Date of Award

May 2015

Degree Type

Thesis

Degree Name

Master of Science

Department

Biological Sciences

First Advisor

Heather A. Owen

Committee Members

Douglas A. Steeber, Dazhong Zhao

Keywords

Arabidopsis, Cals5-2, DEX1, Exine, Pollen Development, Tex2-2

Abstract

The mechanisms behind the construction of the pollen wall are equally elaborate and mysterious. Previous studies primarily used sectioned tissue to elucidate the events involved in proper pollen development. This study proposed and evaluated a protocol for exposing developing microspores to be examined by Scanning Electron Microscopy (SEM). Utilizing this protocol, comparative analyses of the superficial features present at the early, middle, and late tetrad as well as at released microspore stages of the model plants Brassica rapa and Arabidopsis thaliana were conducted. The utility of the technique was then evaluated through the examination of three Arabidopsis pollen wall mutants at multiple developmental stages. The defective in exine formation 1, callose synthase 5-2 and thin exine 2-2 mutant lines are known to exhibit defective exine patterning. The dex1 mutant demonstrates abnormal sporopollenin deposition where aggregates form and coalesce randomly on the microspore surface (Paxson-Sowders et al. 1997). The cals5-2 mutant exhibits aberrant callose synthesis at the microsporocyte stage leading to deposition of globular aggregates and improper exine formation (Dong et al. 2005). The tex2-2 mutant was characterized by the presence of an extremely thin mature exine that lacks patterning (Dobritsa et al. 2011). In addition, this study aimed to further elucidate the role of DEX1 in pollen wall development through indirect immunocytochemistry of developing microsporocytes and tetrad microspores as well as understand how the distribution of this protein may differ in known pollen wall mutants. The methods developed in this study proved effective and allowed for observation of the entire surface of developing microspores with high resolution and three-dimensional qualities. These protocols provided new information in the characterization of pollen wall development in both normal and mutant plants. In addition, it revealed unique features and characteristics of sporopollenin deposition and exine development not readily observed in sectioned tissue nor explicitly described in conventional literature.

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