[Arabidopsis] Selection of transgenic Arabidopsis line in Hygromycin medium

Thomson, James via arab-gen%40net.bio.net (by James.Thomson from ARS.USDA.GOV)
Mon Aug 29 15:04:57 EST 2016


John is correct.  The Hyg selection is very weak.  However, we have
successfully transformed Arabidopsis (Ler).  The selection (20; Sigma
brand) took approximately two weeks and there is a lot of background.  The
trick was looking for expanding primary leaves.  The Arabidopsis that are
sensitive will germinate and expand the cotyledon but fail to develop
primary leaves.  Unfortunately those that are resistant tend to get lost
in the jungle of growing seedlings.  Spreading the seed sparingly helps.
Plants transferred to soil are not very robust but the next generation is
fine.

Good luck

Jim

James Thomson PhD.
USDA-ARS-CIG
Research Geneticist
800 Buchanan Street
Albany, Ca 94710
Fx. 510 559 5815
Ph. 510 559 5721



On 8/29/16, 1:39 AM, "arab-gen-bounces from oat.bio.indiana.edu on behalf of
John Turner (BIO)" <arab-gen-bounces from oat.bio.indiana.edu on behalf of
J.G.Turner from uea.ac.uk> wrote:

>Hi
>
>The phenotype is very weak, and the reason why we turned to kanamycin
>resistance as the marker for transformations. You should see shorter
>roots in the wild type (test plus versus minus hygro on wild type). If
>you dont, then hygro is not working. Is light sensitive.
>
>John
>
>
>John Turner
>Emeritus Professor
>School of Biological Sciences
>University of East Anglia
>Norwich Research Park
>Norwich NR4 7TJ
>England, UK
>
>Mobile Phone 07411429440
>email j.g.turner from uea.ac.uk<mailto:j.g.turner from uea.ac.uk>;
>johngturner2009 from gmail.com
>
>
>________________________________
>From: arab-gen-bounces from oat.bio.indiana.edu
><arab-gen-bounces from oat.bio.indiana.edu> on behalf of MAHMUDUL HASSAN
><mahmudul_bau from yahoo.com>
>Sent: 28 August 2016 03:08:54
>To: arab-gen from net.bio.net
>Subject: [Arabidopsis] Selection of transgenic Arabidopsis line in
>Hygromycin medium
>
>Hi All
>I am a PhD student. I have been trying to select transgenic Arabidopsis
>line in hygromycin medium for the last couple months. I followed this
>paper (A rapid and robust method of identifying transformed Arabidopsis
>thaliana seedlings following floral dip transformation, Plant Methods.
>2006. 2:19. DOI: 10.1186/1746-4811-2-19) but failed to select transgenic
>plants. Both untransformed (Col-0) and Hyg positive plants look like same
>in the selection plate. I used various concentration of Hyg (15, 20, 25)
>but all the time I was unable to differentiate between the susceptible
>and resistant plants. I am using Hyg from Sigma and it was bought
>recently. I am desperately looking for a working protocol for transgenic
>Arabidopsis selection in Hygromycin media.
>
>I would be really grateful if anyone can help me in this regard by giving
>some suggestion for transgenic Arabidopsis selection in Hyg media and
>hopefully any protocol if possible?
>Thank you all
>Regards
>Mahmud Hassan
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