Agrobacterium Protocols by Kan Wang

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 illness. in contrast to many different pathogens, Agrobacterium has the facility to convey DNA to plant cells and completely adjust the plant genome. the invention of this exact characteristic 30 years in the past has supplied plant scientists with a robust software to genetically remodel vegetation for either easy examine 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 good points 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 version of Agrobacterium Protocols used to be released in 1995, just a handful of vegetation might be commonly remodeled utilizing Agrobacterium. Ag- bacterium-mediated transformation is now universal to introduce DNA into many plant species, together with monocotyledon crop species that have been formerly thought of non-hosts for Agrobacterium. such a lot awesome are fresh devel- ments indicating that Agrobacterium can be used to bring DNA to non-plant species together with micro organism, fungi, or even mammalian cells.

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Agrobacterium Protocols

Agrobacterium tumefaciens is a soil bacterium that for greater than a century has been referred to as a pathogen inflicting the plant crown gall ailment. not like many different pathogens, Agrobacterium has the power to carry DNA to plant cells and completely modify the plant genome. the invention of this precise characteristic 30 years in the past has supplied plant scientists with a strong instrument to genetically rework vegetation for either simple study reasons and for agric- tural improvement.

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4. To induce embryogenesis explants are incubated in the dark for 4 wk. For germplasm maintenance explants are transferred directly to a 12-h light/dark regime under 40 µmol/m2/s lighting intensity after 1 wk dark incubation (see Note 8). 5. Explants are maintained on selection medium for 4 to 6 wk. Somatic embryos produced on selection media are transferred to selective RM1 media (RM1 supplemented appropriate selective agent according to the vector used and 500 mg/L of carbenicilin) on which the embryos convert to plants.

4. 0 mL of NAA solution and 5 mL of Peters Nutrient fertilizer to 800 mL of deionized water. 7 as described above and bring to a final volume of 1 L. 0 g of agar prior to autoclaving. 0 mL of thiamine solution after autoclaving when the medium cools down to 50°C (final concentrations listed in Table 1). Fifty milliliters of the medium is prepared and dispensed in magenta boxes. 5 g charcoal/L can be used for rooting. 5. Agrobacterium Media 1. 4 g yeast extract and 10 g mannitol. 0. For solid medium, add 15 g /L agar before autoclaving.

Plantlets are regenerated following successive transfers of the inoculated nodal segment explants onto shoot induction (4 wk), shoot regeneration (4 wk), and shoot multiplication media (4 wk) (see Note 9). 4. Selection and Regeneration of Putative Transformants 1. Selection of putative transformants follows successive transfers of the inoculated explants onto somatic embryo induction (MS8), and embryo maturation (RM1) media supplemented with appropriate selection agents plus carbenicillin (500 mg/L) to kill A.

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