Decoupling of Plant Growth and Accumulation of Biologically Active Compounds in Leaves, Roots, and Root Exudates of Hypericum perforatum L. by the Combination of Jasmonate and Far-Red Lighting.

The plant hormone jasmonic acid (JA) fine tunes the growth-defense dilemma by inhibiting plant growth and stimulating the accumulation of secondary compounds. We investigated the interactions between JA and phytochrome B signaling on growth and the accumulation of selected secondary metabolites in Hypericum perforatum L., a medically important plant, by spraying plants with methyl jasmonate (MeJA) and by adding far-red (FR) lighting. MeJA inhibited plant growth, decreased fructose concentration, and enhanced the accumulation of most secondary metabolites. FR enhanced plant growth and starch accumulation and did not decrease the accumulation of most secondary metabolites. MeJA and FR acted mostly independently with no observable interactions on plant growth or secondary metabolite levels. The accumulation of different compounds (e.g., hypericin, flavonols, flavan-3-ols, and phenolic acid) in shoots, roots, and root exudates showed different responses to the two treatments. These findings indicate that the relationship between growth and secondary compound accumulation is specific and depends on the classes of compounds and/or their organ location. The combined application of MeJA and FR enhanced the accumulation of most secondary compounds without compromising plant growth. Thus, the negative correlations between biomass and the content of secondary compounds predicted by the growth-defense dilemma were overcome.

Genome-wide identification and characterization of R2R3-MYB family in Hypericum perforatum under diverse abiotic stresses.

The R2R3-MYB family is one of the largest families of plant transcription factor playing significant roles in plant growth. Although this gene family has been studied in many species, the R2R3-MYBs in Hypericum perforatum which is the first sequenced species in Malpighiales have not been analyzed. A total of 109 R2R3-MYB genes were identified in H. perforatum and clustered into 36 clades. Gene Ontology analysis revealed that most of the R2R3-MYB genes were involved in biological processes. Four kinds of cis-acting elements were found within the promoter regions, the majority of which were related to the stress responses and plant growth/development. The transcriptome data of different tissues (root, stems, leaves, and flowers) showed that the spatial expression profiles of R2R3-MYBs were different. Also, real-time quantitative PCR analysis revealed that eleven stress-related R2R3-MYB genes showed specific expression patterns under diverse treatments. In addition, sub-cellular localization analysis indicated that five significant proteins HpMYB45, HpMYB48, HpMYB55, HpMYB63, and HpMYB70 were all localized in the nucleus. This study was the first report on identification and characterization of R2R3-MYB gene family in H. perforatum. It facilitated the identification of tissue-preferential and stress-related genes and provided deep insights into the function of R2R3-MYBs in H. perforatum.

Auxin driven indoleamine biosynthesis and the role of tryptophan as an inductive signal in Hypericum perforatum (L.).

In the 60 years since Skoog and Miller first reported the chemical redirection of plant growth the underlying biochemical mechanisms are still poorly understood, with one challenge being the capacity for applied growth regulators to act indirectly or be metabolized to active phytohormones. We hypothesized that tryptophan is metabolized to auxin, melatonin or serotonin inducing organogenesis in St. John's wort (Hypericum perforatum L.). Root explants from two germplasm lines of St. John's wort with altered melatonin metabolism and wildtype were incubated with auxin or tryptophan for 24, 48 or 72 h to induce regeneration. In wildtype, tryptophan had little effect on the indoleamine pathway, and was found to promote primary growth, suggesting excess tryptophan moved quickly through various secondary metabolite pathways and protein synthesis. In lines 4 and 112 tryptophan was associated with modified morphogenesis, indoleamine and auxin levels. Incubation with tryptophan increased shoot organogenesis while incubation with auxin led to root regeneration. The established paradigm of thought views tryptophan primarily as a precursor for auxin and indoleamines, among other metabolites, and mediation of auxin action by the indoleamines as a one-way interaction. We propose that these processes run in both directions with auxin modifying indoleamine biosynthesis and the melatonin:serotonin balance contributing to its effects on plant morphogenesis, and that tryptophan also functions as an inductive signal to mediate diverse phytochemical and morphogenetic pathways.

Melatonin and serotonin: Mediators in the symphony of plant morphogenesis.

Melatonin and serotonin are important signaling and stress mitigating molecules that play important roles across growth and development in plants. Despite many well-documented responses, a systematic investigation of the entire metabolic pathway (tryptophan, tryptamine, and N-acetylserotonin) does not exist, leaving many open questions. The objective of this study was to determine the responses of Hypericum perforatum (L.) to melatonin, serotonin, and their metabolic precursors. Two well-characterized germplasm lines (#4 and 112) created by mutation and a haploid breeding program were compared to wild type to identify specific responses. Germplasm line 4 has lower regenerative and photosynthetic capacity than either wild type or line 112, and there are documented significant differences in the chemistry and physiology of lines 4 and 112. Supplementation of the culture media with tryptophan, tryptamine, N-acetylserotonin, serotonin, or melatonin partially reversed the regenerative recalcitrance and growth impairment of the germplasm lines. Quantification of phytohormones revealed crosstalk between the indoleamines and related phytohormones including cytokinin, salicylic acid, and abscisic acid. We hypothesize that melatonin and serotonin function in coordination with their metabolites in a cascade of phytochemical responses including multiple pathways and phytohormone networks to direct morphogenesis and protect photosynthesis in H. perforatum.

Tamizaje fitoquímico y actividad antibacteriana in vitro de extractos y fracciones de tres especies colombianas del género Hypericum / Phytochemical screening and in vitro antibacterial activity of extracts and fractions from three Colombian species of the genus Hypericum

Introducción: el género Hypericum se caracteriza por poseer gran importancia etnobotánica en diferentes culturas a nivel mundial lo cual ha impulsado los estudios para el aislamiento e identificación de metabolitos bioactivos. Objetivo: realizar la caracterización fitoquímica preliminar y el estudio de actividad antibacteriana in vitro de extractos y fracciones de tres especies colombianas del género Hypericum. Métodos: a partir de las hojas secas de Hypericum myricariifolium Hieron, Hypericum mexicanum L. e Hypericum juniperinum Kunth se obtuvieron los extractos etanólicos, se realizó el tamizaje fitoquímico preliminar y se obtuvieron fracciones de polaridad creciente (hexano, cloroformo, acetato de etilo y metanol). Se evaluó la actividad antibacteriana in vitro de extractos y fracciones por el ensayo de difusión en agar frente a Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 1228) y Escherichia coli (ATCC 8739). A las fracciones y extractos que presentaron mayor inhibición del crecimiento bacteriano se les determinó la CMI por el método de dilución en micropozos (SPOTi). Resultados: el análisis fitoquímico preliminar mostró la posible presencia de esteroides, terpenos, fenoles, flavonoides y quinonas. Los extractos y fracciones de H. mexicanum y H. myricariifolium resultaron activos frente a todas las cepas con halos de inhibición superiores a 15 mm. El extracto de H. juniperinum presentó actividad frente la cepas gram-positivas. El extracto total y la fracción de hexano de H. mexicanum presentaron la mejor actividad frente a S. epidermidis con CMI de 0,05 mg/mL y 0,001 mg/mL respectivamente(AU) Conclusiones: la caracterización fitoquímica preliminar permitió determinar la presencia mayoritaria de metabolitos de tipo fenólico en los extractos de las 3 especies de estudio. Los extractos etanólicos y las fracciones de polaridad baja de estas especies poseen alta actividad inhibitoria frente a las bacterias gram-positivas (S. aureus y S. epidermidis) causantes de un gran número de infecciones cutáneas(AU)

Introduction: The genus Hypericum has been awarded great ethnobotanical importance by various cultures worldwide, which has given an impulse to the conduct of studies aimed at isolating and identifying its bioactive metabolites. Objective: Perform a preliminary phytochemical characterization and study the antibacterial activity in vitro of extracts and fractions from three Colombian species of the genus Hypericum. Methods: Ethanolic extracts were obtained from dry leaves of Hypericum myricariifolium Hieron, Hypericum mexicanum L. and Hypericum juniperinum Kunth. Preliminary phytochemical screening was performed and increasing polarity fractions were obtained (hexane, chloroform, ethyl acetate and methanol). In vitro antibacterial activity of the extracts and fractions was evaluated with the agar diffusion test against Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 1228) and Escherichia coli (ATCC 8739). The MIC was determined for fractions and extracts displaying greater bacterial growth inhibition, using the microwell dilution method (SPOTi). Results: Preliminary phytochemical analysis showed the possible presence of steroids, terpenes, phenols, flavonoids and quinones. Extracts and fractions of H. mexicanum and H. myricariifolium were active against all strains with inhibition haloes above 15 mm. The extract of H. juniperinum was active against gram-positive strains. The total extract and the hexane fraction of H. mexicanum displayed the best activity against S. epidermidis with an MIC of 0.05 mg/mL and 0.001 mg/mL, respectively. Conclusions: Preliminary phytochemical characterization revealed the prevailing presence of phenolic metabolites in extracts of the three study species. Ethanolic extracts and low polarity fractions of these species exhibit high inhibitory activity against gram-positive bacteria (S. aureus and S. epidermidis) responsible for a large number of skin infections(AU)

Acetic acid acts as an elicitor exerting a chitosan-like effect on xanthone biosynthesis in Hypericum perforatum L. root cultures.

KEY MESSAGE: Acetic acid acts as a signal molecule, strongly enhancing xanthone biosynthesis in Hypericum perforatum root cultures. This activity is specific, as demonstrated by the comparison with other short-chain monocarboxylic acids. We have recently demonstrated that Hypericum perforatum root cultures constitutively produce xanthones at higher levels than the root of the plant and that they respond to chitosan (CHIT) elicitation with a noteworthy increase in xanthone production. In the present study, CHIT was administered to H. perforatum root cultures using three different elicitation protocols, and the increase in xanthone production was evaluated. The best results (550 % xanthone increase) were obtained by subjecting the roots to a single elicitation with 200 mg l(-1) CHIT and maintaining the elicitor in the culture medium for 7 days. To discriminate the effect of CHIT from that of the solvent, control experiments were performed by administering AcOH alone at the same concentration used for CHIT solubilization. Unexpectedly, AcOH caused an increase in xanthone production comparable to that observed in response to CHIT. Feeding experiments with (13)C-labeled AcOH demonstrated that this compound was not incorporated into the xanthone skeleton. Other short-chain monocarboxylic acids (i.e., propionic and butyric acid) have little or no effect on the production of xanthones. These results indicate that AcOH acts as a specific signal molecule, able to greatly enhance xanthone biosynthesis in H. perforatum root cultures.

Lanthanum rather than cadmium induces oxidative stress and metabolite changes in Hypericum perforatum.

Physiology, oxidative stress and production of metabolites in Hypericum perforatum exposed to moderate Cd and/or La concentration (10 µM) were studied. La evoked increase in reactive oxygen species, malondialdehyde and proline but suppressed growth, tissue water content, glutathione, ascorbic acid and affected mineral nutrient contents more than Cd while the impact of Cd+La was not synergistic. Similar trend was observed at the level of superoxide dismutase gene expression. Shoot Cd amount increased in Cd+La while only root La increased in the same treatment. Extensive quantification of secondary metabolites revealed that La affected phenolic acids more pronouncedly than Cd in shoots and roots. Flavonols were suppressed by La that could contribute to the appearance of oxidative damage. Procyanidins increased in response to La in the shoots but decreased in the roots. Metabolic responses in Cd+La treatment resembled those of La treatment (almost identically in the roots). Phenylalanine ammonia-lyase activity was mainly suppressed by La. The presence of La also depleted amount of hypericin and expression of its putative gene (hyp-1) showed similar trend but accumulation of hyperforin increased under Cd or La excess. Clear differences in the stem and root anatomy in response to Cd or La were also found. Overall, H. perforatum is La-sensitive species and rather Cd ameliorated negative impact of La.

Hypericin biosynthesis in Hypericum hookerianum Wight and Arn: investigation on biochemical pathways using metabolite inhibitors and suppression subtractive hybridization.

The biochemical pathway to hypericin biosynthesis is presumed to be polyketide synthase (PKS) mediated, but it has not been experimentally validated, and no alternate route (chorismate/o-succinylbenzoate pathway) has been analyzed. We report here our earlier developed auxin inducible culture systems of Hypericum hookerianum as a model, to study the metabolic pathway to hypericin synthesis. Inhibitors of the alternate pathway at varying concentrations showed steady synthesis of total hypericins with means of 2.80±0.22, 18.75±0.01; 16.39±3.75, 29.60±1.90 (mevinolin) 2.53±0.10, 18.12±0.56; 0.14±0.01, 14.28±1.11 (fosmidomycin) and 2.7±0.35, 18.75±0.61; 0.14±0.01, 12.80±1.09 mg g(-1) DW (glyphosate) in the control and auxin-induced shoot and shoot-forming callus cultures, respectively. SSH analysis classified the differentially expressed sequences into protein synthesis (38%), modification (20%), electron transport (9%) and remaining as unclassified (11%) and unknown proteins (22%). Functional annotation of sequences indicates the presence of additional protein components besides PKS activity. Our results demonstrate direct biochemical and molecular evidence of PKS hypothesis of hypericin biosynthesis for the first time.

Effects of polysaccharide elicitors on secondary metabolite production and antioxidant response in Hypericum perforatum L. shoot cultures.

The effects of polysaccharide elicitors such as chitin, pectin, and dextran on the production of phenylpropanoids (phenolics and flavonoids) and naphtodianthrones (hypericin and pseudohypericin) in Hypericum perforatum shoot cultures were studied. Nonenzymatic antioxidant properties (NEAOP) and peroxidase (POD) activity were also observed in shoot extracts. The activities of phenylalanine ammonia lyase (PAL) and chalcone-flavanone isomerase (CHFI) were monitored to estimate channeling in phenylpropanoid/flavonoid pathways of elicited shoot cultures. A significant suppression of the production of total phenolics and flavonoids was observed in elicited shoots from day 14 to day 21 of postelicitation. This inhibition of phenylpropanoid production was probably due to the decrease in CHFI activity in elicited shoots. Pectin and dextran promoted accumulation of naphtodianthrones, particularly pseudohypericin, within 21 days of postelicitation. The enhanced accumulation of naphtodianthrones was positively correlated with an increase of PAL activity in elicited shoots. All tested elicitors induced NEAOP at day 7, while chitin and pectin showed increase in POD activity within the entire period of postelicitation. The POD activity was in significantly positive correlation with flavonoid and hypericin contents, suggesting a strong perturbation of the cell redox system and activation of defense responses in polysaccharide-elicited H. perforatum shoot cultures.

Bioassay-guided fractionation of extracts from Hypericum perforatum in vitro roots treated with carboxymethylchitosans and determination of antifungal activity against human fungal pathogens.

The aim of this study was to individuate, by bioassay-guided fractionation, promising antifungal fractions and/or constituents from Hypericum perforatum subsp. angustifolium in vitro roots. Treatments with chitosan, O-carboxymethylchitosan (CMC) and its derivatives were used to improve xanthone production in the roots. The bioassay-guided fractionation of CMC-treated roots led to the individuation of an ethyl acetate fraction, containing the highest amount of xanthones (6.8%) and showing the best antifungal activity with minimal inhibitory concentration (MIC) values of 53.82, 14.18, and 36.52 µg/ml, against Candida spp., Cryptococcus neoformans and dermatophytes, respectively. From this fraction the prenylated xanthone, biyouxanthone D has been isolated and represented the 44.59% of all xanthones detected. For the first time in the present paper biyouxanthone D has been found in H. perforatum roots and tested against C. neoformans, dermatophytes, and Candida species. The xanthone showed the greatest antifungal activity against C. neoformans and dermatophytes, with MIC values of 20.16, 22.63 µg/ml. In conclusion, the results obtained in the present study demonstrated that CMC-treated Hpa in vitro root extracts represent a tool for the obtainment of promising candidates for further pharmacological and clinical studies.

PAL inhibitor evokes different responses in two Hypericum species.

Accumulation of secondary metabolites (general phenols, naphthodianthrones and phloroglucinol hyperforin) in Hypericum perforatum and Hypericum canariense after application of the inhibitor (2-aminoindane-2-phosphonic acid, AIP) of the pivotal enzyme of general phenylpropanoid pathway (phenylalanine ammonia-lyase, PAL) was studied. Shoots of H. perforatum revealed more expressive growth depression, concomitantly with the inhibition of PAL activity (-60%) and decrease in soluble phenols and individual phenolic acids in response to AIP. Hypericins (hypericin, pseudohypericin and protohypericin) decreased while hyperforin increased in AIP-cultured H. perforatum. On the contrary, growth changes, decreases in soluble phenols and individual phenolic acids were less-visible in H. canariense. This was also reflected in restoration of PAL activity (+330%) and selected flavonoids even increased. Hypericins and hyperforin were present in several orders of magnitude lower amounts in comparison with H. perforatum. Increase in proline indicates potential compensatory antioxidative mechanism if phenols are depleted. Microscopy revealed also differences in secondary xylem formation and lignification between species after exposure to AIP.

[Preliminary study on cultivation of adventitious roots of Hypericum perforatum in bioreactors].

OBJECTIVE: To cultivate adventitious roots of Hypericum perforatum in bioreactors, in order to seek for suitable conditions for adventitious growth. METHOD: The effect of IBA concentration, sugar type and concentration, inoculum volume and air volume of adventitious roots on the cultivation of adventitious roots of H. perforatum was observed in a 5 L air-lift bioreactor. RESULT: Adventitious roots of H. perforatum were cultivated in a MS culture dish. With the increase of IBA concentration, the propagation coefficient of adventitious roots of H. perforatum was on the rise. The IBA concentration ranging between 1.25-1.75 mg x L(-1) was suitable for the growth of adventitious roots. Adventitious roots grew best with sucrose in MS medium, with the propagation coefficient up to 22.15. When sucrose concentration was 30 g x L(-1), fresh weight, dry weight and propagation coefficient reached the maximum value. An adventitious root reactor with an inoculum volume of 20 g was favorable for the growth of adventitious roots. The air volume of reactors of 0.075 vvm (air volume/culture volume per minute) was favorable for the growth of adventitious roots, with the significant increase in the propagation coefficient of adventitious roots. In the amplification experiment, we found that the cultivation conditions of adventitious roots in a 5 L bioreactor was completely applicable to that in 10 and 20 L bioreactors, and adventitious roots grew well in a large bioreactor. CONCLUSION: IBA concentration, sugar type and concentration, inoculum volume and air volume had a significant effect on the growth of adventitious roots.

Chlorogenic acid participates in the regulation of shoot, root and root hair development in Hypericum perforatum.

Chlorogenic acid (CGA), a product of the phenylpropanoid pathway, is one of the most widespread soluble phenolic compounds in the plant kingdom. Although CGA is known to have important roles in plant function, its relevance in plant de novo organogenesis is not yet understood. With a series of experiments, here we show that CGA has a potential role in shoot, root and root hair development. In the first phase of our investigation, we developed an efficient and novel thin cell layer (TCL) regeneration protocol for Hypericum perforatum which could bridge all the in vitro morphogenetic stages between single cell and complete plant. Tissues at different morphogenetic states were analysed for their phenolic profile which revealed that shoot differentiation from callus tissues of H. perforatum is accompanied by the onset of CGA production. Further, the relevance of CGA in de novo organogenesis was deciphered by culturing highly organogenic root explants on media augmented with various concentrations of CGA. Results of this experiment showed that CGA concentrations lower than 10.0 mg l⁻¹ did not affect shoot organogenesis, whereas, higher concentrations significantly reduced this process in a concentration-dependent manner. In spite of the differential concentration-dependent effects of CGA on shoot regeneration, supplementation of CGA did not have any effect on the production of lateral roots and root hairs. Interestingly, CGA showed a concentration-dependent positive correlation with lateral roots and root hairs production in the presence of α-naphthaleneacetic acid (NAA). When the culture medium was augmented with 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia lyase (PAL), induction of shoots, lateral roots and root hairs from the explants was significantly affected. Addition of an optimum concentration of CGA in these cultures partially restored all these organogenic processes.

Enhancing hypericin production of Hypericum perforatum cell suspension culture by ozone exposure.

Accumulation of secondary metabolites is one of the common reactions of plants to ozone exposure in nature. To investigate the effect of ozone on the production of desired compounds of plant cell cultures, we assayed hypericin production of Hypericum perforatum suspension cell cultures treated with different doses of ozone at different culture phases. The results show that hypericin contents of the cells treated with 60 to 180 nL L(-1) ozone are significantly higher than those of the control, showing that ozone exposure may stimulate hypericin synthesis. Hypericin production of the cells treated with ozone at exponential phase is higher than that of lag and stationary phase, which suggests that exponential phase cell cultures are more responsive to ozone exposure than lag and stationary phase cells. The highest hypericin production is obtained by the cells exposed to 90 nL L(-1) ozone at late exponential phase for 3 h, being about fourfold of the control.

Multi-targeted natural products evaluation based on biological activity prediction with PASS.

Natural products found a wide use in folk medicine. Presently, when routine development of new drugs faced a considerable challenge, they become an inspiration and valuable source in drugs discovery. Rather complex and diverse chemical structures of natural compounds provide a basis for modulation of different biological targets. Natural compounds exhibit a multitargeted action that may lead to additive/synergistic or antagonistic effects. Rational design of more safe and potent pharmaceuticals requires an estimation of probable multiple actions of natural products. Our software PASS can perform such estimation. It predicts with reasonable accuracy over 3500 pharmacotherapeutic effects, mechanisms of action, interaction with the metabolic system, and specific toxicity for drug-like molecules on the basis of their structural formulae. We analyzed PASS predictions utilizing PharmaExpert, which performs selection of compounds with multiple mechanisms of action, analysis of activity-activity relationships and drug-drug interactions. The paper describes an application of PASS and PharmaExpert to the evaluation of biological activity of natural compounds including marine sponge alkaloids, triterpenoids of lupane group, and their derivatives. Proposed computer-aided methods can generate combinatorial libraries of macrolides. They help to select the most promising pharmaceutical leads with the required properties. Case study, based on the analysis of biological activity spectra predicted for St John's Wort constituents, clearly demonstrates capabilities of computational methods in the evaluation of multitargeted actions, additive/synergistic and/or antagonistic effects of natural products.

Chitosan enhances xanthone production in Hypericum perforatum subsp. angustifolium cell cultures.

Hypericum perforatum is an important medicinal plant containing numerous biologically active compounds. The effect of chitosan elicitation on xanthone biosynthesis in calli and in cell suspension cultures of H. perforatum subsp. angustifolium was evaluated. Elicited cell cultures showed an increase in xanthone production and a simultaneous decrease in flavonoid production. Chitosan also induced the production of 1,7-dihydroxyxanthone (euxanthone) and cadensin G, which were not detected in either the calli nor the non-elicited cell cultures. 1,7-Dihydroxyxanthone was in part (21%) released in the culture medium.

Production of adventitious roots and secondary metabolites by Hypericum perforatum L. in a bioreactor.

We investigated the effects of different concentrations of auxin and auxin/cytokinin combinations, inoculum sizes and Murashige and Skoog (MS) medium dilutions on biomass and accumulation of total phenols and flavonoids in adventitious roots of Hypericum perforatum. The optimized conditions for the shake flask culture growth and secondary metabolites production were 0.1mg/l kinetin with 1mg/l indole-3-butyric acid (IBA), an inoculum density of 6g/l fresh weight (FW) and 1/2MS in the 5-week old cultures. By inoculating 6g/l of adventitious roots into a 3-l balloon type bubble bioreactor (BTBB) containing l/2 MS medium supplemented with 0.1mg/l kinetin with 1mg/l IBA, 104.2g/l adventitious roots were harvested containing 56.47+/-0.22 mg/g dry weight (DW) total phenolics, 35.01+/-1.26 mg/g DW flavonoids, 0.97+/-0.06 mg/g DW chlorogenic acid and 1.389+/-0.80 mg/g DW hypericin. The results of our study contribute to optimization and development of bioreactor technology for adventitious root cultures of H. perforatum for the production of hypericin.

Effects of different concentrations of benzylaminopurine on shoot regeneration and hypericin content in Hypericum triquetrifolium Turra.

The current study was undertaken to determine the effects of different benzylaminopurine (BAP) concentrations on the accumulation of bioactive hypericin in Hypericum triquetrifolium Turra. via micropropagation. To achieve this objective, seeds of H. triquetrifolium Turra. were cultured on Murashige and Skoog (MS) medium supplemented with a BAP (0.5, 1.0 and 2.0), 3% sucrose and 5.5% agar. Apical tips of axenic germinated seeds were proliferated on a MS medium supplemented with BAP (0.0, 0.5, 1.0 and 2.0 mg L(-1)). The highest shoot number was obtained from a MS medium supplemented with a 2.0 mg L(-1) BAP. Hypericin percentages were found to be highest in a 1.0 mg L(-1) BAP supplemented medium. These results provide the indication that cytokinin BAP can change the chemical composition of H. triquetrifolium Turra.; thereby, seriously impacting the quality and the efficacy of natural plant products produced by an in vitro culture system for aseptic production of hypericin.

[The influence of some elicitors on growth and morphogenesis of Hypericum perforatum callus cultures].

We studied the effects of elicitors, such as mannan, beta-1,3-glucan, ancymidol, and cork crumbs, on morphogenetic and biosynthetic potencies of shoot cultures of Hypericum perforatum L. In the presence of these elicitors, different morphogenetic structures of H. perforatum callus cultures were formed. A correlation was found between the morphogenetic processes and induction of hypericin and pseudohypericin biosynthesis in the callus cultures.

Induction of hypericins in Hypericum perforatum in response to chromium.

Seedlings of Hypericum perforatum were grown with 0.01 and 0.1 mM of chromium added to the nutrient media. A treatment with 0.01 mM Cr(VI) for seven days resulted in an increased production of protopseudohypericin (+135%), hypericin (+38%) and pseudohypericin (+5%). Treatment with 0.1 mM Cr(VI) for two days also caused an increase of protopseudohypericin (+167%), hypericin (25%) and pseudohypericin (+5%). The greatest effect of chromium treatment was observed at a concentration of 0.1 mM for seven days: protopseudohypericin increased +404% and pseudohypericin to +379%. Hypericin was not affected by this treatment.