We use 20 or 25 mg/l hygromycin. Another good news I forgot mention:
you can screen much more seeds per plate in the dark than in light.
We usually screen three to four thousand seeds on each 150 mm plate.
>Your arab-gen net posting with your hygromycin protocol was very
>interesting. I think I'll give it a try. What concentration of Hyg
>do you typically use?
>Date: Wed, 23 Jun 2004 17:20:14 -0700
>To: tanimoto_h at hotmail.com ("Harumi Tanimoto"), arab-gen at net.bio.net>From: Zhiyong Wang <zywang24 at stanford.edu>
>Subject: Re: hygromycin resistance
>>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.
>>Dr. Zhiyong Wang
>Department of Plant Biology
>260 Panama street
>Stanford, CA 94305
>>Phone: 650-325-1521 ext 205
>>>>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?
>>>>Department of Plant and Microbial Biology
>>111 Koshland Hall
>>University of California
>>>>+1 510 643 9204
Dr. Zhiyong Wang
Department of Plant Biology
260 Panama street
Stanford, CA 94305
Phone: 650-325-1521 ext 205