Generation and molecular validation of transgenic soybean (glycine max) lines targeting MRP genes via CRISPR/Cas9

Date

2025-8-15

Author

Söbe, Damla

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Hidden hunger, a vital public health issue affecting billions of people worldwide, is a significant contributor to iron deficiency-related anemia. There are several methods to address this crisis, such as biofortification, a process that increases the micronutrient content of staple crops. However, naturally occurring anti-nutrients in iron-fortified foods can hinder Fe absorption in the human digestive system. Soybean (Glycine max), the most widely produced oilseed, stores a significant amount of Fe within ferritin in its seeds. However, soybean seeds also naturally contain phytate, an anti-nutrient that reduces Fe absorption. Previous mutation breeding efforts aimed at reducing phytate content in soybeans resulted in lines with low phytate content, but they encountered issues with reduced yield and germination. In contrast, studies targeting mutations in some multidrug resistance proteins (MRPs) responsible for phytate storage have demonstrated that these mutations do not negatively affect yield and germination in various plants. Therefore, in this thesis MRP genes of soybean cultivar Arısoy were targeted through CRISPR-Cas9-based gene editing. In contrast to previous mutation breeding efforts, this thesis simultaneously targeted three GmMRP genes (GmMRP3, GmMRP13, and GmMRP19) using CRISPR/Cas9. The thesis includes the design of two different gRNAs targeting the conserved TMD1 and NBD1 domains of the three MRP genes, cloning and validation of these gRNAs into a CRISPR-Cas9 expression vector, optimization, validation and application of Agrobacterium-mediated transformation of soybean, tissue culture optimization, and verifications of transgenic T0 plants through PCR and T7E1 assay.

Subject Keywords

Biofortification, Soybean, Multi-drug Resistance Gene, Phytic acid, CRISPR/Cas9

URI

https://hdl.handle.net/11511/115674

Collections

Graduate School of Natural and Applied Sciences, Thesis