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)?