Abstract
BackgroundSwitchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S. DOE and USDA. Conventional breeding of switchgrass biomass is difficult because it displays self-incompatible hindrance. Therefore, direct genetic modifications of switchgrass have been considered the more effective approach to tailor switchgrass with traits of interest. Successful transformations have demonstrated increased biomass yields, reduction in the recalcitrance of cell walls and enhanced saccharification efficiency. Several tissue culture protocols have been previously described to produce transgenic switchgrass lines using different nutrient-based media, co-cultivation approaches, and antibiotic strengths for selection.ResultsAfter evaluating the published protocols, we consolidated these approaches and optimized the process to develop a more efficient protocol for producing transgenic switchgrass. First, seed sterilization was optimized, which led to a 20% increase in yield of induced calluses. Second, we have selected a N6 macronutrient/B5 micronutrient (NB)-based medium for callus induction from mature seeds of the Alamo cultivar, and chose a Murashige and Skoog-based medium to regenerate both Type I and Type II calluses. Third, Agrobacterium-mediated transformation was adopted that resulted in 50–100% positive regenerated transformants after three rounds (2 weeks/round) of selection with antibiotic. Genomic DNA PCR, RT-PCR, Southern blot, visualization of the red fluorescent protein and histochemical β-glucuronidase (GUS) staining were conducted to confirm the positive switchgrass transformants. The optimized methods developed here provide an improved strategy to promote the production and selection of callus and generation of transgenic switchgrass lines.ConclusionThe process for switchgrass transformation has been evaluated and consolidated to devise an improved approach for transgenic switchgrass production. With the optimization of seed sterilization, callus induction, and regeneration steps, a reliable and effective protocol is established to facilitate switchgrass engineering.
Highlights
Switchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S DOE and USDA
Our results showed clear evidence that vigorously dividing switchgrass calluses originate from the scutellum of mature switchgrass seeds (Fig. 1b), which was generally believed to be competent for Agrobacteriummediated transformation in rice [56]
Medium, which can reduce the prerequisite for Type II callus for Agrobacterium-meditated transformation
Summary
Switchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S DOE and USDA. There are two ecotypes of switchgrass varieties, lowland and upland Lowland types, such as the cultivars Alamo, Kanlow and Timer, grow in the southern U.S and generally result in taller, more robust growing plants displaying a bunching habit, later maturation, larger leaves, and coarser stems. Upland types, such as Blackwell, Carthage, Cave-In-Rock (CIR), Pathfinder, Trailblazer, Dacotah, Shawnee and Caddo, grow in the northern U.S and have different morphological characteristics from the lowland types [8, 9]. Upland types are both tetraploid and octoploid, with octoploid being more predominant [10]
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