preparation of permanent slides of pollen grains by Acetolysis method and study of different pollen characters: study of variation pattern in different taxa.
Answers
1) Collection and preservation of pollen material: It involves collection of anthers from mature flower buds just before opening and anthesis of flowers by using clean forceps and preserving the anthers or sometimes anthers with a portion of filament of stamens in clean glass vials with 70% glacial acetic acid. Some palynologists recommend 70% ethyl alcohol for preserving the anthers in place of glacial acetic acid. In this condition, the anthers packed with pollen can be stored indefinitely.
2) Anthers are transferred to a centrifuge tube and washed thoroughly with distilled water. The anthers are crushed with a glass rod and the mixture is filtered through cheesecloth or a sieve (c. 180 ^m mesh). The lower fraction is recollected in the centrifuge tube.
3) Freshly prepared acetolysis mixture consisting of acetic anhydride and concentrated H2SO4 (sulphuric acid) in the ratio of 9:1 was poured in the centrifuge tube containing washed pollen. Care has to be taken to add conc. H2SO4 to acetic anhydride drop by drop to avoid excess heating. The acetolysis solution reacts violently with water. Glacial acetic acid washes are necessary to replace water with acetic acid, which does not react violently with the acetolysis solution. Although acetolysis procedure varies from palynologist to palynologist, the following procedure seems to be most suitable. From the first to last acetic acid wash, caution has to be taken so that no water comes in contact with the solution. Only distilled water has to be used for washes to avoid any contamination.