By Kan Wang
Agrobacterium tumefaciens is a soil bacterium that for greater than a century has been often called a pathogen inflicting the plant crown gall sickness. in contrast to many different pathogens, Agrobacterium has the power to convey DNA to plant cells and completely modify the plant genome. the invention of this certain characteristic 30 years in the past has supplied plant scientists with a strong software to genetically remodel crops for either simple learn reasons and for agric- tural improvement. in comparison to actual transformation tools equivalent to particle bomba- ment or electroporation, Agrobacterium-mediated DNA supply has an a variety of benefits. one of many positive factors is its propensity to generate unmarried or a low reproduction variety of built-in transgenes with outlined ends. Integration of a unmarried transgene reproduction into the plant genome is much less more likely to set off “gene silencing” usually linked to a number of gene insertions. while the 1st variation of Agrobacterium Protocols was once released in 1995, just a handful of vegetation might be repeatedly remodeled utilizing Agrobacterium. Ag- bacterium-mediated transformation is now ordinary to introduce DNA into many plant species, together with monocotyledon crop species that have been formerly thought of non-hosts for Agrobacterium. so much amazing are fresh devel- ments indicating that Agrobacterium is additionally used to convey DNA to non-plant species together with micro organism, fungi, or even mammalian cells.
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Extra info for Agrobacterium Protocols
The tlp PCR using the primers tlpF/tlpR produces a fragment of 691 bp, whereas the bar PCR using the primers barF/barR produces a fragment of 202 bp (see Fig. 2A). 11. Southern blotting is performed to identify unique transformation events; using 10 µg of total genomic DNA digested with HindIII, run on 1% agarose gel, transferred to a nylon membrane and hybridized using standard protocols with specifically designed bar and TLP DNA probes (4) (see Fig. 2B). 12. Western blotting is performed to ensure recombinant protein expression; using 200 µg of total proteins extracted, run on polyacrylamide gel, transferred to a nylon membrane, and probed with anti-bar and anti-TLP rabbit antibodies using standard protocols (7) (see Fig.
The stock can be stored at 4°C for 6 mo. 2. 6-Benzylaminopurine (BAP, 10 mg/L) solution: The BAP stock solution is made by dissolving in 20% (v/v) 1N NaOH and can be stored at 4°C for 6 mo. 3. Gibberelic acid solution (GA, 10 mg/L): The stock solution for GA is made by dissolving in 20% (v/v) 95% ethanol and can be stored at 4°C for 6 mo. 4. Thiamine (100 mg/L) solution: The thiamine-HCl (100 mg/L) stock is made by dissolving in distilled water. The stock is stored at 4°C for 6 mo. 5. 5 mg/mL stock.
Following wounding explants are transferred onto solid MS8 medium supplemented with 200 µM acetosyringone. 2. 05% (v/v) Tween-20] are applied to the explant tissue, and the tissue is incubated in the dark for 2 d at 24–28°C. Subsequently, the explants are transferred to a new MS8 plate with 200 µM acetosyringone for another 3 d co-cultivation (see Note 6). 3. Following co-cultivation with A. tumefaciens, explants are transferred to selection media (MS8 supplemented appropriate selective agent according to the vector used and 500 mg/L of carbenicillin) (see Note 7).
Agrobacterium Protocols by Kan Wang