Hello,
I am working on setting up Agrobacterium culture in our lab and since we do not have a large centrifuge, I was planning to grow the bacteria on plates (either in YEB agar or LB agar). I am going to perform floral dip on Arabidopsis, and also I will do transient expression (vacuum infiltration) in lettuce. I was planning to try this method (Article An improved method for preparing Agrobacterium cells that si...
) and prepare the dipping solution for Arabidopsis (sucrose/surfactant) directly from the plate. I was wondering what resuspension solution you use for Arabidopsis (I previously used a 5 g/L sucrose and silwet solution) but I have seen some that use Tween-20. What do you use?Additionally I will be doing infiltration of lettuce, and I was wondering what infiltration solution you use. My current plan is to follow the protocol of this paper (Article Transient Protein Expression by Agroinfiltration in Lettuce
) with the following formulation MES buffer (1×): 10 mM MES Hydrate (FW 195.24), 10 mM MgSO4 Hydrate (FW 246.47). Mix in filtered water and adjust pH to 5.5 with NaOH. Sterilize by passing through a 0.2 μM filter. I will resuspend my bacteria with this solution to an OD600 of 0.21. Do you think this would work well?Thank you!
Dear Keegan Pham, for infiltration using Arabidopsis floral dip we use 5% Sucrose (i.e. 50g/l) and 0.05% Silwet (according to the Clough&Bent paper (https://onlinelibrary.wiley.com/doi/full/10.1046/j.1365-313x.1998.00343.x?sid=nlm%3Apubmed), and this works well. Haven't tried other surfactants or the plate method but people used it successfully. If you want to use liquid culture but not so large Volume: for floral dip you can scale down the volume (e.g. by using a flexible plastic bag instead of a larger vessel; or cut away any siliques/ flowers, just keep unopened buds, pipette droplets of transformation solution to buds). Also, you can grow the culture to higher OD and dilute the solution (e.g. OD 2 and dilute to 0.8 so you just need half the vol), it still works well.
For lettuce I have no experience, though I would adjust pH with KOH and OD 0.21 seems a little low, is that what they suggest?
I also use the 5% sucrose .05% silwet with final OD600 of 0.8, this method works on Brassica rapa as well (work in progress), I use liquid culture, I imagine colonies from fresh plates would be fine
I make the solution up in distilled water straight from the tap. I grow my plants in soil and make the bacterial suspension fresh so there isn't a need to sterilize the solution (no time for unwanted microbe growth, and the plants certainly are not sterile)
The lettuce example looks to be an Agro infiltration for transient protein expression in leaves, a bit different from what you are hoping to do
I don't pH my dipping solution but if you do decide to pH adjust yours I'd suggest KOH instead of NaOH, some plants are sensitive to extra Na.
I also second the idea of pipetting on the solution and removing older floral material
good luck!
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