Location: Sugarbeet and Potato Research
Project Number: 3060-21650-001-003-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2018
End Date: Dec 31, 2021
Objective:
(1) Develop a transformation system to deliver gene-editing machinery. (2) Develop an efficient gene-editing process.
Approach:
Meristem transformation methods that have been proven successful in soybean will be adapted to common bean and chickpea. This will involve optimization of growth media, development of protocols for explant isolation, development of new methods to effectively deliver editing molecular machinery, and validation of modified plants. The process is initiated with high-quality mature seed lots with high germination potential and ideally minimal pathogen load. Seeds are surface-sanitized in 20% Chlorox, rinsed, and primed for two hours at room temperature. Seeds are then imbibed in germination medium overnight. Meristem explants are prepared the next day by removing seed coats and cotyledons under sterile conditions. Batch cultures of Agrobacterium inoculum are prepared overnight from glycerol stocks and diluted in inoculation medium. Meristem transformation experiments will be conducted using a beta-glucuronidase reporter system to first assess transient expression which tells us that Agrobacterium is delivering our gene into the correct target tissues. Following optimization of the initial gene targeting, we advance to production of stable transgenic plants. Selection of stable transgenics will be done using spectinomycin, with additional selectable markers considered as needed. Once this first phase is completed, we intend to use collaborator gene targets (e.g. 5 genes) in the final proof-of-technology.
