(e.g. for biolistics in onion epidermis or protoplasts). May transient transformation be achieved this way?
Yes. We have used a binary vector with a 35S promoter to do transient expression analyses in bombarded onion and Arabidopsis leaf tissue. The main reason I believe that most researchers do not use binary vectors for bombardment is that one needs lots of DNA to coat the gold or tungsten particles, and most binary vectors do not have the appropriate origins of replication for ultra-high copy number in E. coli. It is really trivial to get a few hundred micrograms of DNA from a ml of culture if you are using a pUC based plasmid. But once you have the DNA, there is no problem. I would not try bombardment with protoplasts. Instead, use PEG-calcium transformation.
Yoo SD, Cho YH, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc. 2007;2(7):1565-72. PMID: 17585298.
Yes I totally agree with David. I have used a pUC based binary vector to transiently transform rice and jute (Corchorus) embryos as well as, Calli. This protocol need as a lot amount of DNA. But this protocol is suitable for epidermal transformation too. You just have to adjust the distance between the sample and the bombardment. If you are using BioRad machine then there you can adjust the height of the sample at three different heights. epidermal layers are one/two celled thick hence, you might loose a lot of cells due to death occurred by the pressure of bombardment. protoplast transformation is not recommended through this process. Use either Agrobacterium mediated transformation (protoplast derived callus) or PEG-mediated electrophoresis (directly in to protoplasts). Good luck
Hi Tomas. I did use pCAMBIA for epidermal onion cells bombardment and it worked very well. I've never tried bombardment with a protoplast sampe but it doesn't seems to be best option for such a sensitive system.
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