Maize Transformation at Pioneer - Meeting Future Demands for Genome Modification

Although cereal transformation has continued to improve over the years, the process has generally remained constrained to a few genotypes per crop, and methods are still slow and labor intensive. The overall impact has been that cereal transformation, genome modifications and high-throughput transformation remain beyond the reach of almost all academic labs. Recent progress in our labs is rapidly changing this situation for cereals. By focusing on the overexpression of the maize Babyboom (BBM) and Wuschel2 (WUS2) genes, we can routinely produce high transformation frequencies in numerous previously non-transformable maize inbreds. Recent optimization of the promoters driving BBM and WUS2 expression has resulted in direct germination of somatic embryos to recover transgenic plants (eliminating all callus steps), making maize inbreds such as B73 and Mo17 easily transformable using Agrobacterium strain LBA4404. Of even greater import to genome modification methods, this process is largely genotype independent and regenerated 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. We will also describe a new system for Agrobacterium-mediated co-transformation that greatly simplifies the process from vector construction through analysis.