Hello everyone,

I'm a bit baffled about one of my transgenic lines segregation. I made a transgenic construct which encodes a histone deacetylase under a 35s promoter. Did all the verification steps after which it was transferred to A. Tumefaciens with electroporation.

After another round of verification steps, plants with Col-0 background and plants with the histone deacetylase knockout where dipped into the A. Tumefaciens. The plasmid contained tpCRT1, which is norfluorazone resistance. Transgenic and non transgenic seeds were collected from both backgrounds and grown on norflurazon plates. (t1)

The recombination was very efficient for both backgrounds and around 15 accessions for each background where grown further for harvesting.

However when I grew the t2 seeds on norfluorazone plates, nice segregations in the col-0 background where observed (75%), but in the hda background all t2 seeds survived.

I resowed norfluorazone plates with 35s:hda construct in the hda background together with col-0 to antibiotic works, and still 100% of the T2 mutants survived whereas all col-0 seedlings died. 

Why this happens I cannot figure out. I didn't skip a generation by accident or something.. I have thought about multiple insertions, as 2 insertions would give a survival rate of ~92% this is not an option. So it must be 3 or more insertions and even then there would be some seedlings which should not have the resistance. And that is just for 1 accesion, let stand alone 15. The t1 survival rate was approximately 1 in a 100 or so.

Since I don't know what to do with this result I'm going to do the floral dip again in this background line, and see if it still is the same, but it will take a lot of time unfortunately. And I am just too curious to know what is going on so that's why I hope to get some insightful information here..

Thanks in advance!