Does anyone know a good method to perform Agrobacterium-mediated transient transformation of Arabidopsis roots (without having to chop the roots into small pieces)? Please let me know. Thanks.
Hi Simon,
For transient assay, I have done Agroinfiltration on tobacco leaves by using syringe (without needles) to push the Agrobacterium culture into the leaves. I read some articles, researchers also used 'vacuum' methods to suck in the Agrobacteria (see attached paper for one example). I think that you can try the vacuum method. For example, you can carefully dip the roots of Arabidopsis into the Agrobacterium culture/medium in a container (petridish maybe), and put the container into a vacuum chamber (see attached picture) for vacuuming to move the Agrobacterium into the roots. This may work..Good luck on your research.
Hi !
I totally agree with Yau. If you want to transform roots transiently in intact plants, then better to put those plants in petriplates or tubes in such a way that their roots stay inside. then add the buffer media+ Agro culture at an od600 of 0.6 at least to the tubes or plates and see if the roots are dipped into it. Then apply vacuaum as shown in the picture posted by Yau. Vacucum for 30 min. not more than that. This should work. Also, you can use Agrobacterium rhizogene in stead of Agrobacterium tumefascience.
Of course you have to use Agro. rhiz.!
You do not use the roots to get transgenic! You use the plantlets cut off their roots , have them infiltrated, good idea, because they are so thin. Even OD o.1 to be used after checking with 600 wavelength. Of course in a buffer medium. After wards blot them and put them on dishes with medium with low phoshates and nitrates. roots should come out of the stems or even out of the leaves.
You want to transform intact roots transient ly so the best way is to use trans-Zeatin in inoculation medium. I have performed it in various plants. For reference you can see the Science Signalling paper of Stanton B. Gelvin. Good luck.
Dr. Gelvin had developed a protocol:Agrobacterium Transformation
of Arabidopsis thaliana Roots
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