Progress 06/01/20 to 05/31/23
Outputs
Target Audience:The outcome of this project is primarily and immediately relevant and of interest to audiences within the plant science research community. In general, the results of this project and the gained knowledge have been and will be communicated to this audience via presentations at scientific conferences, and through publication of manuscripts in plant science and genetic journals. The results obtained during the duration of the project were presented as posters and oral presentations at the following conferences and events: the 2022 Annual Meeting of the Phytochemical Society of North America (PSNA), Virginia Tech University, Blacksburg, VA (2 posters by graduate students from the PI's and CoPI's labs); 2022 Cannabis Conference, Las Vegas, NV (invited talk by the PI); 2022 Entomological Society of America (ESA) Joint Annual Meeting, Vancouver, BC, Canada (poster presentation by PI); TERPNET 2023 - 15th International Meeting on the Biosynthesis, Function and Synthetic Biology of Isoprenoids, University of California Davis, Davis, CA (invited talk by PI); 12th Annual Conference American Council for Medicinally Active Plants, Charleston, WV (invited talk by PI); 2023 Entomological Society of America (ESA) Annual Meeting, National Harbor, MD (oral presentation by PI). One manuscript (Ahmed et al., 2021) on the genetic transformation of Cannabis sativa has already been published in the Journal of Biotechnology (peer reviewed). We are currently in the process of preparing a manuscript summarizing the results from our studies on the effect of biotic stress on the formation of terpenes and cannabinoids in hemp that will be submitting to a scientific journal soon. In the long run the knowledge gained, and the new methods developed within this project will also target audiences beyond the research community such as breeders that will apply and implement the knowledge and approaches in the development of new hemp varieties. In addition, the results of this project in the future will help to develop recommendations and management strategies for industrial hemp growers to avoid potential spikes in the THC content of their crop. As an effort to inform the general public about this project WVU made a news release at the beginning of the project and the PI has given a number of interviews with media over the course of the project. In 2022 the PI has also been invited to the Cannabis Conference to participate as speaker and panel member in the session 'University Research Roundup: The Latest in Cultivation Science' where he reported on the results of this project and potential consequences and recommendations for hemp growers. Changes/Problems:Due to the Covid crisis that began in 2020, right when this project started, and the resulting campus lock-downs, lab closures, reduced working hours, and difficulties to recruit students, the initial phase in particular as well as the overall progress of the project were significantly affected. The recruiting of a graduate student in the Kovinich lab has been significantly delayed due to the Covid crisis and the associated travel restrictions for international students and respective Covid protocols at York University. Despite opting for a one-year no-cost extension at the end of the second reporting period, these unprecedented circumstances that were beyond our control resulted in the PI and CoPI having to slightly adjust the objectives of the project to achieve a successful outcome. In addition, our metabolite analysis of hemp plants upon stress treatment unexpectedly demonstrated that not only the major cannabinoids are upregulated but also many minor cannabinoid compounds. This however required a time consuming and costly verification of these minor cannabinoid compounds which we had not planned for in the original proposal. Finally, Israt Jahan, the MSc student at York University became severely ill in her second year, forcing her to withdraw from the program, and thus she did not complete her project. What opportunities for training and professional development has the project provided?This project provided training and professional development opportunities for two graduate students, one in the Gutensohn lab at WVU and one in the Kovinich lab at York University. Bikash Deo, the graduate student at WVU, performed stress experiments, metabolite and qRT-PCR, as well as the RNAseq analyses and received training in plant biochemistry, molecular biology and genetics under the guidance of the PI. Israt Jahan, the graduate student at York University, has worked on the development of protocols for the transient and stable transformation of hemp using Agrobacterium and nanoparticle based approaches. In addition, she has designed and cloned multiple DNA constructs that have been used to attempt CRISPR/CAS9 based editing of cannabinoid biosynthetic genes in hemp. In addition, one undergraduate student, Jonathan Morgan, joined the PI's lab in 2020/21 within the framework of the WVU Research Apprenticeship Program (RAP), and assisted in the metabolite analysis of some hemp plants in the project. The undergraduate student received training in basic laboratory skills and in the analysis of terpenes and cannabinoids from hemp plants grown under different nutritional regimes under the guidance of the PI and the graduate student. Dasol Wi, was a Dean's Undergraduate Research Award holder and assisted in developing the Cannabis transformation protocols at York University. How have the results been disseminated to communities of interest?Over the course of this project the results of the project have been disseminated to the research community through poster and oral presentations at multiple conferences including the Annual Conference of the Phytochemical Society of North America 2022, Cannabis Conference 2022, Entomological Society of America Annual Meeting 2022 and 2023, TERPNET 2023 - 15th International Meeting on the Biosynthesis, Function and Synthetic Biology of Isoprenoids, 12th Annual Conference American Council for Medicinally Active Plants 2023. In addition, the results of this project have been and will be communicated to to the research community through publication of manuscripts in peer reviewed scientific journals. One manuscript on our results of the genetic transformation of Cannabis sativa has been published in the Journal of Biotechnology in 2021. In addition, we are currently preparing a manuscript summarizing the results from our studies on the effect of biotic stress on the formation of terpenes and cannabinoids in hemp that will be submitting to a scientific journal later this year. We are also planning to prepare another manuscript on the results of our RNAseq study as soon as the necessary control and follow up experiments are finished. As an outreach effort to inform the general public about this project the PI has attended upon invitation the Cannabis Conference in 2022 and has participated as speaker and panel member in the session ' University Research Roundup: The Latest in Cultivation Science' where he reported on the preliminary results of this project and potential consequences and recommendations for hemp growers. The PI is also regularly working with the Extension Service at WVU and the State Hemp Coordinator at the WV Department of Agriculture to spread the knowledge gained through this project (in particular about pest management) to hemp growers. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: For the proposed analyses the Gutensohn lab at WVU has propagated hemp plants via cuttings and re-rooting from one parental plant grown from seed, thus eliminating any potential genetic variability. In the first part of this project the Gutensohn lab has analyzed the effect of a number of abiotic and biotic factors on the accumulation of cannabinoids and terpenes. Plant material collected from control and treated hemp plants was utilized for the analysis of terpenes and cannabinoids by GC-MS and UHPLC. The identity and quantities of individual compounds were verified by using authentic terpene and cannabinoid standards. Since we hypothesized that plant nutrition could affect formation of terpenes and cannabinoids in hemp, fertilizer applications and the presence/absence of mycorrhizal fungi were tested. Arbuscular mycorrhizal fungi (AMF) were found to indeed interact with roots of hemp plants and thus could influence the uptake of nutrients. These analyses demonstrated that AMF as well as the addition of fertilizer indeed affected the formation of some terpenes and cannabinoids. Likewise preliminary analyses with other abiotic stresses, such as heat and water stress, indicated some effects on the accumulation of cannabinoids and terpenes. However, the effects of these abiotic stress treatments were less pronounced compared to what was observed upon the biotic stress treatments that we have performed in parallel (see below). Thus, considering the unprecedented circumstances due to the Covid crisis and the significantly affected timeline of the project all further efforts and resources were focused on analyzing the effects of biotic stress on the accumulation of cannabinoids and terpenes in hemp. For the analysis of biotic stress hemp plants were initially treated with methyl jasmonate and methyl salicylate to mimic potential attacks by biting-chewing and piercing-sucking pests as well as pathogens. While methyl salicylate essentially had little to no effect, the methyl jasmonate treatment resulted in a significant increase in the content of many cannabinoids including THC and CBD. In addition, the formation of some, but not all, monoterpenes and sesquiterpenes was increased upon methyl jasmonate treatment. Remarkably, similar trends towards increased cannabinoid contents were observed with CBD and seed hemp varieties although the absolute amounts of cannabinoids were respectively different in these hemp varieties. Based on these initial results the larvae of beet armyworm (Spodoptera exigua), a common pest of hemp plants, were used for feeding experiments on hemp plants. The subsequent analyses demonstrated that the accumulation of cannabinoids as well as some mono- and sesquiterpenes was significantly increased by the larval feeding. Remarkably, this study demonstrated that insect herbivory can cause an increase in THC content which would surpass the legal limits for industrial hemp. Moreover, our analysis of cannabinoid formation upon insect herbivory of hemp plants resulted in some unexpected observations. In addition to an increased accumulation of the well-known major cannabinoids, such as CBG, CBD and THC, we also found an increased accumulation of several groups of so-called minor cannabinoids including varinol- and butol-type cannabinoids. While insect lack endocannabinoid receptors, the target of major cannabinoids in mammals, our findings suggest that some of the induced minor cannabinoids might be involved in the defense of hemp against insect herbivory. Objective 2: In parallel to the metabolite analysis the Gutensohn lab has collected plant material from control hemp plants as well as hemp plants upon methyl jasmonate treatment and larval feeding. These plant tissues were subsequently utilized to isolate total RNA. Primers were designed and tested for a selected set of terpene and cannabinoid biosynthetic genes including MVA- and MEP-pathway genes, terpene synthase genes, polyketide pathway genes, and cannabinoid synthase genes (CBGA-, CBDA-, and THCA-synthase). Detailed quantitative RT-PCR analysis of the transcript levels of the selected terpene and cannabinoid biosynthetic genes in control plants and plants upon biotic stresses revealed significant changes in the gene expression. While the expression of MEP pathway genes was upregulated upon biotic stress, no or little changes were found for MVA pathway genes. In line with the metabolite data, the expression of some monoterpene synthase genes was increased. Remarkably our qRT-PCR analyses demonstrated that the transcript levels of several polyketide pathway genes and cannabinoid synthase genes were significantly increased (up to five-fold). These results indicate that genes involved in the biosynthesis of cannabinoids are strongly upregulated upon biotic stress such as insect herbivory. Based on the results of the qRT-PCR analysis RNA isolated from the respective biotic stress treatments have been used for RNAseq analyses to identify new genes involved in the regulation of cannabinoid and terpene biosynthetic genes. The data from the RNAseq analysis are being analyzed for novel genes that are co-expressed with those biosynthetic genes that were found by the qRT-PCR analysis to be upregulated upon the biotic stress. We are particularly focusing on regulatory factors (e.g., transcription factors) as well as biosynthetic genes potentially involved in the formation of the observed minor cannabinoids. These novel candidate genes identified within this seed grant project will now be the foundation of a follow-up project proposal that will aim to study the function of these genes involved in the regulation and biosynthesis of cannabinoids in more detail. Objective 3: For the proposed knock-out of the THCAS gene in hemp through a CRISPR-CAS9 approach, the Kovinich lab at York University designed guide RNAs (gRNAs) to target the biosynthesis gene tetrahydrocannabinolic acid synthase (THCAS). The targets were confirmed in three different hemp varieties by sequencing of genomic DNA. Three DNA constructs were assembled that encode the three gRNAs , the CAS9 enzyme, and the antibiotic resistance marker L-Phosphinothricin (PPT). These constructs were transferred into 327 hemp hypocotyls via an Agrobacterium-mediated transformation method and subsequently 67 calli were selected on PPT medium. Genomic DNA from most of the calli were confirmed to encode the CAS9 gene by PCR and DNA sequencing. However, mismatch cleavage assays and various software algorithms designed to detect CRISPR edits from Sanger sequencing chromatograms failed to detect any edits in the THCAS gene. Similar results were obtained in Cannabis protoplasts and in cucumber protoplasts that were co-infiltrated with our DNA constructs and a construct encoding the THCAS gene. The MSc student leading the project was then working on conducting next generation sequencing of the genomic DNA from calli and designing new gRNAs that may mediate greater editing efficiency.However, she unfortunately became severely ill and had to withdraw from the MSc program at York University. For regeneration of shoots from Cannabis calli, we attempted to reproduce the two most efficient published protocols and variations in culture media components thereof. However, even after nine months of culture on the various media, we did not observe any regeneration of shoot tissues in our three hemp varieties. In collaboration with the Wu lab at the University of Massachusetts at Amherst, the Kovinich lab developed a highly efficient transient transformation protocol for Cannabis. This nanoparticle-mediated genetic transformation protocol provided an advantage over the traditional Agrobacterium mediated method in that it can simultaneously express several DNA constructs in Cannabis trichomes, which are the site of cannabinoid biosynthesis. Thisapproach will be invaluable for studying cannabinoid biosynthesis in the future.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Gutensohn M. and Deo B. (2022) Insect herbivory has significant effects on cannabinoid and terpene levels in industrial hemp (Cannabis sativa). Poster Presentation at the 2022 Annual Meeting of the Entomological Society of America (ESA), November 13-16, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Gutensohn M. and Deo B. (2023) Regulation of the cannabinoid and terpene metabolic network in industrial hemp (cannabis sativa) under biotic stress. Invited talk at the TERPNET 2023 - 15th International Meeting on the Biosynthesis, Function and Synthetic Biology of Isoprenoids, July 31 - August 4, 2023.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Gutensohn M. and Deo B. (2023) Regulation of the cannabinoid and terpene metabolic network in industrial hemp (Cannabis sativa) under biotic stress. Invited talk at the 12th Annual Conference American Council for Medicinally Active Plants, October 18-21, 2023.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Gutensohn M. and Deo B. (2023) Effects of insect herbivory on the terpene and cannabinoid profiles in industrial hemp (Cannabis sativa) and potential biological functions. Oral Presentation at the 2023 Annual Meeting of the Entomological Society of America (ESA), November 5-8, 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Wi D. and Kovinich N. (2022) Plant Regeneration for Genome Engineering of Cannabis. Poster Presentation at the Undergraduate Research Symposium - York University, August 21, 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Kovinich N. (2023) Genetic Transformation of the Pharmaceutical Plant Cannabis sativa using Cationic Polymer-modified Silica Coated Gold Nanoparticles. Poster Presentation at the Nano Ontario Conference, July 16-20, 2023.
|
Progress 06/01/21 to 05/31/22
Outputs
Target Audience:The outcome of this project is primarily and immediately relevant and of interest to audiences within the plant science research community. In general the results of this project and the gained knowledge will be communicated to this audience via presentations at scientific conferences, and through publication of manuscripts in plant science and genetic journals. The results obtained during the currentreporting period were recently presented as two posters by the two graduate students involved in the project, Bikash Deo (WVU) and Israt Jahan (York University), at the Annual Meeting of the Phytochemical Society of North America (PSNA) at Virginia Tech University (with the PI Dr. Gutensohn and CoPI Dr. Kovinich attending as well). While in the lastreporting period one manuscript (Ahmed et al., 2021) on the genetic transformation of Cannabis sativa was published in a peer reviewed scientific journal, we are currently in the process of summarizing the results of our studies on the effect of different biotic stresses on the formation of terpenes and cannabinoids in hemp (which we obtained during the current reporting period) in a manuscript that we will be submitting to a scientific journal this fall. In the long run the knowledge gained and the new methods developed within this project will also target audiences beyond the research community such as breeders that will apply and implement the knowledge and approaches in the development of new hemp varieties. In addition, the results of this project in the future will help to develop recommendations and management strategies for industrial hemp growers to avoid potential spikes in the THC content of their crop. As an effort to inform the general public about this project WVU made a news release at the beginning of the project and the PI has given a number of interviews with media in the last and the current reporting period. The PI recently has also been invited to the Cannabis Conference to participate as speaker and panel member in thesession ' University Research Roundup: The Latest in Cultivation Science' where hereported on the preliminary results of this project and potential consequences and recommendations for hemp growers. Changes/Problems:While the start as well as the progress of the project in the previous reporting perioddue to the Covid crisis and the resulting campus lock-downs, lab closures, reduced working hours and delay inrecruit students was significantly affected,in the current reporting period the project has made good progress. Due to the initial delays to the project we have recently requested and were granted a one-year no-cost extension which now gives us the opportunity continue the project and work on the outstanding parts of the proposed objectives. What opportunities for training and professional development has the project provided?In the current reporting period the project provided training and professional development opportunities for twoPhD graduate students onein the Gutensohn lab at WVU and one in the Kovinich lab at York University. Bikash Deo, the graduate student at WVU, continued to performstress experiments, metabolite and qRT-PCR analyses during the current reporting period and received training in plant biochemistry and molecular biology under the guidance of the PI. The recruiting of a graduate student in the Kovinich lab had been significantly delayed due to the Covid crisis and the associated travel restrictions forinternational students and respective Covid protocols at York University.In the current reporting period Israt Jahan, the graduate student at York University, has worked on the development of protocols for the transient and stable transformation of hemp using Agrobacterium and nanoparticle based approaches. In addition, she has designed and cloned multiple DNA constructs that can now be used for theCRISPR/CAS9 based editing of cannabinoid biosynthetic genes in hemp. How have the results been disseminated to communities of interest?In the current reporting period the results of the project have been disseminated to the research community through the twopresentations at the Annual Conference of the Phytochemical Society of North America (PSNA) at Virginia Tech University. As an outreach effort to inform the general public about this project the PI has recently accepted an invitationto the Cannabis Conference and has participated as speaker and panel member in thesession ' University Research Roundup: The Latest in Cultivation Science' where hereported on the preliminary results of this project and potential consequences and recommendations for hemp growers. What do you plan to do during the next reporting period to accomplish the goals?We are planning to accomplish the following goals in the thirdreporting period of this project as outlined in the original plan: Objective 1:We will continue to treat hemp plants with the different proposed environmental stresses under greenhouse and growth chamber conditions, and will then analyze collected plant material for changes in terpene and cannabinoid accumulation as well as changes in the expression of respective biosynthetic genes. Objective 2: Since our biotic stress treatment experiments have demonstrated quite specific effects on the formation on terpenes and cannabinoids we will now use these treatments for the proposed RNAseq analysis. Considering the different effects of these treatments on the formation of monoterpenes, sesquiterpenes and cannabinoids we expect to be ableto identify transcription factors that are co-regulated with biosynthetic genes involved in the formation of thesedifferent groups of metabolites. Objective 3: A major focus in the Kovinich lab will be on theoptimizationthe shoot and plant regeneration from the transformed calli that we have obtained. Once stable transgenic hemp plants are available these will be utilized for detailed biochemical and genetic analysis to characterize the effects of the knockout and overexpression of the biosynthetic genes. Once candidate regulatory genes have been identified from the RNAseq analysis respective constructs for knockout and overexpression will be designed and subsequently used for transient and stable transformation of glandular trichomes and hemp plants.
Impacts
What was accomplished under these goals?
The following specific goals were achieved for the three proposed objectives of this project in the current reporting period: Objective 1 - Characterize the accumulation of terpenes and cannabinoids in industrial hemp under different abiotic and biotic stress conditions: For the proposed analyses of the effects of various environmental stress conditions on the accumulation of terpenes and cannabinoids in hemp, the Gutensohn lab at WVU has propagated hemp plants via cuttings and re-rooting from one parental plant grown from seed, thus eliminating any potential genetic variability. These hemp plants were treated with jasmonic acid and salicylic acid to mimic potential attacks by pests and pathogens. In addition, the larvae of beet armyworm (Spodoptera exigua), a pest of hemp plants, were used for feeding experiments on hemp plants.Plant material collected from control and treated hemp plants was subsequently utilized to for the analysis of terpenes and cannabinoids by GC-MS and UHPLC. The identity and quantities of individual compounds were verified by using authentic terpene and cannabinoid standards. These analyses demonstrated that the accumulation ofterpene and cannabinoid compounds was affected by the jasmonic acid and salicylic acid treatment as well as the larvalfeeding. However, the three different treatments had quite specific effects on terpene and cannabinoid levels, with jasmonic acid and larval feeding resulting in significant increases of monoterpenes, sesquiterpenes and cannabinoids while thesalicylic acid treatment only affected the sesquiterpene formation. In the current reporting periodthe Gutensohn lab has also analyzed the effectof fertilizer application and the presence of mycorrhizal fungi on the formation of terpenes and cannabinoids inhemp. Arbuscularmycorrhizal fungi (AMF) were found to indeed interact with roots of hemp plants and thus could influence the uptake of mineral nutrients. These analyses have so far demonstrated that AMF as well as the addition of fertilizer significantly affects the formation of terpenes and cannabinoids. However, additional experiments are required using different hemp varieties and fertilizer concentrations before final conclusions can be made. Objective 2 - Characterize the expression level and genetic regulation of key terpene and cannabinoid biosynthetic genes in industrial hemp under different abiotic and biotic stress conditions: Plant material has been collected by the Gutensohn labfrom the control hemp plants as well as hemp plants treated with jasmonic acid, salicylic acid treated and larval feeding andwas subsequentlyutilized to isolate total RNA. Primers were designed for a selectedset of terpene and cannabinoid biosynthetic genes and were tested in preliminary quantitative RT-PCR analysis. Detailed qRT-PCR analysis of the changes in the expression level ofterpene and cannabinoid biosynthetic genes in control plants and plants treated with biotic stresses is currently ongoing. At the same time we are preparing to now useRNA isolated from these treatments for respective RNAseq analyses to identify new genes involved in the regulation of terpene and cannabinoid biosynthetic genes. Objective 3 - Determine the effect of knockout and overexpression of key biosynthetic genes on the accumulation of metabolites in the terpene and cannabinoid biosynthetic network in industrial hemp: For the proposed knock-out of the THCAS gene in hemp through a CRISPR-CAS9 approach, the Kovinich lab at York University has confirmed genomic gRNA targets via sequencing in three different hemp varieties. Subsequently three DNA constructs were assembled encoding different gRNAs that can target the THCAS sites in all three hemp varieties.These different gRNAs were clonedin a CAS9 expression vector that encodes resistance to L-Phosphinothricin (PPT). These constructswere used to transform hemphypocotyls via an Agrobacterium-mediated transformation method and subsequently select calli on selection medium. Genomic DNA isolated from calli was utilized for PCR analysis with THCAS and CAS9 specific primers and subsequent sequencingwhich demonstrated the presence of CAS9.We are currently also further developing our protocolto regenerate shoots from our transformed callus and thus obtain genetically homogenous shoots for the further genetic and biochemical analysis. In addition, the Kovinich lab in collaboration with the Wu lab at the University of Massachusetts at Amherst has developed and further refined a protocol to coupleplasmids to nanoparticles and use these subsequently for the transient transformation of hemp tissues. We are now beginning to use this approachfor the development and testing of knock-out and overexpression constructs for terpene and cannabinoid biosynthetic genes to transiently transform glandular trichomes in hemp.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Deo B., Kovinich N., and Gutensohn M. (2022) Insect herbivory has significant effects on cannabinoid and terpene levels in industrial hemp (Cannabis sativa). Poster Presentation at the Annual Meeting of the Phytochemical Society of North America (PSNA), July 24-28, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Jahan I., and Kovinich N. (2022) Precision genome engineering approaches in Cannabis. Poster Presentation at the Annual Meeting of the Phytochemical Society of North America (PSNA), July 24-28, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Gutensohn M. (2022) Characterization of the terpene-cannabinoid metabolic network and its genetic regulation in hemp. Oral Presentation at the Cannabis Conference, August 23-25, 2022.
|
Progress 06/01/20 to 05/31/21
Outputs
Target Audience:The outcome of this project isprimarily and immediately relevant and of interest to audiences within the plant science research community. In general the results of this project and the gained knowledge will be communicated to this audience via presentations at scientific conferences, and through publication of manuscripts in plant science and genetic journals. Due to the ongoing Covid crisis and associatedrestrictions no presentations were made at scientific conferences in the current reporting period. However, during the current reporting period one manuscript (Ahmed et al., 2021) on the genetic transformation of Cannabis sativa was published in a peer reviewed scientific journal. In the long runthe knowledge gained and the newmethods developed within this project will also target audiences beyond the research community such as breeders that will apply and implement the knowledge and approaches in the development of new hemp varieties. In addition, the results of this project in the future will help to develop recommendations and management strategies for industrial hemp growers to avoid potential spikes in the THC content of their crop. As an effort to inform the general public about this project WVU made a news release at the beginning of the project and the PI has given a number of interviews with media including the magazine 'Hemp Grower' and a local WV TV station. Due to the ongoing Covid crisis and associatedrestrictions no in-person outreach activities were done in the current reporting period. Changes/Problems:Due to theongoing Covid crisis and the resulting campus lock-downs, lab closures, reduced working hours and difficulties to recruit students,the start as well as the progress of the project in the current reporting period was significantly affected. Although the project is now on a good track, we expect that due to the initial delays we will likely have to opt for a no-cost extension (as offered by USDA NIFA at the beginning of the Covid crisis) at the end of the second reporting period. What opportunities for training and professional development has the project provided?In the current reporting period the project provided training and professional development opportunities for one PhD graduate student and one undergraduate student in the Gutensohn lab at WVU.Bikash Deo, the graduate student at WVU funded through this project, performed all stress experiments, metabolite and qRT-PCR analyses during the current reporting period and received training in plant biochemistryand molecular biology under the guidance of the PI. Jonathan Morgan, an undergraduate student majoring in horticulture, joined the Gutensohn labin the fallsemester 2020within the framework of the WVU Research Apprenticeship Program (RAP), and assisted in the metabolite analysis of some hemp plants in the project. The undergraduate student received traning in basic laboratory skills and in the analysis of terpenes and cannabinoids from hemp plants grown under different nutritional regimes (see above) under the guidance of the PI and the graduate student. Due to the ongoing Covid crisis and the associated travel restrictions the international graduate student recruited by the CoPI Dr. Kovinich will only be able to join the lab at York University and to start working on the project by September 2021. Likewise Dr. Kovinich also did not have permission to have undergraduate students work in his lab due to the Covid regulations at York University during the current reporting period. Dr. Kovinich has now recruited an undergraduate student that will join the lab in September 2021 and will be involved in this project. How have the results been disseminated to communities of interest?In the current reporting period the results of the project have been disseminated to the research community through the publication of one manuscript (Ahmed et al., 2021) on the genetic transformation ofCannabis sativain thepeer reviewed Journal of Biotechnology. As an outreach effort to inform the general public about this project WVU made a news release at the beginning of the project and the PI has given a number of interviews with media including the magazine 'Hemp Grower' and a local WV TV station. In addition, the PI has recently been invited to speakat the 2022Annual Conference of the West Virginia Extension Master Gardener Association and will include some results of this ongoing project in the presentation. What do you plan to do during the next reporting period to accomplish the goals?While the start as well as the progress of the project in the current reporting period was significantly affected by the ongoing Covid crisis and the resulting campus lock-downs, lab closures, reduced working hours and difficulties to recruit students working on the project, we hope to be able to work under less challenging and restrictive conditions in the second reporting period. Thus, we are planning to accomplish the following goals in the second reporting period of this project as outlined in the original plan: Objective 1 & 2: The project will greatly benefit from the fact that all methods/protocolls required for the metabolite and qRT-PCR analyses have been fully established. We will continue to treat hemp plants with the different proposed environmental stresses under greenhouse and growth chamber conditions, and will then analyze collected plant material for changes in terpene and cannabinoid accumulation as well as changes in the expression of respective biosynthetic genes. Once one or two stress conditions have been identified that consistently increase the accumulation of cannabinoids (in particular THC) we will then use the isolated RNA for the proposed RNA-Seq analysis to identify transcription factors that are co-regulated with biosynthetic genes. Objective 3: The CoPI Dr. Kovinich will train one undergraduate and one graduate student to conduct tissue culture as well asAgrobacterium- and nanoparticle-mediated genetic transformations of hemp.The graduate student will test the efficiencies of the three selectedgRNAs for the knock-out of the THCAS gene in hemp througha CRISPR-CAS9 by the using hemp protoplasts. The most efficient will then be selected for Agrobacterium- and nanoparticle-mediated genetic transformations. The graduate student will also optimizeshoot regeneration by utilizing theCsGRF3-CsGIF1-GR construct. The undergraduate student will focus on the design and cloningof a GPPS-SSUoverexpressing construct that subsequently will be used for hemp transformation.
Impacts
What was accomplished under these goals?
Despite significant delays and restrictions due to the ongoing Covid crisis, the following specific goalswere achievedfor the three proposed objectives of this project in the current reporting period: Objective 1 -Characterize the accumulation of terpenes and cannabinoids in industrial hemp under different abiotic and biotic stress conditions: For the proposed analyses of the effects of various environmental stress conditions on the accumulation of terpenes and cannabinoids in hemp, the Gutensohn lab at WVU has assembled a collection of several seed/fiber, CBD and CBG hemp varieties. For a first set of analysis these varieties were grown from seeds under greenhouse conditions. These hemp plants were treated with jasmonic acid and salicylic acid to mimic potential attacks by pests and pathogens. Plant material collected from control and treated hemp plants wassubsequently utilized to establish all the methods required for the analysis of terpenes and cannabinoids by GC-MS and UHPLC. The identity and quantities of individual compounds were verified by using authentic terpene and cannabinoid standards. While these analyses demonstratedthat the accumulation of individual terpene and cannabinoid compounds wasindeed affected by the jasmonic acid and salicylic acid treatment, individual plants of the same hemp variety despite showing the same trend upon a stress treatment varied significantly in the absolute amounts of the analyzed terpene and cannabinoid compounds. Since these results suggested a larger genetic variability among individual plants of the same hemp variety, we decided for all further analyses to propagate hemp plants via cuttings and re-rooting from one parental plant grown from seed, thus eliminating thepotential genetic variability. We are now in the process of repeating thejasmonic acid and salicylic acidtreatments with a set of hemp plants derived from cuttings. In addition, we are currently growing a second set of hemp plants derived from cuttings in a growth chamber at WVU and will expose these to different light conditions to analyze the effect of light stress. Moreover, the Gutensohn lab at WVU has acquired adrip-irrigation system and respective moisture sensors, and is currently establishing this systemin the greenhouse in preparation for the drought, nutrition and salinity stress treatments of hemp plants. In a pilot study the Gutensohn lab at WVU also started to analyze the effects of fertilizer application and the presence ofmycorrhizal fungi on the formation of terpenes and cannabinoids in one hemp variety. Objective 2-Characterize the expression level and genetic regulation of key terpene and cannabinoid biosynthetic genes in industrial hemp under different abiotic and biotic stress conditions:Plant material collected by the Gutensohn lab at WVU from the first set of control and jasmonic acid and salicylic acid treated hemp plants (see above) was also utilized to isolate total RNA. Primers were designed for a first set of terpene and cannabinoid biosynthetic genes and were successfully tested in quantitative RT-PCR analysis using the above isolated RNA. Primers for additional candidate genes are currently being designed and tested, thus further completing the available methods and protocols that will be required for the analysis of hemp under various stress conditions. Objective 3-Determine the effect of knockout and overexpression of key biosynthetic genes on the accumulation of metabolites in the terpene and cannabinoid biosynthetic network in industrial hemp:For the proposed knock-out of the THCAS gene in hemp througha CRISPR-CAS9 approach, the Kovinich lab at York University has confirmed genomic gRNA targets via sequencing in three differenthemp varieties. Subsequently three DNA constructs were assembled encoding different gRNAs that can target the THCAS sites in all three hemp varieties. The Kovinich lab has also established a method to produce callus from hemp seedling hypocotyls and has attempted to establish anAgrobacterium-mediated transformation protocol. However, so far the tested hempvarieties have failed to produce shoots according the previously published protocols. To overcome this problem and enhance the frequency of shoot regeneration, an expressionconstruct was assembled that contains a translational fusion of two transcription factors (CsGRF3 and CsGIF1) and a glucocorticoid receptor (GR). In addition, the Kovinich lab in collaboration with the Wu lab at the University of Massachusetts at Amherst has developed a protocol to couple mutiple plasmids to nanoparticles and use these subsequently for the transient transformation of hemp leaves, which was recently published in the Journal of Biotechnology. This approach resulted in the successful and rapid transient expression of multiple transgenes, and can now be used for the development and testing of knock-out and overexpression constructs for terpene and cannabinoid biosynthetic genes in hemp as well as ultimately the isolation of stable transgenic hemp lines.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Ahmed S., Gao X., Jahan A., Adams M., Wu N. and Kovinich N. (2021) Nanoparticle-based genetic transformation of Cannabis sativa. Journal of Biotechnology 326, 48-51.
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