5. Stock, Raum 5X17
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2 sterile benches, 3 growth rooms
Dr. Kenneth W Berendzen
Transformation Unit Protocols
Cell Culture 01.doc
Cell Culture Maintenance (Arabidopsis, BY2)
BY-2 Tobacco Cell Suspension Protoplasts
Arabodopsis Cell Culture Protoplasts
Please cite for the standard protoplast transfection protocol after Feb. 2017:
Mehlhorn D.G., Wallmeroth N., Berendzen K.W., Grefen C. (2018) 2in1 Vectors Improve In Planta BiFC and FRET Analyses. In: Hawes C., Kriechbaumer V. (eds) The Plant Endoplasmic Reticulum. Methods in Molecular Biology, vol 1691. Humana Press, New York, NY.
Please cite for the 96-well transfection protocol:
Berendzen KW, Böhmer M, Wallmeroth N, Peter S, Vesić M, Zhou Y, Tiesler FK, Schleifenbaum F, Harter K. Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry. Plant Methods. 2012 Jul 12;8(1):25.
Transfections performed before Feb.2017 cite:
Schütze K, Harter K, Chaban C (2009) Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in living plant cells. Methods Mol Biol 479: 189-202.
Please cite for tomato transformations:
Wittmann et al. 2015. Plant Pathology. Doi: 10.1111/ppa.12417
|1. The unit offers Agrobacterium-mediated transformation of potato, tobacco and tomato.|
|2. Regenaration and cultivation of transformants.|
|3. PEG-mediated transformation of protoplasts from cell cultures or mesophyll cells (Arabidopsis or tobacco).|
|4. Production of cell cultures (wild type).|
Fig. Z-stack image of protoplasts liberated from Arabidopsis thaliana leaf tissue.
Fig. Examination of the protoplat transformation efficiency by flow cytometric anaylsis (FCA).
Protoplasts isolated from root cell culture are transformed with a 35S::GFP construct (12kb) as a routine control. As an alternative to calculating the transformation efficiency, we have used FCA as a means for detecting cells expressing GFP. The negative population is marked in black. Cells that are strongly expressing GFP have a fluoresence signal that is well above that of the total plant cell population‘s green autofluoresence (cells marked in dark green); cells marked in light green are those with weaker GFP fluoresence emission. In this example 23% of the cells were transformed with GFP, and 50% of those are strongly expressing GFP.
Albert I, Böhm H, Albert M, Feiler CE, Imkampe J, Wallmeroth N, Brancato C, Raaymakers TM, Oome S, Zhang H, Krol E, Grefen C, Gust A, Chai J, Hedrich R, Van den Ackerveken G, Nürnberger T (2015). An RLP23-SOBIR1-BAK1 complex mediates NLP-triggered immunity. Nature Plants, 15140, doi: 10.1038/NPLANTS.2015.140.
J. Wittmann, C. Brancato, K. W. Berendzen and B. Dreiseikelmann (2015).
Development of a tomato plant resistant to Clavibacter michiganensis using the endolysin gene of bacteriophage CMP1 as a transgene.
Plant Pathology DOI: 10.1111/ppa.12417