Progress 07/01/21 to 06/04/24
Outputs
Target Audience:The target audience that was the focus of effort for the project was scientists who are trying to acheive gene editing in crop plants. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Dr. Joanna Jelenska had the opportunity to interact with members of the scientific community through on line meetings of researchers interested in gene editing. She also gained valuable mentoring skills, as she helped mentor a graduate student. Dr. Jessica Morgan contributed to this project during her PhD training and she also gained valuable mentoring skills by working with several undergraduates who learned how to use the nanocarbon fibers for delivery of molecules to plant cells. Dr. Morgan leveraged these skills to obtain a job as one of the science outreach coordinators at the University of Chicago. How have the results been disseminated to communities of interest?Dr. Greenberg and Dr. Jelenska participated in 2021, 2022 and 2023 DOE Genomic Science (GSP) andBiomolecular Characterization and Imaging Science (BCIS) ProgramPrincipal Investigator Meetings. Dr. Jelenska participated in a PlantGENE virtual workshop "Working towards tissue culture-free plant transformation and genome editing", March 7, 2023, workshop "High-throughput screening tools for traits and vector elements", June 29, 2023 and PlantGENE Annual Meeting (virtual), Oct. 24, 2023; Keystone Symposia's eSymposia "Plant Genome Engineering: From Lab to Field" and "Precision Engineering of the Genome, Epigenome and Transcriptome", March 8-10, 2021 for which she was awarded a registration grant; and in several ASPB, MPMI and other plant-related virtual seminars. Project personnel participated in several conferences and presented posters listed below. Also, we published a research article anda JoVE article with detailed descriptions of using nanocarbon fibers (see products). Morgan J, Liu D, Jelenska J, Standaert RF, Morrell-Falvey JL, Goddart J, Hensley D, Retterer ST, Greenberg JT. Understanding Plant Signaling via Innovations in Probe Delivery and Imaging. DOE Biological and Environmental Research Meeting, 2022 (virtual). Jelenska J, Davern SM, Morgan J, McKnight TE, Standaert RF, Morrell-Falvey JL, Mirzadeh S, Greenberg JT. Delivery of biomolecules to plants via carbon nanofiber arrays. Keystone Symposia's eSymposia on Plant Genome Engineering, 2021. Morgan J, Jelenska J, Hensley D, Retterer ST, McKnight TE, Morrell-Falvey JL, Robert Standaert RF & Greenberg JT. Transient Transformation of Plants via Nanofibers. Keystone Symposia's eSymposia on Plant Genome Engineering, 2021. 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 goal of this project was to develop the methodology using nanotechnology for editing intact plants, specifically to deliver editing reagents into lettuce using carbon nanofiber arrays (VACNFs). We produced nanofibers with spacing (10-35 µm) and different lengths (15-50 µm) designed to target cells in different plant species. We used fibers with 10 µm pitch to deliver dyes to lettuce leaves, cotyledons and roots, and we used chips with larger pitch for several fruits such as strawberry, apple and peach. Having functional chips, we used them to for transient transformation of lettuce, Nicotiana benthamiana, Arabidopsis and poplar leaves. These results were published in Morgan JM, Jelenska J, Hensley D, Retterer ST, Morrell-Falvey JL, Standaert RF and Greenberg JT (2022). An efficient and broadly applicable method for transient transformation of plants using vertically aligned carbon nanofiber arrays. Front. Plant Sci.13:1051340. doi: 10.3389/fpls.2022.1051340. In order to deliver biomolecules to shoot meristems and other curved organs, we needed VACNFs with a flexible backing. We produced longer (up to 50-60 µm) fibers and transferred them to flexible films in the Oak Ridge National Laboratory Center for Nanomaterials. We developed a protocol for transient transformation and delivery of biomolecules to curved plant organs using flexible VACNF films, which was published in Morgan JM, Jelenska J, Hensley DK, Li P, Srijanto B, Retterer ST, Standaert RF, Morrell-Falvey JL, and Greenberg JT (2023): Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants. J. Vis. Exp.(197), e65602, doi:10.3791/65602. We used VACNFs to deliver DNA encoding Cas9 and 2 guide RNAs targeting phytoenedesaturase (PDS) to cotyledons and meristems of lettuce Cobham Green and Merlot. We also delivered DNA encoding Cas9 and 4 guide RNAs targeting anthocyanin synthesis (ANS) in red lettuce Merlot. Both targeted genes, when edited, should result in visible phenotypes (white and green plants, respectively), therefore, we were not using any selective agents to obtain transgene-free plants. This approach requires high efficiency of regeneration and optimalization of VACNF-mediated delivery of biomolecules. Trials with standard protocols for meristem isolation from lettuce seeds resulted in damage and death after VACNFs application. We developed a new protocol that uses seedlings and auxin treatment to successfully regenerate multiple viable shoots from meristems, however, the developing shoots were fasciated and the plants did not produce seeds. Regeneration from cotyledons and callus often results in somaclonal variation, which we observed as red and green sectors in regenerated Merlot lettuce. Conditions necessary for red/green phenotype screening (light 200 µmol/m2s, temperature 21ºC) had to be optimized in course of the experiments, because standard growth chamber and greenhouse light (70-150 µmol/m2s) was too low and resulted in high color variation in Merlot lettuce. After delivering editing reagents to cotyledons or meristems in multiple trials, we regenerated ~180 Cobham Green and ~180 Merlot plants. We observed 2 very pale seedlings in Cobham Green treated with editing constructs for PDS, 3 pale Merlot plants treated with editing constructs for PDS and 16 mostly green plants treated with editing constructs for ANS. We also regenerated several mixed (green/red or green with red specks) Merlot plants treated with editing constructs for ANS. We analyzed potentially edited plants and controls by PCR with optimized primer pairs, followed by digestion with restriction enzymes in predicted Cas9 cut sites. We did not detect edited plants using this method. We further analyzed plants by sequencing amplicons and deconvolution analysis, similarly as we did in [1]. We did not detect edition events above noise. Editing in lettuce was reported with very low frequency even when selection was used [2]. Future editing experiments could use using plants that express Cas 9 and/or selection, e.g. editing a knockout of LsNCED4 gene which would allow germination at higher temperature or editing a codon in acetolactate gene (ALS) to obtain resistance to herbicide sulfonylurea. Such modifications should increase editing efficiency and recovery of edited plants. Additionally, we performed preliminary experiments with VACNF-mediated DNA delivery to bean meristems, which are larger and more accessible for VACNF chips than lettuce, however bean is more difficult to regenerate. We also started using newly produced flexible VACNF films that should facilitate delivery to very small, round lettuce meristems. In this project we successfully delivered dyes and DNA resulting in transient transformation of multiple plant species and organs. We produced nanofibers on flexible substrate to improve delivery to curved plant surfaces and we improved regeneration from meristems. This research resulted in two publications and three communications.
Publications
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Progress 07/01/22 to 06/30/23
Outputs
Target Audience:The goal of this project is to develop the methodology using nanotechnology for editing intact plants, specifically to deliver editing reagents into lettuce using carbon nanofiber arrays (VACNFs). We successfully modified the nanotechnology to facilitate delivery of reagents to plants. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Dr. Joanna Jelenska improved her mentoring skills by working closely with PhD student Jessica Morgan on the fiber development and implementation. How have the results been disseminated to communities of interest?We have published our work in peer-reviewed journals and participated in PlantGENE workshops on genome editing. What do you plan to do during the next reporting period to accomplish the goals?In future experiments we will use newly produced flexible VACNF films that should facilitate delivery to very small, round lettuce meristems. We will deliver Cas9 protein and guide RNAs instead of DNA constructs in order to improve editing efficiency. We also consider using plants that express Cas 9. With improved delivery and regeneration protocols, in the future we plan to knockout LsNCED4 gene which will allow germination at higher temperature and to replace a codon in acetolactate gene (ALS) to obtain resistance to herbicide sulfonylurea.
Impacts
What was accomplished under these goals?
We produced nanofibers with spacing (10-35 µm) and different lengths (15-50 µm) designed to target cells in different plant species. We used fibers with 10 µm pitch to deliver dyes to lettuce leaves, cotyledons and roots, and we used chips with larger pitch for several fruits such as strawberry, apple and peach. Having functional chips, we used them to for transient transformation of lettuce, Nicotiana benthamiana, Arabidopsis and poplar leaves. These results were published in [Morgan JM, Jelenska J, Hensley D, Retterer ST, Morrell-Falvey JL, Standaert RF and Greenberg JT (2022). An efficient and broadly applicable method for transient transformation of plants using vertically aligned carbon nanofiber arrays. Front. Plant Sci.13:1051340. doi: 10.3389/fpls.2022.1051340]. In order to deliver biomolecules to shoot meristems and other curved organs, we needed VACNFs with a flexible backing. We produced longer (up to 50-60 µm) fibers and transferred them to flexible films in the Oak Ridge National Laboratory Center for Nanomaterials. We developed a protocol for transient transformation and delivery of biomolecules to curved plant organs using flexible VACNF films, which published in [Morgan JM, Jelenska J, Hensley DK, Li P, Srijanto BR, Retterer ST, Standaert RF, Morrell-Falvey JL, and Greenberg JT (2023). Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants. J. Vis. Exp. e65602, doi:10.3791/65602]. We used VACNFs to deliver DNA encoding Cas9 and 2 guide RNAs targeting phytoenedesaturase (PDS) to cotyledons and meristems of lettuce Cobham Green and Merlot. We also delivered DNA encoding Cas9 and 4 guide RNAs targeting anthocyanin synthesis (ANS) in red lettuce Merlot. Both targeted genes, when edited, should result in visible phenotypes (white and green plants, respectively), therefore, we are not using any selective agents to obtain transgene-free plants. This approach requires high efficiency of regeneration and optimalization of VACNF-mediated delivery of biomolecules. Trials with standard protocols for meristem isolation from lettuce seeds resulted in damage and death after VACNFs application. We developed a new protocol that uses seedlings and auxin treatment to regenerate multiple viable shoots from meristems, however it needs further optimalization as the developing shoots are fasciated. Regeneration from cotyledons and callus often results in somaclonal variation. Conditions necessary for red/green phenotype screening (light 200 µmol/m2s, temperature 21ºC) had to be optimized in course of the experiments, because standard growth chamber and greenhouse light (70-150 µmol/m2s) was too low and resulted in high color variation in Merlot lettuce. After delivering editing reagents to cotyledons or meristems in multiple trials, we regenerated ~180 Cobham Green and ~180 Merlot plants. We observed 2 very pale seedlings in Cobham Green treated with editing constructs for PDS, 3 pale Merlot plants treated with editing constructs for PDS and 16 mostly green plants treated with editing constructs for ANS. We also regenerated several mixed (green/red or green with red specks) Merlot plants treated with editing constructs for ANS. Currently, we are analyzing potentially edited plants by PCR, using multiplexing and next-generation sequencing. Additionally, we performed preliminary experiments with VACNF-mediated DNA delivery to bean meristems, which are larger and more accessible for VACNF chips than lettuce, however bean is more difficult to regenerate.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Morgan, J.M., Jelenska, J., Hensley, D., Retterer, S.T., Morrell-Falvey, J.L., Standaert, R.F., Greenberg. J. T. (2022) An efficient and broadly applicable method for transient transformation of plants using vertically aligned carbon nanofiber arrays. Front. Plant Sci.- Plant Physiology. Published 23 November 2022 https://doi.org/10/3389/fpls.2022.1051340
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Morgan, J.M., Jelenska, J., Hensley, D.K., Li, P. Srijanto, B.R., Retterer, S.T., Standaert, Morrell-Falvey, J.L., Greenberg, J.T. (2023) Using vertically aligned carbon nanofiber arrays on rigid and flexible substrates for delivery of biomolecules and dyes to plants JoVE published 27 June 2023 doi 10.3791/65602
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Progress 07/01/21 to 06/30/22
Outputs
Target Audience:The target audience reached by our efforts are collaborators working on editing (via exchange of emails and zoom meetings), and plant scientists attending several meetings (Gordon Conference, Energy Bioscience and BioImaging sponsored by the Department of Energy). Additional target audiences include students at various stages of education, including students at a nearby highschool, undergraduates studying Plant Development (a course taught by PD Jean Greenberg) that includes a discussion of nanomaterials and gene editing and graduate students at the University of Chicago via our "Allstars" series in which Professors give research talks to PhD students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training activities included active mentoring of project personel at weekly group project updates and monthly individual meetings. Preliminary results were presented by Dr. Jelenskaat Keystone eSymposium"Plant Genome Engineering: From Lab to Field": Delivery of biomolecules to plants via carbon nanofiber arrays. Joanna Jelenska received a registration grant for this symposium.She also participated in2022 Biological Systems Science DivisionGenomic Science Program(GSP) and Bioimaging Science Program (BSP) Annual Principal Investigator Meetings, February 22-24, 2021 and February 28-March 3, 2022;IS-MPMI Congress eSymposium "Molecular Mechanism and Structure", July 12-13, 2021 and Virtual MAS-ASPB. How have the results been disseminated to communities of interest?Lab personnel have visited a local middle school to discuss the goals of the project and discuss plants in general. What do you plan to do during the next reporting period to accomplish the goals?We are collaborating with Richard Michelmore's group to try out nanofibers to deliver editing reagents to lettuce using some of the useful germplasm and constructs that his lab has developed.Current experiments focus on meristem isolation and VACNF-mediated delivery of molecules to lettuce apical and/or floral meristems. Immediate plans are to edit (knockout) genes with visible phenotypes: PDS (phytoenedesaturase) in green lettuce, dsRed in transgenic line expressing this marker and anthocyanin synthesis genes in red lettuce. We will use VACNFs to deliver DNA encoding Cas9 and guide RNAs as well as recombinant Cas9 and synthetic guide RNA. In further future we plan to knockout LsNCED4gene which will allow germination at higher temperature and to replace a codon inacetolactate gene (ALS) to obtain resistance to herbicide sulfonylurea. In these cases, progeny of edited plants can be screened for germination at high temperature and herbicide resistance, respectively.
Impacts
What was accomplished under these goals?
Genome editing promises to enable the development of many useful traits in agronomically important plants such as lettuce. Significant goals are (1) to improve the editing pipeline in a manner that avoids mutagenic events and (2) to promote approaches that not only of achieve loss of function gene variants, but also introduce useful allelic variants that improve traits of interest (e.g. biomass, disease resistance, stress tolerance, etc.). The overall goal of this project is to develop methodology using nanotechnology for editing intact plants. We are developing carbon nanofibers that can be used as a way to deliver molecules for editing lettuce. The first task is to test whether nanotechnology can be used to deliver molecules into lettuce without damaging the plants. Once we show we can use nanotechnology, specifically nanofibers, we will stepwise build up to our gene editing goals. The overarching goal is to create lettuce with improved agronomic qualities through genome editing.Based on our positive findings, it is likely that constructs encoding appropriate editing reagents can be delivered to intact meristems to achieve gene editing, thus bypassing tissue culture and problems with regeneration of recalcitrant varieties or plant species. Ribonuclear particles (RNPs) can also likely be delivered together with DNA encoding allelic variants to achieve allele replacements. Transgene-free editing will be attempted using both tissue that can be regenerated and meristems of germinated embryos that will not require regeneration, but will give rise to edited progeny. The method will be broadly applicable as approach can be applied to any crop with a meristem to be accessed by nanocarbon fibers; this could be especially beneficial for fruit trees for example. The method would provide a rapid method for creating transgene-free edited crops. Our specific accomplishments in the first project period were to build new nanofibers for the project, via the Oak Ridge National Laboratory Center for Nanomaterials. Our new fibers were spaced appropriately such that each cell was only impaled by one fiber. The principle is that biomolecules (eventually editing reagents) are pipetted to the plant tissue and the fibers affixed to a chip are tapped into the tissue to permit entry into the cells via capillary action. We established a protocol for delivery of a fluorescent dye Tamra to lettuce cells using vertically aligned carbon nanofiber (VACNF) arrayswith 10 µm pitch. Tamra was detected inside leaf, cotyledon and root cells 5 min - 1 h after delivery via VACNFs but not when a flat side of a chip without fibers was used. We tested dye delivery to apical meristems; however, meristem cells showed high autofluorescence and specific Tamra signal could not be distinguished. Next, several reporters delivered viaAgrobacteriawere tested in lettuce. Their expression was undetectable in lettuce after 2 days, and much lower than in our test modelNicotiana benthamianaafter 3-4 days. mCherry was chosen for testing VACNF-mediated transient transformation.ForN. benthamiana, which is very amenable to transient transformation withAgrobacteria, mCherry fluorescence was lower with VACNFs DNA delivery than agroinfiltration. The effect was opposite for lettuce, where high signal resulted from VACNF-mediated transformation but very low from agroinfiltration.These results are important because they show we can deliver biomolecules to lettuce successfully with out new nanofiber arrays. These results are a part of a manuscript to be submitted to a special issue on Women in Plant Physiology to be published in Frontiers in Plant Science:Jessica M. Morgan, Joanna Jelenska, Dale Hensley, Scott T.Retterer, Jennifer L. Morrell-Falvey, Robert Standaert and Jean T. Greenberg(2022).An efficient and broadly applicable method for transient transformation of plants using vertically aligned carbon nanofiber arrays.
Publications
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