A Rapid <i>Agrobacterium</i> -Mediated Transformation Method Using Maize B104 Immature Embryos

Maize genetic transformation is a critical tool for functional genomics and crop improvement. Many laboratories, however, continue to face multiple challenges in attempting to achieve routine genetic transformation of maize inbred genotypes. Here, we describe a rapid and robust maize B104 transformation method using immature embryos as explants. This method uses an <i>Agrobacterium</i> ternary vector system, which includes a conventional T-DNA binary vector (pCBL101-RUBY) and a compatible ternary helper plasmid (pKL2299) that carries extra copies of essential virulence genes. The T-DNA binary vector carries the <i>neomycin phosphotransferase II</i> (<i>NptII</i>) gene for selection and a betalain biosynthesis marker, <i>RUBY,</i> for visual screening. We provide step-by-step instructions for immature embryo explant preparation, <i>Agrobacterium</i> infection, tissue culture procedures, and greenhouse care for acclimatization of regenerated plantlets.