Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Target audiences are scientists, faculty, students, crops consultants, farmers, and other members of the public. Changes/Problems:Initial attempts to generate protoplasts from waterhemp cell suspension cultures have not been successful. Different enzymes for cell wall digestion are currently being tested. What opportunities for training and professional development has the project provided?The research specialist on this project was trained and mentored in weed genetics research methods including gene sequence analysis. Professional development for the project director and research specialist on this project included attendance at weed science seminars and participation in organized discussions. How have the results been disseminated to communities of interest?Research results were presented to farmers, crops consultants, and industry personnel at an extension weed science workshop, and herbicide resistance management recommendations based on knowledge of weed genetics were incorporated into an extension publication for dissemination to the public. Aspects of pesticide resistance, including herbicide resistance in weeds, was also incorporated into an invited book chapter. Research activities were described in classroom and online lectures to undergraduate and graduate student. What do you plan to do during the next reporting period to accomplish the goals?Research to develop a protocol for protoplast isolation in waterhemp cell suspension cultures will continue. Gene editing of the ALS gene will continue to be studied in yeast as a model organism, followed by waterhemp using protoplasts derived from cell suspension cultures.
Impacts
What was accomplished under these goals?
North Dakota has at least ten weed species that are resistant to herbicides, and control of herbicide-resistant weeds is estimated to cost $10 to $40 more per acre than standard weed control. In comparison, proactive resistance management strategies are estimated to provide long-term annual economic returns of $20 to $50 per acre. This weed genetics project is developing tools to both facilitate proactive resistance management through genetic diagnostics and to mitigate resistance using emerging genetic technologies. This project is responding to public need by investigating aspects of emerging genetic technologies for pest control that are not yet implemented and for which environmental safety aspects need to be researched. For weed control, these emerging technologies need to be tested under conditions that do not allow genetic material to escape the laboratory. This project has advanced the use of waterhemp tissue cultures that can safely be grown and studied without risk of seeds or pollen escaping containment. Immediate grower need was also addressed by assisting with the development of diagnostic tests for Palmer amaranth and also herbicide resistance in Palmer amaranth and waterhemp. Objective 1: Determine genetic aspects of herbicide resistance. DNA was successfully isolated from waterhemp callus tissue cultures using a cetrimonium bromide (CTAB) DNA extraction procedure. These DNA samples were used to analyze ALS gene sequences using polymerase chain reaction (PCR) amplification followed by direct Sanger sequencing. Sequence analysis confirmed that current waterhemp tissue culture lines have herbicide-susceptible ALS target-site gene sequences. This protocol can also be used in the future to identify ALS target-site resistance mutations in herbicide-resistant cell lines. Objective 2: Develop and utilize a tissue culture system for weedy Amaranthus species. Waterhemp cell suspension cultures were generated from callus tissue culture lines by placing small pieces of callus tissue into 25 ml of sterile liquid suspension media containing 3% sucrose, 2 mg/L 2,4-D as an auxin plant hormone, and 0.5 mg/L 6-benzylaminopurine (BAP) as a cytokinin plant hormone. Cultures were incubated at 25 C on an orbital shaker at 120 rpm, and sub-cultured weekly into fresh liquid media. One waterhemp cell suspension culture was used to evaluate response to the ALS-inhibiting herbicides thifensulfuron-methyl and imazethapyr. To perform these experiments, 12 ml of suspension culture was added to 12 ml of fresh media containing various concentrations of thifensulfuron-methyl or imazethapyr. Final concentrations of herbicide were 0, 1, 10, 100, and 1000 nM. All cultures were grown at room temperature in 125-ml flasks on an orbital shaker at 120 rpm. Culture growth was evaluated by reading OD600 in a spectrophotometer after 7-14 days, and the experiments were repeated. Both herbicides were found to inhibit waterhemp culture growth in a dose-dependent manner. The concentration of herbicide required to reduce growth by 50% was estimated to be approximately 500 pM for thifensulfuron-methyl and approximately 10 nM for imazethapyr. The conditions necessary to remove cell walls from waterhemp cells in suspension culture, in order to generate protoplasts, was investigated. Waterhemp cell suspension cultures were centrifuged to pellet the cells, and the supernatant was replaced with the following filter-sterilized enzyme solution: 0.55 M sorbitol, 0.6-0.8% cellulase R-10, 0.2% hemicellulase, and 0.2% pectinase, pH 5.8. Cells were incubated at room temperature or 30 C for 4-5 hours with gentle agitation, filtered, and washed 6-7 times with resuspension in 0.55 M sorbitol. Digestion of cell walls and the generation of protoplasts was observed by microscopy. Protoplasts were not successfully generated using this protocol. Objective 3: Evaluate the effects of CRISPR-based gene editing in the ALS herbicide target site. Yeast will be used as a model organism to perform initial ALS gene editing experiments. A pCAS plasmid to edit herbicide-susceptible yeast ALS to a resistant form has been prepared using PCR-based cloning, but has not yet been used for gene editing experiments.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2020
Citation:
Christoffers MJ, Sabba RP (2020) The problem of insecticide, fungicide, and herbicide resistance in agricultural crop pests. Pages 261-281 in Gustafson JP, Raven P, Ehrlich P, eds. Columbia, MO: University of Missouri Press
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Ikley J, Christoffers M, Dalley C, Endres G, Gramig G, Howatt K, Jenks B, Keene C, Ostlie M, Peters T, Robinson A, Thostenson A, Hatterman-Valenti H (2020) North Dakota Weed Control Guide, circular W253-20. Fargo, ND: NDSU Extension. 151 p
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