Rapid genotype independent Zea mays L. (maize) transformation via direct somatic embryogenesis

Constitutive expression of the Zea mays L. (maize) morphogenic transcription factors Baby Boom (Bbm) and Wuschel2 (Wus2) in maize can not only greatly increase transformation efficiency but can also induce phenotypic abnormalities and sterility. In an effort to alleviate the pleiotropic effects of constitutive expression, a genome wide search was undertaken to find suitable maize promoters to drive tissue and timing-specific expression of the transformation enhancing genes Bbm and Wus2. A promoter from a maize phospholipid transferase protein gene (Zm-PLTPpro) was identified based on its expression in leaves, embryos, and callus while being downregulated in roots, meristems, and reproductive tissues. When Zm-PLTPpro driving Bbm was transformed into immature maize embryos along with a Wus2 expression cassette driven by the nopaline synthase promoter (Nos(pro)::Wus2) abundant somatic embryos rapidly formed on the scutella. These embryos were individual and uniformly transformed and could be directly germinated into plants without a callus phase. Transformed plants could be sent to the greenhouse in as little as 1 mo and regenerated plants matched the seed-derived phenotype for the inbred and were fertile. However, T1 seed from these plants had poor germination. Replacing Nospro with a maize auxin-inducible promoter (Zm-Axig1(pro)) in combination with ZmPLTP(pro)::Bbm, allowed healthy, fertile plants to be regenerated. Single-copy T1 seed germinated normally and had a predominantly wild-type inbred phenotype. For maize, this callus-free transformation process has worked in all inbred lines tested.