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Antibiotic resistance occurs when the use of antibiotics to treat bacterial infections or to select for transformed bacteria from in vitro cultures becomes ineffective. Although this phenomenon is primarily observed in medicine, it also affects the success of scientific research when bacterial-based genetic transformation experiments are performed. During the agrotransformation of plant cells, tissue, and organs, the acquisition of antibiotic resistance by Agrobacterium spp. has been widely observed, making it difficult to select for the transformed bacteria. The objective of this study was to develop a heat-shock protocol for the transformation of Agrobacterium rhizogenes without the spontaneous generation of antibiotic resistance, to increase its sensitivity and specificity to produce transgenic hairy roots. After the bacterium was transformed, it was cultured in liquid culture medium and plated on solid medium to isolate colonies. The genetic transformation of the bacteria and the plant tissue was verified by PCR and by β-glucuronidase assays. The reproducibility of the method was assessed among the A. rhizogenes strains LBA 9402, A4 and 15834 using streptomycin, kanamycin and ampicillin for selection.

Pioneer native plant species from weathered oil spill-affected sites were selected to study their potential for phytoremediation on the basis of their ecological and phenological changes during the phytoremediation process. Experiments were conducted in field and in greenhouse. In field, native plants from aged oil spill-impacted sites with up 400 g of weathered petroleum hydrocarbons per kilogram soil were selected. In the impacted sites, the principal dominant plant species with potential for hydrocarbons removal were Cyperus laxus, Cyperus esculentus, and Ludwigia peploides. In greenhouse, the phenology of the selected plant species was drastically affected by the hydrocarbons level above 325 g total petroleum hydrocarbons (TPH) per kilogram soil after 2 years of phytoremediation of soils from the aged oil spill-impacted sites. From the phytoremediation treatments, a mix-culture of C. laxus, C. esculentus, and L. peploides in soil containing 325 g TPH/kg soil, from which 20.3 % were polyaromatic hydrocarbons (PAH) and 34.2 % were asphaltenes (ASF), was able to remove up 93 % of the TPH, while in unvegetated soil the TPH removal was 12.6 %. Furthermore, evaluation of the biodiversity and life forms of plant species in the impacted sites showed that phytoremediation with C. esculentus, alone or in a mix-culture with C. laxus and L. peploides, reduces the TPH to such extent that the native plant community was progressively reestablished by replacing the cultivated species resulting in the ecological recovery of the affected soil. These results demonstrate that native Cyperus species from weathered oil spill-affected sites, specifically C. esculentus and C. laxus, alone or in a mix-culture, have particular potential for phytoremediation of soils from tropical wetlands contaminated with weathered oil hydrocarbons.

The effect of recalcitrant hydrocarbons on the fatty acid profile from leaf, basal corm, and roots of Cyperus laxus plants cultivated in greenhouse phytoremediation systems of soils from aged oil spill-impacted sites containing from 16 to 340 g/Kg total hydrocarbons (THC) was assessed to investigate if this is a C18:3 species and if the hydrocarbon removal during the phytoremediation process has a relationship with the fatty acid profile of this plant. The fatty acid profile was specific to each vegetative organ and was strongly affected by the hydrocarbons level in the impacted sites. Leaf extracts of plants from uncontaminated soil produced palmitic acid (C16), octadecanoic acid (C18:0), unsaturated oleic acids (C18:1-C18:3), and unsaturated eichosanoic (C20:2-C20:3) acids with a noticeable absence of the unsaturated hexadecatrienoic acid (C16:3); this finding demonstrates, for the first time, that C. laxus is a C18:3 plant. In plants from the phytoremediation systems, the total fatty acid contents in the leaf and the corm were negatively affected by the hydrocarbons presence; however, the effect was positive in root. Interestingly, under contaminated conditions, unusual fatty acids such as odd numbered carbons (C15, C17, C21, and C23) and uncommon unsaturated chains (C20:3n6 and C20:4) were produced together with a remarkable quantity of C22:2 and C24:0 chains in the corm and the leaf. These results demonstrate that weathered hydrocarbons may drastically affect the lipidic composition of C. laxus at the fatty acid level, suggesting that this species adjusts the cover lipid composition in its vegetative organs, mainly in roots, in response to the weathered hydrocarbon presence and uptake during the phytoremediation process.