RESUMO

The degradation of three antibiotics (sulfamethoxazole, trimethoprim, and ofloxacin) and one synthetic hormone (17 α-ethinylestradiol) was investigated in three in-vitro biotransformation models (i.e., pure enzymes, hairy root, and Trichoderma asperellum cultures) for anticipating the relevance of the formation of transformation products (TPs) in constructed wetlands (CWs) bioaugmented with T. asperellum fungus. The identification of TPs was carried out employing high-resolution mass spectrometry, using databases, or by interpreting MS/MS spectra. An enzymatic reaction with ß-glucosidase was also used to confirm the presence of glycosyl-conjugates. The results showed synergies in the transformation mechanisms between these three models. Phase II conjugation reactions and overall glycosylation reactions predominated in hairy root cultures, while phase I metabolization reactions (e.g., hydroxylation and N-dealkylation) predominated in T. asperellum cultures. Following their accumulation/degradation kinetic profiles helped in determining the most relevant TPs. Identified TPs contributed to the overall residual antimicrobial activity because phase I metabolites can be more reactive and glucose-conjugated TPs can be transformed back into parent compounds. Similar to other biological treatments, the formation of TPs in CWs is of concern and deserves to be investigated with simple in vitro models to avoid the complexity of field-scale studies. This paper brings new findings on the emerging pollutants metabolic pathways established between T. asperellum and model plants, including extracellular enzymes.

RESUMO

Brugmansia candida (syn. Datura candida) is a South American native plant that produces tropane alkaloids. Hyoscyamine, 6ß-hydroxyhyoscyamine (anisodamine), and scopolamine are the most important ones due to their anticholinergic activity. These bioactive compounds have been historically and widely applied in medicine and their demand is continuous. Their chemical synthesis is costly and complex, and thereby, these alkaloids are industrially produced from natural producer plants. The production of these secondary metabolites by plant in vitro cultures such as hairy roots presents certain advantages over the natural source and chemical synthesis. It is well known that hairy roots produced by Agrobacterium rhizogenes infection are fast-growing cultures, genetically stable and able to grow in hormone-free media. Additionally, recent progress achieved in the scaling up of hairy root cultures makes this technology an attractive tool for industrial processes. This chapter is focused on the methods for the induction and establishment of B. candida hairy roots. In addition, the scaling up of hairy root cultures in bioreactors and tropane alkaloid analysis is discussed.

Assuntos

Biotecnologia/métodos , Datura/fisiologia , Raízes de Plantas/fisiologia , Tropanos/metabolismo , Agrobacterium/crescimento & desenvolvimento , Reatores Biológicos , Biotecnologia/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Técnicas de Cultura/instrumentação , Técnicas de Cultura/métodos , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Datura/genética , Datura/crescimento & desenvolvimento , Datura/microbiologia , Desenho de Equipamento , Melhoramento Vegetal/métodos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/fisiologia , Esterilização/métodos , Tropanos/análise , Tropanos/isolamento & purificação

RESUMO

ABSTRACT Momordica charantia (Cucurbitaceae) is an important vegetable and also medicinal crop which produces the bioactive compounds for various biological activities with potential uses in human health. The present investigation relates to elicitors of jasmonic acid (JA) and salicylic acid (SA) to enhance biomass accumulation and phenolic compound production in hairy root cultures of M. charantia. Hairy root cultures were elicited with JA and SA at 0, 25, 50 and 100 μM concentrations respectively. The adding of elicitation to the hairy root cultures on the 15th day of culture and the roots were harvested on day 25. Cultures supplemented with 100 μM JA and SA enhanced the phenolic compounds significantly compared to that of non-elicited hairy root cultures. The biomass of hairy root culture significantly increased by SA whereas decreased in JA elicitation at 100 μM. JA and SA-elicited hairy root cultures significantly produced a higher amount of phenolic compounds (12811.23 and 11939.37µg/g), total phenolic (4.1 and 3.7 mg/g) and flavonoid (3.5 and 3.2 mg/g) contents than non-elicited hairy root cultures (10964.25 µg/g, 2.8 and 2.5 mg/g). JA and SA-elicited hairy root cultures were significantly higher antioxidant activity of DPPH (84 and 78%), reducing potential (0.53 and 0.48), phosphomolybdenum (3.6 and 3.2 mg/g) and ferrous ion chelating assays (80 and 74%) than non-elicited hairy root cultures. The higher antimicrobial and anticancer activity were exhibited in JA and SA-elicited than non-elicited hairy root cultures. This protocol can be developed for the production of phenolic compounds from JA and SA-elicited hairy root cultures.

RESUMO

The aim of this study was to use the, hairy root system for increasing the scopolamine content in Atropa belladonna. Agrobacterium rhizogenes ATCC15834 was utilized to produce hairy roots. The culture was carried out in a 1.5-l bioreactor using the inoculum size of 0.5 g fr. wt of 10-day-old hairy roots and various parameters, including agitation, aeration, conductivity and the consumption of sucrose were evaluated. Results revealed that the highest amount of scopolamine production (1.59 mg/g-1 dry wt) occurred in the bioreactor with aeration and agitation 1.25 vvm (volume per volume per minute) and 70 rpm, respectively. Study of conductivity and the consumption of sucrose showed that the highest amount of sucrose consumption and the highest amount of minerals consumption also was at 1.25 vvm and 70 rpm. Transgenic hairy root lines were confirmed by polymerase chain reaction (PCR).

RESUMO

To develop a successful protocol forAgrobacterium-mediated transformation in plants it is essential to determine the most efficient bacterial strain/plant genotype interaction. In the present work, we evaluated the susceptibility ofEucalyptus grandis ×E. urophylla to fiveAgrobacterium rhizogenes and twelveA. tumefaciens wildtype strains. The results showed different degrees of virulence, according to the strain tested, indicating that transformation of this eucalypt hybrid by Agrobacterium-derived vectors is possible. All developed tumours showed an autonomous growth when transferred to a hormone-free medium. Some of these tumours formed shoots spontaneously, with a normal phenotype. Polymerase Chain Reaction (PCR) and Southern blot analyses were performed to confirm the absence of the oncogenic T-DNA in plants derived from these shooty tumours.