**HELP!** Plant growth medium contamination

[T. Hedley Carr]

My colleague and I are trying to grow Arabidopsis seeds under sterile 
conditions to provide us with tissue for protoplast production and transient 
transformation. Help! We are having great difficulty in getting the seeds 
sterile. We're following an established protocol for sterilisation where the 
seeds are wrapped in filters and passed through several solutions. These are:

70 percent ethanol,  2min.
5% sodium hypochlorite (bleach) + 0.5% SDS, 15min.
sterile water, 5min. Repeated several times.
Dry off seeds and then sow.

All of the above is carried out in a flow hood with great care to avoid 
contamination. The seeds are then grown on half-strength MS10 medium in
90mm petri dishes at 25 degrees C. (Dishes sealed with micropore tape).

We've tried this several times now and cannot eliminate all of the 
contamination, which is primarily fungal. We don't think it's our flow-hood.
The contamination seemes to come from the seeds themselves, so it seems we're 
not sterilising enough. 

Does anybody have a reliable (and preferably quick) seed-sterilisation 
protocol?

Or should we try antimycotics in the growth medium? I notice that SIGMA (who 
else?) do several antimycotics in their plant-culture catalogue and also a 
special antibiotic/antimycotic solution (amphotericin+penicillin+streptomycin)
Would it be wise to try this in the medium? Has anyone else tried this?

Any helpful suggestions would be greatly appreciated.

Thanks in advance, Hedley (Possibly a PhD owner in 2 years time)

Dept. of Biochemistry & Molecular Biology,
University of Leeds, LS2/9JT, UK.
bmbthc at bmb.leeds.ac.uk