* Correspondence: jshch@sdau.edu.cn (J.C.)

Wheat is the second largest food crop in the world, and as living standards improve, the demand for wheat varieties rich in specific nutrients is gradually increasing. Foods with high amylose content have great potential to improve human health, lower glycemic index, and improve gut health.

However, amylose accounts for 15%-26% of the total starch content in common wheat varieties. In this study, we focused on the starch branching enzyme gene TaSBEIIa, using CRISPR/Cas9 gene editing technology to knock out the gene in spring wheat Fielder cultivars. We aim to validate the gene function and develop a new wheat variety with high amylose levels. A total of 68 regenerated seedlings were successfully obtained, with a transformation efficiency of 51.47% and an editing efficiency of 48.53%.

Most of the mutant editing types obtained were 20-58bp large fragment deletions, and a few were single-base insertions. The non-transgenic triple deletion mutants were successfully isolated through screening of progeny, and the amylose content of the mutants increased from 15.41% to 30.47%. The activity of starch branchase (SBE) was found to be significantly lower at 7, 14, and 21 days after flowering compared to the wild type.

The morphology of the mutant starch granules has changed from smooth, spherical, or oval to a highly irregular shape. Most of the A-type starch granules (>10 μm in diameter) now appear sickle-shaped. Currently, gene editing of TaSBEIIa is being conducted on Shannong 44 and Shannong 67, which are new high-quality, high-yield winter wheat varieties. The goal is to develop new non-transgenic wheat germplasm with significantly higher amylose content.

This will enable the production of more cereal products with high amylose content to meet consumer demand. This study also provides a theoretical basis for the feasibility of using gene editing technology to improve the amylose of cereal crops.