Theses and Dissertations at Montana State University (MSU)
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Item Natural variation in camelina nitrogen responses(Montana State University - Bozeman, College of Agriculture, 2023) Gautam, Shreya; Chairperson, Graduate Committee: Chengci Chen; Chaofu Lu (co-chair)Camelina (Camelina sativa L.Crantz) is an oilseed crop with the potential to be planted for biofuel production. It is crucial to select camelina genotypes with higher nitrogen use efficiency (NUE) so that the superior cultivar has higher crop productivity. To select genotypes of camelina that exhibit higher biomass yield and nitrogen use efficiency, two field experiments were conducted in 2021 and 2022 in Sidney, MT with different nitrogen regimes, low (unfertilized) and high (fertilized). Distinct projects were carried out, one of them emphasizing canopy area and normalized difference vegetation index (NDVI), and the other focusing on biomass yield and NUE. The experiments highlighted the response of camelina to nitrogen application and the variation among genotypes. The study identified canopy image analysis effectively differentiated the canopy size and growth rate of camelina genotypes under two nitrogen regimes, demonstrating the influence of nitrogen on camelina growth. The NDVI measurement proved to be useful in evaluating plant health and greenness, offering a time-saving and efficient approach. Additionally, some of the genotypes were identified that exhibited high canopy area, NDVI, and nitrogen use efficiency in both 2021 and 2022, providing potential for enhancing crop productivity. This study reveals the potential to use canopy area, NDVI for biomass yield and nitrogen use efficiency screening in camelina.Item Evaluating the genetic and phenotypic responses of Camelina sativa to heat stress(Montana State University - Bozeman, College of Agriculture, 2023) Smith, Brian Edward; Chairperson, Graduate Committee: Chaofu LuCamelina sativa (L.) Crantz is a low-input oilseed crop with a unique fatty acid profile in its seed oil. Camelina oil can be used in biodiesels, jet fuels, and industrial lubricants. Improving the abiotic stress tolerance of camelina is a crucial step for increasing agronomic viability. Climate change is threatening production of camelina with rising global temperatures and shorter growing seasons. Elucidating the phenotypic and genetic responses to high temperatures is essential for successful breeding of heat tolerant camelina varieties. Three experiments were conducted to understand these responses. Two genotypes, Suneson (MT5) and Pryzeth (MT102) were exposed to a transient 14-day heat stress during the reproductive stage and evaluated for agronomic and seed quality traits along the main stem. Next, a mapping population consisting of 257 recombinant inbred lines (RILs) were grown under the same temperature regimes for 14 days beginning at the onset of the reproductive stage. Finally, reproductive tissues undergoing heat stress from two genotypes with contrasting heat responses, RIL23 and RIL167, were examined with RNA sequencing, and the phenotypes along the main stem were compared. From the phenotype evaluation, both MT5 and MT102 were significantly impacted by heat. Both genotypes experienced reductions in seed and pod size, seed weight, and total oil contents. As reported in other oilseed crops, camelina is negatively affected by heat, characterized primarily by lower yield and reduced oil content. The QTL analysis identified several key gene regions with co-located traits on chromosomes 8, 10, and 12. This demonstrates the ability to identify heat-responsive gene regions via phenotyping along the main stem. The transcriptomes of RILs 23 and 167 contrasted in both sampled tissue types, with RIL23 appearing more responsive to heat. Phenotypic analysis of these genotypes confirmed the transcriptional differences, as RIL23 was more resistant for several traits associated with fertility. These studies provide resources and protocols for future studies that may assist in improving the heat-tolerance of camelina.Item Mapping quantitative trait loci to understand seed size variation in Camelina sativa(Montana State University - Bozeman, College of Agriculture, 2019) King, Kevin Allen; Chairperson, Graduate Committee: Chaofu LuCamelina sativa (L.) Crantz is an emerging Brassica oilseed crop. Camelina oil is high in polyunsaturated C18-fatty acids and its uses range from bio-fuels and bio-lubricants to an animal feed additive and cooking oils. A major breeding objective for camelina is to develop varieties with increased seed size. Understanding the genetics behind seed size variation would help breeders develop varieties that are more robust, easier to plant and harvest, better for oil processing, and could increase oil yield. For this study, a genetic linkage map was created and quantitative traits loci (QTL) were identified for eight agronomic traits using a bi-parental recombinant inbred population created between the two Camelina varieties: 'Suneson,' which has an average seed area of 1.35 mm2, and 'Pryzeth' with an average seed area of 2.24 mm2. Field trials were conducted in 2017 and 2018 in both dryland and irrigated treatments in Bozeman, Montana. Significant QTL were discovered for seed size and other agronomic traits measured, including flowering time, pod size, seed weight, and oil content. The results of this study could lead to marker-assisted breeding for varieties better adapted to modern agriculture.Item Modification of seed fatty acid composition by CRISPR/Cas9 targeting the fatty acid elongase1 in Camelina sativa(Montana State University - Bozeman, College of Agriculture, 2018) Ozseyhan, Mehmet Erkan; Chairperson, Graduate Committee: Chaofu LuThe low-input oilseed crop Camelina (Camelina sativa (L.) Crantz) is known for its high omega-3 (18:3) content, short growth season, and facile gene transformation. Camelina mostly contains unsaturated fatty acids, however its fatty acid composition needs optimization depending on the end uses, for example reduction of unsaturated fatty acid to use as biodiesels, or enhancing omega-3 fatty acid content to use as nutritional supplements. Very long chain fatty acid (VLCFAs, C20-C24), are undesirable for human consumption, and their accumulation in seed oil also needs to be diminished. VLCFAs are produced by the catalytic action of fatty acid elongase1 (FAE1), and Camelina contains three alleles of FAE1 genes (FAE1-A, FAE1-B, and FAE1-C) due to its allohexaploid nature. Recently, VLCFAs in camelina were decreased along with polyunsaturated fatty acids (PUFAs) using the RNA interference (RNAi) technology. A low VLCFA line was also isolated from ethyl methanesulfonate (EMS) induced mutants. Sequencing results indicated that FAE1-B gene was mutated and resulted in 60% reduction in VLCFAs, but other two FAE1 copies were presumably still active in the mutant. To address this multipleallele-knockout-at-once problem, here I investigated the effect of knocking out three alleles of FAE1 genes using CRISPR technology with egg cell-specific Cas9 expression. Due to the germline mutation, homozygous FAE1 knockout mutants were successfully created in a single generation. VLCFA accumulation was significantly decreased from 22% of total fatty acids in wild type to less than 2% in transgenic plants, and the C18 unsaturated fatty acids were improved since 18:1 substrates were diverted to desaturation pathway, rather than elongation. Analysis of the fatty acid composition of four transgenic generations indicated that the mutations that cause low VLCFA genotype were heritable. There was no significant difference observed in seed weight, plant height, total oil content, and seed germination in Cas9-induced mutants compared to the wild type. This study showed that polyploid Camelina can be modified rapidly and effectively through CRISPR/Cas9 to achieve desired fatty acid composition.Item The fatty acid elongase of Physaria fendleri increases hydroxy fatty acid accumulation in transgenic Camelina(Montana State University - Bozeman, College of Agriculture, 2013) Snapp, Anna Rose; Chairperson, Graduate Committee: Chaofu LuPlant oils containing hydroxy fatty acids (HFA) are desirable for a wide variety of applications including lubricants, plasticizers, surfactants, polyesters, paints, sealants, biodiesel, and more. Due to unfavorable agronomic attributes of natural accumulators such as castor and lesquerella, many efforts have been made to produce hydroxyl fatty acids in crop plants. The hydroxy fatty acid synthesis pathway has been extensively studied and key genes such as the castor fatty acid hydroxylase, RcFAH, have been discovered. However, insertion of the RcFAH gene into various Arabidopsis backgrounds under the control of seed specific promoters failed to result in high accumulation of the desired HFA products, highlighting a need for more research to uncover additional constraints and factors affecting the fluxes involved with the accumulation of these unusual fatty acids in seed oil. In this study I investigated the effect of co-expressing a fatty acid elongase gene, LfKCS3, from Lesquerella (Physaria) fendleri along with the castor hydroxylase gene, RcFAH, on accumulation of hydroxyl fatty acids in seed oil of the crop plant Camelina sativa. On its own, wild type camelina contains no hydroxy fatty acids but insertion of the RcFAH gene results in accumulation of around 15% HFA in transgenic camelina, however, addition of the LfKCS3 gene resulted in a significant increase in very long chain 20-carbon hydroxyl fatty acids from <2% to 8%; total hydroxyl fatty acids also increased from 15% to 22% in the highest accumulating lines. The presence of the LfKCS3 enzyme effectively increased total HFA levels at all stages of oil accumulation in developing seeds while also decreasing the amount of these fatty acids left on the phospholipid, phosphatidylcholine. This combination of increased 20- carbon and total hydroxyl fatty acid accumulation along with the decreased HFA levels in phosphatidylcholine indicates that the LfKCS3 gene helps to enhance the flux of HFA out of phosphatidylcholine for incorporation of HFA into triacylglycerol, aiding in relief of the metabolic bottleneck for engineering economically viable levels of these fatty acids in oilseed crops.