We want to use vacuum infiltration for transient expression in tobacco and lettuce. We wonder if anyone could suggest us a good protocol?
Here's how we do it: Cover pots with plastic foil to prevent soil contamination of media after flipping. Dilute Agrobacteria strains to OD600=0.1-0.4 (more might result in unspecific necrosis effects) in infiltration media (MES based or the citric acid based) and add 0.01-0.02% Silwet-L77 (mild surfactant) to break suface tension right before you dip your plants. Draw vacuum until bubbles on the leaf suface appear and then slightly pound the exsiccator to release the bubbles from the surface. Slowly open the valves to raise pressure and check for homogeneous uptake of the liquid in the leaves. Redo just once if necessary.
Side note: A friend of mine built a fish tank like device to do large scale inoculations in N. benthamiana (first pic): http://ebn24.com/index.php?id=36636&L=1
HTH,
Robert
Do you want to vacuum-infiltrate leaf disks, whole leaves, or whole plants?
For tobacco, I think agro-infiltration with A. tumefaciens remains the easiest for transient expression.
Tnx Noémie
I want to vacuum-infiltrate whole leaves and whole plants
Well, this really depends on the vacuum pump you have. I'm just a bit surprised you want to use it for tobacco, since this is more time-consuming than regular syringe-based methods.
For lettuce, can't you infiltrate with syringe?
As mentioned here, It's very simple for tobacco to use a syringe. We use it for tobacco and also for higher plants with no problem. To use a vaccum it's important to know how much pressure you can get with your system.
I fully agree. It is very important to know the power of your pump. You can either break the tissues, or just not get them infiltrated. You can simply try with an A. tumefaciens carrying a GUS construct for all the optimization step (it can be time-consuming!).
For grapevine leaves, I used something like 3 times 3 minutes. What is VERY important is to release the vacuum very slowly, in order not to break the tissues or make them fall out of the solution. What I mean is that the solution enters in the leaves when you release the vacuum.
You must probably have papers for more details, for example in grapevine, rose, or other species. You couldn't find anything?
Here's how we do it: Cover pots with plastic foil to prevent soil contamination of media after flipping. Dilute Agrobacteria strains to OD600=0.1-0.4 (more might result in unspecific necrosis effects) in infiltration media (MES based or the citric acid based) and add 0.01-0.02% Silwet-L77 (mild surfactant) to break suface tension right before you dip your plants. Draw vacuum until bubbles on the leaf suface appear and then slightly pound the exsiccator to release the bubbles from the surface. Slowly open the valves to raise pressure and check for homogeneous uptake of the liquid in the leaves. Redo just once if necessary.
Side note: A friend of mine built a fish tank like device to do large scale inoculations in N. benthamiana (first pic): http://ebn24.com/index.php?id=36636&L=1
HTH,
Robert
Wow, that device seems fantastic! I feel bad when I look back, for me it was a hand-made system, not such a fancy thing. Love it!
I am trying to vacuum-infiltrate some grasses. Can you suggest a method to do it.
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