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[Arabidopsis] Independence of Transformation

Bernd Weisshaar via arab-gen%40net.bio.net (by weisshaa from cebitec.uni-bielefeld.de)
Tue Sep 8 11:32:50 EST 2015


Dear Joseph,

our experience from GABI-Kat is that the border junctions are unique for all "truly independent” lines we selected. If you need to show that you are working with different alleles, sequence the border junctions. Usually, one border is sufficient. If you turn around the argument, you can conclude that if both borders (the upstream and the downstream junction of T-DNA to genome sequence) show identical sequences, it is the same allele that comes from the same insertion event.

The phrase “truly independent” is required, because when working with more than 90.000 lines it can happen that different lines may go back to the same event because of a logistic error. This is what we found, and the very few cases in which identical border sequences were detected all have indications that an error has happened.

So, from our experience, your statement “each individual T1 transformant (from a single seed) can be assumed to be independent” is correct.

Some detail (although with a different focus) can be found here:
http://www.cell.com/molecular-plant/abstract/S1674-2052(15)00357-3

Bests, Bernd


> On 05.09.2015, at 19:56, Joseph Lee Hill Jr <jlh722 from psu.edu> wrote:
> 
> Hi all,
> 
> Sorry in advance for the somewhat naive question.
> Is it overstepping to claim each individual plant isolated from a floral dip
> transformation is an independent transgenic line?
> According to work by Clough & Bent (among others), via floral dip, "T-DNA
> insertion events derived from the same plant are generally independent." I can
> find no guidelines for independence, though. Some journals require multiple
> independent transgenic lines, but I don't believe they require a Southern blot
> or sequencing analysis to actually prove independence. Taken together, that
> makes me feel that it is generally accepted that, when doing Arabidopsis floral
> dip, each transformant can just be assumed to be independent. Would that be a
> correct assessment?
> 
> -Joseph
> 
> 
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Bielefeld University, CeBiTec & Department of Biology
Prof. Dr. Bernd Weisshaar
Chair of Genome Research / Center for Biotechnology
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Germany

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