1. O.D 0.1strain C58C1
2. Antibiotics used: Cefo. 500mg/l, Carbencillin 500mg/l, Timentin 500mg/l, amoxycillin 500mg/l
+ all possible combinations.
3. While making selection medium filter sterilized antibiotics were added at around 40-50 degrees celcius
4. Infection time : just dip in agro suspension followed by blot drying
5. co cultivation period not more than 24 hours. thoroughly washed explants with autoclaved distilled water and antibiotics 3-4 times followed by blotting with filter papers.
not sure whats going wrong!!!!!
Remember that beta-lactam antibiotics are not stable and hydrolyze. We use carbenicillin because it is more stable than ampicilin which looses considerable activity even after a day. Agrobacteria have tolerance to amp/carb, that's why you need to use timentin. Plate should always be prepared freshly. If you continue to experience problems you need to transfer the leaf-discs to fresh plates.
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