Location: Vegetable Crops Research
Project Number: 5090-21000-073-079-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 16, 2022
End Date: Dec 31, 2025
Objective:
Our ongoing work has shown that a mutation in the cucumber CsSVL (Cucumis sativus speckled and virescent leaf) gene confers resistance to multiple foliage pathogens and that, among other genes, a mutation in the gene for an invertase enzyme confers variation in the types and amounts of storage carbohydrates in carrot roots. We will conduct gene editing of CsSVL in cucumber to evaluate its function for multiple disease resistances (MDR), and gene editing of invertase and characterization of other naturally-occurring mutations in carrot genes to evaluate their function for altering carbohydrate profiles in carrot roots.
Approach:
1. We will use the CRISPR/Cas9 system for gene editing to develop knock-out mutants for the cucumber CsSVL gene, and we will use CRISPR-Cas9 ribonucleoprotein complexes (RNPs) for editing the carrot invertase gene. Plasmid constructs carrying the Cas9, gRNA and report gene modules will be assembled in the backbone with the Agrobacterium-mediated plant transformation using the Golden Gate Assembly in cucumber. Different promoter sequences will be tested to optimize the constructs. The utility of RUBY as a reporter will be tested.
2. For genetic transformation, cotyledonary nodes from germinating cucumber seeds and protoplasts of carrots will be used. The cucumber explants will be cocultured with Agrobacterium suspension carrying the plasmid construct with CsSVL. The carrot protoplasts will be transfected with PEG and RNP-RNA complexes.
3. After successful Agrobacterium infection or transfection, explants will be transferred onto shoot initiation growth media containing selective antibiotics. The shoot generation and selection will be performed for a few rounds through subcultures. Rooting from the shoots will be initiated on rooting media for generation of plantlets.
4. Regenerated cucumber plants will be subjected to various tests including validation of target genes through PCR, examination of gene editing efficiency through DNA sequencing, and self-pollination to generate T1 progeny. These experiments will be performed again in T1 plants. Multiple disease resistances of T1 plants will be tested by inoculation with downy mildew, powdery mildew and target leaf spot pathogens in controlled environments. Regenerated carrot plants will be grown to form mature taproots, and the sugar profile of those taproots will be evaluated to confirm editing.
5. In addition to characterizing variation due to editing the carrot invertase enzyme, naturally-occurring variation in diverse carrot germplasm identified by GWAS and QTL analysis will be chromatographically characterized.
