One possibility is that your original plants were not homozygous for the insertion and only some of the progeny will have your GUS reporter construct. You'll have to screen seeds from many parent plants to find one that still has the GUS.

Another possibility is that your reporter gene is present, but not expressed due to gene silencing. Arabidopsis plants will often silence expression from transgene insertions; many of the commercially available T-DNA lines don't retain the herbicide resistance from the T-DNA insertion due to gene silencing.

You can use PCR for the GUS gene to see if your plants have your construct.

Since your positive control did not even show staining, It’s also good to check if your GUS stain solution is still viable or if a component was expired/ missing when you made the solution.

The two previous answers merit follow up. Technical problems can be resolved, and the issue of homozygosity can be addressed by looking at reasonable numbers of progeny plants--16 would be plenty--since not all plants in the population could have segregated as non-transgenic (only 1/4 would be non-transgenic). Other issues can be that the transgene becomes silenced--and there are many studies in the literature that deal with issue. I have been involved in some of these, and other studies are reviewed in our publications, so there's plenty out there to check out.

Hi Brendan,

does your construct also carry a selective gene? If so, did you germinate the seeds on selective medium before testing for GUS activity? That way you should get rid of the homozygous none-transgenics. If the seeds germinate on selective medium, but the seedlings don't show GUS activity you may indeed have silencing in your plants, as suggested above.

Regards,

Klaus

To follow up this issue, the possible roles of the promoter driving GUS gene in this experiment may not be ignored. Try to figure out whether the GUS staining can be detected in leaves , seeds or other plant parts. Seeds are a largely dormant organ and GUS may not be expressed.

Hi Shu Wei,

is it likely, that the tissue specificity of a transgenic line changes? I am aware, that even the 35S promoter is not always a "typical" ubiquitous promoter - I even had a line once with only the stigma showing GUS activity... But, going through the germ line I would expect the transgene to have the same, or diminished (silenced) expression (pattern). Would you expect pattern changes, too?

Regards,

Klaus

Katie A S Burnette Turns out my original seeds were not homozygous, just like you thought. I screened the next generation on hygromycin plates and was able to identify a line I believe is homozygous. Thanks for your help!