Dissertation
IDENTIFICATION AND CHARACTERIZATION OF HERBICIDE TRAITS IN WHEAT AND BARLEY USING TRADITIONAL AND CRISPR/CAS9 TECHNIQUES
Doctor of Philosophy (PhD), Washington State University
01/2017
Handle:
https://hdl.handle.net/2376/111923
Abstract
Herbicide resistance and susceptibility traits are important tools for weed management in crops. However, the number of known genetic traits that confer resistance or susceptibility to herbicides in wheat are limited, justifying the investigation of potential new traits. Germplasm screening was completed on diverse wheat populations for the purposes of identifying new herbicide traits. Potential herbicide resistance traits were discovered for clethodim, (SelectMax®), and indaziflam (Alion®). Separately, a cyctochrome P450-based herbicide sensitivity trait in barley (Hordeum vulgare L.) was investigated for the herbicide fenoxaprop-p-ethyl. CYP71C115, is here characterized and identified as the P450 involved in fenoxaprop-p-ethyl metabolism and thus FXP1 sensitivity to fenoxaprop-p-ethyl. The FXP1 reaction is also characterized. Specific metabolism of fenoxaprop-p-ethyl, and not other ACCase herbicides was demonstrated. Fenoxaprop-p-ethyl and its metabolites were examined by LC/MS after application on wild-type barley cv. Morex, and on a Morex derived FXP1 mutant which lacks CYP71C115. Differential fenoxaprop metabolism was confirmed. Also, a general procedure for CRISPR/Cas9 gene editing is outlined in wheat, targeting CYP71C115 orthologs. In total, 2810 calli were co-cultivated with Agrobacterium tumefaciens, which resulted in regeneration of 12 plants. No transgene or gene edits were detected in the regenerated plants. Possible contributing factors to failure are likely an insufficient number of co-cultivated calli, lack of sufficient specificity in the designed CRISPR guide RNA, and difficulties experienced with antibiotic selection intensity. Further work will be necessary to optimize CRISPR/Cas9 gene editing.
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Details
- Title
- IDENTIFICATION AND CHARACTERIZATION OF HERBICIDE TRAITS IN WHEAT AND BARLEY USING TRADITIONAL AND CRISPR/CAS9 TECHNIQUES
- Creators
- Caleb Carbine Squires
- Contributors
- Ian C Burke (Advisor)Arron H Carter (Committee Member)Michael M Neff (Committee Member)Karen A Sanguinet (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Department of Crop and Soil Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 113
- Identifiers
- 99900581427201842
- Language
- English
- Resource Type
- Dissertation