project on Agrobcterium tumefaciens

[sober]

Hi, I am doing a project on Agrobacterium tumefaciens and i am trying to grow on the potato discs. However, there is no tumor formed on the potato discs even after around 1 month. The absorbance of the bacteria solution is around 0.8 at 600nm. I inoculated around 2 loops of Agrobacterium tumefaciens(ATCC 33970) into 10 ml of LB-broth.

My processes are:
I use russet potatoes and I soak the potato tubers in a beaker of 10% bleach for 15 minutes.
After that, I transfer the potato tubers with a sterile forceps into another beaker of sterile water and soaked the potato tubers in sterile water twice for 15 minutes each time.
Then, i take the potato tubers out with a sterile forceps and I place it on a sterile petri dish and cut out potato cylinders using a sterile cork borer.
Lastly, I cut the cylinder into discs of around 0.5cm using a sterile scalpel.
There are 4 potato discs in 1 petri dish
50 micro-litre of the bacteria solution is induced onto the top of the discs using a micro-pipette.
The petri dish is sealed with parafim and incubated at 26 degree Celsius.

All the work is done in the biosafety cabinet but I am not sure why the tumor is not growing on potato discs. Is it possible that 1 of my steps is incorrect but from what I researched, scientist normally used that to prepare potato bioassay.

Please help. Thank you

p.s.: I have tried the same strain of bacteria on carrot using similar procedure, except that i did not use cork borer and it does work, so I was puzzled.

[deleted-94524]

Hello Sober,

I tried to research why you would not have been successful in transforming the potato. I could not come up with a clear answer, but here are thoughts and informations:
-from what I could gather, carrot transformation seems to be extremely efficient. Is there any reason you would want to do your project on potatoes rather than switching to carrots?
-transformation efficiency depends on the organism that you are working with. For potatoes, I found several references (including those two http://www.banglajol.info/index.php/PTC ... /5440/4252 and http://www.aensionline.com/rjabs/rjabs/2008/16-25.pdf) showing that the efficiency varies a lot even for different cultivars of potatoes. In one of the publication, the rate varied from 7% to 72% depending the species and the protocol. So if your transformation rate is for example as low as 10%, and you work with 20 samples, you would expect on average only 2 transformed samples. So my biggest advice would be to increase your sample size if it was low.
-congratulations for putting a control in your experiment! It was very important to know that your agrobacterium culture worked on carrots because it ruled out the possibility that the problems you were having came from there. From what I looked up, your inoculation protocol has already been used for potatoes. I found another protocol (http://www.aensionline.com/rjabs/rjabs/2008/16-25.pdf) where soaking the explants in the inoculum for 30 minutes worked (again, efficiency ranged from 18% to 37% depending on the cultivar). Depending on the amount of time you have available, if increasing the sample size does not work, you could try this other protocol. But again, if it is possible, I would switch to carrots where you have the experiment set up and working.

I am sorry this is all I have! Don't hesitate to let me know if you have more question and how the experiments are going.

Heloise