Pollen-mediated gene flow from transgenic to non-transgenic switchgrass (Panicum virgatum L.) in the field.

BACKGROUND: Switchgrass is C4 perennial grass species that is being developed as a cellulosic bioenergy feedstock. It is wind-pollinated and considered to be an obligate outcrosser. Genetic engineering has been used to alter cell walls for more facile bioprocessing and biofuel yield. Gene flow from transgenic cultivars would likely be of regulatory concern. In this study we investigated pollen-mediated gene flow from transgenic to nontransgenic switchgrass in a 3-year field experiment performed in Oliver Springs, Tennessee, U.S.A. using a modified Nelder wheel design. The planted area (0.6 ha) contained sexually compatible pollen source and pollen receptor switchgrass plants. One hundred clonal switchgrass 'Alamo' plants transgenic for an orange-fluorescent protein (OFP) and hygromycin resistance were used as the pollen source; whole plants, including pollen, were orange-fluorescent. To assess pollen movement, pollen traps were placed at 10 m intervals from the pollen-source plot in the four cardinal directions extending to 20 m, 30 m, 30 m, and 100 m to the north, south, west, and east, respectively. To assess pollination rates, nontransgenic 'Alamo 2' switchgrass clones were planted in pairs adjacent to pollen traps. RESULTS: In the eastward direction there was a 98% decrease in OFP pollen grains from 10 to 100 m from the pollen-source plot (Poisson regression, F1,8 = 288.38, P < 0.0001). At the end of the second and third year, 1,820 F1 seeds were collected from pollen recipient-plots of which 962 (52.9%) germinated and analyzed for their transgenic status. Transgenic progeny production detected in each pollen-recipient plot decreased with increased distance from the edge of the transgenic plot (Poisson regression, F1,15 = 12.98, P < 0.003). The frequency of transgenic progeny detected in the eastward plots (the direction of the prevailing wind) ranged from 79.2% at 10 m to 9.3% at 100 m. CONCLUSIONS: In these experiments we found transgenic pollen movement and hybridization rates to be inversely associated with distance. However, these data suggest pollen-mediated gene flow is likely to occur up to, at least, 100 m. This study gives baseline data useful to determine isolation distances and other management practices should transgenic switchgrass be grown commercially in relevant environments.

Light and temperature effects on miR156 transgenic switchgrass flowering: A simulated latitudinal study.

The control of flowering in perennial grasses is an important trait, especially among biofuel feedstocks. Lignocellulosic biomass may be increased commensurate with decreased or delayed flowering as the plant allocates energy for stems and leaves harvested for bioenergy at the end of the growing season. For transgenic feedstocks, such as switchgrass (Panicum virgatum L.) grown in its geographic center of distribution, it is foreseeable that regulators may require greatly decreased gene flow frequencies to enable commercialization. Transgenic switchgrass with various overexpression levels of a rice microRNA gene, miR156, when grown in field conditions, holds promise for decreased flowering, yielding high biomass, and altered cell wall traits, which renders it as a potential crossing partner for further breeding with switchgrass lines for decreased recalcitrance. In the current research, we simulated a latitudinal cline in controlled growth chamber experiments for various individual sites from the tropics to cool-temperate conditions which included weekly average high and low temperatures and day lengths over the switchgrass growing season for each simulated site: Guayaquil, Ecuador; Laredo, Texas, USA; and Brattleboro, Vermont, USA. Flowering and reproduction among transgenic lines with low (T-14 and T-35)-to-moderate (T-27 and T-37) overexpression of miR156 were assessed. Lower simulated latitudes (higher temperatures with low-variant day length) and long growing seasons promoted flowering of the miR156 transgenic switchgrass lines. Tropical conditions rescued the flowering phenotype in all transgenic lines except T-27. Higher numbers of plants in lines T-35 and T-37 and the controls produced panicles, which also occurred earlier in the study as temperatures increased and day length decreased. Line T-14 was the exception as more clonal replicates flowered in the cool-temperate (Vermont) conditions. Increased biomass was found in transgenic lines T-35 and T-37 in tropical conditions. No difference in biomass was found in subtropical (Texas) chambers, and two lines (T-14 and T-35) produced less biomass than the control in cool-temperate conditions. Our findings suggest that switchgrass plants engineered to overexpress miR156 for delayed flowering to promote bioconfinement and biomass production may be used for plant breeding at tropical sites.

Field-grown miR156 transgenic switchgrass reproduction, yield, global gene expression analysis, and bioconfinement.

BACKGROUND: Genetic engineering has been effective in altering cell walls for biofuel production in the bioenergy crop, switchgrass (Panicum virgatum). However, regulatory issues arising from gene flow may prevent commercialization of engineered switchgrass in the eastern United States where the species is native. Depending on its expression level, microRNA156 (miR156) can reduce, delay, or eliminate flowering, which may serve to decrease transgene flow. In this unique field study of transgenic switchgrass that was permitted to flower, two low (T14 and T35) and two medium (T27 and T37) miR156-overexpressing 'Alamo' lines with the transgene under the control of the constitutive maize (Zea mays) ubiquitin 1 promoter, along with nontransgenic control plants, were grown in eastern Tennessee over two seasons. RESULTS: miR156 expression was positively associated with decreased and delayed flowering in switchgrass. Line T27 did not flower during the 2-year study. Line T37 did flower, but not all plants produced panicles. Flowering was delayed in T37, resulting in 70.6% fewer flowers than controls during the second field year with commensurate decreased seed yield: 1205 seeds per plant vs. 18,539 produced by each control. These results are notable given that line T37 produced equivalent vegetative aboveground biomass to the controls. miR156 transcript abundance of field-grown plants was congruent with greenhouse results. The five miR156 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) target genes had suppressed expression in one or more of the transgenic lines. Line T27, which had the highest miR156 overexpression, showed significant downregulation for all five SPL genes. On the contrary, line T35 had the lowest miR156 overexpression and had no significant change in any of the five SPL genes. CONCLUSIONS: Because of the research field's geographical features, this study was the first instance of any genetically engineered trait in switchgrass, in which experimental plants were allowed to flower in the field in the eastern U.S.; USDA-APHIS-BRS regulators allowed open flowering. We found that medium overexpression of miR156, e.g., line T37, resulted in delayed and reduced flowering accompanied by high biomass production. We propose that induced miR156 expression could be further developed as a transgenic switchgrass bioconfinement tool to enable eventual commercialization.