Tree Biotechnology Research Articles

Whole genome duplication of wild-type and CINNAMYL ALCOHOL DEHYDROGENASE1-downregulated hybrid poplar reduces biomass yield and causes a brittle apex phenotype in field-grown wild types.

The potential of whole genome duplication to increase plant biomass yield is well-known. In Arabidopsis tetraploids, an increase in biomass yield was accompanied by a reduction in lignin content and, as a result, a higher saccharification efficiency was achieved compared with diploid controls. Here, we evaluated whether the results obtained in Arabidopsis could be translated into poplar and whether the enhanced saccharification yield upon alkaline pretreatment of hairpin-downregulated CINNAMYL ALCOHOL DEHYDROGENASE1 (hpCAD) transgenic poplar could be further improved upon a whole genome duplication. Using a colchicine treatment, wild-type (WT) Populus tremula x P. alba cv. INRA 717-1B4, a commonly used model clone in tree biotechnology research, and hpCAD tetraploids were generated and grown in the greenhouse. In parallel, WT tetraploid poplars were grown in the field. In contrast to Arabidopsis, a whole genome duplication of poplar had a negative impact on the biomass yield of both greenhouse- and field-grown trees. Strikingly, field-grown WT tetraploids developed a brittle apex phenotype, i.e., their tip broke off just below the apex. In addition, the chromosome doubling altered the biomass composition of field-grown, but not of greenhouse-grown tetraploid poplars. More specifically, the lignin content of field-grown tetraploid poplars was increased at the expense of matrix polysaccharides. This increase in lignin deposition in biomass is likely the cause of the observed brittle apex phenotype, though no major differences in stem anatomy or in mechanical properties could be found between di- and tetraploid WT poplars grown in the field. Finally, without biomass pretreatment, the saccharification efficiency of greenhouse- and field-grown WT diploids was not different from that of tetraploids, whereas that of greenhouse-grown hpCAD tetraploids was higher than that of greenhouse-grown diploids. Upon alkaline pretreatment, the saccharification yield of diploids was similar to that of tetraploids for all genotypes and growth conditions tested. This study showed that a whole genome duplication in hybrid WT and hpCAD poplar did neither result in further improvements in biomass yield, nor in improved biomass composition and, hence, saccharification performance.

Overexpression of vesicle-associated membrane protein PttVAP27-17 as a tool to improve biomass production and the overall saccharification yields in Populus trees

BackgroundBioconversion of wood into bioproducts and biofuels is hindered by the recalcitrance of woody raw material to bioprocesses such as enzymatic saccharification. Targeted modification of the chemical composition of the feedstock can improve saccharification but this gain is often abrogated by concomitant reduction in tree growth.ResultsIn this study, we report on transgenic hybrid aspen (Populus tremula × tremuloides) lines that showed potential to increase biomass production both in the greenhouse and after 5 years of growth in the field. The transgenic lines carried an overexpression construct for Populus tremula × tremuloides vesicle-associated membrane protein (VAMP)-associated protein PttVAP27-17 that was selected from a gene-mining program for novel regulators of wood formation. Analytical-scale enzymatic saccharification without any pretreatment revealed for all greenhouse-grown transgenic lines, compared to the wild type, a 20–44% increase in the glucose yield per dry weight after enzymatic saccharification, even though it was statistically significant only for one line. The glucose yield after enzymatic saccharification with a prior hydrothermal pretreatment step with sulfuric acid was not increased in the greenhouse-grown transgenic trees on a dry-weight basis, but increased by 26–50% when calculated on a whole biomass basis in comparison to the wild-type control. Tendencies to increased glucose yields by up to 24% were present on a whole tree biomass basis after acidic pretreatment and enzymatic saccharification also in the transgenic trees grown for 5 years on the field when compared to the wild-type control.ConclusionsThe results demonstrate the usefulness of gene-mining programs to identify novel genes with the potential to improve biofuel production in tree biotechnology programs. Furthermore, multi-omic analyses, including transcriptomic, proteomic and metabolomic analyses, performed here provide a toolbox for future studies on the function of VAP27 proteins in plants.

Fate of forest tree biotechnology facing climate change

Abstract Woody plants have been cultured in vitro since the 1930s. After that time much progress has been made in the culture of tissues, organs, cells, and protoplasts in tree species. Tree biotechnology has been making strides in clonal propagation by organogenesis and somatic embryogenesis. These regeneration studies have paved the way for gene transfer in forest trees. Transgenics from a number of forest tree species carrying a variety of recombinant genes that code for herbicide tolerance, pest resistance, lignin modification, increased woody bio-mass, and flowering control have been produced by Agrobacterium-mediated and biolistic methods, and some of them are undergoing confined field trials. Although relatively stable transgenic clones have been produced by genetic transformation in trees using organogenesis or somatic embryogenesis, there were also unintended unstable genetic events. In order to overcome the problems of randomness of transgene integration and instability reported in Agrobacterium-mediated or biolistically transformed plants, site-specific transgene insertion strategies involving clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) platform offer prospects for precise genome editing in plants. Nevertheless, it is important to monitor phenotypic and genetic stability of clonal material, not just under greenhouse conditions, but also under natural field conditions. Genetically modified poplars have been commercialized in China, and eucalypts and loblolly pine are expected to be released for commercial deployment in USA. Clonal forestry and transgenic forestry have to cope with rapid global climate changes in the future. Climate change is impacting species distributions and is a significant threat to biodiversity. Therefore, it is important to deploy Strategies that will assist the survival and evolution of forest tree species facing rapid climate change. Assisted migration (managed relocation) and biotechnological approaches offer prospects for adaptation of forest trees to climate change.

Efficient Genome Editing in Populus Using CRISPR/Cas12a.

The ability to create targeted mutations using clustered regularly inter-spaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 in support of forest tree biotechnology is currently limited. CRISPR/Cas12a is a novel CRISPR effector protein that not only broadens the CRISPR/Cas targeting range but also enables the generation of large-fragment deletions. In this study, a CRISPR/Cas12a system was evaluated for the induction of targeted mutations in the woody tree poplar (Populus alba × Populus glandulosa). Three Cas12a nucleases, namely, AsCas12a (Acidaminococcus sp. BV3L6), LbCas12a (Lachnospiraceae bacterium ND2006), and FnCas12a (Francisella tularensis subsp. novicidain U112), were used. We knocked out multiple targets of the phytoene desaturase gene 8 (PDS) using the CRISPR/Cas12a genome-targeting system, and the results indicated that the AsCas12a system is the most efficient. We further optimized the co-cultivation temperature after Agrobacterium-mediated transformation from 22 to 28°C to increase the Cas12a nuclease editing efficiency in poplar. AsCas12a showed the highest mutation efficiency, at 70%, and the majority of editing sites were composed of large-fragment deletions. By using this simple and high-efficiency CRISPR/Cas12a system, multiple targets can be modified to obtain multigene simultaneous knockout mutants in tree species, which will provide powerful tools with which to facilitate genetic studies of forest trees.

Technological Advances in Commercial Forestry

Biotechnological tools such as clonal propagation, haploid production, protoplast fusion, marker-assisted selection, and quantitative trait loci mapping are the age-old approaches in commercial forestry. Biotechnology has shown immense guarantee for the subsequent generation of plant breeders to alleviate the rising demand for food, fiber, and wood. The probable social and environmental impacts of the release of transgenic trees become a progressively more debatable issue and call more considerations. The present review is an attempt to summarize how advances in molecular biology i.e., comparative genomics and genome assembly approaches identify numerous polymorphic molecular markers like microsatellite, simple sequence repeats, single-nucleotide polymorphism (SNP), random amplified polymorphic DNA etc. and revolutionize the science of forest tree biotechnology during the last decade. The advent of next-generation sequencing technology identified many microsatellite loci in forest trees. Microsatellite markers play an important role in population genetics, conservation ecology, and phylogenetic analysis. SNP markers provide novel opportunities for the discovery of regulatory elements in the genome and eventually accelerate forest tree breeding.

Ecotechnological solutions to satisfy current demands of forest products, a glance beyond the trees

A significant increase in the demand for wood products, including timber, pulp and biofuels has exerted considerable pressure on the world's remaining natural forests. To expand the forest area by establishing forest plantations has been proposed as a way out to satisfy current and future demands, as well as to mitigate expected increases in atmospheric carbon. Specialized scientists in forestry and tree biotechnology advocate that humanity should rely on biotechnological solutions, particularly GM tree plantations, to achieve sustainable and more productive forest plantations. During the last decade, significant advances have occurred in both molecular methodologies and basic understanding of biotechnological processes associated with timber production. However, important sectors of civil society have showed reticence, even open opposition, towards these eco-technological solutions. This article explores on the one hand the rationale of the academic sector in promoting biotechnological solutions to satisfy current demands of forest products and biofuels, and on the other, analyzes society's opposition towards these solutions. The academic sector needs to understand where the resistance towards the development and establishment of these highly innovative technologies stems. Scientists need to become better at framing the importance of scientific research to citizens, at eliciting public participation and fostering public dialogue about science.

Stimulant Paste Preparation and Bark Streak Tapping Technique for Pine Oleoresin Extraction.

Tapping technique comprises the extraction of pine oleoresin, a non-wood forest product consisting of a complex mixture of mono, sesqui, and diterpenes biosynthesized and exuded as a defense response to wounding. Oleoresin is used to produce gum rosin, turpentine, and their multiple derivatives. Oleoresin yield and quality are objects of interest in pine tree biotechnology, both in terms of environmental and genetic control. Monitoring these parameters in individual trees grown in the field provides a means to examine the control of terpene production in resin canals, as well as the identification of genetic-based differences in resinosis. A typical method of tapping involves the removal of bark and application of a chemical stimulant on the wounded area. Here we describe the methods for preparing the resin-stimulant paste with different adjuvants, as well as the bark streaking process in adult pine trees.

木材と樹木のバイオテクノロジー<br>その現状と将来

主要な農作物における形質改善の場合と同様に，バイオテクノロジーは木材成分の改変や精鋭樹の選抜など，木材の材質改善や樹木の育種にとっても有用である。分子生物学や分子遺伝学，代謝工学などバイオテクノロジー分野の著しい発展により，木材形成や樹木の成長を制御する遺伝子などが同定されている。本稿では，木材や樹木のバイオテクノロジー，特にその基礎研究の現状について手短に触れ，その将来について考えてみたい。

How eco-evolutionary principles can guide tree breeding and tree biotechnology for enhanced productivity.

Tree breeding and biotechnology can enhance forest productivity and help alleviate the rising pressure on forests from climate change and human exploitation. While many physiological processes and genes are targeted in search of genetically improved tree productivity, an overarching principle to guide this search is missing. Here, we propose a method to identify the traits that can be modified to enhance productivity, based on the differences between trees shaped by natural selection and 'improved' trees with traits optimized for productivity. We developed a tractable model of plant growth and survival to explore such potential modifications under a range of environmental conditions, from non-water limited to severely drought-limited sites. We show how key traits are controlled by a trade-off between productivity and survival, and that productivity can be increased at the expense of long-term survival by reducing isohydric behavior (stomatal regulation of leaf water potential) and allocation to defense against pests compared with native trees. In contrast, at dry sites occupied by naturally drought-resistant trees, the model suggests a better strategy may be to select trees with slightly lower wood density than the native trees and to augment isohydric behavior and allocation to defense. Thus, which traits to modify, and in which direction, depend on the original tree species or genotype, the growth environment and wood-quality versus volume production preferences. In contrast to this need for customization of drought and pest resistances, consistent large gains in productivity for all genotypes can be obtained if root traits can be altered to reduce competition for water and nutrients. Our approach illustrates the potential of using eco-evolutionary theory and modeling to guide plant breeding and genetic technology in selecting target traits in the quest for higher forest productivity.

Transgenic woody plants for biofuel

Transgenic trees as a new source for biofuel have brought a great interest in tree biotechnology. Genetically modifying forest trees for ethanol production have advantages in technical challenges, costs, environmental concerns, and financial problems over some of crops. Genetic engineering of forest trees can be used to reduce the level of lignin, to produce the fast-growing trees, to develop trees with higher cellulose, and to allow the trees to be grown more widely. Trees can establish themselves in the field with less care of farmers, compared to most of crops. Transgenic crops as a new source for biofuel have been recently reviewed in several reviews. Here, we overview transgenic woody plants as a new source for biofuel including genetically modified woody plants and environment; main focus of woody plants genetic modifications; solar to chemical energy transfer; cellulose biosynthesis; lignin biosynthesis; and cellulosic ethanol as biofuel.

IUFRO Tree Biotechnology 2011: "From genomes to integration and delivery"

Forest trees have unquestionably entered the genomic era. The updated version of the Populus genome, the recently released Eucalyptus grandis genome and the concerted efforts towards the generation of genome sequences for spruces (Picea sp.) and pines (Pinus sp.) by several groups worldwide, are fueling a multitude of inter-disciplinary studies and applications in sustainable forest production and conservation. Time now calls for the integration of scientific fields with an increased sense of urgency for delivery of effective biotechnologies. The IUFRO (International Union of Forestry Research Organizations) Tree Biotechnology biannual conference has established a solid tradition for over 20 years as the official meeting of the IUFRO working group 2.04.06 – Molecular biology of forest trees. This conference has convened scientists and foresters interested in the genetics, genomics, molecular biology and physiology of forest trees, and the application of this knowledge to tree improvement and conservation. The Tree Biotechnology Conference has undoubtedly been the premiere international forum where the most cutting edge research in tree biotechnology developed both in academia and industry is presented. “From genomes to integration and delivery”, this was the theme chosen for the 2011 edition of the IUFRO Tree Biotechnology Conference, first time to be held in South America. Our intention was to promote a more integrated and applied dialogue on tree biotechnology and genomics, beyond the mainstream discussion of the fundamental advances on the genetic mechanisms that underlie tree phenotypes. In nine scientific sessions some of the current advances of genomics applied to forest conservation, tree physiology, stress response, molecular breeding, in vitro and propagation technologies, wood development and genetically modified (GM) trees were highlighted. With 340 registered participants, the Conference brought to Brazil most of the world’s brain power in forest tree genomics and biotechnology. An outstanding team of international scientists shared their results and visions on the present and future of this fast moving area of forest science, while a brilliant group of young scientist and students delivered a very energetic and diverse collection of high-quality scientific presentations. Forty two countries were represented at the Conference with almost 100 different laboratories from tens of Universities, research institutions and private companies. During the seven days of the Conference 26 invited lectures, 63 oral and 185 poster presentations were delivered, totaling 274 papers made available as extended abstracts into this BMC Proceedings supplement. The special workshop on the hot topic of “Genomic Selection in tree breeding” and the several reports on whole-genome studies, made this conference edition inaugurate a deliberate effort towards a better integration between the quantitative genomics, the “single-gene” and the system biology approaches to more efficiently unravel the complex relationships between genotypes and phenotypes in forest trees. A field trip to the forest plantations, nurseries and mill of VERACEL Cellulose was a definite highlight and a welcome break from the scientific sessions, providing an overview of some of the advances and challenges facing the translation of research into plantation forestry. In closing this introductory statement, acknowledgements are due to the outstanding financial support provided by the competitive grants of the Brazilian Ministry of Science and Technology through the National Research Council (CNPq) and the Ministry of Education through its agency for graduate studies (CAPES). Major support was also provided by EMBRAPA (Brazilian Corporation of Agricultural Research), and VERACEL Cellulose, the host organizations, together with an exceptional suite of private sponsors. Besides the organizations that backed this conference and an active Scientific Committee involved in abstract review a number of people were involved in the organization and logistics. The conference would not have been possible without the valuable contributions of all these players. Given the rewarding feedback received after the Conference, the original goal of providing an exceptional mix of science, social activities and field exploration in a relaxed atmosphere was truly accomplished. The IUFRO Tree Biotechnology Conference 2011 made a significant contribution to advance the forest biotechnology research community one step ahead on the challenging task of moving from gene and genome discoveries to the delivery of valuable technologies into sustainable forestry.

WAT1 (WALLS ARE THIN1) defines a novel auxin transporter in plants and integrates auxin signaling in secondary wall formation in Arabidopsis fibers

WAT1 (WALLS ARE THIN1) defines a novel auxin transporter in plants and integrates auxin signaling in secondary wall formation in Arabidopsis fibers. IUFRO Tree Biotechnology Conference 2011: From Genomes to Integration and Delivery

From genes towards products and the significance of gene delivery

SweTree Technologies is a plant and forest biotechnology company providing products and technologies to improve the productivity and performance properties of plants, wood and fiber for forestry and fiber related industries. In the area of Biotech Trees, we have been testing and developing genes and biotech trees. Since 2002 we have successfully screened well over one thousand genes in our gene testing program. The program has been mainly directed towards increased growth, improved wood properties and stress tolerance. The current outcome of this program will be presented. The primary gene developing program is performed in hybrid aspen for which we have a very efficient transformation protocol. However for practical tree biotechnology the genes and DNA constructs have to be transformed into the best commercial clones. Therefore we have been setting up efficient transformation protocols for both Eucalyptus and Populus elite clones. Another crucial part of a practical tree biotechnology program is to test the GM trees with commercially interesting genes under field conditions. We have initiated field trials and plan to have over 35 different types of transgenic trees in field trials by the end of this year. These field experiments are performed by SweTree or in collaboration with academic partners. The importance and requirements for elite clone transformation and field trials will be discussed in the presentation.

Transgenic biotechnology in forestry: what a long strange trip it's been

Transgenic biotechnology in forestry: what a long strange trip it's been

Hormonally regulated overexpression of Arabidopsis WUS and conifer LEC1 (CHAP3A) in transgenic white spruce: implications for somatic embryo development and somatic seedling growth

Adult conifers are still recalcitrant in clonal propagation despite significant advances in forest tree biotechnology. Plant regeneration through somatic embryogenesis from explants older than mature zygotic embryos is either difficult or impossible to achieve. To investigate if ectopic expression of transcription factors involved in the induction of the embryogenic process would induce somatic embryogenesis in Picea glauca (white spruce) somatic plants, we used the LEAFY-COTYLEDON1 homolog cloned from Picea mariana, CHAP3A, and Arabidopsis thaliana WUS to transform embryonal mass of P. glauca. Ectopic gene expression was induced by 17-beta-estradiol during stages of somatic embryogenesis (early embryogenesis and late embryogenesis) and somatic seedling growth in the transgenics. Of the two transcription factors, only WUS produced severe phenotypes by disrupting the development of somatic embryos on the maturation medium and inhibiting germination. However, none of the transgenes induced ectopic somatic embryogenesis even in the presence of plant growth regulators. Absolute quantitative PCR confirmed the expression of both CHAP3A and WUS in transgenic embryonal mass and in all parts of somatic seedlings. A high expression of the transgenes did not influence expression profiles of any of the ten other transcription factors tested, some of which have been known to be involved in the process of embryogenesis. Implications of these results for further work are discussed.

Functional Characterization of Poplar Wood-Associated NAC Domain Transcription Factors

Wood is the most abundant biomass produced by land plants. Dissection of the molecular mechanisms underlying the transcriptional regulation of wood formation is a fundamental issue in plant biology and has important implications in tree biotechnology. Although a number of transcription factors in tree species have been shown to be associated with wood formation and some of them are implicated in lignin biosynthesis, none of them have been demonstrated to be key regulators of the biosynthesis of all three major components of wood. In this report, we have identified a group of NAC domain transcription factors, PtrWNDs, that are preferentially expressed in developing wood of poplar (Populus trichocarpa). Expression of PtrWNDs in the Arabidopsis (Arabidopsis thaliana) snd1 nst1 double mutant effectively complemented the secondary wall defects in fibers, indicating that PtrWNDs are capable of activating the entire secondary wall biosynthetic program. Overexpression of PtrWND2B and PtrWND6B in Arabidopsis induced the expression of secondary wall-associated transcription factors and secondary wall biosynthetic genes and, concomitantly, the ectopic deposition of cellulose, xylan, and lignin. Furthermore, PtrWND2B and PtrWND6B were able to activate the promoter activities of a number of poplar wood-associated transcription factors and wood biosynthetic genes. Together, these results demonstrate that PtrWNDs are functional orthologs of SND1 and suggest that PtrWNDs together with their downstream transcription factors form a transcriptional network involved in the regulation of wood formation in poplar.

"Post-genomics" research in Eucalyptus in the near future

Forest produces the most abundant biomass resource on earth, and its ecological and commercial importance in maintaining the global environment and human society is irreplaceable. It is an urgent task to establish technological infrastructure to produce forest biomass in a sustainable manner. Forest tree biotechnology plays a pivotal role to achieve this goal. Genus Eucalyptus contains some of the most valuable and the fastest growing tropical tree species in the world. In 2006, the whole genome sequence of poplar, the first woody plant genome, was released. Subsequently, whole genome sequencing projects of two Eucalyptus species have started, and are currently ongoing. Eucalyptus expression-sequence tag (EST) databases are quite useful for identifying Eucalyptus genes which are orthologous to those of poplar and other model plants. In addition, establishing efficient systems for in vitro regeneration of transformed Eucalyptus will be indispensable for genetic improvement of the species. Importantly, these activities must be done to conserve the global environment and benefit the social welfare of local communities. In this review, the current status and “post-genomics” research directions in Eucalyptus biotechnology are outlined.

Integrated Analysis of Transcript, Protein and Metabolite Data To Study Lignin Biosynthesis in Hybrid Aspen

Tree biotechnology will soon reach a mature state where it will influence the overall supply of fiber, energy and wood products. We are now ready to make the transition from identifying candidate genes, controlling important biological processes, to discovering the detailed molecular function of these genes on a broader, more holistic, systems biology level. In this paper, a strategy is outlined for informative data generation and integrated modeling of systematic changes in transcript, protein and metabolite profiles measured from hybrid aspen samples. The aim is to study characteristics of common changes in relation to genotype-specific perturbations affecting the lignin biosynthesis and growth. We show that a considerable part of the systematic effects in the system can be tracked across all platforms and that the approach has a high potential value in functional characterization of candidate genes.

Predictive modelling and integration of functional genomics data for applications in plant biology

Tree biotechnology will soon reach a mature state where it will influence the overall supply of fiber and wood products. Through technological advancements, it may be possible to introduce trees th ...

Tree Biotechnology of Tropical Acacia

To establish a sustainable society, forest biomass or lignocellulosic biomass, which is the most abundantly accumulated biomass resource, plays an important role. Therefore it is critically important to establish systems for sustainable production of forest biomass, especially tropical forest resources. Forest tree biotechnology will play a very important role in such systems. Following the completion of whole genome sequence of Populus, tree bioscience and biotechnology have entered into “post-genomic era”. However, although tropical Acacia is one of the most important tropical plantation tree in Indonesia and Malaysia, its biotechnology is still at a primitive stage. Because the Acacia mangium genome sequence data base is far beyond the ability of university laboratories, it is important to construct expressed-sequence tag (EST) data bases for Acacia. A. mangium EST data bases are quite useful for identifying genes that are possibly involved in the expression of a given trait, and can be exploited to detect Acacia genes which are orthologous to those of other model plants that are involved in secondary xylem formation. In addition, establishing efficient systems for in vitro regeneration of transformed Acacia and identification of genes and metabolites responsible for expression of commercially important traits are indispensable for genetic modification of the species. Importantly, these activities must be done to benefit global environment conservation and social welfare of local communities. In this review, the current status of Acacia biotechnology is outlined.