S2.1 Bob Rutledge and Krystyna Klimaszewska Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du P.E.P.S., Sainte-Foy, Quebec, Canada G1V 4C7 E-mail: brutledge@cfl.forestry.ca In addition to allowing capture, propagation and testing of elite conifer genotypes on a commercial scale, somatic embryogenesis (SE) is a primary enabling technology for the genetic engineering of conifers. Two major restrictions have however, prevented the full commercial potential of conifer SE to be realized. The first is a strong genetic and/or physiological influence that prevents efficient induction of SE from some genotypes of spruce, and from some conifer species such as pines. The second is the inability to induce SE from vegetative tissues taken from mature conifer trees, preventing the ability to clone individual trees with proven elite characteristics. Although many groups have attempted to overcome these limitations, achieving efficiencies required for commercial application has proven elusive. Demonstration that activation of transcription factors such as Arabidopsis LEAFY COTYLEDON1 (LEC1), can induce formation of somatic embryos on vegetative tissues of Arabidopsis, prompted us to examine the potential for manipulation of SE induction via engineering of transcription factors. Functional characterization of a putative LEC1 homologue from black spruce (CHAP3a) was initiated via ectopic expression in transgenic spruce. Changes in both endogenous CHAP3a and transgene expression within embryonal tissue, in addition to dramatic reduction of transgene expression following somatic embryo maturation, likely reflect detrimental effects of CHAP3a ectopic expression. Despite low levels of transgene expression, the frequency of SE induction in transgenic germinants was found to be greater than controls, and transgene activity within secondary cultures was found to return to levels similar to the originating transline. In an attempt to regulate its activity, CHAP3a was fused to the glucocorticoid hormone-binding domain. Although this helped to reduce, it did not eliminate the detrimental effects of ectopic expression. In conclusion, although suggestive that CHAP3a activation can increase SE induction frequency, our results show that effective regulation of transgene activity remains a major challenge.
S2.2 NITROGEN ASSIMILATION AND RECYCLING IN WOODY PLANTS Francisco M. Cánovas, Concepción Avila, Francisco R. Cantón, Fernando Gallardo Departamento de Biología Molecular y Bioquímica, Instituto Andaluz de Biotecnología, Unidad Asociada UMA-CSIC, Universidad de Málaga, 29071, Málaga. E-mail: canovas@uma.es Nitrogen is frequently a limiting factor for plant growth. Consequently, the uptake of inorganic nitrogen and its incorporation into amino acids has been a main area of interest in plant biochemistry and physiology. Most studies have been developed in annual plants and the current knowledge on nitrogen assimilation and metabolism in woody species is still limited, particularly at molecular level. Ammonium, the reduced nitrogen form available for assimilation into aminoacids, is released and reincorporated into metabolism in many processes during growth and development of trees. Thus, the function of enzymes involved in primary assimilation and recycling of ammonium is crucial for the maintenance of tree nitrogen economy. Major research efforts in our laboratory are addressed to study the role of key genes encoding these enzymes, how the genes are expressed and how they are regulated during plant development. Pine genes encoding nitrogen assimilating enzymes have been cloned, characterized and their expression patterns studied. We have shown that ammonium is assimilated in the cytosol of photosynthetic/non-photosynthetic cells of pine and other conifers and two ammonium assimilatory pathways have been proposed [1]. Using transformation approaches, modification of ammonium metabolism has been achieved in poplar by ectopic expression of glutamine synthetase (GS) [2]. Greenhouse studies and field trials of transgenic poplars overexpressing pine cytosolic GS showed that enhanced GS activity in young leaves was correlated with increases in tree growth. The integration of current results with those recently reported in other plants indicate that cytosolic GS plays a central and pivotal role among the enzymes involved in ammonium metabolism and suggests that its manipulation have consequences in plant growth and biomass production. References [1]Suárez MF, Avila C, Gallardo F, Cantón FR, García-Gutiérrez A, Claros MG, Cánovas FM (2002) J Exp Bot, 53, 891-904, [2] Gallardo F, Fu J, Jing ZP, Kirby E:G, Cánovas FM. (2003) Plant Physiol Biochem (in press)
S2.3 GENE EXPRESSION DURING EMBRYO DEVELOPMENT IN CONIFERS Claudio Stasolla. Dept. Biology, University of Winnipeg, MB, R3B-2E9, Canada E-mail: c.stasolla@uwinnipeg.ca Somatic embryogenesis is the process in which embryos, similar in morphology to their zygotic counterparts, are induced to develop in culture from somatic cells. This process represents a suitable model system for investigating factors affecting embryo growth, as many embryos of defined develomental stages can be obtained easily. In spruce the developmental pathway of somatic embryo formation has been described and it consists of a sequence of defined developmental stages corresponding to specific culture treatments. The molecular mechanisms regulating spruce somatic embryo development were investigated in two distinct studies by using cDNA microarray technology. The first study analyzed the effects of polyethylene glycol (PEG) on global gene expression during white spruce embryo development. Inclusion of this compound, which is beneficial for embryo quality, resulted in extensive changes in the transcript levels of genes involved in development, sucrose catabolism, and nitrogen assimilation and utilization. The second study focused on the analysis of transcript levels during early embryogenesis of Norway spruce. Comparative gene expression analysis was conducted between cell lines able to undergo normal embryonic development in culture, and developmentally blocked lines, in which this progression was precluded. This work revealed the existence of possible regulatory genes involved in the progression of embryo development, and stage-specific genes with unique expression pattern in normal lines compared to the developmentally blocked line. Overall, these studies may have important implications in the identification of target genes or metabolic products for improving somatic embryo quality in conifers, through genetic engineering or modification of media during development.
S2.4 REGULATION OF EMBRYO DEVELOPMENT IN CONIFERS Sara von Arnold, Lada Filonova, Maria Fernanda Suarez and Peter Bozhkov Department of plant biology and forest genetics, SLU, PO Box 7080, S-750 07 Uppsala, Sweden. E-mail: Sara.von.Arnold@vbsg.slu.se Somatic embryogenesis of the conifer Norway spruce represents a sequence of specifically regulated developmental stages including proembryogenic mass (PEM), PEM-to-embryo transition and early and late embryogeny. We are using this system for studying the regulation of embryo development. The major gene expression pattern was followed by cDNA array analyses of 373 genes (van Zyl et al., 2003). Genes which are differentially expressed during embryogenesis in Norway spruce include a KNOTTED1-like homeobox gene (HBK2, Hjortswang et al., 2002) and two homeobox genes belonging to the HD-GL2 family (PaHB1 and PaHB2, Ingouff et al., 2003). Endogenous lipophilic ologosaccharides act as signal molecules which stimulates PEM and early embryo development (Dyachok et al. 2002). One of the criteria for embryo pattern formation is binding of the microtubule-associated protein MAP-65 to cortical microtubules (Smertenko et al., 2003). Withdrawal of plant growth regulators triggers the PEM-to- embryo transition providing a synchronous start to the early embryogeny programme and an activation of programmed cell death (PCD). The Norway spruce gene, Mcll-Pa, which encodes a protein with structural homology to mammalian caspases, shows a highly specific expression during PCD (Suarez et al., in preparation). We have also shown that PCD is the major mechanism responsible for elimination of subordinate embryos in polyembryogenic pine seeds (Filonova et al. 2002). Dyachok, Wiweger, Kenne and von Arnold. 2002. Plant Physiol. 128:523-533. Filonova, von Arnold, Daniel, and Bozhkov. 2003. Cell Death and Differentiation 9:1057-1062. Hjortswang, Sundås, Bharathan, Bozhkov and von Arnold. 2002. PPB 40:837-843. Ingouff, Farbos, Wiweger and von Arnold. 2003. J. Exp. Bot. (in press). Smertenko, Bozhkov, Filonova, von Arnold and Hussey. 2003. Plant J. 33:813-824. van Zyl, Bozhkov, Clapham, Sederoff and von Arnold. 2003. Gene Expression Patterns 3:83-91.
S2.5 MOVING ON UP – INSIDE A COMMERCIAL SOMATIC EMBRYO OPERATION Dave Ellis CellFor Inc., Brentwood Bay, B.C., CANADA E-mail: dellis@cellfor.com The world encountered in a typical research lab is completely different from the world involved in the commercialization of a biotechnology product. Cost per unit and the development of scalable systems takes precedent to more intriguing scientific questions like the molecular basis of carbon utilization. This is not to say that the continual input of research results are not needed, rather that these results are now put to the test over many cell lines and with a completely different set of criteria and questions defining their value and use. How do these results aid in year round embryo production? Are they beneficial to loblolly as well as radiata pine? Do the results influence the hundreds of liters of media we have to make/day? Will they save a day, week or month on the typical production cycle? At CellFor, we currently have protocols in place to produce the multi-millions of embryos needed to meet our projections for foreseeable future. Supporting this we have a major ongoing research effort in better understanding the somatic embryo biology to increase efficiency and drive down costs to support our production systems.
S2.6 APPROACHES TO INCREASE EMBRYOGENIC CULTURE INITIATION AND CELL LINE CAPTURE IN LOBLOLLY PINE M. R. Becwar, M. K. Chowdhury, N. S. Nehra, M. R. Rutter, J. J. Clark, M. J. Cook, J. M. Victor, T. J. Stout, A. M. Perry, P. J. Wade, and M. A. Hinchee. ArborGen, PO Box 840001, Summerville, SC 29484 USA E-mail: mrbecwa@arborgen.com Efficient initiation, establishment and capture of cell lines from genetically diverse families is required for successful application of somatic embryogenesis (SE) to clonal and transgenic plant production of loblolly pine. ArborGen is developing ways to address the limitations imposed by recalcitrant families – those that are difficult to capture in the SE process. Here we describe two of these approaches. The first is based on the fact that there typically is a pronounced family by medium interaction during the initiation and pre-cryo culture establishment phases of the SE process. That is, medium modifications often improve initiation in specific families. By using a "battery" (or series) of initiation and establishment media, we have been able to take advantage of the family by medium interaction and capture more culture genotypes from immature seed explants (zygotic embryos) of different families. Using this approach, culture establishment was significantly increased in 5 of 7 recalcitrant families compared to a control medium on at least one of three medium modifications. The best medium modification varied with family. This approach increased capture by 73% overall, and by as much as 190 and 740% among different family-medium combinations compared to the control. The second approach is based on the fact that there are reciprocal effects on initiation and culture establishment. Therefore, how a particular cross is made (A x B or B x A) in terms of female x male parentage very significantly affects culture initiation and establishment. The degree of the reciprocal effect varies among families. Initiation was improved from 50 to 690% just by using a particular parental combination. These two approaches are being combined to further increase embryogenic culture initiation and capture in loblolly pine. This enables ArborGen to access a broader germplasm base for clients, including genetic families that have been considered recalcitrant to the SE process.
S2.7 Marie B. Connett, J. Eric Gulledge, Heather J. Gladfelter, Ryan R. McCormack, Ron T. Kothera, Samantha B. Roberts, Mike R. Becwar and Bob J. Kodrzycki ArborGen LLC, PO Box 840001, Summerville, SC 29484-8401, USA E-mail: mbconne@arborgen.com Pinus taeda is one of the most important forestry species worldwide. While transformation of this species has been reported in several investigations, most transgenic pine tissue has not been regenerable into plants. By use of secondary somatic embryogenesis and examation of the long-term effects of culture manipulations, we identified conditions for transformability that led to improved biolistic transformation efficiency. Southern blots from regenerated transgenic trees showed that 36% of the transgenics have 3 inserts or fewer. Field-grown transgenic trees have normal morphology over three growing seasons. We further improved tissue culture and selection to increase recovery of transformants such that stable transgenic lines were obtained from an average of 72% of the lines in all of 15 elite families tried, and false positives decreased to less than 1%. We extended the above improvements to achieve Agrobacterium transformation of multiple pine species, identification of bottlenecks and steps to address them, and the establishment of field tests with transgenic lines from only elite families. Selection with non-antibiotic marker genes also gave rise to regenerated trees for field tests. Hedges and cuttings from transgenic loblolly pine plants have shown comparable survival to non-transgenics, and transgene expression after two propagation cycles comparable to expression in transgenic promoter::uidA ortets. Transgenic P. taeda and radiata have also been successfully micropropagated for field tests. Additional controlled replicated field tests of transgenic pine have been established each year, for a total of over 400 transgenic pine trees in field trials. These experiments laid the foundation for high-throughput gene testing in a gymnosperm. Initial scale-up provided information on co-transformation efficiency and the degree to which different constructs can differ in throughput, and led to pine trees transgenic for various combinations of 21 constructs with five different lignin biosynthesis genes slated for additional field tests this year.
S2.8 GENOMICS OF EMBRYOGENESIS IN LOBLOLLY PINE John Cairney1, Robin Buell2, Jerry Pullman1, John Quackenbush2 1 - Forest Biology Group, Institute of Paper Science and Technology, Atlanta GA 30318, USA 2 - The Institute for Genomic Research, Rockville, MD 20850, USA E-mail: cairney_john@hotmail.com Embryogenesis proceeds as the result of a controlled program of gene expression. Understanding of embryogenesis is thus assisted by the identification of genes expressed during embryo development, and determination of the function of their encoded proteins and the timing and location of gene expression. Global gene expression assays such as differential display and DNA arrays, in both zygotic and somatic embryos revealed that roughly 5% of genes are actively regulated during embryogenesis. Of the actively regulated genes, approximately one quarter are regulated similarly in somatic and zygotic embryos. Several embryogenesis regulatory genes from Arabidopsis have homologs in pine, thus despite the differences in certain aspects of gymnosperm and angiosperm embryogenesis certain molecular pathways are shared. A recent NSF sponsored program on the Genomics of Lobllly Pine Embryogenesis (https://www.fastlane.nsf.gov/servlet/showaward?award=0217594) has commenced. cDNA libraries from loblolly pine zygotic and somatic embryos, which would represent a combined set of transcripts expressed during all known stages of embryogenesis are being constructed. Randomly selected cDNA clones will be 5’ end sequenced for gene discovery to generate Expressed Sequence Tags (ESTs). which will be clustered and assembled to construct a non-redundant pine EST database (TIGR Gene Index) for identification of unique transcripts. The EST sequence data will be submitted to the Genbank in monthly intervals and will be publicly available as a non-redundant EST data set at TIGR Gene Index web site (http://www.tigr.org/tdb/tgi.shtml). A non-redundant cDNA clone set will be identified and used to develop a 1,000 and 10,000 pine cDNA clone microarray, which will be utilized for characterization of gene expression profiles during various stages of zygtic and somatic loblolly pine embryogenesis . In addition, through collaboration with international laboratories working on closely related species, gene expression during embryogenesis of Slash Pine, Maritime Pine, Norway Spruce, Douglas Fir will be assayed. Somatic embryos of a Larch mutant whose cotyledon number can be altered by medium conditions will be assayed.. These data will provide the first detailed overview of genome-wide gene expression patterns in embryo development of pine. These data would be of significant importance for deciphering embryogenesis and accelerating clonal propagation strategies. The cDNA clones used for EST sequencing and a microarray clone set will be distributed to public via an identified vendor organization. The microarray expression data organized into a publicly accessible pine gene expression database will provide an additional resource for research community. Educational outreach to Undergraduate and High School students will form an important part of this project.
S2.9 FROM SOMATIC EMBRYOS TO GENETIC ENGINEERING OF MARITIME PINE (PINUS PINASTER AIT.) Trontin JF, Harvengt L AFOCEL, Biotechnology Lab (biotech@afocel.fr) - Domaine de l’Etançon – 77370 Nangis, France. E-mail: trontin@afocel.fr Somatic embryogenesis is currently the most promising way to propagate elite clones of maritime pine selected during conventional genetic breeding. Mastering such a powerful method will aid to establish clonal tests and thus improved efficiency of the selection process currently restricted to progenies tests. Moreover, any selected clones from the field clonal tests will be easily and massively deployed from the cryopreserved, juvenile stock of embryonal tissue. Thus, somatic embryogenesis combined with cryopreservation will prevent the selected plant material from depreciation as usually observed for horticultural cuttings subjected to decreased rooting with ageing. Since our initial work, the embryonal-suspensor masses (ESM) initiation from selected seeds, the stabilisation, the multiplication and cryopreservation steps have been significantly improved. Further development and optimisation of the maturation and germination of somatic embryos are still in progress in the frame of our major contribution to the European SEP project (www.sgen.slu.se/sep/index.html). In order to further explore the possibility of reducing the length of the breeding program, AFOCEL is evaluating genetic transformation methods based on microprojectile bombardment and cocultivation of ESM with Agrobacterium. Both hygromycin- and phosphinothricin-based selection methods of transformed cells have been developed. Histochemical (GUS tests) and molecular evidence (diagnostic PCR tests, Southern blots) of genetic transformation have been obtained for ESM lines and regenerated somatic plants grown in the greenhouse. We will present further work revealing the integration pattern of transgenes after biolistic and Agrobacterium cocultivation. We will also present recent data validating our transformation procedure based on Agrobacterium tumefaciens (strain C58pMP90), using pCAMBIA1301 as a valuable vector to transfer genes of interest in maritime pine. This optimized procedure is currently available for research purposes since high transformation yields (up to 60 independent lines/g fresh weight, on average) could be obtained for some ESM lines now considered as models for further developments.
S2.10 D-AMINO ACID SELECTABLE MARKERS Oskar Erikson1, David Clapham2, Magnus Hertzberg3 and Torgny Näsholm1 1 - UPSC, Dept. of Forest Genetics and Plant Physiology, SLU, 901 83 Umeå, Sweden. 2 - Dep. Forest genetics, SLU, Uppsala, Sweden. 3 - SweTreeGenomics AB, Umeå, Seden. E-mail: Oskar.Erikson@genfys.slu.se D-Amino acid selectable markers represent a new class of markers based on genes conferring D-amino acid metabolism. The Escherichia coli gene dsdA, encoding D-serine ammonia-lyase [EC:4.3.1.18] and the yeast Rhodotorula gracilis gene doa1 encoding D-Amino acid oxidase (DAAO), [EC 1.4.3.3] have successfully been used as selectable markers. The rational is that D-amino acids that are toxic to plants are metabolised by the selectable markers into a non-toxic compounds and hence mediate selection of the transgene. D-Serine ammonia-lyase converts D-serine into the products ammonium, pyruvate and water, DAAO catalyses the metabolism of several different D-amino acids into the products ammonium, a corresponding 2-oxo acid and hydrogen peroxide. The release of ammonium can actually support plant growth on D-amino acids in otherwise nitrogen free medium. Further, DAAO represent a novel type of marker-gene that has the unique feature of accomplishing both selection of the transgene and conditional-negative selection depending on the selective agent. The functional basis that allows for dao1 to have this dual selectable capacity is that there is a variation in the toxicity different D-amino acids exert on plants, some affect plant growth at rather low concentration whereas others appears indifferent even at high concentrations. Using D-amino acids that are toxic to plants but metabolised by DAAO into non-toxic compounds enable normal selection of the transgene. On the contrary, using an indifferent D-amino acid that is metabolised into a toxic compound by DAAO enable negative selection of the transgene. The dsdA and dao1 marker has been developed and tested in Arabidopsis with great success. Further, provisional data from selection of Picea abies with the dsdA marker is very promising with similar transformation efficiency to BASTA selection. Moreover, dsdA has been used to generate transgenic poplar and tobacco, but not with ease.
S2.11 IN VIVO TRANSFORMATION OF XYLOGENIC TISSUE IN PINUS RADIATA Kim Van Beveren, Josquin Tibbits, Qing Wang and Gerd Bossinger The University of Melbourne, School of Resource Management at the Forest Science Centre, Creswick, Victoria 3363, Australia E-mail: ksvb@unimelb.edu.au Efforts directed at the molecular manipulation of forest tree species in the past focused on the production of somatic embryos and the transformation of embryonic tissue using a variety of direct and indirect transformation techniques. Long generation times have restricted the possibility of monitoring the effects of inserted genes for the measurement of wood quality traits that appear late in the tree’s development. Consequently, there is a need for alternative systems, which allow the effects and expression of genes in mature wood-forming tissue to be monitored within a reasonable time frame. Towards this end a number of methods were developed for studying such effects in vitro (Leitch & Bossinger 2003). Complementing such systems, we present here progress towards the development of an Agrobacterium mediated in vivo transformation method using the bacterial uid A (GUS) as a reporter gene in xylogenic tissue of Pinus Radiata. Gus activity was observed in vivo 3 to 6 months after inoculation of cambial tissue in 2 to 4-year-old trees, indicative of stable transformation. Following initial screening, results were confirmed using detailed microscopic examination and RT-PCR. Our in vivo transformation system potentially offers an effective means for testing the utility of genes affecting wood traits for the molecular editing of wood quality attributes in this economically important plantation species. Leitch, M.A. and G. Bossinger (2003). In vitro systems for the study of wood formation. In: Molecular Genetics and Breeding of Forest Trees, S. Kumar and M. Fladung eds, Harworth Press, in press.
S2.12 ANALYSIS OF GENE FUNCTION AND STABILITY IN TRANSGENIC POPULUS DELTOIDES Dayton Wilde, Katrina Gause, Carl Huetteman, Shujun Chang, Kim Frampton, Kirk Foutz, and Yuan Zhao ArborGen, Box 840001, Summerville, South Carolina, USA 29484 Email: hdwilde@arborgen.com Eastern cottonwood (Populus deltoides) is being used as a model system to study (1) the function of introduced genes and (2) the stability of transgene expression in serially propagated, field-grown trees. An Agrobacterium-mediated transformation system was developed that produced transgenic trees from 8 of 14 cottonwood clones, the limitation being in regenerability. A commercially planted, female clone was chosen for analysis of transgene function because data were available on its growth performance and other silvicultural characteristics. The frequencies at which transgenic plants were regenerated from leaf explants of this clone were over 50% for two different selectable markers. A high-throughput transformation system was developed to introduce promoters and genes involved in wood quality, enhanced growth, and floral control. Field tests of transgenic cottonwood began in 1998. The effect of serial vegetative propagation on transgene expression was investigated using the cottonwood model system. Six als-transformed lines were chosen that exhibited high levels of resistance to herbicides that target acetolactate synthase (ALS). These lines consisted of two transclones each with als copy numbers of 1, 2, and >2. In each of four years, a new cutting orchard was established with dormant, unrooted cuttings from the previous year’s orchard. Test plantations were established annually with cuttings from (1) the original cutting orchard and (2) the serially propagated orchard established the previous year. To examine als expression, the test plantations were treated with herbicide each spring for two years. To date, no change in herbicide resistance levels has been detected for any of the transgenic lines, indicating stable levels of transgene expression. Gene stability during vegetative propagation is necessary in the commercial production of transgenic tree species for which planting stock is generated by annual cycles of harvest and regrowth.
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