; powder; < 230 mesh (00653 – Sigma). CAS was supported by the Norwegian Research Council (MU:121733). 2000, 9: 471-476. 2 Clearly there is scope for improvements in steps within the floral-dip protocol. "FKB" indicates the PCR product obtained from a known kanamycin-resistant transgenic line; "FRB" indicates the PCR product obtained from a known gentamicin-resistant transgenic line; "WT" represents the negative control using a non-transgenic line. Overall, the transformation rates reported here were similar to the transformation yield of the traditional floral-dip protocol [1, 6, 9]. In pilot experiments, we tried using various concentrations of antibiotics with soil-grown plants carrying different antibiotic-resistance genes. Seed sterilization was as described [1], except that a 33% commercial bleach/Triton X-100 solution was used [33% DanKlorix (this is ~28 g/L sodium hypochlorite; Colgate-Palmolive; Hamburg) and 200 μL/L Triton X-100]. 3. The aliquots were sampled after 0, 5, 21, and 31 days on LB plates with or without KM (50 mg/liter), and CFU were enumerated after 72 h. As shown in Fig.3, the Kmr phenotype in the strain harboring a chromosomally inserted nptII gene was stable, without selection, once the resistance trait had been acquired. The plant DNA, isolated according to Trinker et al. In particular, we note that chromatography sand can be purchased for a lower cost compared to agar substrates. 1. As seen from Fig. Plant Methods Part of If a final volume of 500 mL is required, add the starter 50 mL culture to 200 mL YEBS for ~8 h growth, giving a total volume of 250 mL for each individual culture. PubMed We sought to further simplify the dipping recipe by testing if A. tumefaciens grown in a standard bacterial media (YEBS) (recipe listed below) would suffice for dipping after the supplement of sucrose and Silwet L77. Phosphorus availability, root exudates, and microbial activity in the rhizosphere. Here we show that horizontal transfer of DNA, extracted from transgenic sugar beets, to bacteria, based on homologous recombination, can occur in soil. DNA from conventionally grown sugar beets from adjacent field sites was used as a control. Plant tissue (often leaves) are cut into small pieces, e.g. KM-resistant bacteria, but not nptII-encoded phenotypes (31), are abundant in natural soils (found at levels of 105 CFU/g of soil), suggesting a possible selection of this phenotype in the soil environment. In contrast, the Kms cells were susceptible to the selective effects of the KM. Nucleic Acids Res. Next, we illustrate that A. thaliana lines possessing a double-transformation event can be readily generated by simply by floral-dipping into a mixture of two A. tumefaciens cultures harboring distinct transformation vectors. Furthermore, based on studies using purified DNA and sterile soil conditions, we indicate that homologous recombination, and possible additive integration, of bacterial marker genes harbored in transgenic plants into competent soil bacteria like Acinetobacter spp. A transgene-centered approach to the biosafety of transgenic plants: overview of selection and reporter genes. 2. CD developed the "double dip" sub-protocol. Saturate the sand by pipetting ~10 mL 1/4 MS Basal Salt media (without sucrose) that is buffered and pH-adjusted containing the selecting antibiotic. 2005, 15: 47-54. There are three steps that we find to be both time-consuming and costly. Evolution of the Glx-tRNA synthetase family: the glutaminyl enzyme as a case of horizontal gene transfer. The selective effects of antibiotics in soil. 2004, 101: 12288-12293. Selective effects of KM in soil and soil suspensions. Grow for ~8 hours NOTE:Start the 500 mL culture in the morning and it will be ready for plant dipping just before the end of the working day. When such T1 seed were subjected to double selection, only double transformants were found (Figure 2F). NOTE:under some selection conditions it might be necessary to add a second round of antibiotic treatment. For enumeration of CFU, aliquots were spread on solidified LB medium (19) supplemented with the antibiotics (all at 50 μg ml−1) rifampin (chromosomally encoded resistance), ampicillin (pFG4 encoded resistance), and KM (pFG4-encoded resistance) for selection of transformants and incubated at 30°C for 3 days. Transgenic sugar beets containing a functional nptII gene (16) were used for purification of donor DNA. This led to a substitution of the buffered media. The addition of up to 4.2 mg of KM reduced the CFU numbers less than fivefold, whereas a 1,100-fold drop of the recipient CFU was seen when 8.3 mg of KM was added. Reduced titer of BNYVV in transgenic sugar beet expressing the BNYVV coat protein. C: Growth of a replicate batch of double-dipped A. thaliana seedlings on kanamycin alone. The vectors respectively described were CCR2:LUC-HygR with the hpt-resistance gene conferring plant resistance to hygromycin [11], CAB2:LUC with the nptII-resistance gene conferring plant resistance to kanamycin [12], CCR2:LUC-Gent with the aacCl-resistance gene conferring plant resistance to gentamicin [13], and GI:LUC-Basta with the BAR-resistance gene conferring plant resistance to phosphinotricin [14]. Pour the entire 50 mL culture into 450 mL of YEBS. Plant Methods 5, 3 (2009). We do not retain these email addresses. Doyle MR, Davis SJ, Bastow RM, McWatters HG, Kozma-Bognar L, Nagy F, Millar AJ, Amasino RM: The ELF4 gene controls circadian rhythms and flowering time in Arabidopsis thaliana. The selection and analysis of transformants Using either Agrobacterium or direct gene transfer systems, it is now possible to introduce DNA into virtually any regenerable plant cell type. Tn5 to assess soil fate of genetically marked bacteria: screening for aminoglycoside-resistance advantage and labelling specificity. Based on the above studies with sterile soil microcosms regarding the effects of KM amendment on the population dynamics ofAcinetobacter sp. Rickettsiae and Chlamydiae: evidence of horizontal gene transfer and gene exchange. The image shows seedlings grown on chromatography sand respectively under selection with kanamycin (100 μg/mL), hygromycin (60 μg/mL), gentamicin (125 μg/mL) and phosphinotricin (12.5 μg/mL). strain BD413 cells (19, 23). (19, 20).For filter transformation with purified transgenic plant DNA, 100 μl of a DNA solution (at concentrations of 10, 40, 80, and 160 μg DNA per 160-μl solution) was mixed with the bacterial inoculum. To generate vectors for the identification of double transformation events, the FRB/Nluc and FKBP/CLuc elements were amplified by PCR from original plasmids [15] with KpnI and SacI restriction sites in the primers, and the resultant PCR products were digested with KpnI and SacI and the FRB/NLuc fragment was inserted into the KpnI and SacI sites of pPZP211 [16], and FRB/NLuc was similarly cut and ligated into the KpnI and SacI sites of pPZP221 [16]. Mattanovich D, Ruker F, Machado AC, Laimer M, Regner F, Steinkellner H, Himmler G, Katinger H: Efficient transformation of Agrobacterium spp. https://doi.org/10.1186/1746-4811-5-3, DOI: https://doi.org/10.1186/1746-4811-5-3. CAS T bars, standard deviations. Then pipette or decant off excess liquid media such that the wet sand is no longer muddy. However, we note that KM is able to select for different phenotypes in sterile soil microcosms.