Lignin Characterization of Triploid Clones of Populus tomentosa Carr.

In order to understand the structural characteristics of lignin in triploid clones ofPopulus tomentosa and its changes in the processes of pulping and bleaching, milled wood lignin （MWL）, lignin carbohydrate complex （LCC） and the residual lignin from kraft pulp （KP） and sulfite pulp （SP） were isolated and analyzed by Fourier transform infrared （FTIR） spectrum and ^13C nuclear magnetic resonance （NMR）. The most diagnostic peaks were assigned and the differences were discussed. The spectral patterns reveal that triploid P tomentosa shows the specific features of hardwood from temperate areas, but in the spectrum of FTIR, the strength ratio orAl270 cm^-1 to A1226 cm^-1 is 0.88, higher than the average of hardwood from temperate areas, which will make the lignin delignification more difficult during pulping and bleaching. The LCC from triploid P tomentosa is mainly composed of xyloglucan and glucuronic acid, and other glucides have much lower ratio. In LCC FTIR, there are three peaks at 1 427, 1 329 and 1 046 cm^-1, indicating that both semi-cellulose and cellulose could exist in LCC, and that there might be relationships between cellulose and lignin. Compared with the residual lignin from KP and SP, the condensed structure in KP is more than that in SP.

THE FIBER MORPHOLOGY VARIATION OF TRIPLOID CLONE OF POPULUS TOMENTOSA CARR.AND ITS SUPPOSED HARVESTING AGE FOR THE PAPER INDUSTRY

The fiber morphology variation of triploid clone of Populus tomentosa Carr. and the supposed harvesting age for the paper industry were investigated in this paper. The results shows that in the first few years, 2-4 years for example, the fiber length at breast high grows faster than it does in the later years. But the standard deviation of distribution in fiber length at breast high in mature wood is larger than in the young tree. The technologically harvestable age of triploid clone of Populus tomentosa Carr., BL304 could be 4-7 year old to meet the requirement for paper industry. The distribution of fiber length become concentrated within an annual ring as the tree grows. The test results also show that the cloned poplar has a small Runkel ratio value (less than 0.4, generally about 0.3), and this ratio increases slightly with the tree grows.

Variation of Leaf Characteristics in Populus tomentosa Carr

An investigation was conducted to determine the extent of variations among nine provenances of Populus tomentosa Carr. in terms of leaf characteristics. A total of 263 accessions were studied under field conditions in the National Gene Bank of P. tomentosa in 2003. All of the accessions were characterized by 17 indices from 1 to 2-dimension constructions. Variance analysis of all characteristics showed that there were significant differences among the nine provenances and among individuals within each provenance. This study reveals that the evaluated germplasm appears to have a wide genetic base and high potential for further genetic improvements and it also indicates that abundant gene resources of P. tomentosa have been collected and preserved in the National Gene Bank.

Model of leaf energy distribution and its experimental validation of Populus tomentosa Carr

Leaf temperature of a plant is the result of heat transfer between the plant and its environment. There are many factors that can affect leaf temperature, such as the solar radiation energy, environmental temperature, wind velocity, evaporation on the leaf surface, photosynthesis, respiration and so on, which have different effects on the temperature of leaves. In first instance, we analyzed the heat transfer on leaves of Populus tomentosa Carr. theoretically and constructed a model of energy distribution. We then validated the model by analyzing seven different kinds of one-year-old P. tomentosa leaves experimentally. The result shows that solar radiation is the main energy input and the dominant ways of thermal diffusion are heat transfer between the upper and lower leaf surfaces and evaporation from the leaf surface.

Duplication of Locus Coding of Malate Dehydrogenase in Populus tomentosa Carr

Horizontal starch-gel electrophoresis was used to study crude enzyme extraction from young leaves of 234 clones of Populus tomentosa Cart. selected from nine provenances in North China. Ten enzyme systems were resolved. One hundred and fifty-six clones showing unusual allozyme band patterns at locus Mdh-Ⅰ were found. Three allozyme bands at locus Mdh-Ⅰ were 9：6：1 in concentration. Further studies on the electrophoretic patterns of ground mixed pollen extraction of 30 male clones selected at random from the 156 clones were conducted and it was found that allozyme bands at locus Mdh-Ⅰ were composed of two dark-stained bands and a weak band. Only one group of the malate dehydrogenase （MDH） zymogram composed of two bands was obtained from the electrophoretic segregation of pollen leachate of the same clones. A comparison of the electrophoretic patterns one another suggested that the locus Mdh-Ⅰ coding malate dehydrogenase in diploid species of P. tomentosa was duplicated. The duplicate gene locus possessed three same alleles and was located in mitochondria. The locus duplication of alleles coding malate dehydrogenase in P.tomentosa was discovered and reported for the first time.

Establishment of cell suspension line of Populus tomentosa Carr

Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L^-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subcultured into a MS liquid medium supplemented with 1.5 mg·L^-1 2,4-D. The best subculture medium was MS ＋ 0.8 mg＇L-1 2,4-D ＋ 30 g·L^-1 sucrose with a subculture cycle of seven days.

Incidence and molecular markers of 2n pollen in Populus tomentosa Carr

Microscopic examination and AFLP molecular markers were employed to determine the incidence and related markers of 2n pollen (unreduced pollen) in Populus tomentosa Carr. The parallel and tripolar spindle at metaphase Ⅱ and the absence of cytokinesis at telophase Ⅱ led to the formation of 2n pollen. A group of 298 clones came from their indigenous areas was investigated for the production of 2n pollen based on the pollen size differences, within a clone and between n and 2n pollen. Pollen grains of 224 clones were collected, six of them only produced normal pollen, and the rest produced 2n pollen at different frequencies (0.6% -21.9%). Clones producing six normal and 22 2n pollen were selected for AFLP analysis. Following an initial screening with 55 primer combinations, the E50-M38 (CAT/ACT) primer was identified, which generated a PCR fragment (246 bp) from the normal clones, but not from the 2n pollen producers. In addition, the E31-M50 (AAA/CAT) amplified DNA fragment (204 bp) that was present in 2n pollen producers, and absent in normal clones. Two polymorphic bands were found and they were distinguished between normal and 2n pollen clones. They are very useful as AFLP markers for molecular-assisted selection in triploid breeding of 2n gametes in P.tomentosa.

Comparison and early selection of new clones in Populus tomentosa

In our study, two experimental plantations, respectively, with 24 and 32 new clones of P tomentosa, were established in Weixian County, Hebei Province and Wuzhi County, Henan Province using a completely randomized block design. A comparative study was conducted on the continuous 5-year-old height and diameter at breast height （DBH） of new clones in the two plantations. As well, based on genetic correlation over the years of testing of these clones, a preliminary study of early selection was carried out. Results indicate that the growth traits of the new clones in Weixian were better than those in Wuzhi. The traits show weak correlation between the two plantations. In some stands, the height, DBH and seedling volume of 5-year-old clones presented statistically significant differences among clones. In both plantations, the new clones showed over 0.6 repeatability of height, DBH and volume, as well as larger coefficients of variation （CV）. The fact that these clones achieved the largest repeatability and CV in the second year suggests that these traits are highly controlled by heredity. Thus, based on the growth traits of the second year, the new clones B305, B307, B303, H75, BT18, BT17 and 21J-1 were considered suitable in Weixian. In Wuzhi, the new clones had variable repeatability and CVs in various years and their correlation of growth traits among different years was not high. We conclude that early selection of new clones was not feasible in Wuzhi.

Improvement in salt tolerance of Populus tomentosa from transformation by a mtl-D gene

Transgenic lines were achieved by transforming the E. coli 1-phosphate mannitol dehydrogenase gene （mtl-D） into the Populus tomentosa Cart. genome. An Agrobacterium tumefaciens strain （AGL1）, constructed by cloning mtl-D into the disarmed plasmid pBin438, was used to infect leaves of the clone YW2. The infected leaf discs were cultured on a medium containing 30 mg.L 1 kanamycin and 500 mg.L 1 cefotaxime. Transgenic plantlets regenerated from the infected leaves, rooted on the medium con- taining 30 mg.L 1 kanamycin. PCR and a Southern blotting test verified that the exogenous mtl-D gene had integrated into the trans- formation plants of the P. tomentosa genome. The mannitol content in control plant was 69 gg.gl FW, and the mannitol contents of the transgenic lines T1 to T5 ranged between 103.7 and 289.5 μg·g^-1 FW. Of the shoots of the control plants 20% survived; on the medium containing 0.6% NaCl, 60% and 70% of two transgenic shoots survived on a medium containing 0.8% NaCI.

In vitro regeneration of Populus tomentosa from petioles

A reliable in vitro regeneration procedure for Populus tomentosa is a prerequisite for its trait improvement through genetic transformation. We established a systematic protocol for indirect regeneration of P. tomentosa using in vitro petioles of Chinese poplar cultivar ＇fasta-3＇. A high frequency of callus induction （〉97 %） was obtained from isolated petioles cultured on the modified 1/2MS basal medium supplemented with 0.5 mg/L ZT and 1.0 mg/L NAA, and the tested calli were subsequently plated on 1/2MS basal medium supplemented with 0.25 mg/L BA, 0.25 mg/L ZT, 0.25 mg/L NAA, 0.01 mg/L TDZ, and 0.5 mg/L KT for efficient regeneration of shoots after being cultured for 6 weeks. The regenerated shoots were vigorously rooted on the tested media supplemented with 1.0 mg/ L IBA and 0.5 mg/L NAA. These results can facilitate genetic transformation of P. tomentosa for trait improvements in future.

Selection of female parents with high fertility and high combining abilities for cross-breeding Populus tomentosa

To select maternal parents with high fertility and high combining ability during cross breeding of Populus tomentosa Carr., the ploidy level of 75 female clones was determined using flow cytometry. In addition, genetic variations and phenotypic correlations of seed traits and early growth traits, as well as the general combining ability(GCA) for seedling height(SH) and basal diameter(BD) of all diploid half-sib families were examined. A total of 26 natural triploid clones were identified. For all diploid families, family effects were significant for inflorescence length(IL), thousand kernel weight(TKW), seedling emergence rate(SER), and for SH and BD. There was a positive correlation between TKW and other traits,excluding IL. The seed-bearing coefficient(NS 9 SER)ranged from zero to 32.4%. Clones 3-10-2, 2-8, 3119,3206, and 3-10-1 had the best performance of the GCA for SH and BD. Based on the fertility and GCA for SH and BD, clone 3-10-2 and clone 3119 could be used as female parents for controlled cross-breeding of P. tomentosa.

Genome-wide search for segregation distortion loci associated with the expression of complex traits in Populus tomentosa

Segregation distortion of molecular markers has been reported in a broad range of organisms. It has been detected in an interspecific BC1 Populus pedigree established by controlled crossing between clone ＂LM50＂ （Populus tomentosa） and its hybrid clone ＂TB01＂ （P. tomentosa × p. bolleana）. The study with a total of 150 AFLP markers （approximately 18.9% of the total loci） exhibited significant deviation from the Mendelian ratio （1：1） （p〈0.01）. Twenty-five percent of the markers were mapped on the parental specific genetic linkage maps of clones ＂LM50＂ and ＂TB01＂ with a pseudo-test-cross mapping strategy. Twelve linkage groups had markers with skewed segregation ratios, but the major regions were on linkage groups TLG2, TLG4 and TLG6 in the linkage map of clone ＂LM50＂. We also analyzed the association between distorted loci and expression of complex traits with Mapmaker/QTL software. A total of 16 putative QTLs affecting 12 traits were identified in the distorted regions on seven linkage groups. Therefore we could detect the distribution of skewed loci along the entire genome and identify the association between quantitative traits and segregation loci via genetic mapping in an interspecific BC1 P. tomentosa family. Furthermore, the genetic nature and possible causes of these segregation distortions for differentiation between female and male parents were also discussed.

Isolation, Crystal Structure, and Anti-inflammatory Activity of Sakuranetin from Populus tomentosa

The title compound of sakuranetin, a main active flavanone component, was first isolated from Populus tomentosa Carr. and characterized by X-ray single-crystal diffraction analysis It crystallizes in the monoclinic lattice, space group P21/c with a = 12.8531（12）, b = 5.7141（3）, c = 18.0355（12） A, β = 97.333（8）°, V = 1313.77（16） A^3, Z= 4, Mr = 286.27, C16H14O5,μ（MoKa） = 0.108 mm^-1, Dc = 1.447 g/cm^3, F（000） = 600, the final R = 0.0350 and wR = 0.0859 for 2571 independent reflections （Rint = 0.0246） which were used in all calculations. The molecular crystal structure of sakuranetin shows relative stereochemistry of 5,4′-dihydroxy-7-methoxyflavanone. Intermolecular hydrogen bonds together with the continuous π-π interactions construct the three-dimensional architecture of the title compound. The preliminary bioassay reveals that the title compound exhibits moderate anti-inflammatory activity in vitro against the nitric oxide release.

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 2629 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.

Successful Agrobacterium-mediated transformation of Populus tomentosa with apple SPDS gene

The problem of salinized soils has become one of the most serious constraints to agricultural and forest productivity. With the purpose of enhancing salt stress tolerance of Populus tomentosa, we transformed this tree species with spermidine synthase （SPDS） genes derived from an apple by an Agrobacterium-mediated method. Four transgenic clones were confu＇med by PCR and Southern blot analysis. As well, the expression of introduced SPDS genes was analyzed by real-time quantitative PCR.

Constitutive Expression of Sense & Antisense PtAP3, an AP3 Homologue Gene of Populus tomentosa, Affects Growth and Flowering Time in Transgenic To-bacco

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.

Molecular Cloning of a Cellulose Synthase Gene PtoCesA1 from Populus tomentosa and Its Genetic Transformation in Tobacco

A 3 125 bp cellulose synthase gene, PtoCesA1, which has a 98% identity to PtrCesA1 from Populus tremuloides, was cloned from cDNA prepared from secondary xylem of P tomentosa. Four anti-expression vectors with different fragments of PtoCesAl, named as pBIPF, pBICC1, pBIPR and pBIBR, were constructed. Some traits of transformed tobacco of pBICC1, pBIPR and pBIBR differed from wild types, such as small leaves, ＂dwarf＂ phenotype and thinner xylem and fiber cell walls than wild plants consistent with a loss of cellulose. It indicated that the growth of transgenic tobacco was restrained by the expression of anti-PtoCesA1. Transgenic tobacco was obtained and the contents of cellulose and lignin were analyzed as well as the width and length of fiber cells, and xylem thickness for both transgenic and control plants. Transformed tobacco showed a different phenotype from control plants and it implied that PtoCesA1 was essential for the cellulose biosynthesis in poplar stems.

Cloning and analysis of a new 4CL-like gene in Populus tomentosa

The phenylpropanoid enzyme 4-coumarate： coenzyme A ligase （4CL）, plays a key role in general phenylpropanoid metabolism. Our investigation involves a new 4CL-like gene cloned from Populus tomentosa. This new 4CL- like gene is 1,692 bp and encodes a protein of 552 aa. After analysis of its domain, we found a highly conserved region of a 4CL gene family in this 4CL-like gene. Based on our results, we believe that this gene belongs to the family of 4CL. A recombinant vector, referred to as pET30a-4CL-like, was constructed by connecting this 4CL-like gene fragment to pET30a. After inducing it with IPTG for 3 h, the 4CL-like protein was purified by a Ni-NTA method. This 4CL-like protein has a calculated molecular weight of 60 kDa by SDS-PAGE.