Progress 05/01/10 to 09/30/14
Outputs
Target Audience: Mint industry representatives, mint producers, food and personal product industry, and scientific community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project has substantially contributed to the training of one technician. How have the results been disseminated to communities of interest? Results have been disseminated via presentiation to the Mint Industry research council. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
DNA methylation was demonstrated to be altered at transgenic loci in mint during vegetative propagation. This variation was associated with differences in expression of the transgenes and in plant phenotype. Chromosome doubling was carried out for Black Mitcham peppermint and two polyploid derivatives were obtained and characterized. Neither chromosome doubled type was substantially sexually fertile, indicating that a more substantial genetic approach will be required to generate a sexually fertile and biochmecially favorable (e.g. organoleptically acceptable) mint accession. Work towards that was begun during the last year of the project.
Publications
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: We have evaluated transgene methylation stabilty and herbicide tolerance trait expression. This has indicated that mutliple independent lines have been subject to silencing during clonal propagation and highlight challenges faced when using GMO improvement approaches in this crop. We continue to make progress on working with the sexually reproducing mint species that form the progenitor species complex that gives rise to the various horticulturaly relevant hybrids. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project has engaged one research technician and provided an opportunity to learn both novel molecular techniques for the analysis of DNA methylation and flow cytometric analysis of nuclear DNA content. How have the results been disseminated to communities of interest? The PD has traveled to the Mint Industry Research Council annual meeting and met with the mint industry regional groups for formal research presentations. In Jan 2014 the PD will again travel to the annual meeting of the Mint Industry Research Council. What do you plan to do during the next reporting period to accomplish the goals? We expect to publish our findings on transgene stabilty. We expect to publish our method for high throughput determination of nuclear DNA contents. We will continue to work with the novel polyploid mints and with the sexually fertile mint species.
Impacts
What was accomplished under these goals?
We have flow cytometrically karyotyed the entire Purdue mint collection. We have generated polyploid mint derivatives. We have tested for transgene expression stability.
Publications
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Handcrosses of polyploid mints were completed and seed-like propagules were planted. To date we have not seen germination of any of the seed-like propagules. In addition, we have improved the flow cytometric methods and ported them to be used on a higher throughput flow cytometric analysis machine that is substantially more automated. PARTICIPANTS: Brian Dilkes, PI. Tena Graham, a technician in the Dilkes lab was responsible for the hand crossing seed collection and flow cytometry. She has also taken the lead in interacting with those interested in integrating flow cytometry into their plant genetics research. TARGET AUDIENCES: Fellow scientsits. Plant breeding and improvement companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Dissemination of the method to other laboratories, in particular those working with plant tissues rich in terpenoids and polyphenolic compounds has been successful. Tena Graham has worked both with industry partners and with ecological laboratories in order to allow them to integrate flow cytometric analyses of nuclear DNA into their research programs.
Publications
- No publications reported this period
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: We have had meetings with industry representatives to discuss the possible incorporation of our polyploid mints into flavorings and other essential oil products. This has made industry end-users for the agricultural product aware of the new mint varieties and begun to connect the mint variety improvement efforts at Purdue with the personal products, food, and flavor industries. PARTICIPANTS: Brian Dilkes and Tena Graham participated in the collection and interpretation of the data from the Dilkes lab. Stephen Weller collaborated with us and made available the mint collection, transgenic lines, and expertise in herbicide application and resistance scoring as well as sharing primary data from the initial screening of transgenic events. TARGET AUDIENCES: Molecular Biologists seeking to improve mint crops, flavor, food and personal products industry, mint producers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have identified two polyploid derivatives of Mentha piperita using flow cytometry. Both polyploid individuals have been propagated vegetatively. Both lines were allowed to flower and self pollinate and we have collected fruits. Currently the data from the flow cytometry screens of the Purdue Mentha collection are being organized for dissemination via the Purdue Library digital collections. A publication describing the lines and ploidy levels as well as the phenotypes of the two polyploid Mentha piperita lines is planned for this year to coincide with the warehousing of the flow cytometry data in the Purdue Library digital repository for global open-access dissemination. In addition to the work on mint genome size we have completed our follow-up study on the stability of transgene expression in mint. We have determined that Glufosinate resistance, conditioned by the expression of a bacterial enzyme provided on a transgene, was not stable over the 6 to 10 year study period. Alarmingly, those lines with the highest resistance levels in the first two years did not maintain trait expression. This indicates that either constant monitoring of clones and re-selection will be needed to guarantee trait stability or that additional research will need to be conducted to determine what promotes transgenic trait stability in mint. We tested the hypothesis that DNA methylation would be be found on the transgenes when herbicide resistance is lost. This was confirmed, and we demonstrated that a simple PCR-based molecular test of DNA methylation was sufficient. This suggests that epigenetic silencing may be the culprit in the loss of expression for the herbicide resistance traits. A publication is planned for this year.
Publications
- No publications reported this period
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: We have completed the flow cytometric analysis of the Purdue Mentha collection. This has identified mint plants of a variety of chromosome constitutions, including apparent whole genome duplication of Mentha piperita cv. Black Mitcham, the top accession for flavor and highest acreage and dollar value in the US. We are pursuing a plant variety protection patent for these lines and an invention disclosure has been filed with the Purdue University Office of Technology Commercialization. Flowering was induced in the entire collection (including the polyploid M. piperita) and fruits were collected. We are currently sorting through this material to identify seeds for planting. PARTICIPANTS: Brian Dilkes Tena Graham, a technician in the PD laboratory was responsible for much of the flow cytometry, some plant care, hand crossing, and seed collection. This was Tena's training in flow cytometry and has resulted in significant career development and new skill acquisition. She is now also trained in the application of this technique to Corn, sorghum, and rice. Stephen Weller, a professor at Purdue, is a collaborating faculty who provided plant material and discussed the economic impact of the study. There were no other collaborators or support given to this project. TARGET AUDIENCES: Mint industry - growers and processors PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have filed an invention disclosure. Based on the finding of a whole genome duplicated M. piperita we are testing these plants for phenotypic stability by vegetative propagation of a large number of plants and further testing for sexual fertility by hand crossing.
Publications
- No publications reported this period
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