Development of improved soybean (Glycine max, L. Merill) germplasm through induced and targeted mutagenesis
Soybean (Glycine max L. Merill) is an important oil-producing crop under Fabaceae family and is utilized in various industries. With increasing demands for soybean oil and other soybean products, its production must be increased. Genetic improvement of the crop is important to meet the increasing demands for soybean. A new soybean mutant population was generated through induced Ethyl methanesulfonate (EMS) mutagenesis using the newly released germplasm, JTN-5203 (maturity group V)—a variety that can be cultivated in Tennessee was developed. Initially, EMS concentration was optimized to get the suitable concentration. Seeds were treated with different concentrations of EMS varying from 0 to 150 mM. The optimum concentration obtained was 60 mM and was used to treat bulk JTN-5203 seeds. A total of 1,820 M2 population was produced from 15,000 treated seeds. The M2 population were planted in the field and DNA were extracted from 6,400 individual plants. Extracted DNA were quantified, standardized, and pooled to create a mutant library for high throughput TILLING. Phenotypic variations were recorded in the M2 generation including changes in leaf morphology, plant architecture, and chlorophyll contents. Also, a different mutant germplasm generated through fast neutron (FN) were mass screened for resistance to two different herbicides: glyphosate and metribuzin, which conferred by EPSPS and psbA genes, respectively. With a total of 16,000 FN mutants screened for these herbicides, no herbicide resistant mutant was found. Aside from EMS and FN mutagenesis, site specific mutagenesis using CRISPR/Cas9 system was used in targeting psbA and PDS genes. Guide RNAs specific to the target genes were designed and cloned to a binary vector that contains the Cas9 gene. Soybean cotyledons were transformed with the Cas9 vector through Agrobacterium-mediated transformation. Initial observations showed some putative transformants on shoot induction media. The EMS mutant population will be used for further studies including screening for various traits such as herbicide tolerance, fatty acid profile and other important soybean agronomic traits. Beneficial traits from these mutants can be exploited for future soybean breeding programs. This germplasm can also be used for discovering novel mutant alleles and for functional gene expression analysis using reverse genetics tools such as TILLING.
Mary Jane C Espina,
"Development of improved soybean (Glycine max, L. Merill) germplasm through induced and targeted mutagenesis"
ETD Collection for Tennessee State University.