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.     

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.     

Astragaloside IV and polysaccharide production by hairy roots of Astragalus membranaceus in bioreactors

Hairy roots of Astragalus membranaceus were grown in bioreactors up to 30 l for 20 d. Cultures from a 30 l airlift bioreactor gave 11.5 g l dry wt with 1.4 mg g^–1 astragaloside IV, similar to cultures from 250 ml and 1 l flasks, but greater than yields from a 10 l bioreactor (dry wt 9.4 g l^–1, astragaloside IV 0.9 mg g^–1). Polysaccharide yields were similar amongst the different bioreactors (range 25–32 mg g^–1). The active constituent content of the cells approached that of plant extracts, indicating that large scale hairy root cultures of A. membranaceus has the potential to provide an alternative to plant crops without compromising yield or pharmacological potential.     

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.     

Improvement of phenylethanoid glycosides production by a fungal elicitor in cell suspension culture of Cistanche deserticola

When, on the 15th day of growth, an elicitor from Fusarium solani was added at 40 mg l^–1 to Cistanche deserticola cell suspension cultures, the contents of echinacoside, acteoside and total phenylethanoid glycosides (PeGs) in cultured cells all increased over the next 27 d by over 100% to 15 mg g^–1 dry wt, 9 mg g^–1 dry wt and 57 mg g^–1 dry wt, respectively. The final biomass (1.3 mg dry wt ml^–1) was not affected.     

Production of plumbagin from cell cultures of Plumbago rosea L.

Callus and suspension cultures derived from leaf explants of Plumbago rosea were established and plumbagin, a naphthoquinone, was isolated from them and confirmed by ¹H NMR and electron-ionization mass spectroscopy. Maximum content of plumbagin was obtained in the stationary phase of growth (4.3 mg g^–1 dry cell wt). Media pH, phytohormones and carbon sources were optimized for biomass and plumbagin accumulation. Cell aggregates, measuring 500 m in diam, produced 8.2 g dry cell wt l^–1, but larger aggregates (above 500 m) favored plumbagin accumulation with an yield of 4.5 mg g^–1 dry cell wt.     

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.     

Improved growth of Artemisia annua L hairy roots and artemisinin production under red light conditions

Hairy root cultures of Artemisia annua L were cultivated for 30 days under either white, red, blue, yellow or green light. Red light at 660 nm gave the highest biomass of hairy roots (5.73 g dry wt cells l^–1 medium) and artemisinin content (31 mg arteminsinin g^–1 dry cells) which were, respectively, 17% and 67% higher than those obtained under white light.     

In vitro production of sedative galphimine B by cell suspension cultures of Galphimia glauca

The sedative triterpene, galphimine B (1), was detected in cell suspension-batch cultures of Galphimia glauca. The effect of inoculum size, growth regulators and different concentrations of sucrose, nitrates and phosphates was evaluated. A two-stage batch process for biomass production and accumulation of compound 1 was established. Major cellular growth (15 g l^–1 dry wt) was obtained in the first stage with naphthaleneacetic acid (2 mg l^–1) + kinetin (2 mg l^–1). Adding 4 mg 2,4-dichlorophenoxyacetic l^–1 acid in the second stage resulted in the highest accumulation of 1 (0.21 mg g^–1 dry wt) which was 36% higher with respect to calluses and comparable to that obtained from wild plants.     

Induced accumulation of oleanolic acid and ursolic acid in cell suspension cultures of <Emphasis Type="Italic">Uncaria tomentosa</Emphasis>

Increasing sucrose from 20 to 50 g l⁻¹ in Uncaria tomentosa cell suspension cultures enhanced ursolic acid and oleanolic acid production from 129 ± 61 to 553 ± 193 μg g⁻¹ cell dry wt. The maximal concentration of both triterpenes (1680 ± 39 μg g⁻¹ cell dry wt) was 8 days after elicitation by jasmonic acid, while yeast extract or citrus pectin treatments produced 1189 ± 20 or 1120 ± 26 μg g⁻¹ cell dry wt, respectively. The ratio of ursolic acid:oleanolic acid was constant at 70:30.     

Hairy Root Cultures of <Emphasis Type="Italic">Gynostemma pentaphyllum</Emphasis> (Thunb.) Makino: A Promising Approach for the Production of Gypenosides as an Alternative of Ginseng Saponins

Hairy root cultures of Gynostemma pentaphyllum were established by infecting leaf discs with Agrobacterium rhizogenes. The dry biomass of hairy roots grown in MS medium for 49 days was 7.3 g l⁻¹ with a gypenoside content of 38 mg g⁻¹ dry wt.     

Poly-3-hydroxybutyrate (P3HB) production by bacteria from xylose,glucose and sugarcane bagasse hydrolysate

Fifty-five bacterial strains isolated from soil were screened for efficient poly-3-hydroxybutyrate (P3HB) biosynthesis from xylose. Three strains were also evaluated for the utilization of bagasse hydrolysate after different detoxification steps. The results showed that activated charcoal treatment is pivotal to the production of a hydrolysate easy to assimilate. Burkholderia cepacia IPT 048 and B. sacchari IPT 101 were selected for bioreactor studies, in which higher polymer contents and yields from the carbon source were observed with bagasse hydrolysate, compared with the use of analytical grade carbon sources. Polymer contents and yields, respectively, reached 62% and 0.39 g g^–1 with strain IPT 101 and 53% and 0.29 g g^–1 with strain IPT 048. A higher polymer content and yield from the carbon source was observed under P limitation, compared with N limitation, for strain IPT 101. IPT 048 showed similar performances in the presence of either growth-limiting nutrient. In high-cell-density cultures using xylose plus glucose under P limitation, both strains reached about 60 g l^–1 dry biomass, containing 60% P3HB. Polymer productivity and yield from this carbon source reached 0.47 g l^–1 h^–1 and 0.22 g g^–1, respectively.     

Indigo production in hairy root cultures of Polygonum tinctorium Lour.

The transformed root culture of Polygonum tinctorium Lour. was established by infecting leaf explants with Agrobacterium rhizogenes A4. These cultures were examined for their growth and indigo content under various culture conditions. Among the four different culture media tested, SH medium showed the highest yield for root growth (28 mg dry wt/30 ml) and indigo production (152 g/dry wt). In SH medium, 30 g sucrose l^–1, 2500 mg KNO₃ l^–1, 300 mg NH₄H₂PO₄ l^–1 were the best conditions for indigo production at pH 5.7. The production of indigo in hairy roots slightly increased with the addition of 200 mg chitosan l^–1 (186 g/dry wt) and 20 U pectinase l^–1 (181 g/dry wt).     

Optimization of growth and fatty acid composition of a unicellular marine picoplankton,Nannochloropsis sp., with enriched carbon sources

A unicellular marine picoplankton, Nannochloropsis sp., was grown under CO₂-enriched photoautotrophic or/and acetate-added mixotrophic conditions. Photoautotrophic conditions with enriched CO₂ of 2800 l CO₂ l^–1 and aeration gave the highest biomass yield (634 mg dry wt l^–1), the highest total lipid content (9% of dry wt), total fatty acids (64 mg g^–1 dry wt), polyunsaturated fatty acids (35% total fatty acids) and eicosapentaenoic acid (EPA, 20:53) (16 mg g^–1 dry wt or 25% of total fatty acids). Mixotrophic cultures gave a greater protein content but less carbohydrates. Adding sodium acetate (2 mM) decreased the amounts of the total fatty acids and EPA. Elevation of CO₂ in photoautotrophic culture thus enhances growth and raises the production of EPA in Nannochloropsis sp.     

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.     

Production of a recombinant, immunogenic protein, P64k, of Neisseria meningitidis in Escherichia coli in fed-batch fermenters

P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, codifying for this protein, was cloned in Escherichia coli and the P64k protein was expressed in Escherichia coli K12 W3110 under the control of the tryptophan promoter. The recombinant bacteria were grown in batch or fed-batch cultures. P64k was expressed as an intracellular soluble form at about 40% of the total cellular protein. A final productivity of 215 mg l^–1 h^–1 and 11 g cell dry wt l^–1 were obtained when the fed-batch culture conditions were optimised, compared to 30% of total protein, and a productivity of 76 mg l^–1 h^–1 and 5.1 g cell dry wt l^–1 in batch cultivation.     

Stimulation of artemisinin production in Artemisia annua hairy roots by the elicitor from the endophytic Colletotrichum sp.

Artemisinin content in hairy roots of Artemisia annua was increased from 0.8 mg g^–1 dry wt to 1 mg g^–1 dry wt by using elicitor treatment of mycelial extracts from the endophytic fungus Colletotrichum sp. The increase of artemisinin was dependent on the growth stage of hairy roots as well as on the dose of the elicitor applied. When hairy roots of 23-day-old cultures (later growth phase) were exposed to the elicitor at 0.4 mg total sugar ml^–1 for 4 days, the maximum production of artemisinin reached to 13 mg l^–1, a 44% increase over the control. This is the first report on the stimulation of artemisinin production in hairy roots by the elicitor from an endophytic fungus of A. annua.     

In vitro culture of Montanoa tomentosa for the production of diterpenic acids

Following a solid phase extraction, GC-MS and GC-FID procedures, the production of three kaurane derivatives (grandiflorenic, kaurenoic and monoginoic acids) was detected in callus and cell suspension batch cultures of Montanoa tomentosa. From different hormonal combinations, the addition of 0.5 mg 2,4-dichlorophenoxyacetic acid l^–1 + 2 mg kinetin l^–1 increased the accumulation of total kaurenoids in 6 months old calluses to 2.1 mg g^–1 dry weight and in cell suspensions cultures up to 0.76 mg g^–1 dry weight. Monoginoic acid, which has not been detected before in leaves of wild plants, accumulated in both in vitro systems.     

Manipulating anthocyanin composition in Vitis vinifera suspension cultures by elicitation with jasmonic acid and light irradiation

Jasmonic acid altered the accumulation of major anthocyanins in Vitis vinifera cell culture. Peonidin 3-glucoside content at day three was increased from 0.3 to 1.7 mg g^–1 dry cell wt while other major anthocyanins were increased by smaller increments. By day 14, the content of methylated and acylated anthocyanins (peonidin 3-p-coumaroylglucoside and malvidin 3-p-coumaroylglucoside) was 6.3 mg g^–1 DCW, in response to treatment with jasmonic acid, and comprising 45% (w/w) of total anthocyanins. In comparison, the untreated control culture contained 1.2 mg g^–1 DCW which made up 32% (w/w) of total anthocyanins. Light further enhanced anthocyanin accumulation induced by jasmonic acid elicitation. The content of peonidin 3-glucoside at day 3 was 6.6 mg g^–1 DCW, 22-fold higher than control cultures while the content in response to light irradiation alone was 0.6 mg g^–1 DCW. When a highly pigmented cell line was elicited with jasmonic acid total anthocyanins increased from 9.2 to 20.7 mg g^–1 DCW, but there was no change in the anthocyanin composition.     

Production of crocin using Crocus sativus callus by two-stage culture system

Saffron callus was grown in a two-stage culture on B5 medium supplemented with casein hydrolysate (300 mg l^–1) at 22 °C in dark with naphthalene acetic acid (2 mg l^–1) and 6-benzyladenine (1 mg l^–1) to give maximum biomass (16 g dry wt l^–1), and with indole 3-acetic acid (2 mg l^–1) and 6-benzyladenine (0.5 mg l^–1) for crocin formation. The maximum crocin production (0.43 g l^–1) was achieved by this two-stage culture method, which was three times that by a one-stage method.