Vapor-phase seed sterilization

Andrew Bent afb at plantpath.wisc.edu
Fri Feb 4 15:31:46 EST 2000


An alternative method to add to the seed sterilization commentary:

If you have a large number of tubes (seed batches) to sterilize,
vapor-phase (gas) sterilization is much easier as it requires much less
manipulation.  The recipe is below, and at
http://www.cropsci.uiuc.edu/~a-bent/vapster.html.

NOTE:  We have found that vapor-phase sterilization is not as reliable, so
if contamination is a major problem, do not use this method.   Also, if you
have just a few tubes of seed to sterilize, it is as easy or easier to do
the 50% liquid bleach method.  But if you have a number of tubes to process
independently, the vapor-phase method has been very popular.

_________________________
Vapor-Phase Sterilization of Arabidopsis Seed

The following protocol contains a convenient method for
surface-sterilization of plant seeds. In particular, the method does not
require all of the soaking and rinsing of liquid-phase bleach-sterilization
methods. While this protocol substantially reduces hands-on manipulations,
it does require some incubation time (a few hours to overnight).

We often use this protocol in conjunction with selection of Arabidopsis
transformants on MS medium + antibiotics. See the related  "Simplified
Arabidopsis Transformation Protocol."   This vapor-phase sterilization
protocol was adapted from a version supplied by Maud Hinchee and colleagues
at Monsanto.

Protocol:
1.Obtain a vessel for seed sterilization, typically a dessicator jar. Place
in fume hood.

2.Place seed that is to be sterilized into appropriate resealable
containers (for example,microcentrifuge tubes).

3.Place open containers of seed into a rack or stand inside the dessicator
jar.

4.Place a 250 ml beaker containing 100 ml bleach into the dessicator jar.

5.Immediately prior to sealing the jar, carefully add 3 ml concentrated HCl
to the bleach.

6.Seal jar and allow sterilization by chlorine fumes to proceed for a
period of between three and sixteen hours. The time needed will vary based
on the configuration of seed and the extent to which seed is contaminated.
Three to four hours is often sufficient for reasonablyclean seed. Overnight
is usually acceptable although some seed killing may occur, especially if
seed is not fully mature and dry.

7.Depending on the application, open container in fume hood or in sterile
laminar flow hood, seal microfuge tubes or other seed containers, and
remove surface-sterilized seed for use.

NOTES:
-  Some lab members suspect that sterilization in 15 ml orange-cap tubes is
more likely to result in seed mortality? We welcome your feedback on this
or other aspects of this protocol.
-  Obtain proper approval for transformation work from institutional
authorities. Autoclave and properly dispose of all materials.

- Steve Clough and Andrew Bent


____________________
Andrew Bent
Department of Plant Pathology		Office Phone: (608) 265-3034
University of Wisconsin - Madison		Fax: (608) 263-2626
1630 Linden Drive			Lab Phone: (608) 265-3075
Madison, WI  53706			Office & Lab: 886 Russell Labs
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