Shoot culture dynamics of six Populus clones

Shoot tips of five genotypically diverse Populus clones, P. alba x P. grandidentata 'Crandon,' P. nigra 'Betulifolia' x P. trichocarpa, P. nigra x P. laurifolia 'Strathglass,' P. maximowiczii x P. trichocarpa 'Androscoggin' and P. deltoides x P. nigra 'Eugenei,' were collected from hardwood cuttings, sterilized,and established in vitro. Stable shoot cultures were obtained from all clones except P. deltoides x P. nigra 'Eugenei'. The four poplar clones that formed stable shoot cultures together with a previously established P. tremula 'Erecta' clone were placed as two-node explants on either Murashige and Skoog medium or Woody Plant Medium containing benzyladenine to determine the rate of shoot multiplication, shoot growth and other responses of the clones. All five poplar clones showed rapid shoot multiplication when cultured in the presence of 0.4-1.0 microM benzyladenine on Murashige and Skoog medium, although P. tremula 'Erecta' produced a greater number of healthy shoots when grown on Woody Plant Medium. Individual shoot growth of all clones was more vigorous when the medium contained 0-0.1 microM benzyladenine, and 100% of such shoots rooted ex vitro.     

Water stress, photosynthesis and early growth patterns of cuttings of three Populus clones

Photosynthetic attributes, leaf area and early root growth patterns were studied in three Populus clones to identify traits associated with superior growth potential on sites where water could be a limiting factor. It was found that early root growth and superior leaf area production were more closely related to growth potential than were photosynthetic capacity or carboxylation efficiency. A hybrid clone of Populus nigra var. charkowiensis (syn. P. nigra var. plantierensis) x P. nigra cv. 'Incrassata' (NE308) had more leaf area production and greater root system development in both wet and dry soil than did a P. trichocarpa clone (T6) and a P. balsamifera clone (B3). Despite greater above- and below-ground productivity, plants of clone NE308 had significantly lower photosynthetic capacity and carboxylation efficiency and a slightly higher CO(2) compensation point than plants of clones T6 and B3. Rapid early leaf and root growth appear to be key attributes associated with productivity in these clones regardless of soil water availability.     

Gas exchange characteristics of Populus trichocarpa, Populus deltoides and Populus trichocarpa x P. deltoides clones

Responses of net photosynthesis, dark respiration, photorespiration, transpiration, and stomatal conductance to irradiance, temperature, leaf-to-air vapor density difference (VDD), and plant water stress were examined in two Populus trichocarpa clones (one from a moist, coastal climate in western Washington and one from a dry, continental climate in eastern Washington), one P. deltoides clone, and two P. trichocarpa x P. deltoides clones. Light saturation of photosynthesis in greenhouse-grown trees occurred at about 800 micromol m(-2) s(-1) for P. deltoides, P. trichocarpa x P. deltoides, and the eastern Washington ecotype of P. trichocarpa, but at about 600 micromol m(-2) s(-1) for the western Washington ecotype of P. trichocarpa. Average net photosynthesis (at saturating irradiance and the optimum temperature of 25 degrees C) was 20.7, 18.8, 18.2 and 13.4 micromol CO(2) m(-2) s(-1) for P. deltoides, P. trichocarpa x P. deltoides, and the eastern and western Washington clones of P. trichocarpa, respectively. In all clones, net photosynthesis decreased about 14% as VDD increased from 3 to 18 g H(2)O m(-3). Stomatal conductance decreased sharply with decreasing xylem pressure potential (XPP) in all clones except the western Washington clone of P. trichocarpa. Stomata in this clone were insensitive to changes in XPP and did not control water loss. Complete stomatal closure (stomatal conductance < 0.05 cm s(-1)) occurred at about -2.0 MPa in the eastern Washington clone of P. trichocarpa and around -1.25 MPa in the P. deltoides and P. trichocarpa x P. deltoides clones. Transpiration rates were highest in the P. trichocarpa x P. deltoides clone and lowest in the western Washington clone of P. trichocarpa. The P. deltoides clone and eastern Washington clone of P. trichocarpa had the highest water use efficiency (WUE) and the western Washington clone of P. trichocarpa had the lowest WUE. The hybrids were intermediate. It was concluded that: (1) gas exchange characteristics of eastern and western Washington clones of P. trichocarpa reflected adaptation to their native environment; (2) crossing the western Washington clone of P. trichocarpa with the more drought resistant P. deltoides clone produced plants better adapted to the interior Pacific Northwest climate, although the stomatal response to soil water deficits in the hybrid was conservative compared with that of the eastern Washington clone of P. trichocarpa; and (3) introducing eastern Washington clones of black cottonwood into breeding programs is likely to yield lines with favorable growth characteristics combined with enhanced WUE and adaptation to soil water deficits.     

Spatial distribution of leaf morphological and physiological characteristics in relation to local radiation regime within the canopies of 3-year-old Populus clones in coppice culture

Spatial distributions of leaf characteristics relevant to photosynthesis were compared within high-density coppice canopies of Populus spp. of contrasting genetic origin. We studied three clones representative of the range in growth potential, leaf morphology, coppice and canopy structure: Clone Hoogvorst (Hoo) (Populus trichocarpa Torr. & Gray x Populus deltoides Bartr. & Marsh), Clone Fritzi Pauley (Fri) (Populus trichocarpa Torr. & Gray) and Clone Wolterson (Wol) (Populus nigra L.). Leaf area index ranged from 2.7 (Fri and Wol) to 3.8 (Hoo). The clones exhibited large vertical variation in leaf area density (0.02-1.42 m2 m-3). Leaf dry mass per unit leaf area (DM(A)) increased with increasing light in Clones Hoo and Fri, from about 56 g m-2 at the bottom of the canopy to 162 g m-2 at the top. In Clone Wol, DM(A) varied only from 65 to 100 g m-2, with no consistent relationship with respect to light. Conversely, nitrogen concentration on a mass basis was nearly constant (around 1.3-2.1%) within the canopies of Clones Hoo and Fri, but increased strongly with light in Clone Wol, from 1.4% at the bottom of the canopy to 4.1% at the top. As a result, nitrogen per unit leaf area (N(A)) increased with light in the canopies of all clones, from 0.9 g m-2 at the bottom to 2.9 g m-2 at the top. Although a single linear relationship described the dependence of maximum carboxylation rate (17-93 micromol CO2 m-2 s-1) or electron transport capacity (45-186 micromol electrons m-2 s-1) on N(A), for all clones, Clone Wol differed from Clones Hoo and Fri by exhibiting a higher dark respiration rate at low N(A) (1.8 versus 0.8 micromol CO2 m-2 s-1).     

A method for describing the canopy architecture of coppice poplar with allometric relationships

A multi-scale biometric methodology for describing the architecture of fast-growing short-rotation woody crops is used to describe 2-year-old poplar clones during the second rotation. To allow for expressions of genetic variability observed within this species (i.e., growth potential, leaf morphology, coppice and canopy structure), the method has been applied to two clones: Ghoy (Gho) (Populus deltoides Bartr. ex Marsh. x Populus nigra L.) and Trichobel (Tri) (Populus trichocarpa Torr. & A. Gray x Populus trichocarpa). The method operates at the stool level and describes the plant as a collection of components (shoots and branches) described as a collection of metameric elements, themselves defined as a collection of elementary units (internode, petiole, leaf blade). Branching and connection between the plant units (i.e., plant topology) and their spatial location, orientation, size and shape (i.e., plant geometry) describe the plant architecture. The methodology has been used to describe the plant architecture of 15 selected stools per clone over a 5-month period. On individual stools, shoots have been selected from three classes (small, medium and large) spanning the diameter distribution range. Using a multi-scale approach, empirical allometric relationships were used to parameterize elementary units of the plant, topological relationships and geometry (e.g., distribution of shoot diameters on stool, shoot attributes from shoot diameter). The empirical functions form the basis of the 3-D Coppice Poplar Canopy Architecture model (3-D CPCA), which recreates the architecture and canopy structure of fast-growing coppice crops at the plot scale. Model outputs are assessed through visual and quantitative comparisons between actual photographs of the coppice canopy and simulated images. Overall, results indicate a good predictive ability of the 3-D CPCA model.     

FT-IR imaging and pyrolysis-molecular beam mass spectrometry: new tools to investigate wood tissues

Fourier transform infrared (FT-IR) microimaging spectroscopy and pyrolysis molecular beam mass spectrometry (py-MBMS) were used as rapid analysis tools to evaluate differences in the chemical composition of 1-year-old transgenic aspens. Multivariate analysis of the spectroscopic data sets was used to compare the cell wall composition of nontransformed control to transgenic aspen plants with GRP-iaaM gene and with GRP-iaaM/35S-ACCase gene. Principal component analysis (PCA) was applied to both the FT-IR and py-MBMS spectra, which revealed sample groupings due to differences in chemical composition. Evaluating the PCA loadings allows determination of the chemical features responsible for the groupings. The FT-IR microimaging data was also used to monitor changes in the chemical composition as a function of the distance from the pith to the bark using partial least squares (PLS) analysis. The analysis shows that the changes in the composition of the xylem that occur over one annual growth ring can be monitored with FT-IR microimaging.     

Physiological responses of wild type and putrescine-overproducing transgenic cells of poplar to variations in the form and concentration of nitrogen in the medium

We determined: (a) the physiological consequences of overproduction of putrescine in transgenic poplar (Populus nigra x maximoviczii) cells expressing an ornithine decarboxylase transgene; and (b) effects of variation in nitrogen (N) concentration of the medium on cellular polyamine concentration in transgenic and non-transgenic cells. Cells grown in the presence of supplemental (to the normal concentrations of N sources in the growth medium) and reduced amounts of NH4NO3 and KNO3 were used to study effects on membrane permeability, mitochondrial respiratory activity, protein accumulation, growth rates and changes in cellular polyamine concentration. The N concentration of the MS medium was not a limiting factor for continued overproduction of putrescine in transgenic cells. However, continued supplies of NH4+ and NO3- were required to maintain homeostatic amounts of putrescine in both cell lines. The presence of high amounts of putrescine in transgenic cells had significant effects on the physiological parameters measured. Compared with non-transgenic cells, transgenic cells had greater plasma membrane permeability, less tolerance to NH4NO3, more tolerance to KNO3, and accumulated higher amounts of soluble protein.     

通过RNAi技术抑制杨树c3h基因表达提高糖转化效率

利用克隆得到的毛白杨c3h1基因构建其RNAi抑制表达载体,通过根癌农杆菌介导的叶盘法转化银腺杨无性系84 K,Realtime PCR检测表明其转基因株系323、325和322中c3h1基因表达量较野生型植株分别下调89.04%、82.22%和68.38%;茎横切片组化染色和显微结构观察表明转基因植株木质部发育和木质素沉积方式发生了改变;木质素、纤维素含量测定及苯酚—硫酸法总糖含量与HPLC法可溶性总糖和单糖含量检测结果表明:转基因植株木质素含量平均降低23.00%,最高可达39.71%;酸前处理效率最高提高了41.39%;未经酸处理直接酶解的糖化效率是对照植株的2.34～2.72倍,322株系和323株系比对照植株经酸前处理后再酶解的糖化效率高出81.18%和375.53%。     

Ultraviolet-B radiation alters phenolic salicylate and flavonoid composition of Populus trichocarpa leaves

We investigated foliar phenolic composition of field- and greenhouse-grown Populus trichocarpa Torr. & A. Gray (black cottonwood) ramets subjected to near zero (0x), ambient (1x) or twice ambient (2x) concentrations of biologically effective ultraviolet-B (UV-B) radiation. After a 3-month treatment period, several age classes of foliage samples were harvested and the phenolic compounds extracted, separated by high performance liquid chromatography and identified and quantified by diode-array spectrometry and mass spectrometry. Foliar phenolic concentration was greater in 1x- and 2x-treated tissue than in 0x-treated tissue. Phenolic compounds that increased in response to UV-B radiation were predominantly flavonoids, primarily quercetin and kaempferol glycosides. Enhancement of UV-B radiation from 1x to 2x ambient concentration did not result in further flavonoid accumulation in either greenhouse or field ramets; however, a non-flavonoid phenolic glycoside, salicortin, increased in response to an increase in UV-B radiation from 1x to 2x ambient concentration. Increased salicortin concentrations accounted for at least 30-40% of the total (5%) increase in UV-absorption potential of 2x-treated tissue. Because salicortin and other salicylates are important in plant-herbivore-predator relationships, these increases are discussed in the context of collateral feeding studies. We conclude that enhanced solar UV-B radiation may significantly alter trophic structure in some ecosystems by stimulating specific phenolic compounds.     

Calcium accumulation in the wood of short-rotation cottonwood species: effects on pulp properties

We have found that kraft pulp handsheets, made from a number of hybrid poplar (Populus trichocarpa × Populus deltoides) clones, contain unusual surface deformations caused by vessel element mineral blockages. The blockages were composed mainly of calcium salt deposits that survived chemical pulping and beating processes. Our findings confirm that a natural process of accumulation of calcium salts occurs in certain poplar species. In this article, we describe the implications for the multi-use implementation of these poplars in short-rotation, intensive-culture plantations.     

转蜘蛛杀虫肽与Bt毒蛋白C肽基因小黑杨对舞毒蛾中肠组织结构的影响

采用透射电镜观察舞毒蛾幼虫取食转蜘蛛杀虫肽与Bt毒蛋白C肽融合蛋白基因小黑杨叶片后,其中肠组织的病理变化。结果发现,转蜘蛛杀虫肽与Bt毒蛋白C肽融合蛋白基因小黑杨,能够引起舞毒蛾幼虫中肠组织发生明显的病理变化,致使幼虫中肠组织受到严重破坏。幼虫中肠组织中,柱状细胞、杯状细胞结构发生明显的病理变化,并随着取食的时间和取食量的增加,其病变和破坏的程度逐渐加剧。     

Immunolocalization of (1,4)-beta-galactan in tension wood fibers of poplar

The occurrence and distribution of (1,4)-beta-galactan in wood cells of poplar (Populus trichocarpa Torr. & A. Gray x P. koreana Rehder) were studied by immunolabeling with a monoclonal antibody (LM5) specific to this carbohydrate epitope. Immunofluorescence microscopy revealed exclusive binding of the LM5 antibody to tension wood fibers, indicating the specific presence of (1,4)-beta-galactan in cell walls of this wood cell type. Higher magnifications achieved with the fluorescence microscope and additional immunogold electron microscopy showed that the binding of the LM5 antibody was mainly restricted to a narrow cell wall area between the gelatinous G-layer and the secondary cell wall. This labeling pattern strongly suggests a role of (1,4)-beta-galactan in cross-linking the G-layer and secondary cell wall. Furthermore, the exclusive localization of (1,4)-beta-galactan in tension wood strengthens the view that this carbohydrate epitope can be considered a highly specific marker of reaction wood formation in mechanically stressed trees.     

Transpiration by two poplar varieties grown as coppice for biomass production

Fast-growing tree clones selected for biomass plantations are highly productive and therefore likely to use more water than the agricultural crops they replace. We report field measurements of transpiration through the summer of 1994 from two poplar clones, Beaupré (Populus trichocarpa Torr. & A. Gray x P. deltoides Bartr. ex Marsh.) and Dorschkamp (P. deltoides x P. nigra L.), grown as unirrigated short-rotation coppice in southern England. Stand transpiration was quantified by scaling up from sap flow measurements made with the heat balance method in a sample of stems. Leaf conductances, leaf area development, meteorological variables and soil water deficit were also measured to investigate the response of the trees to the environment. High rates of transpiration were found for Beaupré. In June, when soil water was plentiful, the mean (+/- SD) transpiration rate over an 18-day period was 5.0 +/- 1.8 mm day(-1), reaching a maximum of 7.9 mm day(-1). Transpiration rates from Dorschkamp were lower, as a result of its lower leaf area index. High total leaf conductances were measured for both Beaupré (0.34 +/- 0.17 mol m(-2) s(-1)) and Dorschkamp (0.39 +/- 0.16 mol m(-2) s(-1)). Leaf conductance declined slightly with increasing atmospheric vapor pressure deficit in both clones, but only in Beaupré did leaf conductance decrease as soil water deficit increased.     

欧洲黑杨转基因稳定性及对土壤微生物的影响

利用PCR分析技术 ,分析了田间试验已达 7a的转Bt基因欧洲黑杨的基因稳定性 ,并对其林地和非转基因林地的土壤微生物进行了类群数量分析。PCR分析表明Bt基因仍然存在于转基因植株中 ,转基因植株与对照杂交后代Bt基因分离呈 1∶1比例 ,符合孟德尔遗传分离规律 ,表明基因仍稳定存在。计数结果表明转基因林地中转基因植株与非转基因植株间根系土壤 3大类微生物 (细菌、放线菌和霉菌 )数量无显著差异 ,转基因林地与非转基因林地 (邻近杨树林地和健杨林地 )间的土壤 3大类微生物 (细菌、放线菌和霉菌 )数量无显著差异 ,说明转基因欧洲黑杨对土壤微生物系统尚没有明显的影响。     

No consistent daily variation in DNA methylation detected in Populus nigra leaves by methylation-sensitive amplification polymorphism analysis

DNA methylation, an epigenetic mechanism used by cells to control gene expression, has an important biological role in plant development and environmental fitness. Since plant DNA methylation is closely related to environmental conditions, variation during the day is expected. Here, in genetically identical plants of Populus nigra clone N46, DNA methylation changes in leaves over a 24 h period were detected using the methylation-sensitive amplification polymorphism method. The results showed different DNA methylation patterns in mature poplar leaves: not only in individuals at the same time, but also in samples at each of the six time during the day. In addition,night samples had a higher percentage of methylation than in morning samples. However, no statistically significant differences were found among the samples gathered at different times. Similar results were obtained for three other P. nigra clones with different genetic backgrounds.Real time q PCR showed that the DNA methyltransferase genes Pt-MET1 and Pt-SOM1 involved in CG DNA methylation in poplar were stable over a 24 h period in leaves of P. nigra N46 compared with circadian-controlled genes. That could be part of the reason that methylation of CCGG sites is stable in those leaves. That DNA methylation differed even in genetically identical plants indicates the specificity of DNA methylation changes in their genomes. No statistically significant differences in methylation changes were found between day and night, suggesting that DNA methylation is more stable than expected and is unlikely to be involved in circadian regulation in plants.     

盐胁迫下8个转基因小黑杨株系的抗逆性比较

以3种转基因（codA、betA、lea）小黑杨（populus simonii×P．nigra）为材料,选取XCI、XB1、XB2、XB3、XL1、X15、XL6、XL11共8个株系,非转基因小黑杨为对照,时其进行浓度为0．8％的NaCl胁迫处理,胁迫第6天分别测定叶绿素含量及主要生理指标。结果表明：各转基因株系的平均叶绿素含量高于对照小黑杨的7．9％;POD活性、CAT活性、APX活性都高于对照;膜的损伤程度低于对照;XL1、XL5、XL6、XB4的SOD活性高于对照。总体表现为转基因小黑杨在膜的伤害程度上低于对照,在抗氧化能力上高于对照。     

小黑杨转几丁质酶基因及酶活性

This paper reports a successful transformation of chitinase gene into Populus simonii×P. nigra by Agrobacterium-mediated means. For reducing adventitious buds,the optimal concentration of kanamycin was 40 mg·L~ -1 . Up to 700 mg·L~ -1 of Cefazolin Sodium had no obvious effect on differentiation of leaves. About 20-day-old leaf disc explants were pre-cultured for 3-4 d,then immersed in Agrobacterium suspension for infection for 6-15 min,then co-cultured on non-selection culture medium for 3 d in the dark at ...     

欧洲黑杨转Bt毒蛋白基因植株大田抗虫性测定

近几年,基因工程发展很快,通过基因工程已获得了各种各样的转基因植物,如抗虫转基因农作物的棉花（ＧｏｓｙｐｉｕｍｈｉｒｓｕｔｕｍＬ．）、水稻（ＯｒｙｚａｓａｔｉｖａＬ．）、马铃薯（ＳｏｌａｎｕｍｔｕｂｅｒｏｓｕｍＬ．）、玉米（ＺｅａｍａｙｓＬ．）、小麦...     

Influences of genetic and environmental factors on the concentration of the allergen Cry j 1 in sugi (Cryptomeria japonica) pollen

Pollen from sugi (Japanese cedar, Cryptomeria japonica D. Don), a forest tree species that is widely grown in Japan, causes serious allergic disease. The major allergens from sugi pollen, Cry j 1 and Cry j 2, have been isolated and characterized. It has been reported that Cry j 1 concentration in pollen varies considerably among trees. If Cry j 1 concentration is genetically controlled, the planting of trees with low Cry j 1 concentrations would reduce pollinosis. We investigated genetic and environmental effects on Cry j 1 concentration in eight clones growing at four sites. Concentrations of Cry j 1 in pollen were measured with a monoclonal antibody-based Enzyme Linked Immunosorbent Assay (ELISA). The Cry j 1 concentrations differed significantly among clones and sites, but the site x clone interaction was not significant, suggesting that the Cry j 1 concentration is controlled primarily by genetic factors. We examined correlations between Cry j 1 concentration and temperature and precipitation from July through February. Temperature was not significantly related to Cry j 1 concentration, whereas cumulative precipitation during the 8 months and mean daily precipitation in September showed significant negative correlations with Cry j 1 concentration.     

In vitro anther culture and Agrobacterium-mediated transformation of the AP1 gene from Salix integra Linn. in haploid poplar(Populus simonii × P. nigra)

A reliable,efficient anther culture system,the dominant technique for generating haploid plants in breeding programs,that can be used for generating transgenic poplar plants has been needed.In the present study,therefore,an anther culture system was developed using isolated mid-and late-uninucleate anthers of poplar(Populus simonii x P.nigra).From a combination of SSR and ploidy analyses,six double haploid and two haploid lines were characterized from 86 plants grown from 16 regenerated anther cultured lines.After 48 months of development,two plant lines from the regenerated plants maintained their haploid level in vitro for over 2 years.A number of haploid plants from the different lines weretransferred to soil.The leaves of these transplants were then used as explants for transformation with the APETALA1(AP1) gene using Agrobacterium tumefaciens.Overexpression of AP1 in haploid poplar induced early flowering with obvious petals when ectopically expressed.To our knowledge,this is the first report on changes in flowering time in AP1-trangenic poplar,which is important for elucidating the regulatory mechanism of tree flower development.