Source: TENNESSEE STATE UNIVERSITY submitted to NRP
TITLE: STRENGTHEN PLANT BIOTECH PROGRAM BY INTEGRATING GENOME EDITING AND ARTIFICIAL INTELLIGENCE TECHNOLOGIES IN TOMATO PROJECTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1028535
Grant No.
2022-38821-37339
Cumulative Award Amt.
$500,000.00
Proposal No.
2021-12803
Multistate No.
(N/A)
Project Start Date
May 1, 2022
Project End Date
Apr 30, 2026
Grant Year
2022
Program Code
[EQ]- Research Project
Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209
Performing Department
Agricultural and Environmental
Non Technical Summary
Globally agricultural production and food security are threatened by the warming temperature. Summer tomato production in the US is facing challenges by heat stress (HS)-induced disruption of pollen production and fruit-set and tomato ripening process. This project aims to develop the research/education capacity for producing CRISPR-Cas9-gene edited mutants to be used for developing heat-stress (HS) resilient tomato varieties; it will prioritize at improving the heat tolerance of male gametophytes with a focus on the most HS-sensitive pollen mother cells. The following three objectives will be achieved: Objective 1). To develop the research platform for producing and characterizing CRISPR/Cas9 tomato mutants, which include three modules: a). Procedures and tools for preparation of constructs, regeneration of transgenic lines and validation of Cas9-edited events and mutations at on/off targeted regions using genotyping; b). Single cell-type transcriptomics and proteomics analysis of pollen cells in mutant lines to confirm mutations of the targeted genes at transcript and protein levels; and to identify differential expression in transcripts/proteins between mutant and wild-type lines thus defining the function of the gene mutation related to HS tolerance; c). Developing a AI machine learning (ML) and deep learning (DL) system for phenotyping pollen heat tolerance; and phenotyping of mutant plants; Objective 2). To generate mutant lines for genes affecting PMCs meiotic activities under HS and select for HS tolerance; Objective 3). To enhance 1890 education/training capacity by training graduate and undergraduate students in biotech and AI technologies. Project outcomes will strengthen the Agricultural Biotech program at 1890s.
Animal Health Component
50%
Research Effort Categories
Basic
30%
Applied
50%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011460105030%
2031460106030%
2061460108130%
2061460102010%
Knowledge Area
206 - Basic Plant Biology; 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1460 - Tomato;

Field Of Science
1060 - Biology (whole systems); 1081 - Breeding; 1050 - Developmental biology; 1020 - Physiology;
Goals / Objectives
The goals of the projectare to use the CRISPR/Cas9 gene editing technology to produce heat tolerant traits for tomatoes; and use artificial intelligence (AI) to develop a high throughput pollen phenotyping program; and tobuild the research/training capacity in gene editing and AI at 1890s.The three specific objectives are: 1).To develop a working system for producing and characterizing CRISPR/Cas9 tomato mutants. Two transcription factor (TF) genes including HeatTF1 (Solyc08g016580.1.1, HS-upregulated in pollen mother cells (PMCs)) and HeatTF2 (Solyc06g053350, HS-down-regulated in PMCs) will be the targeted genes to develop the following modules: a) Experimental procedures for producing CRISPR/Cas9 mutant lines; b). Genotyping on/off target mutations; c) A high throughput phenotyping system for pollen heat tolerance using AI machine learning (ML) and deep learning (DL) technologies; d) Phenotyping for heat tolerance; e). Characterization of cell-type specific transcriptomes and proteomes of male gametophytes in mutant lines; f) Integrated data analysis to determine the function of gene mutation in affecting heat tolerance of pollen and other traits. 2). To generate CRISPR/Cas9 constructs/mutant plants for a list of HS-regulated genes and to select heat tolerant mutants. 3). To enhance 1890 education/training capacity using the project platform.
Project Methods
The project is divided into the following two major phases. Phase I will focus on developing the research methods for producing and characterizing CRIPSR/Cas9 edited tomato plants. Phase II aims to produce more mutants with desirable traits.During Phase I, a fewHS-responsive geneswill be targeted to generate CRISPR/Cas9mutant lines. The lab will develop a work-flow for gRNA design,preparation of CRISPR/Cas9 constructs, transformation of tomatoes using the cotyledon-inoculation methods, regeneration of gene-edited plants on culture media supplemented with combinations of auxin and zeatin, genotyping of the mutant lines for the Cas9 gene and targeted gene using Sanger sequencing of PCR products amplified using primers specific to the gene sequences. Positive plants containing Cas9 and desirable edited sequenceof the targeted gene will beselected inT0 generation.The T0 plants will be transplanted into greenhouse and self-pollinated to produce T1 and more advanced generations. These plants will be validated for Cas9 using PCR and Sanger sequencing. Negative Cas9 PCRresults indicate that the Cas9 gene has been segregated out of the plant genome, and these transgene-free plants will be selected for genotyping the targeted gene. For genotyping the on-target mutations, PCR will be performed using primers flanking the gene regions targeted by the gRNA(s) followed by Sanger sequencing. The same genotyping process will continue through multiple generations until homozygous lines for the targeted gene are obtained. Off-target mutation will also be examined.Finally, tomato lines containing desirable edited sequences in the targeted regionswill be selected for phenotyping.Phenotypingis divided into two parts: phenotyping for pollen heat tolerance and phenotyping at a whole plant level. For pollen analysis, a standard procedure for pollen sample preparation and image data collection will be developed usingtomato 'Money Maker' WT plants grown at 25/20oC (12/12 hr; day/night). Pollen will be collected from 10 flowers and suspended in a final volume of 100 µL solution. For pollen viability assay, 10 µL (representing pollen per flower)will be doubled stained with FDA (25 µg/ml final concentration) andPI (20 µg/ml final concentration) and imaged under GFP (green) filter for viable pollen and RFP (red) for non-viable pollen. A master image containing a number of tile images will be generated. For pollen germination assay, the germinated pollens have a head-tail structure and non-germinated ones remain as round structure. Amachine learning (ML) based software tool will be optimized for the sole purpose of automatic tracking and counting of viable, non-viable, and germinated pollens from image tiles. The goal of the custom software tool is to significantly reduce manual labor, processing time, and improve the counting accuracy compared to the generic image processing application, ImageJ. The software development task will be accomplished in two phases. First, we will optimize a recently developed pilot program to track and count pollens using advanced computer vision (CV) and ML techniques. Our program automatically separates the color channels and then uses a sequence of ML and CV routines, including Gaussian image filtering, connected component search, morphological operation such as erosion to eliminate debris, dilation to integrate pixelated pollens, background segmentation, and pollen shape matching. These sequential computing steps will remove complex background, debris and non-pollen artifacts from image samples. The centroids of individual pollens will then be tracked on the pixel coordinate and tagged with a unique number.The pilot program, before any optimization, takes less than 3 s per image tile to yield 100% and ~70% accuracies in counting the viable (green) and nonviable (red) pollens, respectively. Notably, red pollens have a challenging background in the presence of ambiguous non-pollen objects and debris. In the next step, the parameters tuned in sequential computing steps will be optimized across hundreds of image tiles to obtain the best parameter setting for this application. To yield a robust and augmented performance, we will train advanced ML and deep learning (DL) models with individual pollen and non-pollen image samples of various shapes and types. The trained model is expected to classify and locate three objects: viable pollens, non-viable pollens, non-pollen, on the pixel coordinate. The program will label individual pollens and display the count for each of the three objects. For analysis of pollen germination, germinated pollen will be assigned to those with a head-tail (> 25µm in length) fluorescence structure, and those round pollen grains will be assigned as non-germinated pollen. Model development, training, and final application will follow the same procedure as for pollen counts. In the second phase, following the testing and optimization, the program will be packaged into a web-based application with a graphical user interface (GUI) for agricultural researchers and trainees. The GUI will allow users to upload hundreds of images to display the total counts, counts per tile, and tiles with tracked pollen contours.The developed ML program will be used in phenotyping pollen heattolerance. Pollen will be harvested after subjecting plants to 30/24oC, 32/24oC, 34/26oC, 36/ 28oC, 38/ 30oC; the higher temperature (day)will be set from 10am-5pm, and the lower temperature (night) from 5pm to 10 am. The LD50 temperature of viable pollen percentage and pollen germination rate will be determined for the gene-edited lines. The gene-edited plants will also be evaluatedforleaf photosynthetic activity,the total number of flowers in the 3rd-5thclusters,fruit set %= (number of fruits/number of flowers) X 100; and fruit quality. Data fromexperiments described above will be converted into a heat tolerance index (HTI) matrix to compare the heat tolerance level among different mutant lines for individual or multiple traits.HTI will be calculate using the following formula: HTI = (YMutant/YWT) × (HRCMutant/HRCWT); Where YMutantand YWTare data from mutant or WT under HS condition; HRC=Y/YP, where, Y is the data under HS and Yp is data under control condition.The heat tolerant lines will be subjected to single cell type transcriptomic and proteomics analysis.Flower buds will be divided into fourgroups based on the base-tip length after removal of the sepals: 1.5- 3.5 ±0.2 mm (PMC); 4.0±0.2 mm (tetrads); 4.5 ± 0.2 mm (microspore), and immediately prior to open-flower. Laser capture microdissection (LCM) will be used to collect homogenous pollen samples, followed by transcriptome and proteomics analysis. Genes and proteins showing significant differences between gene-edited and WT plants underheat treated conditions will be interpreted as being different due to the effect of gene mutation.A method for integrateddataanalysis will be developed to determine the function of the targeted genes in conferring phenotypic HS tolerance and affecting whole genome expression. First, the DEGs (from the transcriptome analysis) and the DEPs (from the proteomics analysis) will be compared and divided into the following three categories: genes identified in both analysis, genes identified only as DEPs, and genes identified only as DEGs. The affected biological process and role of the edited genes will be developed through bioinformatics analysis.During the second phase of the project, we will generate a large population of gene-edited tomato lines using genes that have been identified from studies of tomato pollen heat tolerance.The project will be used topromote student success by recruting/graduatingPh.D. and MS students students, and train undergraduate students through the collective efforts of themultidisciplinary project team members.

Progress 05/01/24 to 04/30/25

Outputs
Target Audience:The first group of targeted audiences for this project included research faculty and students. Students involved in the project made seven presentations at the 2025 University-wide Research Symposium at Tennessee State University. In addition, graduate students presented their work at national and international conferences through both oral and poster presentations. Faculty members have integrated gene-editing technology into their teaching at both the undergraduate and graduate levels. A total of four graduate students and one undergraduate student participated in the project, gaining valuable skills and knowledge in gene editing, genotyping, tomato genetics, and the selection of transgene-free gene-edited plants. This hands-on experience has prepared them to advance their careers and contribute meaningfully to future research and development in agriculture, biotechnology, and related fields. Results from the project, along with the associated databases, have been published to enable the scientific community to access and utilize these valuable resources. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided significant support for graduate student training and development. It supported the thesis and dissertation research of two graduate students, who successfully graduated with M.S. and Ph.D. degrees in Summer 2025. Two new M.S. students have since been recruited and are currently continuing the research initiated under this project. Additionally, the project has enabled graduate students to attend and present their work at multiple scientific meetings, enhancing their professional development and visibility in the field. In Spring 2025, TSU faculty and three graduate students participated in a one-week proteomics training at the USDA-ARS laboratory, further strengthening the project's capacity for advanced molecular analysis. How have the results been disseminated to communities of interest?The project team, including faculty and graduate students, has delivered seven presentations at national and international conferences, on-campus seminars, and field days focused on tomato growers. These outreach efforts have reached an audience of over 500 individuals, including university faculty, research scientists, students, industry professionals, and tomato growers. One journal article has been published as a result of this work. TSU faculty and graduate students have also conducted multiple presentations for visitors to our labs and introduced the tomato gene-editing system to local farmers during site visits to their operations. Additionally, faculty has incorporated the project as a case study in a graduate-level 'Omics' course to demonstrate the characterization of gene mutations using omics technologies. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete phenotyping for heat stress tolerance and other abiotic stress factors in the existing gene-edited lines, accompanied by comprehensive 'omics' analyses (transcriptomics and proteomics, metabolomics) for selected lines that show promising phenotypic responses. If any of the lines demonstrate significant agronomic or commercial potential, we will initiate preparations to apply for a conditional patent to protect the intellectual property associated with these novel tomato lines.

Impacts
What was accomplished under these goals? During the previous project period, we generated three homozygous CRISPR-Cas9 knockout lines targeting a bHLH transcription factor gene in tomato (accession Solyc01g058670), using the variety 'Black Vernissage'. The mutation introduced a premature stop codon, resulting in the production of a truncated protein lacking the functional domain. Two of the lines were propagated for seed production and used for phenotyping. Phenotypic analyses were conducted to evaluate fruit set, seed size, and plant growth under various abiotic stresses, including salt, drought, aluminum toxicity, acidic pH, and high temperature. In general, the gene-edited lines showed a reduced fruit set rate but larger seed size, and leaf tissues contained higher calcium content compared to wild-type (WT) plants. Tolerance to acidic pH and aluminum (Al) toxicity was assessed using a hydroponic system. Root trait analysis was performed using WinRHIZO imaging, along with measurements of shoot length and fresh biomass. Results showed that the gene-edited lines were more sensitive to acidic pH (pH 4.0) and high Al concentration (70 μM AlCl3). At lower Al concentration (10 μM AlCl3), there were no significant differences in root length or total root area between the gene-edited lines and WT. Electrolyte leakage assays revealed no significant differences at 10 μM AlCl3, but higher leakage in gene-edited linesunder 70 μM AlCl3. Root-tip tissues from the acidic pH and Al treated conditionshave been submitted for transcriptomic and proteomic analysis. Phenotyping for salt stress (200 mM NaCl) and drought stress (by withholding water until leaf wilting) was conducted in pots. Under control conditions, gene-edited lines were taller and showed more vigorous growth compared to WT. However, under salt and drought stress, the gene-edited lines showed higher electrolyte leakage in leaves. Leaf tissues from these treatments have been submitted for proteomic analysis. Phenotyping for heat tolerance is currently underway. Through these efforts, we have successfully developed a comprehensive system for CRISPR-Cas9 gene editing in tomato, including the generation of homozygous mutant lines and multi-stress phenotyping pipelines. This platform will support functional characterization of stress-related genes and the development of improved, stress-resilient tomato varieties. In addition, the CRISPR-Cas9 mutant plants have been generated for additional five genes involved in fruit ripening and secondary metabolite pathways, using three heat-tolerant tomato varieties: 'Black Vernissage', 'LA2662', and 'LA3242'. While these varieties are valuable for research due to their resilience to heat stress, each has traits that limit their suitability for commercial production. Our goal is to develop tomato lines that combine heat tolerance with agronomically desirable characteristics. The gene-edited plants are currently undergoing genotyping/phenotyping analysis. In our initial attempt to knock out the two heat stress-related transcription factors (TFs), Solyc08g016580 and Solyc06g053350, we were unable to successfully disrupt the function of the targeted genes. We are currently preparing new CRISPR/Cas9 constructs with alternative guide RNAs (gRNAs) to generate knock-out lines for both TF genes.

Publications

  • Type: Other Journal Articles Status: Published Year Published: 2025 Citation: Thapa P, Guo J, Pradhan K, Thapa D, Madhavarapu S, Zou J, Potts J, Li H, O'Hair J, Wang C, Zhou S, Yang Y, Fish T, Thannhauser TW. Cell-Type-Specific Heat-Induced Changes in the Proteomes of Pollen Mother Cells and Microspores Provide New Insights into Tomato Pollen Production Under Elevated Temperature. Proteomes. 2025 Mar 25;13(2):13. doi: 10.3390/proteomes13020013.
  • Type: Theses/Dissertations Status: Under Review Year Published: 2025 Citation: Dibya Thapa Magar. Collection of germinating tomato pollen using laser capture microdissection for heat induced proteomes
  • Type: Theses/Dissertations Status: Under Review Year Published: 2025 Citation: Jun Guo. CRISPR/Cas9-Based Gene Editing in Tomato: Platform Development and Phenotypic Characterization Under Stress Conditions
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: J. Guo*, K. Pradhan, D. Jain, J. Potts, H. Li, S. Roy, S. Zhou. (2024). Characterization of Tomato bHLH148 in Aluminum Stress Response Through CRISPR-Cas9 Mutagenesis. Poster presentation American Society of Plant Biology (ASPB) 2024 Conference, June 22-26,2024. Hawaii Convention Center, Honolulu, HI
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: J. Guo*, K. Pradhan, D. Jain, J. Potts, H. Li, S. Roy, S. Zhou. (2025). Developing Gene Editing System for Tomatoes to Generate Stress Tolerant Lines. Presented at Department of Agricultural sciences and Engineering Seminar. Nashville, TN.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Sudhakar, Madhavarapu*, Jing Zou, Jun Guo, Roy Sonali**, Suping Zhou**(2025). CRISPR-Cas9-Based Modification of Tomato Metabolism for Improved Varieties. Presented Poster at TSU Research Symposium, Department of Agricultural sciences and Engineering Seminar. Nashville, TN
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Sudhakar, Madhavarapu*, Jing Zou, Jun Guo, Roy Sonali**, Suping Zhou**(2025) CRISPR-Cas9-Based Modification of Tomato Metabolism for Improved Varieties. HBCU Climate Change Conference, Oral Presentation, New Orleans Louisiana.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Jing zou*, Sudhakar Madhavarapu, Jun Guo. STUDIES OF STIGMA RECEPTIVITY OF TOMATOES DURING TOMATO FLOWER DEVELOPMENTAL PROCESS AND UNDER HEAT STRESS CONDITIONS Agricultural and Environmental Sciences, College of Agriculture. Advisor(s): Suping Zhou. 47th Annual Tennessee State University-Wide Research Symposium. March 24-28,2025. Nashville, TN
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Sudhakar Madhavarapu*. SUPPORTING LOCAL NURSERIES AND GARDEN CENTERS IN MITIGATING THE IMPACT OF THE COVID-19 PANDEMIC Agricultural and Environmental Sciences, College of Agriculture. Advisor(s): Suping Zhou. 47th Annual Tennessee State University-Wide Research Symposium. March 24-28,2025. Nashville, TN


Progress 05/01/23 to 04/30/24

Outputs
Target Audience:1. Graduate and undergraduate students to attact them into the Agricultural Biotechnology fields; 2. High school teachers and students as a means to advertise theAgriculture program and to attrack youth to pursure Agriculture degree; 3. University faculty and students to share the knowlege and technology advancement developed on this project.and to receive their feedbacks; 4. Farmers and the generatl public to seek their support. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this project periods, six graduate students, five from College of Agriculture and one from College of Enginerringhave receiving training in gene-editing, and artificial intelligence (AI) technology. These students were supported to attend conferences. How have the results been disseminated to communities of interest?Five graduate students presented their research results at two conferences: the 46th Annual Tennessee State University-wide Research Symposium in March 2024, and the 2024 1890 Association of Research Directors in April 2024. These conferences were attended by thousands of people, including faculty members and students from 1890 universities, administrators, and business partners. The PIs hosted 10 groups of visitors, including representatives from federal grant management offices such as NIFA, local administration, faculty and graduate students from other universities, parents, high school students and science teachers, non-profit organizations, private businesses like Bayer, and local farmers. What do you plan to do during the next reporting period to accomplish the goals?1. For the gene-edited lines, we will continue phenotyping, conduct cell-specific transcriptomics analysis to identify genes regulated by the transcription factor in the loss-of-function mutant lines, and develop the SWATH-MS proteomics system to assess the effects of the mutation on whole-genome expression. 2. We have three more genes in the pipeline for generating gene-edited plants. The T0 plants with knock-out mutations of the targeted genes will be transplanted to the greenhouse, and homozygous plants will be selected in the T1-T3 generations. 3. We will gradduate one Ph.D. and one MS. students in the next reporting period. Two new MS students will start working in the Fall, 2024. These students will be trained in the gene-editing technology and AI application in tomato project.

Impacts
What was accomplished under these goals? 1. During this report period, we have obtaiend homogenous linesfor mututans of a transcription factor generated using CRISPR-Cas9. Three independent lines have been confirmed to have single mutation, and the Cas9 gene has been segregated out at T3 generation. We are working on characterization of physiological properties of these mutant lines, which include number of seeds/fruits, the size of the seeds, fruit-size, the number of fruits per plant,and fruit quality. These plants are also being evaluated for tolerance to soil salintyand heat stress. 2. The CRISPR-Cas 9 constrcuts for three additional genes have been prepared. Four tomato varieties were inoculated and the cultures are at bud regeneration stage. 3. The AI-poolen counting system has been validated with benchmark data. A manuscript reporting this part of the work has been written andis under internal review. 4. Using laser captture microdissection, we have collected germinatedpollen that were treated with heat stress. These samples are ready for proteomics analysis in the Fall, 2024 when a Barocycler will be installed in our lab. 5.. Two MS students were graduated from the projects in Spring, 2024.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Jun Guo. Advisors: Suping Zhou, Sonali. Roy. Developing Gene Editing System for Tomatoes to Generate Stress Tolerant Lines. 46th Annual Tennessee State University-wide Research Symposium, March 25-29. 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Dibya Thapa Magar*, Shourav Bin Rabbani, Priya Thapa, Kajol Pradhan. Advisor(s): Suping Zhou, Manar Samad Developing an AI-based System for Analysis of Pollen Thermotolerance.46th Annual Tennessee State University-wide Research Symposium, March 25-29. 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Dibya Magar, Jun Guo, Kajol Pradhan, Priya Thapa. Advisors: Suping Zhou, Hui Li, Ted Thannhauser. Proteomics Analysis of Pollen Thermotolerance in Tomato Varieties with Contrasting Heat Tolerance Traits.46th Annual Tennessee State University-wide Research Symposium, March 25-29. 2024.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Magembe EM, Li H, Taheri A, Zhou S, Ghislain M. Identification of T-DNA structure and insertion site in transgenic crops using targeted capture sequencing. Front Plant Sci. 2023 Jul 12;14:1156665. doi: 10.3389/fpls.2023.1156665.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Proteomics Identification of Molecular Mechanisms in Using Essential Oils to Prevent Fruit Decay in Fresh Strawberries. K. Pradhan* and S. Zhou, Department of Agricultural and Environmental Sciences, College of Agriculture, Tennessee State University, Nashville, TN 37209; T. Thannhauser, Y. Yong, and T. Fish, USDA, ARS, Ithaca, NY 14853; and T. Taghavi, Virginia State University, Petersburg, VA 23806. ARD Research Symposium, April 6-9, Nashville, TN.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Proteomics Analysis of Pollen Thermotolerance in Tomato Varieties with Contrasting Heat Tolerance Traits. D.M. Thapa*, J. Guo, K. Pradhan, P. Thapa, H. Li, and S. Zhou, Department of Agricultural Sciences, College of Agriculture, Tennessee State University, Nashville, TN 37209; and T. Thannhauser, USDA, ARS, Ithaca, NY 14853. ARD Research Symposium, April 6-9, Nashville, TN.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Generation and Characterization of CRISPR-Cas9 Gene Edited Tomato Lines. J. Guo*, K. Pradhan, D. Jain, J. Potts, H. Li, S. Roy, and S. Zhou, Department of Agricultural Sciences, College of Agriculture, Tennessee State University, Nashville, TN 37209; and C.Q. Zhang, Alcorn State University, Lorman, MS 39096. Tomato (Solanum lycopersicum) is a globally significant crop, with 200 million tons produced annually. In the U.S. its a $1.3-$1.67 billion industry. Tomato yield and quality are severely affected by suboptimal environmental conditions. In plants, basic helix-loop-helix (bHLH) transcription factors play a role in the responses to a wide array of abiotic and biotic stress factors. The entire tomato genome contains a total of 152 bHLH transcription factors. Previous studies have identified several candidate genes affecting responses to saline and acidic soil conditions and heat stress. This project aims to develop gene-edited lines targeting these candidate genes using CRISPR-Cas9 system and to select for stress tolerant genotypes. Custom-designed guide RNAs (gRNAs) were synthesized and integrated into the pDIRECT_23C binary vector and then transferred into Agrobacterium strain AGL1. Tomato explants were inoculated with Agrobacteria carrying the plasmids, and shoots regenerated on selective culture plates were identified as putative transgenic plants. The integration of T-DNA regions in the tomato genome was verified using PCR analysis of the Cas9 and gRNAs regions. Sequences of the targeted genes were amplified and sequenced using Sanger sequencing. Six individual transgenic lines were confirmed to have insertion/deletion in the gRNA1 targeted region of a bHLH transcription factor (Solyc01g058670.2). These putative gene edited plants (T0) were rooted, transplanted into a greenhouse, and verified for gRNA inserts in the genome and stability of gene mutation. Non-transgenic mutant plants will be selected in T1 and T2 generations, followed by phenotyping for tolerance to soil alkalinity and heat stress. ARD Research Symposium, April 6-9, Nashville, TN.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Developing an AI-Based System for Analysis of Pollen Thermotolerance. D. Thapa*, S.B. Rabbani, P. Thapa, K. Pradhan, J. Guo, M.D. Samad, and S. Zhou, Department of Agricultural and Environmental Sciences, Nashville, TN 37209.ARD Research Symposium, April 6-9, Nashville, TN.


Progress 05/01/22 to 04/30/23

Outputs
Target Audience:During this project period, project directorshave made significant efforts to train the graduate studentson the techniques of developing the gene-editing system. The graduate students are able to use their knowlege and in turn provide training to high school interns. Through the process, these students become very euthusiastic about the research activities. Our collaboraing tomato growers are also very happy to learn thatwe haveproducedthe gene-edited tomatoes(a tangible product). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Five TSU graduate students are involved in the tomato gene-editing project. Three students had received the stipendsto attend CRISPR training sessions offered at regional and national conferences.Four graduate students have participated in developing the machine learning program in which they received mulitple-desciplinary training. How have the results been disseminated to communities of interest?Graduate students have made several presentations to desemminateproject infromationto faculty and students at Tennessee State University. Project director (s) introduced the new technology and project information at national and international events to different groups of audience. What do you plan to do during the next reporting period to accomplish the goals?1. Characteriing the gene-edited tomato lines and select homogygous lines to propagate seeds. 2. Phenotyping for heat tolerance using the customizedpollen count method developed through the project, and phenotyping for other traits. 3. Producing more gene-edited lines.

Impacts
What was accomplished under these goals? Three stress-induciblegenes inclduingBasic Helix Loop Helix (BHLH) (Solyc01g058670.2),Rapid Alkalinization Factor (RAF) (Solyc01g099520.2), Transcription Activator Protein Pur-alpha (PUR) (Solyc01g049680.2) were selected to develop theCRISPR/Cas9 tomato gene editing system. Two gRNAs were designed using the Geneious Prime software, and thencloned onto thepDIRECT_23C vector.The bionary constructs were transferred into Agrobacterium tumefacious AGL1using the electroporation method.Genetic transformation were conducted on four tomato varieties 'Black Vernissage', 'Micro-Tom', 'Money Maker', and 'Brandywine'. All the varieties generated multiple transgenic lines with integration of the selection marker gene. Sequencing results covering the two gRNA targeted regions confirmedthat INDEL inthe gRNA1 region occured only intwo varieties 'Black Vernissage', 'MicroTom'.Three-5independent gene-editedlines were selected for the two varieties.These plants are rooted and they are ready to be transplanted to greenhouse to study the stability/inheritance of the gene-edited events. A new method for pollen counts has been developed. In this method, pollens from a single tomato flower were resuspended in a buffer containing FDA/PI. Pollen suspension (5 ul)were loaded onto a slide and scanned images (with multiple tiles) were generated under a ZEISS microscope. A machine learning program was developed to differentiate pollen from debris based on the shape and separate live and dead pollen based on color (green for live pollen stained with FDA and red for dead pollen stained with PI). Four Master students from Agriculture and Computer Sciences have been working together to develop the program. Their presentation won the first award at TSU research symposium.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Priya Thapa*, Shourav Bin Rabbani*, Kajol Pradhan, Jun Guo, Dibya Thapa Magar, Suping Zhou, Manar Samad, Hui Li, Sonali Roy, "DEVELOPING NEW POLLEN GRAIN COUNT METHOD USING MACHINE LEARNING TO EVALUATE HEAT TOLERANCE OF TOMATOES" (Oral Presentation), 45th Annual University-Wide Research Symposium, Tennessee State University, Nashville, TN, March 28- March 30, 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Jun Guo *, Dybia Jain, Kajol Pradhan. Advisors: Suping Zhou, Sonali RoY. Developing gene editing system for tomatoes to generate stress tolerant lines" along with the information for the student authors and presenters. 45th Annual University-Wide Research Symposium, Tennessee State University, Nashville, TN, March 28- March 30, 2023