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Summary: Wide variation in amenability to transformation and regeneration (TR) among many plant species and genotypes presents a challenge to the use of genetic engineering in research and breeding. To help understand the causes of this variation, we performed association mapping and network analysis using a population of 1204 wild trees of Populus trichocarpa (black cottonwood).To enable precise and high‐throughput phenotyping of callus and shoot TR, we developed a computer vision system that cross‐referenced complementary red, green, and blue (RGB) and fluorescent‐hyperspectral images. We performed association mapping using single‐marker and combined variant methods, followed by statistical tests for epistasis and integration of published multi‐omic datasets to identify likely regulatory hubs.We report 409 candidate genes implicated by associations within 5 kb of coding sequences, and epistasis tests implicated 81 of these candidate genes as regulators of one another. Gene ontology terms related to protein–protein interactions and transcriptional regulation are overrepresented, among others.In addition to auxin and cytokinin pathways long established as critical to TR, our results highlight the importance of stress and wounding pathways. Potential regulatory hubs of signaling within and across these pathways include GROWTH REGULATORY FACTOR 1 (GRF1), PHOSPHATIDYLINOSITOL 4‐KINASE β1 (PI‐4Kβ1), and OBF‐BINDING PROTEIN 1 (OBP1). [ABSTRACT FROM AUTHOR]

Here we report the discovery through activation tagging and subsequent characterization of the BIG LEAF (BL) gene from poplar. In poplar, BL regulates leaf size via positively affecting cell proliferation. Up and downregulation of the gene led to increased and decreased leaf size, respectively, and these phenotypes corresponded to increased and decreased cell numbers. BL function encompasses the early stages of leaf development as native BL expression was specific to the shoot apical meristem and leaf primordia and was absent from the later stages of leaf development and other organs. Consistently, BL downregulation reduced leaf size at the earliest stages of leaf development. Ectopic expression in mature leaves resulted in continued growth most probably via sustained cell proliferation and thus the increased leaf size. In contrast to the positive effect on leaf growth, ectopic BL expression in stems interfered with and significantly reduced stem thickening, suggesting that BL is a highly specific activator of growth. In addition, stem cuttings from BL overexpressing plants developed roots, whereas the wild type was difficult to root, demonstrating that BL is a positive regulator of adventitious rooting. Large transcriptomic changes in plants that overexpressed BL indicated that BL may have a broad integrative role, encompassing many genes linked to organ growth. We conclude that BL plays a fundamental role in control of leaf size and thus may be a useful tool for modifying plant biomass productivity and adventitious rooting. [ABSTRACT FROM AUTHOR]

Genetic engineering (GE) can be used to improve forest plantation productivity and tolerance of biotic and abiotic stresses. However, gene flow from GE forest plantations is a large source of ecological, social and legal controversy. The use of genetic technologies to mitigate or prevent gene flow has been discussed widely and should be technically feasible in a variety of plantation taxa. However, potential ecological effects of such modifications, and their social acceptability, are not well understood. Focusing on Eucalyptus, Pinus, Populus and Pseudotsuga - genera that represent diverse modes of pollination and seed dispersal - we conducted in-depth reviews of ecological processes associated with reproductive tissues.Wealso explored potential impacts of various forms of reproductive modification at stand and landscape levels, and means for mitigating impacts. We found little research on potential reactions by the public and other stakeholders to reproductive modification in forest plantations. However, there is considerable research on related areas that suggest key dimensions of concern and support. We provide detailed suggestions for research to understand the biological and social dimensions of containment technologies, and consider the role of regulatory and market restrictions that obstruct necessary ecological and genetic research. [ABSTRACT FROM AUTHOR]

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 ( At FT). Early flowering was also observed with At FT overexpression from a 409S ubiquitin promoter and under heat induction conditions with Populus trichocarpa FT1 ( Pt FT1) 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. At FT-induced flowers were morphologically normal and produced viable pollen grains and viable self- and cross-pollinated seeds. Many self-seedlings inherited At FT 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. [ABSTRACT FROM AUTHOR]

We studied the effects on plant growth from insertion of five cisgenes that encode proteins involved in gibberellin metabolism or signalling. Intact genomic copies of Pt GA20ox7, PtGA2ox2,Pt RGL1_1, PtRGL1_2 and PtGAI1 genes from the genome-sequenced Populus trichocarpa clone Nisqually-1 were transformed into Populus tremula × alba (clone INRA 717-1B4), and growth, morphology and xylem cell size characterized in the greenhouse. Each cisgene encompassed 1-2 kb of 5′ and 1 kb of 3′ flanking DNA, as well as all native exons and introns. Large numbers of independent insertion events per cisgene (19-38), including empty vector controls, were studied. Three of the cisgenic modifications had significant effects on plant growth rate, morphology or wood properties. The PtGA20ox7 cisgene increased rate of shoot regeneration in vitro, accelerated early growth, and variation in growth rate was correlated with PtGA20ox7 gene expression. PtRGL1_1 and PtGA2ox2 caused reduced growth, while PtRGL1_2 gave rise to plants that grew normally but had significantly longer xylem fibres. RT-PCR studies suggested that the lack of growth inhibition observed in PtRGL1_2 cisgenic plants was a result of co-suppression. PtGAI1 slowed regeneration rate and both PtGAI1 and PtGA20ox7 gave rise to increased variance among events for early diameter and volume index, respectively. Our work suggests that cisgenic insertion of additional copies of native genes involved in growth regulation may provide tools to help modify plant architecture, expand the genetic variance in plant architecture available to breeders and accelerate transfer of alleles between difficult-to-cross species. [ABSTRACT FROM AUTHOR]

Matrix attachment regions (MARs) are thought to buffer transgenes from the influence of surrounding chromosomal sequences, and therefore to reduce transgene silencing and variation in expression. The statistical properties of more than 400 independent transgenic events produced in Populus, with and without flanking MAR elements from the tobacco root gene RB7, were analysed. The expression of two reporter genes in two poplar clones during three phases of vegetative growth, and the association of T-DNA characteristics with expression, was examined. It was found that MARs did not show a consistent effect on transgene expression levels; they had no effect on the green fluorescent protein ( GFP) reporter gene, but reduced expression in the Basta resistance ( BAR) reporter gene by 23%. The presence of MARs reduced expression variability within transformant populations, apparently by reducing the number of silenced or weakly expressing events. Transgene expression was highly stable over vegetative growth cycles that spanned 3 years of growth in the glasshouse and field, but MARs showed no association with the strength of correlations in expression over the years. Nonetheless, MARs increased the correlation in expression between a p35S:: GFP and p rbcS:: BAR transgene linked on the same vector, but the effect was small and varied between the years. The presence of MARs had no effect on the transgene copy number, but was positively associated with T-DNA truncations, as well as with the formation of direct over inverted repeats at the same chromosomal locus. [ABSTRACT FROM AUTHOR]

We tested the efficacy of an attenuation system developed to preclude the deleterious effects of floral promoter::cytotoxin genes on vegetative growth of transgenic sterile plants. We tested the promoter (2.6 kb 5′ region) of the poplar LEAFY gene PTLF driving barstar, combined on the same T-DNA with barstar driven by either the CaMV 35S basal promoter +5 to −72 fragment ( 35SBP), 35SBP fused to the TMV omega element ( 35SBP omega), or the NOS promoter. The unattenuated pPTLF::barnase construct failed to give rise to any transgenic events, suggesting substantial non-reproductive expression from this promoter. The barstar-attenuated constructs enabled transformation, but the rate was reduced by nearly one-third. Four events (7% of attenuated events) had highly abnormal morphology, and were identified during the early phases of propagation; these events had significantly higher barnase:barstar expression ratios based on quantitative RT-PCR. A greenhouse study showed that phenotypically normal attenuated plants grew at the same rate as wild-type and barnase-lacking transgenic plants. A statistically significant positive linear association was found between relative growth rate (RGR) and barstar:barnase ratio in the attenuated events, and graphical analysis suggested a threshold for barstar attenuation of barnase, above which additional levels of barstar did not provide further attenuation. Surprisingly, the appearance and growth rate of the nearly all of the attenuated events were substantially reduced after one or two growing seasons in the field, and the extent of growth reduction was associated with barstar:barnase expression ratio. These results demonstrate the importance of field testing during early phases of research to identify pleiotropic effects of transgenic sterility genes in trees. [ABSTRACT FROM AUTHOR]

Focuses on regulations for genetically engineered organisms. Discrimination in implementation of regulations; Relevance of ability to predict invasiveness of exotic organisms to assignment of homologous genes to risk classes.

Comments on an article by Jim Hancock that suggested a way to improve the process of assigning risk in genetically engineered crops by placing the emphasis on the transgenic trait, the crop host and the presence of wild relatives. Kinds of compartments that can be constructed to allow the breeding process to accelerate in the light of genomics knowledge and genetic engineering; Need for respect for natural selection; Importance of the scale of introduction to the determination of the probability of spread.

Many species of Populus, particularly cottonwoods of sections Aigeiros and Tacamahaca, remain recalcitrant to genetic transformation. We demonstrate that transgenic trees can be readily produced in several poplar genotypes using wild-type Agrobacterium rhizogenes. Hairy roots were produced in a variety of clones that included Populus trichocarpa Torr & A. Gray, Populus deltoides Bartr. ex Marsh., P. trichocarpa x P. deltoides hybrids, and Populus alba L. x Populus grandidentataMichx., some of which were otherwise recalcitrant to regeneration ortransformation. The frequency of hairy roots ranged from 17 to 92%; nearly all hairy roots could be regenerated into transgenic plants. Among 18 transgenic lines that were confirmed by Southern analysis andgrown into small trees, 5 showed severe dwarfism and other phenotypic abnormalities, while the majority grew normally. Agrobacterium rhizogenes appears to be an effective means for tansformation of diverse poplar genotypes and may be useful in genotypes where disarmed strains of Agrobacterium or regeneration protocols are inadequate to produce transgenic plants. [ABSTRACT FROM AUTHOR]

Because cultivation of exotic woody ornamental plants has led to establishment of a number of invasive species, there is considerable interest in breeding methods to reduce the propensity for spread. We review progress in conventional breeding and transgenic biotechnology approaches to producing sterile forms of ornamental woody plants. Conventional forms of inducing sterility, including induction of polyploidy, interspecific hybridization, and mutagenesis, are generally inexpensive and can be applied to a diversity of species at low to moderate cost. They have also been shown to be capable of producing commercially successful cultivars. In contrast, despite a variety of highly promising and rapidly developing approaches using transgenic methods, the inability to efficiently regenerate and genetically transform most ornamental species makes application of these innovations highly problematic. Moreover, because of the fragmented pattern of ornamental nursery ownership, the numerous species and varieties used, and the high regulatory cost for permits to sell most types of transgenic varieties (even when their environmental risk of spread has been reduced by sterility), application of transgenic methods is largely infeasible. A combination of fundamental regulatory reform and expanded biological research on generalized transformation and sterility methods is needed to overcome these barriers. [ABSTRACT FROM AUTHOR]