Kanamycin selection of transgenic Arabidopsis - (Aug/10/2008 )

Hi all,
I made some trangenic Arabidopsis using Agrobacterium and flora dip method. After I got the seeds, I select them in a MS plates with 50mg/ul of kanamycin for 7 days. and Then transfer the green seedlings to another Kanamycin MS plates for second selection to rule out the false positive seedlings. Then after three days, I transfer them to soil. But a week later, all of my seedlings died. Then did not grow after I transfer to soil. My labmate also had this problem.
Does anyone have a clue why they died?
Thanks a lot.

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When I do T1 selection, I normally grow the seeds for about 7 days, then transfer Kan resistant seedlings to ATS plates (no kanamycin) for another 7 days or so before transfering them to soil. Once in the soil I cover them with plastic wrap so the soil doesn't dry out before the roots have time to establish themselves. This gives the seedlings time to recover from the kan selection (which is really not very healthy) and makes it easier for the seedlings to survive. I do not do two rounds of selection. This may increase the number of false positives initially, but you don't necessarily need to screen through all of them. For instance, if I get 20 lines, then I might only look at 5 initially and if they have robust expression then I just store the other ones as backup. I most likely would not look at all 20 lines unless expression was weak or I needed to really confirm what I was seeing.

Hope this helps,
smu

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Thank you smu, I will try your protocol. Did you use RT-PCR or real time PCR to test the expression? Or use regular PCR to amplify the kanamycin gene?

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Normally I first check to see how well the Kan resistance is segregating, just by testing on kan plates. Those that are very resistant (nice green color), I keep and pot to get homozygotes. Sometimes you get seedlings that show spotty light green color and those tend to be weaker, although the correlation is imperfect. (e.g. many of the tDNA lines that were originally made with kan as a marker no longer show kan resistance). Sometimes the spotty green color is all you get, so then I look at more lines or just go with the weaker ones. Once I have some lines to test, then I look for expression of my gene of interest either by visualization (gus or gfp), western, or RT-PCR. I do not test for kanamycin resistance by any molecular means. Basically I only use it in the initial screening process and then to pick homozygotes.

smu

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