Genotype-flexible plant genetic transformation: advances and prospects

Genetic transformation of elite crop varieties remains limited by genotype-specific recalcitrance and dependence on tissue culture. This review provides a comprehensive analysis of current transformation platforms and emerging strategies to overcome these bottlenecks. We examine traditional Agrobacterium-mediated and biolistic methods, then critically assess tissue culture-free approaches including floral-based delivery, cut-dip-budding, in planta injection, and viral vector-mediated transformation systems. A major focus is the deployment of developmental regulators-WUS/BBM, GRF-GIF chimeras, DOF transcription factors, LAX1, PLT, and WIND1-that enhance regeneration efficiency across genotypes. We detail their molecular mechanisms, from chromatin remodeling and auxin gradient establishment to wound-responsive cell reprogramming. Importantly, we address the pleiotropic developmental costs of DR misexpression and review precision control technologies, including promoter optimization and auto-excision systems, that enable transient DRs activity during regeneration while ensuring normal plant development. We propose a roadmap for integrating these advances to achieve genotype-flexible, high-throughput transformation applicable to molecular design breeding.(1).