Development and Validation of Sterility Systems for Trees

The overall goal of this project was to develop and validate sterility systems in poplar with the ultimate goal of fulfilling the basic requirements for commercial use. For this, sterility must be complete and stable over multiple growing seasons, cause no detrimental effects on vegetative growth, and successful transformation events must be identifiable via molecular tests when trees are still juvenile. Because of the inherent difficulties in achieving and demonstrating complete sterility in trees, our approach was to study alternate sterility systems in Arabidopsis and/or early-flowering tree systems. The public benefit from this work is the capacity for containment of genes or exotic forms of trees so they can be of benefit for industry for production of wood, energy, and renewable products, while having minimal impact on wild populations of trees. We tested three methods for engineering sterility: dominant negative mutant (DNM) proteins, floral tissue ablation, and RNA interference (RNAi) to suppress the expression of several floral regulatory genes. The ultimate goal of this work was to produce a number of transgenic poplars that could be outplanted to enable future assessments of the effectiveness of these transgenic sterility methods. Our attempts to produce ablation constructs that did not interfere with tree health were partially successful. Using the poplar LEAFY gene promoter and the barnase/barstar system, we were able to regenerate plants that grew well in the greenhouse, but they showed poor health in the field. Four of seven DNM genes tested were considered promising enough, based on results in Arabidopsis, to produce transgenic poplars. Single, double, and triple RNAi genes were produced and transformed into poplar. Over all, we produced 1,964 PCR-confirmed transgenic events with 19 different kinds of sterility genes and several kinds of control genes. We propagated 5,640, 6,820, and 7,055 trees for each of three test poplar genotypes, and field plantings were begun in Spring of 2003 and will be finished in Spring 2007. Continued field studies and monitoring will be required to establish if any of the approaches we have taken will prove to be safe for tree health, stable, and provide reliable containment

EARLY BUD-BREAK 1 (EBB1) is a regulator of release from seasonal dormancy in poplar trees

Trees from temperate latitudes transition between growth and dormancy to survive dehydration and freezing stress during winter months. We employed activation tagging to isolate a dominant mutation affecting release from dormancy, and identified the corresponding gene EARLY BUD-BREAK 1 (EBB1). We demonstrate through positioning of the tag, expression analysis, and retransformation experiments that EBB1 encodes a putative AP2/ERF transcription factor. Transgenic upregulation of the gene caused early bud-flush, while down-regulation delayed bud-break. Native EBB1 expression was highest in actively-growing apices, undetectable during the dormancy period, but rapidly increased prior to bud-break. The EBB1 transcript was localized in the L1/L2 layers of the shoot meristem and leaf primordia. EBB1-overexpressing transgenic plants displayed enlarged shoot meristems, open and poorly differentiated buds, and a higher rate of cell division in the apex. Transcriptome analyses of the EBB1 transgenics identified 971 differentially-expressed genes whose expression correlated with the EBB1 expression changes in the transgenic plants. Promoter analysis among the differentially expressed genes for presence of a canonical EBB1 binding site identified 65 putative target genes indicative of a broad regulatory context of EBB1 function. Our results suggest that EBB1 has a major and integrative role in reactivation of meristem activity after winter dormancy.Keywords: Bud-break, Phenology, Dormancy, Meristem, Adaptation, Climate change, Populus, Regeneration, AP2/ER

FT overexpression induces precocious flowering and normal reproductive development in Eucalyptus

Eucalyptus trees are among the most important species for industrial forestry worldwide. However, as with most forest trees, flowering does not begin for one to several years after planting which can limit the rate of conventional and molecular breeding. To speed flowering, we transformed a Eucalyptus grandis × urophylla hybrid (SP7) with a variety of constructs that enable overexpression of FLOWERING LOCUS T (FT). We found that FT expression led to very early flowering, with events showing floral buds within 1–5 months of transplanting to the glasshouse. The most rapid flowering was observed when the cauliflower mosaic virus 35S promoter was used to drive the Arabidopsis thaliana FT gene (AtFT). Early flowering was also observed with AtFT overexpression from a 409S ubiquitin promoter and under heat induction conditions with Populus trichocarpa FT1 (PtFT1) under control of a heat-shock promoter. Early flowering trees grew robustly, but exhibited a highly branched phenotype compared to the strong apical dominance of nonflowering transgenic and control trees. AtFT-induced flowers were morphologically normal and produced viable pollen grains and viable self- and cross-pollinated seeds. Many self-seedlings inherited AtFT and flowered early. FT overexpression-induced flowering in Eucalyptus may be a valuable means for accelerating breeding and genetic studies as the transgene can be easily segregated away in progeny, restoring normal growth and form.This is the publisher’s final pdf. The article is copyrighted by Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd. It is published by Wiley Open Access and can be found at: http://onlinelibrary.wiley.com/journal/10.1111/%28ISSN%291467-7652Keywords: Eucalypts, Flowering Locus T, forest biotechnology, breeding, transgenic, genetic engineerin

Genetic containment in vegetatively propagated forest trees : CRISPR disruption of LEAFY function in Eucalyptus gives sterile indeterminate inflorescences and normal juvenile development

Eucalyptus is among the most widely planted taxa of forest trees worldwide. However, its spread as an exotic or genetically engineered form can create ecological and social problems. To mitigate gene flow via pollen and seeds, we mutated the Eucalyptus orthologue of LEAFY (LFY) by transforming a Eucalyptus grandis 9 urophylla wild-type hybrid and two Flowering Locus T (FT) overexpressing (and flowering) lines with CRISPR Cas9 targeting its LFY orthologue, ELFY. We achieved high rates of elfy biallelic knockouts, often approaching 100% of transgene insertion events. Frameshift mutations and deletions removing conserved amino acids caused strong floral alterations, including indeterminacy in floral development and an absence of male and female gametes. These mutants were otherwise visibly normal and did not differ statistically from transgenic controls in juvenile vegetative growth rate or leaf morphology in greenhouse trials. Genes upstream or near to ELFY in the floral development pathway were overexpressed, whereas floral organ identity genes downstream of ELFY were severely depressed. We conclude that disruption of ELFY function appears to be a useful tool for sexual containment, without causing statistically significant or large adverse effects on juvenile vegetative growth or leaf morphology.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1467-7652pm2022BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologyZoology and Entomolog

A tapetal ablation transgene induces stable male sterility and slows field growth in Populus

The field performance of genetic containment technologies–considered important for certain uses of transgenic trees in forestry–is poorly known. We tested the efficiency of a barnase gene driven by the TA29 tapetum-dominant promoter for influencing growth rate and inducing male sterility in a field trial of transgenic hybrid poplar (Populus tremula × Populus tremuloides). When the growth of 18 transgenic insertion events with the sterility transgene were compared to non-transgenic controls after two growing seasons, they grew 40 % more slowly in stem volume, and all but one transgenic event grew significantly more slowly than the control. In contrast, when we compared the growth of transgenic trees containing four kinds of β-glucuronidase (GUS) reporter gene constructs to non-transgenic trees—all of which had been produced using the same transformation method and poplar clone and grown at the same field site—there were no statistically significant differences in growth after three growing seasons. In 2 years where gross pollen release from catkins was monitored and found to be abundant in the control, no pollen was visible in the transgenic trees; microscopy suggested the cause was tapetal collapse and revealed the presence of a very few normal-sized pollen grains of unknown viability. In two additional years when viable, well-formed pollen was microscopically documented in controls, and no pollen could be observed in any transgenic trees. We conclude that this construct resulted in robust and possibly complete male sterility that was stable over 4 years in the field.Keywords: Forest biotechnology, Populus, Pollen, Genetic containment, Risk assessment, Barnase, Genetic engineering, TA29 promoterKeywords: Forest biotechnology, Populus, Pollen, Genetic containment, Risk assessment, Barnase, Genetic engineering, TA29 promote

Methylome reorganization during in vitro dedifferentiation and regeneration of Populus trichocarpa

Background: Cytosine DNA methylation (5mC) is an epigenetic modification that is important to genome stability and regulation of gene expression. Perturbations of 5mC have been implicated as a cause of phenotypic variation among plants regenerated through in vitro culture systems. However, the pattern of change in 5mC and its functional role with respect to gene expression, are poorly understood at the genome scale. A fuller understanding of how 5mC changes during in vitro manipulation may aid the development of methods for reducing or amplifying the mutagenic and epigenetic effects of in vitro culture and plant transformation. Results: We investigated the in vitro methylome of the model tree species Populus trichocarpa in a system that mimics routine methods for regeneration and plant transformation in the genus Populus (poplar). Using methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq), we compared the methylomes of internode stem segments from micropropagated explants, dedifferentiated calli, and internodes from regenerated plants. We found that more than half (56%) of the methylated portion of the genome appeared to be differentially methylated among the three tissue types. Surprisingly, gene promoter methylation varied little among tissues, however, the percentage of body-methylated genes increased from 9% to 14% between explants and callus tissue, then decreased to 8% in regenerated internodes. Forty-five percent of differentially-methylated genes underwent transient methylation, becoming methylated in calli, and demethylated in regenerants. These genes were more frequent in chromosomal regions with higher gene density. Comparisons with an expression microarray dataset showed that genes methylated at both promoters and gene bodies had lower expression than genes that were unmethylated or only promoter-methylated in all three tissues. Four types of abundant transposable elements showed their highest levels of 5mC in regenerated internodes. Conclusions: DNA methylation varies in a highly gene-and chromosome-differential manner during in vitro differentiation and regeneration. 5mC in redifferentiated tissues was not reset to that in original explants during the study period. Hypermethylation of gene bodies in dedifferentiated cells did not interfere with transcription, and may serve a protective role against activation of abundant transposable elements.Keywords: Jacq, Palm Elaeis guineensis, Expression analysis, Genome, Cytosine methylation, Arabidopsis cells, Somaclonal variation, Plants, Tissue culture, Gene