Engineered Crispr Pollen For Bioconfinement In Tomato

Project Director
Liu, W.

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
(N/A)

Non Technical Summary
This proposal is going to address Area 1: Management Practices to Minimize Environmental Risk of GE Organisms of the USDA BRAG Standard Research Program. A problem in tomato is the substantial pollen-mediated gene flow (PMGF) from genetically engineered (GE) tomato to non-GE tomato. Using Arabidopsis as a proof-of-concept research, we seek to genetically engineer tomato producing CRISPR pollen that can destroyseeddevelopment in the non-GE plants after double fertilization. The GE lines will be used for selfing or reciprocal crossing with the non-GE Arabidopsis or tomato plants to test the effect onseed abortion. The best line in tomato will be used for field trials to test phenotypic stability and interference with seed production, and measure the extent of PMGF from the GE to non-GE plants in a real world setting. The expected outcome of this project is the engineered CRISPR pollen in each species, which in turn will highly limit PMGF from GE plants without interference with seed production. It could be used as the background source for genetic engineering of any traits. Therefore, the research directly addresses key barriers to the development of regulatory and best management plans and sustainable production in tomato, which could be applicable to other GE crops.

Animal Health Component

80%

Research Effort Categories

Basic

20%

Applied

80%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
204	1460	1040	80%
204	1460	1080	20%

Knowledge Area
204 - Plant Product Quality and Utility (Preharvest);

Subject Of Investigation
1460 - Tomato;

Field Of Science
1040 - Molecular biology; 1080 - Genetics;

Keywords

pollen-mediated gene flow

tomato

Goals / Objectives
We aim to engineer tomato pollen to express Cas/gRNA that would enable restricting PMGF from GE tomato to non-GE tomato. We will use Arabidopsis to conduct a proof of concept study and then transfer the concept to tomato for real-world applications.1. Generation of GE Arabidopsis and tomato producing CRISPR pollen via genetic engineering.2. Testing of the effect of the CRISPR pollen on endosperm lethality and seed abortion in Arabidopsis and tomato under greenhouse conditions.3. Field trials of the extent of PMGF from the GE tomato to non-GE tomato.

Project Methods
1. Generation of GE Arabidopsis and tomato producing CRISPR pollen via genetic engineering. Stable transgenic Arabidopsis and tomato will be generated to express Cas/gRNA. Phenotypic and molecular analysis will be conducted to examine the GE lines.2. Testing of the effect of the CRISPR pollen on endosperm lethality and seed abortion in Arabidopsis and tomato under greenhouse conditions. The GE lines will be used for selfing or reciprocal crossing with non-GE Arabidopsis or tomato plants to test the effect on seed development andabortion. Seed development will be examined, fruit development will be monitored, fruit and seed numbers will be counted, and fruit size and seed weight will be measured.3. Field trials of the extent of PMGF from the GE tomato to non-GE tomato. The best-performing linein tomato will be used for field trials to test phenotypic stability and interference with seed production, and measure the extent of PMGF from the GE to non-GE plants. Hybrid seed abortion will be monitored in the non-GE plants. Seeds will be collected from the non-GE recipients, followed by seed germination and antibiotic selection. PCR will be used to confirm the presence of the transgenes in the antibiotic-resistant seedlings if any.

Progress 07/01/23 to 06/30/24

Outputs
Target Audience:The target audiences for our project are tomato farmers, tomatocompanies, and others in the private sector interested in pollen-mediated gene flow and gene editing in tomatoand other specialtycrops. We expect that we will develop an efficient delivery approach for limiting pollen-mediated gene flow in tomato, which can be transferred to other selfing and outcrossing species. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have provided traning andmentoring to one graduate student, one postdoc, and one technician via one-on-one mentoring and training. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will finish Objective 1 by generating single-copied, homozygous lines for transgenic Arabidopsis and tomato to provide materials for Objective 2.

Impacts
What was accomplished under these goals? 1. Generation of GE Arabidopsis and tomato producing CRISPR pollen via genetic engineering. For promoter analysis, we have cloned 5 promoters from either Arabidopsis or tomato, and have transformed each of them into tomato by conducting Agrobacterium-mediated transformation. We have obtained 5-10 T0 transgenic tomato lines for each promoter. For gene editing in Arabidopsis and tomato, gRNAs have been designed and their cut efficiencies have been tested in vitro. These gRNAs have been cloned into the destination plasmisds. We're in the process of transforming both species with these destination plasmids. 2. Testing of the effect of the CRISPR pollen on endosperm lethality and seed abortion in Arabidopsis and tomato under greenhouse conditions. N/A. 3. Field trials of the extent of PMGF from the GE tomato to non-GE tomato. N/A.

Publications