Progress 04/01/21 to 05/31/22

Outputs
Target Audience:Because of the nature of this Phase I project, the audience with whom communications about this project will be shared is limited to individuals and companies, as follows: 1. Management team of Kultevat via the Co-PI, R.Beachy, who serves as Chief Science Officer for Kultevat:Beachy will share information with the CEO, CFO and VP for Processing Technology 2. Board of Directors:Beachy will communicate progress of the project with the Kultevat Board of Directors at quarterly meetings 3. Companies with whom Kultevat has a confidentiality agreement and from whom seed-related technologies are licensed Should this Phase I project be successful and a Phase II project follows, other audiences will include USDA regulatory agencies, farmer organizations, and individual farmers. Further details will be provided in the application for the follow-on grant. Changes/Problems:During Phase I of this project, the immediate goal was to develop and apply genetic engineering technologies to alter the architecture of an improved line/variety of TKS using a technology license for this purpose. The technology is a gene derived from TKS that would alter the expression of an extant gene in TKS. The technology was shown effective in other plants, including monocots and dicots, but not previously shown effective in TKS. During this Phase I project, the TKS-specific gene was produced by, and licensed from, a technology company, Performance Plants, Inc. (Kingston, ON, Canada). During this period, the following information has been learned/derived/adapted for use by Kultevat to achieve their goals, i.e., resulting in a; Change in Action: Developing transgenic TKS plants: technologies for developing GM TKS plants was previously described in published and unpublished literature by Dr. J. Silva, the PI of this project, and by other workers in the field. The previously developed techniques were adapted to larger scale to meet the goals of this project. The following includes significant new information and knowledge generated to date. Importance of using axenic plants for transformation reactions. As a result of many experiments, it was learned that greenhouse grown plants are heavily contaminated by microbes, including endophytes and ectophytes. These microbes caused a high level of contamination when tissues derived from these plants were used in plant transformation studies. Ridding explants of microbes proved to be very difficult and led to high levels of failure. The problem was solved by relying on seedlings grown under axenic conditions using methods that are common in such cases. Importance of host genetics in genetic transformation. TKS is not an inbred plant; rather, it is a genomic mixture of selected individuals. We found that some selected lines/varieties were more receptive to transformation than others; two receptive 'lines' were identified after testing six lines. Furthermore, since the plant lines are not inbred, we expected and encountered significant plant-to-plant variability; this made it necessary to transform many different individuals to assure success in producing individual transgenic plants, referred to as an 'event'. After about 6 months into the project, a superior plant line was identified, and frequency of transformation improved substantially thereafter. Transformation and tissue culture processes per se do not alter TKS phenotype. After identifying transformed lines by PCR-based DNA assays, it was determined that more than 7 independent lines, referred to as 'T0' lines, were generated: each has a normal phenotype, and we conclude that the gene does not cause an aberrancy in growth of T0 lines in comparison to non-transgenic plants or to transformed plants that carry an 'empty vector'. The latter is the preferred control for these studies. Furthermore, we tentatively conclude that the act of transformation and plant regeneration, nor tissue culture processes per se, do not cause significant levels of somaclonal variation in the plant lines that were selected, that would give concern going forward. Change in Knowledge: This project required hiring a recent Ph.D. recipient to conduct much of the bench work required. Dr. Laura Gomez was identified and hired as a consequence of conducting an open search for the necessary role. Dr. Gomez received initial training in TKS plant transformation by Dr. Jillian Silva, the PI of the project, with oversight by Dr. Roger Beachy, co-PI and Chief Science Officer of Kultevat. Throughout the conduct of Phase I, Dr. Gomez has been extraordinarily skilled at developing transgenic lines of TKS, and has gained sufficient insight in knowledge of TKS and transformation protocols in an independent manner going forward. What opportunities for training and professional development has the project provided? Nothing Reported 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? Nothing Reported

Impacts
What was accomplished under these goals? Results and updates for the specific objectives of this project as stated are as follows: To establish a robust genetic engineering ('transformation') system for the variety of TKS that is preferred for crop purposes A plant expression vector containing a GUS marker gene was provided through the licensing agreement in place between Kultevat, Inc. and Performance Plants, Inc. (PPI; Kingston ON, Canada). Method optimization and first transformation experiments to introduce this gene construct to TKS began in March, 2021, using the method employed by Dr. Silva as a graduate student. Modifications were introduced to the method to shorten preparation of the Agrobacterium inoculum. Transformation work began by using materials from two different 'lines' of TKS that were provided by Kultevat's plant breeding partner (LionFlex, LLC) that are being used for field production. Plants were grown in the greenhouse at the Donald Danforth Plant Science Center. Several transgenic TKS GUS marker expressing T0 plants were confirmed using standard staining protocols (Figure 1). To construct the intermediate plasmid that harbors the TKS-BET trait (the gene construct per se was obtained by license agreement); and integrate the trait into a disarmed strain of Agrobacterium for use in genetic engineering Expression vectors carrying the TKS-BET trait, along with the empty vector control, were obtained by license agreement and successfully transformed into Agrobacterium tumefaciens using standard techniques. Positively transformed Agrobacterium were confirmed by showing resistance to antibiotic selection, and using colony PCR with primers specific to the transgene cassette and/or gene of interest. To conduct genetic transformation with tissues of TKS Agrobacterium-mediated transformation with expression vectors harboring the TKS-BET trait was used on TKS leaf cuttings from the chosen preferred varieties. Transformations with the BET constructs began in the spring of 2021, using greenhouse grown TKS plants as source material. Initially, growth conditions yielded healthy leaf tissue suitable for transformation. However, it was observed that through the warmer summer months of 2021, into the fall of 2021, insect and fungal populations in the greenhouse grown plants increased. Subsequently, the amount of microbial contamination on transformed tissues was high, leading to lower yields of putative transgenic growing tissues due to contamination loss. In an effort to mitigate putative transgenic tissue loss due to microbial and fungal contamination, a decision was made in early 2022 to grow TKS seedlings axenically in tissue culture Magenta boxes as source materials for genetic transformations. Early results reached at the end of the Phase I grant funding period were positive, and indicate minimal loss from contamination of growing transformed tissues. To regenerate shoots and rooted plants from tissue that is genetically engineered as in (3) Regeneration of plants transformed with the TKS-BET construct has been successful. Kultevat has grown several T0 TKS plants starting from callus tissue regenerated on selection medium, moving to shoot elongation selection medium and finally to rooting medium with selection (Figure 2; rooting medium not shown). After root formation in tissue culture, the selected plantlets were moved to soil to grow and mature in the greenhouse (Figure 3). At end of the Phase I funding period, 7 transgenic plants that carry the BET gene silencing construct had been generated. Work is ongoing after the Phase I period, and additional candidate plants are progressing through tissue culture and plant regeneration and are slated to be transferred to soil in the coming several weeks. To identify genetically transformed tissues that harbor the BET (target) gene by: Conducting genotypic analyses by DNA-based and RNA-based gene expression assays, and selecting for further study those tissues/individuals that harbor and express the BET gene Quality genomic DNA was extracted from putative transgenic and control plants using the commercially available Qiagen DNeasy® Plant Pro Kit. Genotyping PCR using primers specific to the NPTII selection marker gene in the gene constructs was performed. PCR positive plants were identified (Figure 3) and carried through for further analyses. Plant gene expression using semi-quantitiatve RT-PCR techniques with target-gene specific primers is currently underway. Differences in target gene expression will be monitored for correlation with increases in biomass and/or rubber content. Imaging (via light microscopy and by sight) transgenic tissues with altered morphology and/or anatomy of transgenic tissues Two of the confirmed TKS-BET RNAi lines, along with a wildtype control plant, were used to establish microscopy studies to determine if differences in laticifer number and other cell rubber producing architecture could be visualized and quantitated. It is hypothesized that by knocking down the TKS-FVE genes, a delay in flowering will occur and carbon flux will be shifted towards biomass enhancement, and more specifically an increase in the number of laticifer cells in transgenic TKS. Oil Blue NA stain has previously been shown to stain rubber in TKS and other plant species. Root sectioning, staining, and microscopy was completed at the Donald Danforth Plant Science Center Imaging Core Facility under a fee-for-service agreement. Sectioning and staining was successful, with blue laticifer/rubber staining visible in all three plants analyzed (Figure 4). Though there are apparent differences between the samples, no conclusions can be drawn and quantitation cannot be made as these samples are n=1. Future studies with clones of these plants and others will be completed to note any significant differences between control and transgenic plants. A suitable microscopy method has been established and will aid in further characterization of transgenic plants generated through this project, continuing into planned studies for an upcoming Phase II proposal application. Impact Statement The stated goal of this project is to provide a domestic source of natural rubber (NR), i.e., rubber that is produced by U.S. farmers and processors, thus reducing reliance on imported NR. NR is a strategic material that is produced outside of the U.S. and used in a wide range of products, including in clothing and shoes, medical supplies and equipment, small appliances and tools, as well as in the tire industry. Kultevat's goal is, in the mid-term, to develop a farming system to grow improved varieties of Taraxacum kok-saghyz (TKS), a source of NR that is similar in structure to NR produced by rubber trees; and to develop an efficient and cost-effective process to extract and purify NR that is suitable to customers in the U.S. based rubber industry. This will ensure that needs of consumers of products that contain NR are not disrupted by future supply-chain issues. WILL PROVIDE FIGURES IF REQUESTED

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