Transient protoplast assays
NUNBERG, ANDREW NEIL
ann4141 at zeus.tamu.edu
Tue Aug 27 14:36:47 EST 1991
In article <MacMS.6944.16838.karlin at cmgm.stanford.edu>, karlin at CMGM.STANFORD.EDU (Personal Name) writes...
> I would like to be able to do a transient gene expression assay in
>Arabidopsis. Does anyone know of a procedure for doing this in protoplasts
>freshly derived from Arabidopsis leaves? or perhaps in intact seedlings? I have
>tried to adapt the electroporation method of Fromm et al (PNAS, 1985) to
>protoplasts derived from the WS ecotype (by the method of Damm and Willmitzer
>[MGG, 1988]) followed by histochemical staining for GUS activity, but have
>gotten very few GUS+ cells at a wide range of voltages. Any recommendations?
> Thanks, George Karlin-Neumann (karlin at CMGM.stanford.edu)
Electroporation works great if you have enough proto's to work with.
Unfortunately the process kills a good number of them. Another possible problem
is that your culturing media may not be adequate or culturing time maybe
too little or too long.
An alternate to electroporation is PEG. This doesn't kill too many protos.
And i have gotten it to work for other types of protos(not Arab. ones though)
I am interested in transient assays in Arab too, could you send me
your protocal for making proto's and i will send you mine for PEG method of
Andy Nunberg (ann4141 at zeus.tamu.edu)
More information about the Arab-gen