Transformation of elite white poplar (Populus alba L.) cv. ' Villafranca' and evaluation of herbicide resistance

 Transgenic white poplar plants (Populus alba L.) expressing the nptII gene and the bar gene from Streptomyces hygroscopicus have been produced using Agrobacterium tumefaciens-mediated gene transfer. Eleven kanamycin-resistant plant lines were obtained with a transformation frequency of 7%. Successful genetic transformation was confirmed by Southern and northern analyses. The level of resistance to the commercial preparation of phosphinothricin (Basta; Roussel-Hoechst Agrovet) was evaluated by in vitro and in vivo assays. Using in vitro selective conditions for phosphinothricin, only plantlets from four kanamycin-resistant independent lines remained green and continued to grow and root. After transfer to the growth chamber, all selected transgenic lines were shown to be completely resistant to the herbicide Basta with doses equivalent to 6 l ha^–1 (normal field dosage) and were tolerant at concentration of 12 l ha^–1. This is the first report describing the genetic transformation of a P. alba clonal cultivar of commercial interest with a gene of agronomic value. Received: 12 June 1999 / Revision received: 6 March 2000 / Accepted: 7 March 2000     

Cellulose and lignin biosynthesis is altered by ozone in wood of hybrid poplar (Populus tremula × alba)

Wood formation in trees is a dynamic process that is strongly affected by environmental factors. However, the impact of ozone on wood is poorly documented. The objective of this study was to assess the effects of ozone on wood formation by focusing on the two major wood components, cellulose and lignin, and analysing any anatomical modifications. Young hybrid poplars (Populus tremula × alba) were cultivated under different ozone concentrations (50, 100, 200, and 300 l l(-1)). As upright poplars usually develop tension wood in a non-set pattern, the trees were bent in order to induce tension wood formation on the upper side of the stem and normal or opposite wood on the lower side. Biosynthesis of cellulose and lignin (enzymes and RNA levels), together with cambial growth, decreased in response to ozone exposure. The cellulose to lignin ratio was reduced, suggesting that cellulose biosynthesis was more affected than that of lignin. Tension wood was generally more altered than opposite wood, especially at the anatomical level. Tension wood may be more susceptible to reduced carbon allocation to the stems under ozone exposure. These results suggested a coordinated regulation of cellulose and lignin deposition to sustain mechanical strength under ozone. The modifications of the cellulose to lignin ratio and wood anatomy could allow the tree to maintain radial growth while minimizing carbon cost.     

‘Click’ assembly of selective inhibitors for MAO-A

In this Letter, an efficient strategy for the fast construction of 108 compounds library was developed using click chemistry. The fingerprint of inhibitory activity toward MAO-A/B against this library was obtained, and four hit compounds were identified as selective inhibitors toward MAO-A. Docking study was carried out to demonstrate the binding mode between a9 and MAO-A/B, and the result reveals that a9 localized in the ‘aromatic cage’ and oriented to establish π–π stacking interactions with Tyr407, Tyr444 and FAD in MAO-A rather than in MAO-B.     

Plasmodesmatal frequency and radial translocation rates in ray cells of poplar (Populus x canadensis Moench ‘robusta’)

The minimum radial translocation rate of sugars has been determined from the starchaccumulation rate for the wood rays of Populus x canadensis Moench robusta, and related to ultrastructural peculiarities of the cell walls to be passed. The minimum radial flux or flow of sugars through the tangential walls, the pit fields, and per plasmodesma was 80.7 pmol · cm^-2 · s^-1, 400 to 800 pmol · cm^-2 · s^-1, and 1.0 to 1.7 · 10^-7 pmol · plasmodesma^-1 · s^-1, respectively. These values exclude a transmembrane flux mechanism and indicate that the radial translocation in this tissue must proceed via plasmodesmata. In the isolation cells of the ray center we found 39 plasmodesmata per m² of pit field, 8.0 per m² of tangential wall, and 1.98% of the wall occupied by plasmodesmata. Cells of the ray margins show plasmodesmata on only 1.16% of their tangential wall area and thus appear to be slightly inferior for radial translocation. As judged from both the observed plasmodesmatal frequencies and the translocation rates, the ray parenchyma cells are comparable to cells specialized in short-distance translocation.Abbreviations CCR contact-cell row - IC isolation cell - ICR isolation-cell row     

Biochemical,immunochemical, and ultrastructural studies of protein storage in poplar(Populus × canadensis ‘robusta’) wood

The seasonal changes in protein content have been followed in the wood of Populus × canadensis Moench robusta, both biochemically and electronmicroscopically at the cellular level. In the storage-parenchyma cells of the twig wood, 4–6 g · mg^–1 DW protein were deposited in the fall, parallel to the yellowing of leaves, and mobilized completely again during the outgrowth of buds in the spring. Environmental impacts on the leaves, e.g. a fungal attack and mechanical injury by a hurricane, were found to affect protein deposition in the wood considerably. Accumulation of protein bodies in the fall and their disappearance from the cells in the spring proceeded parallel to the changes in protein content measured biochemically, proving that these organelles are the main sites of protein storage in the wood parenchyma cells. Using immunogold labelling and an anti-32-kDa poplar storage-protein antibody the protein bodies were shown to be the exclusive sites of storage of a 32-kDa polypeptide. Transient changes in protein content were also observed during fall and winter. Because these changes coincided with changes in protein-body structure and with changes in the population of vesicles and-or tubular membrane cisternae of the cells, an exchange of nitrogen compounds from the storage pool into the structural protein of membranes possibly takes place during these periods. The structural events observed during proteolysis in spring are very similar to those found in seeds. The possible roles of small cytoplasmic vesicles found within protein bodies during proteolysis and of multimembraneous vacuolar compartments during membrane retrieval are discussed.Abbreviations DW dry weight - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Dedicated to Professor Dr. Dr. Hans Marquardt on the occasion of his 80th birthdayThe valuable technical assistance of Miss Sabine Karg and Miss Astrid Diercks is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft.     

The isolation of tissue culture ofPopulus alba L. ‘Pyramidalis’

Tissue culture was isolated from the stem ofPopulus alba L. ‘pyramidalis’. Callus formation was observed since November till March (1974),i.e. till the formation of calluses suitable for further subeultivation. The most vigorous growth was obtained with the callus culture cultivated on the nutrient medium of DIAZ-COLONet al. (1972) on which more than 11 g of fresh matter was produced after 30 d at the end of the first year of cultivation in darkness, with inoculum weight 1.5-1.8 g. A mild decrease in growth rate of the tissue culture was observed after the first year of cultivation. When illuminated, the originally yellow calluses turned green. The morphological and anatomical structure of the callus culture is also described and cell shape and cell size evaluated.     

PCD hallmarks in cell suspension cultures of white poplar (Populus alba L., cv. “Villafranca”) challenged with cadmium

Abstract

The response to oxidative stress was investigated in suspension cultures of Populus alba L. “Villafranca” exposed to cadmium. Cell death was demonstrated by Evans Blue staining. Although DNA laddering was not detected, the nuclear morphology evaluated by DAPI revealed irregularly stained granular nuclei derived from chromatin condensation, a programmed cell death hallmark.     

The effect of methyl salicylate on the induction of direct and indirect plant defense mechanisms in poplar (Populus × euramericana ‘Nanlin 895’)

The objective of this study was to investigate the effect of different concentrations of methyl salicylate (MeSA) on direct defense and indirect defense in poplar cuttings (Populus × euramericana ‘Nanlin 895’). Four defense-related enzyme activities, such as superoxide dismutase (SOD, EC1.15.1.1), oxydoreductases peroxidase (POD, EC1.11.1.7), catalase (CAT, EC1.11.1.6), and polyphenol oxidase (PPO, EC 1.14.18.1), were measured. The results showed that the SOD activities were induced by 1.0 and 10.0 mM MeSA and the POD activities were induced by MeSA. The CAT activity was induced at low concentrations of MeSA but was inhibited by high concentrations, and the PPO activity was affected by 0.1, 1.0, and 10.0 mM MeSA. Furthermore, the volatiles were detected using gas chromatography–mass spectrometry and gas chromatography. Twenty-one volatile compounds were tentatively identified in the emissions from plant leaves. Of these volatile compounds, (Z)-3-hexen-1-ol and cis-3-hexenyl acetate emissions were the highest. (Z)-3-Nonen-1-ol, (E)-2-hexen-1-ol, 1-octanol, (E,Z)-3,6-nonadien-1-ol, β-ionone, and hexadecanamide were six compounds that were only produced after treatment by 10.0 mM MeSA. These results showed that direct and indirect defense mechanisms in poplars were induced by MeSA.     

Implementation of a ‘standard’ LIMS

This article is an extract of a presentation given at the LIMS conference in the Hague June 4, 1997 [S.M. Fransen et al., Presentation given at the conference LIMS 97, June 4 1997 in the Hague.]. It describes the project steps for the LIMS project in the Danish company Novo Nordisk, with focus on vendor evaluation, validation and implementation of a ‘Standard’ LIMS software. The main project steps are described and experiences gained are discussed.     

Metabolic ‘engines’ of flight drive genome size reduction in birds

The tendency for flying organisms to possess small genomes has been interpreted as evidence of natural selection acting on the physical size of the genome. Nonetheless, the flight–genome link and its mechanistic basis have yet to be well established by comparative studies within a volant clade. Is there a particular functional aspect of flight such as brisk metabolism, lift production or maneuverability that impinges on the physical genome? We measured genome sizes, wing dimensions and heart, flight muscle and body masses from a phylogenetically diverse set of bird species. In phylogenetically controlled analyses, we found that genome size was negatively correlated with relative flight muscle size and heart index (i.e. ratio of heart to body mass), but positively correlated with body mass and wing loading. The proportional masses of the flight muscles and heart were the most important parameters explaining variation in genome size in multivariate models. Hence, the metabolic intensity of powered flight appears to have driven genome size reduction in birds.     

Protein bodies in ray cells of Populus x canadensis Moench ‘robusta’

Light- and electron-microscopical investigations revealed distinct intravacuolar protein aggregates of 0.3–0.8 m in diameter in ray cells of poplar during the dormant season. In semi-thin sections, these bodies showed positive protein staining and enzymatic digestibility with pepsin, indicating their proteinaceous nature. Morphometric measurements showed such protein bodies in 7–13% of the area of the ray-cell lumen. This amount corresponded with the protein content of the wood determined biochemically, e.g. 2.0–5.0 g·mg^-1 dry weight. Polyacrylamide gel electrophoresis of the total protein fraction extracted from wood showed prominent polypeptide species with an apparent molecular weight of 30–32 kilodaltons. The results indicate considerable protein storage in ray cells, especially in the form of protein-storage vacuoles.Abbreviation DW dry weight     

RAPD Linkage Mapping in a Populus adenopoda×P. alba F1 Family

Random amplified polymorphic DNAs(RAPDs) were used to construct linkage maps of the parents of a Populus adenopoda Maxim. x P. alba L. Fl family. A set of 620 random oligonucleotide primers were screened and 128 primers were selected to generate RAPD markers within a sample of 80 Fl progenies. A total of 333 segregating loci [ (326( 1:1 ) ,7(3:1 ) ] were identified. Among the 326 1:1 segregating loci (238 loci from P. adenopoda and 88 loci from P. dba),36 loci (26 loci in P. adenopoda and 10 loci in P. dba) were found distorted from the normal 1:1 ratio. Altogether 290 loci segregating 1:1 (testcross configuration) were used to construct parent-specific linkage maps,212 for P. alba and 78 for P. adenopoda. The resulting linkage maps consisted of 189 marker loci in 20 groups (four or more loci per group), 6 triples and 16 pairs for P. dba, which cover the map distance about 2 402.4 cM, and 41 linked marker loci for P. adenopoda which cover map distance about 479.4 cM. Further study is warranted to locate some important quantitative trait loci (QTLs) based on the maps.     

Structure and seasonal distribution of litterfall in young plantations of Populus ‘Tristis#1’

Litterfall was sampled in 3- to 7-year-old irrigated and fertilized plantings of Populus Tristis#1 in northern Wisconsin. Leaves accounted for more than 90 per cent of the total litter and reached an asymptotic value of about 4,000 to 4,200 kg/ha. Leaf litterfall culminated in late September or first half of October. Inadequate light appeared to be responsible for an early (June) leaf fall in dense plantings. The maximum cumulative leaf area index ranged from 8.4 to 8.7. The pattern of foliation and defoliation was similar: it started with small leaves in the lower canopy and ended with large leaves of the upper canopy. Longevity of the small leaves was shorter than that of the large leaves. Leaf size was positively affected by fertilization but was not affected by a discontinuation of irrigation by mid-July during the droughty 1976. The specific leaf weight ranged from 2.6 mg/cm² in the lower canopy to 10.2 mg/cm² in the upper canopy. The specific leaf weight of litter leaves was 4 to 38 per cent lower than that of green leaves of the same size.     

Evaluation of heterologous promoters in transgenic Populus tremula × P. alba plants

The pattern and expression level of β-glucuronidase (gus) reporter gene regulated by six heterologous promoters were studied in transgenic Populus tremula × P. alba plants obtained by Agrobacterium-mediated transformation. Binary vector constructs used contained the following promoter sequences: the CaMV35S from cauliflower mosaic virus; its duplicated version fused to the enhancer sequence from alfalfa mosaic virus; CsVMV from cassava vein mosaic virus; ubiquitin 3 from Arabidopsis thaliana (UBQ3); S-adenosyl-L-methionine synthetase (Sam-s) from soybean; and the rolA from Agrobacterium rhizogenes. Histochemical staining of root, stem and leaf tissues showed phloem and xylem-specific gus expression under rolA promoter, and constitutive expression with the other putative constitutive promoters. Quantitative GUS expression of 10 – 15 independently transformed in vitro grown plants, containing each promoter, was determined by fluorimetric GUS assays. The UBQ3-gus fusion induced the highest average expression level, although an extensive variation in expression levels was observed between independent transgenic lines for all the constructs tested.     

Effects of drought stress on photosynthesis,growth and root structure of transgenic PtPIP2;8 poplar 84K (Populus alba × P. glandulosa)

为研究水通道蛋白PtPIP2;8基因功能, 了解其不同表达水平的转基因84K杨(Populus alba × P. glandulossa)应对干旱胁迫的响应, 该文以转PtPIP2;8 84K杨抑制表达株系(抑制表达)、野生型(WT)和转PtPIP2;8 84K杨超表达株系(超表达)为试验材料, 测定PtPIP2;8表达水平、根系导度、光响应曲线、气体交换参数、生长及根系形态指标。结果显示: (1) WT植株PtPIP2;8仅在根系表达; 超表达植株PtPIP2;8除在根部显著表达外, 在茎和叶片中也显著表达; 抑制表达植株PtPIP2;8仅在根部有微量表达, 表达量分别是WT和超表达植株的1/20和1/80。(2)根系结构分析发现, 超表达植株总根长、总根表面积、总根体积、总根尖数显著低于WT和抑制表达植株, 根系导水率显著高于WT和抑制表达植株, 表明PtPIP2;8参与了植物根系水分运输, 提高了水分运输效率。(3)正常水分条件下, 抑制表达植株苗高、叶面积显著低于WT和超表达植株, 根冠比显著高于WT和超表达植株。干旱胁迫后, 抑制表达植株净光合速率(P_n)、气孔导度(G_s)下降幅度小, 仍能维持较高的P_n。气体交换参数显示抑制表达植株P_n、G_s日变化为“单峰”型, 属气孔因素引起的净光合速率下降;WT和超表达植株P_n、G_s日变化为“双峰”型, 干旱胁迫后, 抑制表达植株P_n略微下降, WT和超表达植株P_n均下降, 尤其是13:00、15:00下降显著, 表明WT和超表达植株对干旱胁迫更加敏感, 干旱对其影响更大。(4)干旱胁迫后, 抑制表达植株相对生长速率、总生物量降低的最少, 根冠比最高; 总根表面积、总根体积、总根尖数显著高于WT植株。表明PtPIP2;8直接参与水分运输并提高水分运输效率, 其转化影响了植株根系发育和生长。超表达植株根系发育的下降和叶面积的增大减弱了它的抗旱性, 而抑制表达植株矮小, 降低的叶面积, 增加的根系生长和根冠比提高了它的抗旱能力。从研究结果来看, 水通道蛋白提高了水分跨膜运输效率, 而非水通道蛋白导水机制对干旱有较强的耐受性。     

Haploid plants from anther cultures of poplar (Populus × beijingensis)

Homozygous genotypes are valuable for genetic and genomic studies in higher plants. However, obtaining homozygous perennial woody plants using conventional breeding techniques is currently a challenge due to a long juvenile period, high heterozygosity, and substantial inbreeding depression. In vitro androgenesis has been used to develop haploid and doubled haploid plants. In the present study, we report the regeneration of haploid lines of poplar (Populus × beijingensis) via anther culture. Anthers at the uninucleate stage were induced to produce callus using three basic media. Two auxins (naphthalene acetic acid [NAA] and 2,4-dichloro-phenoxyacetic acid [2,4-D]), and two cytokinin (kinetin [KT] and 6-benzyladenine [BA]) were tested to explore the influence of plant growth regulators on callus response. H medium (Bourgin and Nitsch 1967) supplemented with 1.0 mg/L NAA and 1.0 mg/L KT induced the highest rate of callus formation. When callus obtained from anthers were subcultured in MS medium containing 1.0 mg/L BA and 0.2 mg/L NAA, followed by transfer to half-strength MS medium supplemented with indole-3-butyric acid (0.2–0.5 mg/L), the formation of regenerated plantlets increased dramatically. Inclusion of gibberellic acid (0.02–0.2 mg/L) in addition to a combination of BA (0.6 mg/L)-NAA (0.2 mg/L) in the culture medium resulted in enhanced frequency of shoot development, as well as greater internode elongation. Ploidy analysis of 580 regenerated plants, using both flow cytometry and chromosome counting, revealed 10.3 % haploid and 1.0 % triploid plantlets. The remaining plantlets were all diploid.     

‘Kumanasamuha’ a new genus of dematiaceae

Summary Kumanasamuha a new genus of Dematiaceae is described, collected growing as saprophyte on unidentified wood, from Pakhal forest, withK. sundara as type.