火炬松(Pinus taeda L.)合子胚愈伤组织的器官发生和植株再生

以火炬松(Pinus taeda L.)的成熟合子胚为外植体在附加NAA和BA的TE培养基上诱导产生了淡黄色、疏松、有光泽的颗粒状愈伤组织。经过愈伤组织的保持和增殖培养及不定芽原基的诱导培养后,进行了不同激素、低温处理和蔗糖浓度对不定芽分化的影响实验。结果表明,在附加0.5 mg·L^(-1)IBA和2 mg·L^(-1)BA的TE培养基上,愈伤组织上的不定芽分化频率最高达62.15%。不定芽分化的最佳低温处理时间是5—6周,最佳蔗糖浓度是25—30 g·L^(-1)。不定芽经伸长培养后取高于1cm的小苗用于生根。在附加IBA、BA和GA_3的TE培养基上不定芽的生很频率最高达46%。

Agrobacterium-mediated transformation and assessment of factors influ-encing transgene expression in loblolly pine (Pinus taeda L.)

This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for β-glucuronidase (GUS) and neomycin phosphotransferase (NPTII). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.

Genetic transformation of Pinus taeda by particle bombardment

A protocol is presented for genetically engineering loblolly pine (Pinus taeda L.) using particle bombardment. This protocol enabled the routine transformation of loblolly pine plants that were previously difficult to transform. Mature zygotic embryos were used to be bombarded and to generate organogenic callus and transgenic regenerated plants. Plasmid pB48.215 DNA contained a synthetic Bacillus thuringiensis (B.t.) cryIAc coding sequence flanked by the double cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (Nos) terminator sequences, and the selectable marker gene, neomycin phosphotransferase II (nptII) controlled by the promoter of the nopaline synthase gene was introduced into loblolly pine tissues by particle bombardment. The transformed tissues were proliferated and selected by kanamycin resistance conferred by the introduced NPTII gene. Shoot regeneration was induced from the kanamycin-resistant callus, and transgenic plantlets were then produced. The presence of the introduced genes in the transgenic loblolly pine plants was confirmed by polymerase chain reactions (PCR) analysis, by Southern blot analysis, and insect feeding assays. The recovered transgenic plants were acclimatized and then established in soil.

Influences of antibiotics on plantlet regeneration via organogenesis in loblolly pine (Pinus taeda L.)

Three antibiotics ampicillin, carbenicillin, and cefotaxime were evaluated for their effects on induction, growth, and differentiation of organogenic calli, as well as rooting of regenerated shoots of three loblolly pine (Pinus taeda L.) genotypes. Of the antibiotics administered, cefotaxime maximally increased the frequency of callus formation and growth rate of organogenic calli, carbenicillin maximally increased the frequency of shoot regeneration and the average number of adventitious shoots per piece of organogenic callus, ampicillin maximally decreased the rooting frequency of regenerated shoots and mean number of roots per regenerated shoot, in comparison with antibiotic-free media. Compared with the control, ampicillin minimally increased the frequency of callus formation, cefotaxime minimally increased the frequency of shoot regeneration, and carbenicillin mini-mally decreased the rooting frequency of regenerated shoots in three loblolly pine genotypes tested. All three antibiotics in-creased the frequencies of callus formation and shoot regeneration, and reduced the rooting frequency of regenerated shoots suggested that the establishment of an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integra-tion of foreign genes into loblolly pine need to select a suitable antibiotic. This investigation could be useful for optimizing genetic transformation of conifers.

Phylogenetic Relationship of an Extremely Endangered Species, Pinus squamata (Pinaceae) Inferred from Four Sequences of the Chloroplast Genome and ITS of the Nuclear Ribosomal DNA

Pinus squamata X. W. Li, a recently-described species of the Pinaceae, is an extremely endangered pine with only 32 individuals in the wild. This species was thought to be intermediate between P. bungeana Zuec. ex Endl. of subgen. Strobus and P. yunnanensis Franch. of subgen. Pinus by the original author. We made an effort to address the question of the phylogenetic relationship of this peculiar and important species in the context of the major groups of the genus Pinus by using sequences of the rbcL, matK,genes, rpl20-rps18 spacer, trnV intron of the chloroplast genome and the nuclear ribosomal ITS region. The results of the separated analysis and the combined analysis of the four cpDNA sequences and ITS sequence indicated that P. squamata was a stable member of subsect. Gerardianae and P. gerardiana Wall. was the closest species of P. squamata phylogenetically. As a result, the causes of the distribution pattern of subsect. Gerardianae were also discussed.

The impact of the environmental factors on the photosynthetic activity of common pine (Pinus sylvestris) in spring and in autumn in the region of Eastern Siberia

The taiga coniferous forests of the Siberian region are the main carbon sinks in the forest ecosystems.Quantitatively, the size of the carbon accumulation is determined by the photosynthetic productivity, which is strongly influenced by environmental factors. As a result,an assessment of the relationship between environmental factors and photosynthetic productivity makes it possible to calculate and even predict carbon sinks in coniferous forests at the regional level. However, at various stages of the vegetative period, the force of the connection between environmental conditions and the productivity of photosynthesis may change. In this research, correlations between the photosynthetic activity of Scots pine(Pinus sylvestris L.) with the environmental conditions were compared in spring and in autumn. In spring, close positive correlation of the maximum daily net photosynthesis was identified with only one environmental factor. For different years, correlations were for soil temperature(rs= 0.655,p = 0.00315) or available soil water supply(rs= 0.892,p = 0.0068). In autumn within different years, significant correlation was shown with two(temperature of air and soil; rs= 0.789 and 0.896, p = 0.00045 and 0.000006,respectively) and four factors: temperature of air(rs=0.749, p = 0.00129) and soil(rs= 0.84, p = 0.00000),available soil water supply(rs= 0.846, p = 0.00013) and irradiance(rs= 0.826, p = 0.000001). Photosynthetic activity has a weaker connection with changes in environmental factors in the spring, as compared to autumn.This is explained by the multidirectional influence of environmental conditions on photosynthesis in this period and by the necessity of earlier photosynthesis onset, despite the unfavorable conditions. This data may be useful for predicting the flow of carbon in dependence on environmental factors in this region in spring and in autumn.

Adaptation of pine wood nematode,Bursaphelenchus xylophilus,early in its interaction with two Pinus species that differ in resistance

Pine wilt disease(PWD)is one of the most devastating diseases of Pinu s spp.and is caused by the pine wood nematode(PWN),Bursaphelenchus xylophilus(Steiner&Buhrer)Nickle.To study adaptation of PWN to survive in hosts that differ in resistance,we examined the self-regulatory characteristics of PWN at the biological and molecular levels early in the interaction.Two-year-old susceptible Pinus thunbergii and resistant Pinus taeda were selected for this experiment,and changes in PWNs after inoculation were assessed.q RT-PCR was used to detect changes in genes related to PWN pathogenicity and detoxification.The results showed that the migration and reproductive abilities of PWNs in P.thunbergii were stronger than those of PWNs in P.taeda.After 7 d,the number of nematodes in P.thunbergii was approximately 3.2-fold higher than in P.taeda.After 15 d,the number of nematodes in P.thunbergii was approximately twofold higher than that in P.taeda.Because PWN can adjust its sex ratio after infection,we compared the sex ratio of uninoculated PWNs,to that in the two pine species.In P.thunbergii,the female to male ratio first decreased and then stabilized over time;in P.taeda first decreased and then increased.Relative fat accumulation in PWNs increased significantly after the PWNs entered the tree body;the accumulation rate in P.thunbergii was higher than in P.taeda at 7 d,but lower after 15 d.Scanning electron microscopy(SEM)showed significantly more bacteria on the surface of PWNs in P.taeda compared with PWNs in susceptible P.thunbergii.At 12 h after inoculation,the expression of genes related to cell-wall degradation(Bx Beta1-4 and Bxpel1),effectors(Bx CDP1,Bx Sap B1),and active oxygen metabolism(Bxy-ctl-1 and Bx GST3)was 2–6×higher in the resistant pine than in the susceptible one.In contrast,in PWNs,the expression of autophagy-related genes Bx ATG1 and Bx ATG16 was 1.5–2 times higher in P.thunbergii than in P.taeda.These results indicate that the interaction between PWNs and pine trees with different resistance levels elicits a series of physiological and molecular adaptations that affect nematode reproduction and virulence.This study will help elucidate the adaptive mechanisms of PWN in different pine trees.

Growth response of broom (Cytisus scoparius) growing with and without radiata pine (Pinus radiata) seedlings to different P levels in soils

A study was carried out to test the effects of three rates of TSP （triple superphosphate） （0, 50, and 100 mg·kg^-1 P） on growth of broom with and without radiata pine seedlings and to determine the relationships between P concentrations in the broom shoot and dry matter yields with soil plant-available P （Bray-2 P）. A bulk sample of soil was collected from Kaweka forest at soil depth of 0-10 cm, in New Zealand on March 11, 2001. The forest area was not supplied with fertiliser at least 30 years. The results show that TSP application increased P avail- ability in the soil. The P availability concentration in soil of broom with radiata pine seedlings was higher than that in soil of broom alone. Bray-2 P concentrations had a significant logarithmic relationship with Pcon- centrations of broom shoot and an exponential relationship with dry matter weight of whole broom plant.

Bio-productivity of Pinus sylvestris L.var. mongolica plantation on west Kerqin sandy land

The biomass and net primary production of Mongolian scotch pine (Pinus sylvestris L. var mongolica) plantations of west Kerqin sandy land were measured. According to average standard trees, the biomass, netprimary production and their distributions of trunk, bark, branch, leaf and root of 16-year-old stand were analyzed.The regressive equation for the estimation of each organ biomass was established through djmensional analysis.Preferable equation with higher precision was selected. The study results showed that the total biomass of theforest community was 62.023 t/hm2 and net primary production was 5.045 V(hm2. a). which indicates that thecommunity of plantation possesses high bio-productivity.

Assessment of early survival and growth of planted Scots pine(Pinus sylvestris) seedlings under extreme continental climate conditions of northern Mongolia

Environmental factors play vital roles in successful plantation and cultivation of tree seedlings.This study focuses on problems associated with reforestation under extreme continental climatic conditions.The objectives were to assess relative seedling performance(survival and growth)with respect to plantation age,and to analyze the influence of specific climatic factors during the early stages of Scots pine(Pinus sylvestris L.)plantations.The study was carried out in reforested areas of the Tujyin Nars region of northern Mongolia on six Scots pine plantations ranging from 5 to 10 years.In each of the six plantations,five 900 m^2 permanent sample plots were established and survival rates and growth performance measured annually over 7 years.Results show high variation in survival among the plantations(p<0.001,F=29.7).Seedling survival in the first year corresponded directly to the number of dry days in May.However,survival rate appeared to stabilize after the second year.The insignificant variation of height categories throughout the observation period indicated low competition among individuals.Two linear mixed-effect models show that height and radial growth were best explained by relative air humidity,which we consider to be a reliable indicator of site-specific water availability.Insufficient amounts and uneven distribution of rainfall pose a major threat during the first year of plantation establishment.Humidity and water availability are decisive factors for a successful seedling plantation.This highlights the impact of drought on forest plantations in northern Mongolia and the importance of developing climate resilient reforestation strategies.

Seasonal Changes in Copper and Cobalt Concentrations of PinusnigraL.,Cedrus libani and Cupressus arizonica Leaves to Monitor the Effects of Pollution in Elazig (Turkey)

The aim of this study is to examine seasonal changes in Cu and Co concentrations of three plant species for monitoring the effects of pollution in Elazig,Turkey.For this purpose,the leaves of the Pinus nigra L.,Cedrus libani and Cupressus arizonica together with soil samples were collected from different points depending on traffic intensity,nearness the city center and cement factory as well as control location during different months of the year.Flame atomic absorption spectrophotometer(FAAS) was used for measurement of the metals in clear digests after the dry ashing method.Copper and Co concentrations were in the ranges from 1.3to 2.6mg.kg-1 and<LOD to 0.26mg.kg-1 for Pinus nigra L.,1.2to 4.7mg.kg-1 and<LOD to 0.41mg.kg-1 for Cedrus libani and 1.5to 4.8mg.kg-1 and<LOD to 0.42mg.kg-1 for Cupressus arizonica,respectively.The levels observed for Cu and Co in the soil ranged from 12to 38mg.kg-1 and 6.0to 17mg.kg-1,respectively.

Transformation and Expression of Choline Monooxygenase(CMO) Gene in Embryogenic Tissue of White Pine(Pinus strobus)

A transformation procedure of choline monooxygenase(CMO) gene, involved in stress tolerance, was established in white pine embryogenic tissue by using A. tumefaciens C58/pMP90. The CMO cDNA fragment(1.3 kb) was generated by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) with primers based on the report sequence of CMO in gene bank. A chimerical gene composed of the cauliflower mosaic virus (CaMV) 35S promoter fused to CMO cDNA and β-glucuronidase (GUS-marker gene) was transferred into Ti-derived disarmed binary vector pBI121. The new vector, p35SCMOp, was transferred into Agrobacterium tumefaciens C58/pMP90 by freeze-thaw method. Somatic embryogenesis (SE) initiation of Pinus. Strobus L. and Pinus.Koraiensis Sieb. et Zucc. depended on the manipulation of plant growth regulator (PGR) concentrations in the GLH culture medium. Transgenic embryos and regenerated plants of two Pine species were produced after co-culture of embryogenic tissue with the disarmed strain of A. tumefaciens C58/pMP90/ p35SCMOp and selected on medium containing 25mg/L kanamycin. The transformed embryogenic tissue was initially confirmed by histochemical GUS assay followed by PCR. One copy of T-DNA was detected by transgenic lines analysis in Pinus. Strobus L. and transgenic plants were regenerated for two species using modified protocols for maturation and germination of somatic embryos.

Microscopic Characterisation of Pinus sylvestris Cell Structures under Compression Loading

This study is mainly focused on the 3D mechanical cell deformations of 20 × 20 × 60 mm sized softwood specimens under 35 - 40 MPa compression loading at room temperature of 20?C. The moisture content of the specimens was 6% - 7%. The data of microscopic images were measured and compared in terms of the permanently degenerated individual cell structures each in micro-scale . 3D cell deformations of tissues were observed with a magnification of (×100) - (×1500) and in the range of 3.0 - 5.0 kV voltage under the SEM microscope. The specimens were examined under magnification and photographed before and after the compression loading applied parallel to the grain angles to the wood samples. Specimens were painted with gold liquid (12 × 12 × 12 mm sized specimens) in obtaining the SEM images. Under the SEM, these specimens were photographed and lengths between the cell walls ranged between 15 to 40 micrometers. In this study, relative deformations of pinewood cells were determined statistically considering the percentage permanent deformation under the compression loading. It was performed by using knowledge of structural mechanics, considering the measurement of permanent deformation in honeycomb-pinewood structure material.

速生抗病火炬松Pinus taeda苗的磁化培育和早期筛选技术研究

研究表明,利用磁场处理火炬松种子能显著提高种子发芽率、苗木抗病性和促进苗高生长,并以浸种后的冷藏期处理效果最好;利用病原菌的毒素粗提液可进行抗病苗的早期筛选。

Enhanced Somatic Embryogenesis and Plant Regeneration from Embryogenic Cultures Derived from Mature Loblolly Pine Zygotic Embryos by Suspension Culture and Medium Selection

Mature zygotic embryos of three families of loblolly pine ( Pinus taeda L.) were cultured on callus induction medium containing 8?mg·L -1 2,4 dichlorophenoxyacetic acid (2,4 D), 4?mg·L -1 6 benzyladenine (BA), 4?mg·L -1 kinetin (KT), 500?mg·L -1 casein hydrolysate, and 500?mg·L -1 glutamine for 9 weeks, callus was formed on cotyledons, hypocotyls, and radicles of mature zygotic embryos. Callus was sub cultured on the callus proliferation medium with 1 6?mg·L -1 2,4 dichlorophenoxyacetic acid (2,4 D), 0 8?mg·L -1 6 benzyladenine (BA), 0 8?mg·L -1 kinetin (KT) for 9 weeks. White translucent, glossy, mucilaginous embryogenic callus containing embryogenic suspensor masses (ESM) and immature somatic embryos was obtained, and the highest frequency of explants forming embryogenic callus was 16 9%. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus proliferation medium. Liquid cultures containing embryogenic suspension masses and immature somatic embryos were transferred to medium containing abscisic acid (ABA), polyethylene glycols (PEG), or activated charcoal for enhancing the production of cotyledonary somatic embryos. After mature somatic embryos were cultured on medium containing indole butyric acid (IBA), gibberellic acid (GA 3), BA, and activated charcoal and being lowered sucrose concentration for 4～12 weeks, somatic embryos germinated to form regenerated plantlets. Seventy one regenerated plantlets were transferred to a perlits∶peatmoss∶vermiculate (1∶1∶1) soil mixture, and 23 plantlets survived in the field.

Sloboda树高生长模型在火炬松人工林中的应用研究

地位指数是立地质量相对于具体树种的数量化反映,也是森林经营和收获预估的重要依据.为了编制福建省火炬松多形地位指数,根据收集的火炬松人工林优势木树干解析资料,采用Sloboda树高生长数学模型,利用遗传算法拟合出火炬松优势高生长模型.精度检验表明:用Sloboda树高生长模型拟合的火炬松人工林多形地位指数曲线可以应用于林业生产实践.

NaCl胁迫下盐松不同种源种子的发芽特性

研究从国外引进的盐松种子的耐盐发芽特性,用0%、0.25%、0.5%、0.75%、1%和1.25%6种不同浓度的NaCl溶液对9个种源盐松种子进行处理,同时以国内樟子松种子作对照进行发芽试验。结果表明:随着NaCl浓度的增大,各种源种子的发芽率、发芽势和发芽指数均呈下降趋势,初始萌发时间和平均发芽时间呈上升趋势。不同种源种子的萌发对NaCl胁迫的反应不同,引种盐松种子的耐盐能力优于国内樟子松。聚类分析表明:耐盐能力最好的种源是754、N8和17245。

Studies on Parameters Affecting Embryogenesis of Protoplasts Isolated from Suspension of Embryogenic Cells in Loblolly Pine

Protoplasts of embryogenic suspension cells of loblolly pine (Pinus taeda L).were isolated at exponential growth stage.Influences of various concentrations of basal medium,levels of BA,and concentrations of inositol on the differentiation of embryonal suspensor mass (ESM),early stage somatic embryos (ESE) ,and lae stage somatic embryos (LSE) were investigated .A study of the effect of various concentrations of LP basal medium sowed that the optimal basal medium concentration of ESM,ESE,and LSE differentiation was 1.25 LP medium.The effects of various levels of BA and inositol showed that the optimal concentrations of BA for the formation of ESM,ESE and LSE were 4 mg/L ,2mg/L and 1mg/L,respectively ,and the optimal concentrations of inositol for the ESM ,ESE and LSM formation were 400mg/L,800mg/L and 1,200mg/L,respectively.

Regeneration of transgenic loblolly pine expressing genes for salt tolerance

Salinity stress is one of the most serious factors limiting the distribution and productivity of crops and forest trees. The detrimental effects of salt on plants are a consequence of both a water deficit resulting in osmotic stress and the effects of excess sodium ions on critical biochemical process. A novel approach to improve salt tolerance has been established by using the technology of plant genetic transformation and using loblolly pine (Pinus taeda L.) as a model plant. Mature zygotic embryos of loblolly pine were infected with Agrobacterium tumefaciens strain LBA 4404 harbouring the plasmid pBIGM which carrying the mannitol-1-phosphate dehydrogenase (Mt1D) and glucitol-6-phosphate dehydrogenase (GutD). Organogenic transgenic calli and transgenic regenerated plantlets were produced on selection medium containing 15mg/L kanamycin and confirmed by Southern blot analysis of genomic DNA. Salt tolerance assays demonstrated that the salt tolerance of transgenic calli and regenerated plantlets were increased. These results suggested that an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and this could be useful for the future studies on engineering breeding of conifers.

Somatic embryogenesis and peroxidase activity of desiccation tol-erant mature somatic embryos of loblolly pine

White, translucent, glossy mucilaginous callus was initiated from the mature zygotic embryos explants on callus induction medium with 2,4-D, BA, and kinetin in the 3-9th week of culture. This type of callus induction occurred at a lower fre-quency with either a-naphthaleneacetic acid (NAA) or IBA (both 8 mg/L). White, translucent, glossy mucilaginous callus was embryogenic and mainly developed from the cotyledons of the mature zygotic embryo. Somatic embryos were formed on dif-ferentiation medium. Desiccation tolerance can be induced by culturing somatic embryos of loblolly pine (Pinus taeda L.) on medium supplemented with 50 mm abscisic acid (ABA) and/or 8.5% polyethylene glycol (PEG6000). Scanning electron micros-copy of desiccated somatic embryos showed that the size and external morphology of the desiccation tolerant somatic embryos recovered to the pre-desiccation state within 24-36 h, whereas the sensitive somatic embryos did not recover and remained shriveled, after the desiccated somatic embryos had been rehydrated. Peroxidase activity of desiccated somatic embryos in-creased sharply after 3 days of desiccation treatment, and desiccation tolerant somatic embryos had higher peroxidase activity compared to sensitive somatic embryos. Higher peroxidase activity of desiccation tolerant somatic embryos was possibly ad-vantage of catalyzing the reduction of H2O2 which was produced by drought stress, and protecting somatic embryos from oxida-tive damage.