladonna and hairy roots were investigated. As hairy roots cultures are able to keep a stable production of alkaloids over long periods of subculturing, they are considered as an interesting option for the study of alkaloid biosynthesis. A hairy roots culture of Atropa belladonna was established by transformation with Agrobacterium rhizogenes strain AR15834. The results of our study showed that a rise in KNO3 concentration caused a decline in hairy roots growth, and had a remarkable effect on the alkaloid content. The alkaloid concentrations obtained in the hairy roots were 3-20 times higher than that in the plants at 35 mM of KNO3. Increasing the nitrate concentration in the medium of hairy roots also improved the hyoscyamine/scopolamine ratio, while it increased the scopolamine/hyoscyamine ratio in the studied plants.

Growth of hairy-root cultures in various bioreactors for the production of secondary metabolites.

Agrobacterium rhizogenes-mediated hairy-root cultures are a very promising alternative to the biotechnological exploitation of plant cell cultures. Their characteristic capacity for secondary-metabolite production, inherent genetic stability reflected in stable productivity and the possibility of genetic manipulation to increase biosynthetic capacity have initiated a considerable interest, both as a fundamental research tool and as a source of valuable products. One of the most important limitations for the commercial exploitation of hairy roots is the development of technologies for large-scale culture. Though these roots have been grown in various bioreactors--stirred-tank, bubble-column, airlift or submerged, trickle-bed and nutrient mist--the question as to which of these alternatives can be successfully and economically scaled-up has yet to be definitively answered. This present minireview highlights various perspectives of hairy-root cultures, describes a comparative scale-up study and discusses various aspects of these cultures when grown in various bioreactors for the production of secondary metabolites.

[Genetic aspects of the interrelation between alkaloids and chemical elements in Atropa belladonna L. and Glaucium flavum Grantz. plants].

The variability of the contents of tropane and isoquinoline alkaloids, ashes, Na, K, Ca, Mg, Fe, Mn, Cu, Zn, Co, Mo, Cr, Al, Ba, V, Ni, Sr, Cd, Pb, J, and Ag was studied in individual plants of the industrial population of belladonna (Atropa belladonna L.) and yellow horned poppy (Glaucium flavum Crantz.). Numerous linear and nonlinear correlations of isoquinoline and tropane alkaloids with ashes and mineral elements were revealed by means of correlation and regression analyses. Alkaline earth elements (especially Sr and Ba) were shown to have a major role in the regulation of tropane alkaloid accumulation in belladonna leaves. K and Ni were of particular importance in the aerial part of yellow horned poppy. These elements at the suboptimal concentrations were most favorable for isoquinoline alkaloid accumulation in yellow horned poppy. Analytical mathematical models were derived for the regulation of alkaloid metabolism in test plants by some mineral elements (Ba, Mg, Al, Sr, Ni, Mn, and K). Our results indicate that the interrelation between alkaloids and elements in these plants is genetically determined.