A new enzyme­based method for the treatment of fragile pollen grains collected from herbarium material

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Abstract
Although the majority of systematic palynologists use Erdtman’s acetolysis method or the slightly modified method of Reitsma for the preparation of pollen grains for LM and SEM observations, these methods have some major drawbacks. Especially within monocots, pollen grains are often thin­walled, and therefore tend to collapse even after a mild acetolysis, as experienced, for instance, in Cyperaceae and Dioscoreaceae, among other groups. Very satisfying results often are obtained by the use of fresh material. Unfortunately, this is not available in most cases, especially when working on tropical taxa—hence making herbarium material indispensable. We present a comparison between three major methods for the treatment of fragile pollen (KOH/CPD­treatment, glutaraldehyde/CPD­treatment, and a mild acetolysis) and introduce a new enzyme­based method using cellulase and pectinase. All four methods are tested on three different monocotyledons of three different orders: Allium ursinum L. (Liliales), Asparagus officinalis L. (Asparagales), and Tamus communis L. (Dioscoreales). The properties and results of each method are then critically assessed. The new method yields satisfying results especially on the hydration state and shape of the pollen grains.

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