B5 Liquid Medium Research Articles

Rosmarinic Acid Content and Antioxidant Activity in Ehretia asperula Zollinger et Moritzi Cell Suspension Cultures

Background: Ehretia asperula Zollinger et Moritzi is a medicinal tree abundant in rosmarinic acid, the primary phenolic component, with numerous beneficial biological properties, including antioxidant, antibacterial, antiviral, anti-allergy, anti-arthritis, asthma and anti-cancer. Methods: The cell suspension cultures of E. asperula Zollinger et Moritzi derived from the leaf in vitro callus were established in liquid B5 medium added with 0.4 mg/L NAA, 0.1 mg/L BA and 45 g/L glucose. After four weeks, 50 mg/L of chitosan was given to the cell suspension cultures to stimulate rosmarinic acid (RA) production after a 48-hour treatment period. RA content was analyzed using the HPLC and spectrophotometry method after four weeks and 48 hours of chitosan treatment. In addition, the antioxidant capacity of the extracts from E. asperula Zollinger et Moritzi was also tested using DPPH free radical scavenging assay. Result: The RA content and antioxidant capacity of E. asperula Zollinger et Moritzi’ extracts from the leaf in vitro greater than callus derived from the leaf in vitro greater than field-grown leaf greater than biomass of cell suspension cultures. These results suggested a strong correlation between RA concentration and antioxidant capacity. The use of E. asperula Zollinger et Moritzi cell suspension cultures with chitosan as an elicitor for RA production and evaluation of antioxidant activity is presented in this study for the first time. Our results suggest that cell suspension cultures and others may be a good source of RA, an antioxidant compound.

Sugar beet cells cellular and extracellular events taking place in response to drought and salinity

Salt and drought stress are important abiotic factors that negatively affect plant growth and yield. To understand how these stress factors affect metabolism at the cellular level, we analyzed cation concentrations and expression of cellular and extracellular proteins, as well as their functions and types. Cells of the industrially important halophyte sugar beet were exposed to 300 mM NaCl and 600 mM mannitol as stressors in modified Gamborg B5 liquid medium (PG0). Severe stress altered the intracellular concentrations of the most measured cations. The cellular proteome revealed that both stressors provoked significant differential regulation of 110 cellular proteins. About 80% of the identified proteins were classified in metabolism, energy, or cell rescue, defense and virulence categories. We identified several novel proteins that respond to stress, including a member of the bZIP family of transcription factors, a member of the glycine-rich RNA-binding proteins, and the K+ channel beta subunit. Among extracellular proteins we found previously unreported stress-responsive proteins, a beta-xylosidase and an isoform of chitinase. The obtained results indicate that salt and drought stress disturbed the concentrations of cellular cations and the affected expression of cellular and extracellular proteins in sugar beet cells.

Auxin sensitivity improves production of rosmarinic acid in transformed hairy roots of <em>Lavandula angustifolia</em>

Hairy root culture is a promising approach to improve production of plant secondary metabolites. The genes, which are located in T-DNA of a root-inducing plasmid, regulate auxin sensitivity of hairy roots. Therefore, this study was aimed to improve the growth and rosmarinic acid production of Lavandula angustifolia hairy roots. Lateral branches of hairy roots were transferred to ½ MS and ½ B5 liquid media. To assess auxin sensitivity, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) with four different concentrations (0, 0.1, 0.5 and 1 mg/l) were also applied. The growth of hairy roots in ½ MS medium was two-fold higher than in ½ B5 medium. In addition, both auxins were found to significantly improve the growth of hairy roots whereas non-transformed roots stopped growing in the presence of the auxins. The highest dry weight and rosmarinic acid production of hairy roots were obtained from ½ MS medium supplemented with IBA irrespective of its concentration. As a result, the hairy roots grown in ½ MS medium supplemented with IBA produced the maximum amount of rosmarinic acid (7.98 mg/g dry weight of hairy roots). This first report of rosmarinic acid production in L. angustifolia hairy roots provides new insights into the auxin sensitivity of L. angustifolia hairy roots.

Rosmarinic acid production of Ehretia asperula Zollinger & Moritzi cell suspension cultures: effects of cell aggregate size, glucose, and chitosan

Rosmarinic acid (RA) is an ester of caffeic acid and 3,4-dihydroxy phenyl lactic acid with anti-oxidant, anti-bacterial, anti-viral, and anti-cancer properties. It has been found in some plants of the Boraginaceae and Lamiaceae families. Ehretia asperula Zollinger & Moritzi is a medicinal plant, widely distributed in some countries such as China, Vietnam, Myanmar, and Thailand. Cell suspensions were obtained by transferring 1 g of friable callus into 20 mL of B5 liquid medium supplemented with 0.4 mg/L NAA and 0.1 mg/L BA, on orbital shaker at 90 rpm in the dark. The subculture was carried out after each of 21 days along with removing brown cell aggregates. To investigate the effects of cell aggregation, glucose, and chitosan on cell biomass and RA biosynthesis, the yellow cell suspensions were transferred to a new liquid medium. The results showed that non-sieved cell aggregates produced the highest biomass and RA content of E. asperula Zollinger & Moritzi cell suspension cultures. Glucose concentrations had direct effects on RA production. An addition of 45 mg/L glucose showed a significant (1.198 fold) increase in RA yield compared with control (30 mg/L) and other concentrations. On the other hand, the optimum concentration of chitosan for enhancing RA content was found at 50 mg/L after 48 hours of treatment, with an RA concentration 1.17 times greater than the control. These findings suggested that the using of cell clusters which size more than 0.125 mm, the addition of appropriate concentrations of sugar and chitosan could stimulate RA biosynthesis of E. asperula Zollinger & Moritzi cell suspensions

Rosmarinic acid production in cell suspension cultures of Ehretia asperula Zollinger & Moritz

Plant cell cultures provide an alternative means for producing secondary compounds in food, cosmetic and pharmaceutical industries. Ehretia asperula Zollinger & Moritzi is used as a traditional medicine for the treatment of liver detoxification, ulcers, tumors, inflammation and enhancing the body's resistance in Vietnam. The study was carried out to select suitable callus line for cell suspension cultures of E. asperula Zollinger & Moritzi and investigate the effects of inoculum size, rotation speed and naphthalene acetic acid (NAA) on the proliferation of cell suspension cultures. In addition, the influence of light intensity on the growth and rosmarinic acid (RA) biosynthesis of cell suspension was also surveyed. After 4 weeks of culture, the white to pale yellow friable callus expanded significantly with a fresh weight (FW) of 0.788 g and a high RA content of 2.062 mg/g FW. An appropriate medium for cell proliferation was the liquid B5 medium, which contained 30 g/l glucose, 0.1 mg/l benzyl adenine (BA) and 0.4 mg/l NAA. The results also demonstrated that a 1:20 ratio (w/v) inoculum size, darkness and rotation speed of 90 rpm were the optimal conditions for the proliferation and RA accumulation to 188.217 mg/l in 4 weeks of culture. These findings showed that E. asperula Zollinger & Moritzi cell suspension cultures could be a potential alternative approach for RA production in vitro.

The direct and indirect transformation methods on expressing a recombinant Dermaseptin peptide in tobacco transgenic hairy root clones

Despite the increasing demand for production of recombinant proteins, only plant cell cultures are considered as the efficient systems for producing eukaryotic valuable products. Although plant cells are grown in the straightforward and simple conditions, optimization of culture conditions is crucial for a higher yield. In the present study, the establishment of transgenic tobacco hairy roots (HRs) expressing a Dermaseptin B1 recombinant antimicrobial peptide (C2-B1) is examined. Direct and indirect transformation (DT and IT) methods were applied to compare the amount of the recombinant peptide production. The integration, transcription and expression of the transgene in the HRs were confirmed by PCR, semi-quantitative RT-PCR and western blot analysis, respectively. The C2-B1 level was quantified by ELISA analysis of HRs extracts. The results of this study showed that the longer the inoculation and co-culture period, the higher the transformation efficiency. Antimicrobial activity by of crude protein extract from transgenic HR clones showed a significant (P ≤ 0.01) difference between HRs derived from DT and IT methodes methodsmethodes . Transgenic clones derived from both DT and IT methodes and grown in liquid medium showed a difference in terms of total protein content significantly. HRs derived from IT methodmethod produced a significantly higher amount of C2-B1 recombinant peptide in liquid B5 medium supplemented with sucrose.

Establishment of hairy root cultures by Agrobacterium rhizogenes mediated transformation of Trachyspermum ammi L. for the efficient production of thymol.

Trachyspermum ammi is an important medicinal plant that contains a bioactive compound namely thymol. In the study, T. ammi was transformed by Agrobacterium rhizogenes strains. Seedling stem explants were inoculated with A. rhizogenes strains A4, LBA 9402, ATCC 15834, and the effect of different co-cultivation media along with incorporation of acetosyringone (100 µM) was evaluated comparatively on the frequency of hairy root induction. The polymerase chain reaction using rolB and virD specific primers was served to confirm the putative transformed hairy roots. All strains established hairy root with various frequencies, among which strain ATCC 15834 was significantly the most efficient strain for hairy root induction (84.3%). Half-strength B5 medium and incorporation of acetosiryngone (100 µM) were also significantly optimal for hairy root induction. Hairy roots culture induced by ATCC 15834 using half-strength B5 liquid medium supplemented with 30 g L-1 sucrose indicated the highest accumulation of biomass (99.05 g L-1 FW and 10.95 g L-1 DW) and thymol content (11.30 mg g-1 DM) at 20days. Nearly 4.9-fold and 5.3-fold increment of biomass and thymol accumulation was observed, respectively, at 20days in comparison with the untransformed control roots. The results showed the high potential of T. ammi hairy roots for the biosynthesis of thymol.

CELL SELECTION WITH BARIUM IONS FOR OBTAINING GENETICALLY MODIFIED TOBACCO

Introduction. The salinity is one of the most aggressive environmental factors. It provokes the wide range of pathological changes in the plant tissues. The drastic decrease of K + cations in plant cells due to salt toxicity is the main feature of injury. The lands with secondary salinization increase. Fresh water in many regions transforms to the product of deficit even to the public. So the problem of obtaining plant forms with elevated levels of salt tolerance becomes extremely significant. Genetic effects that increased the genotype tolerance abilities are the aims of various investigations. Cell selection with heavy metal ions is the appropriate biotechnology for obtaining plant forms that challenged the salinity. The scientific interest to Ba 2+ ions is due to its interaction with K + cations. There was shown that Ba 2+ interrupted the K + inward transport Purpose. The obtaining glycophyte-derived salt tolerant forms (cell lines – regenerants – progeny )via cell selection. Methods. Selective systems with lethal doses of barium ions (Ba 2+ ) for obtaining tobacco cell forms tolerant to salt stress are proposed and elaborated. The minimum of Ba 2+ doses that eliminate wild type cell population was established as lethal doses. Primary calli cultures were initiated from tobacco leaves on B5 cultural agar medium. Cell suspension was cultivated in liquid B5 medium. Cell suspension (wild type) was placed between two layers of agar selective medium with the addition of lethal doses of barium ions (“plating procedure”). Only Ba-resistant cells survive under such stress pressure. The survived cells formed primary minicolonies. Such colonies are considered to be tobacco Ba-resistant cell lines (Ba-RCL). Ba-RCL grew at Ba 2+ ions presence during 3 passages . Then callus was cut and transferred to fresh media: basal medium (normal conditions) and selective media (stress conditions). Resistant cell variants were tested under lethal salinity. Salt stress was simulated by the addition of sodium chloride or sodium sulfate. There were established three variants of selective systems: medium with the addition of Ba 2+ cations, (stress I); cultural media with the addition of salinity (stress II, III). Genetic basis of combined stress resistance was confirmed via media rotations. The changes were: normal conditions → stresses I, II, III; stresses I, II, III → normal conditions; stress I → stress II or stress III or other way roads. The type of cultural medium and the number of passages were always free. As proliferation marker calli relative fresh mass growth (RFW, Δm) was used. Regenerants and plants of R1 seed progeny were cultivated in vitro at presence of sea water salts. Result. Tobacco cel lines with combined resistance to lethal ion and chloride and sulfate stresses were obtained via cell selection with Ba 2+ cation. Ba-RCL maintained their viability under any stress pressure. The calli relative fresh mass growth (RFW, Δm) was always positive. Regenerants from selected cell lines and R1 seed progeny were cultivated at presence of sea water salts (2,5%) in vitro. During such cultivations tested plants formed new roots and leaves. After transfer to control (salt free) medium plants adapted to normal conditions. Originality of the investigation and its priority was in the promotion of cell selection with Ba 2+ cations for obtaining tobacco variants tolerant to lethal salinity. Conclusion . Cell selection with heavy metal ions is the perspective approach for obtaining genetically modified plant forms. Barium cation is appropriate agent for selection variants with higher tolerance to salinity. Tobacco is a classic glycophyte. From such initial tissues tobacco plant forms (cell lines → regenerants → progeny) that survive under lethal salinity were obtained. This approach is suitable for other genotypes.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves.

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 ± 1 °C. After 3-4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 µg mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Comparison of the Metabolic Behaviors of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea ( Camellia sinensis L.) Leaves.

The use of an in vitro cell suspension to study insecticide metabolism is a simpler strategy compared to using intact plants, especially for a difficult matrix such as tea. In this study, a sterile tea leaf callus was inoculated into B5 liquid media with 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and Kinetin (KT, 0.1 mg L-1). After 3-4 subcultures (28 days each), a good cell suspension was established. Utilizing these cultures, the metabolic behaviors of six insecticides, including two organophosphates (dimethoate, omethoate) and four neonicotinoids (thiamethoxam, imidacloprid, acetamiprid, and imidaclothiz) were compared. The results showed that thiamethoxam, dimethoate, and omethoate were easily metabolized by tea cells, with degradation ratios after 75 days of 55.3%, 90.4%, and 100%, respectively. Seven metabolites of thiamethoxan and two metabolites of dimethoate were found in treated cell cultures using mass-spectrometry, compared to only two metabolites for thiamethoxam and one for dimethoate in treated intact plants.

Influence of Different Carbohydrates on Flavonoid Accumulation in Hairy Root Cultures of Scutellaria baicalensis

Carbohydrate sources play important roles in energy and growth of plants. Therefore, in this study, we investigated the optimal carbohydrate source in hairy root cultures (HRCs) of Scutellaria baicalensis infected with Agrobacterium rhizogenes strain R1000. The hairy roots were cultured in half-strength B5 liquid medium supplemented with seven different carbohydrates sources (sucrose, fructose, glucose, galactose, sorbitol, mannitol and maltose), each at a concentration of 100 mM, in order to identify the best carbon sources for the production of major flavones, such as wogonin, baicalin and baicalein. Sucrose, galactose and fructose markedly influenced the production of major flavones and were therefore chosen for subsequent experiments. HRC growth and flavone accumulation were examined following culture with 30, 100 and 150 mM sucrose, galactose and fructose, respectively. From these data, 150 mM sucrose was found to be the optimal carbon source for the enhancement of baicalein production and growth of S. baicalensis HRCs. Fructose caused the greatest increase in baicalin accumulation. Additionally, galactose was the optimal carbon source for wogonin production. These results provide important insights into the optimal growth conditions, particularly the appropriate carbohydrate source, for S. baicalensis.

Improved production of chlorogenic acid from cell suspension cultures of <i>Lonicera macranthoids</i>

Purpose: To evaluate the potential of Lonicera macranthoides Hand. -Mazz. Yulei1 suspension culture system for enhanced production of the main secondary metabolite, chlorogenic acid.Methods: The callus of L. macranthoides Hand.-Mazz. “Yulei1” was suspension cultured in B5 liquid medium supplemented with different plant growth regulators. Biomass accumulation was calculated by weight method and chlorogenic acid production was measured using high performance liquid chromatography (HPLC). HPLC was carried out on C18 analytical column at 35 °C and the detection wavelength was set at 324 nm.Results: The results showed that maximum accumulation of biomass and chlorogenic acid were achieved 15 days after culture growth. The optimized conditions for biomass accumulation and chlorogenic acid production were 50 g/L of inoculum on fresh weight basis, B5 medium supplemented with plant growth regulators, 30 - 40 g/L sucrose and initial medium pH of 5.5. Maximum accumulation of chlorogenic acid and biomass were observed when the culture medium was supplemented with 2.0 mg/L6-BA. Optimal accumulation of chlorogenic acid was observed using combination of hormones 2.0 mg/L 6-Benzyladenine (BA) + 0.5 mg/L2, 4-Dichlorophenoxyacetic acid (2,4-D), while optimal accumulation of biomass was observed with 2.0 mg/L 6-BA + 2.0 mg/L2, 4-D. In addition, phenylalanine also contributed to the synthesis of chlorogenic acid at a concentration > 50 mg/L.Conclusion: Cell suspension cultures of L. macranthoides Hand.-Mazz. “Yulei1” have successfully been established. The findings provide a potential basis for large scale production of chlorogenic acid using cell suspension cultures of L. macranthoides.Keywords: Lonicera macranthoides, Cell suspension culture, Chlorogenic acid, Phenylalanine, Optimization

Production of naphthoquinone derivatives using two-liquid-phase suspension cultures of Alkanna orientalis

An in vitro two-liquid-phase suspension culture system of Alkanna orientalis was established in order to evaluate the ability of cultured cells to produce naphthoquinone derivatives. Initially, callus tissues were prepared from cotyledon explants of A. orientalis on the solidified B5 medium supplemented with 1 mg/L 2,4-dicholorophenoxyacetic acid (2,4-D) and 2 mg/L kinetin (kin). Subsequently, fragile calli were transferred to liquid B5 medium with the same growth regulators for the initiation of cell suspension culture. Thereafter, the two-liquid-phase suspension culture was established using M9 medium and liquid paraffin as the absorbent. The presence of liquid paraffin efficiently induced the production of pigments by the cultured cells. The n-hexane extract of proliferated cell suspension culture was studied by analytical HPLC. Then, the main secondary metabolites were separated by preparative HPLC and their structures were elucidated by UV, 1H and 13C-NMR spectroscopy. Accordingly, alkannin/shikonin, alkannin/shikonin acetate, deoxyshikonofuran and a dimmer of alkannin/shikonin were identified as the main chemical compounds in the extract. On the basis of our results, the two-liquid-phase suspension culture led to the effective induction of naphthoquinone derivatives. These findings draw attention to the potential of A. orientalis cell suspension cultures in the production of naphthoquinone which may be considered as a source for the biosynthesis of these secondary metabolites for medicinal or industrial uses.

Establishment and characterization of a Satureja khuzistanica Jamzad (Lamiaceae) cell suspension culture: a new in vitro source of rosmarinic acid.

An in vitro approach to the production of rosmarinic acid (RA), a medicinally important caffeic acid ester, in a cell suspension culture (CSC) of Satureja khuzistanica Jamzad (Lamiaceae) has been investigated for the first time. The CSC was established from friable calli derived from shoot tip explants in Gamborg's B5 liquid medium supplemented with 30g/L sucrose, 20mg/L L-glutamine, 200mg/L casein hydrolysate, 5mg/L benzyladenine (BA) and 1mg/L indole-3-butyric acid (IBA). The effect of nitrogen source (KNO3 and (NH4)2SO4) and their different concentrations on the fresh and dry weight (g/L), as well as RA content (mg/g dry weight) were measured. CSC growth measurements indicated a maximum specific cell growth rate of 1.5/day, a doubling time of 7.6days and a high percentage of cell viability (96.4%) throughout the growth cycle. Maximum cell fresh weight (353.5g/L), dry weight (19.7g/L) and RA production (180.0mg/g) were attained at day 21 of culture. Cell growth and RA content were affected by nitrogen deficiency. Media containing 8.3mM of total nitrogen (¼ of B5 standard medium) led to a minimum cell fresh weight (243.0g/L), dry weight (17.4g/L) and RA content (38.0mg/g) after 21 days. The established CSC provided useful material for further optimization experiments aimed at a large-scale production of RA.

Effects of media and carbon sources on growth and canthin-6-ones accumulation in hairy root cultures of Eurycoma harmadiana

Eurycoma harmandiana Pierre belongs to Simaroubaceae family which is a Thai traditional medicinal plant used for intermittent fever (Malaria). The major bioactive compounds of the these plants are quassinoids and canthin-6-one alkaloids which possessed cytotoxic, antimalarial, and aphrodiasiac[1 – 2]. In this study, the effects of various basal media and carbon sources on growth and canthinone alkaloids production in hairy root cultures of E. harmadiana was investigated. The effect of basal media on the hairy root biomass was no significantly different, while the highest amount of alkaloids, 9-hydroxycanthin-6-one (5.75 mg/g dry weight) and 9-methoxycanthin-6-one (5.87 mg/g dry weight), could be obtained in B5 liquid medium. At fructose of 3% (w/v), the production of 9-methoxycanthin-6-one (5.74 mg/g dry weight) is promoted to gain the highest yield, while 3% (w/v) mannitol gave the highest amount of 9-hydroxycanthin-6-one (4.94 mg/g dry weight), compared to other carbon sources tested. This study suggests that the successful production of 9-hydroxycanthin-6-one and 9-methoxycanthin-6-one in vitro cultures has a potential in large-scale production using bioreactor technology.

High-frequency Plant Regeneration from Cultured Flower Bud Receptacles of Allium hookeri L.

Allium hookeri L. (Alliaceae family) is an important ethnomedicinal plant native to the Himalayan region of Asia. The aim of this research was to establish a high-frequency plant regeneration system for in vitro propagation of A. hookeri. Among the tissue types examined, receptacle explants derived from immature flower buds showed the highest regeneration rate of shoots (<TEX>$93.33{\pm}4.63%$</TEX>), roots (<TEX>$76.67{\pm}7.85%$</TEX>), and calli (<TEX>$80.00{\pm}7.43%$</TEX>) when cultured on Gamborg B5 (B5) medium containing <TEX>$10{\mu}M$</TEX> 6-benzylaminopurine (BA) + <TEX>$1{\mu}M$</TEX> naphthalene acetic acid (NAA), <TEX>$0.5{\mu}M$</TEX> BA + <TEX>$5{\mu}M$</TEX> NAA, and <TEX>$1{\mu}M$</TEX> BA + <TEX>$10{\mu}M$</TEX> NAA, respectively. Shoot multiplication was superior when cultured in liquid rather than on solid medium and relatively high concentrations of BA, ranging from 5 to <TEX>$10{\mu}M$</TEX>. Efficient bulblet formation following root induction from shoot clumps was achieved with culture in liquid B5 medium containing 7% (w/v) sucrose. Regenerated bulblets were successfully acclimatized to ex vitro conditions with a greater than 95% survival rate. By this method, a maximum of 62 plantlets per receptacle could be propagated within 9 weeks of initial culture. The in vitro propagation system established in this study will promote A. hookeri biotechnology, including large-scale production of healthy and aseptic clones, preserving parental genotypes with desirable traits, and genetic manipulation to enhance medicinal value.

Establishment of hairy root lines and analysis of iridoids and secoiridoids in the medicinal plant Gentiana scabra.

BackgroundGentiana scabra is commonly known as ‘Longdan’ is an important herb in traditional Chinese medicines, commonly used for the treatment of inflammation, anorexia, indigestion and gastric infections. Iridoids and secoiridoids are main bioactive compounds which attributed to the pharmacological properties of this plant. The use of hairy root cultures as an excellent alternative for the production of pharmaceutically important metabolites in less time period with ensured quality of raw materials.ResultsAn efficient hairy root culture system of Gentiana scabra and influence of different plant growth regulators (PGRs) on the production of gentiopicroside, swertiamarin and loganic acid constituents were described. Leaf explants were infected with Agrobacterium rhizogenes, which induced hairy roots up to 21%. The transformed hairy root lines were confirmed by PCR using rolB and rolC gene-specific primers. Among various solid and liquid media, B5 liquid medium resulted maximum root biomass (36- fold higher) in 4-weeks. Quantitative analysis showed loganic acid was 6.6- fold higher in the presence of zeatin (1 mg/l) and gentiopicroside accumulation was 1.8- fold higher in the presence of naphthaleneacetic acid (NAA, 1 mg/l), as compared to the roots of plants grown in greenhouse. On the other hand, 1.4- and 2.5- fold higher gentiopicroside and swertiamarin were observed in the presence of 1.0 mg/l NAA as compared to commercial Gentiana herb No. 2. The result also showed iridoid and secoiridoid contents affected greatly by age, physiology and growing environment of the plant.ConclusionsThe use of hairy root cultures is an excellent alternative to harvesting natural or in vitro grown plants to produce pharmaceutically important metabolites in less time with ensured quality.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-55-17) contains supplementary material, which is available to authorized users.

Rosmarinic acid biosynthesis in callus and cell cultures of Agastache rugosa Kuntze

Callus and cell suspension cultures of Agastache rugosa were established to investigate the production of rosmarinic acid in vitro. For callus induction 2,4-D treatment was more effective than that of NAA. The best callus induction rate (92%) and callus growth A. rugosa were obtained in MS medium containing 2 mg/l 2, 4-D. In cell suspension culture of A. rugosa, the maximum growth (7.7 g/l) and the highest rosmarinic acid production (11.5 mg/g) were attained in the liquid B5 medium supplemented with 2 mg/l 2,4-D and 0.1 mg/l BAP at 10 days after culture. The present results demonstrate that cell culture of A. rugosa might be an alternative approach for the production of rosmarinic acid. Key words: Agastache rugosa, callus, cell suspension, rosmarinic acid.

Molecular and chemical characterization of plants regenerated from Ri-mediated hairy root cultures of Rauwolfia serpentina

Hairy root lines were induced from leaf explants of Rauwolfia serpentina known to contain high levels of reserpine (0.0882 % DW) content. Out of five high yielding hairy root lines, three (R1, R14 and R15) exhibited spontaneous regeneration of shoots after 6–8 weeks in liquid B5 medium. Excised regenerated shoots underwent robust shoot proliferation when cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l naphthanleneacetic acid (NAA) and 1.0 mg/l 6-benzyladenine. When shoots were transferred to a root induction medium, consisting of MS basal medium and 1.0 mg/l NAA, all rooted within 2–3 weeks. Of a total of 45 plants developed from three different hairy root lines, 30 were successfully acclimatized and transferred to the green house. Almost 90 % of these plants grown in the green house showed no observed phenotypic differences, while 10 % were stunted and grew poorly, in comparison to non-transformed plants. Phenotypic assessment of regenerated plants for plant length, number of nodes and intermodal lengths, number of leaves per node, leaf color, leaf size, number of flowering shoots, flower size, fruit size, lateral root branching and root biomass was conducted. Polymerase chain reaction and Southern blot hybridization revealed that all plants derived from hairy roots carried the Ri TL-DNA fragment. Moreover for plants derived from transgenic hairy root line R14, presence of more than a single transgene copy number was observed, and this might have contributed to observed abnormal phenotypes. Analysis of reserpine content revealed that roots of regenerated plants had similar levels (0.0889 % DW) to those of their corresponding hairy roots.

Benzothiadiazole (BTH) activates sterol pathway and affects vitamin D3 metabolism in Solanum malacoxylon cell cultures

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a particularly efficient inducer of systemic acquired resistance (SAR), was developed as an immunizing agent to sensitize various crop species against pathogen infections. Recent works highlighted its activating effect on different metabolic pathways, concerning both primary and secondary metabolites. In this study, we investigated the effect of BTH treatment on sterol levels and vitamin D(3) metabolism in Solanum malacoxylon cultures. Calli of S. malacoxylon were incubated in Gamborg B5 liquid medium alone or added with 50μM BTH for different times (one, two or three cycles of light). Histocytochemical investigations performed on our experimental system using 3,3'-diaminobenzidine (DAB) for hydrogen peroxide (H(2)O(2)) detection and phloroglucinol for lignin staining showed that BTH causes H(2)O(2) accumulation and lignin deposition in treated calli. Gas chromatographic analysis of principal cell membrane sterols (β-sitosterol, campesterol, stigmasterol) showed that BTH transiently increases their cellular levels. Callus cultures were found to contain also cholesterol, 7-dehydrocholesterol, the putative precursor of vitamin D(3), and the hydroxylated metabolites 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)]. BTH treatment enhanced 7-dehydrocholesterol while reduced cholesterol. HPLC analysis of sample extracts showed that BTH does not affect the cell content of vitamin D(3), though results of ELISA tests highlighted that this elicitor moderately enhances the levels of 25(OH)D(3) and 1α,25(OH)(2)D(3) metabolites. In conclusion, BTH treatment not only causes cell wall strengthening, a typical plant defence response, as just described in other experimental models, but in the same time increases the cellular level of the main sterols and 7-dehydrocholesterol.