Ana M. Giulietti

The influence of different biotic and abiotic elicitors on the production and profile of tropane alkaloids in hairy root cultures of Brugmansia candida.

Hairy root cultures of Brugmansia candida produce the tropane alkaloids scopolamine and hyoscyamine. In an attempt to increase productivity, several biotic and abiotic elicitors were tested. Salicylic acid increased significantly the release of both alkaloids (2- to 12-fold) and it also acted positively on specific production without altering the production profile. AgNO(3) increased significantly scopolamine release (3-fold) and both alkaloids accumulation (5- to 8-fold) in the roots, thus favoring the production of scopolamine (up to 2-fold). The inhibiting effects of AgNO(3) and salicylic acid on ethylene could be partly responsible for these responses. Yeast extract incremented the intracellular content of both alkaloids (ca. 3-fold), but particularly increased the release of scopolamine (7-fold). CaCl(2) had little effect on accumulation or release of either alkaloid. CdCl(2) acted positively on the release of both alkaloids (3- to 24-fold), but was highly detrimental to growth. Hairy roots of B. candida are therefore susceptible to elicitation by biotic and abiotic elicitors, with variations in the kinetics of induction and the extent of release of each metabolite, thereby also exerting different effects on the alkaloid profile.

Phytoremediation of 2,4-dichlorophenol by Brassica napus hairy root cultures

We have obtained hairy root cultures of Brassica napus with high biomass and genetic stability which produce peroxidases, enzymes involved in biodegradation processes. In this work, these hairy root cultures were used to study the removal of 2,4‐dichlorophenol (2,4‐DCP), a common contaminant in industrial effluents that is highly toxic for human and aquatic life. The optimum conditions to obtain high efficiency in the removal process were established. Roots were able to remove 2,4‐DCP from aqueous solutions containing 100–1000 mg/l, in the presence of H2O2 concentrations ranging from 5 to 10 mM. After a short period of incubation (15 min), high removal efficiencies were achieved (91–94%) and maximal removal, of approx. 97–98%, was obtained with 1 h of reaction. High removal efficiencies (93–95%) were observed in a broad pH range (pH 3–9), reaching 98–99% in the range pH 4–8. Moreover, roots could be re‐used, almost for six consecutive cycles, to remove 2,4‐DCP. The oxidation catalysed by peroxidases would be the main mechanism involved in this process. The results suggest that these cultures could be useful tools for phytoremediation.

Artemisia annua L.: dedifferentiated and differentiated cultures

Dedifferentiated and differentiated tissue cultures ofArtemisia annua L. for artemisinin production were carried out. The calluses were initiated on MS medium supplemented with sucrose (30 g l-1), myoinositol (100 mg l-1) and RT vitamins. The auxins used were naphtaleneacetic acid (NAA), indoleacetic acid (IAA), indolebutyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-d). These were added to the basal medium either singly or in combination. The best results were obtained with 2.4-d (4.5 μM : μ0.02 d-1) and NAA (5.4 μM : μ 0.06 d-1). Cell suspensions were established on the same media without agar. Suspension cultures showed different morphological characteristics according to the plant growth regulator supplied. Organized cultures were initiated from callus obtained on 2,4-d (4.5 μM) and from bud cultures. Medium containing 6-benzylaminepurine (BA) (8.9 μM)+NAA (0.54 μM); Zeatin (45.62 μM)+NAA (5.37 μM) or BA (8.9 μM) stimulated both organogenesis in callus (frequency of induction =50%) and semi-organized tissue in shoot buds. BA (13.32 μM)+NAA (1.08 μM) or BA (13.32 μM) only stimulated multiple shoot cultures (frequency of induction =80%). Regarding artemisinin content, while the values obtained were 1.13 and 0.78 mg gDW-1 in primary callus, artemisinin was not detected in cell suspension and only traces of it were found in multiple shoot cultures.

Artemisinin production by Artemisia annua L.-transformed organ cultures

Abstract Transformed shoot and root cultures of Artemisia annua L. were established by infection with Agrobacterium tumefaciens strain T37 and A. rhizogenes LBA 9402. These cultures were able to grow in a hormone-free medium and produce artemisinin during prolonged subcultures. Several clones of transformed shoots were obtained and the average artemisinin content found in them was approximately 0.018 ± 0.004% dry weight (DW). Changes in Ca2+, Mg2+, and PO43− concentration of the RT vitamin complex medium (MSRT) did not induce an increase in artemisinin production. The addition of plant growth regulators such as GA3 improved the artemisinin production of 300–400% of the control value. Transformed root cultures showed a strong morphological instability and accumulated low levels of artemisinin (0–0.01% DW).

A simple and rapid micro-Kjeldahl method for total nitrogen analysis

A fast and simple micro-method for total nitrogen analysis in complex fermentation media is presented. Samples containing up to 50 ug nitrogen are digested in tubes with sodium selenite (in sulphuric-phosphoric mixture) plus hydrogen peroxide during one hour at 380°C. Ammonium liberated is measured without extraction by Berthelot colorimetric reaction.

Phytoremediation potential of the novel atrazine tolerant Lolium multiflorum and studies on the mechanisms involved.

Atrazine impact on human health and the environment have been extensively studied. Phytoremediation emerged as a low cost, environmental friendly biotechnological solution for atrazine pollution in soil and water. In vitro atrazine tolerance assays were performed and Lolium multiflorum was found as a novel tolerant species, able to germinate and grow in the presence of 1 mg kg(-1) of the herbicide. L. multiflorum presented 20% higher atrazine removal capacity than the natural attenuation, with high initial degradation rate in microcosms. The mechanisms involved in atrazine tolerance such as mutation in psbA gene, enzymatic detoxification via P(450) or chemical hydrolysis through benzoxazinones were evaluated. It was demonstrated that atrazine tolerance is conferred by enhanced enzymatic detoxification via P(450). Due to its atrazine degradation capacity in soil and its agronomical properties, L. multiflorum is a candidate for designing phytoremediation strategies for atrazine contaminated agricultural soils, especially those involving run-off avoiding.

Cupriavidus pampae sp. nov., a novel herbicide- degrading bacterium isolated from agricultural soil

A bacterial consortium able to degrade the herbicide 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB) was obtained from an agricultural soil of the Argentinean Humid Pampa region which has a history of long-term herbicide use. Four bacterial strains were isolated from the consortium and identified as members of the genera Cupriavidus, Labrys and Pseudomonas. A polyphasic systematic analysis was carried out on strain CPDB6(T), the member of the 2,4-DB-degrading consortium able to degrade 2,4-DB as a sole carbon and energy source. The Gram-negative, rod-shaped, motile, non-sporulating, non-fermenting bacterium was shown to belong to the genus Cupriavidus on the basis of 16S rRNA gene sequence analyses. Strain CPDB6(T) did not reduce nitrate, which differentiated it from the type species of the genus, Cupriavidus necator; it did not grow in 0.5-4.5 % NaCl, although most species of Cupriavidus are able to grow at NaCl concentrations as high as 1.5 %; and it was able to deamidate acetamide, which differentiated it from all other species of Cupriavidus. DNA-DNA hybridization data revealed low levels of genomic DNA similarity (less than 30 %) between strain CPDB6(T) and the type strains of Cupriavidus species with validly published names. The major cellular fatty acids detected were cis-9-hexadecenoic (16 : 1ω7c) and hexadecanoic (16 : 0) acids. On the basis of phenotypic and genotypic characterizations, strain CPDB6(T) was recognized as a representative of a novel species within the genus Cupriavidus. The name Cupriavidus pampae sp. nov. is proposed, with strain CPDB6(T) (=CCUG 55948(T)=CCM-A-29:1289(T)) as the type strain.

Effect of carbon and nitrogen sources on growth and solasodine production in batch suspension cultures of Solanum eleagnifolium Cav.

The effect of inoculum size, carbon sources (fructose, glucose, maltose, sucrose), nitrate and ammonia on solasodine production by Solanum eleagnifolium Cav. was studied. The specific growth rate was estimated to be 0.15–0.20 d-1 with all sugars tested at a concentration of 90 mM. Sucrose (180 mM) produced the highest biomass value (about 2.8 mg DW ml-1) while the lowest one was produced by maltose. Although solasodine productivity values after 11 days of culture were similar for all sugars tested, the maximum values of productivity (0.9 mg g-1 d-1) were achieved after 6 days of culture with sucrose (180 mM). Solasodine productivity of cultures conducted with a large inoculum (20% w/v fresh material) was double that with a small inoculum (10% w/v fresh material).

Effect of jasmonic acid and aluminium on production of tropane alkaloids in hairy root cultures of Brugmansia candida

Hairy root cultures of Brugmansia candida (Solanaceae), a South American plant which produces scopolamine and hyoscyamine, were exposed to different elicitors (jasmonic acid (JA) and aluminum chloride (AlCl3)) in order to increase their productivity and/or stimulate their liberation. Hairy roots of 19-day old cultures (exponential phase) were exposed to these elicitors for 24 and 48 hours. The effects on alkaloid accumulation and release into the medium were evaluated. JA was tested at 2.5 and 25 m g/ml. After 24 hours, JA promoted the release of hyoscyamine (~1200%) when the highest concentration was used. Therefore, the positive effects seen with JA could possibly be attributed in part to ethanol (EtOH), the solvent in which the acid was dissolved. At the lowest concentration tested, JA promoted an increase on scopolamine accumulation (30%) after 48 hours of exposure. When exposed to AlCl3 for 48 hours and at concentrations of 25 and 250m M, scopolamine and hyoscyamine accumulation increased in the roots (43-83%). After 48 hours of treatment with the highest concentration of AlCl3, release of scopolamine into the medium increased approximately 150%

Influence of chitosan, acetic acid and citric acid on growth and tropane alkaloid production in transformed roots of Brugmansia candida Effect of medium pH and growth phase

The effects of chitosan, acetic acid and citric acid on production and release of hyoscyamine and scopolamine in hairy root cultures of Brugmansia candida were studied. Chitosan and acetic acid were tested at different concentrations and also at different media pH values. At pH 5.5, and at certain concentrations, acetic acid and chitosan increased the content of root scopolamine and hyoscyamine, and promoted the release of both alkaloids. Lowering the pH to 3.5 and 4.5 reduced the accumulation of both alkaloids in the roots, but at a pH of 4.5, their release increased significantly. Acetic and citric acid stimulated the release of scopolamine and hyoscyamine.