Vapor-Phase Sterilization of Arabidopsis Seed
Steve Clough and Andrew Bent, University of Illinois at Urbana-Champaign.
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.
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.
Chlorine gas is poisonous to humans - work with proper ventilation.
- Obtain a vessel for seed sterilization, typically a dessicator
jar. Place in fume hood.
- Place seed that is to be sterilized into appropriate resealable containers (for example, microcentrifuge tubes), marked with pencil or with ink known to be sufficiently chlorine-resistant such as black Sharpie.
- Place open containers of seed into a rack or stand inside the dessicator
- Place a 250 ml beaker containing 100 ml bleach into the dessicator
- Immediately prior to sealing the jar, carefully add 3 ml concentrated
HCl to the bleach.
- 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 reasonably
clean seed. Overnight is usually acceptable although some seed killing
may occur, especially if seed is not fully mature and dry.
- 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.
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