Honors College Thesis


Exploring maize pollen with microscopy: confocal, fluorescence and time-lapse imaging Public Deposited

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  • Pollen, the male product of the flower, is a crucial component in plant reproduction, as it transmits the sperm cells to the female structures, enabling fertilization and subsequent seed production. A major transition of the pollen life cycle, triggered upon pollination of female floral tissue, is from a dormant pollen grain to an actively growing pollen tube. Many of the genetic and cellular mechanisms that enable pollen to accomplish this transition, and thus successfully deliver the sperm cells, are not well understood. My thesis research encompassed a number of microscopic approaches to investigate this portion of maize pollen development. Maize is a useful model for this project, as pollen is readily collectible, can be germinated in vitro, and has a number of genetic tools available. First, I validated several fluorescent protein fusions, tagged either YFP (fluorescing yellow) or mCherry (fluorescing red), revealing internal structures of maize pollen (e.g., the cytoskeleton) via confocal microscopy. Notably, I showed that combination of a YFP-tubulin and a nuclear-localized mCherry fusion in the same pollen grain clearly revealed both sperm cells and the large vegetative nucleus. Using a different combination of fluorescent proteins, I showed that YFP-ROP2 fusion localized similarly to a cytoplasmic mCherry in the pollen tube, raising doubts about a hypothesis predicting ROP2 localization to the pollen tube tip. Second, I worked out conditions for using fluorescent dyes (calcofluor white, propidium iodide) to reveal pollen cell wall features. A new methodology needed to be devised to deliver dye to the dry pollen grain without inducing the hydration (rapid absorption of water) that triggers pollen tube germination. I found that pollen could be dyed without hydration by adding a minimum amount of propidium iodide to a large pollen sample suspended in paraffin oil. Finally, I used time-lapse microscopy to image the entire developmental transition: from dry pollen grain, through hydration, to opening of the pore and formation of the papilla (the first sign of cellular expansion), and finally the initiation of a rapidly growing pollen tube. Using quantitative analysis of growth rate, I tested the hypothesis that papilla formation via hydration is a separate process from active, rapid pollen tube growth. My data suggest that the two processes show growth at different rates, consistent with the hypothesis. The collected data help me propose a working model for maize pollen tube germination.
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