The stock centers do NOT use kanamycin selection, they use a PCR-based assay and then just raise bulk populations of seeds.  You'll probably get an F2 or F3 population.  Even if the stock says it's homozygous, check for yourself.  Trust me, it's well worth your time.

Short version: never trust a line from a stock center, do all of your own verification.

Longer version: in theory, the kanamycin selection will work well and only let plants with your T-DNA insertion of interest grow.  In practice, the kanamycin resistance gene is often silenced so it's no longer useful as a selectable marker.  Also, MANY of the T-DNA lines have more than one T-DNA insertion so you might be selecting for a T-DNA in another location in the genome.  

You will want to use a PCR assay to verify the presence of the T-DNA insertion in your gene of interest.  

As a heads up, be aware that about 20% of T-DNA lines have chromosomal translations associated with the T-DNA mutagenesis.  The only reliable way to test is "old school" genetic mapping.  

Article Chromosomal translocations are a common phenomenon in Arabid...

Thank you Katie. I already sowed seeds from SALK lines in culture media + kanamycin so in the next days I am checking via PCR whether they carry the T-DNAs. But now, I've got these CSHL lines which got me confuse. It seems they have two resistance genes (kan and nam) but I am not sure whether I should grow them in media + kan + nam or media + kan (and no nam), because it's not clear for me if the nam is a compound used for selection only in the first steps of generation or if I am supposed to select the "putative" transgenic plants with nam before carrying a PCR. And so, do not want to loose my seeds before clearing this. 

I always used just the PCR assay.  You don't need to try the resistance markers, especially with the risks that the genes are silenced or from T-DNAs in another insertion site.  And you can use PCR when the plants are ANY size (just germinated to setting seeds).  You only need a tiny piece of leaf tissue for DNA.

As others have said it is quite common for the antibiotic resistance genes to become silenced meaning that you might be killing plants which contain the T-DNA insert, in addition to this some lines contain multiple T-DNA inserts and this means that even if you do get resistance it may be from an unexpected T-DNA insert in a different place in the genome. The PCR assay is the standard protocol for selecting stock centre lines such as SALK an SAIL. Extract genomic DNA from the plants, a quick and dirty extraction will do. Then you use a 3 primer system, which some people do all in one reaction but I prefer to split into 2 paired reactions. Reaction 1 has a forward and reverse primer for the gene supposed to contain the T-DNA insert (these are designed by you), Reaction 2 has a primer contained within the T-DNA insert and the gene reverse primer. For salk lines LBb1.3 is commonly used (with sequence ATTTTGCCGATTTCGGAAC). In this system when you run your gel you will get a band just in reaction 1 if the plants don't contain the insert, only in reaction 2 if they do have the T-DNA and one in each if the plants are heterozygous, Even if you select on antibiotics first you really should check by PCR before investing any time characterising these plants. A quick and easy primer design tool (and further explanation) can be found here http://signal.salk.edu/tdnaprimers.2.html

http://signal.salk.edu/tdnaprimers.2.html

Thank you guys. I happened to find that NAM is not necessary and only KAN if marker selection is on one`s sight. But, yes, I am going to test throug PCR whether plants are T-DNA lines or not. I designed primer with T-DNA express and ordered.

If there's such a high divergence whithin Tms I'd suggest you run a gradient PCR at first.  Then, whether is still not working, order other primer with a similar Tm. Actually, keep working with primers whose Tms are around 60

Also, I suppose you did not run the LBb1.3+LP. You should that reaction too, as T-DNAs can be inserted in the oposite orientation

Hi Leonardo Cardoso Alves

I hope your experiment with SALK lines went well. I just want to know, how many days SALK lines generally take to germinate in MS Agar medium. How long Do you keep for stratification?. Do you add GA? Thanks in advance.@ Leonardo Cardoso Alves

Hi Mr Muthusamy. Based on the lines I have studied the germination time can vary, specially if the knocked gene should be active in the early development. But, in general, I'd say you should see something in about 3-7 days. I used 1/2 MS medium, with no GA but sucrose 3% added instead. For stratification: 2 days at 4oC in the dark (you can wrap the plate in aluminium foil) is enough. Hope it helps.

Thanks a lot . I am waiting to see the germination. All the best