Location: Crops Pathology and Genetics Research
Project Number: 2032-22000-016-066-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Nov 1, 2019
End Date: Apr 30, 2022
Objective:
1. Development of green flourescent protein (GFP) expressing Phytophthora citricola (P. citricola), which will enable observation of the pathogen in plant tissues.
2. In vitro evaluation of walnut transgenic lines for Phytophthora resistance in tissue culture.
3. Evaluation of walnut transgenic lines for Phytophthora resistance in the greenhouse.
4. Development of almond somatic embryos.
5. Development of an almond transformation system.
Approach:
1) Vectors developed for Phytophthora species will be modified if necessary and amplified in the lab. Agrobacterium-mediated transformation protocols and electroporation protocols will be optimized for P. citricola and transformants generated. Transformants of P. citricola expressing GFP will be tested for virulence on walnut seedlings. inoculation substrate will be mixed with soil and added to pots to which walnut seedlings will be transplanted. Controls will receive sterile substrate.
2) Transgenic somatic embryo lines carrying HIGS constructs previously created will be propagated using standard walnut tissue culture techniques. Embryos will be cultured in media containing plant hormones to induce shoot development. Shoots will then be transferred to individual containers and maintained until they can be tested for resistance to Phytophthora. Transgenic lines of walnut will be inoculated with a GFP-expressing P. citricola. Disease progression inside the plant tissues will be monitored using a fluorescence microscope. Transgenic lines showing strong inhibition of in planta pathogen growth will be selected.
3) Walnut lines that show increased Phytophthora resistance (small or no necrotic lesions) will be propagated and regenerated to whole plants in pots. Transformant lines showing reduced crown and root rot will then be evaluated in the field.
4)Perform crosses of almond and peach varieties. Pollinate Mission almond with pollen from peach varieties that have been proven tolerant to nematodes and other important pathogens.
5)Multiply somatic embryos to obtain sufficient material for the transformation. Conduct Agrobacterium-mediated genetic transformation of somatic embryos with HIGS constructs. Disease resistance, the length of necrotic tissue, fluorescence through microscopic observation of pathogen progression inside the plant tissues will be evaluated.
