[Arabidopsis] Re: Embryogenic culture

[Anbu]

On Jun 29, 4:28 pm, punamaya Maharjan <puna... from hotmail.com> wrote:
>   Dear Sir/ madam
>  I would like to post my query in this page .
>
> Thank you .
> Puna
>
>  "Query"
>
>     Dear
> arab-gen users,
>
> I am posting a query related to
> the problem of contamination in embryogenic cultures. If anybody has idea to overcome it, I highly appreciate any kind of your comments.
>
> I
> followed the method described in the paper by Mordhorst et al., 1998.  As mentioned in the Mordhorst et al., 1998, seeds were
> surface sterilized for 10 sec in 70% ethanol followed by a 10- min incubation
> in commercial bleach (final concentration 2% sodium hypochlorite, containing
> 0.3% Tween 20), washed 9 times with sterile water, dried on filter paper, and
> stored at room temperature before use. Then 30 seeds were incubated in 20 ml  MS-4 (pH 5.8)
> induction medium [Murashige and Skoog salts containing 2% (w/v) Sucrose , 4.5 uM 2,4-D,
> and 10 mm MES
> [2-(N-morpholino)-ethanesulonic
> acid]. After a cold treatment of 4 d at 4°C, cultures were kept on a rotary
> shaker (100 rpm) at 25°C in the light or darkness. For seed incubation, I used
> the 250 ml flasks instead of 190-ml Greiner plastic Containers (Alphen a/d Rijn, The
> Netherlands) mentioned in Mordhorst et al., 1998. I have tried with
> different sterilization buffers like 1) 5 % Clorax, 1% SDS 2) 0.05 % TritonX-100
> followed by absolute ethanol washing.  Still I am getting the contamination problem
> just 1 week after transferring the flasks from cold room.
>
>  Does anyone have the idea to overcome the
> problem?  Does this experiment need
> specific 190-ml Greiner plastic Containers
> (Alphen a/d Rijn, The Netherlands). I tried to find this container also but I
> could not get the information about it. Would you please give me
> suggestion?  I thank you in advance for
> your reply.
>
>  Puna
>
> _________________________________________________________________
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Dear Puna,
      I am suggesting you the following,
  1. Remove infected seeds before starting sterilization procedure.
  2.  Use autoclaved filter paper
  3. Use minimal amount of seeds

I hope this will be very helpful to you.


looking forward to hear from you