Graduate Thesis Or Dissertation


Evaluation of transgenes for stress tolerance, lignin modification, and growth enhancement in poplar Public Deposited

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  • We evaluated genes previously identified from a large scale functional genomics screen for their potential value to help enhance carbon sequestration in planted trees. We used poplar as a model tree species because of its abundant genetic variation, ease of gene transfer, and availability of large databases for genomic, anatomical, physiological, and silvicultural traits. Enhancement of carbon sequestration can be achieved by modifying many aspects of plant growth and development, including rate of growth, stress tolerance, and partitioning of assimilated carbon into compounds that are resistant to rapid decomposition, such as lignin. The selected genes were placed under a constitutive promoter and transferred to poplar via Agrobacterium transformation, then clonally propagated and evaluated in the greenhouse. We tested 15 genes (15 different plasmids constructs) in greenhouse trials. For each construct, we tested 10-15 events (i.e., unique gene insertions into the genome) with 3-8 ramets per event (replicate plants). The traits analyzed included: growth rate, plant morphology, fertilizer response, lignin concentration, and drought, salt, and shade tolerance. Treatment conditions for the abiotic stress treatments were based on preliminary experiments using non-transgenic plants that examined the response of the transformed host poplar (Populus tremula x P. alba INRA 717-1B4) to a variety of salt, drought, fertilizer, and shade levels in potted plants. The abiotic treatment levels identified and used in the transgenic trials were -2 MPa soil moisture potential for drought tolerance; 275 mM saline solution for salinity tolerance; 60% shade or 40% light for shade tolerance; and 0, 300, 600, and 1,200 ppm NPK fertilizer rates for fertilizer efficiency. Constructs were considered to give preliminary evidence for an effect of interest when at least two events showed a similar modified phenotype of biological interest and the individual events were statistically significant (P < 0.05) or substantially different from controls; or, all events pooled were significantly different from controls. We tested eight constructs for improved interaction of plants with their abiotic environment. Drought construct #13 (At glycosyl hydrolase protein) exhibited an increase in biomass accumulation and growth in 4 out of 12 events. The salinity tolerance constructs (At hydrolase and Zea mays SF16 calmodulin-like family protein) provided no detectable improvement in tolerance. Both of the constructs for shade tolerance (At zinc finger protein and At salicylic acid carboxyl methyltransferase) imparted shade tolerance characteristics, including lower SR and taper. Construct #11 (At zinc finger protein) imparted significantly higher relative growth rate (RGR) for 2 out of 9 events. Despite the lack of fertilizer x event interactions, fertilizer efficiency construct #16 (At myb protein) imparted strong and significant growth enhancement. We screened and scored two constructs (construct #41 – At ARR (Arabidopsis response regulator) protein and construct #44 – At DEAD/DEAH box gene, DNA/RNA helicase) for their ability to increase lignin accumulation in stems and roots via the Weisner reaction. Accumulation of lignin for the transgenic events was higher in stem tissues compared to root tissues, and caused a significant increase in the concentration of lignin in stems, with both constructs. Growth was also modified by both constructs, but in one case it was increased (construct #41) and in the other it was reduced (construct #44). We evaluated five constructs for growth modification. Of the constructs tested for stature or growth rate modification, only #7 (Triticum aestivum bHLH family protein) showed a significant effect of interest. Twenty percent (2 out of 10) of the events in construct #7 showed a significant increase in growth, RGR, and biomass accumulation. The analyses provide evidence that several transgenes—all of unknown physiological function and derived from other plant species—can provide increased growth, biomass accumulation, and/or RGR, but that the results are highly variable and event-dependent. This indicates that these genes may be useful in promoting carbon sequestration from planted or wild stands, but require extensive testing to identify useful candidate constructs and genes, even at the primary stage of greenhouse analysis.
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