Polysaccharide synthesis by Phanerochaete chrysosporium during degradation of kraft lignin

Summary Phanerochaete chrysosporium (Sporotrichum pulverulentum) produced an extracellular glucan type polysaccharide when grown in a chemostat under nitrogen limitation. When cells were transferred to a standing mode of cultivation in the presence of excess glucose (6 gl^–1), the amount of non-glucose total carbohydrates in the culture increased from 0.58 gl^–1 to 1.76 gl^–1 during 15 day experiments. The change in total carbohydrates was due to an increase in extracellular and cell-bound glucan type polysaccharide. This increase occured simultaneously with formation of mycelial mats and appearance of ligninolytic activity. When the cultures were agitated under atmospheric oxygen rather than 100% O₂, their non-glucose total carbohydrate content increased to 2.15 gl^–1 in 4 days. The excess polysaccharide formation had an inhibitory effect on lignin degradation as more lignin was degraded by cells with lower polysaccharide content. The lignin that was associated with cells after the degradation had stopped could be further degraded by new active cells.     

Taxol production in suspension cultures of Taxus baccata

The response of Taxus baccata (PC2) to basic manipulations of culture conditions is described. Suspension cultures of Taxus baccata (PC2) were maintained at 25°C on a modified B5 medium with two-week transfers. Under these conditions, no taxol^® is formed. However, if the cells are left in the same medium for 7 or more additional days, taxol is produced and released (ca. 90%) into the extracellular medium. Levels as high as 13 mg 1^–1 extracellular taxol were achieved in shake flask cultures and taxol was the primary taxane formed representing between 50 and 80% of total taxane in the medium. The cells are sensitive to changes in culture conditions and cultures cycle through periods of high (13 mg 1^–1) and low (<0.1 mg 1^–1) levels of taxol production during extended culture. Picloram was the most effective of the auxins tested with respect to cell growth but it suppressed taxol production. Addition of fructose to moderately-productive cultures (ca. 4 mg 1^–1) improved taxol production, but cultures in a high producing state did not respond. Glucose suppressed taxane production. Two isoprenoids (geraniol and pinene) had a modest effect on taxol production when added to cultures at 10 mg 1^–1.®|Taxol is a registered trademark of Bristol Meyer Squibb for paclitaxel     

Improved yields of the extracellular domain of the epidermal growth factor receptor produced using the baculovirus expression system by medium replacement following infection

The extracellular domain of the epidermal growth factor receptor (EGFR) was expressed using the baculovirus expression vector system. The maximum level of the EGFR extracellular domain secreted into the medium in Sf-9 (Spodoptera frugiperda or fall armyworm) cell batch culture was approximately 2.5 g ml^–1. In order to increase this yield, a process was developed that included the following sequence of steps: batch growth to maximum cell density, infection of the cells with recombinant virus, and replacement of spent medium. By using this process, the specific yield of recombinant protein, which in batch culture drops when infection is carried out at densities greater than 3 × 10⁶ cells ml^–1, can be maintained at a maximum in cultures infected at densities of 10⁷ cells ml^–1 or greater. The process, when applied to 3-1 and 11-1 bioreactor cultures, allowed a maximum volumetric yield of triple the maximum value attainable in batch culture. Spent-medium analysis indicates that medium replacement provides certain nutrients that could otherwise be limiting for recombinant protein production.     

Pinoresinol and matairesinol accumulation in a Forsythia × intermedia cell suspension culture

A newly established Forsythia × intermedia cell suspension culture was shown to accumulate (+)- and (–)-pinoresinol as well as matairesinol. The influence of the sucrose content of the culture medium and of the cultivation time on pinoresinol and matairesinol accumulation was evaluated. The highest pinoresinol yield was achieved from cells grown in medium containing 6% sucrose for 12 ± 2 days with levels of 0.6–0.8 mg g^–1 dry weight and an average enantiomeric composition of 75 ± 5% (+)-pinoresinol. The highest matairesinol amount was reached in the same medium at the 14th ± 2 culture day with levels of 1.0–2.7 mg g^–1 dry weight. To our knowledge, this is the first report on pinoresinol accumulation in Forsythia × intermedia plants or cell suspension cultures.     

Growth characteristics of Sanguinaria canadensis L. Cell suspensions and immobilized cultures for production of benzophenanthridine alkaloids

Summary Sanguinaria canadensis L. plants were harvested from a local forest and calli were initiated from leaf explants. The production of benzophenanthridine alkaloids (i.e. sanguinarine, sanguilutine, sanguirubine, chelerythrine, chelilutine and chelirubine) by S. canadensis cell grown in modified B5 and IM2 media was compared to the alkaloid content of rhizomes. Sanguinarine accounted for approximately 80% of the total alkaloid content of cultured cells (1.3%,g g^–1) while sanguinarine and sanguirubine accounted for 70% of rhizome alkaloids (9.0%, g g^–1). Sanguinarine, chelirubine and chererythrine were the only known alkaloids detected in cultured S. canadensis cells. Maximum alkaloid production of cultures performed using B5 medium, containing half the original nitrate concentration, was observed following extracellular nitrate and sugar depletion. The scale-up of this culture was successfully performed in a 2-1 immobilization bioreactor. The consumption of sugar and nitrate as well as the oxygen (OTR) and carbon dioxide (CTR) transfer rates of the immobilized cell culture were monitored for 15 days. The maximum sugar and nitrate consumption rates were 1.8 g l^–1 per day and 2.3 mm per day respectively. The maximum OTR and CTR of the immobilized cell culture were 0.8 mmol O₂ l^–1 h^–1 and 0.95 mmol CO₂ l^–1 h^–1 respectively. The sanguinarine yield of this culture reached 1.0% based on biomass dry weight (g g^–1 dw) by day 15.     

Feasibility study on the use of hyperosmolar medium for improved antibody production of hybridoma cells in a long-term,repeated-fed batch culture

To test the feasibility of using hyperosmolar medium for improved antibody production in a long-term, repeated fed-batch culture, the influence of various culture conditions (serum concentration and cultivation method) on the hybridoma cells' response to hyperosmotic stress resulting from sodium chloride addition was first investigated in a batch culture. The degree of cell growth depression resulting from hyperosmotic stress was dependent on serum concentrations and cultivation methods (static and agitated cultures). Depression of cell growth was most significant in agitated cultures with low serum concentration. However, regardless of serum concentrations and cultivation methods used, the hyperosmotic stress significantly increased specific antibody productivity (q _MAb). Increasing osmolality from 284 to 396 mOsm kg^–1 enhanced the q_MAb in agitated cultures with 1% serum by approximately 124% while the similar osmotic stress enhanced the q _MAb in static cultures with 10% serum by approximately 153%. Next, to determine whether this enhanced q_MAb resulting from hyperosmotic stress can be maintained after adaptation, long-term, repeated-fed batch cultures with hyperosmolar media were carried out. The cells appeared to adapt to hyperosmotic stress. When a hyperosmolar medium (10% serum, 403 mOsmkg^–1) was used, the specific growth rate improved gradually for the first four batches and thereafter, remained constant at 0.040±0.003 (average ± standard deviation) hr^–1 which is close to the value obtained from a standard medium (10% serum, 284 mOsmkg^–1) in the batch culture. While the cells were adpating to hyperosmotic stress, the q_MAb was gradually decreased from 0.388×10^–6 to 0.265×10^–6 g cell hr^–1 and thereafter, remained almost constant at 0.272±0.014× 10^–6 g cell^–1 hr^–1. However, this reduced q _MAb after adaptation is still approximately 98% higher than the q_MAb obtained from a standard medium in the batch culture.The authors would like to thank Dr.M. Kaminski for providing the hybridoma cell line used in this study. This work was supported by the Korea Science and Engineering Foundation.     

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.     

Extracellular Protease as a Reversible Adhesion Regulator in Pseudomonas fluorescens

The investigation of growth dynamics and protein content in a batch Pseudomonas fluorescens culture grown in a synthetic medium with glucose as the sole source of carbon and energy showed that cells reversibly adhere to the walls of the cultivation flask during the first 2–3 h of growth. Over this time period, the total protein content of free and bound cells increased exponentially at a rate of 0.25 h^–1, the fraction of proteins in cells being almost the same (60–70%). The protein content in the medium increased from 3 to 50 mg/l, reaching about 30% of the total protein of the culture. The addition of the exponential culture liquid filtrate to the medium together with the inoculum led to the complete inhibition of cell adhesion and a drastic activation of proteolysis, with a concurrent release of more than 80% of cellular proteins into the medium. After 3–5 h of growth, the concentration of extracellular proteins decreased to the control level. Exogenously added proteinase K inhibited cell adhesion, the effect being more pronounced for R-type than for S-type cells. The hypothesis is discussed that the short-term reversible adhesion of cells is regulated with the involvement of a mixture of hydrocarbons, which inactivate the functional activity of bacterial adhesins, and proteases, which digest these adhesins.     

Protoplasts of Gelidium robustum (Rhodophyta)

Viable protoplasts were isolated from apices of the agarophyte Gelidium robustum (Gardn.) Hollenb. & Abb. using a combination of commercial cell-wall degrading enzymes and extracellular wall-degrading enzymes isolated from a marine bacterium. The protoplasts were approximately 8–15 µm in diameter, liberated mainly from the surface cell layers and from cells at the distal ends of medullary filaments. The bacterial enzyme alone was not sufficient to liberate significant numbers of protoplasts. Maximum yield was 9 × 10⁵ protoplasts/g tissue (wet wt.). Optimum osmolality occurred between 1750–1950 mOs kg^–1; yield and viability were severely diminished at osmolalities less than 1350 mOs kg^–1. Viability, as determined by flurorescein diacetate staining and Evans Blue exclusion 1 hr after removal from the enzyme solution, was approximately 80–95%. Roughly 80% of the cells did not show Calcofluor fluorescence, while 40% stained positively for the presence of sulfated polysaccharides. Cell wall regeneration was observed with inconsistent reproducibility, and no cell division was observed when the protoplasts were placed in culture medium.Dedicated to the memory of Professor Michael Neushul.     

Rhizobium extracellular structures in the symbiosis

The extracellular and surface polysaccharides produced by Rhizobium species constitute a composite macromolecular interface between the bacterial cell and its environment. Several of these polysaccharides are involved in the complex series of interactions leading to the establishment of an effective Rhizobium-legume symbiosis. Extracellular heteropolysaccharides (EPSs) are found in culture supernatants, while capsular polysaccharides adhere to the cell surface. Cyclic (1–2)--d glucan is a periplasmic oligosaccharide that has also been found in the culture supernatants of some strains. The lipopolysaccharides (LPSs), which form part of the outer membrane and contain the O-somatic antigens, comprise the other major group of extracellular polysaccharides. In this review we will describe the major Rhizobium extracellular structures and their role in symbiosis with leguminous plants.The authors are with the Departamento de Microbiologia y Parasitologia, Facultad de Farmacia. Universidad de Sevilla, 41012 Sevilla, Spain     

The degradation and utilization of wheat-straw cell-wall monosaccharide components by defined ruminal cellulolytic bacteria

Cell walls (CW) of untreated wheat straw and sulphur-dioxide (SO₂)-treated wheat straw were used as model substrates for the hydrolysis and utilization of CW carbohydrates by pure cultures or pair-combinations of defined rumen bacterial strains. Fibrobacter succinogenes S85 and BL2 strains and their co-cultures with D1 were the best degraders of CW among ruminal cultures, solubilizing 37.2–39.6% of CW carbohydrates of untreated straw and 62.2–74.5% of SO₂-treated straw. Complementary action between Butyrivibrio fibrisolvens D1 and the F. succinogenes strains was identified with respect to co-culture growth and carbohydrate utilization. However, the extent of CW solubilization was determined mainly by the F. succinogenes strains. In both substrates, utilization of solubilized cellulose by F. succinogenes S85 and BL2 monocultures was higher than that of xylan and hemicellulose: 96.5–98.3%, 34.4–40.5% and 33.5–36.2%, respectively. Under scanning electron microscopy visualization, S85 and BL2 cells of the co-cultures comprised the most dense layer of bacterial cell mass attached to and colonized on straw stems and leaves, whereas D1 cells were always nearby. Stems and leaves of the untreated straw were less crowded by attached bacteria than that of the SO₂-treated straw. In both materials, the cell surface topography of S85 and BL2 bacteria attached to CW particles was specified by a coat of characteristic protuberant structures, polycellulosome complexes.     

Indole-3-acetic acid in the culture medium of two axenic green microalgae

In the view of the facts that algal extracts have been used in agriculture asa source of plant growth stimulating agents and IAA has been shown to bepresent in the extracts, a study was planned to establish whether or notaxenic algae can produce IAA. Evidence is provided for extracellular IAAproduction during culture of two axenic green microalgae. IAAidentification was based on co-chromatography with the standard, analysisof UV and fluorescent spectra, and gas chromatography – selectedion-monitoring mass spectrometry. HPLC analyses showed that underthe experimental conditions the amounts of IAA released to the mediumby Scenedesmus armatus and Chlorella pyrenoidosa weregenerally low. IAA tended to occur in Scenedesmus armatus culturemedium at higher concentrations than in that of Chlorellapyrenoidosa. In fast-growing cultures of Scenedesmus armatus,constantly aerated with CO₂/air mixture, the concentration of IAAcalculated per cell was less than in the slow-growing cultures.     

Mass cultivation of the nitrogen-fixing cyanobacteriumGloeotrichia natans,indigenous to rice-fields

Gloeotrichia natans, a nitrogen fixing cyanobacterium common in rice fields in the Philippines, was used for studies to establish key features of its physiology and potential production in outdoor cultures. Under optimal growth conditions (38 °C, pH 8.0, no carbon enrichment) the specific growth rate of rice-field isolate was 0.076 h^–1. The pH of the medium (between 6.5 and 9.0) did not influence the growth rate, but it did affect phycobiliprotein content, as reflected by a change in colour. At pH 7.0 the culture was green-brown, with phycobiliproteins constituting up to 10% of the total protein, while at pH 9.0 the culture was brownish-black and the pigment content was as high as 28% of the total protein. In outdoor cultures the specific growth rate was related directly to cell density in the range of 0.7–1.5 g dry weight 1^–1 at a rate of stirring of 30 rpm, and inversely related to cell density at half this rate. At a stirring of 30 rpm, daily production of outdoor cultures harvested to maintain cell densities of 0.7, 1.15 andw 1.5 g 1^–1 were 14.7, 17.1 and 18.1 g m^–2 d^t, respectively. This rate of production was maintained for more than 45 days. Phycobiliprotein content in the culture kept at a density of 1.5 g 1^–1 reached 14% of the total biomass.     

Secretion of an antibacterial factor during resuscitation of dormant cells inMicrococcus luteus cultures held in an extended stationary phase

A high proportion ofMicrococcus luteus cells in cultures starved for 3–6 months in spent medium following growth to stationary phase in batch culture lost the ability to grow and form colonies on agar plates, but could be resuscitated from dormancy by incubation in liquid medium containing supernatant taken from the late log phase of viable cultures of the same organism (Kaprelyants et al. 1994). In the present work, we found that during the first 50–70 h of such resuscitation the dormant cells actually divide for 10–17 generations in lactate minimal medium containing yeast extract whilst remaining nonculturable on agar plates. Further incubation results in a decrease in the total cell number in liquid medium. The addition of viable (culturable)Micrococcus luteus cells in concentrations of up to 10⁴ ml^–1 to test tubes containing either resuscitating cells or supernatant from these cultures revealed the excretion of a factor or factors which inhibited the proliferation of otherwise viable cells. The maximum production of this factor took place after some 96 h of incubation of starved cells in resuscitation medium. Supernatant from late logarithmic phase batch cultures ofM. luteus abolished the antibacterial effect of starved cultures incubated in resuscitation medium. It is concluded that the stimulating effect of viable cells, and of supernatant taken from batch cultures, on the resuscitation of dormant cells might be connected in part with overcoming the activity of an antibacterial factor causing self-poisoning of dormant cells during their resuscitation.     

Physiological activity of immobilized cells of Claviceps fusiformis during long-term semicontinuous cultivation

Summary The 550-day semicontinuous cultivation of Claviceps fusiformis immobilized in calcium alginate is documented. The vegetative mycelium from seed or from early-production submerged culture is the best choice for immobilization. No extracellular glucans are produced by immobilized cells. Immobilized spores give low yields of clavine alkaloids. Alginate concentrations in a range of 2%–4% do not influence yield and spectrum of alkaloids. The cytoplasm of the immobilized cells becomes condensed (after 3 days), polysaccharides disappear, and centres of lipid synthesis are formed in the cytoplasm. After 60 days the cells harbour a great number of lipid particles, mitochondria are diminishing and their cristae partly disappear, indicating a decreased respiration capacity. After 350–500 days the volume of most cells is increased many times and the cells are filled with large oval bodies of electrondense material. Chloramphenicol protects immobilized cultures against bacterial contamination.     

A simple combined method for determination of β-1,3-glucan and cell wall polysaccharides in diatoms

A new method is described for the combined determination of -1,3-glucan and cell wall polysaccharides in diatoms, representing total cellular carbohydrate. The glucan is extracted by 0.05 mol l^–1 H₂SO₄ at 60 °C for 10 min, and the cell wall polysaccharides are subsequently hydrolyzed by 80% H₂SO₄ at 0–4 °C for 20 h. Each carbohydrate fraction is determined by the phenol-sulphuric acid method. The method has been demonstrated for axenic cultures of the marine diatom Skeletonema costatum and natural marine phytoplankton populations dominated by diatoms. Cellular glucan and cell wall polysaccharides were determined with standard deviations of 1–3% and 2–5%, respectively.     

Accumulation of acyclic polyols and trehalose as related to growth form and carbohydrate source in the dimorphic fungi Mucor rouxii and Candida albicans

Yeast (Y) and hyphal (H) cells of Mucor rouxii and Candida albicans were cultivated in liquid media containing different carbon nutrient sources (glucose, fructose, ribose), and their free acyclic polyol and trehalose contents determined using capillary gas liquid chromatography (TMS- and OAc-derivatization). Irrespective of growth form and C-source, the fraction of the water-soluble neutral components of the cellular mass of the cultures — highly homogeneous with regard to the respective cell form produced — contained glycerol, ribitol and arabitol, in addition to trehalose. The polyols contributed 0.5–2% to the biomass of M. rouxii and 1.5–6% to that of C. albicans; the values for trehalose ranged from 0.2–11% in the former and 1–3.5% in the latter species. Mucor contained higher amounts of ribitol and arabitol in H cells and larger quantities of trehalose and glycerol in Y cells. In Candida, too, hyphae always exhibited higher ribitol contents, whereas arabitol attained higher levels in yeasts under almost any conditions — regardless of the type of medium (synthetic vs. complex), stage of culture (early vs. late log-phase) and strain used. Glycerol concentration was not correlated with the growth form; trehalose contents tended to be higher in Y cells. Taking into account the facts that C. albicans and certain Mucor species are agents of opportunistic infections and are invasive mainly in the filamentous form, and that the prospective hosts do not accumulate either of these carbohydrates, the possibility is considered of using trehalose- and polyol-metabolizing enzymes as targets for designing antifungal drugs.     

Plant regeneration of rose (Rosa hybridia) from embryogenic cell-derived protoplasts

Culture conditions are described for sustained cell division and plant regeneration from protoplasts of rose (Rosa hybrida L. `Sumpath'). Protoplasts were enzymatically isolated from 2-week-old embryogenic cell suspension cultures. Freshly isolated protoplasts were plated as a thin layer onto protoplast culture medium (half-strength 21 Murashige and Skoog's medium containing 60 g l^–1 myo-inositol, 4.4 M BA, and 1.4 M 2,4-D) at a density of 5×10⁴ protoplasts ml^–1. The plating efficiency reached 3.9% after 2 weeks of culture. However, few protoplasts underwent cell division when cultured in protoplast culture medium in which 60 g l^–1 myo-inositol was replaced with the same osmolarity of 90 g l^–1 mannitol, indicating that myo-inositol is essential for sustained cell division of protoplasts. Colonies were formed after 8 weeks of culture at a frequency of 0.2%. Colonies were then transferred to colony culture medium (0.4% Gelrite-solidified protoplast culture medium) and maintained by subculturing at 4-week intervals to form embryogenic calluses. Upon transfer to half-strength MS basal medium, embryogenic calluses gave rise to numerous somatic embryos. Somatic embryos were transferred to half-strength MS basal medium containing 48 mg l^–1 ferric ethylenediamine di-(o-hydroxyphenylacetate), where they subsequently developed into plantlets at a frequency of 30.9%. The plantlets had the same chromosome number of 2n=3x=21 as the source plant. They were successfully transplanted to potting soil and grown to maturity in a greenhouse.     

Initiation and growth characteristics of suspension cultures of Calendula officinalis cells

Callus cultures of marigold (Calendula officinalis L.) were induced on Murashige and Skoog medium with different concentrations of auxin (dichlorophenoxyacetic acid (2,4-D) or indole-3-acetic acid (IAA) and cytokinin (kinetin or 6-(,-dimethylallylamino)purine (2iP). Of all hormone combinations used in the medium, two were the most efficient in promoting callus development: 1.81 M (0.4 mg l^–1) 2,4-D and 1.85 M (0.4 mg l^–1) kinetin (0.4d–0.4k culture) or 0.45 M (0.1 mg l^–1) 2,4-D and 2.02 M (0.5 mg l^–1) 2iP (0.1d–0.5p culture). These combinations were selected to induce cell suspension cultures. The suspension cultures were maintained under light or dark conditions. The light stimulated cell aggregation in the cultures. In both cultures cells were undifferentiated under darkness, whereas in the light, rhyzogenesis was observed in 0.1d–0.5p culture. The cell growth and protein and oleanolic acid contents were determined. Initially, biomass production was similar under light and dark conditions, but after 7–8 months from the induction the cell growth was reduced by approximately 30% in the light, whereas the cell growth of the cultures maintained under darkness did not reveal any changes. The presence of oleanolic acid was detected in the suspension cultures kept in darkness. This compound reached two quantitative peaks: in the lag and stationary phases –- beyond the active growth phase of the culture cycle and its concentration was several times higher in 0.1d–0.5p culture than that in 0.4d–0.4k culture. It was for the first time that callus and suspension cultures were induced from the marigold plant.     

Anthraquinones production,hydrogen peroxide level and antioxidant vitamins in Morinda elliptica cell suspension cultures from intermediary and production medium strategies

The effects of medium strategies [maintenance (M), intermediary (G), and production (P) medium] on cell growth, anthraquinone (AQ) production, hydrogen peroxide (H₂O₂) level, lipid peroxidation, and antioxidant vitamins in Morinda elliptica cell suspension cultures were investigated. These were compared with third-stage leaf and 1-month-old callus culture. With P medium strategy, cell growth at 49 g l^–1, intracellular AQ content at 42 mg g^–1 DW, and H₂O₂ level at 9 mol g^–1 FW medium were the highest as compared to the others. However, the extent of lipid peroxidation at 40.4 nmol g^–1 FW and total carotenoids at 13.3 mg g^–1 FW for cultures in P medium were comparable to that in the leaf, which had registered sevenfold lower AQ and 2.2-fold lower H₂O₂ levels. Vitamin C content at 30–120 g g^–1 FW in all culture systems was almost half the leaf content. On the other hand, vitamin E content was around 400–500 g g^–1 FW in 7-day-old cultures from all medium strategies and reduced to 50–150 g g^–1 FW on day 14 and 21; as compared to 60 g g^–1 FW in callus and 200 g g^–1 FW in the leaf. This study suggests that medium strategies and cell growth phase in cell culture could influence the competition between primary and secondary metabolism, oxidative stresses and antioxidative measures. When compared with the leaf metabolism, these activities are dynamic depending on the types and availability of antioxidants.Abbreviations AQ Anthraquinone - DW Dry cell weight - FW Fresh cell weight - G Intermediary medium - M Maintenance medium - MDA Malondialdehyde - P Production medium - ROS Reactive oxygen species - TBA Thiobarbituric acid - t_d Doubling time