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Extra resources for Agrobacterium Protocols, Second Edition: Volume II (Methods in Molecular Biology Vol 344)
As a result of these cultivar-dependent differences a variety of tissues including auxiliary buds (17–19), apical leaves (10), and floral meristems (20) have been used for Agrobacterium-mediated transformation of cassava. Briefly, embryos are induced from callus tissues and directly inoculated with Agrobacterium for transformation. When using friable embryogenic callus (FEC), embryos are initiated and then transferred from Murashige and Skoog (MS)–based medium to Greshoff and Doy (21) basal medium supplemented with a high concentration of the auxins 4amino-3,5,6-trichloropicolinic acid (picloram) or 2,4-dichlorophenoxyacetic acid (2,4-D) to initiate massive secondary embryo production.
3 g) and sucrose (20 g) are dissolved in 800 mL of de-ionized water. 88 mL of copper sulfate solution, 16 mL of 2,4-D solution, and 50 mg of casein hydrolysate. 0 g of Phytagel prior to autoclaving. After autoclaving, add 10 mL of B5 vitamin solution when the medium cools down to about 50°C (see Note 3). 3. 0 mL of NAA solution are dissolved in 800 mL of de-ionized water. 7 as described above and the final volume is brought to 1L. Add 2 g of phytagel and autoclave. Add 10 mL of GA solution and 10 mL of thiamine solution after autoclaving when the medium has cooled to 50°C (final concentrations listed in Table 1) (see Note 4).
Cultures are incubated at 28°C. 2. After 2 d, single colonies are used to inoculate 20 mL of liquid YM medium supplemented with 200 µM acetosyringone, 100 mg/L streptomycin, 6 mg/L tetracycline (or the antibiotics used depend on the plasmid used), and incubated at 28°C on a shaker at 250 rpm for 2 d. 3. 5. 4. 5 × 109 cfu/mL) using a spectrophotometer. 5. At this stage the bacteria are ready for inoculating the explants. 3. Explant Inoculation and Cocultivation 1. Following wounding explants are transferred onto solid MS8 medium supplemented with 200 µM acetosyringone.