Progress 05/01/24 to 04/30/25
Outputs
Target Audience:The target audiences reached by our efforts during this reporting period include fellow scientists and students, through efforts including seminars at professional conferences and institutional speaking events, laboratory instruction and formal classroom instruction. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Guest lecture about genetic modification to study cannabinoid biosynthsis in industrial hemp given by a graduate student to an audience of undergarduate students in the Virginia Tech School of Plant and Environmental Science Crop Physiology and Ecology course. Laboratory instruction to teach molecular biology, bioinformatics, plant tissue culture, cell biology, and biochemistry to two graduate students and two undergraduate research assistants. Participation in and poster presentation at the 5th Annual Mid-Atlantic Synthetic Biology Symposium for one graduate student. Posters were presented by both graduate students at local symposia organized by the School of Plant and Envirtonmental Sciences and the Translational Plant Science Center. How have the results been disseminated to communities of interest?Poster presentations and seminars given at professional conferences and invited institutional seminar series. Conversations with local (Virginia-based) hemp growers and breeders. What do you plan to do during the next reporting period to accomplish the goals?Clone and over-express additional transcription factor and cannabinoid biosynthetic genes. Generation of additional stable transformants (cell suspension and potentially whole plants) with inducible overexpression of candidate transcription factors. Gene regulatory network analysis using transient transformation of protoplasts and RNA-seq analyses. Further metabolite analyses in transgenic cultures using LC-MS. Publish two or more papers in peer-reviewed journals; one on the involvement of the AP2L1 transcription factor in the regulation of cannabinoid and flavonoid biosynthetic genes, one on a newly developed cell-suspension transformation procedure, and potentially more work in the down-stream gene regulatory networkanalysis of two transcription factors using theTARGET system and cannabinoid biosynthesis in heterologous systems.
Impacts
What was accomplished under these goals?
Cloning of seven transcription factor genes as well as five cannabinoid biosynthetic pathway component genes from industrial hemp genotypes. Repeated generation of a cell suspension culture with stable conditional overexpression of a candidate transcription factor as well as a control cell suspension with stable conditional overexpression of green fluorescent protein. RNA-sequencing analysis of the effects of conditional overexpression of a transcription factor, specifically looking at the regulation of cannabinoid biosynthetic genes but actually finding significant effects on flavonoid biosynthesis. We are currently preparing a manuscript and expect submission to a peer-reviewed journal before the end of the Spring semester. Further use of Yeast-1-hybrid, qRT-PCR, and transient transformation of protoplasts assays to validate RNA-seq results. We have validated two flavonoid biosynthetic genes identified by our RNA-seq analysis and verified by qRT-PCR in independently generated transgenic cell-suspension lines.Targeted metabolite analyses (LC-MS) of cannabinoid and flavonoid production in transgenic cell suspension cultures, both in cells and spent medium. Establishment of a novel method for cell-suspension transformation with Agrobacterium tumefaciens. We have shown that we can successfully transform cell-suspension during culture in flasks by simply co-cultivating with Agrobacterium carrying a 35S::RUBYexpression cassette.
Publications
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Progress 05/01/23 to 04/30/24
Outputs
Target Audience:The target audiences reached by our efforts during this reporting period include fellow scientists and students, through efforts including seminars at professional conferences and institutional speaking events, laboratory instruction and formal classroom instruction. Changes/Problems:One of the two graduate students working on this project was awarded a two-semester scholarship, meaningthat we have spent less money so far than originally projected and can either extend the period of work or add to the personnel working on this project. What opportunities for training and professional development has the project provided?Guest lecture about genetic modification to study cannabinoid biosynthsis in industrial hemp given by a graduate student to an audience of undergarduate students in the Virginia Tech School of Plant and Environmental Science Crop PHysiology and Ecology course. Laboratory instruction to teach molecular biology, bioinformatics, plant tissue culture, cell biology, and biochemistry to two graduate students and one undergraduate research assistant. One-week plant transformation and regeneration workshop attendance for one graduate student at theWisconsin Crop Innovation Center. How have the results been disseminated to communities of interest?Poster presentations and seminars given at professional conferences and invited institutional seminar series. Conversations with local (Virginia-based) hemp growers and breeders. What do you plan to do during the next reporting period to accomplish the goals?Clone additional transcription factor and cannabinoid biosynthetic genes. Generation of additional stable transformants (cell suspension and potentially whole plants) with overexpression of candidate transcription factors. Gene regulatory network analysis using transient transformation of protoplasts and RNA-seq analyses. Further metabolite analyses in transgenic cuyltures using LC-MS. Publish two or more papers in peer-reviewed journals.
Impacts
What was accomplished under these goals?
Cloning of several transcription factor genes as well as a number of cannabinoid biosynthetic pathway component genes from industrial hemp genotypes. Generation of a cell suspension culture with stable conditional overexpression of a candidate transcription factor as well as a control cell suspension with stable conditional overexpression of green fluorescent protein. RNA-sequencing analysis of the effects of conditional overexpression of a transcription factor, specifically looking at the regulation of cannabinoid biosynthetic genes. Establsihment of Yeast-1-hybrid, qRT-PCR, and transient transformation of protoplasts assays to validate RNA-seq results. Targeted metabolite analyses (LC-MS) of cannabinloid production in transgenic cell suspension cultures, both in cells and spent medium.
Publications
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