Progress 07/15/19 to 03/14/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:The major problem in approach was an inability to regenerate rice shoots from the bombarded explants. This obviously hindered our ability to determine whether the proposed novel approaches to drive plastid transformants toward homoplasmy would in fact be successful. What opportunities for training and professional development has the project provided?The project provided experience for scientists and technicians to develop tissue culture protocols and to learn about plastid transformation technology, and provided an opportunity for the scientists to present their work to the internal Benson Hill R&D team. How have the results been disseminated to communities of interest?Results and ongoing experimental work were presented to the Benson Hill R&D group. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Multiple vectors were designed, constructed, and used for bombardment of rice immature embryos and other explants. The resulting rice tissue was monitored for the presence of GFP-derived fluorescence based on the presence of a GFP-aadA fusion gene in the transformation vectors. While multiple callus pieces exhibited what appeared to be GFP-derived fluorescence, none of these calli were able to generate shoots that could be screened for plastid transformation and ultimately for homoplasmy. Multiple different tissue culture approaches were taken to try to regenerate shoots but unfortunately no shoots were produced from the rice tissue.
Publications
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Progress 07/15/18 to 03/14/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:The major problem encountered was an inability despite multiple attempts at tissue culture protocol modifications to regenerate plastid-transformed rice plants, which precluded further molecular characterization of any rice plastid transformants. What opportunities for training and professional development has the project provided?The project offered opportunities for professional development for molecular cloning of, and transformation with, plastid transformation vectors, and discussion among molecular and plant transformation technicala personnel regarding plastid transformation technologies. How have the results been disseminated to communities of interest?The results were presented to internal Benson Hill R&D groups. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The envisioned plastid transformation constructs were designed and used for tobacco and rice transformation. Tobacco transformation experiments were abandoned when it was realized that the envisioned GFP visual selection strategy would not be efficient enough to provide meaningful data. The research work was therefore focused on rice where it was anticipated that greater value would be developed if the project was successful. Biolistic transformation of various rice explants was attempted and various tissue culture modifications were attempted to try to regenerate a rice plastid transformant shoot. While multiple rice callus pieces were observed with GFP fluorescence presumed to come from the plastid transformation vector, likely indicating a successful transformation event, none of these callus pieces was able to successfully regenerate a shoot for further molecular characterization.
Publications
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Progress 07/15/18 to 07/14/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:Tobacco plastid transformation has been deprioritized in favor of a focus on rice transformaiton because it was determined that the tobacco transformation experiments added minimal value to the overall project goals. Multiple site-specific nucleases are being tested in parallel in rice transformation vectors in an attempt to maximize the likelihood of identifying one or more approaches to produce homoplasmic rice plastid transformants. What opportunities for training and professional development has the project provided?The project has resulted in the development of transformation protocols for rice plastids in-house and professional development through the various project planning discussions among Benson Hill team members. Internal presentations by the scientists performing those experiments have also provided professional development opportunities. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Molecular characterization of the rice shoots developed to date will be a high priority. Additionally, further explants will be bombarded while molecular assessment is performed in an attempt to generate further putative rice plastid transformants.
Impacts
What was accomplished under these goals?
Multiple transformation vectors for plastid transformation of tobacco and rice have been designed and synthesized. One insertion site in tobacco was targeted, while two different insertion sites were targeted in rice. Initial tobacco transformations were attempted, and several putative transformants were identified using aadA and spectinomycin selection. Because of difficulties in identifying GFP expressors in the absence of antibiotic selection, it was determined that assessing the novel technologies under development in tobacco would be very difficult and as a result, rice transformation has been prioritized while deprioritizing tobacco transformation. Various rice explants have been bombarded with a total of six different plastid transformation vectors. A number of shoots have been identified that are growing on streptomycin-containing medium, some of which show visible fluorescence under a microscope, indicating likely insertion of the plastid transgenes. Molecular characterization of these shoots will be performed in the near future to assess whether the transformation constructs have been inserted at the intended sites and if so, whether these plants are heteroplasmic or homoplasmic.
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