TWENTY YEARS OF GENETIC MODIFICATIONS IN FORESTRY

Genetsko modificiranje, genetsko transformiranje ili genetski inženjering skup je tehnika koje pružaju mogućnost dodavanja poželjnih obilježja u superiorne geno tipove biljaka, putem prijenosa gena između različitih nesrodnih vrsta, rodova, pa čak i carstava, što se inače ne događa u prirodi. S druge strane, klasičnim se oplemenjivanjem putem selekcije i križanja između jedinki koje pripadaju istoj ili srodnim vrstama također izmjenjuju i ističu određena svojstva radi poboljšanja vrsta prema ljudskim mjerilima, ali je takav prijenos gena moguć i u prirodi. S genetskim modificiranjem na drvenastim biljkama započelo se 1987. godine na topolama (Fillatti i dr. 1987). To je prva studija u kojoj je uspješno regenerirano genetski modificirano tkivo šumske vrste drveća. Svojstva koja su najčešće predmet genetske modifikacije kod biljaka jesu sastav lignina, tolerancija na herbicide, otpornost na bolesti i štetnike te kontrola cvatnje. Osim samog postupka genetskih transformacija, terenski pokusi genetski modificiranoga šumskog drveća predstavljaju vrlo važan segment u cjelokupnom lancu istraživanja. Testiranje izmijenjenih svojstava u prirodnim okolišnim uvjetima pridonosi boljem shvaćanju interakcije okolišnih utjecaja i transformiranih svojstava. Iako se procjenjuje kako postoji preko 600 terenskih nasada s genetski modificiranim drvenastim vrstama širom svijeta, u šumarstvu gotovo nema komercijalne uporabe takvih plantaža. U hrvatskom šumarstvu ne postoje naznake takva korištenja biotehnologije. Međutim, praćenje svjetskih trendova važno je za implementaciju inozemnih iskustava u zakonsku regulativu te za stjecanje dovoljnih količina znanja za provedbe mjera sigurnosti i opreza prilikom takvih istraživanja.Genetic modification, genetic transformation or genetic engineering are synonyms for technique that permits adding desirable traits into superior genotypes by gene insertion between different species, families and even kingdoms which dose not occur in nature. On the other hand, traditional breeding relies primarily on selection and crosses within species or within closely related genera which exist in nature, to emphasize certain characteristics. But this method has no control over additional genetic material being incorporated within desired phenotype. Genetic modification is defined as use of recombinant DNA and asexual gene transfer methods to alter the structure or expression of specific genes and traits (FAO, 2004). The first report of genetic modified trees was in 1987 on Populus sp. (Fillati et al. 1987). For the first time it was successfully regenerated transformed tissue of forest tree species. Applications of genetic modification in forest tree species include lignin modification, herbicide tolerance, disease and pest resistance and flowering control, so called target traits. Besides genetic modification techniques, field trial experiments are also very important step in genetic transformation. After first successful regeneration of transformed tissue, in the next 15 years more than 210 field trials of genetically modified trees were established in 16 countries worldwide. The great majority of field trial experiments occur in the United States (FAO, 2004). According to the newest assessment there are over 600 field trials with genetically modified trees in the world (Strauss, IUFRO Conference 2011). But there are hardly any commercial use of genetic modified forest trees. The rapid development of genetic engineering it will attempt to meet global demand for forest products, biofuels, to restore threatened species, and to protect future forests from invasive pests and climate change. When used responsibly, society and the environment can benefit from advanced biotechnology (Anon. 2010). There is no indication for using biotechnology or genetic modification in Croatian forestry. But gathering existing knowledge and capacity building can only contribute for implementation of world knowledge into national legislation, and prepare experts for future development

Analytical Py-GC/MS of genetically modified poplar for the increased production of bio-aromatics

Genetic engineering is a powerful tool to steer bio-oil composition towards the production of speciality chemicals such as guaiacols, syringols, phenols, and vanillin through well-defined biomass feedstocks. Our previous work demonstrated the effects of lignin biosynthesis gene modification on the pyrolysis vapour compositions obtained from wood derived from greenhouse-grown poplars. In this study, field-grown poplars downregulated in the genes encoding CINNAMYL ALCOHOL DEHYDROGENASE (CAD), CAFFEIC ACID O-METHYLTRANSFERASE (COMT) and CAFFEOYL-CoA O-METHYLTRANSFERASE (CCoAOMT), and their corresponding wild type were pyrolysed in a Py-GC/MS. This work aims at capturing the effects of downregulation of the three enzymes on bio-oil composition using principal component analysis (PCA). 3,5-methoxytoluene, vanillin, coniferyl alcohol, 4-vinyl guaiacol, syringol, syringaldehyde, and guaiacol are the determining factors in the PCA analysis that are the substantially affected by COMT, CAD and CCoAOMT enzyme downregulation. COMT and CAD downregulated transgenic lines proved to be statistically different from the wild type because of a substantial difference in S and G lignin units. The sCAD line lead to a significant drop (nearly 51%) in S-lignin derived compounds, while CCoAOMT downregulation affected the least (7-11%). Further, removal of extractives via pretreatment enhanced the statistical differences among the CAD transgenic lines and its wild type. On the other hand, COMT downregulation caused 2-fold reduction in S-derived compounds compared to G-derived compounds. This study manifests the applicability of PCA analysis in tracking the biological changes in biomass (poplar in this case) and their effects on pyrolysis-oil compositions

Mechanical characterisation of the developing cell wall layers of tension wood fibres by Atomic Force Microscopy

Abstract Trees can generate large mechanical stresses at the stem periphery to control the orientation of their axes. This key factor in the biomechanical design of trees, named “maturation stress”, occurs in wood fibres during cellular maturation when their secondary cell wall thickens. In this study, the spatial and temporal stiffening kinetics of the different cell wall layers were recorded during fibre maturation on a sample of poplar tension wood using atomic force microscopy. The thickening of the different layers was also recorded. The stiffening of the CML, S 1 and S 2 -layers was initially synchronous with the thickening of the S 2 layer and continued a little after the S 2 -layer reached its final thickness as the G-layer begins to develop. In contrast, the global stiffness of the G-layer, which initially increased with its thickening, was almost stable long before it reached its final maximum thickness. A limited radial gradient of stiffness was observed in the G-layer, but it decreased sharply on the lumen side, where the new sub-layers are deposited during cell wall thickening. Although very similar at the ultrastructural and biochemical levels, the stiffening kinetics of the poplar G-layer appears to be very different from that described in maturing bast fibres. Highlight New insights into the changes in mechanical properties within the cell wall of poplar tension wood fibres during maturation have been obtained using atomic force microscopy

Different routes for conifer- and sinapaldehyde and higher saccharification upon deficiency in the dehydrogenase CAD1

In the search for renewable energy sources, genetic engineering is a promising strategy to improve plant cell wall composition for biofuel and bioproducts generation. Lignin is a major factor determining saccharification efficiency and, therefore, is a prime target to engineer. Here, lignin content and composition were modified in poplar (Populus tremula 3 Populus alba) by specifically down-regulating CINNAMYL ALCOHOL DEHYDROGENASE1 (CAD1) by a hairpin-RNA-mediated silencing approach, which resulted in only 5% residual CAD1 transcript abundance. These transgenic lines showed no biomass penalty despite a 10% reduction in Klason lignin content and severe shifts in lignin composition. Nuclear magnetic resonance spectroscopy and thioacidolysis revealed a strong increase (up to 20-fold) in sinapaldehyde incorporation into lignin, whereas coniferaldehyde was not increased markedly. Accordingly, ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a more than 24,000-fold accumulation of a newly identified compound made from 8-8 coupling of two sinapaldehyde radicals. However, no additional cinnamaldehyde coupling products could be detected in the CAD1-deficient poplars. Instead, the transgenic lines accumulated a range of hydroxycinnamate-derived metabolites, of which the most prominent accumulation (over 8,500-fold) was observed for a compound that was identified by purification and nuclear magnetic resonance as syringyl lactic acid hexoside. Our data suggest that, upon down-regulation of CAD1, coniferaldehyde is converted into ferulic acid and derivatives, whereas sinapaldehyde is either oxidatively coupled into S'(8-8) S' and lignin or converted to sinapic acid and derivatives. The most prominent sink of the increased flux to hydroxycinnamates is syringyl lactic acid hexoside. Furthermore, low-extent saccharification assays, under different pretreatment conditions, showed strongly increased glucose (up to +81%) and xylose (up to +153%) release, suggesting that down-regulating CAD1 is a promising strategy for improving lignocellulosic biomass for the sugar platform industry

Genome-Wide Analysis of LIM Gene Family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa

In Eukaryotes, LIM proteins act as developmental regulators in basic cellular processes such as regulating the transcription or organizing the cytoskeleton. The LIM domain protein family in plants has mainly been studied in sunflower and tobacco plants, where several of its members exhibit a specific pattern of expression in pollen. In this paper, we finely characterized in poplar six transcripts encoding these proteins. In Populus trichocarpa genome, the 12 LIM gene models identified all appear to be duplicated genes. In addition, we describe several new LIM domain proteins deduced from Arabidopsis and rice genomes, raising the number of LIM gene models to six for both species. Plant LIM genes have a core structure of four introns with highly conserved coding regions. We also identified new LIM domain proteins in several other species, and a phylogenetic analysis of plant LIM proteins reveals that they have undergone one or several duplication events during the evolution. We gathered several LIM protein members within new monophyletic groups. We propose to classify the plant LIM proteins into four groups: αLIM1, βLIM1, γLIM2, and δLIM2, subdivided according to their specificity to a taxonomic class and/or to their tissue-specific expression. Our investigation of the structure of the LIM domain proteins revealed that they contain many conserved motifs potentially involved in their function