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Help to sterilize unknown seed - (Dec/26/2013 )

I have to do tissue culture of the seed (given an unknown seed, name as seed x)

i culture it 3 times already but damaged du to contamination, what can i do to avoid the contamination and make the seed germinate?

 

first technique: put under running tap water - 15 min

under laminar flow: put with 70% ethanol for 3 min

                               50% clorox-10 min

2nd sterilization 95 % ethanol- 1/2 min

25% clorox- 5min

results: contaminated

 

 2nd technique: seed soaked overnight

run under running tapwater- 30 min

70% ethanol- 1 1/2 min

50% clorox with tween 20- 10 min

 result: contaminated 

 

the seed picture- in the attachment


Attached Image

Attached Image

-ckzimase-

the methods you use seem ok.

2 reasons that might be causing the problems:

 

- your technique working in the hood is not good enough (which is a known problem when working with seeds with people that do not do this on a daily base, I am not sure whether you are a student or used to this?)

 

- contamination in the seeds themself, under the "skin".

 

There are many protocols to sterilise seeds, so you might want to look for an alternative, but yours seem (at first sight) ok.

-pito-

i used to do sterilization, but this seed is given as unknown, i dont know the exact protocol to use. yes, i thought i saw some fungus inside the seed (in the second picture of the attachment)

-ckzimase-

If there is a fungus inside it, you will need to do some other stuff. But I dont know the seed, could it be possible to remove the outer protective layer and then sterilize it and let it germinate? If there is an external layer that is contaminated, this might be a option.

Other options: just grow the plants with the intact seed, without sterilisating it and then work from the plant material....

-pito-