Embryogenic culture

[punamaya Maharjan]

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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[Eve Wurtele]

it might help to reduce the number of seeds you are using in each flask.
one bad seed contaminates the whole flask.

also, have you tried controls in which you do the same protocol, but do not
add ANY seeds- and then put on shaker.

this will help you rule out something to do with flasks, media, etc.

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[Anbu]

On Jun 29, 4:28�pm, punamaya Maharjan <puna...@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
>
> _________________________________________________________________
> More than messages�check out the rest of the Windows Live�.http://www.microsoft.com/windows/windowslive/

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