Rapid genotype "independent" Zea mays L. (maize) transformation via direct somatic embryogenesis

Abstract

Constitutive expression of the Zea mays L. (maize) morphogenic transcription factors Baby Boom (Bbm) and Wuschel2 (Wus2) in maize can not only greatly increase transformation efficiency but can also induce phenotypic abnormalities and sterility. In an effort to alleviate the pleiotropic effects of constitutive expression, a genome wide search was undertaken to find suitable maize promoters to drive tissue and timing-specific expression of the transformation enhancing genes Bbm and Wus2. A promoter from a maize phospholipid transferase protein gene (Zm-PLTP_pro ) was identified based on its expression in leaves, embryos, and callus while being downregulated in roots, meristems, and reproductive tissues. When Zm-PLTP_pro driving Bbm was transformed into immature maize embryos along with a Wus2 expression cassette driven by the nopaline synthase promoter (Nos_pro ::Wus2) abundant somatic embryos rapidly formed on the scutella. These embryos were individual and uniformly transformed and could be directly germinated into plants without a callus phase. Transformed plants could be sent to the greenhouse in as little as 1 mo and regenerated plants matched the seed-derived phenotype for the inbred and were fertile. However, T1 seed from these plants had poor germination. Replacing Nos_pro with a maize auxin-inducible promoter (Zm-Axig1_pro ) in combination with Zm-PLTP_pro ::Bbm, allowed healthy, fertile plants to be regenerated. Single-copy T1 seed germinated normally and had a predominantly wild-type inbred phenotype. For maize, this callus-free transformation process has worked in all inbred lines tested.

Keywords: BabyBoom; Direct embryogenesis; Maize transformation; Wuschel.

Figure 2.

Gene expression profile for Zm-PLTP and Zm-UBI. Values are the average gene expression across samples grouped by major tissue category (unit: parts per million sampled reads or the numerical equivalent transcripts per million). Black bars represent the 95% confidence interval around mean). All samples contained multiple bio-replicates except for pericarp and stalk

Figure 1.

T-DNA composition for vectors used in corn transformation experiments. A depicts the Zm-PLTP_PRO::ZsGREEN reporter construct, while B and C depict the arrangement of Wus2, Bbm, Hra, and ZsYELLOW expression cassettes in PHP79065 and PHP79066

Figure 3.

Zm-PLTP_PRO::ZsGREEN expression in Zea mays L. in the pairs of subsidiary cells adjacent to the guard cells, and in the small, single cork cells observed between the elongated epidermal cells in leaves (A), in silk hairs (B and C), and in across-section of an immature T1 embryo (D)

Figure 4.

Rapid development of single somatic embryos of Zea may L. on the surface of recently transformed zygotic immature maize embryos shown under bright field microscopy for Fast-Flowering Mini-Maize at 7 d after infection (A). Green fluorescence of somatic embryos at progressive durations after Agrobacterium tumefaciens infection are shown for pioneer inbred PH184C (at 4 d (B), 7 d (C), and 10 d (D) after infection ). E, F Germinating transgenic plantlets using the public inbred Mo17 at 14 and 24 d (respectively) after A. tumefaciens infection showing 1–2 plantlets developing from each of the originally infected immature embryos

Figure 5.

Histology of Zea mays L. somatic embryo development after A. tumefaciens-mediated transformation of immature embryos with a T-DNA containing the expression cassettes Zm-Axig1_pro::Wus2 plus Zm-PLTP_pro::Bbm. The earliest morphological change observed were transverse or oblique cell divisions (arrow, A), continuing to divide and become multicellular (arrows, B) and growing into early globular pro-embryos (arrow, C) with some being subtended by what appeared to be a suspensor (double arrow, C). In the developing somatic embryos, multiple mitotic figures were often observed in the same cross-section (arrows, D). In E, multiple independent somatic embryos were observed in close proximity, and as somatic embryos continued to develop, the embryonic meristem could be observed to develop (arrows, F, G). Scale bar: A 12 μm; B–D 25 μm; E, F 50 μm; G 100 μm

Figure 6.

SbS™ data from nine single copy Zea mays L. events produced in inbred PHR03 showing the flanking sequences between the left border (yellow) and chromosomal DNA. Chromosome assignments are based on B73 models which may not be representative of this inbred. In some cases, the T-DNA insertion integrated into a repetitive region and chromosome location could not be accurately assigned

Figure 7.

Using vortexing to separate germinating Zea mays L. somatic embryos. At 20 d post Agrobacterium tumefaciens infection, all the developing tissue (A) was transferred to liquid and vortexed (B). The tissue was distributed back onto germination medium. The number of recovered T0 plantlets was high (C) and from a total of 45 originally transformed zygotic embryos, a total of over 380 independent transgenic plantlets were recovered (D)