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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