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Nuclease-producing bacteria in soil cultivated with herbicide resistant transgenic white poplars
No full textThis study was carried out using soil cultivated, under greenhouse conditions, with transgenic white poplars expressing the
bar gene for tolerance to the Basta® herbicide. The occurrence of extracellular nucleolytic activity was monitored in soil samples collected
at four different times over a 26-month period. The fraction of nuclease producing bacteria (NPB) ranged from 62.5 to 100%
of the total culturable bacterial population. The DNA-methyl green plate assay allowed to distinguish five groups of bacteria showing
increasing levels of extracellular DNase activity. The NPB isolates were classified by 16S rDNA sequence analysis as members of the
Bacillus, Brevibacillus, Microbacterium, Pseudomonas and Stenotrophomonas genera. For each genus, NPB isolates were cultured in
liquid medium and the nucleolytic activity during different growth phases was monitored. Production of extracellular nucleases was
observed only during the mid-exponential growth phase of the Brevibacillus, Microbacterium and Stenotrophomonas isolates, while no
activity was evidenced for isolates classified within the Bacillus and Pseudomonas genera
PCD hallmarks in cell suspension cultures of white poplar (Populus alba L., cv 'Villafranca') challenged with cadmium
No full textIl promotore 35SCaMV: uno studio per valutare la stabilità ed il livello di espressione dei transgeni bar e StSy nelle piante.
No full textTransgene stability in response to the growth/dormancy cycle in white poplars engineered with the 35SCaMV-bar and 35SCaMV-StSy constructs
No full textDNA extraction from soil: comparison of different methods using spore-forming bacteria and the swrAA gene as indicators
Full text linkSoil microcosms seeded with spores of a tracer organism (Bacillus subtilis strain PB5332) were used to test five different
DNA extraction protocols hereby indicated as A, B, C, D and E. The representativity of DNA samples obtained from each procedure was
evaluated by PCR amplification of the swrAA gene, unique to PB5332 strain, followed by Southern hybridization with a gene-specific
probe. A significant improvement of DNA extraction from spores was obtained using grinding under liquid N2 associated with sodiumdodecyl
sulphate (SDS)-based lysis in presence of 1% hexadecyltrimethylammonium bromide (CTAB; protocol C). The same procedure
was tested on soil samples from two distinct greenhouse trials carried out with genetically modified white poplars (Populus alba L.)
expressing the StSy gene for resveratrol production and the bar gene for Basta�� tolerance, respectively. The representativity of DNA
samples recovered from the greenhouse soil was assessed using three spore-forming bacteria (SFB) as tracer organisms. The tracers
(SFB-1, SFB-2 and SFB-3) were previously isolated from the same trials classified as members of the genus Bacillus. All the tested
DNA samples produced the expected amplification products, indicating the presence at the soil level of the tracers and confirming the
reliability of the optimized DNA extraction protocol
Downregulation of the CaMV35S- driven expression of StSy and bar transgenes at the onset of dormancy in poplar.
No full textSoil persistence of DNA from transgenic poplar
Full text linkThe presence of recombinant DNA in soil cultivated with white poplars (Populus alba L.) expressing either the
bar transgene for herbicide tolerance or the StSy transgene for resveratrol production, respectively, was investigated
in a greenhouse over a 20-month period. The bar trial included the transgenic lines 5P56 and 6EA22P56
and the untransformed line, while the StSy trial was established with the transgenic lines 5EAC1 and 12EAC1
and with the untransformed line. All the transgenic poplars harbored the nptII marker gene. Plantlets were
cultivated in pots, and soil samples were mixed in order to obtain composite pools which were used for molecular
analyses. The 35SCaMV-bar (1504 bp), 35SCaMV-StSy (1403 bp) and NosP-nptII (1188 bp) sequences were
detected in total DNA extracted from soil samples taken at different times after planting, using PCR/Southern
blot hybridization. Microcosm experiments, carried out to assess the effects of temperature and DNA purity on
transgene persistence, revealed only a partial correlation between the intensity of hybridization signals and the
parameters tested
Evaluation of the expression level of the endogenous marker poUBI gene for studies on transgene stability in bar and StSy GM poplars
No full textThis work reports on the isolation and molecular characterization of the poUBI cDNA encoding polyubiquitin
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
Occurrence of multiple metal-resistance in bacterial isolates associated with transgenic white poplars (Populus alba L.)
No full textThe 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 ‘Villafranca’) 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 the StSy 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 plantgrowth-
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 metalresistant
phenotype, was detected in the HRB-1c cells
Agrobacterium tumefaciens-mediated transformation of barrel medic (Medicago truncatula L.) by an ipt-type Multi-Auto-Transformation (MAT) vector system
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