protocol: flower-dip transformation of Arabidopsis
When plants have been growing for 4-6 weeks and have flowers it's time to do a flower dip!
We followed the procedure outlined in the open access paper Logemann et. al. "An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transfromation protocol."First, YEB lawn plates of the Agrobacteria transformed with each of our transformation vectors were scraped and the cells were resuspended in 50mL conical tubes containing 30 mL of YEB or LB.
This suspension of Agrobacteria is then mixed with 120mL of the surfactant sucrose solution. Per 120 mL dip solution:
- 6 grams sucrose
- 36 uL of L-77 surfactant (PDF info)
- 120 mL of sterile water
The flower pot is then simply turned upside down and the flowers swished in the surfactant/sucrose/agrobacterium solution for ten seconds
The dipped plants are then kept in a humidified chamber for 2-4 days and dipped for a second time in freshly made bacterial solution after 4-7 days. After the second dip plants are monitored for several weeks as the seed pods form and dry, at which point the seeds are harvested.
protocol: harvesting Arabidopsis seeds
Once the seed pods have dried, it is very easy to pull them off of the stem and onto a piece of paper.
To separate the seeds from the pods, pass everything collected through a small sieve.
Seeds are now ready to be dried further. Place seeds in a coin envelope (make sure to seal all seams with tape to ensure that no seeds are lost) and place the sealed envelope in a sealed container with humidity absorbing rocks for one week at 4 degrees Celsius.
protocol: seed sterilization
Once seeds are dry they must be sterilized before planting. Place seeds in a 1.5ml eppendorf tube and suspend in 70% ethanol plus 0.1% Triton-X detergent. Allow the seeds to fall to the bottom of the tube, pure off the ethanol and replace with 95% ethanol. Do this twice, then replace with water just up to the top of the seeds. Keep the seeds at 4 degrees in the dark for 3 days to cold shock them into thinking they've survived the winter, preparing them for germination.
recipe: selective plates
Autoclave 1X Murashige and Skoog with 0.7% agar in deionized water. Once agar has cooled to approximately 50 degrees Celsius, add 150 microgarms/ml carbenicillin and either 50 microgarms/ml kanamycin for nptII selection, or 5mg/Liter glufosinate (Basta) for pat selection.
protocol: seed plating
Using a pasteur pipette pick up seeds from the eppendorf tube and spot them at even intervals across a selective agar 15cm petri dish. Seal the dish with parafilm and place under constant bright light until the seeds sprout and untransformed sprouts are selected against.
After a couple days, all of the seeds will sprout.
A couple days after that, only the plants that have been stably transformed with the selectable marker will survive the selection. Four days after plating, seedlings that integrated the pat glufosinate (Basta) resistance marker began to grow larger leaves while untransformed seedlings began to die.
Here is a seedling transformed with our brazzein expression construct: