Fast transgenic Arabidopsis selection by using GFP - Interesting topic (Sep/20/2008 )
Recently I am working on hygromycin selection of GFP fusion protein transgenic plants. I am selecting T1 seeds. I have not done PCR to confirm the T-DNA yet. But I took tiny leaves from several plants to fluorescent microscope. Nothing except the autofluorescence.So I think there may be a lots of faulse positive there. And it takes a lot of time. So I look up in the literature, found some paper present a fast way to select. They expressed GFP in seeds and can select transgenic seeds directly by looking for GFP. It takes only a few minutes. But here is the question, will 35S promoter::GFP-protein seeds also can be selected this way? I checked hundreds of seeds under fluorescent microscope and only found autofluorescence. Some seeds seems to be brighter, but not significant. Since 35S promoter is universally expressed in transgenic plants, so the seeds should also have this GFP. But why can't select the transformants? If it can, everybody should already do this, right?
I hope you may be also interested in this topic if you have ever done hygromycin selection.
The paper discribe the method are:
Analysis of an activated ABI5 allele using a new selection method for
transgenic Arabidopsis seeds
Fluorescent Screening of Transgenic Arabidopsis Seeds
Blackwell Publishing Ltd.
Seed-expressed fluorescent proteins as versatile tools for
easy (co)transformation and high-throughput functional
Acctually, in the paper you cite, they did not use GFP but beta-glucosidase enzyme assay. So, they have expressed beta-glucosidase enzyme and then simply "poured" some substrate (MUGluc) on seeds. After few mnutes, enzyme cleaves MUGluc and releases fluorescent product that can be detected.
I'm wondering if your GFP is expressed in seeds at all... And if so, if its fluorescence is strogn enough to be visible...
Perhaps the safest way would be the classical way - germinate, harvest and prove with PCR (or northern). After that, collect seeds form positive plants, be careful not to cross-polinate them with non-transformed plants, and in second generation you should have 1/4, 1/2, 1/4 ratio of homo, hetero, WT... After that you could try northern.
Or as I said before, colect anthers form transformats, try anther tissue culture, treat with colhicine and hope to get transformed, homozygous dihaploids...
BTW, how are you getting your transformants? A. tumerfaciens, protoplasts-CaCl2, gun? M.
You should also keep in mind that even though your protein is expressed off a strong, constitutive reporter, your protein may not be very stable and hence might not be that easy to see. Did you try using a confocal microscope to see expression? Often you can see things under a confocal that you might not be able to see under a regular flurorescent microscope.