Genetic transformation of loblolly pine using mature zygotic embryo explants by Agrobacterium tumefaciens

Introduction1Genetictransformationinconifershasthepotentialtoallowtheselectiveimprovementofindividualtraitsinelitecloneswhilestillmaintainingtheexistingcombinationofgenesresponsibleforthesuperiorphenotype(Charestetal.1991;Jamesetal.1996;Walteretal.1999).Atpresent,althoughconsiderableresearchefforthasbeendevotedtothegeneticengineeringofconiferspecies(Sederoffetal.1986;Bekkauoietal.1988,1990;Robertsonetal.1992;Bomminenietal.1993;Shinetal.1994;Klimaszewskaetal.1997),ithaslaggedbehindadvancesma…     

Genetic transformation of loblolly pine using mature zygotic embryo expiants byAgrobacterium tumefaciens

Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of β-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected withAgrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of β-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficientAgrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.     

Seasonal variation in gene expression for loblolly pines (Pinus taeda) from different geographical regions

In developing xylem, gene expression levels vary in different genotypes, at different stages of development, throughout a growing season, and in response to stresses. Commercially important characteristics such as wood-specific gravity are known to differ with seed source. For example, when grown on a common site, the specific gravity of Arkansas loblolly pine (Pinus taeda L.) trees is greater than that of Louisiana loblolly pine, and Texas loblolly pines have a greater specific gravity than loblolly pines from the Atlantic coast. A microarray analysis was performed to examine variation in gene expression among trees from different geographical sources when grown on a common site, and seasonal variation in gene expression in each seed source. We used microarrays containing 2171 expressed sequence tags (ESTs) with putative functions of interest, selected from several loblolly pine xylem partial cDNA libraries and a shoot tip library. Genes with significant variation in expression for each factor were identified. Many genes preferentially expressed in latewood compared with earlywood were for proteins involved in cell wall biosynthesis. Variation in gene expression among trees from the two seed sources in each growing season suggests that there may be more differences between South Arkansas trees and South Louisiana trees in latewood than in earlywood. Variation in gene expression among trees from different regions may reflect adaptation to different environments.     

多重复干旱循环对北美短叶松和黑云杉苗木气体交换及水分利用效率的影响

本文研究了多重复干旱循环对１年生北美短叶松（ＰｉｎｕｓｂａｎｋｓｉａｎａＬａｍｂ．）和黑云杉（Ｐｉｃｅａｍａｒｉａｎａ［Ｍｉｌ］Ｂ．Ｓ．Ｄ．）苗木的气体交换速率及水分利用效率的影响。结果表面，多重干旱循环对它们的气体交换（Ｃｓ，Ｐｎ，Ｔｒ）有显著影响（Ｐ＜０．５），而对其水分利用效率（ＷＵＥ）影响不大（Ｐ＞０．１）。尽管北美短叶松的气孔对轻度干旱胁迫不如黑云杉敏感，但是它对中度及严重干旱胁迫的敏感程度却高于黑云杉。在轻度及中度干旱胁迫下，北美短叶松的光合作用主要受非气孔因素的影响，而黑云杉则主要受气孔因素的影响。解除干旱胁迫后，黑云杉的气孔敏感性、光合能力及水分利用效率的恢复都要比北美短叶松更快．我们认为，延迟脱水是北美短叶松的主要耐旱机理，而忍耐脱水则是黑云杉重要的耐旱途径。轻度的干旱胁迫锻炼可以帮助北美短叶松在更严重的干旱胁迫下保持固有而较强的耐旱能力。然而，通过多重复干旱循环锻炼后黑云杉在改善耐旱能力的强度方面则大于北美短叶松     

Drought effects on water use efficiency, freezing tolerance and survival of Eucalyptus globulus and Eucalyptus globulus × nitens cuttings

Genetic improvement of Eucalyptus genotypes for drought and frost resistance is essential for successful intensive management of commercial plantations. Understanding the physiological mechanisms that relate water use and frost resistance for highly deployed genotypes may allow for better prediction of their future performance, genetic selection and seedling management for site specific purposes. We studied whether instantaneous water use efficiency (WUE _i ) may serve as drought, freezing and photoinhibition tolerance predictor by studying its response on six E. globulus clones (Eg1–Eg6) and four E. globulus × E. nitens hybrid seedlings (Egn1–Egn4) under drought and irrigated (control) treatments. Net photosynthesis (A) and transpiration (E) were studied using a gas exchange system in order to calculate WUE _i (A/E). Simultaneous chlorophyll a fluorescence measurements were performed to assess the non photochemical quenching components. Frost tolerance of plants under control and drought treatments were evaluated by measuring temperatures that exert 50% photoinactivation of photosystem II. Finally, drought tolerance was evaluated by plant survival within each genotype after rehydration. Our results showed significant genotype variability in the rate of soil and xylem water potential decrease during drought. While most of the genotypes reached ?4.0 MPa in about 35 days of drought, genotypes Eg6 and Egn4 required 56 days of drought to reach this xylem water potential. WUE _i exhibited significant differences among genotypes and irrigation treatments. Genotypes Eg5 and Egn4 increased their WUE _i between 70 and 80% after drought. This was associated with a more conservative control of water loss at the stomatal level combined with maintenance of relatively higher rates of net photosynthesis than the other genotypes under drought conditions. Plants exposed to drought were more freezing tolerant than control plants, having in average 3°C lower LT₅₀ than well irrigated ones. There was no a clear correlation between WUE _i and drought tolerance or drought-induced photoinhibition, however WUE _i was inversely correlated with LT₅₀. Our results suggest that WUE _i is not suitable by itself to select drought tolerant genotypes, but may provide evidence for discarding drought sensitive genotypes. In addition, it could provide valuable information to select for freezing tolerance.     

Preliminary Studies on Establishment of Genetic Transformation System in Loblolly Pine

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杨树抗黑斑病相关基因表达谱分析

利用黑斑病的高抗无性系美洲黑杨I-69及黑斑病的高感无性系欧美杨I-45为材料建立的2个cDNA文库,随机挑取cDNA克隆进行5'端EST序列测序,共获得有效序列20 023条.序列经聚类分析和拼接后,共获得10 816个Unigene,其中包括3 734个Contig,7 082个独立的Singleton.被注释的8 853个具有同源性匹配序列基因中,按照GO的分子功能、生物过程和细胞组分3个不同分类角度进行分类.在具有功能注释的8 853个Unigene中,选出330个与完成全基因组测序的毛果杨序列进行BLAST分析,结果发现有177个抗病相关候选基因出现在282个Unigene中,其中135个分布于杨树的18个不同连锁群上,其他42个基因位于还没定位的scaffolds上.所测定的这些EST序列为后期在基因组水平上研究杨树黑斑病的水平抗性遗传机制及进一步的相关基因发现奠定基础.

Abstract：

In an attempt to elucidate the molecular mechanism for resistance of black spot disease in poplar,gene expression profiles in leaves of Populus deltoides'Lus'(I-69/55)and P.euramericana'I-45/51',which were inoculated with the pathogen Marssonina brunnea f.sp.brunnea,were analyzed based on expressed sequence tags (ESTs).A total of 20 023 valid ESTs from the 5'terminal ends derived from corresponding cDNA libraries of the two poplar species were sequenced.Cluster analysis of the 20 023 sequences yielded 10 816 tentative unigenes,including 3 734 contigs and 7 082 singletons.All tentative unigenes were classified by Gene Ontology functional categories.To find resistance-associated candidate genes and locate them on poplar genome,330 unigenes was chosen from 8 853 annotated tentative unigenes,and their BLAST alignment was conducted with Populus trichocarpa assembly,1 77 related candidate resistant genes were found,and they presented in 282 unigenes.Among these genes,there were 1 35 genes located on 18 different linkage groups of poplar genome,and 42 genes located on the different scaffolds.This study supplied a resource of candidate genes for further exploring the genetic mechanism for the host horizontal resistance to the pathogen Marssonina brunnea at the whole genome range,and provided important information for further gene discovery.     

Tree-growth analyses to estimate tree species' drought tolerance

Climate change is challenging forestry management and practices. Among other things, tree species with the ability to cope with more extreme climate conditions have to be identified. However, while environmental factors may severely limit tree growth or even cause tree death, assessing a tree species' potential for surviving future aggravated environmental conditions is rather demanding. The aim of this study was to find a tree-ring-based method suitable for identifying very drought-tolerant species, particularly potential substitute species for Scots pine (Pinus sylvestris L.) in Valais. In this inner-Alpine valley, Scots pine used to be the dominating species for dry forests, but today it suffers from high drought-induced mortality. We investigate the growth response of two native tree species, Scots pine and European larch (Larix decidua Mill.), and two non-native species, black pine (Pinus nigra Arnold) and Douglas fir (Pseudotsuga menziesii Mirb. var. menziesii), to drought. This involved analysing how the radial increment of these species responded to increasing water shortage (abandonment of irrigation) and to increasingly frequent drought years. Black pine and Douglas fir are able to cope with drought better than Scots pine and larch, as they show relatively high radial growth even after irrigation has been stopped and a plastic growth response to drought years. European larch does not seem to be able to cope with these dry conditions as it lacks the ability to recover from drought years. The analysis of trees' short-term response to extreme climate events seems to be the most promising and suitable method for detecting how tolerant a tree species is towards drought. However, combining all the methods used in this study provides a complete picture of how water shortage could limit species.     

Preliminary studies on establishment of genetic transformation system in loblolly pine

A transformation system was established for loblolly pine (Pinus taeda L.) mature zygotic embryos usingAgrobacterium tumefaciens. The gene coding for the β-glucuronidase (GUS) gene was introduced into loblolly pine tissues and its transient expression was detected with histochemical staining. The influences of different genotypes.Agrobacterum concentrations. and cocultivation time on GUS expression and kanamycin resistant callus and shoot regeneration were investigated. The results showed that the highest GUS expression frequency (16.3%) and shoot regeneration frequency (78%) were obtained from genotype 9–1003 withAgrobacterium concentration decreased 9 times and cocultivation time of 56 hours respectively. GUS expression was obtained in all genotypes tested. The successful expression of the GUS gene in different genotypes suggested that it will be a useful transformation system for loblolly pine. (Responsible Editor: Chai Ruihai)     

Changes in protein synthesis during drought conditioning in roots of jack pine seedlings (Pinus banksiana Lamb.)

The impact of drought conditioning on the ability of eight-week-old jack pine (Pinus banksiana Lamb.) seedlings to withstand drought was assessed. Two progressive cycles of drought conditioning significantly increased the survival of seedlings subjected to a subsequent prolonged drought. The in vivo accumulation of several root membrane proteins during drought conditioning was correlated with an increase in seedling survival. A group of root proteins, ranging in molecular mass from 43 to 47 kDa, increased accumulation during one cycle of drought conditioning and to a lesser extent during two cycles of drought conditioning. The accumulation of several low molecular mass membrane and soluble proteins also increased during drought conditioning, suggesting that these proteins may play an important role in the enhancement of drought tolerance. In vitro translation studies showed a general increase in the abundance of protein products encoded by mRNAs from drought-conditioned seedlings. Although the majority of the in vitro translation products appeared in both control and drought-conditioned seedlings, one mRNA encoding a 15 kDA translated protein was more prominent during the second cycle of drought conditioning.     

Leaf-level gas-exchange uniformity and photosynthetic capacity among loblolly pine (Pinus taeda L.) genotypes of contrasting inherent genetic variation

Variation in leaf-level gas exchange among widely planted genetically improved loblolly pine (Pinus taeda L.) genotypes could impact stand-level water use, carbon assimilation, biomass production, C allocation, ecosystem sustainability and biogeochemical cycling under changing environmental conditions. We examined uniformity in leaf-level light-saturated photosynthesis (A(sat)), stomatal conductance (g(s)), and intrinsic water-use efficiency (A(sat)/g(s) or δ) among nine loblolly pine genotypes (selected individuals): three clones, three full-sib families and three half-sib families, during the early years of stand development (first 3 years), with each genetic group possessing varying amounts of inherent genetic variation. We also compared light- and CO(2)-response parameters between genotypes and examined the relationship between genotype productivity, gas exchange and photosynthetic capacity. Within full-sib, half-sib and clonal genotypes, the coefficient of variation (CV) for gas exchange showed no consistent pattern; the CV for g(s) and δ was similar within clonal (44.3-46.9 and 35.5-38.6%) and half-sib (41.0-49.3 and 36.8-40.9%) genotypes, while full-sibs showed somewhat higher CVs (46.9-56.0 and 40.1-45.4%). In contrast, the CVs for A(sat) were generally higher within clones. With the exception of δ, differences in gas exchange among genotypes were generally insignificant. Tree volume showed a significant positive correlation with A(sat) and δ, but the relationship varied by season. Individual-tree volume and genotype volume were positively correlated with needle dark respiration (R(d)). Our results suggest that uniformity in leaf-level physiological rates is not consistently related to the amount of genetic variation within a given genotype, and δ, A(sat) and R(d) were the leaf-level physiological parameters that were most consistently related to individual-tree and genotype productivity. An enhanced understanding of molecular and environmental factors that influence physiological variation within and between loblolly pine genotypes may improve assessments of genotype growth potential and sensitivity to global climate change.     

Responses of Prunus ferganensis,Prunus persica and two interspecific hybrids to moderate drought stress

Prunus ferganensis (Kost. & Riab) Kov. & Kost, a close relative of cultivated peach (Prunus persica (L.) Batsch.), is native to arid regions of central Asia. A distinguishing feature of P. ferganensis is its prominent, elongated, unbranched pattern of leaf venation. To determine whether the long-vein trait could be used as a marker in breeding for drought tolerance, we investigated the association between this trait and the leaf morphological and physiological parameters related to drought response in P. ferganensis, P. persica and two interspecific hybrids, one with the long-vein trait (BY94P7585) and one without (BY94P7589). The four genotypes were grafted onto "Guardian" peach rootstock and half of the plants were assigned to a drought treatment in which irrigation was limited to 25-50% of the evapotranspiration (ET) rate measured in the remaining well-watered plants, which were irrigated to runoff daily. The drought treatment reduced photosynthesis and leaf conductance by 49-83% and reduced total leaf area per plant by 17-24%, but generally did not affect mid-morning leaf water potential. Leaf gas exchange did not differ among genotypes in either treatment. Sorbitol accumulated in mature leaves in response to drought, but neither its amount nor its metabolism varied systematically with climatic adaptation among genotypes. Accumulation of transport sugars was highest in P. ferganensis, indicating that growth reduction may represent an important strategy for coping with drought in this species. Prunus ferganensis and the hybrids had higher ET than P. persica, and seemed to use water opportunistically, maintaining high gas exchange rates and consequently high ET when water was available, and avoiding low water potentials through stomatal closure as soil water declined. Leaf size (cm(2) leaf(-1)) and specific leaf area (cm(2) g(-1) dry mass) were lower in P. ferganensis and the hybrids than in P. persica. We conclude that the long-vein trait is not a reliable marker for drought tolerance, but leaf traits of P. ferganensis such as size and specific leaf area may be useful in P. persica breeding programs targeting drought tolerance.     

Relationship between the incidence of pine wilt disease and the drainage area

We examined the relationship between the incidence of pine wilt disease and moisture conditions in the stand level ofPinus densiflora Sieb. et Zucc. forests in the warm-temperate zone of the western part of Japan. For this analysis, pine trees killed by pine wilt disease were distinguished from the suppressed trees by their position in the layer of the pine forest stand. The drainage area, which is small in the upper part and large in the lower part of the slope, was adopted for representing the moisture conditions in the soil of the slope. The percentage of the pine trees killed by pine wilt disease increased as the size of the drainage area increased. This result suggested that the incidence of pine wilt disease tended to be high in areas with moist conditions. Pine trees attacked by the pinewood nematode die from extensive water deficit due to tracheid cavitations. Pine wilt disease mainly emerges in the summer when the soil water conditions become especially severe, and the radical water stress is thought to accelerate the disease. It was assumed that pine trees in the plots with the small drainage area resisted the influence of the attack of the pinewood nematode because pine trees in the plots with the small drainage area encountered long-term water stress and acquired drought tolerance. Pine trees in the plots with the large drainage area were presumed to be well established in the moist conditions and not to have acquired drought tolerance. The drought tolerance of pine trees was thought to be an important factor in resistance to pine wilt disease.     

Unravelling and managing fusiform rust disease: a model approach for coevolved forest tree pathosystems

Fusiform rust disease remains the most destructive disease in pine plantations in the southern United States. Our ongoing research is designed to identify, map, and clone the interacting genes of the host and pathogen. Several resistance (R) genes have been identified and genetically mapped using informative pine families and single‐spore isolate inoculations. In addition, we are mapping the first of many expected corresponding avirulence (Avr) genes in the fungal pathogen. The Avr genes condition avirulence/virulence and avirulence is required for an incompatible reaction (i.e., no‐gall development) to take place within an inoculated tree that carries resistance at the corresponding R gene. We provide an overview of our methodology for identifying and mapping R and Avr genes, an update of our current progress, and a brief discussion of two approaches for predicting R gene genotypes of uncharacterized parental trees and for estimating the efficacy of specific pine genotypes at various planting locations. This paper emphasizes the critical importance of controlled genetic materials of both the host and pathogen for elucidating the genetic nature of resistance and virulence in coevolved forest pathosystems.     

Genotypic variation in drought response of silver birch (Betula pendula): leaf water status and carbon gain

To assess genotypic variation in drought response of silver birch (Betula pendula Roth), we studied the plasticity of 16 physiological traits in response to a 12-14-week summer drought imposed on four clones in two consecutive years. In a common garden experiment, 1-year-old clonal trees from regions with low (550 mm year(-1)) to high rainfall (1270 mm year(-1)) were grown in 45-l pots, and leaf gas exchange parameters, leaf water potentials, leaf osmotic potentials and leaf carbon isotope signatures were repeatedly measured. There were no clonal differences in leaf water potential, but stomatal conductance (gs), net photosynthesis at ambient carbon dioxide concentration, photosynthetic water-use efficiency, leaf carbon isotope composition (delta13C) and leaf osmotic potentials at saturation (Pi0) and at incipient plasmolysis (Pip) were markedly influenced by genotype, especially gs and osmotic adjustment. Genotypes of low-rainfall origin displayed larger osmotic adjustment than genotypes of high-rainfall origin, although their Pi0 and Pip values were similar or higher with ample water supply. Genotypes of low-rainfall origin had higher gs than genotypes of high-rainfall origin under both ample and limited water supply, indicating a higher water consumption that might increase competitiveness in drought-prone habitats. Although most parameters tested were significantly influenced by genotype and treatment, the genotype x treatment interactions were not significant. The genotypes differed in plasticity of the tested parameters and in their apparent adaptation to drought; however, among genotypes, physiological plasticity and drought adaptation were not related to each other. Reduction of gs was the first and most plastic response to drought in all genotypes, and allowed the maintenance of high predawn leaf water potentials during the drought. None of the clones exhibited non-stomatal limitation of photosynthesis. Leaf gs, photosynthetic capacity, magnitude of osmotic adjustment and delta13C were all markedly lower in 2000 than in 1999, indicating root limitation in the containers in the second year.     

Co-occurring tree species show contrasting sensitivity to ENSO-related droughts in planted dipterocarp forests

This study explores the diversity in sensitivity to drought of moist tropical forest tree species. Yearly tree growth records collected over a ten-year period in two one-hectare 70-year-old damar agroforest plots in Sumatra are analysed. These agroforests are mixed tree plantations, dominated by Shorea javanica K. & V., a dipterocarp tree cultivated and tapped for its commercially valuable resin (damar). Many indigenous fruit tree species grow in these agroforests, as well as timber tree species originating from the nearby natural forest. During the census period the multi-species stands were subjected to three El Nino Southern Oscillation (ENSO)-related droughts (1994, 1997 and 2002). At the tree community level, these droughts were associated with a marked decrease in radial stem growth. Multilevel modelling was used to explore the relative contribution of species, tree size and individual tree characteristics to the observed response to drought.All tree species appeared to be sensitive to drought but the amplitude of the response varied significantly across species. Predicted species mean decrease in stem radial growth rate on drought years (i.e. years with 6 months with less than 50 mm/month rainfall) ranged from less than 5% to more than 80%. Shared species were ranked consistently between plots indicating that the results were robust. Stem diameter significantly affected tree sensitivity to drought in two species only, but in opposite ways: in S. javanica, larger trees appeared to be less sensitive while the opposite was true for Lansium domesticum, an abundant fruit tree. Individual tree sensitivity to drought contributed significantly albeit to a small extent to the overall response to drought. This individual tree effect did not show any pattern of spatial correlation and hence could not be related to topographic features. It is likely to reflect the individual's unique history and genotype.