hygromycin resistance

[Zhiyong Wang]

You will love hygromycin selection if you follow the following 
procedure. The key is to grow your seedlings in the dark. After you 
sow the seeds and treat them in cold for a couple of days, put the 
plates in light for 4-12 hr to promote germination, then put the 
plates in the dark (wrap up with foil and put in your drawer is good 
enough). Grow for 5 days (start from the beginning of light 
treatment, longer growth in the dark will reduce the 
greening/recovery in light). You will find hyg sensitive seedlings 
lying on medium with very short hypocotyls and open dotyledons (look 
like those severe det and cop mutants), and hyg resistant seedlings 
will be standing up tall like normal dark-grown seedlings (with long 
hypocotyls and closed cotyledons). Keep the plate in weak light (on 
you bench) for a day or two to allow hygR seedlings to green up and 
recover (strong light sometimes bleach the etiolated seedlings, 
particularly old ones). You can grow them longer in growth chamber to 
get bigger seedlings and them transfer the tall seedlings to soil. 
Whatever vector you use, you won't misscore the hygromycin resistant 
seedlings, unless your transgenic plants have a 
de-etiolated-in-the-dark phenotype. Have fun.

Zhiyong

Dr. Zhiyong Wang
Staff Member
Department of Plant Biology
Carnegie Institution
260 Panama street
Stanford, CA 94305

Phone: 650-325-1521 ext 205
Fax: 650-325-6857
><x-flowed>Hi,
>
>I am having trouble growing Arabidopsis plants with hygromycin resistance.
>After selecting on hygromycin and transfering resistant plants to soil, most
>plants do not survive.  I have spoken to other researchers, some of whom
>have had the same trouble and some who have not. It seems that it may depend
>on the vector used and/or the gene being expressed from the vector.
>
>Can anybody tell me more specifically what it is that determines how
>resistant transformants are for  particular vector?  If not, could you let
>me know whether or not you have had trouble with hygromycin resistance in
>the past and what vector you used?
>
>Thanks,
>
>Mimi Tanimoto
>
>Department of Plant and Microbial Biology
>111 Koshland Hall
>University of California
>Berkeley
>CA 94720
>
>+1 510 643 9204
>
>_________________________________________________________________
>Express yourself with cool new emoticons http://www.msn.co.uk/specials/myemo
>
>---
></x-flowed>


--
Dr. Zhiyong Wang
Staff Member
Department of Plant Biology
Carnegie Institution
260 Panama street
Stanford, CA 94305

Phone: 650-325-1521 ext 205
Fax: 650-325-6857