Arabidopsis transformation by floral dipping（拟南芥转化）
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Steak the Agrobacterium clone you want to use for plant transformation onto an LB plate containing selective antibiotics (in our case: Kanamycin 20 mg/l, Gentamycin 15 mg/l). Grow at 28℃ for 2-3 days.
(If you are not using this plate straight away for transformation, you can wrap it with cling film and keep it on your bench in our coolish lab for a couple of days. Agro cells from plate store in the cold room tend to grow as lumps in liquid culture.)
Step 2. 2 days before floral dipping
Take a generous scoop of the cells and resuspend them in 10 ml LB containing selective antibiotics in a 50 Falcon (blue-capped) tube. Grow at 28℃ with shaking (200-220 rpm) overnight.
Step 3. 1 day before floral dipping
Take about 6 ml of the culture to inoculate 500 ml LB containing selective antibiotics in a sterile 2 litre Erlenmeyer flask. Grow at 28℃ with shaking (200 rpm) overnight.
Step 4. Floral dipping
First prepare everything for the transformation.
-Make inoculation medium: 5% sucrose (=50 g/l), 0.025% Triton X-100 (=250 µl per litre) in H2O (or in 1/2 strength ATS medium). You need about 300 ml for each Agro culture.
-Get the plants for transformation from the greenhouse. Label each pot with the name of the clone or construct that you are going to transform and with the transformation date.
Scecond distribute the Agro culture into 2 × 250 ml Nalgene centrifuge bottles. Spin down the cells (12 min in Jouan benchtop centrifuge, 3000 rpm). As soon as the centrifuge has stopped, open the bottles and decant the supernatant back into the Erlenmeyer flask. The supernatant has to be treated as bacterial waste (autoclave or add 10% bleach).
Use a 25 ml pipette to transfer about 20 ml of the inoculation medium to each centrifuge bottle. Resuspend the cells, first by mixing them with the tip of the pipette, then by pipetting up and down. When they are resuspended, combine the cells from both centrifuge beakers in a 500 ml glass Weck jar and make the volume up to about 250-300 ml with more inoculation medium.
Third dip the inflorescences of the Arabidopsis plants into the Agro suspension for about 30 sec. Place the pots with the dipped plants into a green tray. Use one tray for each clone to be transformed, to avoid cross-contamination. Make sure the pots are well watered. Cover the tray with a perspex lid. If the plants are too tall and touch the lid, lay the pots on their side. Bring trays back to the greenhouse and leave lids closed overnight.
The inoculation medium and the weck jar used for dipping also have to be treated as bacterial waste (autoclave or use 10% bleach). Wipe your workspace and anything that has come into contact with the bacteria with 70% ethanol.
Step 5. 1 day after floral dipping
One day after dipping, remove the perspex lids form the dipped plants. If pots were laid on their side, stand them upright again. Make sure the plants are well watered during the next days.
(Step 6. 1 week after the floral dipping. Optional
To increase the transformation efficiency, the plants can be re-dipped seven days after the first dipping. People do this less and less because the transformation efficiency is generally quite good with one dip only.)
Step 7. Maybe 2-3 weeks after floral dipping
Visit your plants in the greenhouse every now and then to check they are well watered. When the plants have stopped flowering and the first siliques start to ripen and get yellow, put a paper bag over the shoots for each pot to prevent any loss of potentially transformed seed. The plants will now mature over the next two weeks. If most of the siliques inside the bag are yellow, put a “do not water” label into the pot. The plants will now dry during the next 1-2 weeks. Then cut off the bag with all the shoots and seeds inside. Dry for one more week in the lab. Then the seeds should not be dormant any more and can be plated on agar to select for transformants.
