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-Pltppro), 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.”
Highlights
The transcription factors Baby boom (Bbm) and Wuschel2 (Wus2), which are referred to as morphogenic genes, greatly enhance maize transformation (Lowe et al 2016)
The T-DNA in all four constructs carried a CRE expression cassette under the control of the Zm-Globulin1 (Glb1) promoter (Liu et al 1998), to enable auto-excision of the morphogenic genes and the CRE gene flanked by directly oriented loxP sites (Fig. 1), which has been used previously for excision of the morphogenic genes Zm-Wus2 and Zm-Bbm (Chu et al 2019)
When the test vectors were compared with the control plasmid in the two inbreds, the SC frequency ranged between 2 and 4% for the control, while no SC events were recovered from ZmPltppro::Bbm
Summary
The transcription factors Baby boom (Bbm) and Wuschel (Wus2), which are referred to as morphogenic genes, greatly enhance maize transformation (Lowe et al 2016). Spatio-temporal regulation of morphogenic gene expression by the maize Pltp promoter stimulate rapid (3–7 d post infection), and direct formation of somatic embryos from immature scutella. Axig1pro::Wus plus Pltppro::Bbm has been shown by Lowe et al (2018) to stimulate somatic embryo formation in B73, Mo17, and Fast Flowering Mini Maize (FFMM germplasm developed by McCaw et al 2016). While resultant T0 plants that contained a single copy (SC) of the T-DNA with Bbm and Wus were fertile and had a normal phenotype, ectopic expression of transcription factors can have subtle pleiotropic effects (Lowe et al 2016, 2018). Expression of CRC (a fusion between two transcription factors, known as R and C1, which activates the anthocyanin pathway) in maize, induces the expression of hundreds of genes
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