Occurrence of multiple metal-resistance in bacterial isolates associated with transgenic white poplars ( Populus alba L.)

The occurrence of multiple metal-resistance was assessed in two bacterial collections, named Herbicide Resistant Bacteria (HRB) and Nuclease-Producing Bacteria (NPB) respectively, consisting of 15 and 11 isolates obtained from a loamy sand cultivated with transgenic white poplars (Populus alba L., cv 'Vilafranca') engineered for herbicide resistance. A third collection of 11 bacterial isolates, named Leaf-Associated Bacteria (LAB), obtained from the leaves of transgenic white poplars expressing theStSy gene for resveratrol production and from untransformed plants was evaluated. Resistance to Cd, Co, Cu, Pb and Zn was tested. As for the HRB collection, nine different phenotypes were monitored, which included tetra-, tri- and double-resistance. Tri- and double-metal resistance occurred also within the NPB and LAB collections. In both cases five different phenotypes were recovered. An additional investigation was carried out on the HRB-1c isolate, resistant to Cd, Co, Pb and Zn, which was previously demonstrated to produce indoleacetic acid, a plant-growth-promoting trait. Colorimetric assays, performed on the cell-depleted medium of HRB-1c liquid cultures grown in presence of heavy metals, confirmed that this trait was not affected. A 19-kb plasmid, possibly involved in the maintenance of the multiple metal-resistant phenotype, was detected in the HRB-1c cells

Evaluation of the expression level of the endogenous marker poUBI gene for studies on transgene stability in bar and StSy GM poplars

Abstract This work reports on the isolation and molecular characterization of the poUBI cDNA encoding polyu-biquitin from white poplar (Populus alba L. cv 'Villafranca'). Expression analysis was performed on different poplar organs and tissues, at different developmental stages and in relation to the growth/dormancy cycle. Information concerning the steady-state level of poUBI transcripts in planta are required to better evaluate the possible use of this gene as endogenous marker for studies on long-term transgene stability in genetically modified white poplars

Spore-forming bacteria in soil cultivated with GM white poplars: isolation and characterization

The impact of transgenic white poplars (Populus alba L. cv. ‘Villafranca’) was assessed on the soil aerobic spore-forming bacteria (SFB). The genetically modified poplars, expressing either the StSy gene for resveratrol production or the bar gene for herbicide tolerance, were cultivated in greenhouse. The occurrence of SFB was monitored in soil samples collected at eight different timepoints over a two-year period. The total culturable bacterial population of the StSy and bar trials underwent significant seasonal fluctuations in the range of 106–2.5 × 108 CFU/g dry soil and of 104–5 × 108 CFU/g dry soil, respectively. Changes occurred also within the culturable SFB population with size varying at 103–5 × 104 CFU/g dry soil and 102–2 × 105 CFU/g dry soil in the StSy and bar trials, respectively. No significant differences in the size of the total and SFB culturable populations were observed when comparing each transgenic line with the nontransformed control line while seasonal shifts of soil bacterial populations were evident in both trials. The culturable SFB fraction included three isolates (SFB-1, SFB-2 and SFB-3) classified by 16S rDNA sequence analysis as members of the Bacillus genus. According to the reported data, cultivation of both herbicide- resistant and resveratrol-producing GM white poplars did not affect the culturable SFB population at the soil leve

Unraveling the response of plant cells to cytotoxic saponins: role of metallothionein and nitric oxide

A wide range of pharmacological properties are ascribed to natural saponins, in addition to their biological activities against herbivores, plant soilborne pathogens and pests. As for animal cells, the cytotoxicity and the chemopreventive role of saponins are mediated by a complex network of signal transduction pathways which include reactive oxygen species (ROS) and nitric oxide (NO). The involvement of other relevant components of the saponin-related signaling routes, such as the Tumor Necrosis Factor (TNF)α, the interleukin (IL)-6 and the Nuclear Transcription FactorκB (NFκB), has been highlighted in animal cells. By contrast, information concerning the response of plant cells to saponins and the related signal transduction pathways is almost missing. To date, there are only a few common features which link plant and animal cells in their response to saponins, such as the early burst in ROS and NO production and the induction of metallothioneins (MTs), small cysteine-rich, metal-binding proteins. This aspect is discussed in the present paper in view of the recent hypothesis that MTs and NO are part of a novel signal transduction pathway participating in the cell response to oxidative stress

The Effects of Vitamins on Micropropagation of Desiree and Mozart Potatoes (Solanum Tuberosum L.)

An attempt was done to achieve a micropropagation protocol by determining the most optimal types and concentrations of vitamins including thiamine (B1), nicotinic acid (B3), pyridoxine (B6) and folic acid to induce shoot formation and rooting in vitro, as replacement vitamins in the culture medium. The four vitamins were included in the medium at two concentrations and the control treatment without vitamins. The highest number of shoots per explant (2.50 shoots/ explant) was achieved from the addition of 0.3 mgl-1thiamin to Desiree cultivar grown on MS medium. Whereas, the highest mean length of shoots was recorded in the case of adding nicotinic acid at 3.0 mgl-1 to Mozart cultivar grown on MS medium. The highest number of leaves 10.50 leaves/ explant) was obtained from Mozart cultivar grown on a medium enriched with 0.6 mgl-1 pyridoxine. The highest number of roots (7.00 roots/ explant) was achieved from the control treatment from Desiree potato cultivar. The longest roots (12.50 cm) were produced by Desiree potato cultivar grown on MS medium supplemented with 0.5 mgl-1 thiamin. Mozart potato cultivar performed better than Desiree cultivar in concern to mean length of shoots, number of leaves, number of roots and mean length of roots. No significant differences were found between the both cultivars in concern to the number of shoots and roots

Evaluation of the expression level of the endogenous marker poUBI

Abstract This work reports on the isolation and molecular characterization of the poUBI cDNA encoding polyu-biquitin from white poplar (Populus alba L. cv 'Villafranca'). Expression analysis was performed on different poplar organs and tissues, at different developmental stages and in relation to the growth/dormancy cycle. Information concerning the steady-state level of poUBI transcripts in planta are required to better evaluate the possible use of this gene as endogenous marker for studies on long-term transgene stability in genetically modified white poplars

Spatial expression of DNA topoisomerase I genes during cell proliferation in Daucus carota

The spatial expression of carrot (Daucus carota L.) top1 genes encoding the two isoforms of the enzyme DNA topoisomerase I (EC 5.99.1.2) was investigated. In situ hybridization analysis performed with a probe recognizing both top1 transcripts provided evidence that in explanted hypocotyls induced to proliferate in vitro by the addition of the growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D), the mRNA accumulation parallels the proliferation of provascular cells of the stelar cylinder. During somatic embryogenesis, the histological distribution of top1 transcripts was strongly evident at the stage of torpedo-shaped embryos, but gene expression was not only restricted to meristematic regions. When the spatial localization was extended to carrot vegetative apices and the investigation was carried out with specific probes for top1a and top1b, both transcripts preferentially accumulated in tissues having mitotic activity

DNA profiling, telomere analysis and antioxidant properties as tools for monitoring ex situ seed longevity

Background and Aims The germination test currently represents the most used method to assess seed viability in germplasm banks, despite the difficulties caused by the occurrence of seed dormancy. Furthermore, seed longevity can vary considerably across species and populations from different environments and studies related to the eco-physiological processes underlying such variations are still limited in their depth. The aim of the present work was the identification of reliable molecular markers that might help monitoring seed deterioration. Methods Dry seeds were subjected to artificial aging and collected at different time points for molecular/biochemical analyses. DNA damage was measured using the RAPD (Random Amplified Polymorphic DNA) approach while the seed antioxidant profile was obtained using both the DPPH (1,1-diphenyl, 2-picrylhydrazyl) assay and the Folin Ciocalteu reagent method. Electron Paramagnetic Resonance (EPR) provided profiles of free radicals. Quantitative RealTime-Polymerase Chain Reaction (QRT-PCR) was used to assess the expression profiles of the antioxidant genes MT2 (Type 2 Metallothionein) and SOD (Superoxide Dismutase). A modified QRT-PCR protocol was used to determine telomere length. Key Results The RAPD profiles highlighted different capacities of the two Silene species to overcome DNA damage induced by artificial aging. The antioxidant profiles of dry and rehydrated seeds revealed that the high-altitude taxon Silene acaulis was characterised by a lower antioxidant specific activity. Significant up-regulation of the MT2 and SOD genes was observed only in the rehydrated seeds of the low-altitude species. Rehydration resulted in telomere lengthening in both Silene species. Conclusions Different seed viability markers have been selected for plant species showing inherent variation of seed longevity. RAPD analysis, quantification of redox activity of non enzymatic antioxidant compounds and gene expression profiling provide deeper insights to study seed viability during storage. Telomere lengthening is a promising tool to discriminate between short- and long-lived species