Chemical potential of Aphelandra sp. cell cultures

Six different callus lines and three different suspension culture lines were established from plants of two Aphelandra species (Acanthaceae). All established lines were analyzed for secondary metabolite accumulation. A discrepancy between secondary metabolites accumulated in the plants and in the cell cultures could be observed. All established Aphelandrasp. cell cultures produced verbascoside (acteoside) as the major extractable metabolite. Time course experiments were carried out to investigate the relationship between cell growth and verbascoside production. In the present study it was shown that verbascoside accumulation was growth dependent and positively related to the presence of 2,4-D in the medium. The conditions in which verbascoside represents ca. 18% of cell culture weight have been defined. Free polyamines were detected in the cell culture lines cultivated in MS liquid medium (cysteine 10 mg l^-1, thiamine 1 mg l^-1, 2,4-D 1 mg l^-1, kinetin 0.2 mg l^-1 and sucrose 30 g l^-1). Putrescine and spermidine accumulated within 8 days to a maximum of 8.4 μmol g^-1 of dry wt and 2.6 μmol g^-1 of dry wt respectively and thereafter their concentration decreased rapidly. There was no evidence for the presence of spermine or any other type of free or conjugated polyamines in the tested cell culture lines. This revised version was published online in June 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">Cupressus</Emphasis> callus and cell suspension cultures: Effect of seiridins on their growth and sensitivity

Summary Cupressus macrocarpa and C. arizonica were examined for callus and cell culture production in vitro. Both species produced callus on agar-solidified MSCY medium supplemented with vitamins, antioxidants, 0.14 μM kinetin (KIN), and 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures of both species were established in liquid MSCY medium. Seiridin (SE) and iso-seiridin (ISE), two phytotoxic butenolides produced by Seiridium cardinale, S. cupressi, and S. unicorne, the causal agents of many canker diseases of cypress, were tested on callus or cell suspension cultures. In the medium without other plant growth regulators (PGR), SE promoted cell proliferation of cypress better than ISE, for callus initiation, callus maintenance, and cell suspension cultures. The growth rates of cypress callus tissues and suspension cultures of both cypress species on media containing 50–150 μM SE or ISE were measured. At concentrations of 50 μM and higher, growth rates increased exponentially with the SE concentration. A comparison with KIN and 2,4-D indicated that 50 μM SE promoted growth of callus tissues and cell suspension cultures more than 100 μM ISE. SE can also interact with, or counteract, KIN and 2,4-D. It was demonstrated that SE could replace KIN in the medium for C. arizonica. SE could be involved in cell enlargement and proliferation processes. The less susceptible cypress species (C. arizonica) had a high content of terpenoids than that of the more susceptible species (C. macrocarpa). SE could be a useful tool as a phytohormonal-like regulator to manipulate physiological changes at the cellular level and as an elicitor of sensitivity or tolerance of cypress germplasm to the phytotoxin.     

Establishment of <Emphasis Type="Italic">Cyperus aromaticus</Emphasis> cell suspension cultures for the production of Juvenile hormone III

Cell suspension cultures of Cyperus aromaticus were established from the yellow friable callus derived from the root explants of in vitro plantlets. Four callus cell lines were selected based on their growth index from two populations of callus cultures originated from the mother plants grown in two different locations. The selected four cell lines (Z1, Z6, P4, P9) showed uniform cell growth but produced different amounts of juvenile hormone III (JHIII). The Z1 cell line possessed fast-growing characteristics, produced a high JHIII content, and was chosen as the elite cell line for an optimization study of C. aromaticus cell suspension cultures. An inoculum cell mass of 0.3 g from 12-d cultures in 30 ml culture medium was found to be the optimum inoculum size and culture age for establishing the cell suspension culture of C. aromaticus. MS basal medium supplemented with 4.5 mg/l 2,4-D and 5.5 mg/l NAA was found to be the best medium for production of maximum cell biomass and JHIII. These results indicated that JHIII can be produced from suspension culture of C. aromaticus using a single-stage cell-culture system.     

Osmotic stress induced-capsaicin production in suspension cultures of Capsicum chinense Jacq.cv. Naga King Chili

The influence of osmotic stress on capsaicin production was investigated in cell suspension cultures of Capsicum chinense Jacq.cv. Naga King Chili, a chili species native to Northeastern India. The sterilized seeds were germinated in Murashige and Skoog medium. Two-week-old hypocotyls were excised from in vitro germinated seedlings and implanted in MS medium containing 2, 4-dichlorophenoxyacetic acid (2?mg/l), and Kinetin (0.5?mg/l) for callus induction. Capsaicin production in the suspension cultures was significantly affected using sucrose, mannitol, and NaCl in the medium. Stoichiometric analysis with different combinations of sucrose and non-sugar osmotic agent (NaCl) showed that osmotic stress was an important factor for enhancing capsaicin production in cell suspension cultures of C. chinense. The capsaicin content of 1,644.1???g?g^?1 f.wt was recorded on day 15 in cultures grown in MS medium containing 87.64?mM sucrose in combination with 40?mM NaCl. However, osmotic stress treatment at 160?mM NaCl with sucrose resulted in lowering capsaicin accumulation and separation of cell wall from their cytoplasm, under microscopic observation.     

Ginkgolide B production in cultured cells derived from Ginkgo biloba L. leaves

Callus cultures and cell suspension cultures derived from Ginkgo biloba L. leaves produced ginkgolidc B. In cell suspension cultures, the production reached a maximum by the 13th day of subculture and followed by a sharp decrease. The medium of Murashige and Skoog induced the highest ginkgolide B content in cultures while the medium of Schenk and Hildebrandt promoted cell growth. For the maximal production of ginkgolide B, cells were cultured in Murashige and Skoog medium modified to contain 1.0 mg/l of -naphthaleneacetic acid, 0.1 mg/1 of kinetin, 30 g/1 sucrose and 1.25 mM potassium phosphate with a molar ratio of ammonium to nitrate ions of 1 3.Abbreviations B5 Gamborg et al (1968) medium - GKB Ginkgolide B - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic aicd - SH Schenk and Hildebrandt (1972) medium     

Elicitation with methyl-jasmonate stimulates peruvoside production in cell suspension cultures of <Emphasis Type="Italic">Thevetia peruviana</Emphasis>

Thevetia peruviana is a small tree that produces several compounds with pharmaceutical application, among which peruvoside could be highlighted. However, these compounds are produced in low concentration in the plant, making it important to develop strategies such as plant cell culture and elicitation to obtain higher quantities of the desired product. In this work, cell suspension cultures of T. peruviana were established in four different culture media: Murashige–Skoog (MS), half Murashige–Skoog (half MS), Schenk–Hildebrandt (SH), and Gamborg (B5) to study their effect on cell growth. Cell growth kinetics were studied in SH medium, and the extracellular peruvoside production during the culture time was determined. The best culture medium for the establishment of cell suspension cultures was MS with a growth index of 3.17 ± 0.2 g g⁻¹ inoculum. The cell growth kinetics showed the four characteristic growth phases of a cell culture (lag, exponential, stationary, and death), and during none of these phases was it possible to observe peruvoside production. The elicitor effect of methyl-jasmonate (MeJ) was studied in cell suspension cultures established in SH medium. The effect of MeJ concentration and the time in which it should be applied were determined. The best results were obtained at a concentration of 100 mg l⁻¹ of MeJ applied at the beginning of the culture, which induced a peruvoside production of 8.93 mg l⁻¹ medium. The current results are the first report of an in vitro peruvoside production system.     

Culture of Isolated Single Cells from <Emphasis Type="Italic">Taxus</Emphasis> Suspensions for the Propagation of Superior Cell Populations

Single cells isolated from aggregated Taxus cuspidata cultures via enzymatic digestion were grown in suspension culture. High seeding density (4×10⁵ cells/ml) and the addition of cell-free conditioned medium were essential for growth. Doubling the concentration of the nutrients [ascorbic acid (150 g/l), glutamine (6.25 mm), and citric acid (150 g/l)] had no effect on single cell growth or viability. A specific growth rate of 0.11 days⁻¹ was achieved, which is similar to the observed growth rate of aggregated Taxus suspensions. The biocide, Plant Preservative Mixture, added at 0.2% (v/v) to all single cell cultures to prevent microbial contamination, had no significant effect on growth or viability. Following cell sorting, single cell cultures can be used to establish new cell lines for biotechnology applications or provide cells for further study.     

Oxytocin influences the production of glycyrrhizin from cell cultures of Abrus precatorius Linn.

Oxytocin, a peptide animal hormone, was used as a growth regulator to test its effect on biomass accumulation and production of secondary plant constituent glycyrrhizin in the cell cultures of Abrus precatorius. Glycyrrhizin is an important phytoconstituent of liquorice which is widely used in the pharmaceutical and food industries. Cell suspension cultures of A. precatorius were developed from leaf explant of in vitro germinated plant in Murashige and Skoog medium supplemented with 30 g/l sucrose, 1 mg/l naphthalene acetic acid and 1 mg/l kinetin. The influence of oxytocin on biomass accumulation as well as on the production of glycyrrhizin was observed in the cell cultures of A. precatorius. Treatment of A. precatorius cell cultures with 100 μg/l oxytocin, improved glycyrrhizin production up to 34.27 mg/l on the dry cell weight basis third day after oxytocin treatment, which is over four times that of the control cultures, simultaneously nearly two fold increase in the biomass 2 days after the oxytocin treatment was recorded over the control cultures.     

不同种类无机盐对灰毡毛忍冬“渝蕾1号”悬浮培养体系中细胞生长和绿原酸含量的影响

为提高灰毡毛忍冬"渝蕾1号"悬浮培养体系中绿原酸的含量,该研究探讨了B_5培养基中不同浓度的无机盐对灰毡毛忍冬"渝蕾1号"悬浮培养细胞生物量及绿原酸含量的影响,通过在悬浮培养体系中添加不同浓度的无机盐,采用重量法测定灰毡毛忍冬"渝蕾1号"悬浮培养细胞的生物量及采用高效液相色谱法测定绿原酸的含量。结果表明:当硝态氮和铵态氮配比与B_5培养基中硝态氮和铵态氮配比一致时,即NO_3~-/NH_4~+摩尔比值为13∶1时,培养体系有利于细胞的生长和绿原酸的积累。当KNO_3浓度为3.5 g·L~(-1)时,细胞生物量达到最大,为19.26 g·L~(-1);当KNO_3在较低浓度(0.5 g·L~(-1)和1.5 g·L~(-1))时,积累较多的绿原酸。NO_3~-的两项研究结果均与对照浓度(2.5g·L~(-1))有一定的差异。另外,对(NH_4)_2SO_4来说,在高于对照浓度0.134 g·L~(-1),即浓度为0.268 g·L~(-1)时,生物量和绿原酸含量都达到了最大。P、Ca、Mg三种矿质元素的研究结果表明,当Na H_2PO_4·2H_2O浓度为0.10 g·L~(-1)、Ca Cl_2的浓度为0.20 g·L~(-1)时,细胞的生长和绿原酸的积累均可达到最大值;而对Mg~(2+)来说,低浓度促进细胞的生长,高浓度促进绿原酸的积累。兼顾细胞生物量和绿原酸含量两个指标,需选择适中的浓度。这些结果均与对照浓度有一定的差异。这说明灰毡毛忍冬"渝蕾1号"悬浮细胞所需无机盐的浓度与B_5培养基无机盐的浓度有一定的差异,选择适宜的浓度可促进其悬浮细胞的生长及次生代谢产物绿原酸的积累。该研究结果为绿原酸的工业化生产打下了基础。     

Rollinia mucosa cell suspension cultures: Establishment and growth conditions

Different types and concentrations of plant growth regulators were tested in order to obtain the best callus and cell suspension culture growth conditions of Rollinia mucosa (Jacq.) Baill. (Annonaceae). Picloram was shown to be the most efficient for induction and production of friable calluses, independent of the concentration used. Cellular morphology and viability, fresh and dry weights, pH and medium sugar concentration were determined for cell suspension cultures. Dissimilation curves were used for the characterization of the growth of cell suspension cultures. Picloram provided the most rapid growth and produced the highest biomass, with little variation in morphology (differentiated cells). It also provided the highest dissimilation, when compared with cell suspension cultures maintained in media with 2,4-D or NAA + BA + GA₃. Stable cell suspension cultures can be established in MS medium supplemented with 20.8 μM picloram. This revised version was published online in August 2006 with corrections to the Cover Date.     

Production of a plasmin inhibitory substance by Scopolia japonica suspension cultures

A potent inhibitory agent against human plasmin, fibrinolytic proteinase, has been found in the extracts of callus tissue of Scopolia japonica. Effects of cultural conditions on cell growth and production of the plasmin inhibitory substance by this cell line in suspension cultures were examined in MurashigeSkoog's medium. More than l.5 mg of the inhibitor, as t-amino cyclohexane carboxylic acid, a synthetic plasmin inhibitor, were observed to accumulate per ml of medium containing 0.83 g of NH₄NO₃ and 7.6 g of KNO₃ per liter as well as suitable levels of growth hormones. Addiction of antibiotics and deformers were examined in preliminary tests for large scale cultivation. Semicontinuous culture on a small scale in a glass cylinder, was also tested and growth rate of 1.29 g/liter/day (by dry wt) was obtained. Plasmin inhibitory activities in the extracts of the results intact plant and in cultured cells of S. japonica were compared and the results indicated that cell suspension culture was superior to extraction the natural plant for inhibitor production.     

Growth and production characteristics of permeabilized Coleus blumei cells in immobilized fed-batch culture

Summary Permeabilized Coleus blumei cells were cultivated in an immobilized state to study the effect of dimethyl sulfoxide (DMSO) concentrations and growth regulators on cell growth and rosmarinic acid (RA) production characteristics. Luffa (the fibrous skeleton of mature fruit of Luffa cylindrica) was a good support matrix for cell immobilization because of its high void volume. Maximum cell loading capacity was 1.33 g dry cell weight (DCW)/g dry Luffa. The experiments were done in shake flasks with no free medium. The medium was supplied in a fed-batch mode to avoid the flotation of Luffa pieces. The sucrose in the medium was completely hydrolyzed to glucose and fructose without any sugar accumulation in the medium. The cell viability was slightly higher in the cells on top of the Luffa than those in the middle. Cell growth rate and rosmarinic acid (RA) production were approximately half that obtained in cell suspension cultures. Cell yield (g DCW/g glucose) was similar to that of cell suspension cultures. The absence of growth regulators did not promote an increase of RA production but did decrease the cell mass. The second step preconditioning with 0.5% DMSO did not improve the cell's adaptability to higher DMSO concentrations and the cell mass did not increase with 2.5% DMSO.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

Identification and production of flavonoids in a cell suspension culture of Scutellaria baicalensis G.

A high yielding cell line of Scutellaria baicalensis G. has successfully been developed to produce flavonoids. Major components of the flavonoids were identified as baicalin and wogonin-7-O-glucuronic acid by a series of instrumental analyses using UV, IR, MASS, and NMR. After 12 days in suspension culture, the cell growth reached 14 g ^DW/l, and baicalin and wogonin-7-O-glucuronic acid were obtained in concentrations of 2.9 g/l and 1.07 g/l, respectively. The culture temperature was found to be an important parameter for improving production yield of the flavonoids. The yield of baicalin was observed to increase to 4.2 g/l by shifting the temperature from 30 °C to 25 °C after 72 h of suspension culture.Abbreviations DW cell dry weight - FW cell fresh weight - 2,4-D 2,4-dichlorophenoxyacetic acid - PSH medium phytohormone added Schenk and Hildebrandt medium - FPM a modified Schenk and Hildebrandt medium for flavonoid production     

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     

Development of callus and suspension cultures of potato resistant to NaCl and mannitol and their response to stress

Callus and suspension cultures adapted to various concentrations of NaCl or mannitol were developed from the cultivated potato Solanum tuberosum cv. Desire. Growth of the calli was less inhibited by mannitol than by iso-osmotic concentrations of NaCl. Reduction of growth by both NaCl and mannitol was considerably lower in osmotically adapted calli than in non-adapted ones. Salt-adapted suspension cultures that grew in the medium to which they had been originally adapted had a shorter lag in growth as well as a shorter time required to achieve the maximum growth, as compared with non-adapted cells. Suspension cultures adapted to NaCl concentrations higher than 150 mM were obtained only after preadaptation to osmotic stress. Adaptation of these cells was found to be stable. Accumulation of Na⁺ was lower and level of K⁺ was more stable in osmotically adapted than in non-adapted calli, when both were exposed to salt. Potassium level in NaCl-adapted calli exposed to saline medium was lower than that in non-adapted calli in standard medium. The maximum of Cl^– and Na⁺ accumulation was reached at higher external salt concentration in salt-adapted than in non-adapted suspension cultures. In both callus and suspension cultures, Cl^– accumulated more than Na⁺. Potassium level decreased more in non-adapted than in NaCl-adapted suspension cultures. The decrease of osmotic potential in osmotically adapted calli exposed to mannitol and in salt-adapted calli and suspension cultures exposed to salt was correlated to the increase of the external concentration. Such a correlation was not found in osmotically adapted calli exposed to salt. Non-electrolytes were found to be the main contributors to the decrease is osmotic potential in both callus and suspension cultures.     

Establishment of a system of high-frequency embryogenesis from long-term cell suspension cultures of rice (Oryza sativa L.)

Summary Suspension cultures which maintained embryogenic potency for more than 18 months were established from excised immature embryos of rice (Oryza sativa L. cv. Konansou). The cultures were subcultured every three days in N6 medium supplemented with proline (10 mM), casein hydrolysate (300 mg/l), sucrose (30 g/l) and 2,4-D (1 mg/l). The frequency of embryogenesis from the embryogenetic suspension cultures reached about 90% when cell clusters (about 1 mm in diameter) were transferred to a solid medium which consisted of N6 medium, NAA (1 mg/l), kinetin (5 mg/l), sucrose (30 g/l) and Gelrite (2 g/l). When smaller clusters of cells (approximately 200–400 m in diameter) were transferred to a liquid medium which consisted of salts of N6 medium diluted with an equal volume of water plus sucrose (45 g/l), NAA (0.01 mg/l) and 4-PU (0.1 mg/l) at a cell density of 13 clusters/ml in 2 ml of medium, somatic embryogenesis was initated at high frequency (about 50%). Morphological evidence is provided to demonstrate that the regeneration occurred via embryogenesis. This is the first report of high-frequency embryogenesis in suspension cultures of rice cells.     

Secoiridoid content of Blackstonia perfoliata in vivo and in vitro

Summary This study reports the analysis of secondary metabolites of gentiopicrin, swertiamarin, and sweroside in shoot and root cultures of yellow wort (Blackstonia perfoliata), which were initiated from seeds, grown on Murashige and Skoog (MS) medium. Shoot cultures of B. perfoliata were inoculated with suspension of Agrobacterium rhizogenes strain A4M70GUS and hairy roots appeared at the infected sites after 3 wk of inoculation. Tips of adventitious roots of B. perfoliata were grown on hormone-free MS medium and three clones of the transformed roots regenerated shoots spontaneously. Gentiopicrin, swertiamarin, and sweroside were detected in both roots and shoots of B. perfoliata in vitro and in vivo, but gentiopicrin was found to be the major compound. The concentration of growth regulator in the medium affected the production of secoiridoids in B. perfoliata in vitro, where the level of gentiopicrin was higher in plants grown in the presence of indole-3-butyric acid, but the presence of 6-benzylaminopurine was inhibitory to secoiridoid production.     

The formation of flavonoids in cell suspension cultures ofPrunus x yedoensis matsum

Two lines of the red and pale yellow cell suspension cultures, prepared fromPrunus x yedoensis Matsum. callus induced by Murashige and Skoog's (1962) basal medium supplemented with 2, 4-dichlorophenoxyacetic acid (2, 4-D, 1.0 mg/l), kinetin (0.1 mg/l) and sucrose (30 g/l), were maintained on Schenk and Hildebrandt medium as modified by Mitchell and Gildow (1975). The red cell suspension culture produced cyanidin 3-monoglucoside, 5, 4′-dihydroxy-7-methoxyisoflavone 4′-glucoside (prunetrin), isoquercitrin, catechin, epicatechin, and procyanidin B-1, B-2, B-3 and B-4, while the pale yellow cells produced only a small amount of catechin and epicatechin as main flavonoids. These flavonoid compounds found in the red cell culture were present also in maturePrunus leaves. Maximum growth and maximum amount of total phenol and proanthocyanidin (procyanidins) were obtained with 0.3 mg/l of both 2,4-D and kinetin. Maximum concentration of anthocyanin was also obtained with 0.3 mg/l 2, 4-D regardless of kinetin concentration. Accumulation of proanthocyanidin was markedly stimulated by low concentrations of phosphate, which reduced growth by about half, and also by high concentrations of inorganic nitrogen. Production of both anthocyanin and proanthocyanidin was reduced by lowered nitrogen levels. Cell growth and production of all phenolics were inhibited when ammonium ion replaced nitrate in the medium.     

Growth and anthocyanin production by carrot suspension cultures grown under chemostat conditions with phosphate as the limiting nutrient

Chemostat cultures of carrot suspension cultures, where growth was limited by the concentration of phosphate in the input medium, were achieved by replacing a fixed proportion of the culture with fresh medium at daily intervals. In the range 0.05–0.30mM phosphate in the input medium and at a specific growth rate of 0.357 days^?1, steady-state culture density but not anthocyanin in the cells was strictly proportional to the input phosphate concentration with no intercept. At a phosphate concentration of 0.10mM and growth rates from 0.105 to 0.430 days^?1, the steady-state culture density could not be described by Monod's model of chemostat cultures, but could be described by Nyholm's model. The steady-state levels of anthocyanin were not strictly proportional to the steady-state biomass under all conditions, showing that anthocyanin production is not completely growth associated.