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Purpose-grown trees will be part of the bioenergy solution in the United States, especially in the Southeast where plantation forestry is prevalent and economically important. Trees provide a “living biomass inventory” with existing end-use markets and associated infrastructure, unlike other biomass species such as perennial grasses. The economic feasibility of utilizing tree biomass is improved by increasing productivity through alternative silvicultural systems, improved breeding and biotechnology. Traditional breeding and selection, as well as the introduction of genes for improved growth and stress tolerance, have enabled high growth rates and improved site adaptability in trees grown for industrial applications. An example is the biotechnology-aided improvement of a highly productive tropical Eucalyptus hybrid, Eucalyptus grandis × Eucalyptus urophylla . This tree has acquired freeze tolerance by the introduction of a plant transcription factor that up-regulates the cold-response pathways and makes possible commercial plantings in the Southeastern United States. Transgenic trees with reduced lignin, modified lignin, or increased cellulose and hemicellulose will improve the efficiency of feedstock conversion into biofuels. Reduced lignin trees have been shown to improve efficiency in the pre-treatment step utilized in fermentation systems for biofuels production from lignocellulosics. For systems in which thermochemical or gasification approaches are utilized, increased density will be an important trait, while increased lignin might be a desired trait for direct firing or co-firing of wood for energy. Trees developed through biotechnology, like all transgenic plants, need to go through the regulatory process, which involves biosafety and risk assessment analyses prior to commercialization.

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Caimey, J. 1996, Gene expression under water deficit in loblolly pine (Pinus taeda): Isolation and characterization of cDNA clones, -Physioi. Plant. 97: 139-148,Poly(A*) RNA was extracted from roots of loblolly pine (Pinus taeda L.) seedlings subjected to gradual and prolonged water deficit. This RNA was used to constmct a cDNA library, A number of cDNA clones were isolated whose expression was induced by water deficit. Four of these cDNA clones, designated pLP2, pLP3, pLP4 and pLP5, were characterized further. Each of these pine genes has unique characteristics either in sequence or pattem of expression. The protein encoded by pLP2 shows 91% identity to S-adenosylmethionine synthetase from a range of plants. The protein encoded by pLP3 is similar to a tomato protein induced by water deficit and during fruit ripening, but the pine protein possesses a unique 34-amino-acid region near the aniino terminal. The LP4 protein is similar to stellacyanin, a copper-binding protein in the Japanese lacquer tree, but possesses a proline/serine-rich caiboxy terminus not found in other plants. Clone pLP5 encodes a novel glycine-rich protein with similarity to both silk fibroin and the rat chondroitin core protein, but the putative pine protein is distinct from previously characterized glycine-rich proteins. Transcript levels of the four genes rose under moderate water deficit stress and then declined as stress became severe (1 month without water), with the exception of pLP5 mRNA, which remained at elevated levels even under severe stress. The possible roles of the encoded proteins in cell wall reinforcement are discussed.

Pollen elimination provides an effective containment method to reduce direct gene flow from transgenic trees to their wild relatives. Until now, only limited success has been achieved in controlling pollen production in trees. A pine (Pinus radiata) male cone-specific promoter, PrMC2, was used to drive modified barnase coding sequences (barnaseH102E, barnaseK27A, and barnaseE73G) in order to determine their effectiveness in pollen ablation. The expression cassette PrMC2-barnaseH102E was found to efficiently ablate pollen in tobacco (Nicotiana tabacum), pine, and Eucalyptus (spp.). Large-scale and multiple-year field tests demonstrated that complete prevention of pollen production was achieved in greater than 95% of independently transformed lines of pine and Eucalyptus (spp.) that contained the PrMC2-barnaseH102E expression cassette. A complete pollen control phenotype was achieved in transgenic lines and expressed stably over multiple years, multiple test locations, and when the PrMC2-barnaseH102E cassette was flanked by different genes. The PrMC2-barnaseH102E transgenic pine and Eucalyptus (spp.) trees grew similarly to control trees in all observed attributes except the pollenless phenotype. The ability to achieve the complete control of pollen production in field-grown trees is likely the result of a unique combination of three factors: the male cone/anther specificity of the PrMC2 promoter, the reduced RNase activity of barnaseH102E, and unique features associated with a polyploid tapetum. The field performance of the PrMC2-barnaseH102E in representative angiosperm and gymnosperm trees indicates that this gene can be used to mitigate pollen-mediated gene flow associated with large-scale deployment of transgenic trees.

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Development of transgenic cell lines or organisms for industrial, agricultural, or medicinal applications involves inserting DNA into the target genome in a way that achieves efficacious transgene expression without a deleterious impact on fitness. The genomic insertion site is widely recognized as an important determinant of success. However, the effect of chromosomal location on transgene expression and fitness has not been systematically investigated in plants. Here we evaluate the importance of transgene insertion site in maize and soybean using both random and site-specific transgene integration. We have compared the relative contribution of genomic location on transgene expression levels with other factors, including cis-regulatory elements, neighboring transgenes, genetic background, and zygosity. As expected, cis-regulatory elements and the presence/absence of nearby transgene neighbors can impact transgene expression. Surprisingly, we determined not only that genomic location had the least impact on transgene expression compared to the other factors that were investigated but that the majority of insertion sites recovered supported transgene expression levels that were statistically not distinguishable. All 68 genomic sites evaluated were capable of supporting high-level transgene expression, which was also consistent across generations. Furthermore, multilocation field evaluation detected no to little decrease in agronomic performance as a result of transgene insertion at the vast majority of sites we evaluated with a single construct in five maize hybrid backgrounds.

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