Antisense expression of a caffeic acidO-methyltransferase ofStylosanthes humilis in transgenic poplar: Effect of expression onO-methyltransferase activity and lignin composition

Poplar (Populus tremula) was transformed with a construct carrying an antisense caffeic acidO-methyltransferase (COMT) cDNA (pOMT8) from a tropical pasture legume,Stylosanthes humilis. pOMT8 shows 83% overall homology to the corresponding COMT gene (pPCLA) of poplar. Of the 200 putatively-transformed plants regenerated on selective media after co-cultivation of poplar stem explants withAgrobacterium tumefaciens harbouring a CaMV 35S-antisensepOMT8 construct, a subset of 20 plants were randomly chosen for further analysis. PCR and Southern blot analysis demonstrated the stable integration of T-DNA into the genome of these plants. Antisense expression ofpOMT8 resulted in reductions in total COMT activity in the majority of the transgenic plants with the lowest total COMT activities (61–70% of untransformed control plants) being observed in four transgenic plants. The composition of lignin in transgenic plants was also changed, as detected by reductions in the content of syringyl units using infrared spectroscopy. However, no changes were found in the amount of insoluble lignin in transgenic plants as compared to untransformed control plants. These results indicate the potential of thepOMT8 gene to partially suppress COMT activity and modify the composition of lignin in transgenic poplar. This work was partly supported by General Management of Turkish Pulp and Paper Mills.     

Assessment of Populus wood chemistry following the introduction of a Bt toxin gene

Unintended changes in plant physiology, anatomy and metabolism as a result of genetic engineering are a concern as more transgenic plants are commercially deployed in the ecosystem. We compared the cell wall chemical composition of three Populus lines (Populus trichocarpa Torr. & A. Gray x Populus trichocarpa Bartr. ex Marsh., Populus trichocarpa x Populus nigra L. and Populus deltoides x Populus nigra) genetically modified to express the Cry3A or Cry3B2 protein of Bacillus thuringiensis (Bt) with the cell wall chemistry of non-transformed isogenic control lines. Three genetically modified clones, each represented by 10 independent transgenic lines, were analyzed by pyrolysis molecular beam mass spectrometry, gas chromatography/mass spectrometry and traditional wet chemical analytical methods to assess changes in cell wall composition. Based on the outcome of these techniques, there were no comprehensive differences in chemical composition between the transgenic and control lines for any of the studied clones.     

Spring aphid-parasitoid (Hom., Aphididae,Hym., Braconidae) associations and interactions in a Mediterranean arable crop ecosystem,including <Emphasis Type="Italic">Bt</Emphasis> maize

Conspecific and heterospecific associations of aphid parasitoids (Hymenoptera, Braconidae, Aphidiinae) were used to determine the interactions between crops forming the main arable crop ecosystem (wheat, alfalfa, isogenic and [Bt Bacillus thuringiensis] transgenic maize) in an irrigated area in Catalonia, Spain. Species composition and instars of parasitized aphids (mummies) were used to detect the parasitoid population movements between the crops and proved interactions (alternation) between the crops, including transgenic maize. Parasitoid interactions were species-dependent, appearing in the movements between alfalfa-wheat-maize in the individual species. Aphidius ervi Hal., A. rhopalosiphi DeSt.-Perez, A. uzbekistanicus Luzh., Lysiphlebus testaceipes (Cress.) and Praon volucre (Hal.) were classified as key-stone species in the target agroecosystems in the area. No differences were found in aphid-parasitoid associations on Bt transgenic maize and isogenic maize for aphid species composition and associated parasitoid guild.     

Pattern recognition as a tool to discriminate softwood and hardwood bark fractions with different particle size

The aim of this study was to characterize the chemical composition of different granulometric fractions obtained by milling of the bark of two softwood [Norway spruce, Picea abies (L.) Karst., and Scots pine, Pinus sylvestris L.] and two hardwood species (birch, Betula pendula Roth, and eucalypt, Eucalyptus globulus Labill.), and to discriminate between them on the basis of their chemical composition content via pattern recognition techniques [principal component analysis (PCA), cluster analysis (CA), and discriminant analysis (DA)]. Bark chemical composition differed between species, and chemical variables could be used to differentiate between them. Size reduction yields granulometric fractions that are not chemically homogeneous and that can also be discriminated. Therefore, potential applications of bark in valorization programs have to carefully consider the species-specific composition and their size reduction patterns. PCA and CA were adequate tools to characterize the different bark fractions within each species. DA allowed identifying the bark samples according to species and independently from particle size. Pattern recognition statistical methods were shown to be useful tools to analyse bark fractions and chemically discriminate species and fractions.     

Surface deterioration of moso bamboo (<Emphasis Type="Italic">Phyllostachys pubescens</Emphasis>) induced by exposure to artificial sunlight

The effect of ultraviolet-visible light irradiation on changes in surface chemistry and morphology of moso bamboo (Phyllostachys pubescens Mazel) was investigated. Fourier transform infrared (FT-IR) and FT-Raman spectroscopy were used in combination to study chemical changes induced by exposure to artificial sunlight (xenon lamp) for up to 160 h, and the resulting physical changes of cell walls of bamboo surfaces were examined by scanning electron microscopy (SEM). FT-IR results showed that significant changes occurred in the lignin component as indicated by considerable decreases in the intensities of the characteristic aromatic lignin peak at 1512 cm⁻¹ and other associated bands. This was accompanied by formation of new carbonyl groups at 1735 cm⁻¹, resulting in photooxidation of bamboo surfaces. The photosensitive nature of bamboo lignin was also demonstrated by FT-Raman analysis, in which obvious decreases in intensities of Raman bands at 1604 and 1630 cm⁻¹ mainly derived from lignin and free and esterified p-coumaric and ferulic acids were observed. SEM micrographs of the irradiated cross sections of bamboo revealed that significant damage occurred to the fiber walls, whereas the parenchyma cells exhibited slight distortion and some cracks occurred in the cell walls. The structures of cell corners and middle lamellae were nearly intact after irradiation.     

Chemical composition and variability of cork from Quercus suber L.

Summary The chemical composition of cork from Quercus suber L. was determined for virgin cork and for reproduction cork. Average chemical composition of virgin cork is: ash 0.7%, total extractives 15.3%, suberin 38.6%, lignin 21.7% and polysaccharides 18.2%. The carbohydrate composition shows that glucose represents 50.6% of all monosaccharides, xylose 35.0%, arabinose 7.0% and galactose and mannose, respectively, 3.6% and 3.4%. Reproduction cork has a composition similar to virgin cork. For the cases studied, reproduction cork had however less extractives and a higher amount of suberin. Significant differences were found for the chemical composition of virgin cork in relation to location of extractives and polysaccharide content. The between-tree variability was large and a variation within the tree was also recorded.The author is grateful for the help of collaborators throughout the chemical analysis of the many samples: Joaquina Ferreira, A. Velez Marques, M. Lurdes Ferreira, Rosa Amorim and Célia Constâncio.The work was financially supported by the Instituto de Ciência e Tecnologia dos Materiais (ICTM), Lisbon, Portugal     

Assessment of rhizospheric microorganisms of transgenic Populus tomentosa with cowpea trypsin inhibitor ( CpTI ) gene

To have a preliminary insight into biosafety of genetically transformed hybrid triploid poplars (Populus tomentosa × P. bolleana) × P. tomentosa with the cowpea trypsin inhibitor (CpTI) gene, two layers of rhizospheric soil (from 0 to 20 cm deep and from 20 to 40 cm deep, respectively) were collected for microorganism culture, counting assay and PCR analysis to assess the potential impact of transgenic poplars on non-target microorganism population and transgene dispersal. When the same soil layer of suspension stock solution was diluted at both 1:1 000 and 1:10 000 rates, there were no significant differences in bacterium colony numbers between the inoculation plates of both transgenic and non-transgenic poplars. The uniform results were revealed for both soil layer suspension solutions of identical poplars at both dilution rates except for non-transgenic poplars at 1:10000 dilution rates from the same type of soil. No significant variation in morphology of both Gram-positive and Gram-negative bacteria was observed under the microscope. The potential transgene dispersal from root exudates or fallen leaves to non-target microbes was repudiated by PCR analysis, in which no CpTI gene specific DNA band was amplified for 15 sites of transgenic rhizospheric soil samples. It can be concluded that transgenic poplar with the CpTI gene has no severe impact on rhizospheric microorganisms and is tentatively safe to surrounding soil micro-ecosystem. [Supported by the National Project in Transgenic Plant and Application (Grant No. J2002-2003)]     

Cloning and RNAi construction of a LEAFY homologous gene from Populus tomentosa and preliminary study in tobacco

PtLFY, a LEAFY (LFY) gene, was cloned from Populus tomentosa (LM50) by PCR. Sequencing analysis indicated that PtLFY was 2 629 bp long, composed of three exons and two introns and encoded 378 amino acids. The splice donor sites and the splice acceptor sites were in identical positions to the LFY and its homologues. The amino acid sequence inferred was 68%-99% homologous to those of LFY and its homologues by blast analysis in GenBank. The Southern blot analysis indicated that there was a single copy of the PtLFY gene in genomic DNA of male and female P. tomentosa (LM50 and 5082). The pBI121-Ptalfy (reverse)-intron-Ptlfy-GUS-nos was constructed using RNA interference (RNAi) technique and verified by PCR and digestion identification and transformed into tobacco. Some transgenic tobacco plants were obtained by PCR and PCR-Southern identification. The growth was generally repressed in transgenic tobacco plants compared with wild-type ones and some phenotypic differences were observed. [Supported by the National Natural Science Foundation of China (Grant No. 30371175) and Postdoctoral Foundation of China (Grant No. 2002032041)]     

基于多元素含量的统计学方法鉴别我国不同产地核桃

[目的]对我国不同产地核桃进行鉴别,为核桃的原产地保护提供一定的基础数据和理论依据。[方法]应用电感耦合等离子体质谱(ICP-MS)测定全国8个核桃主产省128份核桃样品中35种元素含量,结合单因素方差分析、主成分分析(PCA)和线性判别分析(LDA),建立判别模型,对核桃产地进行鉴别。[结果]表明:核桃中主要微量营养元素为Fe、Zn、Cu和Ni等,重金属(Pb、Cd和As)及稀土元素含量较低;单因素方差分析结果表明,不同地区核桃样品中元素组成差异显著(P0.05);主成分分析表明,不同地区核桃样品中的特征元素为稀土元素以及Co、Fe、Rb、Zn、Tl、Cu、Cd、Ba、Sm、Sc、Mo和Ti等元素;应用线性判别分析建立了不同产地核桃判别模型,8个省份核桃整体判别正确率为99.2%,同时,应用线性判别分析建立了核桃地理标志产品与非地理标志产品判别模型,6种核桃地理标志与非地理标志产品整体判别正确率为95.7%。[结论]通过测定核桃中多种元素含量,结合主成分分析(PCA)和线性判别分析(LDA)等方法,可对不同产地核桃进行鉴别。     

Potential for genetic improvement of oil production in Melaleuca alternifolia and M. linariifolia

Variation in oil yields from plantations and natural stands of Melaleuca alternifolia (Maiden and Betche) Cheel indicates considerable potential for improving plantation production through breeding. Some populations of the more widely distributed species, M. linariifolia Sm., produce a similar leaf oil and may provide opportunities to expand the genetic base of plantations. Growth and oil traits were assessed for 60 half-sib families, representing three chemical forms of Melaleuca alternifolia, and 10 half-sib families of the terpinen-4-ol rich chemical form of M. linariifolia, in a provenance/progeny trial in northern New South Wales, Australia. Differences in oil composition and coppicing between the terpinen-4-ol rich forms of the two species were significant (P < 0.05), while differences in growth traits and leaf oil yield were not significant. The difference in oil composition between northern and southern populations of M. linariifolia was as great as differences between the two species, suggesting the two regions should be considered distinct races for breeding purposes.Narrow-sense heritabilities in M. alternifolia of 0.67 for oil yield, 0.25 for plant dry weight, and 0.27 for coppice indicate substantial improvement would follow selection for single traits. However, negative genetic correlations between oil yield and plant dry weight indicate it is not possible to simultaneously achieve major gains in the two traits. Using combined index selection with a restriction on plant dry weight, expected gains of 17% in oil yield and 14% in coppicing are predicted from one generation of selection at an intensity of one tree in ten.     

Identification of <Emphasis Type="Italic">CpTI</Emphasis> gene integration for 2-year-old transgenic poplars at DNA level

The putative transgenic hybrid triploid poplars [(P. tomentosa × P. bolleana) × P. tomentosa] with CpTI gene have been outplanted in test field for 2 years. Although the authors’ previous studies have proved that they are highly resistant to 3 species of poplar-threatening insect pests and contain high content of CpTI protein in foliage, incorporation status of foreign CpTI gene in poplar genome is uncertain. In this present study, the incorporation of foreign CpTI gene in genome of 5 transgenic poplars was confirmed by PCR and Southern blotting analysis. DNA amplification showed that there were clear DNA bands of about 450bp specific to CpTI gene in transgenic lanes, while no corresponding band in non-transgenic lane was observed. Correspondingly, clear DNA hybridization signals and no signal were exhibited on film for DNA Southern blotting analysis in transgenic lanes and non-transgenic lane, respectively, which further confirmed the stable integration of foreign CpTI gene in genome of 2-year-old transgenic poplar.     

Use of transmittance near-infrared spectroscopy to predict the mechanical properties of 1- and 2-year-old transgenic aspen

The investigation of genetically modified trees requires rapid and reliable techniques to measure the mechanical properties at an early age in order to give timely feedback to forest geneticists. In this study, transmittance near-infrared (NIR) spectroscopy was used to predict the green mechanical properties of 1- and 2-year-old transgenic and wild-type aspen. Green modulus of elasticity (MOE) in three-point bending and green ultimate compression strength (UCS) parallel to the grain were predicted from the NIR spectra of dry wood meal pellets. Green UCS had strong correlation (R ² = 0.91) and green MOE had good correlation (R ² = 0.78) with the spectra. The latter could be explained by the moderate correlation of MOE with the lignin content of the transgenic samples, suggesting that besides chemical composition MOE also depends on anatomical properties. The ratio of performance to deviation value suggested that the calibration model of both UCS (2.94) and MOE (1.91) could be used for screening.     

Amplified Fragment Length Polymorphism (AFLP) Based Genetic Diversity Studies in Betel Vine (Piper betle L.)

Betel vine has been identified as an important cash crop in India. It is a vegetatively propagated plant and more than 100 landraces (cultivars) are cultivated in India. They are often named after the localities where they are grown. To assess the molecular genetic diversity among 10 betel vine cultivars, amplified fragment length polymorphism (AFLP) markers were used. Five AFLP primer combinations produced 323 bands of which 155 were polymorphic, leading to a 47.99% polymorphism. Average polymorphic information content (PIC) was 0.14 over all primer combinations. Genetic similarity was calculated from the Jaccard's similarity coefficient (r) value for all accessions which varied from 0.39 to 0.81, with an average of 0.60 implying a medium level of genetic variation between investigated cultivars. Cluster analysis divided the cultivars into two main clusters where M2 and M3 were closely related but F3 was separate from all others. The cluster analysis based on AFLP data indicated that cultivars were not grouped according to their geographic place of collection but rather by genetic relatedness. The principle coordinate analysis (PCA) of the markers was linked to the dendrograms generated by employing UPGMA.     

Fourier transform-infrared spectroscopy as a new method for evaluating host resistance in the Dutch elm disease complex

Resistance of elms (Ulmus spp.) to the pathogenic fungus Ophiostoma novo-ulmi Brasier depends on chemical and anatomical factors that confine the spread of the pathogen in the vascular system of the host. This study focused on detecting chemical differences in 4-year-old Ulmus minor Mill. seedlings before and after inoculation with a virulent O. novo-ulmi isolate. According to symptom development over 60 days, the trees were divided into resistant (0-33% wilting) and susceptible (67-100% wilting) groups. Histochemical tests and Fourier transform-infrared (FT-IR) spectroscopy analysis were performed on transverse sections of 2-year-old twigs, 2 days before and 40 days after inoculation. Although histochemical tests did not clearly discriminate susceptible from resistant elms, chemical differences between resistant, susceptible and control trees were detected by FT-IR. The average spectrum for resistant tree samples had higher absorbance peaks than the spectra from the susceptible and control samples, indicating increased formation of lignin and suberin. The roles of lignin and suberin in the resistance of the elms against O. novo-ulmi and the usefulness and sensitivity of the FT-IR technique for analyzing metabolic changes caused by pathogens in plants are discussed.     

Constitutive expression of sense &; antisense <Emphasis Type="Italic">PtAP3</Emphasis>, an <Emphasis Type="Italic">AP3</Emphasis> homologue gene of <Emphasis Type="Italic">Populus tomentosa</Emphasis>, affects growth and flowering time in transgenic tobacco

To analyze the function of PtAP3, an APETALA3 （AP3） homologue gene isolated from Populus tomentosa Carr., the full length sequence （1797 bp） and a fragment （870 bp） of PtAP3 were fused to a CaMV 35S promoter of pBI121 to generate the sense and antisense constructs of PtAP3. These constructs were transformed into tobacco by Agrobacterium infection of leaf disks and selection on kanamycin medium. Some sense and antisense transgenic tobacco plants were obtained by PCR and Southern blot analysis. Great phenotypic differences in transgenic tobacco plants were observed. Almost all of sense PtAP3 to transgenic tobaccos showed a higher growth rate than those of antisense transformants and a few developed pregnancy earlier than wild type seedlings and antisense transformants under the same conditions.     

徐州出土汉代棺木用材树种鉴定及其化学性质

木材是人类日常生活的常见材料,也是从古至今人类文明与民族文化的重要载体。伴随着考古发掘工作的进行,来自不同地区、不同朝代的大量木质文物出土并需要持续保护,因此掌握出土木质文物所用的木材树种情况,并研究其化学组分的降解状态,可为制定合理的木质文物修复和保护方案提供科学依据。通过对徐州万达汉墓墓群中1号与4号墓出土棺木进行取样和对标准三切面的切片进行观察,根据三切面显微构造特征对棺木所用木材树种进行鉴定,经与标准切片比对,结果表明M1c、M1oc、M4c、M4oc棺木树种分别为楠木(Phoebe sp.)、硬木松(Pinus sp.)、梓木(Catalpa sp.)、榉木(Zelkova sp.)。进一步采用荧光显微镜与傅里叶变换红外光谱、X射线衍射相结合的方法,分析棺木的主要化学组分降解情况。结果表明:与现代健康材相比,古木在长期埋藏过程中,木材的主要多糖类化学组分纤维素和半纤维素降解严重,纤维素结晶度降低;古木各类型细胞中木质素的自发荧光效应均减弱,且红外光谱分析表明木质素化学结构发生改变;古木在埋藏过程中木材的整体构造保存较好,但主要化学组分发生了降解,这与其地下饱水低氧的埋藏环境有很大关系。     

Durability of isocyanate resin adhesives for wood V: changes of color and chemical structure in photodegradation

The effect of ultraviolet (UV) light irradiation on the color and chemical structure of water-cured polymeric diphenylmethane diisocyanate (PMDI) was investigated using a UV long-life fade meter. Control treatment was performed without UV light irradiation using a thermohygrostat for comparison. Two kinds of resin were used in this study: that to which only water had been added, and resin to which a small amount of polyol and water had been added. In addition, lauan (Shorea spp.) wood was used as a reference. The photodegradation of the resins over a period of up to 300 h was observed using a colorimeter and Fourier transform infrared (FT-IR) spectroscopy. When the resins were treated with UV light, the color difference (ΔE ^* ab) of the resins increased signifi cantly in a short time, and then reached a near-constant value. For lightness, L ^* decreased rapidly for a few hours and then decreased gradually. The color darkened compared with that of the wood used. When treatment was performed without UV light irradiation, ΔE ^* ab and ΔL ^* of the resins showed negligible change. Based on the results of FT-IR analysis, severe degradation such as cleavage of the main chemical bond was hardly observed under UV light irradiation irrespective of the type of resin. Part of this report was presented at the 54th Annual Meeting of the Japan Wood Research Society, Sapporo, Japan, and at the 6th International Wood Science Symposium, Bali, Indonesia     

The effect of Sirex noctilio infestation and fire damage on the chemical composition of South African-grown Pinus patula pulpwood

The infection and association between the wood wasp Sirex noctilio and the fungus Amylostereum areolatum is responsible for large-scale tree mortality in the Midlands of the KwaZulu-Natal province in South Africa. An exploratory investigation on the effect of the infestation of trees by the wood wasp and its associated fungus on the chemical composition of Pinus patula pulpwood was undertaken. Various tree classes representing different levels of physiological growth stress from this infestation were compared. Together with the above stress agents, fire damage to P. patula trees was also considered as a possible cause of changes in pulpwood chemical composition. Chemical analyses to determine the Seifert cellulose and Klason lignin contents as well as the levels of water- and solvent-soluble extractives were conducted using published Tappi standard methods. The results indicated only negligible differences between infestation levels (tree classes) with respect to Seifert cellulose and Klason lignin contents. In contrast, highly significant differences were observed for solvent- and water-borne extractives. It is evident from the results that P. patula trees engage a defence strategy to counter the effects of the infestation and the resulting physiological stress. The results of the chemical analyses suggest that trees should not be harvested any younger than the intended rotation age of 12 years and at the time of harvesting all the biomass, including the infected wood, should be sent to the pulp mill. Fire-damaged trees can be utilised in the same way as healthy trees when applying the TMP process provided the charcoal on the outer bark is removed. The usefulness of a biplot to simultaneously display the various tree classes and their chemical composition is illustrated.     

Allelopathy assessments for the environmental biosafety of the salt-tolerant transgenic Eucalyptus camaldulensis, genotypes codA12-5B, codA 12-5C, and codA 20C

Allelopathy tests were conducted on salt-tolerant transgenic eucalyptus trees conferring bacterial codA gene in the designated net-house conditions under Type II use (contained use) of the Japanese law on environmental biosafety aiming for Type I (field use) application. Three transgenic and corresponding nontransgenic genotypes were employed for four different tests: (1) sandwich bioassay; (2) soil germination method; (3) gas chromatography (GC) for volatile substances from the plants; and (4) high-performance liquid chromatography (HPLC) on phenolic compounds from fresh leaves, which are the primary allelopathic substances on the species. The simple approaches, the bioassays, indicated no significant difference between the transgenic and nongenetically modified genotypes. There was no qualitative difference between the transgenic and nontransgenic lines by GC or HPLC. Absence of any quantitative difference was suggested by repetitive examination and subsequent analysis of variance assessments with the chromatographic methods and bioassays. Moreover, it was also indicated that bioassays should be the primary assessment method for allelopathy in considering the simplicity, speed, low cost, and reproducibility of these methods. Overall, substantial equivalence was considered on the three transgenic genotypes with codA gene when compared with the nontransgenic Eucalyptus camaldulensis lines. The experiments supported the application to isolated field testing of the transgenic Eucalyptus camaldulensis genotypes as the first case and experience in Japanese regulatory approval processes Type I Use for the deliberate release to the environment.