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

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

Nuclease-producing bacteria in soil cultivated with herbicide resistant transgenic white poplars

This 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

Soil persistence of DNA from transgenic poplar

The 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

This 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