Use of non-integrating Zm-Wus2 vectors to enhance maize transformation

Abstract

The use of Baby boom (Bbm) and Wuschel2 (Wus2) has made maize transformation more efficient across an increasingly wide range of inbreds. However, the benefits have come with the requirement of excising these transformation helper components to enable plant regeneration, which adds size to the T-DNA, and complexity to the transformation system. A new system with the advantages of smaller size and simplicity for the selectable marker gene-containing T-DNA is described. First, expression of Zm-Wus2 alone driven by the maize Pltp promoter (Zm-Pltp pro), was determined to be sufficient to induce rapid somatic embryo formation from the scutella of maize immature embryos. It was also demonstrated that co-infecting with two strains of Agrobacterium, one with a Wus2 expression cassette, and the other with a combination of both selectable and visual marker cassettes, produced transformed T0 plants that contained only a single copy of the selectable marker T-DNA, without the integration of Wus2. Furthermore, the process was optimized by varying the ratio of the two Agrobacterium strains, and by modulating Wus2 expression to enable high-frequency recovery of selectable marker-containing T0 plants that did not contain Wus2. Several factors may have contributed to this outcome. Wus2 expression in localized cell(s) appeared to stimulate somatic embryogenesis in neighboring cells, including those that had integrated the selectable marker. In addition, in cells in which the Wus2 T-DNA did not integrate but the selectable marker T-DNA did, transient Wus2 expression stimulated somatic embryo formation and regeneration of stable T0 plants that contained the selectable marker. In addition, augmenting the Pltp promoter with three viral enhancer elements to increase Wus2 expression stimulated embryogenesis while precluding their regeneration. The phenomenon has now been designated as "altruistic transformation."

An Efficient Gene Excision System in Maize

“…Therefore, it is desirable to generate transgenic plants free from morphogenic genes. Previously, a method using a non-integrating Wus2 gene approach recovered fertile T0 plants free-off morphogenic genes, however this method needed a plant selectable marker gene (SMG) for regenerating events ( Hoerster et al., 2020 ). Here we report an approach that allows excision of both the morphogenic gene and the SMG used in transformation at the same time.…”

An efficient gene excision system in maize

“…Briefly, maize immature embryos (1.5-2 mm) were harvested and used for Agrobacterium- mediated transformation, using the media, selection and regeneration methods described previously (Lowe et al, 2018;Chu et al, 2019;Hoerster et al, 2020). All media recipes are described by (Lowe et al, 2018;Chu et al, 2019;Hoerster et al, 2020). For selection, 0.1 mg/L imazapyr was supplemented to somatic embryo formation medium or 150 mg/L G418 was substituted for imazapyr.…”

“…All transformations were done using the thymidine auxotrophic Agrobacterium tumefaciens strain LBA4404 THY-containing pVIR9 (Anand et al, 2018) at OD550 of 0.5. The conditions for Agrobacterium suspension culture preparation following embryo isolation and infection has been previously described (Lowe et al, 2018;Hoerster et al, 2020). Two selectable markers were used in experiments: HRA (Green et al, 2009), a sulfonylurea herbicide resistance marker, driven by the sorghum Als promoter for selection with 0.1 mg/L imazapyr in culture medium, or the Ubipro::NPTII gene for selection with 150 mg/L G418 in culture medium.…”

“…All molecular analysis and transgene copy number determination methods were previously described (Wu et al, 2014;Lowe et al, 2016;Hoerster et al, 2020). qPCR data was used to confirm recombinase-mediated excision based on the absence the transgenes flanked by the loxP sites, determine the copy number of structural genes outside the excision DNA, and to screen for the presence of Agrobacterium binary construct backbone integration.…”

“…Therefore, excising the morphogenic genes is desirable for regenerating healthy plants, for transgene testing and commercial product development. Previously a method using a non-integrating Wus2 gene expression approach recovered fertile T0 plants free-off morphogenic genes, however this method needed a plant selectable marker gene (SMG) for regenerating events (Hoerster et al, 2020). Here we report an approach that allows excision of both the morphogenic gene and the SMG used in transformation at the same time.…”