Plant Science & Landscape Architecture Theses and Dissertations
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- ItemRE-RO(O/U)TING: Reconfiguring mobilities and materialities through the design of a green-blue infrastructure corridor in Baltimore, Maryland(2022) Martin, Bryn; Ellis, Christopher; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This thesis engages with Ecological Urbanism through a literature review and design project. Through a literature review which reads ecological urbanism with other contemporary social theory, the thesis raises a question: how might an approach centering the materiality of landscape and its relation to mobility inform the interdisciplinary work of translation and interpretation which is central to ecological urbanism? This approach is examined through a design project examining the landscape of the lower Jones Falls, a small, culverted urban river. The project profiles some of the past and present mobilities and materialities shaping this urban environment. These observations inform a design project envisioning a daylighting of the culverted Jones Falls as a focal intervention in the reimagination of an urban expressway corridor as a twenty-first century ecological mobility corridor: a landscape of green and blue spaces, ecological infrastructure multimodal streetscapes, and a reinvigorated public realm.
- ItemDEVELOPING AND EXPANDING CRISPR-CAS PLANT GENOME ENGINEERING SYSTEMS(2022) Malzahn, Aimee Alyssa; Qi, Yiping; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In order to advance plant biology and speed up crop breeding, researchers have used genome engineering tools in their research. Genome engineering with CRISPR has revolutionized agriculture by providing an easy, fast, and accessible tool to induce desirable mutations. This thesis works on addressing problems in the application of CRISPR for plant genome engineering. CRISPR systems are adopted from bacterial immune systems and consists of a Cas endonuclease and a guide RNA (or crRNA). Cas variants have different characteristics and exploring natural variants can provide systems with enhanced or new applications. The first aim in this thesis is to demonstrate novel LbCas12a for genome editing in Arabidopsis. To overcome the temperature sensitivity of LbCas12a, a heat treatment regime was developed. In order to expand LbCas12a use beyond genome editing, a transcription repression system was developed and used successfully for multiplexed repression of two homologs of EDS1. Two crRNA processing systems were compared, and results suggest that either can be used successfully in Arabidopsis. The second aim is to improve Cas9 and Cas12a editing outcomes by creating Cas-effector fusions. Cas9 and Cas12a were fused with six different exonucleases and compared at three targets in rice protoplasts. Several Cas-exonuclease fusions resulted in an increase in editing efficiency and the production of larger deletions. The Cas-exonuclease fusions’ editing efficiency differed between Cas9 and Cas12a, along with the deletion profile. Additionally, chromatin modulating peptides were fused to Cas9, which resulted in higher editing efficiency without altering deletion profiles. These engineered Cas proteins can be used to create unique editing outcomes, and paired with an increased editing efficiency, could be used to target difficult-to-edit target sites for gene knockout and cis-regulatory elements for fine-tuning gene expression. In summary, this work explored new Cas variant LbCas12a, developed multiplex gene repression systems, and compared engineered fusion Cas9 and Cas12a proteins for increased editing efficiency and larger deletions. The developed and improved CRISPR systems expand the number of available targets, improve efficiency, demonstrate novel editing outcomes, and enable multiplexed transcriptional regulation in plants.
- ItemDEVELOPING AND IMPROVING CRISPR-BASED GENOME EDITING TECHNOLOGIES IN PLANTS(2022) Sretenovic, Simon; Qi, Yiping; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Earlier genome editing technologies were developed based on programmable nucleases including zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), both requiring tedious protein engineering. By contrast, clustered regularly interspaced short palindromic repeats (CRISPR) systems, such as CRISPR-Cas9, has revolutionized the genome editing field in the past decade due to ease of guiding Cas9 endonuclease to the target site by programmable guide RNAs. However, not every target site can be edited due to Cas9 endonucleases’ recognition of so-called protospacer adjacent motif (PAM) when binding to the target site. For example, the PAM for the widely used SpCas9 is NGG (N=A, C, T or G). This drastically limits targeting scope in the genomes. Thus, researchers have developed engineered Cas9 variants recognizing more relaxed PAMs and tested them in mammalian cell lines. Repair of Cas9 endonuclease-induced double strand breaks through non-homologous end joining (NHEJ) DNA repair pathway typically generates unspecified insertions and deletions, which is a missed opportunity for introducing precise edits. To confer precise genome editing, CRISPR-Cas9 derived base editors and prime editors have been developed. In this work, expanding the plant genome editing scope with engineered Cas9 variants, improving precise cytosine and adenine base editing in plants as well as demonstrating prime editing in plant cells were pursued. The technologies were tested in the model crop, rice, in transiently transformed protoplasts and stably transformed T0 lines. Findings suggest that engineered Cas9 variants can drastically expand the targeting scope for targeted mutagenesis and base editing in plants. Additionally, newer genome editing technologies such as base editors and prime editors can be applied in plants to achieve precise genome editing with varying efficiencies. These validated and useful CRISPR-Cas9 genome editing toolkits have been deposited to the public vector depository, Addgene. Adoption of these genome editing technologies by plant scientists and breeders will enable basic research discoveries and accelerate breeding of next generation crops, ensuring global food security amidst climate change and increasing global population.
- ItemInvestigating the hyperdiversity of fungal endophytes in wild Rubiaceae tropical plants and coffee plantations.(2022) Castillo Gonzalez, Humberto; Yarwood, Stephanie A; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fungal endophytes are an essential component of a plant’s microbiome, their effect spreads to fitness, disease dynamics, stress tolerance, water acquisition and nutrient uptake. Plant ecosystems, from natural forest to plantations bear the indelible signature of its presence. The current investigation was designed to understand the diversity of endophytes in the Rubiaceae family, in plants associated to natural and managed ecosystems. The effect of location, leaf developmental stage, tissue type, host genotype, and anthropogenic interference was evaluated through amplicon sequencing. Costa Rica served as base for the sample collection. Leaves and sapwood from a variety of tropical plant species were collected in old-growth natural forests and foliar tissue from domesticated coffee plants were sampled in two plantations under different management. Fungal diversity was assessed by metabarcoding using the ITS2 nrDNA region fITS7 – ITS4, and library sequencing was completed by Ion Torrent. We identified a hyperdiversity of endophytes inhabiting these plants and were able to isolate a total of 659 fungi from coffee leaves. This investigation provides relevant information about overall community composition, the ecological drivers of community assemblage and the characteristics of the fungal endophytic communities, including potential interactions among the identified taxa. Endophytes may harness the potential to transform agriculture and conservation science, however we currently lack the knowledge to engineer microbial communities through breeding or management. It is essential to continue the efforts on understanding community functions and dynamics, and how host, endophyte interactions, and other ecological and human- related mechanisms influence their diversity in both forest species and agronomically important crops.
- ItemMULTIFACETED APPROACHES TO CONTROL FUSARIUM HEAD BLIGHT IN WHEAT: GENETIC MAPPING, MECHANISTIC STUDIES, AND FUNGICIDE EFFICACY ANALYSES(2022) Singh, Lovepreet; Rawat, Nidhi; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fusarium Head Blight (FHB), caused by the fungal pathogen Fusarium graminearum, is the second most important disease of wheat globally. This doctoral research contributes toward improving the two most widely used FHB management strategies: development of resistant cultivars and the use of fungicides. Given the quantitative nature of FHB resistance, pyramiding of multiple quantitative trait loci (QTLs) is required to develop resistant cultivars. Marker-assisted selection using diagnostic Kompetitive Allele-Specific PCR (KASP) assays is a cost and time-efficient approach. Toward this end, a previously identified resistance QTL from a moderately resistant soft red winter wheat cultivar Jamestown located on chromosome 1B was genetically mapped and diagnostic KASP assay was developed for it. Furthermore, a robust gene specific KASP marker was developed and validated for Fhb1, the most widely used QTL for resistance against FHB. Previously, a Pore-forming toxin-Like (PFT) gene was identified as the major underlying gene for Fhb1-mediated resistance in wheat. In the present research, functional characterization of PFT was done toward improving the understanding of the molecular mechanisms of FHB resistance. Ectopic expression of PFT in Arabidopsis conferred a broad-spectrum resistance against multiple fungal pathogens. PFT protein was purified from heterologous expression in Nicotiana benthamiana and used for various bioassays to elucidate the biological function of its individual domains. The experimental evidence supported our working hypothesis of PFT’s atypical mechanism of resistance. Working on the chemical control options for FHB, the efficacy and appropriate timing of application was investigated of a newly recommended fungicide for FHB-‘Miravis Ace.’ Overall, the knowledge and resources developed in this research would contribute towards improved integrated disease management of FHB in wheat.