Expression of Bbm and WUS2 Extends Monocot Transformation into New Target Explants and Genotypes

While transformation methods for monocot crops continue to improve, the process remains confined to one or two genotypes per species, often with poor agronomics, and efficiencies that place these methods beyond the reach of most academic labs. To rectify these issues, we have focused on overexpressing the maize Babyboom (BBM) and Wuschel2 (WUS2) genes, producing high transformation frequencies in numerous previously non-transformable maize inbreds. For example, the Pioneer inbred PHH5G is totally recalcitrant to Agrobacterium transformation. However, when BBM and WUS2 are expressed, transgenic calli are recovered often without chemical selection from over 40% of the starting explants, with most producing healthy, fertile plants. Recently, we have optimized expression of our BBM and WUS cassettes in such a way that we can eliminate all callus steps and obtain transformants via direct germination of somatic embryos, making maize inbreds such as B73 and Mo17 easily transformable using Agrobacterium strain LBA4404. This process is largely genotype independent and transgenic plants can be sent to the greenhouse in less than half the time of conventional methods. Another limitation for many monocots is the intensive labor and greenhouse space required to supply immature embryos for transformation. As a new alternative to immature embryos, we use BBM and WUS2 to recover transgenic events directly from either embryo slices from mature seed or leaf segments from seedlings in a variety of Pioneer inbreds, routinely recovering healthy, fertile T0 plants. Finally, we demonstrate that the maize BBM and WUS2 genes stimulate transformation in sorghum immature embryos, sugarcane callus, indica rice callus and japonica leaf tissue.