Involvement of NADPH oxidase-mediated H<Subscript>2</Subscript>O<Subscript>2</Subscript> signaling in PB90-induced hypericin accumulation in <Emphasis Type="Italic">Hypericum perforatum</Emphasis> cells

PB90 is a novel protein elicitor isolated from Phytophthora boehmeriae. Here, we report that treatment of PB90 stimulates hypericin production and hydrogen peroxide (H₂O₂) generation in Hypericum perforatum L. cells and demonstrate that H₂O₂ is essential for PB90-induced hypericin production. To further study the source of PB90-triggered H₂O₂, we have investigated activities of plasma membrane NADPH oxidase in Hypericum perforatum L. cells subjected to PB90 treatment. It is revealed that treatment of the cells with PB90 significantly increases NADPH oxidase activity. NADPH oxidase inhibitors suppress not only the PB90-stimulated NADPH oxidase activity but also the PB90-triggered H₂O₂ generation and PB90-induced hypericin production, showing that NADPH oxidase is involved in PB90-triggered H₂O₂ generation and hypericin production. Moreover, the suppression of NADPH oxidase inhibitors on PB90-induced hypericin production can be reversed by H₂O₂, although H₂O₂ per se has no effects on hypericin production of the cells. Together, the data demonstrate that PB90 may induce hypericin production of H. perforatum cells through the NADPH oxidase-mediated H₂O₂ signaling pathway.     

Activity of antioxidant enzyme during <Emphasis Type="Italic">in vitro</Emphasis> organogenesis in <Emphasis Type="Italic">Crocus sativus</Emphasis>

The effect of various hormonal combinations on regeneration of shoots and roots from meristem-derived callus of Crocus sativus L. and activities of antioxidant enzymes have been studied. The most efficient regeneration occurred with 1.0 mg dm⁻³ 1-naphthaleneacetic acid (NAA) + 1.0 mg dm⁻³ thidiazuron and 1.0 mg dm⁻³ NAA + 2.0 mg dm⁻³ kinetin. For sprouting, regenerated shoot were subcultured on Murashige and Skoog medium containing 1.0 mg dm⁻³ NAA + 1.0 mg dm⁻³ benzylaminopurine (BAP). Protein content and superoxide dismutase activity decreased in regenerated shoots and roots and increased in sprouting shoots, while catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) activities increased during organogenesis and decreased in sprouting shoots. High CAT and PPO activities were detected in regenerated roots, whereas high POX activity was observed in regenerated shoot.     

Cryopreservation of embryogenic cell suspensions of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> L. (G) Don.

The present study describes the potential of in vitro grown adventitious roots of Hypericum perforatum L. commonly known as St. John’s wort at low nutrient and auxin levels in the liquid medium for micropropagation. Roots were regenerated from shoot-derived callus on MS medium containing 4.0 mg l⁻¹ Indole-3 acetic acid (IAA). IAA and Indole-3 butyric acid (IBA) were equally effective for the induction of roots from shoot cultures. Half strength MS medium containing 1.0 mg l⁻¹ IAA was most found suitable for culturing roots in liquid medium. A total biomass of 4.13 ± 0.67 g comprising 226 ± 34.4 shoots and shoot buds along with roots was obtained per culture starting with 200 mg roots inoculum. Pretreatment with kinetin (2.0 mg l⁻¹) enhanced the shoot multiplication. Shoots proliferated profusely from excised roots in static liquid medium supported with glass bead matrix. Growtek^™ vessel was found suitable and cost effective system for high throughput plantlet production. In vitro grown roots regardless of their source of origin were an excellent and easy to handle source of explant for aseptic production of plantlets without loosing the morphogenetic potential over the generations. The plants exhibited 84–99% similarity among themselves through RAPD. The in vitro shoots produced can either be multiplied or rooted perpetually, and alternatively they can also be explored for the in vitro production of hypericin and hyperforin.     

Effects of plant growth regulators and elicitors on production of secondary metabolites in shoot cultures of <Emphasis Type="Italic">Hypericum hirsutum</Emphasis> and <Emphasis Type="Italic">Hypericum maculatum</Emphasis>

We investigated the effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 6-γ,γ-dimethylallylaminopurine (2iP), thidiazuron (TDZ) and α-naphthaleneacetic acid (NAA)], modified Murashige and Skoog (MS) medium containing 10 mM NH₄ ⁺ and 5 mM NO₃ ⁻ and supplemented with 2iP, BA, Kin and NAA (MSM medium), and two elicitors [jasmonic acid (JA), and salicylic acid (SA)], on plant growth and accumulation of hypericins (hypericin and pseudohypericin) and hyperforin in shoot cultures of Hypericum hirsutum and H. maculatum. Our data suggested that culture of shoots on MS medium supplemented with BA (0.4 mg l⁻¹) or Kin (0.4 mg l⁻¹) enhanced production of hypericins in H. maculatum and hyperforin in H. hirsutum. Hypericins and hyperforin concentrations decreased in both species when TDZ (0.4 mg l⁻¹) was added to the MS medium. Also, TDZ induced hyperhydric malformations and necrosis of regenerated shoots. Cultivation of H. maculatum on MSM medium resulted in approximately twofold increased production of hypericins compared to controls, and the growth of H. hirsutum shoots on the same medium led to a 6.16-fold increase in hyperforin production. Of the two elicitors, SA was more effective in stimulating the accumulation of hypericins. At 50 μM, SA enhanced the production of hypericin (7.98-fold) and pseudohypericin (13.58-fold) in H. hirsutum, and, at 200 μM, enhanced the production of hypericin (2.2-fold) and pseudohypericin (3.94-fold) in H. maculatum.     

Expression of a KDEL-tagged dengue virus protein in cell suspension cultures of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> and <Emphasis Type="Italic">Morinda citrifolia</Emphasis>

Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined after 28 days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1 mg/l kinetin, where the regeneration frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25 mg/l induce callus and shoot formation at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin.     

Sucrose-induced osmotic stress affects biomass,metabolite, and antioxidant levels in root suspension cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L.

In this study, we investigated the influence of initial sucrose concentration on the accumulation of biomass, phenols, flavonoids, chlorogenic acid, and hypericin in adventitious root cultures of Hypericum perforatum L. Cultures were initiated in shake flasks by using half-strength Murashige and Skoog (MS) medium, 1.0 mg l⁻¹ indolebutyric acid (IBA), 0.1 m g l⁻¹ kinetin, and different concentrations 0, 1, 3, 5, 7, or 9% in w/v) of sucrose and were maintained in darkness. The medium supplemented with 3% (w/v) sucrose resulted in the optimum biomass accumulation, but higher sucrose concentrations (5, 7, and 9%) inhibited biomass accumulation due to the relatively higher osmotic pressure. However, the amount of total phenols, flavonoids, chlorogenic acid, and total hypericin was increased with the roots grown in the medium supplemented with 5, 7, and 9% (w/v) sucrose. The antioxidant potential of methanolic extract [1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid; ABTS) radical scavenging activities] of H. perforatum adventitious roots was also assessed and correlated with the metabolite accumulation. Cultures maintained with higher initial sucrose concentration (5, 7, and 9% w/v) showed increased accumulation of phenols, flavonoids, chlorogenic acid, and total hypericin, and this might be due to the osmotic stress at elevated sucrose concentrations. To verify the effect of osmotic stress on lipid peroxidation, the levels of hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and proline were determined in the adventitious roots and the results revealed a marked increase in the concentrations of these compounds. These results suggest that optimal adventitious root biomass could be achieved in the MS medium with 3% (w/v) sucrose and increased sucrose concentration resulted in osmotic stress and, in turn, induces the accumulation of secondary metabolites.     

Effects of cytokinins and elicitors on the production of hypericins and hyperforin metabolites in Hypericum sampsonii and Hypericum perforatum

Hypericum perforatum L. (St. John’s wort) and Hypericum sampsonii Hance are medicinal plants used in China in the treatment of viruses and other disorders. In the current study, we investigated the effects of cytokinins 6-benzylaminopurin (BA), zeatin (ZT) and thidiazuron (TDZ) on plant growth and production of hypericins (pseudohypericin and hypericin) and hyperforin. Our data suggested that culture of H. perforatum in modified MS (Murashige and Skoog) medium, with a 50% reduction in ammonium nitrate and potassium nitrate, and supplemented with BA (0.44 μM) and indolebutyric acid (IBA, 0.049 μM), resulted in increased production of hypericins. Similar results were noted with H. sampsonii with minor changes to the medium (0.46 μM ZT and 0.049 μM IBA). There were approximately 2.95-, 2.62-fold increases in H. perforatum pseudohypericin and hypericin production by TDZ (0.45 μM) induction compared to the controls. No enhancement of hypericins and hyperforin production was elicited by TDZ in H. sampsonii. The elicitor methyl jasmonate (MJA, 50 μM) and its analog, 2,3-dihydroxypropyl jasmonate (DHPJA, 50 μM), were also used in H. perforatum and H. sampsonii shoot culture to increase secondary metabolite production, eliciting an increase in the production of hypericins and hyperforin. While leaf senescence and biomass inhibition were observed in cultures induced by MJA, no such effects were observed with DHPJA.     

Shoot regeneration and free-radical scavenging activity in <Emphasis Type="Italic">Silybum marianum</Emphasis> L.

The morphogenic potential and free-radical scavenging activity of the medicinal plant, Silybum marianum L. (milk thistle) were investigated. Callus development and shoot organogenesis were induced from leaf explants of wild-grown plants incubated on media supplemented with different plant growth regulators (PGRs). The highest frequency of callus induction was observed on explants incubated on Murashige and Skoog (MS) medium supplemented with 5.0 mg l⁻¹ 6-benzyladenine (BA) after 20 days of culture. Subsequent transfer of callogenic explants onto MS medium supplemented with 2.0 mg l⁻¹ gibberellic acid (GA₃) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) resulted in 25.5 ± 2.0 shoots per culture flask after 30 days following culture. Moreover, when shoots were transferred to an elongation medium, the longest shoots were observed on MS medium supplemented with 0.5 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA, and these shoots were rooted on a PGR-free MS basal medium. Assay of antioxidant activity of in vitro and in vivo grown tissues revealed that significantly higher antioxidant activity was observed in callus than all other regenerated tissues and wild-grown plants.     

Multiple shoot induction and plant regeneration of the endangered species <Emphasis Type="Italic">Crepis novoana</Emphasis>

In vitro culture is a useful tool in the ex situ conservation of rare, endemic, and threatened plant species. Crepis novoana (Compositae) is an endangered endemic in northwestern Spain. Use of in vitro culture tools is necessary due to the poor conservation status of populations of the species. The systems of in vitro propagation developed for this species in the present study were caulogenesis from leaf explants and growth of axillary buds from shoots. Explants were produced by placing fragments of leaves on Murashige and Skoog medium (MS) supplemented with 2.22 μM 6-benzyladenine (BA) and 2.69 μM naphthaleneacetic acid (NAA); caulogenesis was induced in 80% of explants, with development of a mean number of 2.48 shoots per explant. Axillary bud development from shoots was highest with MS supplemented with 4.44 μM BA and 0.54 μM NAA, resulting in production of a mean number of 49.77 shoots per explant. Immersion of the basal side of shoots in a solution of 5.37 mM NAA for 30 s yielded 90% success in the production of rooted shoots. Plantlets were well acclimatized, and almost 100% of plants transferred to soil recovered successfully.     

An efficient in vitro plantlet regeneration of <Emphasis Type="Italic">Cryptocoryne wendtii</Emphasis> and <Emphasis Type="Italic">Cryptocoryne becketti</Emphasis> through shoot tip culture

An efficient micropropagation protocol was established for Cryptocoryne wendtii and Cryptocoryne becketti using shoot tips explants. Multiple shoots were induced from shoot tip explants of both species cultured on agar-gelled as well as liquid MS medium supplemented with 0.5 mg/L BA and 0.2 mg/L IBA (proliferation medium). The multiple shoots of both the species formed on agar-gelled as well as liquid medium were vigorously growing with well-developed roots and leaves after 4 weeks of culture. Highest number of multiple shoots was obtained from shoot tip explants of both the species cultured in liquid proliferation medium after 4 weeks of culture. The shoot tip explants of C. wendtii and C. becketti, that were cultured in liquid proliferation medium (2 weeks) followed by culturing on agar-gelled proliferation medium (2 weeks) also produced the multiple shoots. Shoot tips cultured on agar-gelled medium produced the least number of multiple shoots after 4 weeks of culture. Histological study did not show any abnormalities in the leaves of in vitro plantlets of both the species, cultured in agar-gelled and liquid proliferation medium. The leaves of the in vitro plantlets formed normal mesophyll cells and vascular bundles. More than 95% of the acclimatized plantlets grew vigorously without any morphological abnormalities.     

Variation in the contents of pseudohypericin and hypericin in Hypericum perforatum from Lithuania

Hypericin and hypericin-like substances are considered the main active compounds in Hypericum perforatum L. (Hypericaceae). In this work pseudohypericin and hypericin of H. perforatum collected in Lithuania were quantified. Studies on accumulation dynamics and between-accession variation of the contents of these secondary metabolites were carried out by high performance liquid chromatography (HPLC). The data were statistically processed with ANOVA and PCA. Significant difference between pseudohypericin and hypericin content in floral budding and full flowering stages was detected. The highest amounts of the secondary metabolites were observed in the flowering stage. The study revealed evident within population variations in H. perforatum. Mean concentrations of pseudohypericin and hypericin among accessions varied from 3.45 to 6.82 mg/g and from 1.17 to 2.59 mg/g, respectively. Accessions of H. perforatum showed remarkable differences in chemical composition depending on the provenance of plants.     

A rapid in vitro propagation of red sanders (<Emphasis Type="Italic">Pterocarpus santalinus</Emphasis> L.) using shoot tip explants

An efficient protocol has been developed for the in vitro propagation of Pterocarpus santalinus L. using shoot tip explants which is a valuable woody medicinal plant. Various parts of this plant are pharmaceutically used for the treatment of different diseases. Multiple shoots were induced from shoot tip explants derived from 20 days old in vivo germinated seedlings on 1:1 ratio of sand and soil after treating with gibberellic acid (GA₃). The highest frequency for shoot regeneration (83.3%) with maximum number of shoot buds (11) per explant was obtained on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l of 6-benzylaminopurine (BAP) along with 0.1 mg/l of thidiazuron (TDZ) after 45 days of culture. A proliferating shoot culture was established by repeatedly subculturing the original shoot tip explants on fresh medium after each harvest of the newly formed shoots. Sixty percent of the shoots produced roots were transferred to rooting medium containing MS salts and 0.1 mg/l indole-3-butyric acid (IBA) after 30 days. About 73.33% of the in vitro raised plantlets were established successfully in earthen pots. Random amplified polymorphic DNA (RAPD)-based DNA fingerprinting profiles were generated for the first time using shoot tip explants of this species and confirmed that there was no genetic variability. This protocol might be helpful for the mass multiplication of P. santalinus in the future.     

Defence‐Related Enzymes in Soybean Resistance to Target Spot

Target spot, caused by the fungus Corynespora cassiicola, has become a serious foliar disease in soybean production in the Brazilian Cerrado. Information in the literature regarding the biochemical defence responses of soybean to C. cassiicola infection is rare. Therefore, the objective of this study was to determine the biochemical features associated with soybean resistance to target spot. The activities of chitinases (CHI), β‐1‐3‐glucanases (GLU), phenylalanine ammonia‐lyases (PAL), peroxidases (POX), polyphenol oxidases (PPO) and lipoxygenases (LOX), as well as the concentrations of total soluble phenolics (TSP) and lignin‐thioglycolic acid (LTGA) derivatives, were determined in soybean leaves from both a resistant (FUNDACEP 59) and a susceptible (TMG 132) cultivar. The target spot severity, number of lesions per cm² of leaflet and area under the disease progress curve were significantly lower for plants from cv. FUNDACEP 59 compared to plants from cv. TMG 132. The GLU, CHI, PAL, POX and PPO activities and the concentration of LTGA derivatives increased significantly, whereas LOX activity decreased significantly on the leaves infected by C. cassiicola. Inoculated plants from cv. FUNDACEP 59 showed a higher PPO activity and concentrations of TSP and LTGA derivatives at 4 and 6 days after inoculation compared to plants from cv. TMG 132. In conclusion, the results of this study demonstrated that the defence‐related enzyme activities increased upon C. cassiicola infection, regardless of the basal level of resistance of the cultivar studied. The increases in PPO activity and concentrations of TSP and LTGA derivatives, but lower LOX activity, at early stages of C. cassiicola infection were highly associated with soybean resistance to target spot.     

Shoot induction and regeneration using internodal transverse thin cell layer culture in <Emphasis Type="Italic">Sesamum indicum</Emphasis> L.

An efficient protocol for shoot regeneration was developed for sesame (Sesamum indicum L.) internodes using the transverse thin cell layer (tTCL) culture method. The frequency of shoot regeneration and the number of adventitious buds produced from regenerated shoots depend significantly on explant age, thickness of the tTCL sections, and the phytohormones supplemented to the culture medium. A combination of 6-benzyladenine (2.0 mg l⁻¹) and α-naphthaleneacetic acid (0.5 mg l⁻¹) was found to be the best phytohormone combination for shoot bud induction, with the maximum number of shoots obtained when the tTCL sections were 0.5–1.0 mm thick and derived from 4- to 6-week-old seedlings of sesame. Well-developed shoots were rooted on MS medium without phytohormones, and 80% of the regenerated plantlets were successfully established in soil.     

Biochemical responses associated with resistance to bacterial stalk rot caused by Dickeya zeae in maize

Bacterial stalk rot (BSR) of maize caused by Dickeya zeae is an important disease in northwest region of India. In the current study, eighty maize lines were evaluated for resistance against this disease. Of these, 20 were moderately resistant, 25 were moderately susceptible and the rest were highly susceptible to BSR. Six lines from each set were randomly selected. Activities of three antioxidant enzymes, viz. phenylalanine ammonia lyase (PAL), peroxidase (POX) and polyphenol oxidase (PPO) were analysed from these three sets of maize lines representing different levels of resistance. A trend of elevated activity of PAL, POX and PPO was observed in all the three sets of maize lines. The results showed significantly more activity of these three enzymes in moderately resistant than highly susceptible maize lines. The activity of PAL and PPO peaked after 48 hr and of POX after 72 hr of challenge inoculation by D. zeae in all the maize lines. The activity of these enzymes further correlated negatively with disease development. Our results show that PAL, POX and PPO play an important role in contributing towards resistance in maize against BSR.     

The Effect of Cork Pieces on Pseudohypericin Production in Cells ofHypericum perforatumShoots

The stimulating effect of cork pieces on hypericin and pseudohypericin biosyntheses was studied in cells of shoots regenerated from the callus cultures of St. John's wort (Hypericum perforatumL.). The addition of the cork matrix slightly stimulated shoot growth and enhanced pseudohypericin biosynthesis about threefold (to 0.4 mg/g dry wt). Pseudohypericin production increased proportionally with the amount of cork material added (from 1 to 4 mg/ml of growth medium). Further increase in the amount of cork pieces inhibited both pseudohypericin production and shoot growth. Organic and aqueous extracts of cork pieces did not affect the production of these substances.     

In vitro plant regeneration from seedling explants of Stereospermum personatum D.C.: a medicinal tree

Padar (Stereospermum personatum, family Bignoniaceae) is a well-known medicinal tree. Its complete regeneration occurred through shoot bud culture in vitro. The seeds germinated sequentially on plastic trays and polyethylene bags for 21 days served as explants source. Nodal segments from the seedlings were established on MS medium supplemented with 4.44 μM BA, in which 86.6% nodes showed shoot bud elongation. Then, nodal segments from the developed shoots were cultured on MS medium with several BA concentrations; best shoot multiplication was obtained with 0.44 μM BA. In a second experiment where PVP was added to proliferation medium, nodal segments from developed shoots produced maximum 2.78 shoots per node. The nodal segments showed shoot multiplication up to seventh subculture on. Finally, shoots were rooted on MS medium with 2.46 μM IBA. The plants transferred to net pots containing coco-peat were acclimatized in green house, where more than 80% plants survived and grew normally.     

Efficient regeneration and antioxidant potential in regenerated tissues of <Emphasis Type="Italic">Piper nigrum</Emphasis> L.

The organogenic potential and antioxidant potential (1, 1-diphenyl-2-picrylhydrazyl-scavenging activity) of the medicinal plant Piper nigrum L. (black pepper) were investigated. Callus induction and shoot regeneration were induced from leaf explants of potted plants cultured on MS medium supplemented with different plant growth regulators. The best callogenic response was observed on explants cultured for 30 days on MS medium supplemented with either 0.5 or 1.5 mg l⁻¹ 6-benzyladenine (BA) + 1.0 mg l⁻¹ α-naphthaleneacetic acid. Subsequent transfer of the callogenic explants onto MS medium supplemented with 1.5 mg l⁻¹ BA + 1.0 mg l⁻¹ gibberellic acid (GA₃) achieved 85% shoot organogenesis after 30 days of culture. The maximum number (7.2) of shoots/explant was recorded for explants cultured in MS medium supplemented with 1.0 mg l⁻¹ BA. Following the transfer of shoots to an elongation medium, the longest shoots (5.4 cm) were observed on MS medium supplemented with 1.0 mg l⁻¹ BA + 1.0 mg l⁻¹ GA₃. The elongated shoots were rooted on MS medium supplemented with different concentrations of indole butyric acid. An assay of the antioxidant potential of the in vitro-grown tissues revealed that the antioxidant activity of the regenerated shoots was significantly higher than that of callus and the regenerated plantlets.     

Effect of TDZ on plant regeneration from mature seeds in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

Pea (Pisum sativum L. cv. Espace) seeds directly cultured on thidiazuron (TDZ)-containing medium formed high numbers of shoots. The number of shoots per seedling depended on the concentration and duration of the TDZ treatment. The best treatment was 12-wk incubation on MS medium supplemented with 4 mg/l TDZ followed by 4-wk culture on MS medium supplemented with 0.5 mg/l benzylaminopurine (BA) and produced more than 400 shoots/seedling. Isolated shoots rooted at a high frequency on MS medium containing 2–3 mg/l indole-3-butyric acid and 2 mg/l α-naphthalene acetic acid. In addition to the formation of shoots, bud-containing tissues (BCT) were formed at the cotyledonary nodes, shoot nodes, tendrils, stipules, and internodes. The BCT from the cotyledonary nodes and the shoot nodes was maintained in its pure state on MS medium supplemented with 4 mg/l TDZ by repeated culture. Shoot development was accomplished when the BCT were left on MS medium supplemented with 4 mg/l TDZ without subculture prior to transfer onto MS medium supplemented with 0.5 mg/l BA.     

An in vitro approach to investigate medicinal chemical synthesis by three herbal plants

Ever since regulatory changes introduced herbals into mainstream supermarkets and pharmacies, there has been an explosion of demand for herbal plants and extracts which can be used to improve human health and well being. Science still lacks a basic mechanistic understanding of how environmental triggers regulate phytochemical accumulation, but this gap can be bridged using in vitro models to examine herbal species responses. For St. John's wort (Hypericum perforatum), uniform in vitro shoot cultures were set up as a parallel to a previously established sand culture system for investigation of physical and chemical environmental factors that control hypericin accumulation. Cytokinin supplementation of shoot culture medium resulted in a proliferation of abundant leaf glands with enhanced levels of hypericin, as compared to controls. Cell cultures of echinacea (Echinacea angustifolia) were established, and hydrophilic pharmacological components (caffeic acid derivatives) were detected. A protocol of rigorous explant pretreatment, and use of newly emerging vegetative shoots permitted establishment of axenic kava (Piper methysticum) callus, which was used to regenerate roots (organogenesis). Kavapyrone synthesis was achieved in both undifferentiated cell cultures and in cultured roots, although at lower levels than found in in vivo root systems. The predominance of kavain and methysticin in both forms of the in vitro cultures was parallel to the relative proportions from kava roots in vivo. The cell and organ cultures of all three herbal medicinals provide advantageous, easily-manipulated models to decipher environmental controls of phytochemical biosynthesis.