Common use of Selection of transformed calli Clause in Contracts

Selection of transformed calli. 1. Transfer the infected calli to a sterile 50 ml tube containing 35 ml sterile distilled water. 2. Shake the tube for a few seconds and pour out water. Repeat washing 6 times. 3. For the final wash, add carbenicillin or cefotaxime at the concentration of 500 mg/ml. 4. Transfer calli onto autoclaved Kimwipes paper to remove extra water. 5. Culture calli on the semi-solid N6-P medium supplemented with 250 mg/ml carbenicillin or cefotaxime at 28 °C under cold fluorescent light for 7 days. Each 100 x 20 mm dish contains 30 ml N6-P medium, 30 calli on each dish. Dishes are sealed with surgical tape. 6. Subculture calli on the N6-P medium supplemented with 250 mg/ml carbenicillin or cefotaxime and 100 mg/ml paramomycin for 16 days. The Gelrite is replaced by Agarose (7-8 g/l) in the N6-P medium. 7. Subculture calli on the N6-P medium supplemented with 250 mg/ml carbenicillin or cefotaxime and 100 mg /ml paramomycin for another 16 days. Use agarose to replace Gelrite in N6-P medium. Paramomycin-resistant calli should start to be visible at the end of the 2nd selection. 8. Note: fast-growing transformed cells must be visible during second selection (Fig. 6). Non-transformed wild-type calli must be completely killed by selection (Fig. 7).