Hi guys,
I am recently doing arabidopsis transformation by floral dipping. However, after dipping, the flowers were dead. Does anybody know what happened? Here is my protocol: 5% sucrose, 0.05% silwet L-77. after overnight culture, agrobacteria were centrifugted and directly resuspended in 1 volume transformation medium. I dipped solution onto flowers by pipett. After finished, let plants still grow in the same condition under light. By the way, the initial OD is about 2, so whether is it high? Should the plants kept in dark for 24h?
Cheers,
Feng
Hi Feng,
Here is the link for a protocol that i used to use
http://www.plantpath.wisc.edu/fac/afb/protocol.html
You can reduce the amount of silwet down to .005%. Also, the concentration of Agro seems a bit high (a final OD of 0.8 is standard).
I used to keep the plants overnight in lidded trays lines lined with damp paper towels (just on the benchtop, not in the greenhouse), perhaps that might help?
Amy
After dipping, let plants at dark for 48 h prior to let them grow at light to avoid flowers necrosis
You need to incubate the plants in the dark (and humid) overnight after dipping. Too close proximity can burn the flowers, also after the dipping and dark incubation. Further, do not use continuous light after dipping and growth for seed set. You will get seeds but no transformants.
I would suggest reducing your sucrose and silwet concentrations, as well as not letting drops of the sucrose solution dry on the plants.
Our standard method is pretty similar to a recent Plant Methods article (see below) and provides good to excellent results. Plant damage is never a factor unless the plants are dipped for too long, or the excess solution is not allowed to drip off.
Good luck!
** Quick floral dip prep:
1) Obtain ~thick lawn plate of Agro (as above) and a 50ml tube containing 30 ml LB
2) Pour 2-3 ml of LB onto plate with Agro lawn.
3) Let sit on bench for a 10-15 seconds for media to soften lawn
-- or a little longer if plates grew for several days
4) Scrape the plate using a razor blade, cover slip, or other device to suspend the lawn
5) Tilt plate and pipet up and down to suspend bacteria
-- you can also use more media from the 50ml tube to wash down the plate
6) Use P1000 tip to move suspended lawn to the 50 ml tube
*
For the dip:
7) Prepare as much 3-4% sucrose with 0.03% Silwet as needed (120 ml per Agro culture)
8) Using a cylinder, measure out 120 ml sucrose/silwet solution
9) Pour into a 1 gallon Ziploc bag
10) Add the 30 ml of resuspended Agro culture and mix
11) Dip inflorescences into solution (10-12 seconds is plenty). Laying the bag down helps.
-- agitate infloresences and gently press contents to ensure full coverage in flowers.
12) Remove plant, let excess solution drip off (give a little shake if necessary)
13) Lay plant horizontal in a tray, mist an opaque lid with water and cover the plants
14) Incubate in the dark overnight (16-24hr)
15) Return to vertical and grow plants as normal
-- for higher efficiency, repeat dip 4-5 days later with new culture.
-- double dipping allows transformed segment identification by 2nd curve in stem.
See: Plant Methods 2006, 2:16 doi:10.1186/1746-4811-2-16
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