Phylogeny of the Grass Family.
Relationships based largely on Vicentini et al. (2008) and Christin et al. (2009), showing multiple origins of C₄ photosystems. S. viridis is an NADP-ME subtype C₄ grass that is closely related to the bioenergy feedstock switchgrass (NAD-ME subtype), the grain crop foxtail millet, and the agricultural weed guinea grass (PCK). The C₄ photosynthetic systems in this Setaria/Urochloea/Panicum (SUPa clade, indicated with a yellow star) arose independently from the NADP-ME family members of the Andropononeae (maize, sorghum, sugarcane, and M. x giganteus). Dashed lines show clades with multiple subtypes.

Regeneration of S. viridis.
Several steps in the regeneration process are shown, including the following.
(A) Callus formation induced from germinating S. viridis seeds.
(B) Initial shoot regeneration from callus.
(C) Young shoot after regeneration.
(D) Plantlets after transfer to rooting media.
(E) Rooted plant.
(F) Regenerated plants at flowering.

A. tumefaciens–Mediated Transformation of S. viridis.
(A) Developing shoots grown on selective media following cocultivation with A. tumefaciens. Image shows nontransformed (left) and GUS-positive (right) shoot.
(B) Mature leaf tissue from three independent GUS-positive transformants and a nontransgenic control (leftmost sample). A detailed protocol for S. viridis transformation is provided in Supplemental Methods online, and constructs used are shown in Supplemental Figure 2 online.

Transient A. tumefaciens–Mediated Transformation of S. viridis.
S. viridis (accession A10) seedlings were grown on MetroMix 360 soil mix under a 12-h light/dark cycle with high relative humidity (75%) at a constant temperature of 23°C. The plants were watered as needed and every 3rd day with 20-10-20 fertilizer. Twelve days after germination, leaf 4 of a healthy S. viridis seedling was inoculated with A. tumefaciens strain AGL1 carrying the pPTN469 vector (Sattarzadeh et al., 2010) at a concentration of 0.05 (OD₆₀₀). The images show transient expression of a plastid-targeted YFP fusion protein. A detailed protocol for transient transformation is provided in the Supplemental Methods online, and constructs used are shown in Supplemental Figure 2 online.
(A) Low magnification fluorescence image showing abundance of transformed cells (arrows). The picture was taken using a Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems) using ×63 oil submerged objective lens. For the YFP signal (yellow), leaf tissue was excited with a 514-nm laser, and emitted light was collected from 525 to 575 nm. Autofluorescence (red) was captured from 650 to 789 nm. Image was compiled using Leica image software LAS-AF (version 1.8.2.) and Adobe Photoshop CS3 version 9.0.2 (Adobe Systems).
(B) Confocal reconstruction of leaf section showing transformed mesophyll cells (arrows). Chlorophyll autofluorescence is red and cell walls counterstained blue. bs, bundle sheath cell; m, mesophyll cells. Leaves were fixed in 2.5% paraformaldehyde, embedded in 7% low melting point agarose, cryosectioned, and counterstained with calcofluor white as described (Goldshmidt et al., 2008). The image was taken with a Carl Zeiss LSM 710 laser scanning microscope. A 514-nm laser excitation and 520- to 550-nm prism filter set was used to detect YFP emission (yellow), a 405-nm laser excitation and 475- to 500-nm prism filter set used for calcofluor white emission (blue), and a 488-nm laser excitation and 650- to 700-nm prism filter set used for detection of the chlorophyll emission (red). Subsequently, the confocal z-stack was reconstructed using Bitplane Imaris 7 software.