While transformation of the major monocot crops is currently possible, the process typically 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 laboratories. Here, we report a transformation approach involving overexpression of the maize (Zea mays) Baby boom (Bbm) and maize Wuschel2 (Wus2) genes, which produced high transformation frequencies in numerous previously nontransformable maize inbred lines. For example, the Pioneer inbred PHH5G is recalcitrant to biolistic and Agrobacterium tumefaciens transformation. However, when Bbm and Wus2 were expressed, transgenic calli were recovered from over 40% of the starting explants, with most producing healthy, fertile plants. Another limitation for many monocots is the intensive labor and greenhouse space required to supply immature embryos for transformation. This problem could be alleviated using alternative target tissues that could be supplied consistently with automated preparation. As a major step toward this objective, we transformed Bbm and Wus2 directly into either embryo slices from mature seed or leaf segments from seedlings in a variety of Pioneer inbred lines, routinely recovering healthy, fertile T0 plants. Finally, we demonstrated that the maize Bbm and Wus2 genes stimulate transformation in sorghum (Sorghum bicolor) immature embryos, sugarcane (Saccharum officinarum) callus, and indica rice (Oryza sativa ssp indica) callus.

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.

Modern maize hybrids often contain biotech and native traits. To-date all biotech traits have been randomly inserted in the genome. Consequently, developing hybrids with multiple traits is expensive, time-consuming, and complex. Here we report using CRISPR-Cas9 to generate a complex trait locus (CTL) to facilitate trait stacking. A CTL consists of multiple preselected sites positioned within a small well-characterized chromosomal region where trait genes are inserted. We generated individual lines, each carrying a site-specific insertion landing pad (SSILP) that was targeted to a preselected site and capable of efficiently receiving a transgene via recombinase-mediated cassette exchange. The selected sites supported consistent transgene expression and the SSILP insertion had no effect on grain yield. We demonstrated that two traits residing at different sites within a CTL can be combined via genetic recombination. CTL technology is a major step forward in the development of multi-trait maize hybrids.

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Development of transgenic cell lines or organisms for industrial, agricultural, or medicinal applications involves inserting DNA into the target genome in a way that achieves efficacious transgene expression without a deleterious impact on fitness. The genomic insertion site is widely recognized as an important determinant of success. However, the effect of chromosomal location on transgene expression and fitness has not been systematically investigated in plants. Here we evaluate the importance of transgene insertion site in maize and soybean using both random and site-specific transgene integration. We have compared the relative contribution of genomic location on transgene expression levels with other factors, including cis-regulatory elements, neighboring transgenes, genetic background, and zygosity. As expected, cis-regulatory elements and the presence/absence of nearby transgene neighbors can impact transgene expression. Surprisingly, we determined not only that genomic location had the least impact on transgene expression compared to the other factors that were investigated but that the majority of insertion sites recovered supported transgene expression levels that were statistically not distinguishable. All 68 genomic sites evaluated were capable of supporting high-level transgene expression, which was also consistent across generations. Furthermore, multilocation field evaluation detected no to little decrease in agronomic performance as a result of transgene insertion at the vast majority of sites we evaluated with a single construct in five maize hybrid backgrounds.