Leaching and transformability of transgenic DNA in unsaturated soil columns.

Unsaturated soil columns were used to examine the transport of the plasmid pLEPO1 and plant DNA (transplastomic tobacco DNA), both carrying an antibiotic resistance gene (aadA gene), and the capacity of bacteria to incorporate the gene in their genome after its passage through the soil. Soil columns containing a top leaf layer had sterile water percolated through them at a rate of 0.5mLh(-1). DNA from column leachate water was extracted and analyzed. Quantitative measurements included total DNA concentrations in the water and the transformation frequencies of Acinetobacter sp. BD413 by DNA in the column effluent. Qualitative measurements included the relative degradation of DNA after passage in the columns by agarose gel electrophoresis and the potential of effluent DNA to transform bacteria, leading to the production of antibiotic-resistant bacteria. The presence of aadA gene in the leachate water of soil columns suggests the mobility of DNA in unsaturated soil medium. The extent of DNA degradation was found to be proportional to its residence time in the soil column while a fraction of DNA was always able to incorporate into the Acinetobacter genome under all conditions studied. These results suggest that biologically active transgenic DNA might be transported downward by rain in unsaturated soils.

Extracellular plant DNA in Geneva groundwater and traditional artesian drinking water fountains.

DNA, as the signature of life, has been extensively studied in a wide range of environments. While DNA analysis has become central to work on natural gene exchange, forensic analyses, soil bioremediation, genetically modified organisms, exobiology, and palaeontology, fundamental questions about DNA resistance to degradation remain. This paper investigated on the presence of plant DNA in groundwater and artesian fountain (groundwater-fed) samples, which relates to the movement and persistence of DNA in the environment. The study was performed in the groundwater and in the fountains, which are considered as a traditional artesian drinking water in Geneva Champagne Basin. DNA from water samples was extracted, analysed and quantified. Plant gene sequences were detected using PCR amplification based on 18S rRNA gene primers specific for eukaryotes. Physicochemical parameters of water samples including temperature, pH, conductivity, organic matter, dissolved organic carbon (DOC) and total organic carbon (TOC) were measured throughout the study. The results revealed that important quantities of plant DNA can be found in the groundwater. PCR amplification based on 18S rDNA, cloning, RFLP analysis and sequencing demonstrated the presence of plant DNA including Vitis rupestris, Vitis berlandieri, Polygonum sp. Soltis, Boopis graminea, and Sinapis alba in the water samples. Our observations support the notion of plant DNA release, long-term persistence and movement in the unsaturated medium as well as in groundwater aquifers.

Release and leaching of plant DNA in unsaturated soil column.

Little information is available on the process of DNA release from plants and neither is there much information to be found regarding DNA transport in the vadose zone. Unsaturated soil columns were used to examine the release and transport of DNA content in the leaf of tomato variety Palmiro, which was introduced into the soil columns after being dried at 35 degrees C for 3 days. Soil columns were leached with sterile water at a rate of 0.5mLh(-1) for 104 days. DNA from column leachate water was extracted and analysed. Both quantitative and qualitative measurements of extracted DNA were taken. Tomato gene sequences were detected using PCR amplification based on tomato specific 18S primers. The concentration of total DNA in soil column leachate water ranged from 6.7 to 50.4microgL(-1). Genomic analysis using agarose gel electrophoresis showed degradation of DNA after its passage through unsaturated soil columns. The presence of tomato genes in the leachate water of soil columns suggests a potential release of tomato DNA from leaves and potential transport of DNA over considerable distances in water-unsaturated soil. Consequently, transport of plant DNA in vadose zone is indicated, and there is a risk that the DNA may reach the groundwater.

Kinetics of mass and DNA decomposition in tomato leaves.

This laboratory study investigated the kinetics of leaf and DNA content decomposition in two varieties of tomato (Palmiro and Admiro) after incubation in soil for 35 days. Results revealed that the decrease of dry matter in leaves in both varieties did not follow a single exponential function and was better described by a double exponential model. Composite half-decrease times were 3.4 and 2.4 days for Palmiro and Admiro respectively. The same pattern was observed for DNA mass loss, although this was closer to a single exponential model with composite half-decrease times of 1.5 and 1.4 days. Genomic analysis showed that DNA in dried leaves at room temperature (not inoculated in the soil), remains intact or presents a weak degradation, and DNA extracted from leaves inoculated in non-sterile soil showed degradation after two days. These results indicate that before release an important quantity of DNA may be degraded inside plant tissues during decomposition in soil.