An investigation into Ploidy and Polyploidy in the Sphagnum genus, a Screening of Micropropagated Material and Wild Material

Dronfield, Samuel Peter (2025) An investigation into Ploidy and Polyploidy in the Sphagnum genus, a Screening of Micropropagated Material and Wild Material. Masters by Research thesis (MSc), Manchester Metropolitan University.

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Abstract

Ploidy and polyploidization plays an essential role in the evolutionary diversification of plants. An evolutionary trait that not only continues the plants habitats and functions. but can initiate the rebuilding of habitats, biodiversity and sequester atmospheric carbon. The Sphagnum genus is no exception to this and perhaps one of most understood when it comes to its genetic advantages. Investigations of Sphagnaceae ploidy and genome sizes using flow cytometry are predominantly featured in studies analysing a wide range of bryophytes. Studies analysing Sphagnaceae specifically are few and protocol of extraction and analysis tend to be nondisclosed. The potential of polyploidy and its presence in non-hybridized Sphagnum has both evolutionary and climatic benefits, although it’s theorised that natural polyploidy is not apparent throughout Sphagnum species. This study was devised to firstly, to create a competent protocol incorporating nuclei extraction and flow cytometry that would be relatively easy to follow and efficient. Secondly the study wanted to gain a wider understanding of ploidy levels in micropropagated material for better application to Sphagnum farming practices. Thirdly the potential of synthetic polypoidal inducement using a known herbicide was to be investigated to attempt to achieve a polypoidal state in diploid species or a general increase in ploidy in haploid species. Lastly, species from various origin where to be assessed to distinguish regional difference in species across Europe. This study offers a comprehensive protocol including nuclei extraction, flow cytometric preparation, and an acquisitional gating strategy to ascertain Sphagnum nuclei with low background debris. Nine species of micropropagated Sphagnum species where analysed and recorded. Standards have been outlined in haploid and diploid species which can be used as a reference when analysing Sphagnum material for future studies A wide screening of oryzalin induced material at various thresholds of higher oryzalin (120 μm) displayed characteristics initiating large fluctuations in in relative fluorescence when compared to a non-induced standard indicating that oryzalin did influence diploid specimens. Further subculturing, secondary oryzalin inoculation trials, and cell sorting techniques, could result in a whole genome duplication from diploid to polyploid. Regional diversity was seen in S. fallax with a diploid variant identified from a German source but may indicate fixed heterozygosity due to the lack of natural polyploid populations. This study provided the first whole genome duplication (GWD) using oryzalin In S. squarrosum resulting in a diploid variant.