Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation

Abstract

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.

Figure 1.

Early Growth Response at 7 d Showing Morphogenic Stimulation of Non-Cell-Autonomous Wus2 and Cell-Autonomous Bbm Gene Delivery into the Scutellum of 18-DAP Embryos. (A) Introduction of Ubi_pro:moGFP:pinII alone (control). Single green fluorescent cells were observed on the scutellum surface. (B) to (D) nos_pro:Wus2:pinII cobombardment with the moGFP cassette. Foci of fluorescing cells appeared to enlarge slightly but remained confined to the tips of elongating protrusion (B) or formed a file of fluorescing cells from the tip as the protrusions continued to elongate (arrows in [C] and [D]). (D) A light micrograph and epifluorescence micrograph from the cyan filter set were superimposed onto each other with no other changes made to the data. (E) Ubi_pro:Bbm:pinII cointroduced with moGFP. Green fluorescent multicellular clusters were observed. (F) Cobombardment of the Wus2, Bbm, and moGFP expression cassettes. High growth stimulation was observed with a mixed phenotype exhibiting attributes from both Bbm and Wus2.

Figure 2.

Ectopic Expression of Bbm and Wus2 Increased Transformation Frequencies in Four Maize Inbreds. Immature embryos from inbreds PHN46, PH581, PHP38, and PHH5G were transformed using Agrobacterium in which the T-DNA contained no Bbm or Wus2 (pPHP24600), Bbm alone (pPHP24955), or Bbm and Wus2 (pPHP35648). For each inbred, significant differences between treatments are indicated by letter designations determined using penalized logistic regression analysis (P = 0.05). ND, not determined.

Figure 3.

nos_pro:Wus2:pinII Plus Ubi_pro:Bbm:pinII Containing T-DNA Used for DNA Gel Blot Analysis in the T1 Generation. Red opposing arrows indicate approximate locations of qPCR primers for YFP and moPAT for copy number determinations and PCR primers to detect the absence of the Bbm cDNA sequence and the formation of the newly formed junction across the one remaining loxP site after excision. Locations of EcoRI (red numbers on top) and HindIII (black numbers on bottom) restriction sites used in DNA gel blot analysis are also shown.

Figure 4.

Representative DNA Gel Blot Analysis of Four Transgenic PHH5G Inbred T1 Plants Derived from Each of Four Independent Transgenic Events. For all blots, P1 = pPHP35648 plasmid, while P2 = pPHP35648-excised (pPHP35648 after in vitro CRE-mediated excision). M, molecular weight ladder; P1 + NT, wild-type PHH5G genomic DNA spiked with control plasmid; NT, genomic DNA from the nontransgenic, wild-type plant. PC1, positive control unexcised; PC2, positive control excised. For event 1, DNA was inadvertently left out of lane 6. (A) T1 plant samples digested with EcoRI and probed for YFP; a single 3.2-kb band indicates intact gene sequence. (B) T1 plant samples digested with EcoRI and probed for moPAT; a single 1.5-kb band indicates intact gene sequence. (C) T1 plant samples digested with EcoRI and probed for CRE; the absence of a single 2.4-kb band indicates that CRE had been excised. (D) T1 plant samples digested with EcoRI and probed for Bbm; two bands result from hybridization to the endogenous genomic Bbm sequence and were also present in the two nontransgenic control lanes (labeled “NT” and “P1 plus NT”). The absence of a single 2.8-kb band indicates that Bbm had been excised. (E) T1 plant samples digested with EcoRI and probed for Wus2; numerous bands resulting from hybridization to the endogenous genomic Wus2 sequence were also present in the two nontransgenic control lanes (labeled “NT” and “P1 plus NT”). The absence of a single 1.8-kb band indicates that Wus2 had been excised. (F) T1 plant samples digested with HindIII and probed for moPAT; single bands larger than 3.1 kb and differing in size between the lines derived from the four events indicate that all four events were independent and single copy.

Figure 5.

Transformation of Alternative Explants Derived from Maize Seed or Seedlings. (A) to (D) To prepare mature embryo sliced for transformation, the mature embryo was cut out of the kernel (red box in [A]), and parallel cuts were then made to produce 300- to 400-μm-thick sections (black arrows) in which the scutellum (SC) and embryo axis (EA) could be discerned (B). After Agrobacterium-mediated transformation (strain LBA4404 with pPHP54733), transient ZS-GREEN1 expression was observed predominantly in the embryo axis (C), and after 2 to 3 weeks, callus could be observed growing from this region (D). (E) to (H) To prepare leaf tissue for transformation, a segment of the leaf whorl was removed from individual PHH5G seedlings (E) and cut into ∼1-mm segments. After Agrobacterium transformation (strain AGL1 with pPHP54733) and culture for 7 to 14 d, a dispersed pattern of multicellular cell clusters was clearly observed emerging from the leaf surface (F), and with continued culture multiple independent events were often observed arising from the surface of a single leaf segment under light microscopy (G) or under epifluorescence illumination (H). (I) to (L) Early observation of ectopic cell division patterns in the leaf were made by comparing cross sections of leaves from a nontransformed seedling leaf (I) with those of leaves transformed via Agrobacterium strain AGL1 delivering a T-DNA containing BSV_pro:GUS:pinII + nos_pro:Wus2:pinII +Ubi_pro:Bbm:pinII (pPHP46344), which revealed numerous GUS-staining multicellular foci (J) 7 d after infection. Within this population of growing multicellular foci, there were two types of division patterns: with all dividing cells within the cluster transformed based on GUS staining (K) or with GUS-staining cells (arrow in [L]) appearing to stimulate ectopic cell division in neighboring cells (double arrow in [L]).