Establishment of Tissue Culture and Genetic Transformation Systems Using Mature Embryos of Bread Wheat.

Genetic transformation is a core technology for wheat molecular breeding. Transformation efficiency and genotype compatibility determine the pace of targeted genetic improvement. Mature embryos are readily accessible but are associated with low transformation efficiency and strong genotype dependence. Using mature wheat embryos as explants, we screened eight wheat varieties representing three ecological types to develop genotype-adapted tissue culture systems. Treatments A5 (4 mg/L 2,4-D + 2 mg/L Dicamba), A7 (4 mg/L 2,4-D + 6 mg/L Dicamba), and A8 (4 mg/L 2,4-D + 8 mg/L Dicamba) were optimal for callus induction in winter, semi-winter, and spring wheat, respectively. WF3 (a 1/2 MS-based medium) was broadly applicable to callus differentiation, whereas IBA produced the highest rooting efficiency in regenerated plantlets. The optimal transformation conditions comprised ultrasound treatment (2.5 min) followed by vacuum infiltration (5 min), supplementation of the infection solution with PEG6000 (250 mg/L) and Tween20 (0.01%), and an OD600 of 0.6-0.8. Transformation efficiencies using mature embryos of the semi-winter wheat varieties YX288 and ZM578 were 2.5% and 1.8%, respectively. Using this transformation platform, the dwarfing genes Rht12 in ZM578 and Rht8 in YX288 were targeted by CRISPR/Cas9-mediated editing. Multiple single-base substitutions at the target sites were detected in ZM578 rht12 mutants, which exhibited significantly reduced height, increased tiller number, and a compact plant architecture. However, no successfully edited rht8 lines were obtained in YX288. These findings indicate that this study established a reliable mature-embryo transformation system for wheat and successfully validated gene editing in the semi-winter variety ZM578. The system provides a technical platform for the functional analysis of key genes and molecular breeding in bread wheat.