[Arabidopsis] GUS staining in Alfalfa

JP Fonseca via arab-gen%40net.bio.net (by zepedrof from gmail.com)
Thu Oct 8 10:17:01 EST 2009


Hi Lisa,

Not very familiar with Alfafa. I have observed GUS staining in Arabidopsis
before by Agroinfiltrating leaves with a needleless syringe containing my
GUS reporter constructs. Then allow transformed samples to rest for ~ 2 days
to allow GUS expression and then incubate samples in GUS staining solution
overnight at 37 C.
Next day washed several times with ethanol 70% to remove clorophyll and...
"voila!"...blue GUS spots observed.
Hope it works in Alfafa.

JP Fonseca





On Wed, Oct 7, 2009 at 5:12 PM, LISA M KOCH <lmkoch from wisc.edu> wrote:

> We've been trying to do GUS staining in mature Alfalfa plants using a
> standard protocol of  acetone fix / phosphate buffer &FeCNs & Xgluc /
> incubate at 37 / EtOH clear if necessary.   This always worked very well for
> me in arabidopsis, but in alfalfa, the stain does not seem to permeate -
> even in constitutive GUS plants, only the blade-cut-edges will stain,
> leading me to believe this is a permeability issue.
>
> We tried the X-gulc method with cycling vacuum infiltration (which didn't
> work either) but I'm not sure what level of low pressure we achieved (we
> just used our speedvac).   We've also attempted a fluorometric assay with
> MUG, which didn't give great results, and may return to that idea, but
> visualizing the stain would be much more convenient for us.
>
> Does anyone have any helpful suggestions on GUS staining permeability
> (besides looking at younger plants - this is not an option for this
> project)?
> Thanks!
> Lisa Koch
>
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