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

Constitutive expression of the <i>Zea mays</i> L. (maize) morphogenic transcription factors <i>Baby Boom</i> (<i>Bbm</i>) and <i>Wuschel2</i> (<i>Wus2</i>) 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 <i>Bbm</i> and <i>Wus2</i>. A promoter from a maize phospholipid transferase protein gene (<i>Zm</i>-<i>PLTP</i>_<i>pro</i> ) was identified based on its expression in leaves, embryos, and callus while being downregulated in roots, meristems, and reproductive tissues. When <i>Zm-PLTP</i>_<i>pro</i> driving <i>Bbm</i> was transformed into immature maize embryos along with a <i>Wus2</i> expression cassette driven by the nopaline synthase promoter (<i>Nos</i>_<i>pro</i> ::<i>Wus2</i>) 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 <i>Nos</i>_<i>pro</i> with a maize auxin-inducible promoter (<i>Zm-Axig1</i>_<i>pro</i> ) in combination with <i>Zm-PLTP</i>_<i>pro</i> ::<i>Bbm</i>, 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.