GUS staining in Alfalfa

[LISA M KOCH]

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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[Jose Pedro Fonseca]

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@...> 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
>
> _______________________________________________
> Arab-gen mailing list
> Arab-gen@...
> http://www.bio.net/biomail/listinfo/arab-gen
>

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