Date of Award

2022

Publisher

North Dakota State University

Document Type

Dissertation

Degree Awarded

Doctor of Philosophy (PhD)

Program

Plant Pathology

Department

Plant Pathology

College

Agriculture, Food Systems and Natural Resources

Faculty Advisor

Zhong, Shaobin

Description

Plant transformation is a valuable tool for gene characterization and crop improvement. However, wheat transformation is greatly impacted by genotype dependency for successful regeneration of the transgenic plants. For improving disease resistance in wheat, targeting host genes for disease susceptibility has become one of the most attractive strategies. This study aimed to 1) identify the QTL associated with a genetic transformation-related trait (plant regeneration capability from calli derived from immature embryos); 2) targeting the wheat Tsn1 gene for susceptibility to tan spot disease, using CRISPR/Cas9 vector cassette delivered by Agrobacterium and particle bombardment mediated transformation; 3) targeting wheat TaHRC gene (encoding for reticulum histidine-rich calcium binding protein) at the Fhb1 locus involved in the resistance/susceptibility to Fusarium head blight (FHB) resistance, using haploid induction coupled with the CRISPR/Cas9 genome editing technology. Using phenotypic and genotypic data from a mapping population of 186 recombinant inbred lines derived from the cross between wheat cultivar Bobwhite and wheat line PI 277012, two QTL controlling plant regeneration capability in Bobwhite were detected on chromosome 1A and 6D. To target the Tsn1 gene in wheat, two and 30 T0 transgenic plants were generated from Fielder through the Agrobacterium-mediated transformation and Bobwhite by the biolistic transformation, respectively. However, no Tsn1 mutant was identified by ToxA infiltration on 1176 T1 transgenic plants. Previous studies revealed two allelic forms of the TaHRC gene with TaHRC-S controlling susceptibility and TaHRC-R resistance to FHB, and deletion of TaHRC-S renders wheat FHB resistance. To target TaHRC, pollens of the transgenic hybrid corn variety Hi-II with high expression of Cas9 and gRNA were used to pollinate emasculated spikes of wheat variety Dayn with TaHRC-S and TaHRC-R. After embryo rescue, of the 82 haploid plants screened, 12 plants were identified having mutations at the target sites of TaHRC-S allele and 2 plants at the target site of TaHRC-R allele. Doubled haploid plants are being generated from these gene-edited haploid plants and will be evaluated for FHB resistance. This study may facilitate our understanding of genetics of transformation-related traits and provide a novel approach for improving disease resistance in wheat.

Rights

NDSU policy 190.6.2

Rights Link

https://www.ndsu.edu/fileadmin/policy/190.pdf

Handle Identifier

https://hdl.handle.net/10365/33519

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