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.     

Production of insect juvenile hormone III and its precursors in cell suspension cultures of the sedge, Cyperus iria L.

 Juvenile hormones (JHs) are sesquiterpenoids that regulate metamorphosis and reproduction in most insect species. There has been one report of an insect JH in plants: JH III, methyl-10R,11-epoxy-3,7,11-trimethyl 2E, 6E-dodecadienoate, has been identified in two sedge species, Cyperus iria L. and C. aromaticus (Ridley) Mattf and Kük. This is the first report of callus and cell suspension cultures derived from C. iria. Farnesol and methyl farnesoate, two biosynthetic intermediates of JH III in insects, as well as JH III have been identified in suspension culture cell extracts by gas chromatography-mass spectroscopy. These cultures thus provide a useful in vitro model to investigate the biosynthesis of JH III in the sedge, C. iria. Received: 30 October 1998 / Revision received: 11 February 1999 / Accepted: 3 March 1999     

In vitro metabolism of juvenile hormone III and juvenile hormone III bisepoxide by Drosophila melanogaster and mammalian cytosolic epoxide hydrolase

In vitro metabolism of juvenile hormone III (JH III) and juvenile hormone III bisepoxide was investigated using purified mouse liver cytosolic epoxide hydrolase (cEH) and cell fractions from Drosophila melanogaster. JH III was metabolized faster than JH III bisepoxide by epoxide hydrolase activity in D. melanogaster cell fractions and by cEH. After incubation with JH III bisepoxide, all cell fractions and cEH produced epoxy-diol, cis- and trans-tetrahydrofuran-diols, and tetraol as metabolites. An increase in the concentration of cEH resulted in an increase in the proportion of tetraol as a JH III bisepoxide metabolite but this trend was not observed in the D. melanogaster cell fractions. Differences between cell fractions in the metabolism of JH III and JH III bisepoxide suggests the presence of juvenile hormone epoxide hydrolase isozymes.     

Improved paclitaxel and baccatin III production in suspension cultures of Taxus media

A cell suspension culture of Taxus media was established from a stable callus line of this species. The growth rate and production of paclitaxel and baccatin III of this cell suspension were significantly increased during the shake flask culture in its respective optimum media for cell growth and product formation, which were selected after assaying 24 different culture media. The highest yields of paclitaxel (2.09 mg L(-1)) and baccatin III (2.56 mg L(-1)) in the production medium rose (factors of 7.0 and 3.0, respectively) in the presence of methyljasmonate (220 microg g(-1) FW). When the elicitor was added together with mevalonate (0.38 mM) and N-benzoylglycine (0.2 mM), the increase in the yields of paclitaxel and baccatin III was even higher (factors of 8.3 and 4.0, respectively). Thereafter, a two-stage culture for cell suspension was carried out using a 5-l stirred bioreactor running for 36 days, the first stage being in the cell growth medium until cells entered their stationary growth phase (12 days) and the second stage being in the production medium supplemented with the elicitor and two putative precursors in the concentrations indicated above. Under these conditions, 21.12 mg L(-1) of paclitaxel and 56.03 mg L(-1) of baccatin III were obtained after 8 days of culture in the production medium.     

Polysaccharide elicitors enhance anthocyanin and phenolic acid accumulation in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

The effects of yeast extract and selected polysaccharide elicitors on secondary metabolite production, particularly of anthocyanin and phenolic acid, in cell suspension cultures of Vitis vinifera were investigated. All elicitors either maintained or promoted cell growth in culture. Overall, secondary metabolite production in V. vinifera cell suspension cultures responded differently to different elicitors. Chitosan, pectin, and alginate enhanced production of anthocyanin within 13 days of culture with levels of 2.5-, 2.5-, and 2.6-fold increase, respectively, over that of control. Chitosan, alginate, and gum arabic significantly promoted accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol, in V. vinifera cultures, as well as in the culture medium. Intracellular phenolic acid production was significantly enhanced by alginate and chitosan, with 1.7- and 1.5-fold levels, respectively, of that of control. Extracellular phenolic acid production was also significantly increased in the presence of chitosan and gum arabic, with levels of 3.3- and 1.7-fold higher, respectively, than those of control. In addition, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was enhanced in the presence of elicitors, and this was positively correlated with increased accumulation of anthocyanin in V. vinifera cell suspension cultures.     

Influence of endophytic fungal elicitation on production of inophyllum in suspension cultures of <Emphasis Type="Italic">Calophyllum inophyllum</Emphasis> L.

The influence of dried cell powder and culture filtrates of endophytic fungi on production of inophyllum in cell suspension cultures of leaf- and stem-derived callus of Calophyllum inophyllum was investigated. Two fungi, Nigrospora sphaerica and Phoma spp., endophytic to C. inophyllum, were isolated from leaf tissues, and were identified by both 18S rRNA gene amplification and sequencing. Elicitation of suspension cultures of both callus types of C. inophyllum with dried cell powder and culture filtrates of both fungi consistently elicited production of inophyllum A, B, C, and P. In comparison to stem-derived callus, suspension cultures of leaf-derived callus enhanced production of most inophyllum. Of the four inophyllum studied, the highest production of inophyllum A, C, and P was achieved in elicited suspension cultures of leaf-derived callus. Suspension cultures of stem-derived callus enhanced production only of inophyllum B. When suspension cultures of leaf-derived callus were elicited with 40 mg dried cell powder of Phoma spp., a level of 751-fold (6.84 mg/100 g elicited biomass) of inophyllum A was produced, compared to control. Whereas, a level of 414-fold (6.22 mg/100 g elicited biomass) of inophyllum B was produced when suspension cultures of stem-derived callus were elicited with 20 mg dried cell powder of N. sphaerica. When compared to control, a 10% culture filtrate of N. sphaerica in suspension cultures of leaf-derived callus elicited inophyllum C and P production by 928-fold (7.43 mg/100 g elicited biomass) and 750-fold (1.5 mg/100 g elicited biomass), respectively.     

The effect of conditioned medium obtained from <Emphasis Type="Italic">Scenedesmus subspicatus</Emphasis> on suspension culture of <Emphasis Type="Italic">Silene vulgaris</Emphasis> (<Emphasis Type="Italic">Caryophyllaceae</Emphasis>)

The effect of culture filtrate (conditioned medium, CM) containing cell exudates obtained from green alga, Scenedesmus subspicatus, on cell suspension of dicotyledonous plant Silene vulgaris was examined. The addition of diluted CM to the modified MS medium, supplemented with dicamba and BAP, stimulates cell biomass production. The biomass was composed of association of single non-dividing cells, cells during mitosis stage and cellular aggregates. Silene cells began mitotic divisions earlier in the presence of CM in medium when compared to control treatments. Results of performed bioassay showed that some factor or factors released by green alga to the culture medium could be responsible for sustained proliferation of phylogenetically distant species cells. Although it is still unclear which culture constituent influenced most the mitotic response of Silene suspension, results point at versatile stimulatory character of green alga exudates in higher plant cell culture.     

条件培养液对红豆杉细胞Paclitaxel生产的促进作用

在两步法红豆杉（Taxus chinensis)细胞悬浮培养体系的生产阶段,加入从生长阶段悬浮培养物中制得的条件培养液（conditioned Medium,CM)既能促进细胞的生长,又能提高紫杉醇（paclitaxel)的产率,解决了生产培养时,细胞生长受抑制的问题,特别是,取自生长12天的细胞悬浮培养物的CM按体积分数为25％添加到新鲜生产培养基中时,可使细胞紫杉醇最高产量达28．5mg/L,细胞干重达32．3g/L,分别是对照的2．4倍和2．2倍,对CM中的蔗糖,果糖,NO3－和PO4－3等的含量的进行了分析。     

Establishment of anthocyanin-producing cell suspension cultures of Cleome rosea Vahl ex DC. (Capparaceae)

The effects of different levels of Murashige and Skoog (MS) basal medium, 2,4-dichlorophenoxyacetic acid (2,4-D), and sucrose on anthocyanin production and biomass accumulation of cell suspension cultures of Cleome rosea were investigated. Cultures were established in liquid MS medium containing 30 g l⁻¹ sucrose and supplemented with 0.90 μM 2,4-D. Proliferating cell suspension cultures achieved the highest growth capacity, a fourfold increase in biomass accumulation, following subculture at the exponential growth phase, 14–18 days of culture. Moreover, the presence of 2,4-D was essential for anthocyanin production and biomass accumulation. On the other hand, increasing levels of sucrose above 30 g l⁻¹ resulted in a drastic reduction in biomass accumulation. Anthocyanin production was highest in cell suspension cultures grown on half-strength MS medium (1/2 MS), 30 g l⁻¹ sucrose, and 0.45 μM 2,4-D. These cell suspension cultures were mainly composed of small aggregates of spherical cells with similar morphology observed in anthocyanin-producing and non-producing cultures. Moreover, microscopic analysis of anthocyanin-producing cultures showed the presence of mixtures of non-pigmented, low-pigmented, and high-pigmented cells.     

Solasodine production from cell culture of <Emphasis Type="Italic">Solanum hainanense</Emphasis> Hance

Stem explants of Solanum hainanense Hance plantlets were cultured on Murashige and Skoog solid medium, containing 3% (w/v) sucrose, supplemented with 0.1 mg/L benzylaminopurine (BAP) and 1.0 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) for callus production. To establish the cell suspension culture, 3 g of fresh callus were cultured in 50 mL of the same medium, but without a solid agent, at an agitation speed of 120 rpm. Every 15 mL of culture was sub-cultured in fresh MS liquid medium for maintenance. The cell biomass of S. hainanense reached a maximum value of 18.47 g after 4 weeks of culture on the same MS medium, but with the sucrose content increased to 4%, at an agitation speed of 150 rpm, with 20 mL of inoculum. Analysis via high performance liquid chromatography (HPLC) showed that the solasodine content in the cell suspension after 4-weeks old (121.01 mg/g) was higher than that of in planta 1-year old roots (20.52 mg/g) by approximately 6-fold.     

Induction,characterization, and NMR-based metabolic profiling of adventitious root cultures from leaf explants of <Emphasis Type="Italic">Gynura procumbens</Emphasis>

Gynura procumbens is a medicinal plant used in South East Asia to treat various ailments such as rash, hemorrhoids, inflammation, and diabetes. In order to develop a large-scale culture system for G. procumbens biomass containing bioactive compounds, adventitious root cultures were initiated from leaf explants. Murashige and Skoog (MS) media containing different compositions of indole-3-butyric acid (IBA), 1-naphthalene-acetic acid (NAA), and combinations of both plant growth regulators (PGRs) were evaluated for root induction. A combination of 3 mg/l NAA + 1 mg/l IBA gave the highest root induction (48%) as compared to other PGRs treatments after 9 weeks of incubation period. Subsequently, the adventitious roots were established in liquid culture containing MS medium and the combination of 3 mg/l NAA + 1 mg/l IBA. A study on the medium strength, sucrose concentration, pH, and light versus dark was conducted to optimize the in vitro culture conditions. The results showed that differences in MS medium strength from half to double strength, and light or dark condition did not significantly affect the biomass production, while the initial medium pH of 5.5 and 2% w/v sucrose concentration were most suitable for the root culture growth. Nuclear magnetic resonance (NMR) spectroscopy was performed to characterize the metabolite content in the root cultures of G. procumbens. Among the elucidated metabolites were some phenylpropanoids identified as caffeic acid, chlorogenic acid, and 3,5-di-O-caffeoylquinic acid which might be the bioactive compounds associated to the folk use of this plant.     

Genetic transformation of <Emphasis Type="Italic">Solanum chrysotrichum</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and the production of antifungal saponins

Solanum chrysotrichum (Solanaceae) synthesizes a family of six antifungal spirostanol saponins designated as SC-1 to SC-6. The production of saponins by wild-type plants is variable depending on the environmental conditions. In order to develop an in vitro system for the sustained production of these saponins, transformed cell suspension cultures of S. chrysotrichum were established from nodal explants of 3-mo-old plantlets by infecting with the Agrobacterium tumefaciens strain C58/pBI12. From these cultures, kanamycin-resistant and phytohormone-independent cell suspension line C58 5.1.1 was obtained. PCR and Southern blot analyses were used to confirm the integration of the wild-type T-DNA into the plant genome. Batch cultures of the C58 5.1.1 cell line were grown in phytohormone-free MS liquid medium for 25 d. First-order growth kinetics and the production of the antifungal saponins (SC-2, SC-3, and SC-4) were determined by dry weight and quantified by HPLC, respectively, from the cells as well as the culture medium. Based on the cell biomass, the specific growth rate was 0.09 d⁻¹ and the yield of SC-2 reached 5.5% of dry weight, representing 40 times higher amount than that produced in plant leaves. SC-3 was recovered with a maximum yield of 0.9% of dry weight, whereas SC-4 was accumulated at 1.1% of dry weight. Saponins SC-2 and SC-3 were also excreted into the culture medium in low concentrations.     

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.     

Cell growth stimulating effect of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> spores and their potential application for Chinese hamster ovary K1 cell cultivation

In this work, water-soluble extracts of Ganoderma lucidum spores (Gls), a Chinese medicinal herb that possesses cell growth stimulating function, were found to be an effective growth factor for Chinese hamster ovary (CHO) cell cultivation. The Gls extract was prepared and supplemented to CHO K1 cell culture media with various serum levels. Our results obtained from both the static culture and the spinner-flask suspension culture showed that use of small-amount Gls extract effectively promoted cell growth and suppressed cell apoptosis induced by serum deprivation with normal cell cycle maintained in a low-serum medium. The low-serum medium containing 1 % (v/v) fetal bovine serum (FBS) and 0.01 % (w/v) Gls extract showed a comparable performance on both cell growth and fusion protein productivity with the conventional CHO culture medium containing 10 % (v/v) FBS and a commercial serum-free medium. This is the first study of the potential of Gls extracts for use as an alternative cell growth factor and nutrient for CHO cells. The findings have presented a new approach to economic cultivation of CHO cells for therapeutic protein production.     

Enhanced taxol production and release in Taxus chinensis cell suspension cultures with selected organic solvents and sucrose feeding

Suspension culture of Taxus chinensis cells was carried out in aqueous-organic two-phase systems for the production and in situ solvent extraction of taxol (paclitaxel). Three organic solvents, hexadecane, decanol, and dibutylphthalate, were tested at 5-20% (v/v) in the culture liquid. All of these solvents stimulated taxol release and the yield per cell, though decanol and higher concentrations of the other two solvents depressed biomass growth significantly. Ten percent dibutylphthalate was the optimal solvent for improving taxol production and release with minimal cell growth inhibition. The time of solvent addition to the culture also affected taxol production, with the addition during the late-log growth phase being most favorable. By feeding sucrose to the culture near the stationary growth phase, the cell growth and taxol production period was extended from 27 to 42 days. The combining of the two-phase culture and sucrose feeding increased the taxol yield by about 6-fold compared with the single-phase batch culture, to 36.0 +/- 3.5 mg/L, with up to 63% taxol released. This study shows that in situ solvent extraction combined with nutrient feeding is an effective process strategy for production and recovery of secondary metabolites in plant cell suspension culture.     

In vitro production of gymnemic acid from cell suspension cultures of Gymnema sylvestre R. Br

Gymnema sylvestre is an important medicinal plant that bears bioactive compound namely gymnemic acids. The present work deals with the optimization of a cell suspension culture system of Gymnema sylvestre for the production of biomass and gymnemic acid, which has anti‐diabetic properties. We investigated the effect of inoculum densities (2.5–20.0 g/L), the strength of the Murashige and Skoog (MS) medium (0.25–2.0), carbon source (sucrose, glucose, fructose, maltose), and the concentration of the sucrose (1–8% w/v) to determine their effects on biomass accumulation and production of gymnemic acid. Overall, 10 g/L of inoculum density, full‐strength MS medium supplemented with 2,4‐dichlorophenoxy acetic acid (2.0 mg/L) and Kinetin (0.1 mg/L), and 3% w/v sucrose was found best for the accumulation of biomass and gymnemic acid content (9.95 mg/g dry weight). The results of the current study will be useful for bioprocess and biochemical engineers for large‐scale production of gymnemic acid in cell culture.     

Establishment of Croton stellatopilosus suspension culture for geranylgeraniol production and diterpenoid biosynthesis

Diterpenoids in higher plants are biosynthesized from isoprene units obtained from two distinct pathways: the mevalonate pathway and the deoxyxylulose phosphate pathway. The metabolic partitioning of both pathways in plant species is dependent upon the type of culture. In order to study the diterpenoid biosynthesis in Croton stellatopilosus cell culture, callus culture was firstly induced from C. stellatopilosus young leaves in Murashige and Skoog (MS) medium in the presence of 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg/l benzyladenine (BA), 3% (w/v) sucrose and 0.8% (w/v) agar. The suspension culture was further induced from its callus in the same medium without gelling agent. Detection of diterpenoid accumulation by gas chromatography-mass spectrometry revealed that a cell culture could accumulate a low amount of geranylgeraniol (GGOH) and a high content of fatty acids and phytosterols. To improve the GGOH production, the culture conditions were optimized by medium manipulation in terms of hormonal factors. The growth rates of cell cultures were similar in all kinds of media. The GGOH production curve indicated that GGOH plays an important role as a primary metabolite in the cell culture. The optimum medium for GGOH production was MS medium supplemented with 2.0 mg/l 2,4-D and 2 mg/l BA that could produce GGOH with a yield of 1.14 mg/g FW.     

Co-production of biomass and metabolites by cell retention culture of <Emphasis Type="Italic">Leuconostoc citreum</Emphasis>

Cell retention culture of lactic acid bacterium Leuconostoc citreum was carried out in a fermentor equipped with an internal ceramic filtration system to co-produce biomass and metabolites. The filtration system was composed of porous ceramic filter module with pore size of 0.1 μm and total surface area of 330 cm². High cell density cultivation of L. citreum was achieved within the fermentor, while extracellular metabolites such as mannitol and d-lactic acid were produced through the filter with high productivities. In batch culture of L. citreum using a medium containing 50 g/L of glucose and 100 g/L of fructose, the maximum optical density (OD) monitored at 660 nm was 13 with 65 g/L of mannitol and 38 g/L of lactic acid. In cell retention culture of L. citreum with dilution rate of 0.07 h⁻¹, OD increased to 75, which was 6 times higher than that in batch culture. The concentrations of mannitol and lactic acid increased to 85 and 45 g/L, respectively, and were maintained throughout the cultivation to 105 h. By increasing dilution rate to 0.13 h⁻¹, the productivities of mannitol and lactic acid increased to 8.5 and 4.2 g/L/h, respectively, which were 2.7 to 3 times higher than those in batch culture, suggesting that cell retention culture using internal filtration system is highly effective for co-production of useful cell biomass and various extracellular metabolites.     

Identification of methyl farnesoate from in vitro culture of the retrocerebral complex of adult females of the moth, Heliothis virescens (Lepidoptera: Noctuidae) and its conversion to juvenile hormone III

Gas chromatographic-mass spectral analysis of extracts obtained from in vitro culture of isolated retrocerebral complexes obtained from adult females of the moth Heliothis virescens resulted in identification of methyl farnesoate as well as juvenile hormone III (JH III) but not JH III acid. Inhibition of JH biosynthesis by incubation of tissue in synthetic Manduca sexta allatostatin (Manse-AST, pGlu-Val-Arg-Phe-Arg-Gln-Cys-Tyr-Phe-Asn-Pro-Ile-Ser-Cys-Phe-COOH) reduced production of these chemicals to negligible levels. However, incubation of tissue in the presence of Manse-AST plus farnesol resulted in production of significant amounts of both methyl farnesoate and JH III. Tissue incubated in the presence of Manse-AST plus methyl farnesoate produced only JH III. The results indicated that methyl farnesoate is naturally produced by the corpora allata of adult females of Heliothis virescens. However, tissue incubated in the presence of Manse-AST plus JH III acid also produced JH III in amounts equivalent to that produced by tissue incubated with methyl farnesoate. Thus, both methyl farnesoate and JH III acid could serve as a precursor for biosynthesis of JH III.     

Characterization of aggregate size in Taxus suspension cell culture

Plant cells grow as aggregates in suspension culture, but little is known about the dynamics of aggregation, and no routine methodology exists to measure aggregate size. In this study, we evaluate several different methods to characterize aggregate size in Taxus suspension cultures, in which aggregate diameters range from 50 to 2,000 μm, including filtration and image analysis, and develop a novel method using a specially equipped Coulter counter system. We demonstrate the suitability of this technology to measure plant cell culture aggregates, and show that it can be reliably used to measure total biomass accumulation compared to standard methods such as dry weight. Furthermore, we demonstrate that all three methods can be used to measure an aggregate size distribution, but that the Coulter counter is more reliable and much faster, and also provides far better resolution. While absolute measurements of aggregate size differ based on the three evaluation techniques, we show that linear correlations are sufficient to account for these differences (R ² > 0.99). We then demonstrate the utility of the novel Coulter counter methodology by monitoring the dynamics of a batch process and find that the mean aggregate size increases by 55% during the exponential growth phase, but decreases during stationary phase. The results indicate that the Coulter counter method can be routinely used for advanced process characterization, particularly to study the relationship between aggregate size and secondary metabolite production, as well as a source of reliable experimental data for modeling aggregation dynamics in plant cell culture.