Effect of chitosan elicitation and media components on the production of anthraquinone colorants in madder (Rubia akane Nakai) cell culture

To increase the production of anthraquinone colorants in madder (Rubia akane Nakai) cell culture, the effects of elicitation on the colorant production were investigated. Chitosan was the best biotic elicitor among nine plant derived and microbial derived polysaccharides. When elicited with 25 mg/L chitosan, the total production was increased approximately two times in a seven-day culture as compared to that in the unelicited cells. Anthraquinone production was increased in proportion to the contact period up to day 3. Maximum anthraquinone colorants were obtained with 3-day treatment of chitosan. During chitosan elicitation, the total production was increased 1.3 times in MS medium containing galactose as compared to that containing sucrose. The degree of deacetylation in chitosan and the use of growth regulator or addition of precursor did not affect the production of anthraquinone colorants. When madder cells were elicited at optimum condition, anthraquinone concentration and specific anthraquinone content increased 1.3 times (0.69 g/L) and 2.2 times (0.32 g/g DCW), respectively.     

Optimization of culture conditions and continuous production of chitosan by the fungi,Absidia coerulea

The production of chitosan from the mycelia ofAbsidia coerulea was studied to improve cell growth and chitosan productivity. Culture conditions were optimized in batch cultivation (pH 4.5 agitator speed of 250 rpm, and aeration rate of, 2 vvm) and the maximum chitosan concentration achieved was 2.3 g/L under optimized conditions. Continuous culture was carried out successfully by the formation of new growth spots under optimized conditions, with a chitosan productivity of 0.052 gL⁻¹ h⁻¹, which is the highest value to date, and was obtained at a dilution rate of 0.05 h⁻¹. Cell chitosan concentrations reached about 14% in the steady state, which is similar to that achieved in batch culture. This study shows that for the continuous culture ofAbsidia coerulea it is vital to control the medium composition.     

Asiaticoside production from centella (Centella asiatica L. Urban) cell culture

Petiole explants of centella plants (Centella asiatica L. Urban) were cultured on Murashige and Skoog (MS) solid medium containing 20 g/L sucrose, supplemented with 1.0 mg/L benzylaminopurine and 1.0 mg/L naphthaleneacetic acid for callus production. To establish a cell suspension culture, 2 g of fresh callus was cultured in 50 mL of the same medium but without solid agent at a 100 rpm agitation speed. Every 2 g of culture was subcultured in fresh MS liquid medium for maintenance. After 24 days of culture at a 120 rpm agitation speed, the centella cell biomass reached a maximum of 9.03 g/50 mL on the same MS medium with 30 g/L sucrose and a 3 g inoculum size. A high performance liquid chromatography analysis showed that asiaticoside content in 24-day old suspension cultured cells (45.35 mg/g dry weight) was significantly higher (4.5 fold) than that of in planta leaves (10.55 mg/g dry weight).     

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.     

Alginate promotes production of various enzymes by Catharanthus roseus cells

When 1.0 g/l of alginate was added to a Catharanthus roseus L. cell culture, many proteins were released from the cells as detected by SDS polyacrylamide gel electrophoresis. In particular, production and/or release of 5′-phosphodiesterase (5′-PDase), catalase and chitinase by C. roseus L. cells were promoted by the addition of alginate. The promotive effects of alginate on 5′-PDase production were observed for various C. roseus cell lines and similar results were obtained when different alginates with various mannuronate/guluronate ratios and viscosities were used. In contrast, agar, agarose, and chitosan did not promote 5′-PDase production. The promotion of 5′-PDase production was not due to cell mutation, the alginate acted rather as a kind of elicitor. During 82 subcultures (577 days) in Murashige and Skoog medium containing 1.0 g/l of alginate, production and release of 5′-PDase by C. roseus L. cells were promoted without inhibition of cell growth. Received: 27 February 1997 / Revision received: 10 July 1997 / Accepted: 20 July 1997     

In vitro production studies with a wild-type Helicoverpa baculovirus

The potential use of a wild-type Helicoverpa baculovirus as a biopesticide, using insect cell culture for its production, has been investigated. A Helicoverpa zea cell line was adapted to grow in suspension culture using a serum-free medium, SF900II and serum supplemented SF900II. The serum supplemented cells were infected with a wild-type nuclear polyhedrosis virus of Helicoverpa armigera (HaNPV), at different stages of growth, in conditioned and tresh medium, to determine the effect of cell density on polyhedra production. Cultures infected at low cell densities, produced similar yields of virus (20–40 PIB/cell), irrespective of medium conditions. However, in infections which occurred at high cell densities, there was a 16-fold improvement in cell specific yields, when the spent medium was renewed with fresh medium prior to infection. Results indicated that only 60–70% of the viable cells in a culture produced polyhedra as a result of infections.     

Promotion effect of alginate on chitinase production by Wasabia japonica

Summary When alginate (1.0 g/l) was added to Wasabia japonica cell culture, cell growth was slightly inhibited (11–17%) but both the chitinase production and the specific chitinase productivity increased. Similar results were also observed when chitosan (1.0 g/l), which is well known as an elicitor, was added to the culture. These results suggest that alginate act as a kind of elicitor. Promotion effect of alginate on chitinase production was more remarkable when low molecular weight alginate (oligomer) was used. In comparison with free cells, addition of alginate to W. japonica protoplast culture resulted to 3 times increase in the chitinase productivity.     

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     

Improvement of catechin productivity in suspension cultures of tea callus cells

In the suspension cultures of tea callus cells, C.sinensis cv. Yabukita, the effects of the culture conditions, such as culture period and light irradiation, on cell growth and catechin production were investigated. The production of flavonoids (catechins + proanthocyanidins) was promoted by inoculating the cells into the fresh medium at the culture period giving the maximum flavonoid content in the cells. The cultivation under light irradiation was repeated several times by inoculating the cells with the maximum flavonoid content. The flavonoid production was significantly increased without inhibiting the cell growth. We obtained the maximum flavonoid production, 1.5 g/dm(3) medium, and the maximum content, 150 mg/(g of dry cell weight (DCW)). The latter value was larger than that in the leaves of the tea plant.     

聚乙二醇对东北红豆杉培养细胞的

在改良的B     

低密度和条件培养对红豆杉细胞生长的影响

红豆杉种胚来源的细胞,在改良Ｂ５液体培养基中继代培养的临界接种密度为鲜重４０ｇ／Ｌ．低密度培养下,１０－１６ｄ的条件培养液（ＣＭ）与新鲜培养液按５７：４３的比例混合时,能显著缩短细胞生长的延迟期,提高生长率,１００Ｌ生物反应器中,按４５．５％体积分数添加条件培养液,在鲜重２７ｇ／Ｌ低接种密度下培养５周,生物量增长９倍,达干重１４．３ｇ／Ｌ．对内源植物激素、精胺、维生素和氨基酸等的比较分析表明,吲哚     

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.     

Demonstration of a bubble-free annular-vortex membrane bioreactor for batch culture of red beet cells

Summary A putative bioreactor, which exploits Taylor-Couette (annular vortex) flow and a gas-permeating membrane, has been constructed and used to culture red beet (Beta vulgaris L.) cells. The cell growth was followed indirectly as sugar uptake by the cells from the medium. The ultimate fresh mass concentration of 93g/l is regarded as proof-of-concept.     

Photoautotrophic potato cells: Transition from heterotrophic to autotrophic growth

Cells of potato (Solanum tuberosum L.) were obtained which were capable of photoautotrophic growth in liquid suspension culture under a photon flux density of 90–110 μmol m^?2 s^?1 PAR and in an atmosphere enriched with 2% CO₂. These photoautotrophic cells contained between 100 to 200 μg Chl (g fresh weight)^?1 and fixed CO₂ at a maximum rate of 16 μmol CO₂ (g fresh weight)^?1h^?1. In order to obtain cells capable of photoautotrophic growth it was necessary to adapt highly chlorophyllous heterotrophic cells (>50 μg Chl (g fresh weight)^?1) for growth in medium with 2.5 g sucrose 1^?1 (photomixotrophic cells). The photomixotropic cells had a Chl content of ca 100 μg Chl (g fresh weight)^?1 and were capable of photosynthetic activity which allowed them to survive after sugars had been depleted from the medium. It was from the photomixotrophic cells that cells capable of photoautotrophic growth were obtained. Heterotrophic cells initially established in liquid medium with 25 g sucrose I^?1 from chlorophyllous callus contained about 50 to 150 μg Chl (g fresh weight)^?1. However, after 5 to 10 passages the Chl content decreased to a maximum of 15 μg Chl (g fresh weight)^?1. These cells could not be adapted to photomixotrophic or photoautotrophic growth. These cells also were not able to regain Chl or initiate high rates of CO₂ fixation during the stationary phase of growth as did photomixotrophic cells or chlorophyllous heterotrophic cells. The loss of Chl exhibited by the cells during adaption to heterotrophic growth could be attributed at least in part to unbalanced growth (when cell division and growth exceeds Chl accumulation). Sucrose appeared to have an inhibitory effect directly on photosynthesis independent of Chl accumulation.     

Production of diosgenin by hairy root cultures ofTrigonella foenum-graecum L.

Hairy root cultures ofTrigonella foenum-graecum L. were established withAgrobacterium rhizogenes strain A₄. The hairy roots produce diosgenin, an important spirostanol for the semi-synthesis of steroid hormones. Fourteen different liquid media were investigated. The fastest growth was obtained in McCown's woody plant (WP) medium supplemented with 3% sucrose; the highest diosgenin content was observed in half-strength WP medium with 1% sucrose (0.040% dry weight), which represents almost twice the amount detected in the 8-month-old non-transformed roots (0.024%). A time-course study in WP liquid media supplemented with 3% sucrose was undertaken. In these conditions, 17 g diosgenin/g fresh weight were produced. The influence of cholesterol, medium pH and chitosan on diosgenin production was tested. The addition of 40 mg/l chitosan elevated the diosgenin content to three times that found in non-elicited hairy roots.Abbreviations MS Murashige and Skoog (1962) medium - WP McCown's woody plant medium     

Establishment of callus and cell suspension culture of Scrophularia striata Boiss.: an in vitro approach for acteoside production

In the present study, a protocol was optimized for establishment of callus and cell suspension culture of Scrophularia striata Boiss. as a strategy to obtain an in vitro acteoside producing cell line for the first time. The effects of growth regulators were analyzed to optimize the biomass growth and acteoside production. The stem explant of S. striata was optimum for callus induction. Modified Murashige and Skoog medium supplemented with 0.5 mg/l naphthalene acetic acid + 2.0 mg/l benzyl adenine was the most favorable medium for callus formation with the highest induction rate (100 %), the best callus growth and the highest acteoside content (1.6 μg/g fresh weight). Incompact and rapid growing suspension cells were established in the liquid medium supplemented with 0.5 mg/l naphthalene acetic acid + 2.0 mg/l benzyl adenine. The optimum time of subculture was found to 17–20 days. Acteoside content in the cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The maximum content of acteoside (about 14.25 μg/g cell fresh weight) was observed on the 17th day of the cultivation cycle. This study provided an efficient way to further regulation of phenylethanoid glycoside biosynthesis and production of valuable acteoside, a phenylethanoid glycoside, on scale-up in S. striata cell suspension culture.     

Increasing productivity of Spirulina platensis in photobioreactors using artificial neural network modeling

Although production of microalgae in open ponds is conventionally practiced due to its economy, exposure of the algae to uncontrollable elements impedes achievement of quality and it is desirable to develop closed reactor cultivation methods for the production of high value products. Nevertheless, there are several constraints which affect growth of in closed reactors, some of which this study aims to address for the production of Spirulina. Periodic introduction of fresh medium resulted in increased trichome numbers and improved algal growth compared to growth in medium that was older than 4 weeks in 20 L polycarbonate bottles. Mixing of the cultures by bubbling air and use of draft tube reduced the damage to the growing cells and permitted increased growth. However, there was better growth in inclined cylindrical reactors mixed with bubbling air. The oxygen production rates were very similar irrespective differences in the maintained cultures densities. The uniformity in oxygen production rate suggested a tendency towards homeostasis in Spirulina cultures. The frequency of biomass harvest on the productivity of Spirulina showed that maintenance of moderate culture density between 0.16 and 0.32 g/L resulted in about 14% more productivity than maintaining the cell density between 0.16 and 0.53 g/L or 48% more than by daily harvest above 0.16 g/L. An artificial neural network based predictive model was developed, and the variables useful for predicting biomass output were identified. The model could predict the growth of Spirulina up to 3 days in advance with a coefficient of determination >0.94.     

Immobilization of Nicotiana tabacum plant cell suspensions within calcium alginate gel beads for the production of enhanced amounts of scopolin

Scopolin-producing cells of Nicotiana tabacum were immobilized within Ca-alginate gel beads. Free cell suspensions accumulated scopolin within cytoplasmic compartments and cell disruption was necessary to recover scopolin. On the contrary, immobilized plant cells excreted considerable amounts of scopolin. Scopolin diffused throughout the gel matrix and reached the culture media. A large fraction of produced scopolin could then be recovered from the culture medium without disrupting cells. Immobilized N. tabacum cells produced more scopolin than free cell suspensions did (3.8 mg/g fresh weight biomass [into the culture media] versus 0.2 mg/g fresh weight biomass [intracellular]). Variation of the immobilization conditions revealed a marked influence on the behavior of N. tabacum plant cells: production of scopolin and enhanced excretion, cell growth, and morphological aspect of plant cell colonies. This excretion phenomenon could be used advantageously at an industrial production level.     

Induction of lignans and phenolic compounds in cell culture of Linum album by culture filtrate of Fusarium graminearum

Cell suspension cultures of Linum album Kotschy ex Boiss. have been reported to produce anticancer podophyllotoxin and its related lignans. In the present study, we investigated the effect of culture filtrate of Fusarium graminearumon growth, and lignan and phenolic compounds in L. album cell culture. After 7 days of pre-culture, the cells were treated with 1% (v/v) of the culture filtrate. Cell growth was reduced, while phodophyllotoxin and lariciresinol production was stimulated reaching a maximum 0.0187 mg/g fresh weight (FW) and 0.0136 mg/g FW 5 days after the treatment, respectively. Also, our results provide evidence that the culture filtrate of F. graminearum can be effective on phenylalanine ammonia-lyase activity and phenolic compounds.     

Photoautotrophic Growth and Photosynthesis in Cell Suspension Cultures of Chenopodium rubrum

A method is described for growing cell suspension cultures of Chenopodium rubrum photoautotrophically for prolonged periods of time. By using a two-tier culture vessel the growth medium with the cells was separated from the CO₂ reservoir. Definite CO₂ concentrations were established by a K₂CO₃/KHCO₃ buffer. Photoautotrophic growth in C. rubrum cell suspension cultures was correlated with the CO₂ level. At 0.5% CO₂ the cell cultures contained 68 μg chlorophyll/g fresh weight and showed an increase in fresh weight of about 80% in 18 days. At 1% CO₂ an increase in fresh weight of 165% in 18 days was observed. The chlorophyll content rose up to 84 μg/g fresh weight. The photoautotrophic growth was also greatly influenced by the 2,4-D content of the medium. Cell growth was enhanced by lowering the auxin concentration. Best growth was attained (210% increase in fresh weight) at 10^?8M 2,4-D. The photosynthetic activity of the cells was measured by the light dependent ¹⁴CO₂ incorporation. At 0.5% CO₂ the cell suspensions assimilated about 100 μmol CO₂/mg chlorophyll × h. In the presence of 1% CO₂ the light driven assimilation was raised up to 185 μmol CO₂/mg chlorophyll × h. In both cases, the dark incorporation of CO₂ was merely 1.8% of the values obtained in light.