Ecological impacts of trees with modified lignin

Few experiments have yet been performed to explore the potential ecological impacts of genetic modification in long-lifespan species such as trees. In this paper, we review the available data on GM trees with modified lignin focussing on the results of the first long-term field trials of such trees. These trials evaluated poplars expressing antisense transgenes to reduce the expression of the lignin biosynthesis genes cinnamyl alcohol dehydrogenase (CAD) or caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) with the aim of producing trees with improved pulping characteristics. The trees were grown for 4 years at two sites in France and England, and their ecological impacts and agronomic performance were assessed. Modifications to lignin in the poplars were maintained over the 4 years of the trial. The trees remained healthy throughout and growth was normal. The lignin modifications had no unexpected biological or ecological impacts. Interactions with leaf-feeding insects, microbial pathogens and soil organisms were unaltered although the short-term decomposition of transgenic roots was slightly enhanced. Investigation of the ecological impacts of the GM trees was curtailed by the early termination of the field trial when it was attacked and largely destroyed by anti-GM protestors. To supplement our work on the decomposition of GM plant materials with modified lignin, we have therefore turned to the study of transgenic tobacco lines where we can perform more comprehensive and controlled analyses of the biological and ecological effects of lignin-gene suppression.     

Poplar genetic engineering: promoting desirable wood characteristics and pest resistance

Worldwide biomass demand for industrial applications, especially for production of biofuels, is increasing. Extended cultivation of fast growing trees such as poplars may contribute to satisfy the need for renewable resources. However, lignin, which constitutes about 20–30 % of woody biomass, renders poplar wood recalcitrant to saccharification. Genetic engineering of the enzymes of the lignification pathway has resulted in drastic decreases in lignin and greatly improved the carbohydrate yield for ethanol fermentation. While uncovering key enzymes for lignification facilitated rapid biotechnological progress, knowledge on field performance of low-lignin poplars is still lagging behind. The major biotic damage is caused by poplar rust fungi (Melampsora larici-populina), whose defense responses involve lignification and production of phenolic compounds. Therefore, manipulation of the phenylpropanoid pathway may be critical and should be tightly linked with new strategies for improved poplar rust tolerance. Emerging novel concepts for wood improvement are discussed.     

Topochemical studies on modified lignin distribution in the xylem of Poplar (Populus spp.) after wounding

BACKGROUND AND AIMS: Information on the influence of wounding on lignin synthesis and distribution in differentiating xylem tissue is still scarce. The present paper provides information on cell modifications with regard to wall ultrastructure and lignin distribution on cellular and subcellular levels in poplar after wounding. METHODS: Xylem of Populus spp. close to a wound was collected and processed for light microscopy, transmission electron microscopy and cellular UV microspectrophotometry. Cell wall modification with respect to lignin distribution was examined at different stages of wound tissue development. Scanning UV microspectrophotometry and point measurements were used to determine the lignin distribution. KEY RESULTS: Xylem fibres within a transition zone between differentiated xylem laid down prior to wounding and the tissues formed after wounding developed distinctively thickened secondary cell walls. Those modified walls and cell corners showed, on average, a higher lignin content and an inhomogeneous lignin distribution within the individual wall layers. CONCLUSIONS: The work presented shows that wounding of the xylem may induce a modified wall architecture and lignin distribution in tissues differentiating at the time of wounding. An increasing lignin content and distinctively thickened walls can contribute to improved resistance as part of the compartmentalization process.     

Wood of trees: Cellular structure,molecular formation,and genetic engineering

Wood is an invaluable asset to human society due to its renewable nature, making it suitable for both sustainable energy production and material manufacturing. Additionally, wood derived from forest trees plays a crucial role in sequestering a significant portion of the carbon dioxide fixed during photosynthesis by terrestrial plants. Nevertheless,with the expansion of the global population and ongoing industrialization, forest coverage has been substantially decreased, resulting in significant ...     

林木抗旱的渗透调节及其基因工程研究进展

与其它植物一样,林木对干旱的适应和抵御机理是多种多样的,渗透调节是其中重要的一个方面。在通过渗透调节抵御干旱胁迫的过程中,起作用的有一些小分子渗透调节物质,如脯氨酸、甜菜碱等;也有果聚糖、海藻糖和蔗糖等可溶性糖类;Lea蛋白在很多植物抵御干旱胁迫过程中也起到了重要作用。本文介绍了林木抗旱性的渗透调节机理及基因工程研究进展,并探讨了林木抗旱基因工程改良中面临的问题及改进措施。     

Plant fibers in the paper industry

For 20 centuries, vegetable fibers from various sources have been converted into thin sheets for use in trade, communications, law, and even for shelter. Cotton and linen rags were the first fibrous raw materials to acquire widespread status in paper making, and they are still needed for specialty products. Increasing demands for paper ultimately surpassed the availability of rags, so that other sources of fiber were sought. About the year 1800, numerous other materials of agricultural origin began to come into use somewhat temporarily. Although woods have become the prime source of paper making fibers during the last 100-125 years, annual plant fibers retain their importance. The technical feasibility of non-woody materials is substantiated by more than 300 paper mills throughout the world that use such raw materials. Both necessity and special properties account for their use. The potential for various nonwoody fibrous plants is being investigated intensively by the United States Department of Agriculture’s Agricultural Research Service, to discover new crops that might hare practical significance in the national agricultural program and in the commercial production of paper.     

Metabolic engineering of p-hydroxybenzoate in poplar lignin

Ester-linked p-hydroxybenzoate occurs naturally in poplar lignin as pendent groups that can be released by mild alkaline hydrolysis. These ‘clip-off’ phenolics can be separated from biomass and upgraded into diverse high-value bioproducts. We introduced a bacterial chorismate pyruvate lyase gene into transgenic poplar trees with the aim of producing more p-hydroxybenzoate from chorismate, itself a metabolic precursor to lignin. By driving heterologous expression specifically in the plastids of cells undergoing secondary wall formation, this strategy achieved a 50% increase in cell-wall-bound p-hydroxybenzoate in mature wood and nearly 10 times more in developing xylem relative to control trees. Comparable amounts also remained as soluble p-hydroxybenzoate-containing xylem metabolites, pointing to even greater engineering potential. Mass spectrometry imaging showed that the elevated p-hydroxybenzoylation was largely restricted to the cell walls of fibres. Finally, transgenic lines outperformed control trees in assays of saccharification potential. This study highlights the biotech potential of cell-wall-bound phenolate esters and demonstrates the importance of substrate supply in lignin engineering.     

Slime problems in the paper and board industry

 The latest trends in the paper industry have been towards manufacturing by a neutral or alkaline process, greater consumption of secondary fibres and the closing-up of the process water systems. Under these conditions of papermaking, the problems of deposits, corrosion and odours due to the microbiological activity increase considerably and therefore, runnability and production problems occur. To help our understanding of the current situation in the paper industry, this paper presents a review of the microorganism sources, the consequences of the microbiological activity upon the actual systems of manufacturing paper and board, and the current state of the different alternatives for its prevention and control, as well as the future trends to address environmental considerations. Received: 20 March 1996/Received revision: 23 May 1996/Accepted: 28 May 1996     

Transient host range selection for genetic engineering of modified vaccinia virus Ankara

Recombinant vaccinia viruses are extremely valuable tools for research in molecular biology and immunology. The extension of vaccinia vector technology to replication-deficient and safety-tested virus strains such as modified vaccinia virus Ankara (MVA) have made this versatile eukaryotic expression system even more attractive for basic and clinical research. Here, we report on easily obtaining recombinant MVA using stringent growth selection on rabbit kidney RK-13 cells. We describe the construction and use of new MVA vector plasmids that carry an expression cassette of the vaccinia virus host range gene, K1L, as a transient selectable marker. These plasmids allow either stable insertion of additional recombinant genes into the MVA genome or precisely targeted mutagenesis of MVA genomic sequences. Repetitive DNA sequences flanking the K1L gene were designed to remove the marker gene from the viral genome by homologous recombination under nonselective growth conditions. The convenience of this new selection technique is demonstrated by isolating MVA recombinants that produce green fluorescent protein and by generating MVA deletion mutants.     

Recent advances in the genetic transformation of trees

As the commercial production of transgenic annual crops becomes a reality in many parts of the world, many people wonder if the genetic engineering of perennial trees will allow their eventual commercialization. Not long ago, trees were considered to be recalcitrant material for most molecular biology techniques, including genetic transformation. However, transgenes for shortening the juvenile phase or for phytoremediation purposes have now been incorporated, and the alteration of lignin biosynthesis and increased cellulose accumulation in forest trees have also been accomplished. For long-lived tree species, new questions arise regarding the stability of integration and expression of foreign genes. Biosafety considerations, including transgene dispersion through the pollen and advances in strategies to avoid this, are also important.     

Microbiologic problems associated with closed process water systems in the paper industry

Summary Process water from the entirely closed water system of a wastepaper reprocessing mill was examined for its content of different groups of microorganisms. Comparison with waste-paper reprocessing mills with nonclosed systems and concomitant higher specific water consumption showed that system closing is associated with a marked increase in the number of anaerobic microorganisms; Bifidobacteria were found to be the most frequently represented bacteria.The development of the microflora in the entirely closed water system was studied after total replacement of the process water with fresh water. Constant levels of anaerobic microorganisms were recorded after a lag period of 15 days. Gas chromatographic analyses showed that the process water from the entirely closed system contains significant amounts of metabolic products of anaerobic microorganisms.     

Monitoring genetic diversity in tropical trees with multilocus dominant markers

Since no universal codominant markers are currently available, dominant genetic markers, such as amplified fragment length polymorphism (AFLP), are valuable tools for assessing genetic diversity in tropical trees. However, the measurement of genetic diversity (H) with dominant markers depends on the frequency of null homozygotes (Q) and the fixation index (F) of populations. While Q can be estimated for AFLP loci, F is less accessible. Through a modelling approach, we show that the monolocus estimation of genetic diversity is strongly dependent on the value of F, but that the multilocus diversity estimate is surprisingly robust to variations in F. The robustness of the estimate is due to a mechanistic effect of compensation between negative and positive biases of H by different AFLP loci exhibiting contrasting frequency profiles of Q. The robustness was tested across contrasting theoretical frequency profiles of Q and verified for 10 neotropical species. Practical recommendations for the implementation of this analytical method are given for genetic surveys in tropical trees, where such markers are widely applied.     

Genetic engineering of trees: progress and new horizons

Genetic engineering of trees to improve productivity, wood quality, and resistance to biotic and abiotic stresses has been the primary goal of the forest biotechnology community for decades. We review the extensive progress in these areas and their current status with respect to commercial applications. Examples include novel methods for lignin modification, solutions for long-standing problems related to pathogen resistance, modifications to flowering onset and fertility, and drought and freeze tolerance. There have been numerous successful greenhouse and field demonstrations of genetically engineered trees, but commercial application has been severely limited by social and technical considerations. Key social factors are costly and uncertain regulatory hurdles and sweeping market barriers in the form of forest certification systems that disallow genetically modified trees. These factors limit and, in many cases, preclude field research and commercial adoption. Another challenge is the high cost and uncertainty in transformation efficiency that is needed to apply genetic engineering and gene editing methods to most species and genotypes of commercial importance. Recent advances in developmental gene-based transformation systems and gene editing, if combined with regulatory and certification system reform, could provide the foundation for genetic engineering to become a significant tool for coping with the increasing environmental and biological stresses on planted and wild forests.     

The control of microbiological problems in the paper industry

The aqueous environment of the paper or board machine is ideal for the growth of many types of organisms. These can have a major impact on mill profitability by causing production downtime, product rejects and operational difficulties. Treatment of these problems first requires accurate definition, followed by a good understanding of the system. In-depth analysis allows the deposition succession to be summised, which in turn will allow a correct choice of control strategy. This may require treatments to include both biocide and chemical deposit control. The benefits of biocides and combination programmes are discussed.     

Application of enzymes in the pulp and paper industry

The pulp and paper industry processes huge quantities of lignocellulosic biomass every year. The technology for pulp manufacture is highly diverse, and numerous opportunities exist for the application of microbial enzymes. Historically, enzymes have found some uses in the paper industry, but these have been mainly confined to areas such as modifications of raw starch. However, a wide range of applications in the pulp and paper industry have now been identified. The use of enzymes in the pulp and paper industry has grown rapidly since the mid 1980s. While many applications of enzymes in the pulp and paper industry are still in the research and development stage, several applications have found their way into the mills in an unprecedented short period of time. Currently the most important application of enzymes is in the prebleaching of kraft pulp. Xylanase enzymes have been found to be most effective for that purpose. Xylanase prebleaching technology is now in use at several mills worldwide. This technology has been successfully transferred to full industrial scale in just a few years. The enzymatic pitch control method using lipase was put into practice in a large-scale paper-making process as a routine operation in the early 1990s and was the first case in the world in which an enzyme was successfully applied in the actual paper-making process. Improvement of pulp drainage with enzymes is practiced routinely at mill scale. Enzymatic deinking has also been successfully applied during mill trials and can be expected to expand in application as increasing amounts of newsprint must be deinked and recycled. The University of Georgia has recently opened a pilot plant for deinking of recycled paper. Pulp bleaching with a laccase mediator system has reached pilot plant stage and is expected to be commercialized soon. Enzymatic debarking, enzymatic beating, and reduction of vessel picking with enzymes are still in the R&D stage but hold great promise for reducing energy. Other enzymatic applications, i.e., removal of shives and slime, retting of flax fibers, and selective removal of xylan, are also expected to have a profound impact on the future technology of the pulp and paper-making process.