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Additional info for Agrobacterium Protocols, Second Edition: Volume II (Methods in Molecular Biology Vol 344)
The stem pieces are then rinsed three times with sterilized distilled water and blotdried on sterile No. 1 Whatman filter paper. 5. The stem pieces are cultured by inserting the basal stem portion into Magenta boxes containing 50 mL semi-solid MS shoot multiplication medium (4E). 6. Cultures are incubated at 26°C under a 12-h light/dark regime provided by coolwhite fluorescent lamps (40 µmole/m2/s) until young actively growing apical leaves emerge (about 3 wk after culture). 7. ). The in vitro plants can supply apical leaves for up to 3 mo.
The culture is transferred to a sterile tube and spun at 700–1000g for 15 mins and then pellet resuspended in 15 mL of MS20. 6. At this stage, 5 mL of the culture can be taken and used to make 1 mL glycerol stocks, 650 µL of culture medium to 350 µL of 50% Glycerol (v:v) and the samples flash frozen and stored at −80°C. 3. Inoculation, Co-Cultivation, and Regeneration 1. 5-mm wide excised from 3- to 4-wk old potato stock plants (see Note 9). 2. Explants (up to 30 per plate) are placed into Petri dishes containing 15 mL of MS20 to avoid dessication (see Note 10).
Are transferred to embryo maturation medium (RM1) and incubated at 26oC under a 12-hr light/dark regime. 2. 05% (v/v) Tween-20. 3. The germinated somatic embryos are then cut into pieces approxi 25–50 mm2 and are wounded using a sterile 38-mm long BD1M needle prior to inoculation with Agrobacterium (see Fig. 1). 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.