杨树转基因研究进展及展望

杨树是重要的栽培树种,也是研究林木基因工程的重要模式植物。杨树转基因研究可以打破种属限制,具有高效性和专一性的特点,是对杨树进行遗传改良的重要手段。为了更好的进行该方面的工作,本文介绍了杨树转基因涉及到的抗虫、抗除草剂、木材材性改良、抗逆、抗病、激素调控、开花调控和植物修复等应用领域的研究进展及现状,分析了影响杨树农杆菌转化效率的主要因素,并探讨了杨树转基因研究存在的问题及发展方向,希望能为后期从事林木基因工程研究的科研工作者提供依据。     

Agrobacterium-mediated transformation of lombardy poplar (Populus nigra L. var.italica Koehne) using stem segments

Genetically transformed lombardy poplar (Populus nigra L. var.italica Koehne) plants were regenerated by co-cultivation of stem segments withAgrobacterium tumefaciens strain LBA4404 that harbored a binary vector (pBI121) which included genes for β-glucuronidase (GUS) and neomycin phosphotransferase. Successful transformation was confirmed by the ability of stem segments to produce calli in the presence of kanamycin, histochemical and fluorometric assays of GUS activity in plant tissues, and Southern blot analysis.     

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)     

Treatment of poplar callus with ferulic and sinapic acids I: incorporation and enhancement of lignin biosynthesis

Ferulic acid (FA), tetradeuteroferulic acid (DFA), sinapic acid (SA), or heptadeuterosinapic acid (DSA) was exogenously supplied to poplar (Populus alba L.) callus. Administration of FA or SA increased the lignin content of the callus to about twice that of the control callus. Gas chromatographic analysis of the alkali hydrolysate of the cell wall residue revealed that only a trace amount of SA was bound to the cell wall, and the amount of FA was less than 2% of the total callus lignin. Thioacidolysis of the DFA-treated callus indicated that DFA is effectively converted to both coniferyl and sinapyl alcohols and then incorporated into the corresponding lignin. Incorporation of DSA into syringyl lignin or guaiacyl lignin was not observed, but yields of syringyl lignin thioacidolysis products were markedly increased by DSA treatment of the callus. These results suggest that SA may not be a precursor of sinapyl alcohol and syringyl lignin per se, but it may induce or enhance the biosynthesis of syringyl lignin in poplar callus.     

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.     

Comparison of gene delivery methods for transformation ofRobinia pseudoacacia L. by Ti-plasmid

The binary vector plasmid pIG121-Hm carrying the chimeric neomycin phosphotransferase (npt II), β-glucuronidase (gusA) and hygromycin phosphotransferase (hpt) genes was delivered intoRobinia pseudoacacia L. callus using theAgrobacterium tumefaciens-mediated transformation method and particle gun transformation method. It was determined that adding 10 mg/L of acetosyringone toAgrobacterium infection medium and callus-Agrobacterium co-culture medium could increaseAgrobacterium-mediated transformation efficiency. In contrast, particle gun transformation could successfully deliver plasmid DNA into the callus, but its transformation efficiency was lower and gene expression was transient in comparison with theAgrobacterium-mediated method, suggesting that substantially stronger promoters might be required.     

An improved transformation system for Lombardy poplar (Populus nigra var. italica)

We report an improved transformation system for Lombardy poplar (Populus nigra var. italica). A new binary vector, pBF2, with 11 unique restriction enzyme sites and the normal neomycin phosphotransferase II (NPTII) gene was constructed for the transformation of Lombardy poplar. Genetically transformed adventitious shoots were directly regenerated after cocultivation of stem segments with Agrobacterium tumefaciens EHA105 that harbored a binary vector with genes for the NPTII and enhanced green fluorescent protein. Successful transformation was confirmed by fluorescence microscopy, immunoblotting and Southern blotting analyses, and resistance to kanamycin and geneticin. This transformation system requires less time than our previous method for the regeneration of transgenic shoots. When explants were incubated on a smedium containing dithiothreitol, the transformation frequency increased to approximately 20%.     

Changes through time in traits of poplar clones in selection trials

Growth, survival, stem characteristics, resistance to wind, and canker disease of 51 poplar clones were evaluated at age 9 and 10 years, and compared to measurements at age 3 in two trials on the Lower Valley of the Colorado River, Argentina. About 30% of the clones in both trials had large changes in growth rankings at age 9 and 10 compared to rankings at age 3. Canker incidence increased about 4-fold between age 3, and measurements at age 9 and 10. Stem form was in general satisfactory, and wind incidence was almost nil. In trial 1, clones 588–1, Conti 12, 582–41, and 582–5 occupied the upper positions in the growth rankings at age 3 and 10, and the clone 588–1 ranked first both in growth and canker incidence in 1998. Stem growth trajectories of clones with similar growth rates did not differ among clones with, and without canker disease. In trial 2, the range in growth among clones was narrower than in trial 1, and canker disease, although of minor incidence, was present in most clones at age 9. Some of the P. deltoides clones in the trials had similar growth, and less canker disease than the Euroamerican clones commonly grown in the region, but progressive canker incidence should be addressed with additional selection trials.     

Steady-state nutrition of hybrid poplar grown from un-rooted cuttings

Fertilizing plantations of fast-growing tree crops, in manners which supply nutrients at rates that match plant demand and maintain stable internal plant nutrient ratios, can maximize biomass production and carbon sequestration while reducing fertilizer wastage and pollution. Our objectives were to determine nutrient ratios of common hybrid poplar (Populus trichocarpa Torr. and Gray × Populus deltoides Marsh.) (T × D) clones under steady-state nutrition, and to determine if incremental additions of fertilizer were more successful than conventional (evenly-split) fertilizer additions in maximizing biomass production and inducing stable nutrient ratios. Un-rooted cuttings of three T × D clones (49–177, DTAC-7, 15–29) were grown under a conventional regime and a modified-exponential fertilization regime at three application rates (1.8, 3.7 or 7.4 g N plant⁻¹). Above- and below-ground biomass and nutrient concentrations were measured after one growing season. There were few differences in total plant biomass between conventional and modified-exponential fertilization regimes, but for one clone, biomass accumulation equivalent to the highest rate under the conventional regime was achieved with the medium rate of the modified-exponential regime. Stable nutrient ratios (at conditions consistent with steady-state nutrition) were: 100N:14P:50K (49–177), 100N:13P:49K (DTAC-7) and 100N:12P:60K (15–29).     

Relationship between trophic component of different poplar strains and occurrence ofSaperda poplnea

An experimental area of poplar was established in Songyuan of Jilin Province in 1999 for testing the resistance of different poplar stains toSaperda poplnea. Incidence ofS. poplnea on ten poplar strains were investigated, and the main trophic component of branches of these poplar trees were measured and analyzed in April 2001. The results showed that there existed significant difference in population size ofS. poplnea on different poplar strains, and the branches of these poplar strains have significant difference in nutrient component and content of amino acids. The population size of this pest had a significantly positive correlation with dissolvable total sugar and water content but had no significant correlation with content of total nitrogen and protein nitrogen. Foundation Item: This paper is part of the subject of integrated control of longicorn of poplar for mainly focusing on pest resistance, which was supported by Jilin Provincial Forestry Department (96-010). Biography: Gao Chang-qi (1949-), Research Fellow, Doctorate Tutor, Jilin Provincial Academy of Forestry Science, Changehun 139931, P. R. China. Responsible editor: Chai Ruihai     

应用单拷贝核基因标记研究我国欧洲山杨自然居群的遗传多样性

[目的]对分布于我国新疆地区欧洲山杨自然居群的遗传多样性与遗传分化进行研究。[方法]对4个欧洲山杨自然居群共72个个体的6个单拷贝核基因标记(sing-copy nuclear markers)进行扩增与测序,并计算相应的遗传多样性和遗传分化参数。[结果]表明:比对后6个基因的长度在459 bp到747 bp之间,平均多态核苷酸位点个数为14,遗传多样性指数π和θ_w分别达到了0.004 8和0.004 4,基因多样性指数为0.73,以上结果表明欧洲山杨表现出较高的遗传多样性水平。Tajima中性检验结果均不显著,表明所用6个单拷贝核基因均符合中性进化假设。AMOVA分析表明89.72%的遗传变异存在于居群内,居群间的平均遗传分化指数F_st为0.10,居群间平均基因流(Nm)为3.235。[结论]高度异交,高碱基突变率及超长距离的基因流机制能够很好的解释我国新疆地区欧洲山杨表现出的较高的遗传多样性水平与较低水平的遗传分化。     

匈牙利丹努波（Danube）河和梯沙（Tisza）河间沙丘洋槐——银白杨混交林林分结构和产量研究

作者采用新的研究方法,研究了不同配比的洋槐(Robinia pseudoacacia L.)/银白杨(Populus alba L)混交林林分结构和产量。通过逐株测量并采用材积表确定了各树种的主林分结构和产量因子。测定了混交林树种以及纯林不同树种的蓄积量。发现林分总蓄积量和和树种混交比例密切相关。与纯林对照组相比,混交林蓄积在16年龄时的相对剩余为1.24-1.55。研究证明,如果2树种都具有初期快速生长率的特点和相同的轮伐年龄,可以进行混交造林,并相互有利。图4表2参15。     

A comparison of pre-planting treatments on hardwood cuttings of four hybrid poplar clones

Rooting and early growth of four hybrid poplar clones (Populus spp.) planted in a greenhouse were examined after applying 40 pre-rooting treatment combinations to dormant cuttings. Treatments included 2 cutting lengths (5 and 10 cm), 5 soaking times (0, 2, 4, 8, and 12 days), and 4 dips (chitosan, rooting hormone powder, liquid rooting hormone added to the soaking water, and none). Significant differences in both rooting percentages and growth were shown between clones after 7 weeks of growth. Ten cm cuttings had 29% greater rooting success, 28% more above-ground growth, and 12% lower root/shoot ratios than 5 cm cuttings. Cuttings planted without soaking had the lowest rooting success, at less than 45% on average. Commercial rooting hormones decreased the number of rooted cuttings but increased root/shoot ratios. For optimal rooting, we recommend using 10 cm cuttings, soaked for 2 days in water (4 days for the Jackii10 clone) without any additional dipping/hormone substance.     

杨树不同无性系长江中游地区造林对比试验

将收集到的20个杨树优良无性系,在湖北省长江中游平原地区的监利县进行了造林对比试验.通过10a的试验选出适宜在湖北省长江中游平原栽培的长江杨、鲁山杨、303杨、中汉17、375杨5个优良无性系.以材积为主要参考指标,所选出的优良无性系生长量比对照提高20％以上.本试验为湖北省长江中游平原地区杨树人工林经营提供了无性系选择依据.     

Effects of thinning on growth of six tree species in north-temperate forests of Lithuania

This paper summarises the results from 35 years-observed thinning experiments on 256 permanent sample plots in 10–60 year-old stands of ash, aspen, birch, oak, pine and spruce in Lithuania. Thinning enhanced crown projection area increment of residual trees. The largest effect was observed in stands of aspen and birch (growth increase by 200%), followed by ash and oak (over 100%), and spruce and pine (about 80%). Thinning also promoted dbh increment, especially in younger stands, and the increase of dbh increment was positively correlated with the thinning intensity. The strongest reaction was exhibited by oak and aspen, while ash, birch and conifers reacted to a lower extent. Low and moderate intensities of thinning stimulated volume production in younger stands while the opposite was observed in older stands with increasing removals. Spruce stands exhibited relatively strongest increase of volume increment and pine, –the weakest, while the effect on deciduous species was intermediate. The results demonstrate that significant increase in volume increment is achievable with thinning of only young forest stands, e.g. 10–20 year-old pine, birch and ash, or 10–30 year-old oak, aspen and spruce.     

Isolation and characterization of a novel anionic peroxidase cDNA found in poplar (Populus nigra) suspension cultured cells

A cDNA clone fromPopulus nigra L. var.italica Koehne, denoted PCY2-6, coding for an anionic peroxidase has been isolated, cloned, and characterized. PCY2-6 is 1160 bp long; and its deduced product, PnC26, contains 343 amino acid residues. The mature protein has a calculated isoelectric point of 4.09. The protein contains two motifs typical of peroxidase and 10 potentialN-glycosylation sites. PnC26 is therefore classified as an anionic peroxidase. The mRNA of the PCY2-6 gene family was detected in immature and mature leaves and in two parts of current-year stems: the shoot tip and the older stem. The mRNA of PCY2-6 gene family was found to localize in the phloem and cortex of the current-year stems. We therefore conclude that expression of the PCY2-6 gene family is related to bark development.     

Effect of cutting position on rooting and shoot growth of two poplar clones

One-year-old shoots from stooling beds of Populus × deltoides Walker and Populus jackii Northwest were collected in late fall to determine the effect of cutting position on rooting and growth of hardwood poplar cuttings. Cuttings with a minimum diameter of 9.0 mm were obtained from basal, middle and distal locations on the poplar shoot. Rooting and shoot growth were assessed in the greenhouse and under field conditions. Walker poplar cuttings collected from basal portions of the shoot and planted in the field had 87.6% rooting and 103.8 cm growth compared to 78.8% and 103.4 cm and 67.6% and 88.8 cm for middle and distal locations respectively. For Northwest rooting and growth under field conditions was 84.4% and 94.7 cm for basal, 78.4% and 90.5 cm for mid and 64.4% and 84.3 cm for distal locations. Rooting in the greenhouse was superior to the field. Walker cuttings had fewer roots and buds per cutting than Northwest, however, growth of Walker in the field was superior.     

Development of oak and beech seedlings planted under varying shelterwood densities and with different site preparation methods in southern Sweden

Effects of site preparation, shelterwood density and planting depth on the survival and growth of planted beech and oak seedlings were studied. Experiments were performed in one oak and one beech stand in southern Sweden. Two areas with different densities of shelterwood and one clearcut were established in each stand. Growth, damage and survival of the planted seedlings were observed for three years. Soil water potential was recorded weekly and radiation and soil temperatures were recorded continuously during the growing seasons.Neither site preparation methods nor planting depth affected oak seedling growth, when planting was carried out on fresh clearcuts or in shelterwoods, while growth of beech seedlings was positively affected by mounding. Growth of oak seedlings was inhibited by the shelterwood treatments. In beech seedlings, growth was lowest in dense shelterwood, while there was no difference in growth between seedlings on the clearcut area and in the shelterwood of low density. Differences in growth may be explained by differences in radiation and soil water potential.When planting was carried out on a one-year-old clearcut, site preparation improved the subsequent growth of oak and beech seedlings.     

Mulch and hexazinone herbicide shorten the time longleaf pine seedlings are in the grass stage and increase height growth

Herbaceous plant control with mulchor hexazinone herbicide influenced planted longleafpine (Pinus palustris Mill.) seedling totalheight on a silt loam site in central Louisiana. Thesite had been sheared and windrowed in 1991 and rotarymowed before three treatments were established in arandomized complete block design: (1) Untreatedcheck: no herbaceous plant control after planting;(2) Five mulches: on each plot, five randomlyassigned mulches were placed around seedlings; themulches were either a mat of cotton, hemlock andpolyester, pine straw, woven polypropylene, orperforated polyethylene; and (3) Hexazinone: theherbicide hexazinone at 1.12 kg active ingredient/hawas annually sprayed in the first two growing seasonsover the rows of unshielded seedlings. The longleafseedlings were planted in February 1993.After three growing seasons, seedlings on the mulchand hexazinone treatments were taller than those onthe check plots. About 59% of the mulched andhexazinone treated seedlings had grown out of thegrass stage (at least 12 cm tall) compared to 17% ofthe check seedlings. After five growing seasons, thepercentage of longleaf pine seedlings out of the grassstage was similar on all treatments and averaged 87%. However, these better growing pines were taller on themulch and hexazinone treatments (a 142-cm average)than on the checks (78 cm). Pine straw was anineffective mulch probably because the straw smotheredthe seedlings. The longleaf saplings were tallestwhen the perforated polyethylene mat was used.