Rosmarinic acid production in hairy root cultures of Agastache rugosa Kuntze

Rosmarinic acid, an important phenolic active compound, is one of the main active constituents of Agastache rugosa Kuntze and has astringent properties, antioxidant capacity, anti-inflammatory activity, antimutagenic ability, antimicrobial capacity, and antiviral properties. To investigate in vitro production of rosmarinic acid, we established a hairy root culture of A. rugosa by infecting leaf and stem explants with Agrobacterium rhizogenes R1000, and tested the growth and rosmarinic acid production of these cultures. Hairy roots were cultured in Murashige and Skoog liquid medium and maximum growth (14.1 g dry wt/l) was attained after 14 days of culture, at which time the content of rosmarinic acid was 116 mg/g dry wt. The present results demonstrate that hairy root culture of A. rugosa is a valuable alternative approach for the production of rosmarinic acid.     

Establishment of Salvia officinalis L. hairy root cultures for the production of rosmarinic acid

Shoots of Salvia officinalis, a medicinally important plant, were infected with Agrobacterium rhizogenes strains ATCC 15834 and A4 which led to the induction of hairy roots in 57% and 37% of the explants, respectively. Seven lines of hairy roots were established in WP liquid medium under light and dark conditions. The transformed nature of the root lines was confirmed by polymerase chain reaction using rolB and rolC specific primers. Transformed root cultures of Salvia officinalis showed variations in biomass and rosmarinic acid production depending on the bacterial strain used for transformation and the root line analyzed. Both parameters (growth and rosmarinic acid content) of ATCC 15834-induced lines were significantly higher than the A4-induced lines. The maximum accumulation of rosmarinic acid (about 45 mg g(-1) of dry weight) was achieved by hairy root line 1 (HR-1) at the end of the culture period (45-50 days). The level was significantly higher than that found in untransformed root culture (19 mg g(-10 of dry wt).     

Influence of the carbon source on growth and rosmarinic acid production in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei were characterized with respect to growth and rosmarinic acid formation in media with different sugars and various sugar concentrations. Sucrose is the sugar with the highest stimulating effect on growth and rosmarinic acid accumulation, followed by glucose and fructose. The sugar alcohol mannitol cannot be metabolized by the plant cells. Sucrose is cleaved into glucose and fructose by the Coleus cells. Sucrose concentrations from 1 to 5% have an increasing positive effect on growth and rosmarinic acid synthesis in the cell cultures with a maximum rosmarinic acid content of 12% of the dry weight in medium with 5% sucrose; in medium with 6% sucrose rosmarinic acid accumulation obviously did not reach its highest level in the culture period of 14 days. A very high yield of rosmarinic acid (2 mg ml^-1 suspension) could also be achieved by maintaining a sucrose concentration of 2% during the whole culture period. The start of rosmarinic acid synthesis by the cell cultures seems to be regulated by the growth limitation when a nutrient, e.g. phosphate is depleted from the medium. The rate of rosmarinic acid accumulation is related to the amount of carbon left in the medium when growth ceases.Abbreviations RA rosmarinic acid     

The Action of Fusicoccin Alone and with Some Plant Growth Substances on Tobacco Tissue Cultures

The interaction of fusicoccin, a terpenoid glucoside produced by Fusicoccum amygdali Del., and some plant growth regulating substances, i.e., indole-3-acetic acid, kinetin, 2,4-dichlorophenoxy-acetic acid and abscisic acid, was investigated on pith and sub-cultured callus cultures of Nicotiana tabacum L. Addition of fusicoccin alone to the basal medium, either in the light or in the dark, caused a fairly good development of tobacco callus. When fusicoccin and kinetin were simultaneously added to the culture medium, the callus growth increased. However, fusicoccin in combination with indole-3-acetic acid caused limited callus development: the tissue appeared brown and reduced in volume. Addition of fusicoccin with 2,4-dichlorophenoxyacetic acid stimulated growth of callus and chlorophyll was formed under light. Finally, abscisic acid did not interfere with the effect of fusicoccin on the callus growth.     

Rosmarinic acid production by Lavandula vera MM cell-suspension culture

The time courses of growth and rosmarinic acid production by Lavandula vera MM cell suspension were investigated. The uptake of the main nutrients (sucrose, nitrogen, phosphorus, K, Ca, Mg) was followed during cultivation and the data on the physiology of the L. vera MM cell culture are presented. It was established that the cell culture synthesizes rosmarinic acid during the linear phase of growth for a relatively short period (between the 4th and 8th days of cultivation). The influence of sucrose concentration in the nutrient medium on cell growth and accumulation of rosmarinic acid by L. vera MM cell culture was investigated. The results showed that 7% sucrose in the nutrient medium ensured a steady growth of the cell suspension and increased the yield of rosmarinic acid (29.2 g/l dry biomass and 507.5 mg/l rosmarinic acid compared to 13.0 g/l dry biomass and 68.6 mg/l rosmarinic acid for the control cultivation with 3% sucrose). Received: 17 September 1996 / Received revision: 31 January 1997 / Accepted: 1 February 1997     

The Effects of Agitated Liquid Medium on in vitro Cultures of Hevea brasiliensis

The initiation and prolonged growth of callus, from stem explants of young plants of Hevea brasilienies on solid medium yielded a heterogeneous callus, with areas which are the result of compact growth interspersed with brown necrotic tissue and soft white tissue formations. Subculturing this callus (O callus) to agitated liquid medium and returning it to solid medium resulted in the production of a homogeneous friable and rapidly growing callus (Rl callus) The two established lines O and Rl have remained stable over one year in culture and differ in gross morphology, anatomy, growth and auxin content. Both were maintained on Murashige and Skoog's medium, with 2 mg/1 2,4-D and 0.5 mg/I kinetin. R 1 but not O showed enhanced growth at the lower 2,4-D level of 0.2 mg/l: both lines failed to continue growing when 2,4-D was omitted. It is suggested that the changes resulting from subculture in agitated liquid medium are related to those undergone by callus cultures which become habituated. Thus the Rl callus line is regarded as partially habituated. Subculture in agitated liquid medium also resulted in the production of large numberr of polyploid cells but these did not persist over the long periods of subsequent growth on agar medium, Enhanced auxin production by the establihed Rl callus line was thus observed in the absence of a detectable level of polyploidy.     

Accumulation of Glutamine by Suspension Cultures of Symphytum officinale

A callus was induced from the veins of a leaf of Symphytum officinale, comfrey, on a medium containing the inorganic elements reported by Murashige and Skoog with addition of 3% sucrose, 0.5 mg/liter 2,4-D and 0.3~3.0 mg/liter kinetin.

Suspension cultures of this cell line obtained from the callus were shown to accumulate a large amount of L-glutamine intracellularly, The effect of growth hormones and nutrients on accumulation of the amino acid has been examined in suspension cultures. The most suitable concentrations of 2,4-D and kinetin for glutamine accumulation were 0.3 mg/liter each. The presence of potassium nitrate as a nitrogen source was beneficial for growth and ammonium nitrate stimulated the accumulation of glutamine. High levels of these nitrogen sources in the medium were required for obtaining a high level of glutamine. The concentration of glutamine accumulated reached to approximately 20% of dry cell weight when S. officinale was incubated in the medium containing 0.495 % of ammonium nitrate and 0.570% of potassium nitrate which corresponded to three times higher levels than those in a Murashige and Skoog’s medium.

Most of the amino acid was found intracellularly but a small amount was excreted into the medium in the later stages of the incubation. Addition of a cationic surfactant, cetyltrimethylammonium bromide, to the cultures caused to increase the amount of the amino acid in the culture filtrate.

The contents of free amino acids in leaves of S. officinale were compared with those in the callus. The level of glutamine in the callus was 260 times higher than that in the intact plant.     

Effects of Abscisic Acid on Phenolic Content and Lignin Biosynthesis in Tobacco Tissue Culture

A significant depression of callus growth resulted from low concentrations of abscisic acid (ABA) added to the medium recommended by Linsmaier and Skoog. Low concentrations also decreased the chlorogenic acid and lignin content of the callus, and generally decreased amounts of scopolin and scopoletin in the tissue. Gibberellic acid (GA₃) stimulated callus growth in a low concentration (0.1 mg/1) and inhibited growth at a high concentration (10.0 mg/1). Both levels of GA₃ increased scopoletin accumulation in tobacco callus. A high concentration of GA₃ increased the accumulation of scopolin and chlorogenic acids, whereas a low concentration decreased the amounts of these two phenolic compounds. In comparison with the control, lignin synthesis was stimulated by a low GA₃ concentration, but a high GA₃ concentration did not have a significant effect. Both low and high concentrations of GA₃ overcame ABA inhibition of growth and lignin synthesis, and partially reversed ABA inhibition of scopoletin production. However, GA₃ did not reverse the inhibitory effect of ABA on scopolin production. The low concentration of GA₃ overcame the inhibition of chlorogenic acid production resulting from a 0.01 mg/1 concentration of ABA, but this was the only reversal of chlorogenic acid inhibition resulting from addition of GA₃ to the medium.     

Genotype and explant-type dependent morphogenesis and silicon response of common reed (Phragmites australis) tissue cultures

The effects of silicon on the growth and development of Phragmites australis (Cav.) Trin. Ex Steud. (common reed) stem nodal and root embryogenic calli were investigated. Silicon is considered to be a beneficial or quasi-essential nutrient for several Gramineaceous plants, including reed. Seven callus lines of four geographical locations (genotypes 1-4) within Hungary were investigated. Callus lines 1A, 2A and 3A were produced from stem nodal explants, while lines 1B, 2B, 3B and 4 were produced from roots. For the assay of silicon-dependent growth of callus lines of identical genotype but originating from different explants, we measured the increase of fresh weight of lines 1A and 1B. The studied developmental parameters were the increase of the number of somatic embryos (for callus lines 1A and 1B) and plant or root production from somatic embryos (for all genotypes/callus lines). Silicon was added to the culture medium as sodium silicate. In control cultures, plant or root regeneration from embryogenic calli was strongly genotype- and explant type-dependent. Stem nodal explants developed plants on regeneration medium in case of callus lines 2A and 3A, while line 1A produced roots only. All root derived calli developed roots on regeneration medium. Silicon stimulated the growth of both stem nodal and root calli (callus lines 1A, B) however, the concentration optima were different. Somatic embryogenesis of root calli, but not of stem nodal calli, was stimulated by silicate at low concentrations. However, for both of these callus lines, root development was stimulated by silicon. It had genotype-dependent influences on plant regeneration: while stimulation was observed in case of callus line 2A, inhibition occurred for line 3A. Root morphogenesis on calli was significantly influenced by silicon and depended on the callus line studied. Root production was stimulated on callus lines 1A, B and 2B, while in case of callus line 3B, it was significantly inhibited. The morphogenetic effects of Si were similar for different explants of the same geographical origin, i.e. plant or root production was similarly stimulated or inhibited by this element. We can conclude that the effects of Si on plant or root development depend on reed genotype used for callus induction. Its effect on growth and somatic embryogenesis depends on the explant type used for callus production. This is the first detailed report on the role of silicon in plant vegetative development and morphogenesis of a Gramineaceous plant.     

Effect of auxin on alkaloids, K+ and free amino acid content in cultured tobacco callus

Callus cultures derived from the petiole of Nicotiana tabacum L. cv. Burley 21 were grown at 25°C in the dark on two different basal media containing: (1) 11.5 μ M α-naphthaleneacetic acid and 1 μ M kinetin, and (2) 1 μ M α-naphthaleneacetic acid and 1 μ M kinetin. The contents of alkaloids, K⁺ and free amino acids of callus tissues were determined. The tissues were also examined microscopically for organization when organogenesis was not apparent. The first medium limited nicotine synthesis and stimulated its N-demethylation to nornicotine. The second medium stimulated nicotine synthesis and limited tissue growth. Although significantly higher concentrations of K⁺ were observed in calli grown on the high-auxin medium, both cultures were K⁺ deficient. The fact that the low-auxin medium limited K⁺ uptake to a higher degree would account for the lower growth observed in calli cultured on this medium, and it is possible that the effect of auxin concentration on nicotine production may be mediated through its effects on K⁺ uptake by cells of the culture. The free amino acid concentration increased in the calli grown on the low-auxin medium. Glutamic acid and proline, known as initial precursors of nicotine, increased significantly. Histological examination showed that the occurrence of meristematic areas in calli without organogenesis promoted nicotine synthesis. The relation between the accumulation of nicotine and formation of roots or shoot-buds is discussed.     

Medium composition and methyl jasmonate influence the amount and spectrum of secondary metabolites in callus cultures of Zanthoxylum stenophyllum Hemsl.

Callus cultures of Zanthoxylum stenophyllum were initiated in vitro and the effect of growth regulators and elicitors was tested both upon callus growth and secondary metabolite production. On a medium containing naphthaleneacetic acid, kinetin, and 2,4-dichlorophenoxyacetic acid, a yellowish and friable callus was obtained from 90% of cotyledon explants. Callus growth and secondary metabolite accumulation was followed after sub-culturing the established callus culture on different media containing various hormonal combinations. Results indicate that medium containing naphthaleneacetic acid and a higher concentration of 2,4-dichlorophenoxyacetic acid gave the highest stimulation of growth. Addition of an organic nitrogen source also had a positive effect on growth. Rapid HPLC screening of methanol extractable secondary metabolites from calluses showed that phytohormones and nutrients were able to modify the chromatographic pattern of compounds. Calluses grown on the medium giving the highest stimulation of growth show a reduced accumulation of some secondary products, but not all. In response to elicitation by methyl jasmonate, metabolite production was different for the different classes of compounds, and hormonal composition of the culture medium influenced the response. Thus, results confirm the importance of the reciprocal interactions between hormones, nutrients, and elicitors when attempts are made to enhance secondary metabolite accumulation in in vitro cultures.     

Increased saturated phospholipid in cultured cells grown with linoleic acid

Summary We found that fetal bovine serum supplementation of culture medium provided limited quantities of linoleic acid, an essential fatty acid, to cells grown in culture (2.8 ± 0.3% of total fatty acids in 12 lots). Supplementation of the medium with additional linoleic acid resulted in altered phospholipid acyl composition in cells of two established lines, A549, a putative model of the pulmonary Type II epithelial cell, and SIRC, a line derived from rabbit corneal epithelium. In particular, linoleic acid supplementation induced a relative increase in disaturated choline phosphoglycerides of 33 and 36%, respectively, in cells of the two lines. This observation may be relevant to design of media for primary culture of Type II cells, in which disaturated phospholipid synthesis is used as an index of differentiated function (surfactant production). Linoleate supplementation did not alter growth or size (protein content) of cells of either line and caused a slight increase in accumulation of neutral lipid, in the form of cytoplasmic droplets, in A549 cells. Supplementation of cell cultures with equivalent concentrations of the nonessential fatty acids palmitic and oleic acid did not significantly alter the growth, morphologic appearance, or lipid composition of the cells. However, it was demonstrated in cells of one line that palmitic acid supplementation temporarily stimulated synthesis of disaturated choline phosphoglyceride from radiolabeled choline. This work was supported by Grants HL-24817 and HL-21251 from the National Institutes of Health, USPHS, and by a grant from the Alexandrine and Alexander L. Sinsheimer Fund.     

Fungal elicitor preparations and methyl jasmonate enhance rosmarinic acid accumulation in suspension cultures of Coleus blumei

bstract Suspension cultures of Coleus blumei (Lamiaceae) treated with either an elicitor preparation from the culture medium of the phytopathogenic oomycete Pythium aphanidermatum or with methyl jasmonate enhanced accumulation of rosmarinic acid approximately threefold. The specific activities of phenylalanine ammonia lyase and rosmarinic acid synthase were also enhanced after addition of the fungal elicitor. The addition of methyl jasmonate transiently increased activities of phenylalanine ammonia lyase and hydroxyphenylpyruvate reductase, whereas the activity of rosmarinic acid synthase was not stimulated and the activity of tyrosine aminotransferase was slightly and constantly enhanced. Methyl jasmonate stimulated rosmarinic acid accumulation not only when added directly to the culture medium, but also when it could reach the cells only via the gas phase. Received: 2 April 1997 / Revision received:16 June 1997 / Accepted: 15 September 1997     

Rosmarinic acid

Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. It is commonly found in species of the Boraginaceae and the subfamily Nepetoideae of the Lamiaceae. However, it is also found in species of other higher plant families and in some fern and hornwort species. Rosmarinic acid has a number of interesting biological activities, e.g. antiviral, antibacterial, antiinflammatory and antioxidant. The presence of rosmarinic acid in medicinal plants, herbs and spices has beneficial and health promoting effects. In plants, rosmarinic acid is supposed to act as a preformed constitutively accumulated defence compound. The biosynthesis of rosmarinic acid starts with the amino acids L-phenylalanine and L-tyrosine. All eight enzymes involved in the biosynthesis are known and characterised and cDNAs of several of the involved genes have been isolated. Plant cell cultures, e.g. from Coleus blumei or Salvia officinalis, accumulate rosmarinic acid in amounts much higher than in the plant itself (up to 36% of the cell dry weight). For this reason a biotechnological production of rosmarinic acid with plant cell cultures has been proposed.     

Selection of cell lines for the production of rosmarinic acid from cell suspension cultures of Orthosihon stamineus benth

Summary Cell suspension cultures of Orthosiphon stamineus were established from friable calluses produced from leaf pieces of in vitro plantlets that were derived from nodal segments of the mother plants collected from three different geographical locations. Eight lines were eventually selected after seven subculture cycles based on the growth characteristic (plant height) of the plantlets from the three locations: two fast-growing lines (>5.1 cm tall), three intermediate-growing lines (3.1–5.0 cm tall), and three slow-growing lines (<3.0 cm tall). All eight lines grew well in liquid Murashige and Skoog medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.4 μM 1-naphthaleneacetic acid (NAA). All cell lines exhibited the same growth pattern but produced different maximum cell biomass when cultured in this medium. The time of harvesting the plant cells from the culture medium and the geographical source of the original plant material were both found to affect the production of rosmarinic acid (RA) in cell cultures. Two cell lines were successfully selected and identified to produce high amounts of RA. These cell lines were a fast-growing cell line from Air Itam, Penang and an intermediate-growing cell line from Relau Agriculture Research Centre, Penang which could produce 5% [(w/w) dry weight] and 4.5% [(w/w) dry weight] of RA, respectively.     

氮碳源对冬凌草再生植株生长及次生代谢产物的影响

以冬凌草再生植株为研究材料,通过改变培养基中蔗糖浓度及NO3-/NH4+比例,研究冬凌草再生植株的生长状况及植株体内次生代谢物的积累规律.结果表明:3%蔗糖有利于冬凌草再生植株的生长和冬凌草甲素的积累,5%蔗糖有利于迷迭香酸的积累;NO3-/NH4+比例过高或过低均不利于再生植株的生长和次生代谢产物的积累,二者比例为2∶1时再生植株生长和次生代谢产物积累最佳.说明不同蔗糖浓度及NO3-/NH4+比例对冬凌草再生植株的生长和次生代谢物合成有明显的影响.     

High yield production of salidroside in the suspension culture of Rhodiola sachalinensis

Salidroside has been identified as the most potent ingredient of the Chinese medicine herb, Rhodiola sachalinensis. Since the natural supply of this herb is rapidly decreasing, we established a compact callus aggregate (CCA) strain and culturing system for high yield salidroside production. Several callus strains induced from the explants originated from root, stem, leaf and cotyledon of R. sachalinensis were established and screened for rapid growth rate, high salidroside content and easy propagation in suspension culture condition. The CCA strain was established from a callus strain initiated from the cotyledon. The kinetics of dry weight accumulation and cellular salidroside content in various culture conditions for the strain was determined. For high salidroside production, the optimal inoculum amount was 10% and the optimal concentration for 6-benzylaminopurine and indole-3-butyric acid added in the liquid medium was 5 and 2.5 mg l-1, respectively. The acidic culture medium and a faster shaking speed favored the salidroside accumulation. The addition of 2,4-D, in the liquid MS medium and the utilization of L-tyrosol for chemical feeding enhanced salidroside production. Using a proper combination of culture condition and treatment, salidroside accumulation could reach 57.72 mg g-1 dry weight, that was 5-10-fold higher than that detected in field-grown plants. The corresponding salidroside yield was 555.13 mg l-1, a level suitable for cost effective commercial production to compensate the natural resource shortage of R. sachalinensis.     

茶条槭悬浮培养体系的建立与没食子酸合成的优化条件

初步建立茶条槭（Acer ginnala）细胞悬浮培养体系：以茶条槭子叶为外植体,接种于WPM培养基中,对茶条槭愈伤组织进行诱导和继代培养。悬浮培养中,每代增长指数达到11．6,没食子酸含量达到1．518％。通过对比NT、IS、WPM、B5和MS培养基所含成分对茶条槭愈伤组织悬浮培养的影响,综合考虑悬浮细胞的生长速率和有效成分的含量,确定WPM为基本培养基。WPM培养基大量元素的浓度对细胞的生长和没食子酸的积累有显著影响,其浓度越高,促进作用越明显。3倍浓度的大量元素最有利于没食子酸的积累。蔗糖浓度为10g·L^-1最适于没食子酸的积累,浓度为30g·L^-1最适于茶条槭细胞生长和没食子酸合成。     

Bud endophytes of Scots pine produce adenine derivatives and other compounds that affect morphology and mitigate browning of callus cultures

Endophytes are found in meristematic bud tissues of Scots pine ( Pinus sylvestris L.) especially prior to growth, which would suggest their involvement in growth of the bud. To test this hypothesis, production of phytohormones by two bacterial ( Methylobacterium extorquens , Pseudomonas synxantha ) and one fungal endophyte ( Rhodotorula minuta ) was studied by mass spectrometry. The most common gibberellins, auxins, or cytokinins were not detected in the fractions studied. Instead, M. extorquens and R. minuta produced adenine derivatives that may be used as precursors in cytokinin biosynthesis. A plant tissue culture medium was conditioned with the endophytes, and pine tissue cultures were started on the media. Tetracycline inhibited callus production, which was restored on the endophyte-conditioned media. In addition, conditioning mitigated browning of the Scots pine explants. However, a decrease in tissue size was observed on the endophyte-conditioned media. Addition of adenosine monophosphate in the plant culture medium restored callus production and increased growth of the tissues, but had no effect on browning. Therefore, production of adenine ribosides by endophytes may play some role in the morphological effect observed in the pine tissues.     

Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures

The effect of explant age, plant growth regulators and culture conditions on somatic embryogenesis and rosmarinic acid production from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS) medium supplemented with 1.8–18 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) or 10.5–21 μm 1-naphthalenacetic acid and 6-benzyladenine. Only explants from young plants (with six to eight leaves) responded to the culture treatments and, in general, low light intensities (50 μmol m^–2 s^–1) favoured callus formation and induction of somatic embryos. Somatic embryos were further developed on the same medium. Heart- and torpedo-shaped embryos (1–2 mm long) were subcultured on a growth-regulator-free MS medium for maturation. Maximum rosmarinic acid accumulation in S. officinalis and S. fruticosa callus cultured on 4.5 μm 2,4-D and 4.5 μm Kin was 25.9 and 29.0 g/l, respectively. Received: 17 January 1997 / Revision received: 26 May 1997 / Accepted: 30 June 1997