[Arabidopsis] Landsberg erecta and kanamycin: usefull replies

Tine Beunens via arab-gen%40net.bio.net (by tine.beunens At psb.ugent.be)
Tue Nov 14 02:42:43 EST 2006


Here are some usefull replies which I got to my question.


Dear all,

I have a T-DNA insertion line in the ecotype Landsberg erecta. I seem
unable to select these plants on kanamycin though.
All of my wild type Ler continue to grow on kanamycin, while Col0
plants, which I put on the same plate as a test, are staying small and
are clearly sensitive.
I am using MS medium with 0.75% plantagar. The problem is not the
kanamycin or the plates (since Col0 are sensitive).
All of my transgenic plants are also staying alive, while I didn't
expect all of them to be homo- and heterozygous for the insert.
Has anybody had a similar experience with the Landsberg erecta ecotype
(all Kana-segregation is going fine with Col0 ecotpye)

Kind regards,

Tine

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Dear Tine,

It has been a long time ago that I used Ler for kanamycin selection, but
I never had problems in selecting for kanamycin resistance. I do not
recall seedlings were a lot smaller, but mostly they were yellow. It was
a while ago though. The most obvious explanation for your observation
would be that your wild type is not really wild type, but transgenic.
PCR for the NPTII gene might tell it?

Kind regards,

Mark Aarts

Dr. Mark G.M. Aarts 
Laboratory of Genetics, Wageningen University 
Arboretumlaan 4 
6703 BD Wageningen 
The Netherlands 
phone: +31-317-485413 
fax: +31-317-483146
[1]www.gen.wur.nl/uk



-------------------------------------------------------------------------

Hi Tine,



we've got a similar problem with a T-DNA insertion line we obtained from
Syngenta. This line is in a Col-0 background, and when we tried to
complement the mutation, we discovered, that all of the plants were
already Kanamycin resistant...

We've no explanation for this, but I'm sure, that when you repeat the
experiment with new (i.e. newly bought) Ler wild-type, it will be
kanamycin sensitive.



Best regards,



--
Andreas Timper

LMU München

Botanisches Institut

Menzinger Str. 67

80638 München


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Hi Tine,

I've used Landsberg (or mutants in a Landsberg background) a lot for
transformation and Kanamycin selection, and never saw anything like you
describe. Wildtype Ler is equally sensitive (and resistant if
transformed with the appropriate T-DNA) as Columbia in my experience.

Are you certain your "wild-type" Ler is not contaminated (maybe screen
by PCR for the NPTII)?

Cheers,

Tony

-----------------------------------------------------------------

I had actually ordered primers for the nptII gene yesterday, so I will check wh
ether my wild type is truly wild type.
Personally I don't think that's the problem, although it definitely needs to be
 checked. I think the only solution will be add more Kana, since none of my tra
nsgenic plants are dying either and I was expected that. I will inform you on t
he results.

Thanks!

Tine

--
==================================================================
Tine Beunens                                   Tel: 32 (0)9 3313922
DEPARTMENT OF PLANT SYSTEMS BIOLOGY            Fax:32 (0)9 3313809
GHENT UNIVERSITY/VIB,    Technology Park 927, B-9052 Gent, Belgium
Vlaams Interuniversitair Instituut voor Biotechnologie         VIB
[2]mailto:tine.beunens At psb.ugent.be                  [3]http://www.psb.ugent.be
==================================================================

References

   1. http://www.gen.wur.nl/uk
   2. mailto:tine.beunens At psb.ugent.be
   3. http://www.psb.ugent.be/


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