Morphogenic Regulator-Mediated Transformation of Maize Inbred B73

Maize B73 is a reference genome and has long been a major resource for genetics and molecular biology research. We have developed an efficient B73 transformation protocol by enabling somatic embryogenesis through differential co-expression of maize morphogenic regulators BBM and WUS2. We describe a successful protocol that utilizes Agrobacterium tumefaciens strain AGL1 harboring binary vector PHP78891 that comprises a BBM and WUS2 expression cassette as well as a green fluorescent protein (GFP) reporter cassette. The PHP78891 vector also contains, within the T-DNA region, a CRE/lox recombination system flanking the CRE/BBM/WUS2 co-expression cassette driven by the desiccation inducible RAB17 promoter that allows removal of the BBM/WUS2 cassette. Introduction and co-expression of BBM and WUS2 induced direct somatic embryogenesis (SE) in non-regenerable maize B73 from immature embryo explants. Removal of the CRE/BBM/WUS2 cassette is essential to allow regeneration to fertile plants. The GFP expression cassette outside the lox excision sites is retained in the transgenic plant genome, allowing subsequent phenotypic analysis of calli and regenerated transgenic events. This transformation system enables a selectable marker-free transformation process by taking advantage of BBM/WUS2-induced SE as a developmental selection system. © 2018 by John Wiley & Sons, Inc.