Our standard practice is to dip twice, with the four-day interval separating the two interventions. For research applications that are unlikely to be grown in the field, strong herbicide- or antibiotic resistance is commonly used. 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. CFU of KM-sensitive recipients (open symbols) and KM-resistant transformants (filled symbols) of Acinetobactersp. One limitation in the identification of transgenic A. thaliana lines after floral dip is that the seeds are often internally contaminated with the A. tumefaciens line used. 3. Google Scholar, de la Riva GA, González-Cabrera J, Vázquez-Padrón R, Ayra-Pardo C: Agrobacterium tumefaciens: a natural tool for plant transformation. 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. Finally, we report an alternative method of transformant selection on chromatography sand that does not require surface sterilization of seeds. Harrison SJ, Mott EK, Parsley K, Aspinall S, Gray JC, Cottage A: A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation. 2001, 19: 227-233. Transgenic Res. 2006, 2: 16-. Article Once your seeds from your T0 plants are dry, it’s time to select for transformants. Since the numbers of transformants generated in soil is expected to be very low, a selective advantage for their enrichment and, hence, environmental significance is needed. Cite this article. (19, 20). In particular, it was noticed that pre-washing the sand improved the selection ability. This led to a substitution of the buffered media. Thank you for sharing this Applied and Environmental Microbiology article. strain BD413 with a chromosomally inserted nptII gene (19). SJD wrote the paper. Since the number of recipient CFU was similar in both studies (4 × 109 to 5 × 109), our 20-fold-higher number of transformants was also reflected in the correspondingly higher transformation frequency of 5.7 × 10−7 on solidified medium compared to that of 2.0 × 10−8 obtained in liquid culture (5). Plant Mol Biol. strain BD413 cells (19, 23). Natural transformation and restoration of a 317-bp internal deletion in the nptII gene inAcinetobacter sp. cells from 109 CFU/g of soil to below detection. T bars, standard deviations. 00653) or dry silicon dioxide (Fluka catalogue no. 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. Seeds were plated on 1% agar containing MS medium (including vitamins) and kanamycin at a concentration of 50 … T bars, standard deviations. 2001, 51 (Pt 1): 89-103. Transgenic sugar beets containing a functional nptII gene (16) were used for purification of donor DNA. The latter control indicated that transformation did not occur on the selective plates. 1. Manage cookies/Do not sell my data we use in the preference centre. Zhang X, Henriques R, Lin SS, Niu QW, Chua NH: Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. The Biotechnology and Biological Sciences Research Council funded work in the Millar group (BBSRC award E015263) and the Hall group (BBSRC award (F005318). strain BD413(pFG4) Kms; lane 3, Acinetobacter sp. 2002, 419: 74-77. Sequencing PCR DNA amplified directly from a bacterial colony. Walter M, Chaban C, Schutze K, Batistic O, Weckermann K, Nake C, Blazevic D, Grefen C, Schumacher K, Oecking C: Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. F: Multiplex genomic PCR of FRB and FKBP sequences in nine lines selected on both antibiotics. The above two sands are our favorites, but by no means do these limit potential substrate choices. statement and Based on this mechanism, several laboratory studies have been conducted to elucidate the potential for plant-harbored resistance determinants to be taken up by naturally occurring bacterial recipients (2, 3, 21, 28). The soil suspension was incubated at 27°C with shaking (180 rpm) for 24 h before sampling and enumeration of CFU. 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.