Protocol for Genome Editing to Produce Multiple Mutants in Wheat

Area of Science:

• Plant genetics
• Molecular biology
• Agricultural science

Background:

• Hexaploid wheat (Triticum aestivum) is a vital global crop.
• Efficient methods for generating gene mutants are crucial for understanding gene function and improving crop traits.
• Existing genome editing techniques may require optimization for polyploid species like wheat.

Purpose of the Study:

• To develop a robust protocol for generating multiple recessive mutants in hexaploid wheat cv. Fielder.
• To enable the creation of all combinations of single, double, and triple gene knockouts.
• To facilitate accelerated generation advancement for faster mutant production.

Main Methods:

• Utilized Agrobacterium-mediated transformation to deliver CRISPR/Cas9 components.
• Employed three sub-genome-specific primer sets for targeted genome editing.
• Incorporated embryo culture techniques to accelerate generation advancement.

Main Results:

• Successfully generated transgene-free single, double, and triple mutants in hexaploid wheat.
• The protocol allows for the production of all possible allelic combinations.
• Accelerated generation advancement significantly reduced the time required for mutant generation.

Conclusions:

• The described protocol provides an efficient method for producing diverse mutants in hexaploid wheat.
• These mutants are valuable resources for functional genomics studies and breeding programs.
• This technique contributes to advancing crop improvement strategies in wheat.