Controlling GRF4 ‐ GIF1 expression for efficient, genotype‐independent transformation across wheat cultivars

SUMMARY Wheat is a staple crop critical for global food security, and its continuous genetic improvement is essential to meet the demands of a growing population. Efficient, genotype‐independent transformation is a major bottleneck in wheat functional genomics and gene editing. The growth regulating factor (GRF)–GRF‐interacting factor (GIF) fusion technology enhances regeneration efficiency and broadens the range of transformable cultivars, but constitutive expression can reduce fertility and spikelet number. Here, we present an optimised Agrobacterium ‐mediated wheat transformation protocol incorporating GRF4‐GIF1 , tested across multiple tetraploid and hexaploid cultivars. Transformation efficiency was improved through adjustments in selection pressure, zeatin concentration, and promoter choice, with GRF4‐GIF1 consistently enabling successful transformation across genotypes. Tissue‐specific promoters and heat‐inducible excision strategies effectively minimised pleiotropic effects, such as reduced fertility, while maintaining high transformation rates. This refined system provides a robust and versatile platform for gene function studies and gene editing, advancing genotype‐independent wheat transformation and supporting breeding efforts to improve crop productivity, resilience, and nutritional value.