The summery : Seeds sterilization

Byung-Hoon Kim byung-hoon.kim at uni-tuebingen.de
Fri Feb 4 13:19:31 EST 2000


Hi again,

Thank you for over 30 replies to my question on seeds sterilization.
For the people who want to know how other poeple do,  I summerized the
answers.

Most of the methods were combinations of the following steps :

(1) 70-100% EtOH (or 70-100% isopropanol),  up to 5 min
(2) 10-100% commercial bleach (or sodium hypochlorite solution) with
detergent (0.01-0.1% TritonX-100 or Tween20),
                           10% bleach : up to 30 min
                         100% bleach : up to 10 min
(3) washing with 70-100% EtOH
(4) washing with H2O
(5) let the seeds dry

The most popular method is  :  (1)->(2)->(4)
Some people do                   :  (2)->(3, optional)->(4)
More or less rare cases are  :  (80% EtOH, 20% bleach,  for  15
min)->(wash 3X 100% EtOH)->(5)
                                               (80% isopropanol for 15
min)->(4)->(plate the seeds)->(let them dry)

Most of the people who are using above methods have had no problem with
fungal contamination, but the sterilization should be done in a large
volume of the above solutions in order to get hight sterilization
efficiency.

                            Up to 2000 seeds : in Eppi (1.5 ml) tubes
                            Over 2000 seeds : 15 ml or larger tubes

Sometimes there are agrobacterial contaminations which can be eliminated
by using one of the following things on the plates.

                            Timentin (50-200 mg/l)
                            Vancomycin (500 mg/l)
                            Cefotaxime (200 mg/l)


But there were some COMMENTS on FUNGAL CONTAMINATIONS :

'The fungus is probably in the seed and you can't kill them with surface
sterilization. Do you see fungus on your plants (inflorescence)?
This is mostly an indication that the growth room is too humid. Further
adding Timentin to the medium will reduce contaminating growth but fungi
mostly will grow happily on it.'

'These infections probably result from fungal spores that are included
during seed formation within the seed coat. They grow only after seed
germination. You will not get rid of them by any surface sterilization.
To avoid this problem in future experiments: infiltrated plants should
be growing at low humidity during seed formation, not to promote fungal
growth on the plants. The only thing you can do with the seed you have:
plate them at very low density, check the plates everyday, and cut out
infected seeds from the agar as soon as you see them, to rescue the
others.'

'I only have fungal infection if there are soil particles mixed with my
seeds which I try to avoid.'

=============================================


Thanks for all the answers again.

Best wishes
Byung-Hoon Kim
-------------------------------------
Center for Plant Molecular Biology
Tuebingen, Germany
-------------------------------------

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