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

Chelsea R Johnson,Mark F Davis,Reginald J Millwood,Yuhong Tang,Zeng-Yu Wang,Geoffrey B Turner,Yi Sang,C Neal Stewart,Jiqing Gou,Robert W Sykes

doi:10.1186/s13068-017-0939-1

Abstract

BackgroundGenetic 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.ResultsmiR156 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.ConclusionsBecause 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.

Highlights

Genetic engineering has been effective in altering cell walls for biofuel production in the bioenergy crop, switchgrass (Panicum virgatum)
Results miR156 overexpression levels affect flowering timing and reproductive effort The medium overexpression lines (T27 and T37) had notably decreased numbers of flowers that were produced in a delayed floral transition phase (Figs. 2, 3)
Exploiting gene regulation by manipulating miRNAs could be useful in the sustained use of genetically engineered biofuel feedstocks to enhance desired traits such as abiotic and biotic stress responses, biomass yield, and lignin content [24,25,26,27,28,29,30]. miR156 targets the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor family, which is involved in many plant developmental processes including the vegetative to reproductive phase developmental transition [32,33,34,35, 47]

Summary

Introduction

Genetic engineering has been effective in altering cell walls for biofuel production in the bioenergy crop, switchgrass (Panicum virgatum). 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. Transgene flow from switchgrass will likely need to be severely curtailed to facilitate the commercialization of transgenic varieties [6, 9] This situation is especially pertinent in the eastern United States where switchgrass is endemic and common [12]. The delay or elimination of flowering itself could promote simultaneous improvements for a transgenic biomass crop such as switchgrass: it could decrease or eliminate pollen while simultaneously increase vegetative biomass [8, 18]

Methods

Results

Discussion

Conclusion