Field trial detects incomplete barstar attenuation of vegetative cytotoxicity in Populus trees containing a poplar LEAFY promoter::barnase sterility transgene
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We tested the efficacy of an attenuation system developed to preclude the deleterious effects of floral promoter::cytotoxin genes on vegetative growth of transgenic sterile plants. We tested the promoter (2.6 kb 5′ region) of the poplar LEAFY gene PTLF driving barstar, combined on the same T-DNA with barstar driven by either the CaMV 35S basal promoter +5 to −72 fragment (35SBP), 35SBP fused to the TMV omega element (35SBP omega), or the NOS promoter. The unattenuated pPTLF::barnase construct failed to give rise to any transgenic events, suggesting substantial non-reproductive expression from this promoter. The barstar-attenuated constructs enabled transformation, but the rate was reduced by nearly one-third. Four events (7% of attenuated events) had highly abnormal morphology, and were identified during the early phases of propagation; these events had significantly higher barnase:barstar expression ratios based on quantitative RT-PCR. A greenhouse study showed that phenotypically normal attenuated plants grew at the same rate as wild-type and barnase-lacking transgenic plants. A statistically significant positive linear association was found between relative growth rate (RGR) and barstar:barnase ratio in the attenuated events, and graphical analysis suggested a threshold for barstar attenuation of barnase, above which additional levels of barstar did not provide further attenuation. Surprisingly, the appearance and growth rate of the nearly all of the attenuated events were substantially reduced after one or two growing seasons in the field, and the extent of growth reduction was associated with barstar:barnase expression ratio. These results demonstrate the importance of field testing during early phases of research to identify pleiotropic effects of transgenic sterility genes in trees.
KeywordsAblation Biosafety Containment Biotechnology Forestry Gene flow Genetic engineering Trees Sterility Genetic modification Barnase Barstar LEAFY
We are grateful to Jace Carson, Edward Leber, and Elizabeth Jaeger for their technical assistance. We also thank the Consortium for Plant Biotechnology Research, Inc. (agreement GO12026-157); the US Department of Energy’s Biomass Program [through contract #85X-ST807V with Oak Ridge National Laboratory (managed by UT-Battelle, LLC, for the US Department of Energy under contract DE-AC05-00OR22725)]; and companies supporting the Tree Genomics and Biosafety Research Consortium, based at Oregon State University, for their financial support.
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