An efficient transformation method for genome editing of elite bread wheat cultivars

Frontiers in Plant Science

Plant Science

Kanwarpal S. Dhugga¹  Ana I. R. Castillo¹  L. Ruben B. Hernandez¹  Akshaya K. Biswal¹  Juan M. Debernardi² 

[1] International Maize and Wheat Improvement Center (CIMMYT), Texcoco, Mexico;Plant Transformation Facility, University of California, Davis, Davis, CA, United States;

关键词: wheat;    transformation;    GRF4-GIF1;    particle bombardment;    gene editing;    CRISPR-Cas;    Lr67;    MLO;   

DOI  :  10.3389/fpls.2023.1135047

 received in 2022-12-31, accepted in 2023-04-17,  发布年份 2023

来源: Frontiers

【 摘 要 】

An efficient genetic transformation protocol is necessary to edit genes for trait improvement directly in elite bread wheat cultivars. We used a protein fusion between a wheat growth-regulating factor 4 (GRF4) and its interacting factor (GIF1) to develop a reproducible genetic transformation and regeneration protocol, which we then used to successfully transform elite bread wheat cultivars Baj, Kachu, Morocco, Reedling, RL6077, and Sujata in addition to the experimental cultivar Fielder. Immature embryos were transformed with the vector using particle bombardment method. Transformation frequency increased nearly 60-fold with the GRF4-GIF1-containing vectors as compared to the control vector and ranged from ~5% in the cultivar Kachu to 13% in the cultivar RL6077. We then edited two genes that confer resistance against leaf rust and powdery mildew directly in the aforementioned elite cultivars. A wheat promoter, TaU3 or TaU6, to drive the expression of guide RNA was effective in gene editing whereas the OsU3 promoter failed to generate any edits. Editing efficiency was nearly perfect with the wheat promoters. Our protocol has made it possible to edit genes directly in elite wheat cultivars and would be useful for gene editing in other wheat varieties, which have been recalcitrant to transformation thus far.

【 授权许可】

Unknown   
Copyright © 2023 Biswal, Hernandez, Castillo, Debernardi and Dhugga