We do not retain these email addresses. Lane 1 from the left side, 1-kb ladder (Gibco-BRL); lane 2, Acinetobacter sp. The latter control indicated that transformation did not occur on the selective plates. Circles in A-C indicate antibiotic resistant plants. The plant DNA, isolated according to Trinker et al. Plant Cell. The soil suspension was incubated at 27°C with shaking (180 rpm) for 24 h before sampling and enumeration of CFU. Selective effects of KM in soil and soil suspensions. Following a 24-h transformation period at 20 or 30°C, the microcosms were sampled as described previously (20), and CFU were enumerated after a 72-h incubation period at 30°C. 2002, 419: 74-77. Then pipette or decant off excess liquid media such that the wet sand is no longer muddy. There are three steps that we find to be both time-consuming and costly. Article Although negative selective effects of this high KM amendment were seen for the Kmr transformants, with a 540-fold reduction in CFU from 0 to 42 mg of KM added per g of dry soil (Fig. Google Scholar. If assistance is needed on how to generate a construction for plant transformation, or how to transform A. tumefaciens with this vector, we direct the reader to [8, 10, 19]. Transformations occurring with this efficiency were below our limit of detection (Table 1), and the identification of such events in nonsterile soil, using the same experimental setup as for the sterile soil, would require screening of approximately 20,000 agar plates. Google Scholar. The recipient inoculum was prepared as described by Nielsen et al. 2004, 40: 419-427. After 24 h of incubation in soil microcosms at a temperature of 30°C, both nutrient solutions A and B were able to generate transformants of Acinetobacter sp. Enter multiple addresses on separate lines or separate them with commas. NOTE:Room temperature growth on a rotating lab shaker is possible if an incubator is not available. As many labs use LB media, we tested the suitability of this media variation. However, only high amounts of KM reduced the number of recipients substantially over a 24-h period. Reactivation of the selective effects of KM, … We provide a description of a bacterial-growth media that supports direct dipping and plant transformation after the trivial addition of surfactant, thereby eliminating the need to exchange bacteria from growth media to a buffer. The low frequency and randomness of transgene integration into host cells, combined with the significant challenges of recovering whole plants from those rare events, makes the use of selectable marker genes routine in plant transformation experiments. Numbers of replicates and repeats were as described for the soil transformations. Sequence comparisons of genes isolated from wild plants and bacteria have indicated that horizontal gene transfer has occurred naturally between them (13, 32). Plant Biotechnol J. The latter addition is close to the upper limit of administering liquid KM in soil and equals 100 μl of a 100-mg ml−1 KM stock solution per 1.2 g (dry weight) of soil. 2). strain BD413 cells in soil and liquid soil suspension. 1. Plant Mol Biol. As seen from Fig. 6) Segegration analysis of resistance can be done in the same way if you plant seeds on soil and count the number of surviving germinated seedlings. Article A. tumefaciens strains ABI and GV3101 harboring the CCR2:LUC-HygR transgene were cultured on YEBS or LB liquid media as indicated. D: An expanded view of a robust seedling growing on both gentamicin and kanamycin. strain BD413(pFG4) isolated from soil after addition of increasing amounts of kanamycin to soil microcosms (a) or liquid soil suspensions (b). NOTE:Alternative chromatography sands can work, but the specific type of sand matters a great deal, as some sands were found to be entirely unsuitable for certain antibiotic-selection regimes. 4. Natural transformation and restoration of a 317-bp-deleted nptII gene in Acinetobacter sp. We found that after saturated growth in YEBS media of A. tumefaciens cell line ABI harboring the CCR2:LUC transgene [11], the addition of both 25 g per L of sucrose and 200 μL Silwet L77 per L of culture supported robust transfomation (Figure 1). In contrast, the Kms cells were susceptible to the selective effects of the KM. Restoration of a 317-bp-deleted nptIIgene in Acinetobacter sp. Amanda M Davis, Anthony Hall, Chiarina Darrah contributed equally to this work. Nat Protoc. Logemann E, Birkenbihl RP, Ulker B, Somssich IE: An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol. DNA from conventionally grown sugar beets from adjacent field sites was used as a control. Move plates to a growth cabinet, as typical for agar selection. Transformation can now be performed at very large scales leading to near saturating mutagenesis [2, 3]. 2008, 427: 91-100. Identify transformed A. tumefaciens cells from an agar plate. 1992, 4: 1075-1087. Transgenic sugar beets containing a functional nptII gene (16) were used for purification of donor DNA. Chemotactic response to amino acids of fluorescent pseudomonads isolated from spinach roots grown in soil with different salinity levels. Plant Methods These studies have, however, not been able to demonstrate uptake of such determinants, nor have studies of bacteria obtained from soil samples from field trials with transgenic plants (8, 25). Both resistant strains were inoculated (six replicates) in separate aliquots of 5 ml of nonselective LB medium and incubated at 27°C with agitation (180 rpm). Horizontal gene transfer from transgenic plants to terrestrial bacteria—a rare event? Inoculation of GN-Biolog plates, quantified on a Biolog MicroLog station (Biolog Microlog3; Biolog, Inc, Hayward, Calif.), with transformants also revealed that their metabolic pattern was indistinguishable from the one obtained from the inoculant strain. Appropriate antibiotics were added to the respective cultures. NOTE: as previously noted [1], plants can be re-dipped every 3–5 d resulting an increase in transformation percentage, but a modest decrease in total seed yield. K.M.N. strain BD413 cells (19, 23). As another example, we transformed the above A. tumefaciens cell line GV3101 harboring the CCR2::LUC construct into a variety of A. thaliana accessions. The phytotoxicity of toxoflavin was effective on broad ranges of monocots and dicots. The selected transgenic seedlings can now be easily removed with forceps and transferred to soil for further growth. It has also allowed the systematic study of gene function through transgenic approaches. SJD conceived the experiments. Curr Biol. Experimental studies by our groups (21) and others (2, 3, 28) have confirmed the low probability of integration of transgenes in recipient bacteria if DNA homology is not present. 2004, 40: 428-438. Nevertheless, there is room to improve this protocol. Here, we present our simplified methods that are robustly effective in the generation and selection of transgenic A. thaliana plants. Unigen and Department of Botany, Norwegian University of Science and Technology, 7491 Trondheim, Norway; Research Institute for Plant Protection, IPO-DLO, 6700 GW Wageningen, The Netherlands; and, Institute for Plant Virology, Microbiology and Biosafety, Federal Biological Research Centre for Agriculture and Forestry, (BBA), 38104 Braunschweig, Germany, Sign In to Email Alerts with your Email Address. Not surprisingly, the rate of double transformation events is less frequent than single-transformation events, by a factor of ~4–8 (data not shown). Begleitende sicherheitsforschung zur freisetzung gentechnisch veränderter petunien, Examination of the putative horizontal gene transfer from transgenic plants to agrobacteria. Results a. Seedlings from Arabidopsis thaliana Col-0 T1 seed obtained from plants subjected to floral dip transformation using Agrobacterium tumefaciens strain GV3101 harbouring the binary plasmid pBINPLUS. The selection of transgenic plant cells has traditionally been accomplished by the introduction of an antibiotic or herbicide-resistant gene, enabling the transgenic cells to be selected on media containing the corresponding toxic compound. 1, saturation for transformation was reached at a concentration of approximately 100 μg of DNA per 4 × 109 recipient CFU per filter. 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. Cite this article. The sequences were amplified by the method described by Hofmann and Brian, (10) with primer set P1 and P2: P1 (1236), 5′ TGC TAA AGG AAG CGG AAC 3′; P2 (2929), 5′ AGG TCA ACA GGC GGT AAC 3′ (7). NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. Transformations on filters and in soil microcosms. by electroporation. Use appropriate antibiotics for bacterial selection of the Ti-plasmid and the introduced transformation vector.