Anthocyanin production in selected cell lines of grape (Vitis vinifera L.)

Summary Anthocyanin production of two lines ofVitis vinifera cell cultures, i.e., 5.4 and 13.1, which were obtained from the same starting material after 20 and 37 mo. of clonal selection, respectively, was investigated. Cell suspension cultures of lines 5.4 and 13.1 maintained an anthocyanin content of 0.44 ± 0.15 and 1.02 ± 0.31 mg·g⁻¹ fresh weight during 50 and 32 weekly maintenance subcultures, respectively. Under anthocyanin-promoting culture conditions, both lines showed an enhancement of their anthocyanin level by approximately fourfold. While line 5.4 accumulated peonidin 3-glucoside and cyanidin 3-glucoside in decreasing order, line 13.1 accumulated primarily peonidin 3-p-coumaroylglucoside with lesser amounts of malvidin monoglucoside. Results show that while the anthocyanin content was improved during the course of repeated selections, the anthocyanin composition was modified markedly favoring the accumulation of more metabolically-advanced anthocyanins.     

Accumulation of peonidin 3-glucoside enhanced by osmotic stress in grape (Vitis vinifera L.) cell suspension

Cell cultures of grapes, Vitis vinifera L. cv Gamay Fréaux were grown under different conditions of external osmotic potential induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Addition of 82 mM mannitol or increasing sucrose concentration to 132 mM had similar effects on repressing growth. Cyanidin 3-glucoside, peonidin 3-glucoside and peonidin 3-p-coumaroylglucoside are three main anthocyanins of Vitis cells. Increasing osmotic potential from –0.43 MPa to –0.8 MPa in the medium resulted in a significant intracellular accumulation of anthocyanin especially peonidin 3-glucoside in the pigmented cells. High osmotic potential appears to stimulate the methylation of anthocyanins. Osmotic potential is an important culture factor and may be useful in the controlling of anthocyanin production and composition.     

Production of ajmalicine and ajmaline in hairy root cultures of Rauvolfia micrantha Hook f., a rare and endemic medicinal plant

Hairy roots of Rauvolfia micrantha were induced from hypocotyl explants of 2–3 weeks old aseptic seedlings using Agrobacterium rhizogenes ATCC 15834. Hairy roots grown in half-strength Murashige & Skoog (MS) medium with 0.2 mg indole 3-butyric acid l^–1 and 0.1 mg -naphthaleneacetic acid l^–1 produced more ajmaline (0.01 mg g^–1 dry wt) and ajmalicine (0.006 mg g^–1 dry wt) than roots grown in auxin-free medium. Ajmaline (0.003 mg g^–1 dry wt) and ajmalicine (0.0007 mg g^–1 dry wt) were also produced in normal root cultures. This is the first report of production of ajmaline and ajmalicine in hairy root cultures of Rauvolfia micrantha.     

Increased taxane accumulation in callus cultures of Taxus cuspidata and Taxus × media by some elicitors and precursors

In Taxus cuspidata callus, vanadyl sulfate (10 mg l^–1) induced a high (146 g g^–1 dry wt) production of 10-deacetylbaccatin III in comparison to 7 g g^–1 dry wt of the control. The content of paclitaxel in this species increased from 16 g g^–1 to 74 g g^–1 dry wt when 20 mg phenylalanine l^–1 was used. In T. media, p-aminobenzoic acid induced the highest content of 10-deacetylbaccatin III (481 g g^–1 dry wt) versus 181 g g^–1 in the control. Paclitaxel increased from 89 to 139 g g^–1 dry wt after adding chitosan (20 mg l^–1) to the cultures.     

Enhanced colchicine production in root cultures of Gloriosa superba by direct and indirect precursors of the biosynthetic pathway

Excised root cultures of Gloriosa superba reached 7.5 g dry wt l^–1 and accumulated 240±40 g colchicine g^–1 cell dry wt after 4 weeks growth. While all precursors (except trans-cinnamic acid) enhanced colchicine content of root cultures without adversely affecting root growth, treatment with p-coumaric acid + tyramine (each at 20 mg l^–1) increased colchicine content to 1.9 mg g^–1 cell dry wt.     

Glucosylation of flavonoids in petals of Petunia hybrida

During the biosynthesis of anthocyanins in Petunia hybrida, the 3-hydroxyl group is glucosylated. Their supposed biosynthetic precursors, the dihydroflavonols, are glucosylated at the 7 or 4 positions. The question arose of whether these glucosides or the aglucones act as a substrate in anthocyanin synthesis. Using isolated flower buds of white flowering mutants that were blocked in an earlier step of biosynthesis, it was found that anthocyanin-3-glucosides and dihydroquercetin-7-glucoside were synthesized if dihydroquercetin, dihydroquercetin-7-glucoside, or dihydroquercetin-4-glucoside were used as precursors in these experiments. Intracellular dihydroquercetin-glucosides were not used as a substrate for anthocyanin synthesis. The results are explained by deglucosylation of dihydroquercetin-glucosides during uptake by isolated flower limbs. Dihydroquercetin-7-glucoside, formed intracellularly, is not available as a precursor for anthocyanins. We conclude that the aglucone form of dihydroquercetin acts as a substrate in anthocyanin biosynthesis.Abbreviations dHO dihydroquercetin - dHQ-7=g dihydroquercetin-7-glucoside - dHQ-4-g dihydroquercetin-4-glucoside     

Selection of anthocyanin-accumulating potato (Solanum tuberosum L.) cell lines from calli derived from seedlings produced by gamma-irradiated seeds

Callus cell lines of potato (Solanum tuberosum L. cv. Zarevo) were obtained from seedlings germinated from gamma-irradiated seeds (200 Gy). Some of these cell lines produce red-violet pigments which were identified as acylated anthocyanins. The major anthocyanin was determined to be peonidin 3-O-[6-O-(4-O-E-p-coumaroyl-rhamnosyl)-glucoside]-5-O-glucoside (peonanin). Single cell-derived protoclones from non-pigmented protoplasts sometimes also gave rise to pigmented cell clusters thus indicating that the changes in the expression of the anthocyanin pathway can also occur after the stage of initial callus induction.     

Induction of astaxanthin formation by reactive oxygen species in mixotrophic culture of Chlorococcum sp.

The induction of astaxanthin formation by reactive oxygen species in mixotrophic culture of Chlorococcum sp. was investigated. H₂O₂ (0.1 mM) enhanced the total astaxanthin formation from 5.8 to 6.5 mg g^–1 cell dry wt. Fe²⁺ (0.5 mM) added to the medium with H₂O₂ (0.1 mM) further promoted astaxanthin formation to 7.1 mg g^–1 cell dry wt. Similarly, Fe²⁺ (0.5 mM) together with methyl viologen (0.01 mM) promoted astaxanthin formation to 6.3 mg g^–1 cell dry wt. In contrast, an addition of KI (1 mM), a specific scavenger for hydroxyl radicals (OH), together with H₂O₂ (0.1 mM) and Fe²⁺ (0.5 mM), to the medium decreased astaxanthin formation to 1.8 mg g^–1 cell dry wt. KI (1 mM) also inhibited the enhancement of carotenogenesis by superoxide anion radicals (O₂ ^–), with a decrease of astaxanthin formation to 1.7 mg g^–1 cell dry wt. This suggested that O₂ ^– might be transformed to OH before promoting carotenogenesis in Chlorococcum sp.     

Effects of high ammonium concentrations on growth and anthocyanin formation in grape (Vitis vinifera L.) cell suspension cultured in a production medium

Cultivating Vitis vinifera cell suspensions in a production medium which is characterized by high sucrose and low nitrate concentrations (132 mM and 6.25 mM respectively) repressed growth but enhanced the intracellular accumulation of anthocyanins, especially peonidin 3-glucoside. Increasing the ammonium concentration of the production medium from 2 to 8–16 mM increased growth and decreased the accumulation of anthocyanins and peonidin 3-glucoside specifically. Instead, peonidin 3-p-coumaroylglucoside accumulated. At 24 mM ammonium concentration, growth was inhibited and accumulation of peonidin 3-p-coumaroylglucoside was significant (p<0.05) and represented 42% of total anthocyanins after 12 days of culture compared with 19% in the production medium with 2 mM ammonium.Contribution Number 217.     

Bioreactor seaweed cell culture for production of bioactive oxylipins

Liquid cell suspension cultures derived from marine plants have the potential to biosynthesize novel biomedicinal compounds in a controlled environment. Of particular interest are the eicosanoids and related oxylipins emanating from the 15-lipoxygenase manifold of the arachidonic acid cascade, which is active in the brown algaLaminaria saccharina. Filamentous cell clumps ofL. saccharina isolated from female gametophytes were cultured in an illuminated bubble-column bioreactor in GP2 artificial seawater nutrient medium at 13 °C and air flow rate of 0.35 L air min^–1 L^–1 culture (vvm). Growth kinetics and biomass productivity data were obtained as a function of incident light intensity (2.4 to 98mol photon m^–2 s^–1) and initial cell density (27 to 149 mg DCW L^–1). Maximum cell densities exceeded 1200 mg DCW L^–1 after a 20 day cultivation time at optimal conditions of 98mol photon m^–2 s^–1 and 118 mg DCW L^–1 initial cell density. Qualitative analysis of chloroform/methanol extracts of the cell culture biomass by GC-MS confirmed the presence of the hydroxy fatty acids 13-HODTA and 13-HOTE, the likely products of 15-lipoxygenase catalyzed oxidation of linoleic or linolenic acids.     

In vitro rosmarinic acid accumulation in sweet basil (Ocimum basilicum L.)

Leaf-derived suspension cultures of sweet basil, Ocimum basilicum L. accumulated rosmarinic acid up to 10 mg g^–1 dry wt, a value up to 11 times higher than in callus cultures or in leaves of donor plants. Immobilized cells accumulated less than 15 g rosmarinic acid g^–1.     

Accumulation of Cd2+ and Pb2+ in the suspension cultures of Agave amaniensis and Costus speciosus and the determination of the culture's growth and phytosteroid content

Cell suspension cultures of Agave amaniensis and Costus speciosus were grown in media containing Cd^{2 +} up to 25 and 20 mg l^–1, respectively, and Pb²⁺ up to 40 mg l^–1. The cultures hyper-accumulated Cd²⁺ up to 900 and 530 g g^–1 and Pb²⁺ up to 1390 and 1170 g g^–1 dry wt. in their respective biomasses. Increasing Pb²⁺ up to 30 mg l^–1 increased the biomass production and total sitosterol content of Costus speciosus by up to 1.7- and 1.3-fold, respectively.     

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     

Improved stability and expression of a recombinant shuttle plasmid in Escherichia coli during fedbatch cultivation

The effect of higher cell densities on the expression and segregational stability of a recombinant E. coli- B. subtilisshuttle plasmid coding for carboxymethylcellulase (CMCase) activity, was studied in E. coli DH5. Of the various feeding policies adopted for maximal expression and stability, exponential feeding resulted in the highest biomass of 15g dry cell weight (DCW) l^–1 and plasmid stability of 45%. A CMCase activity of 11400 Uml^–1 was achieved as compared to 230 Uml^–1 during batch cultivation. In the case of other feeding strategies viz., constant feeding, linear feeding or intermittent feeding, the plasmid stability varied between 20% to 60%. Biomass achieved ranged from 5.0 g DCW l^–1 to 9.0 g DCW l^–1 and enzyme activities were between 2550 Uml^–1 and 6000 Uml^–1.     

Changes in amino acid content of an algal feed species (Navicula sp.) and their effect on growth and survival of juvenile abalone (Haliotis rubra)

The growth and survival of juvenile Haliotis rubra, when fed with the diatom Navicula sp. cultured in f/2 medium containing combined nitrogen at 24.71 mg NO₃-N L^–1 (high), 12.35 mg NO₃-N L^–1 (standard) or 2.47 mg NO₃-N L^–1 (low), were compared in a 33-day trial. The alga in the low nitrogen medium contained 37% less total amino acid than that in the high and standard nitrogen media. There was a slightly greater reduction in essential amino acids (40%) compared to non-essential amino acids (35%). Juvenile abalone feeding on Navicula grown in medium with low nitrate and lower total amino acid content grew more slowly than when fed on the same species grown in standard or higher nitrogen medium with a higher amino acid content. The growth rate of juveniles was highest (43 m d^–1) in the high nitrate treatment followed (40 m d^–1) by the standard nitrate treatment and lowest (31 m d^–1) in the low nitrate treatment. The survival of the juveniles was also effected by the diet. Survival was better in the high and standard nitrogen media (88%) than the low nitrogen medium (75%). The results suggest that in order to achieve uniformity in nutritional quality of diatoms and good growth of abalone juveniles in commercial abalone nurseries, the nitrogen concentration in tanks should be monitored and additional nitrate added to provide an optimum concentration of between 2 and 12 mg NO₃-N L^–1.     

Efficient micropropagation of <Emphasis Type="Italic">Ensete ventricosum</Emphasis> applying meristem wounding: a three-step protocol

A highly efficient three-step protocol for in vitro propagation of Ensete ventricosum (enset) was developed that consisted of initiation, bud proliferation, and shoot elongation and rooting stages. At the initiation stage, it was crucial to use shoot tips (5–8 mm) with subtending corm tissues as explants to obtain growth. The addition of 0.5–1% (w/v) activated charcoal to the medium was essential to prevent phenol exudation which otherwise leads to the loss of cultures. During the bud proliferation stage, modified MS macronutrients and micronutrients together with a combination of cytokinins (1.6 M naphthaleneacetic acid, 4.4 M 6-benzylaminopurine, 23.2 M kinetin, 22.6 M N⁶ 2-isopentyladenine) was used. This novel composition of macronutrients was based on the analysis of leaf nutrient content of glasshouse-grown enset sprouts. Multiple bud formation on the enlarged corm tissue was induced only when the meristem region was wounded before transfer to the bud proliferation medium. Up to 75 healthy shoots per explant were produced, whereas unwounded explants produced, only one to two shoots per explant. A third stage with a low concentration of cytokinin enabled shoot elongation as well as root development. The plantlets were acclimatized with 100% success and they showed no apparent phenotypical deviation.Abbreviations BAP 6-Benzylaminopurine - IBA Indole-3-butyric acid - DW Dry weight - EV Ensete ventricosum medium - 2-iP N⁶ 2-Isopentyl adenine - NAA -Naphthaleneacetic acid     

Formation of phenylethanoid glycosides by Cistanche deserticola callus grown on solid media

The optimal growth of Cistanche deserticola callus and formation of phenylethanoid glycosides (PeG) was at 25°C with light irradiation intensity of 24 mol m^–2 s^–1 on solidified B5 media supplemented with 0.5 mg 6-benzylaminopurine l^–1, 10 mg gibberellin l^–1, 800 mg casein hydrolysate l^–1 and 20 g sucrose l^–1. After 30 d culture, the biomass reached 15.5 g dry wt callus l^–1 medium and its PEG content was 10.7% (w/w). The PeG content was 42%–127% higher than those in explants.     

Screening yeast cells for absorption of selenium oxyanions

Certain yeast cells on solid nutrient medium produced colonies surrounded by a light zone of selenite absorption. This screening procedure resulted in the selection of 22 strains out of 200 isolates with different Se⁴⁺-absorbing capacity ranging from 16 to 98.8 g Se⁴⁺ g^–1 l^–1 h^–1. The highest rate of Se⁴⁺ elimination from the Na₂SeO₃ solution was observed with an oval shaped, cream pigmented fermentative yeast, tentatively called Candida sp. strain MS4. This strain was isolated from wastewater and found to accumulate selenium oxyanions. Se⁴⁺ uptake involved both inactive and active phenomena. The amounts of selenium (initial concentration 2 mg Se⁴⁺ l^–1) removed from aqueous solution by inactive and active phenomena were 667 g Se⁴⁺ g^–1 l^–1, and 1580 g Se⁴⁺ g^–1 l^–1, respectively. The strain also removed selenate inactively (135 g Se⁶⁺ g^–1 l^–1).     

Degradation of polyaromatic hydrocarbons by Burkholderia cepacia 2A-12

A new strain of bacterium degrading polyaromatic hydrocarbons (PAHs), Burkholderia cepacia 2A-12, was isolated from oil-contaminated soil. Of three PAHs, the isolated strain could utilize naphthalene (Nap) and phenanthrene (Phe) as a sole carbon source but not pyrene (Pyr). However, the strain could degrade Pyr when a cosubstrate such as yeast extract (YE) was supplemented. The PAH degradation rate of the strain was enhanced by the addition of other organic materials such as YE, peptone, glucose, and sucrose. YE was a particularly effective additive in stimulating cell growth as well as PAH degradation. When 1 g YE l^–1, an optimum concentration, was supplemented into the basal salt medium (BSM) with 215 mg Phe l^–1, the specific growth rate (0.30 h^–1) and Phe-degrading rate (29.6 mol l^–1 h^–1) were enhanced approximately ten and three times more than those obtained in the BSM with 215 mg Phe l^–1, respectively. Both cell growth and PAH degradation rates were increased with increasing Phe and Pyr concentrations, and B. cepacia 2A-12 had a tolerance against Phe and Pyr toxicity at the high concentration of 730–760 mg l^–1. Through kinetic analysis, the maximum specific growth rate ( _max) and PAH degrading rate ( _max) for Phe were obtained as 0.39 h^–1 and 300 mol l^–1 h^–1, respectively. Also, _max and _max for Pyr were 0.27 h^–1 and 52 mol l^–1 h^–1, respectively. B. cepacia 2A-12 could simultaneously degrade crude oil as well as PAHs, indicating that this bacterium is very useful for the removal of oils and PAHs contaminants.     

Immobilisation of lipases by adsorption and deposition: high protein loading gives lower water activity optimum

Two different immobilisation techniques for lipases were investigated: adsorption on to Accurel EP-100 and deposition on to Celite. The specific activities were in the same order of magnitude, 2.9 (mol min^–1 mg protein) when Celite was used as support and 2.3 (mol min^–1 mg^–1 protein) when Accurel EP-100 was used as support, even if the amount of lipase loaded differed by 2 orders of magnitude. Immobilisation on Accurel EP-100 was the preferred technique since 40–100 times more protein can be loaded/per g carrier, thus yielding a more active catalyst. The water activity profiles in lipase catalysed esterification were influenced by the amount of protein adsorbed to Accurel EP-100. Higher protein loading (40 mg g^–1) resulted in a bell-shaped water activity profile with highest specific activity (6.1 mol min^–1 mg^–1 protein) at a _w=0.11, while an enzyme preparation with low protein loading (4 mg g^–1) showed highest specific activity at a _w=0.75.