Date of Award

2020

Degree Type

Thesis

Degree Name

Master of Science in Biological and Environmental Sciences (MSBES)

Specialization

Cell & Molecular Biology

Department

Cell & Molecular Biology

First Advisor

Albert P. Kausch

Abstract

Agrobacterium-mediated transformation of sorghum (Sorghum bicolor) requires minimally 9-months from wild type immature embryo explants to T0 seeds. More efficient methods for sorghum transformation are necessary to conduct routine transgenesis for genome editing purposes in this important crop. With this in mind, there were two primary objectives to this thesis. The first was to evaluate and possibly improve upon methods for characterizing putative transformants once produced through Agrobacterium-mediated transformation of sorghum. The second was to evaluate and possibly improve upon transformation efficiencies in sorghum using an available Cas9 construct that would provide, long-term, a platform in sorghum for gene editing purposes. The first objective was addressed by evaluating a previously generated transgenic sorghum line designed to improve overall grain yield through the introduction of a maize silkless gene (sk1) construct. The maize silkless gene was first used in maize by Hayward et al., 2016 to produce transgenics that conferred feminization on maize male flower by down regulating the jasmonic acid synthesis pathway. The same construct SK1ΔSVL:Citrine:SVL used in Hayward et al., 2016 was used to transform sorghum based on the understanding of high homology between the sorghum and maize genomes. Through a series of analyses, the presence and expression of SK1ΔSVL:Citrine:SVL was confirmed, but the predicted phenotype of flower feminization and improved yields was not observed in T1 transgenic sorghum lines. The second objective was designed to introduce into sorghum a vector containing Cas9 to test the stable expression of Cas9 for genome editing in transgenic lines. The first step towards this objective was to generate and characterize the required transgenic lines and appropriate controls. The pNG111-ZmUbi::TaCas9_PvUbi::1GFP construct was used to integrate TaCas9 into the wild type BTx430 sorghum genome and molecularly characterize these events. The pNG108PvUbi::1GFP construct served as a negative control for pNG111 since it is lacking the TaCas9 cassette. Both constructs contain constitutively expressed mGFP which is detected as a visible reporter, and the bar gene served as a selectable marker conferring resistance to the herbicide bialaphos. In addition, transgenic lines for both constructs were molecularly characterized by PCR, Southern blot analysis, and the ‘paint assay’ to detect the functional expression of the bar gene. Functional analysis of the stably integrated TaCas9 will be conducted in future studies. By analyzing the newly developed transgenic sorghum lines with constitutively expressed TaCas9, we hope to contribute to the development of a new platform for genome editing in sorghum.

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