Mechanical
separation of aborted and normal pollen grains
--Gurjal Madhavi Reddy and Ed Coe
It is known from Bryan Kindiger's work
(Kindiger, Beckett and Coe, manuscript in preparation) that heterozygous
and homozygous B-A translocations produce two types of pollen grains -
a starch-filled normal class and a smaller starchless aborted class that
is different for each translocation. Separation of aborted and normal grains
would pave the way for further work on the haploid genome. Separation of
aborted pollen and normal pollen was done using a series of sieves with
openings 297, 210, 177, 150, 125, 106, 88, 62 and 53 u. The pollen obtained
from hypoploids as well as heterozygous B-A translocations, containing
50% and 20-25% aborted pollen grains respectively, was allowed to pass
through the series of sieves placed in decreasing order of their pore size.
Sifting was done by gently rocking the sieves allowing the pollen grains
to separate on the basis of size.
It was observed that separation of aborted
and normal pollen was better with pollen dried at room temperature than
with vacuum dried pollen or fresh pollen. Separation of aborted and normal
pollen is good for pollen that has been left at room temperature for 3-4
hrs after collection as the aborted starchless grains shrivel faster than
the normal starch filled pollen grains, increasing the difference in size.
Further removal of normal pollen from the fraction containing 85-90% aborted
pollen obtained upon sifting was achieved by subjecting the pollen mixture
to vibrations using a seed counter. The last fraction so collected contained
95% aborted pollen grains. Not all of the aborted pollen grains present
in the original starting fraction were recovered by this method, but the
separated fraction had 95-97% aborted pollen grains, good enough separation
to expect to get DNA for further analysis.
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