Problem with Arabidopsis seeds sterilization

Jim Tokuhisa tokuhisa at ice.mpg.de
Wed Feb 2 06:04:23 EST 2000


At least one important correction needs to be mentioned regarding the
message thread "Problem with Arabidopsis seeds sterilization"

ARABIDOPSIS IS  NATURALLY SENSITIVE TO HYGROMYCIN.
Hygromycin will work only if Arabidopsis is transformed with a
plant-expressed hygromycin phosphotransferase.

>Hi Byung-hoon Kim, I have same experience with you. Something on your
>plate after sterilization might be agrobacteria kept in seed coat.
>Hygromysin work to kill them in my experiment.
>Good luck.

Seed sterilization might be more effective if a 5-20% bleach solution
(0.5-2% sodium hypochlorite) is used rather than potassium chloride.

>Hello everyone,

>I sterilized Arabidopsis seeds which have been transformed by vacuum
>infiltration. But one week after growing on the selection plates I
found
>a lot of fungi on the plates. I repeated it again and again, but I
>couldn't get rid of them.
>The sterilization methode was :
>       3000 - 4000 seeds per 1.5 ml Epp. tube
>       shaking for 5 min, RT, in (95% EtOH and 0.1% Triton X-100)
>        shaking for 5 min, RT, in (Potassium chloride solution with one
>drop of Tween 20)
>       washing 3 times with sterile H2O
>
>Could anyone out there give me a tip?
>Thanks in advance for any comment
>
>Byung-Hoon Kim


--
Jim Tokuhisa
Max Planck Institut für Chemische Ökologie
Carl-Zeiss-Promenade 10
D-07745 Jena
Germany

Phone:  +49-3641-64-36-51
FAX:  +49-3641-64-36-50
E-mail:  tokuhisa at ice.mpg.de


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