Denis Lachance

Stable genetic transformation of Picea mariana (black spruce) via particle bombardment

Stable genetic transformation ofPicea mariana (black spruce) was obtained via particle bombardment into two target tissues, mature cotyledonary somatic embryos and suspensions from embryonal masses, with the Biolistic PDS-1000/He device. Seven transgenic embryogenic cell line were obtained from the mature cotyledonary somatic embryos after secondary somatic embryogenesis from two different cell lines (R4F14 and 119794-014). The suspension culture from embryonal masses produced five transgenic cell lines from one cell line (R4F14). Expression of the introduced β-glucuronidase (GUS) and neomycin phosphotransferase II (NPT II) genes was detected by histochemistry and fluorometry, and by ELISA in 10 of the lines. Two lines showed only NPT II gene expression. Four of the five lines obtained after bombardment of suspensions of embryonal masses showed lower levels of expression of GUS and NPT II. The integration of the foreign genes was confirmed by polymerase chain reaction analyses and Southern hybridization for GUS and NPT II, and complex hybridization patterns were observed. The 12 transgenic lines obtained had a typical embryogenic morphology and were capable of maturation and germination. Over 40 transgenic trees were regenerated from one of the transgenic lines, and they have a normal phenotype.

Regeneration of transgenic Picea glauca, P. mariana, and P. abies after cocultivation of embryogenic tissue with Agrobacterium tumefaciens

SummaryTransgenic plants of three Picea species were produced after coculture of embryogenic tissue with the disarmed strain of Agrobacterium tumefaciens C58/pMP90/pBIV10 and selection on medium containing kanamycin. In addition to the nptII selectable gene (conferring resistance to kanamycin), the vector carried the uidA (β-glucuronidase) marker gene. Transformation frequencies were dependent on the species, genotype, and post-cocultivation procedure. Of the three species tested, P. mariana was transformed at the highest frequency, followed by P. glauca and P. abies. The transgenic state of the embryogenic tissue was initially, confirmed by histochemical β-glucuronidase (GUS) assay followed by Southern hybridization. One to over five copies of T-DNA were detected in various transgenic lines analyzed. Transgenic plants were regenerated for all species using modified protocols for maturation and germination of somatic embryos.

Characterization of a poplar NIMA-related kinase PNek1 and its potential role in meristematic activity

Meristems are sites of undifferentiated cell division, which carry on developing into functional organs. Using the two‐hybrid system with a poplar 14‐3‐3, we uncovered poplar NIMA‐related kinase 1 (PNek1) as an interacting protein. PNek1 shows high homology to the mammalian NIMA‐related kinases, which are thought to be involved in cell cycle progression. Using a synchronized poplar cell suspension, we observed an accumulation of PNek1 mRNA at the G1/S transition and throughout the G2‐to‐M progression. Moreover, PNek1‐GFP fusion protein localized in the cytoplasm and in both the nuclear and nucleolar regions. Overexpression of PNek1‐GFP in Arabidopsis caused morphological abnormalities in flower and siliques. Overall, these results suggest that PNek1 is involved in plant development.

Large-scale screening of transcription factor–promoter interactions in spruce reveals a transcriptional network involved in vascular development

This research aimed to investigate the role of diverse transcription factors (TFs) and to delineate gene regulatory networks directly in conifers at a relatively high-throughput level. The approach integrated sequence analyses, transcript profiling, and development of a conifer-specific activation assay. Transcript accumulation profiles of 102 TFs and potential target genes were clustered to identify groups of coordinately expressed genes. Several different patterns of transcript accumulation were observed by profiling in nine different organs and tissues: 27 genes were preferential to secondary xylem both in stems and roots, and other genes were preferential to phelloderm and periderm or were more ubiquitous. A robust system has been established as a screening approach to define which TFs have the ability to regulate a given promoter in planta. Trans-activation or repression effects were observed in 30% of TF–candidate gene promoter combinations. As a proof of concept, phylogenetic analysis and expression and trans-activation data were used to demonstrate that two spruce NAC-domain proteins most likely play key roles in secondary vascular growth as observed in other plant species. This study tested many TFs from diverse families in a conifer tree species, which broadens the knowledge of promoter–TF interactions in wood development and enables comparisons of gene regulatory networks found in angiosperms and gymnosperms.

Transient gene expression and stable genetic transformation into conifer tissues by microprojectile bombardment

Genetic tranformation technologies are essential to programs of molecular biology and genetic engineering. Although significant efforts in conifer molecular biology have been initiated since the late 1980s, so far only a limited number of genes have been cloned [1]. Conifers are by far the most difficult plant group for this type of study because of their large genomes and lengthy life cycles. Furthermore, progress has been hindered by the present inefficiencies in gene transfer methods and tissue culture protocols for certain species such as pines. Nevertheless, there is a significant body of literature on gene transfer in several conifer species using Agrobacterium-mediated transformation and other protocols of direct DNA transfer.

Evaluation of microprojectile-mediated DNA delivery and reporter genes for genetic transformation of the Mediterranean cypress (Cupressus sempervirens L.)

Abstract Embryonal-suspensor tissue (EST) of Mediterranean cypress (Cupressus sempervirens L.) was tested for microprojectile-DNA delivery (by the PDS-1000/He device) for different subculture periods (9, 15, and 21 days) using the plasmid vectors pRT99GUS [containing the β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes, and the CaMV 35S promoter], pBI426 (with a GUS::NPT II fusion gene under the control of a duplicated 35S RNA promoter), and pCGUδ0 (containing the GUS gene with the ubiquitin intron, under the control of the sunflower ubiquitin promoter). The relative strengths of the promoters as determined by GUS assays were sunflower ubiquitin>35S-35S-AMVE>35S. The highest expression level was observed when 15-day-subcultured EST was bombarded with the pCGUδ0 gene construct, which also showed high activity of the chloramphenicol acetyltransferase and NPT II genes. Green fluorescent areas were observed on EST when bombarded with the p35S-GFP plasmid, carrying the gene for the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria.

In vivo function of Pgβglu-1 in the release of acetophenones in white spruce

Eastern spruce budworm (Choristoneura fumiferiana Clemens) (ESBW) is a major forest pest which feeds on young shoots of white spruce (Picea glauca) and can cause landscape level economic and ecological losses. Release of acetophenone metabolites, piceol and pungenol, from their corresponding glycosides, picein and pungenin, can confer natural resistance of spruce to ESBW. A beta-glucosidase gene, Pgβglu-1, was recently discovered and the encoded enzyme was characterized in vitro to function in the release of the defensive acetophenone aglycons. Here we describe overexpression of Pgβglu-1 in a white spruce genotype whose metabolome contains the glucosylated acetophenones, but no detectable amounts of the aglycons. Transgenic overexpression of Pgβglu-1 resulted in release of the acetophenone aglycons in planta. This work provides in vivo evidence for the function of Pgβglu-1.

Scientific Research Related to Genetically Modified Trees

Over the last decade, we have witnessed impressive advances in tree molecular biology and the consolidation of tree genomics. We have essentially moved from a small portfolio of genes focusing on a specific genetic trait to large databases including thousands of genes and their respective expression profiles. In 2006, we saw the publication of the first genomic sequence of a tree, the model tree species Populus trichocarpa. Though, not surprisingly, much progress has been made with Populus, impressive research results have also been realized in more recalcitrant coniferous species such as pines and spruces.