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Selection of transgenic Arabidopsis line in Hygromycin medium
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MAHMUDUL HASSAN
Aug 28, 2016, 5:24:34 PM8/28/16
to arab...@magpie.bio.indiana.edu
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
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
John Turner (BIO)
Aug 29, 2016, 2:01:49 PM8/29/16
to arab...@magpie.bio.indiana.edu, MAHMUDUL HASSAN
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.t...@uea.ac.uk<mailto:j.g.t...@uea.ac.uk>; johngtu...@gmail.com
________________________________
From: arab-gen...@oat.bio.indiana.edu <arab-gen...@oat.bio.indiana.edu> on behalf of MAHMUDUL HASSAN <mahmud...@yahoo.com>
Sent: 28 August 2016 03:08:54
To: arab...@net.bio.net
Subject: [Arabidopsis] Selection of transgenic Arabidopsis line in Hygromycin medium
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http://www.bio.net/biomail/listinfo/arab-gen
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.t...@uea.ac.uk<mailto:j.g.t...@uea.ac.uk>; johngtu...@gmail.com
________________________________
From: arab-gen...@oat.bio.indiana.edu <arab-gen...@oat.bio.indiana.edu> on behalf of MAHMUDUL HASSAN <mahmud...@yahoo.com>
Sent: 28 August 2016 03:08:54
To: arab...@net.bio.net
Subject: [Arabidopsis] Selection of transgenic Arabidopsis line in Hygromycin medium
Arab-gen mailing list
Arab...@net.bio.net
http://www.bio.net/biomail/listinfo/arab-gen
Thomson, James
Aug 31, 2016, 1:26:25 PM8/31/16
to John Turner (BIO), arab...@magpie.bio.indiana.edu, MAHMUDUL HASSAN, Thomson, James
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...@oat.bio.indiana.edu on behalf of
John Turner (BIO)" <arab-gen...@oat.bio.indiana.edu on behalf of
This electronic message contains information generated by the USDA solely for the intended recipients. Any unauthorized interception of this message or the use or disclosure of the information it contains may violate the law and subject the violator to civil or criminal penalties. If you believe you have received this message in error, please notify the sender and delete the email immediately.
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...@oat.bio.indiana.edu on behalf of
John Turner (BIO)" <arab-gen...@oat.bio.indiana.edu on behalf of
ravipal...@gmail.com
Aug 31, 2016, 2:44:12 PM8/31/16
to bionet-genom...@moderators.isc.org
Hi Mahmud,
We do the following after following the procedures and rationale described in Harrison et al 2006 (https://plantmethods.biomedcentral.com/articles/10.1186/1746-4811-2-19):
1) Sterilize seeds and plate them.
2) Incubate the plates in 4C for 3 days in dark (plates covered with foil).
3) Remove foil and place plates in the growth chamber for 5-6 hours (in light).
4) Then, place plates in dark for 3 days (a drawer is practical). Resistant seedlings would elongate further and can begin to be distinguished from susceptible seedlings.
5) Move the plates to the growth chamber. Hygromycin resistant seedlings can be more reliably scored over the next few days.
Best Regards
Ravi Palanivelu
Associate Professor
School of Plant Sciences
University of Arizona
Tucson, AZ 85721
Web: http://www.ag.arizona.edu/research/ravilab
We do the following after following the procedures and rationale described in Harrison et al 2006 (https://plantmethods.biomedcentral.com/articles/10.1186/1746-4811-2-19):
1) Sterilize seeds and plate them.
2) Incubate the plates in 4C for 3 days in dark (plates covered with foil).
3) Remove foil and place plates in the growth chamber for 5-6 hours (in light).
4) Then, place plates in dark for 3 days (a drawer is practical). Resistant seedlings would elongate further and can begin to be distinguished from susceptible seedlings.
5) Move the plates to the growth chamber. Hygromycin resistant seedlings can be more reliably scored over the next few days.
Best Regards
Ravi Palanivelu
Associate Professor
School of Plant Sciences
University of Arizona
Tucson, AZ 85721
Web: http://www.ag.arizona.edu/research/ravilab
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