[Arabidopsis] Re: Independence of Transformation (answer compilation)

Joseph Lee Hill Jr via arab-gen%40net.bio.net (by jlh722 from psu.edu)
Tue Sep 22 12:44:16 EST 2015


I'm compiling a list of highlights from the replies to my inquiry, for
posterity's sake (those ungrateful jerks).

First, my summary:
I was already aware that the female gamete was the target of floral dip
transformation, which would logistically make each T1 plant independent. My
worry in asking this question was if there was any evidence to support the
(paranoid?) worry that a gamete progenitor cell could be transformed, then
resulting in multiple female gametes possessing the same transformation event.
While the early publications on the cell-type target of T-DNA insertion all
point to the female gamete only...as they say "absence of evidence is not
evidence of absence". However, the consensus seems to be that pre-gamete
transformation does not occur or is rare enough to ignore. Empirically, data
from GABI-kat best puts my mind at ease, as they have screened nearly 100,000
transformants and found them all to be independent (note and ref below).
Papers outlining the targeting of agro transformation:
Ye GN, Stone D, Pang SZ,Creely W, Gonzalez K, Hinchee M. Arabidopsis ovule is
the target for Agrobacterium in planta vacuum infiltration transformation.
Plant J. 1999; 19:249-57.

Bechtold, N, Jaudeau, B, Jolivet S,Maba B, Veson D, Vosin R, Pelletier G The
maternal chromosome set is the target of the T-DNA in the in planta
transformation of Arabidopsis thaliana. Genetics. 2000; 155:1875-87.Desfeux,
C., Clough, S. J., & Bent, A. F. (2000). Female reproductive tissues are the
primary target of Agrobacterium-mediated transformation by the Arabidopsis
floral-dip method. Plant Physiology, 123(3), 895–904. doi:10.1104/pp.123.3.895

Some replies to my question:* It seems like ovules or young zygotes are
transformed, so indeed each plant originating from a floral dipped plant will
be an independent transgenic line. So far, as far as I know, no evidence of the
contrary has appeared.
* You are correct.  It is safe to assume that each transformant plant (seed
off of the originally dipped plant) is an independent transformant.
That is what referees will be assuming too.
* Transformation occurs in the ova.  So no two T1 seedlings are the same.  When
the journal asks for independent lines, 2 different T1’s from the same dipped
(=T0) plant are fine.


*Our experience from GABI-Kat is that the border junctions are unique for all
"truly independent” lines we selected.
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:
<a target="_new"
href="http://www.cell.com/molecular-plant/abstract/S1674-2052%2815%2900357-3">http://www.cell.com/molecular-plant/abstract/S1674-2052(15)00357-3</a>


Kleinboelting, N., Huep, G., Appelhagen, I., Viehoever, P., Li, Y., & Weisshaar, B. (2015). The structural features of thousands of T-DNA insertion sites are consistent with a double-strand break repair based insertion mechanism. Molecular Plant. doi:10.1016/j.molp.2015.08.011

-Joseph

On Sat, Sep  5, 2015 01:56 PM, "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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