dark germination

jkreps at biochem.umass.edu jkreps at biochem.umass.edu
Mon Jan 29 13:07:05 EST 1996

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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