dark germination

Tue Jan 30 13:01:57 EST 1996

Dear Jim M. and Others,

I too have germinated Arab. seeds in liquid culture, but the cultures were
shaking to enhance oxygen exchange.  And, I have successfully used
surface-sterilized seeds, stored in H2O at 4C for over 30 days.  I think the
wording in my original posting was slightly inaccurate.  I should have used
the term suffocated instead of drowned.  My thinking is that seeds that sit
in water, on a plate at RT may not get enough oxygen and fail to germinate.
The two examples cited below may represent the oxygen rich environment of a
shaking liquid culture and seeds soaking at 4C in H2O may not be respiring
enough to be affected by reduced oxygen.  

Joel A. Kreps
Biochem and Mol. Bio.
UMass Amherst 

>In regards to the drowning seed hypotheses that have been floating 
>around, we have successfully germinated our seeds in liquid media and I 
>routinely cold-treat my seeds by soaking them at 4 degrees in water for 
>up to a week.  Neither treatment affects germination frequency.
>Jim Masucci
>Dear Network,
>Although this thread is a couple of weeks old, I thought I would contribute
>some recent observations that may be of interest to others.  This month I
>had the following experience growing etiolated seedlings on MS Agar.  I had
>harvested some seeds  on Jan. 3 (C24 transgenics) from plants that had been
>drying for 14 days.  I stored the seeds in glass vials in a cardboard box at
>4C for 5 days.  I poured MS Agar (0.8%) + 3% Sucrose and Vit/Glycine and let
>the plates dry in the hood for 48 hours.  I surface-sterilized the seeds
>according to the method in the Valvekens, 1988 PNAS paper and immediately
>plated out the seeds onto the agar plates, minimizing, somewhat, the amount
>of water left on each plate.  I then wrapped each plate with parafilm and
>aluminum foil, the plates were kept in a light-tight box at 4C for 72 hours
>at which time I transferred them to a light-tight box at room temp and put
>one set out under constant light. The control plates under constant light
>had nearly 100% germination efficiency.  Six days later I went to harvest
>the etiolated seedlings and found that maybe 30% of the seeds had germinated
>and there was considerable moisture on the plates.  I put some of the
>"etiolated plates" under constant light and the seeds that had not
>germinated yet, still didn't, indicating that they had died.  I had used the
>imbibition/plating technique several times with previously good results.  I
>was very surprised at the amount of moisture/condensation that I saw on the
>"etiolated plates".  Under the assumption that the failure to germinate was
>a moisture problem, I transferred my seed stocks to a bottle containing
>dryrite-like material and stored them at 4C.  I poured new MS agar plates,
>allowed them to dry for three days, including leaving the hood on during the
>day and inverting the plates (things I had not done before).  I again
>surface sterilized the same 2 batches of C24 gen.background seeds (that had
>now sat at 4C for 17 days) and plated them out as before only this time I
>was careful to remove essentially all of the excess water (produced by
>placing the seeds, ~1000/150 mm plate, out with a pasteur pipette).  This
>new batch of plates was handled exactly as before and today is the sixth day
>and of the four plates I have harvested so far, each has about 80 to 90%
>germination efficiency.  The bottom line in this case, I believe, is that
>Arab. seeds can drown on agar plates.
>Joel A. Kreps
>Dept. of Biochem. and Mol. Bio.

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