GENERATION OF A TRANSGENIC VECTOR FOR THE PURPOSE OF TRANSFORMING CHARCOAL ROT RESISTANCE INTO SOYBEANS (GLYCINE MAX)

Charcoal rot, caused by Macrophomina phaseolina, is among the most devastating fungal pathogens affecting G. max, particularly in drought-susceptible regions such as the midwestern United States. Traditional agricultural practices have included crop rotation and irrigation, which have proved ineffective or economically unfeasible. Charcoal rot infects over 500 other species of plants and can overwinter as dormant sclerotia which can germinate once soybeans are re-planted and infect young plants. As a result, transgenic solutions have offered a promising alternative for the development of disease-resistant soybeans. This thesis documents the construction of a transgenic vector to express BOZO, a glucanase endogenous to Glycine max, which has previously been shown to exhibit antifungal properties. Previous attempts utilized a constitutive CAMV35S promoter, which may have led to soybean seedling lethality due to BOZO overexpression. This project used an ethanol-inducible ethanol promoter to regulate BOZO expression, minimize phytotoxicity, and maintain antifungal properties. An unorthodox recombinant PCR program was utilized to assemble BOZO with a Nos terminator to prevent read-through expression of downstream genes and precise expression of BOZO. The construct was cloned into the pTRAkt-alcR-alcR-AH-Cel5A vector, placing it just upstream of ethanol-inducible AlcA-min35S promoter. Clone JB5, confirmed via Sanger sequencing and bioinformatic analysis, was the highest fidelity BOZO-Nos construct, free from mutations and frameshifts. This thesis illustrates transgene assembly and provides a framework for future research on the development of inducible, disease resistant plants. For disease-endemic regions, the successful generation of the vector has implications for the use of inducible-expression systems in plants, as well as a clearer path toward sustainable agricultural practices.