[Medicago] Gus staining in Alfalfa

medicago from net.bio.net via medicago%40net.bio.net (by medicago from net.bio.net)
Fri Oct 9 12:29:21 EST 2009


Dear Lisa,

My lab has done lots of GUS work with alfalfa, both histochemical and 
enzymatic assays. You are right, the technique you used for Arabidopsis 
does not transfer well to alfalfa. (I could give you quite an extensive 
list of things that don't transfer!) You need to cut the leaves into 
smaller sections, if possible. Whole leaves are almost impossible to stain 
successfully. The key is to do the vacuum infiltration correctly. The speed 
vac is probably pulling a sufficient vacuum, but it is releasing the vacuum 
too slowly. The release needs to be very fast, so that the stain permeates 
into the leaf tissues. I suggest using a small side arm flask attached to a 
vacuum pump. Put a large rubber stopper on top of the flask opening but not 
inside the opening. Pull the vacuum for several minutes while swirling the 
leaf pieces in the stain. You should see little air bubbles coming out of 
the leaf pieces. Then, pull off the stopper quickly. You should see the 
leaf pieces turn darker green as the stain infiltrates. You may need to 
repeat the infiltration process several times to get the stain into the 
center of the leaf piece. Incubate at 37 C checking frequently so that over 
staining does not occur. I suggest putting the leaf pieces and stain into a 
multiwell plate that can be put under a dissecting microscope.

Feel free to give me a call to discuss the assay further. I may be able to 
offer other tricks depending on the objectives of your experiment.

Debby Samac
(612) 625-1243


On Oct 8 2009, medicago from oat.bio.indiana.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 & 
> Tx100 / 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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-- 
Deborah Samac
USDA-ARS Research Plant Pathologist
1991 Upper Buford Circle
495 Borlaug Hall
St. Paul, MN 55108



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