Sandra I. Pitta-Alvarez

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.

Advantages and limitations in the use of hairy root cultures for the production of tropane alkaloids: Use of anti-auxins in the maintenance of normal root morphology

SummaryBrugmansia candida hairy roots, obtained by infection withAgrobacterium rhizogenes LBA 9402, exhibit, after subculturing in liquid media, a tendency towards dedifferentiation. It has been found that the following strategies can be applied to inhibit this dedifferentiation and preserve normal root morphology: (a) lowering both the mineral and sucrose concentration in the media employed so as to diminish osmotic stress (a condition to which these roots appear to be particularly susceptible); (b) employing antiauxins in appropriate concentrations; and (c) maintaining the hairy roots on solid media prior to use in production processes in liquid media. The first strategy suggested does not favor alkaloid productivity, but in this case a two-step method could be attempted: biomass with normal root morphology could be obtained in a first stage using low sucrose concentrations, and in a second stage, sucrose could be increased in order to achieve higher productivity. In all the clones ofB. candida obtained, alkaloid production was biased towards scopolamine.

Occurrence of cadaverine in hairy roots of Brugmansia candida

The polyamine, cadaverine, was detected in transformed root cultures of Brugmansia candida (syn. Datura candida), a Solanaceae which produces the tropane alkaloids scopolamine and hyoscyamine. To the best of our knowledge, this is the first time that the existence of this uncommon polyamine has been detected in a Datura species. Cadaverine, however, could not be found in the whole plant. The occurrence of cadaverine in hairy roots could be a consequence of either the transformation or a response to stress. Also, cadaverine could be participating in other secondary pathways rather than to the tropane alkaloids. The common polyamines, putrescine, spermidine and spermine were also observed.

Biotransformation of hydroquinone by hairy roots ofBrugmansia candida and effect of sugars and free-radical scavengers

Hairy roots ofBrugmansia candida were used to bioconvert hydroquinone into arbutin. The highest bioconversion, with the lowest damage to the cells, was attained when concentrations of 20–40 mg/L hydroquinone were used. Sugars (sucrose, glucose, mannitol, and sorbitol) at concentrations of 30–120 g/L enhanced bioconversion, and, of these, sucrose was the most effective. Two different free-radical scavengers were also tested: sodium benzoate and gallic acid. The first one diminished biotransformation efficiency; gallic acid did not affect biotransformation at all. Preliminary permeabilization treatments tested failed to liberate arbutin into the medium, and provoked a total loss in cell viability.

Effects of gibberellin GA7 on kinetics of growth and tropane alkaloid accumulation in hairy roots of Brugmansia candida

SummaryBrugmansia candida, an indigenous South American plant, produces the tropane alkaloids scopolamine and hyoscyamine, which are widely employed in medicine as anticholinergic agents. In this research, hairy roots of Brugmansia candida, obtained through infection with Agrobacterium rhizogenes LBA 9402, were employed to produce these tropane alkaloids in vitro. The effects of different concentrations of GA7 on kinetics of growth and alkaloid accumulation on two different hairy root clones of B. candida were analyzed, and the influence of GA7 on the number of new branches and rates of elongation was also studied. On clone 7A, GA7 at concentrations of 10−4, 10−1, and 1 mg/l increased the exponential growth rate. Levels of 10−1 and 10−4 mg/l GA7 elevated the scopolamine/hyoscyamine (S/H) ratios in the early phases of growth, but the sum of scopolamine plus hyoscyamine per flask (S + H) decreased during that period. When 1 mg/l GA7 was used, the highest S/H ratios were observed in late exponential/early stationary phases, but the highest S + H totals were obtained in mid-exponential phase. GA7 at levels of 10−1 and, especially, 1 mg/l exerted a positive effect on formation, emergence, and rate of elongation of lateral roots (clone 7A). On clone 7B, levels of 10−1 and 1 mg/l GA7 did not alter significantly the exponential growth rate. GA7 in concentrations of 10−1 mg/l induced increases in both S/H ratio and S + H totals in late phases of growth.

In vitro shoot culture and antimicrobial activity of Berberis buxifolia Lam

Berberis buxifolia Lam., known as “Calafate”, is a plant native to Argentina that exhibits antimicrobial activity. This biological activity is attributed to the isoquinoline alkaloid berberine. The aim of this research was to test the antimicrobial properties of different extracts of this species, taking berberine as the reference molecule, and to examine if the expression of bacterial multidrug resistance (MDR) efflux pumps could be responsible for possible resistance mechanisms. To this end, a wild-type and a mutant strain of Staphylococcus aureus with a defective MDR efflux pump were used and the minimum inhibitory concentrations of the extracts were determined. The studies were carried out with infusions of in vivo shoots and “Calafate” commercial tea, as well as with the media derived from shoot cultures incubated with different plant growth regulators (thidiazuron, picloram, and jasmonic acid). As far as antimicrobial activity is concerned, all the extracts tested were significantly more effective than berberine standard. “Calafate” commercial tea and shoot tea had inhibitory concentrations similar to the one observed for ampicillin standard. The media from the shoot cultures, however, were significantly more effective than all the others, particularly the one derived from jasmonic acid, suggesting the presence of compounds that could be acting synergistically with berberine. There were no differences in antimicrobial activity against the wild-type and the mutant S. aureus; no definite conclusions could be drawn concerning the relationship between MDR pumps and possible pathogen resistance to extracts of B. buxifolia.

Novel biotechnological approaches to obtain scopolamine and hyoscyamine: the influence of biotic elicitors and stress agents on cultures of transformed roots of Brugmansia candida

Scopolamine and hyoscyamine are tropane alkaloids employed in medicine as antispasmodics, preoperative medication and for the treatment of motion sickness. Since their chemical synthesis is difficult and expensive, these compounds are still extracted from plants that belong to several species of the Solanaceae. In this research, a biotechnological system for the production of both alkaloids was employed. Hairy roots of Brugmansia candida (syn. Datura candida), obtained through transformation with the soil pathogen Agrobacterium rhizogenes, were cultured in vitro. In an attempt to increase the productivity of the cultures, they were exposed to biotic elicitors (hemicellulase and homogenized B. candida roots) and stress agents (CuSO4). It was observed that hemicellulase had a positive effect on scopolamine (2‐fold increase) and, especially, hyoscyamine accumulation (4‐fold increase) in the roots. The homogenized roots also had a positive effect on alkaloid accumulation in the roots throughout the experiment (approximately 2.5‐fold). The main effects of CuSO4 were seen in the release of alkaloids to the medium, particularly at 24 and 48 (6 to 10‐fold increase).

Hairy roots of Brugmansia candida that grow without agitation: biotechnological implications.

Hairy roots of Brugmansia candida that grew without agitation were obtained. Kinetics of growth and production of the tropane alkaloids scopolamine and hyoscyamine, with and without agitation, were studied. The exponential growth rate was higher in the roots that were exposed to shaking (0.13 d−1) than in the nonagitated ones (0.09 d−1). The specific production and the levels per flask of both alkaloids were enhanced without shaking. The use of these roots in large‐scale productions could be economically advantageous. It remains to be seen if the data obtained in shake flasks can be extrapolated to large‐scale bioreactors.

Development of agroclimatic zoning model to delimit the potential growing areas for macaw palm (Acrocomia aculeata)

The growing biodiesel production requires the use of new technologies and alternative feedstocks to maintain the growing demand of this biofuel. The macaw (Acrocomia aculeata) is a palm native to Argentina whose fruits present high oil content. Due to its tolerance to prolonged drought, it is a promising crop for biodiesel and biokerosene production. The aim of this work was to design an agroclimatic zoning model to define the potential growing areas from macaw in Argentina. To define the agroclimatic suitability to produce oil, it was necessary to identify the requirements, limits, and biometeorological tolerance for this palm. In order to define the agroclimatic fitness of this crop in Argentina, the meteorological data corresponding to the period 1981–2010 were employed. The agroclimatic indices were integrated in a Geographic Information System. The maps were superimposed and the overlapping regions delineated the agroclimatic zoning. The agroclimatic zonation classified zones with homogeneous characteristics responding to bioclimatic requirements of this species, resulting in optimal, very suitable, suitable, and nonsuitable areas for macaw cultivation. The authors designed an agroclimatic zoning model based on bibliography. This model can be used in any part of the world, employing the same agroclimatic indices presented in this work.