Source: UNIV OF MINNESOTA submitted to NRP
GENETIC TRANSFORMATION AND GENE-EDITING FOR GRAIN LEGUMES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1025823
Grant No.
2021-67013-34565
Cumulative Award Amt.
$290,000.00
Proposal No.
2020-05657
Multistate No.
(N/A)
Project Start Date
Apr 1, 2021
Project End Date
Mar 31, 2023
Grant Year
2021
Program Code
[A1191]- Agricultural Innovation through Gene Editing
Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
Plant & Microbial Biology
Non Technical Summary
Grain legumes constitute major sources of plant dietary protein for human and feed consumption. Currently, generating novel traits in these crops is largely limited to classical mutagenesis and genomics-assisted breeding methods. Gene-editing offers great potential for accelerating crop improvement through efficient and precise genetic modifications. Two key technological barriers, lack of efficient reagent delivery methods and poor plant regeneration rates, have impeded use of gene editing in these crops. Agrobacterium-mediated transformation has been the preferred method to deliver gene editing reagents in plant species, but has shown little success in grain legumes due to the host range limitation and strong genotype-dependency. Our laboratories have been developing direct delivery methods using biolistics or nanoparticles to overcome these limitations. Efficient gene editing has been achieved by directly delivering CRISPR/Cas9 DNA or ribonucleoproteins via the biolistic-based approach. Moreover, we recently developed a new method to improve plant regeneration using developmental regulators (DRs), such as the Baby Boom (BBM) and Wuschel (Wus) genes, in soybean. In this proposal, we aim to extend our direct delivery and improved plant regeneration methods to two grain legumes, common bean and chickpea. By combining these methods with CRISPR/Cas9 reagents, we will expand the use of genetic transformation and gene editing in grain legumes.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
100%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20114111080100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1411 - Beans (fresh, fresh-processed);

Field Of Science
1080 - Genetics;
Goals / Objectives
The goal of this project is to expand the use of gene-editing technology to agriculturally-important minor crops, such as grain legumes, that are currently not amenable to genetic engineering techniques.The key objectives include:Objective 1. Develop direct reagent delivery methods for genetic transformation andgene editing in grain legumes 1a. Develop the biolistic-based delivery method1b. Deliver gene editing reagents using the biolistic approach1c. Develop the nanoparticle-based direct delivery methodThis objective is to develop a biolistic and nanoparticle mediated direct delivery method for two grain legume species, common bean and chickpea. The CRISPR/Cas9 system will be tested and optimized using this approach to achieve efficient gene editing in somatic cells.Objective 2. Improve plant transformation and gene editing for grain legumes2a. Test developmental regulators (DRs) to enhance transformation efficiency2b. Combine developmental regulators and CRISPR/Cas9 to improve regeneration and gene-editingIn this objective, we aim to improve plant regeneration for common bean and chickpea by testing and optimizing developmental regulators. Our ultimate goal is to combine the functional DRs with the gene editing system to overcome technological barriers of expanding the use of gene editing in grain legumes.
Project Methods
The methods used to achieve each objective are:1a. Develop the biolistic-based delivery methodTo test the biolistic method in different tissues from common bean and chickpea, a series of DNA constructs with the green fluorescent protein (GFP) and the firefly luciferase (Luc) reporter genes will be built. These will include three constitutive promoters, the 35S promoter, the RuBisCO small subunit (rbcS) promoter from chickpea, and the soybean Ubiquitin (Ubi) promoter. The GFP and Luc constructs driven by each promoter will be co-bombarded to identify the best candidate for each tissue. Two different genotypes of common bean, Sedona and G19833 (the reference genome), and chickpea, 'ICCC37' and CDC Frontiers (the reference genome), will be used for the biolistic bombardment. The critical parameters, including bombardment medium composition, DNA/gold particle ratio, and the pressure and distance within the high-pressure helium driven system, will be optimized for each tissue.1b. Deliver gene editing reagents using the biolistic approachFollowing the optimization of the biolistic delivery method using the reporter genes, the objective in this sub-aim is to test the gene editing reagents. Our laboratories have developed the multiplexed CRISPR/Cas9 gene editing system in many plant species including soybean. To test the efficiency of this system in common bean and chickpea, the CRISPR/Cas9 expression constructs will be built with the promoters identified in the aim 1a. For gene targets, we will choose two genes, the PDS gene and the Fatty Acid Desaturase 2 (FAD2) gene for the following reasons. First, knocking out these genes gives rise to the easy-to-score phenotypes. The PDS mutants display the albino phenotype, as shown in our rice study, that can serve as a visual marker to quickly assess gene editing efficiency; the FAD2 mutants could lead to fatty acid composition alteration in seeds that can be readily assayed using the methods developed by the Co-PI, Dr. Robert Stupar. Second, Several CRISPR gRNAs for these genes have been designed and demonstrated with high efficiency in soybean by our laboratories and other groups. Further analysis showed that the sequences of these genes are highly conserved between soybean, common bean and chickpea with only 2-3 bp variations in the gRNA target sites. New gRNAs will be redesigned with these small variations to target the PDS and FAD2 genes in common bean and chickpea.The CRISPR/Cas9 constructs will contain the three expression components, Cas9, CRISPR gRNA and the GFP reporter gene. In this proposal, we will also include the Trex2 gene in our gene editing construct for the test. The individual construct will be delivered into the same tissues tested in the aim 1a using the optimized biolistic approach. The GFP reporter gene in each construct will allow for rapid assessments of the success of biolistic delivery. The GFP positive tissues will be cultured for 72 hours, and then subjected to genomic PCR across the targeted sites. Our laboratory has developed a protocol to assess the gene editing efficiency by using PCR amplicon next generation sequencing technology (Amplicon-NGS). Each PCR amplicon from the transformed tissues will be sequenced using Illumina pair-end sequencing.1c. Develop the nanoparticle-based direct delivery methodThe biolistic delivery approach can cause tissue damage and is often disruptive to both the transgene and genome. These limitations can be overcome by the emerging nanoparticle-based technology. With the collaboration with Dr. Markita Landry laboratory at University of California, Berkeley, we are testing SWNTs to deliver the GFP reporter genes in tobacco and soybean using their developed protocol. In this sub-aim, we will extend this effort to common bean and chickpea. The DNA constructs containing the GFP reporter genes and gene editing components from aims 1a and 1b will be delivered into both meristematic and non-meristematic tissues used in the prior aims. After 72-hour transformation, microscopy, biochemical and molecular characterization will be conducted on the transformed tissues to assess transformation and gene editing efficiencies.2a. Test developmental regulators to enhance transformation efficiencyAlack of efficient plant regeneration methods is another major technological barrier for expanding the use of gene editing in grain legume crops. The traditional tissue culture techniques involve the exogenous application of plant growth hormones, and show low regeneration efficiency in many grain legumes. To improve plant regeneration efficiency in these agriculturally important crops, we propose to extend the developmental regulator-based method developed in soybean to two grain legumes, common bean and chickpea. In this sub-aim, we will test four DRs, BBM, Wus, STM and Lec1, for promoting plant regeneration in common bean and chickpea. The coding sequences of each gene will be deduced from the soybean genome, directly synthesized and cloned under the control of promoters as described in aim 1a. These DR-expressing DNA constructs will be delivered into both meristematic and non-meristematic tissues, including embryonic apex, cotyledon, hypocotyl, and leaf segments, using both biolistic and SWCNT approaches optimized in aims 1a and 1c. Each DR will be tested individually or in combination. The whole transformation procedure will be conducted without selection. The regenerated shoots will be transferred to a root regeneration medium. The well-rooted plantlets will be transplanted to soil and grown to maturity to collect T1 seeds. Genomic PCR, Southern blot assays and Sanger sequencing will be used to test the presence of transgenes in both T0 and T1 plants.2b. Combine developmental regulators and CRISPR/Cas9 to improve regeneration and gene-editingIn this sub-aim, we will combine the selected DRs with CRISPR/Cas9 to create gene edited mutants in common bean and chickpea. The DNA constructs will be built by cloning the DRs identified from aim 2a with the CRISPR/Cas9 construct tested in aim 1b with the reporter gene. Using the optimized biolistic and SWCNT approaches from the previous aims, these constructs will be delivered into the explants of the tissues showing the highest regeneration frequency in aim 2a. The regenerated shoots will be assessed for transgene integration and gene editing efficiency using the molecular assays described above. The shoots harboring gene edited mutations will be transplanted into soil and grown to maturity. The T1 progeny plants will be genotyped to identify transmission of the transgene and gene edited events in the next generation. If necessary, plants with heritable mutations will be further propagated and segregated to identify transgene-free homozygous mutants in the next generation.

Progress 04/01/21 to 03/31/23

Outputs
Target Audience:Over the course of this project, the first target audience group will be the researchers in the plant research community. It includes not only the researchers who are working on grain legumes, but also anyone who is interested in harnessing the power of plant biotechnology for crop improvement. The second target audience group will be the grain legume breeding community. The technological breakthroughs from this project can expand the use of gene-editing technology and expedite the development of agronomically important traits in these underdeveloped grain legumes. Last but not least, the third target audience will be the general public. Gene editing technologies have not been effectively demonstrated in most grain legumes largely due to the lack of efficient plant transformation methods. Gene-edited grain legume plants produced in this research will be a good educational resource to promote public awareness and acceptance of the gene-editing technology. Changes/Problems:This is a 2-year project that started from 04/01/2021 to 03/31/2023. The majority of thisproject was carried out during the Covid-19 pandemic which caused significant delays in carrying out Objective 1c and Objectives 2a and 2b. In addition, the Postdoctoral researcher. Dr. Suhas Shinde from Texas Tech University, left the position for an industrial job in Oct 2022. This caused additional delays to conduct Objective 2a and 2b and the slow expenditure from Texas Tech University. What opportunities for training and professional development has the project provided?In this 2-year project, two postdoctoral researchers, Vidhya Raman (University of Minnesota) and Suhas Shinde (Texas Tech University), were recruited and trained. They had the opportunity to perform grain legume tissue culture and CRISPR-mediated gene editing. Dr. Raman focused on the common bean while Dr. Shinde worked on chickpeas. This collaborative project between the 2 institutes also provided training for them to gain project planning, organization, and management skills through monthly meetings. These soft skills will be valuable for their future research career. How have the results been disseminated to communities of interest?The promoters and vectors used in this research have been included in the vector toolkit that was published as:Chamness JC, Kumar J, Cruz AJ, Rhuby E, Holum MJ, Cody JP, Tibebu R, Gamo ME, Starker CG, Zhang F, Voytas DF. An extensible vector toolkit and parts library for advanced engineering of plant genomes. Plant Genome. 2023 Jun;16(2):e20312. doi: 10.1002/tpg2.20312. The vector can be requested through Addgene. 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. Develop direct reagent delivery methods for genetic transformation andgene editing in grain legumes (1a.) Develop the biolistic-based delivery method: In this 2-year period, as reported in the first year annual report, the gene gun-mediated (biolistic) transformation protocol was successfully established for the cotyledon and embryonic axis explant for common bean and chickpea. To test the promoter activity, we cloned mGFP6 reporter gene under Glycine max Ubiquitin promoter (names as GmUbi::GFP) and Cicer arietinum rubisco promoter (named as CarbcS::GPF). Additionally, a constitutive promoter 35S was used as a control. These plasmid constructs were used for biolistic transformations. The plasmid DNAs (1 μg each) were coated separately with 1 μM gold microcarrier particles. Then the coated micro-carrier was mounted in the Biolistic® PDS-1000/He particle delivery system (Bio-Rad Laboratories, USA) for the transformation. The microcarrier (particle) bombardments were carried out at helium pressure 1100 psi, with the distance between the macro-carrier and target tissue being 3 cm. The GFP expression in transformed tissues (embryonic axis and cotyledons) was observed every 24 hrs after transformation. Among GmUBI, CarbcS and 35S promoters, both GmUBI and 35S promoters showed higher GFP activity (visible as GFP foci), however, CarbcS promoter failed to show any GFP activity. (1b.) Deliver gene editing reagents using the biolistic approach: following the optimization of the biolistic delivery method using GFP reporter gene, we initiated a experiment to test the gene editing reagents. To test efficiency of this system, the CRISPR/Cas9 expression was built with GmUBI promoter. To achieve the targeted gene knock-out (KO), we chose to target the phytoene desaturase (PDS) gene in common bean and chickpea. Successful KO of the PDS gene would lead to the albino phenotype. Using homology search we identified the PDS homologous gene. Two guide RNAs were then designed for both species. These gRNAs were cloned under CmYLCV promoter and the biolistic transformation will be carried out via co-bombardment with GmUBI::Cas9 and FMV::Trex2 plasmid vectors. This approach, involving co-expression of an exonuclease Trex2 (Three Prime Repair Exonuclease 2) with CRISPR/Cas9, resulted in an increase of gene editing frequencies by 1.3-fold on average (Zhang lab; Weiss et al. 2020). The final assembled construct was transformed on chickpea embryonic axis and young seedlings. We observed several albino sectors on the explant transformed with the CRISPR construct while the no CRISPR vector control did not show white sectors. This result indicated that the gene editing reagents were successfully delivered to the legume explant tissues using the biolistic approach. (1c) Develop the nanoparticle-based direct delivery method: the nanoparticle was made and shared by Dr. Markita Landry lab from UC Berkeley. The nanoparticle-based delivery was performed using the protocol developed by Dr. Landry lab. The construct containing GmUbi::GFP was delivered as the reporter gene into the cotyledon of common bean. Unfortunately, no GFP expression was observed after many attempts of transformation. Objective 2. Improve plant transformation and gene editing for grain legumes (2a) Test developmental regulators to enhance transformation efficiency: the developmental regulator genes namely Wuschle (WUS), Baby-Boom (BBM), Shoot-meristemless (STM), WOX5 (WOX5), and PLETHORA3 (PLT3) were cloned from either soybean (GmWUS, GmWOX5, and GmBBM) or Arabidopsis (AtSTM and AtPLT3). These genes were further sub-cloned into plasmid vectors under high (GmUBI, 35S) or low (NOS) activity promoters to fine tune the gene expression and induce stem cell activity in meristematic or non-meristematic tissues. Currently, we are testing the effect of individual developmental regulator (GmWOX5, GmBBM, GMWUS, AtPLT3 and AtSTM) using embryonic axis explant using the biolistic approach established in Objective 1a. This experiment was initiated on the first quarter of 2023 and currently all transformed tissues are in tissue culture phase. We have not obtained the result on the effect of individual developmental regulators for common bean and chickpea transformation. (2b) Combine developmental regulators and CRISPR/Cas9 to improve regeneration and gene-editing: we have not initiated this experiment as we are still waiting for the data from Objective 2a. As soon as the optimal combination of developmental regulators are identified we will combine them with the CRISPR constructed that were tested in Objective 1b to achieve improved plant regeneration and gene editing.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Chamness JC, Kumar J, Cruz AJ, Rhuby E, Holum MJ, Cody JP, Tibebu R, Gamo ME, Starker CG, Zhang F, Voytas DF. An extensible vector toolkit and parts library for advanced engineering of plant genomes. Plant Genome. 2023 Jun;16(2):e20312. doi: 10.1002/tpg2.20312. Epub 2023 Mar 9. PMID: 36896468.


Progress 04/01/21 to 03/31/22

Outputs
Target Audience:The first target audience group will be the researchers in the plant research community. It includes not only the researchers who are working on grain legumes, but also anyone who is interested in harnessing the power of plant biotechnology for crop improvement. The second target audience group will be the grain legume breeding community. The technological breakthroughs from this project can expand the use of gene-editing technology and expedite the development of agronomically important traits in these underdeveloped grain legumes. Last but not least, the third target audience will be the general public. Gene editing technologies have not been effectively demonstrated in most grain legumes largely due to the lack of efficient plant transformation methods. Gene-edited grain legume plants produced in this research will be a good educational resource to promote public awareness and acceptance of gene-editing technology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two postdoctoral researchers, Vidhya Raman (University of Minnesota) andSuhas Shinde (Texas Tech University),were recruited to work on this project. This project provided training opportunities for both of them to perform grain legume tissue culture and CRISPR-mediated gene editing. I anticipate 1-2 publications could be yielded from this project with them as leading authors. Working on this collaborative project between 2 institutes would also provide training for them to gain project planning, organization, and management skills. These soft skills will be valuable for their future research career. How have the results been disseminated to communities of interest?The results are still preliminary to be disseminated to communities of interest. The postdoc researcher, Vidhya is planning to participate the ASPB conference in the summer of 2022 to present a poster. What do you plan to do during the next reporting period to accomplish the goals?The project is on track to achieve the proposed goals. We don't anticipate any problemsfollowing the proposed plan.

Impacts
What was accomplished under these goals? Objective 1. Develop direct reagent delivery methods for genetic transformation and gene editing in chickpea (CDC-Frontier and Desi cultivar) (1a.) The gene gun-mediated (biolistic) transformation protocol was successfully established for the cotyledon and embryonic axis explant for common bean and chickpea. To test the promoter activity, we cloned mGFP6 reporter gene under Glycine max Ubiquitin promoter (names as GmUbi::GFP) and Cicer arietinum rubisco promoter (Chakrabarti et al. 2016; named as CarbcS::GPF). Additionally, a constitutive promoter 35S was used as a control. These plasmid constructs were used for biolistic transformations. The plasmid DNAs (1 µg each) were coated separately with 1 µM gold microcarrier particles. Then the coated micro-carrier was mounted in the Biolistic® PDS-1000/He particle delivery system (Bio-Rad Laboratories, USA) for the transformation. The microcarrier (particle) bombardments were carried out at helium pressure 1100 psi, with the distance between the macro-carrier and target tissue being 3 cm. The GFP expression in transformed tissues (embryonic axis and cotyledons) was observed every 24 hrs after transformation. Among GmUBI, CarbcS and 35S promoters, both GmUBI and 35S promoters showed higher GFP activity (visible as GFP foci), however, CarbcS promoter failed to show any GFP activity. (1b.) Deliver gene editing reagents using the biolistic approach Following the optimization of the biolistic delivery method using the GFP reporter gene, we initiated an experiment to test the gene-editing reagents. To test the efficiency of this system, the CRISPR/Cas9 expression was built with theGmUBI promoter. For target gene knock-out (KO), we chose thephytoene desaturase (PDS) gene from both species. Using homology serach we identified the PDS gene and two guide RNAs were designed for both species. These gRNAs were cloned under the CmYLCV promoter and the biolistic transformation will be carried out via co-bombardement with GmUBI::Cas9 and FMV::Trex2 plasmid vectors. This approach, involving co-expression of an exonuclease Trex2 (Three Prime Repair Exonuclease 2) with CRISPR/Cas9, resulted in an increase of gene editing frequencies by 1.3 fold on average (Zhang lab; Weiss et al. 2020). Additionally, to understand the gene-editing activity, various promoters will be used to express PDS gRNAs. 1c. Develop the nanoparticle-based direct delivery method: Nothing to report for this task as we focused on the biolistic-based transformation method, which appeared to work well. Objective 2. Improve plant transformation and gene editing for grain legumes 2a. Test developmental regulators to enhance transformation efficiency: The developmental regulator genes namely Wuschle (WUS), Baby-Boom (BBM), Shoot-meristemless (STM) were cloned from either soybean (GmWUS and GmBBM) or Arabidopsis (AtSTM). These genes have beenfurther sub-cloned into plasmid vectors under high (GmUBI) or low (NOS) activity promoters to fine-tune the gene expression and induce stem cell activity in meristematic or non-meristematic tissues. 2b. Combine developmental regulators and CRISPR/Cas9 to improve regeneration and gene-editing Upon the success of objective 2a, this objective will be initiated for chickpea andcommon bean.

Publications