Gentamycin selection of Arabidopsis seeds - What

joel.kreps at syngenta.com joel.kreps at syngenta.com
Mon Jul 30 17:43:51 EST 2001


Dear Yulan,

I have used the following conditions to select for Arabidopsis seedlings
transformed with pPZP221:

Put out surface sterilized seeds onto MS agar containing 3% sucrose with 200
ug/ml carbecillin and 75 ug/ml of active Gentamycin (consult supplier for
actual potency, we typically get 65 to 75% effective gentamycin); stratify
the seeds for 72 hrs at 4C in the dark; transfer plates to 12 hr Light, 12
hr dark, 100 uEi cool white light, 22C; by day 7 all seedlings are still
green and have expanded cotyledons; by day 10 only transgenic seedlings have
true leaves and their cotyledons are as fully expanded as similar seedlings
grown without gentamycin, sensitive seedlings will never grow further and
their cotyledons do not fully expand while truly resistant seedlings will
continue to grow normally in the presence of the gentamycin.  The gent
selection is not as obvious as Kan or herbicides on soil but it can still
work.

sincerely,

joel

Joel A. Kreps, Ph.D.
Senior Staff Scientist
Plant Health Department
Torrey Mesa Research Institute, Syngenta
3115 Merryfield Row, Rm 267
San Diego, California 92121
USA

tel: 1-858-812-1071
fax: 1-858-812-1105
joel.kreps at syngenta.com


-----Original Message-----
From: ligeng [mailto:ligeng.ma at yale.edu]
Sent: Thursday, July 26, 2001 3:46 PM
To: arab-gen at net.bio.net
Subject: Re: Gentamycin selection of Arabidopsis seeds - What


Pls try to use 200-300ug/ml, even 400ug/ml. I have ever tested At by this
concertrations. Try! Further discussion will be welcomed.

My email: kangdingming at yahoo.com or kdm at pku.edu.cn

Yulan Cheng wrote:

>   Hi, all,
>
>   If anyone has any experience on the selection of transformants (Vacuum
>   infiltration transformation) by gentamycin, please give me a help.  I am
>   using plant transformation vector pPZP222 and I have tried up to 100
>   ug/ml  gentamycin, and the plates are still covered with green and
>   healthy seedlings.
>
>   Thanks  in advance!
>
>   Yulan
>
>   ---

---




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