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. not like many different pathogens, Agrobacterium has the facility to bring DNA to plant cells and completely regulate the plant genome. the invention of this particular characteristic 30 years in the past has supplied plant scientists with a strong software to genetically remodel crops for either simple examine reasons and for agric- tural improvement. in comparison to actual transformation equipment corresponding to particle bomba- ment or electroporation, Agrobacterium-mediated DNA supply has a number 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 replica into the plant genome is much less more likely to set off “gene silencing” usually linked to a number of gene insertions. whilst the 1st version of Agrobacterium Protocols used to be released in 1995, just a handful of vegetation may be normally remodeled utilizing Agrobacterium. Ag- bacterium-mediated transformation is now accepted 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 contemporary devel- ments indicating that Agrobacterium is also used to carry DNA to non-plant species together with micro organism, fungi, or even mammalian cells.
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Additional info for Agrobacterium Protocols
38119). 2. Agrobacterium Strain and Constructs 1. Agrobacterium LBA4404 (Invitrogen) was used in cassava transformation. 2. The main vector backbone used in our laboratory is pKYLX (see Note 1). 3. 45 µm Cameo 25ES filter (Fisher Scientific) attached to a 60 mL BD syringe (Fisher Scientific) and are stored at 4°C. Plant hormones and antibiotics are supplied by Sigma-Aldrich. 1. 02 mg NAA in 1 L of ddH2O. The NAA stock is made in 20% (v/v) 1 N NaOH. 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.
4. Preparation of Auxiliary Buds 1. 1. 2. Buds are excised using a No 10 surgical blade. A small incision is made on the surface of the excised bud. 20 Msikita et al. Fig. 2. Overall scheme of Agrobacterium-mediated transformation of cassava. Explant types are shown in bold. 3. At this stage the buds may be used to initiate callus cultures (Fig. 2). To do so, the buds are cultured on MS8 medium supplemented with 16 mg/L (MS16, two times the regular amount) 2,4-D. 4. ). 2. Agrobacterium Preparation 1.
The embryos are transferred every month onto hormone-free MS with selection for regeneration (see Fig. 1G). 6. The tiny plantlets or small shoots are transferred to Magenta boxes containing hormone-free MS medium plus 10 mg/L PPT and 300 mg/L timentin (see Fig. 1H). Roots are induced quickly after transfer of shoots to larger containers, if they are not present prior to the initial transfer (see Fig. 1I). 7. The fully rooted plantlets are transferred to potting medium in 9 × 9 cm pots and placed in a growth chamber maintained at 26°C, 85% relative humidity, and a 16 h photoperiod (450 µmol/m2/s) where they grow into full plants (see Fig.
Agrobacterium Protocols by Kan Wang