Location: Small Grain and Food Crops Quality Research
Project Number: 3060-21650-002-032-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2021
End Date: Dec 31, 2024
Objective:
(1) Optimize germline transformation systems for common bean and chickpea genotypes to increase the overall transformation efficiencies; (2) Test the optimized protocols across additional genotypes; and (3) Investigate and optimize gene editing systems for target genes using the optimized transformation protocols.
Approach:
Meristem-based transformation methods that have been proven successful in soybean and preliminarily in common bean and chickpea will be optimized via protocol optimization of factors such as growth media, development of protocols for explant isolation and storage, 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, then imbibed in germination medium overnight. Meristem (embryonic axis) explants are prepared the next day by removing seed coats and cotyledons under sterile conditions. Meristem transformation experiments are conducted using reporter constructs containing GUS and/or RFP visual reporter genes and aadA (encoding resistance to spectinomycin) on the T-DNA. The GUS/RFP reporter genes allow detection of plant tissues containing and expressing those genes either transiently after infection or stably in regenerating tissues. Following optimization of the initial DNA delivery steps, we advance to production of stable transgenic plants. Selection of stable transgenics is done using spectinomycin, with additional selectable markers and selection systems investigated for optimization of selection strategies. Once the common bean and chickpea transformation methods are optimized for a cultivar within a species, we will introduce editing constructs containing different editing components to assess the efficiency of different editing systems in other genotypes of that species and work toward optimization. The editing enzyme (and promoters) will be dicot optimized, and the target site verified within that species. Likely targets for initial proof-of-concept work would be a gene affecting leaf carotenoid or chlorophyll synthesis so that there would be a visible white leaf phenotype upon successful editing.
