Reconstructing tissue culture to improve Agrobacterium-mediated transformation of maize

Abstract The ability to insert, delete, or modify genetic information is crucial for mechanistic studies and biotechnological applications. However, efficient genetic transformation remains a major bottleneck for research in maize and many other crops. Here, we report an optimized Agrobacterium -mediated transformation platform based on systematic reconstruction of tissue culture handling in the maize inbred line A188. Refinement of callus induction, selection, and regeneration substantially improved recovery of transgenic plantlets. To distinguish independent T-DNA insertion events, we developed TAFLP (T-DNA Amplified Fragment Length Polymorphism), a simple and inexpensive assay that amplifies T-DNA flanking sequences and can be performed using standard laboratory equipment. Our enhanced transformation pipeline was also applicable to the inbred line B104 as well as to hygromycin and G418 selection systems, demonstrating broad utility of our method. We validated the platform for CRISPR/Cas9 mutagenesis and reporter line generation. Using this approach, we isolated new loss-of-function alleles of MAC1 and ACOZ1 and generated reporter lines for analysis of meiotic protein dynamics. Together, these results provide a broadly applicable framework for improving maize transformation efficiency and recovering independent transgenic and genome-edited events.