Production of Transgenic Tobacco Plants
Movie of Agrobacterium tumefaciens infecting a plant A. tumefaciens movie
First day of lab
On the first day of this experiment you will incubate pieces of sterile tobacco leaves with a disarmed control strain of A. tumefaciens that does not contain a foreign gene (LBA4404) and an engineered strain that carries the NPTII and the gusA gene (LBA4404/pBI121). All steps must be done in the laminar flow hoods with great care to keep everything STERILE!
- Clean the hood with 70% ETOH. Unwrap a sterile tile taking care not to touch its upper surface with your hands. Obtain a sterile tobacco plant, 3 MS4 plates, blotting paper, and the control bacterial culture from the supply area.
- Flame sterilize all tools carefully and completely, as demonstrated by your instructor.
- From the sterile tobacco plant cut 1-3 leaves (depending on size) and place them on your sterile tile. To score the leaves before cutting segments, gently make incisions along each leaf perpendicular to the long axis without cutting all the way to the margin. The scoring of the leaf tissue will help the leaf tissue lie flat on the tissue culture media rather than curling up and preventing good contact with the media.
- Cut the leaves into 1 cm square segments avoiding large veins and mix them together to randomize. Work quickly but carefully. You will need approximately 4-6 segments for the control plate and 8-12 for the two experimental plates.
- Transfer 4- 6 segments to a small "dipping" dish containing the control bacterial culture, LBA4404. If possible, you should work with the control culture first and, after completing the control plate, use the genetically modified strain to create 2 experimental plates. Make sure there is only one bacterial strain in the hood at a time to avoid cross-contamination. Make sure all surfaces of the leaf segments come into contact with the bacteria. "Dip" for 1 minute.
- Gently blot leaf segments between sterile filter paper.
- Label the bottom of one plate with the strain used first (LBA4404 Control), your initials, lab section, date, and medium (MS4).
- Place leaf segments with the upper surface down onto the MS4 plate.
- If you think there is any chance that your work surface (the tile) has become contaminated and is no longer sterile: obtain a new sterile tile. If you are sure your tile is still sterile you can continue with the one you are using.
- Exchange the control bacterial culture for the experimental one (LBA4404/pBI121), obtain new filter paper, place your tools in the ethanol jar and flame sterilize them again.
- Label the bottom of 2 MS4 plates with the name of the experimental strain, LBA4404/pBI121 and the other information needed (name, lab section, date, medium).
- Prepare and dip 10-12 leaf segments in the bacterial culture LBA4404+ pBI121 as before and distribute the leaf segments onto those 2 additional MS4 plates.
- Seal all 3 plates with a stretched strip of parafilm and incubate at 25° C under continuous light for 3 days.
Each student should end up with 2 plates of leaf segments incubated with LBA4404/pBI121 and 1 control plate incubated with LBA 4404. Make sure the 3 plates are clearly labeled with your initials, the date and which Agrobacterium strain was used: LBA 4404 (control) or LBA4404/pBI121 (genetically modified).