Problem with Arabidopsis seeds sterilization

Suk-whan Hong sukwhan at U.Arizona.EDU
Wed Feb 2 12:07:12 EST 2000

Hi Byung-Hoon Kim. This is Suk-Whan again. If what is on your plates for
selection of transformants is white ( or milky) things, that might be a
agrobacteria. I think that some agrobacteria reach into seed somehow
after vacuum infiltration and survive to the next generation. So, if my
thought is right, those agrobacteria might not be killed by
sterilization of seed coat. Because they are inside seed coat, they
could be removed after planting your seeds on the plates. What I used to
kill them is vancomycin (500mg/L). My seeds are placed on the growth
media containing selection antibiotic and vancomycin (500mg/L). I'm very
sorry to confuse you.

Suk-Whan Hong.

On 1 Feb 2000, BHKim wrote:

>  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
>  ---


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