Dissertations and Theses @ UNI

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Open Access Thesis

Keywords

Arabidopsis thaliana--Genetic engineering; Herbicide resistance;

Abstract

Plant genetic engineering requires a marker gene to differentiate transformed cells. To test the csr1-2 allele as a selectable marker for the shoot apical meristem (SAM), we cloned a genomic fragment from Arabidopsis thaliana mutant CS3102 which contained the coding sequence and promoter. We then evaluated the utility of this allele on two model systems, Arabidopsis and tobacco, with soybean as the positive control. Initial dose-response experiments were conducted using a seedling plate assay to determine the lethal dose of imazapyr. For the mutant Arabidopsis thaliana CS3102, a concentration of 500 μM imazapyr was required to overcome the resistance phenotype and kill 100% of the seedlings. At the lower dose of 50 μM, the herbicide killed all of the tobacco and soybean seedlings. Wildtype (WT) Arabidopsis thaliana Columbia seedlings were all killed at an even lower dose of 5 μM. Next, we used biolistics to target the SAM. Experiments using the visible marker gene β-glucuronidase showed transient expression in the epidermal cells of the soybean SAM, indicating our methods were capable of delivering transgenes. Experiments with csr1-2 did not identify any transformed plants, suggesting this allele is of limited utility for SAM biolistics of Arabidopsis and tobacco. Selection with imazapyr did not produce transformants after testing 305 soybean seedlings, 670 tobacco seedlings, and 1,000 Arabidopsis thaliana Columbia seedlings. Future research to replace the native Arabidopsis thaliana promoter with a constitutive promoter could improve the utility of csr1-2 as a selectable marker by increasing expression of the resistance gene.

Year of Submission

2020

Degree Name

Master of Science

Department

Department of Biology

First Advisor

Marek Sliwinski, Chair, Thesis Committee

Date Original

7-2020

Object Description

1 PDF file (ix, 86 pages)

Language

en

File Format

application/pdf

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