Increasing Total Lipid Content in Pennycress (Thlaspi Arvense L.) Utilizing CRISPR-Cas9 and Transgenic Approaches
Date of Award
Master of Science (MS)
School of Biological Sciences
Thlaspi arvense (pennycress) is a member of the Brassicaceae family currently under development as an oilseed winter cash cover crop for the U.S. Midwest. Planted in the fall at the time of corn or soybean harvest, pennycress establishes vegetative leaves before going dormant for the winter. Pennycress plants then flower in early spring and mature in mid May to early June in time for seed harvest followed by planting soybeans or corn. Hence, pennycress along with corn and soybeans can constitute e.g. a corn-pennycress-soybean rotation resulting in the production of three crops in two years. The winter annual life cycle of pennycress along with its relatively small diploid genome and ease of mutagenesis provide the foundation for rapidly domesticating pennycress into a new oilseed winter cash cover crop called CovercressTM to be used for a variety of fuel, food, and feed purposes. Wild-type species of pennycress, similar to its Brassica relatives, rapeseed and canola (Brassica napus, Brassica rapa, and Brassica juncea), carinata (Brassica carinata), and camelina (Camelina sativa L.), produce high levels of seed oil and protein. On average, wild pennycress seeds produce about 34% oil in the form of triacylglycerides (TAG) and 20% protein (seed dry-weight basis). Here, we generated and characterized loss of function mutations in three Biotin Attachment Domain-Containing (BADC) genes (BADC1, BADC2, and BADC3) as well as in LDAP-Interacting Protein (LDIP), utilizing CRISPR-Cas9 gene editing to knockout putative repressors of fatty acid biosynthesis (the BADCs) and of lipid droplet formation (LDIP). The overall goal of this work was to test genetic changes to pennycress that we hypothesized would result in increased seed oil content without compromising plant fitness including seed yields. We also generated transgenic pennycress lines designed to overexpress the FATTY ACID EXPORT1 (FAX1) gene, which in Arabidopsis thaliana has been shown to positively regulate fatty acid biosynthesis. No significant differences in total seed triacylglyceride amounts, compared to wild type, were observed in the various badc1, badc2, and badc3 single mutants generated, in a badc1 badc3 mutant allele combination which is likely partial loss of function, in ldip knockout alleles, or in FAX1 overexpression mutant lines. Some of the CRISPR-induced badc3 mutant lines had associated stunted growth and maturation delays, the basis of which remains to be determined. While larger lipid droplets were observed in ldip157 allele seeds, no increase in triacylglyceride content was observed. FAX1 overexpression lines showed an increase in FAX1 gene expression but no increase in seed oil content. Some of the badc and ldip mutant lines including mutant combinations were determined to be CRISPR construct-free and are undergoing field testing to determine if the seed oil content mirrors that of plants grown in growth chambers. The generation of these mutant resources and initial genotypic and phenotypic characterizations sets the stage for deciphering how fatty acid biosynthesis regulation differs in pennycress compared to other Brassicas, and how genetic manipulations may increase seed triacylglyceride levels thereby making Covercress (domesticated pennycress) a more profitable oilseed cash cover crop.
Marchiafava, Damiano, "Increasing Total Lipid Content in Pennycress (Thlaspi Arvense L.) Utilizing CRISPR-Cas9 and Transgenic Approaches" (2021). Theses and Dissertations. 1559.
Imported from Marchiafava_ilstu_0092N_12071.pdf