[Medicago] Gus staining in Alfalfa

medicago from net.bio.net via medicago%40net.bio.net (by medicago from net.bio.net)
Fri Oct 9 14:04:19 EST 2009

While tissue penetration is part of the problem, the major complication
is interference by chlorophyll; the effect varies with species and leaf
age. Etiolation or extraction of chlorophyll with ethanol will allow
better assessment of promoter activity.

The attached paper "The dark side of green fluorescent protein" from my
lab (Zhou et al 2005, New Phytol 168:313-322) should be of interest.


Timothy C. Hall Ph.D. 
Distinguished Professor and Director 
Institute of Developmental and Molecular Biology 
407 Biological Sciences Building West 
MS 3155 Texas A&M University 
College Station, TX 77843-3155 

Phone: 979-845-7728; Fax 979-862-4098 
tim from idmb.tamu.edu 
Original Message-----
From: medicago-bounces from oat.bio.indiana.edu
[mailto:medicago-bounces from oat.bio.indiana.edu] On Behalf Of
medicago from oat.bio.indiana.edu
Sent: Friday, October 09, 2009 12:29 PM
To: medicago from oat.bio.indiana.edu
Subject: Re: [Medicago] Gus staining in Alfalfa

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
>Medicago mailing list
>Medicago from net.bio.net

Deborah Samac
USDA-ARS Research Plant Pathologist
1991 Upper Buford Circle
495 Borlaug Hall
St. Paul, MN 55108

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