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

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.