Tomato Transformation protocol - Interactive Resource Center番茄的遗传转化协议交互资源中心.doc

BTI Tomato Transformation Protocol – last updated 8/1/06 From Joyce van Eck, Boyce Thompson Institute, Cornell University. Email Joyce if questions: jv27@cornell.edu. A) Preparation of Plant Material Sterilize seed a. Immerse seed in 20% Clorox for 20 min. (100 seeds~380mg.) b. Rinse well with sterile Milli-Q water (2 or more times). 2) Sow seed in Magenta boxes containing 1/2 MSO (approximately 30 seeds/box). Prepare feeder layer (one day prior to cutting cotyledons). Pipet 2 ml of a one-week- old NT1 suspension culture onto a KCMS medium plate. (NT1 cells are subcultured weekly (2:48) in KCMS liquid medium). Cover suspension with a sterile 7 cm Whatman filter Culture in dark, overnight 4) One day prior to inoculation with Agrobacterium, cut cotyledons from 6 – 8-day-old seedlings or the appropriate germination time for your material. It is very important that the first true leaves have not emerged. Place seedling on a sterile paper towel moistened with sterile water b. Excise cotyledon at petiole and cut tips off. Cut in half if size of cotyledon is 1 cm. Use curved scalpel blades because they cause less damage than straight blades. c. Place explants on feeder plates adaxial side down Culture 25C, 16 hr photoperiod. B) Agrobacterium Streak Agrobacterium onto LB selective media. Incubate 24 - 48 hrs at 28C. Select 4, single well-formed colonies from the plate and transfer to 50 ml of YM selective medium. Culture in a shaking incubator 250 rpm at 28oC for 24 hours. After 24 hrs, check the OD600. Optimum OD600 = 0.6 –0.7. If OD600 1.0, dilute the culture until the OD600 reading is below 0.5 and grow for another hour. Check the OD600 reading periodically. Centrifuge at 8000 rpm (Sorvall centrifuge, SS34 rotor) for 10 minutes at 20C. Pour the supernatant into a graduated cylinder and record the volume. Discard the supernatant. Resuspend the pellet in the same amount of MS-0,2% liquid medium by vortexing. Inoculum is ready to use. C) Transformation Incuba