A New Micropropagation Technology of Tilia amurensis:In VitroMicropropagation of Mature Zygotic Embryos and the Establishment of a PlantRegeneration System

Tilia amurensis is an economically valuable broadleaf tree species in Northeast China.The production of highqualityT.amurensis varieties at commercial scales has been greatly limited by the low germination rates.Thereis thus a pressing need to develop an organogenesis protocol for in vitro propagation of T.amurensis to alleviate ashortage of high-quality T.amurensis seedlings.Here,we established a rapid in vitro propagation system forT.amurensis from mature zygotic embryos and analyzed the effects of plant growth regulators and culture mediain different stages.We found that Woody plant medium(WPM)was the optimal primary culture medium formature zygotic embryos.The highest callus induction percentage(68.76%)and number of axillary buds induced(3.2)were obtained in WPM+0.89μmol/L 6-benzyladenine(6-BA)+0.46μmol/L kinetin(KT)+0.25μmol/Lindole-3-butryic acid(IBA)+1.44μmol/L gibberellin A_(3)(GA_(3)).The multiple shoot bud development achievedthe highest percentage(83.32%)in the Murashige and Skoog(MS)+2.22μmol/L 6-BA+0.25μmol/L IBA+1.44μmol/L GA_(3).The rooting percentage(96.70%)was highest in 1/2 MS medium+1.48μmol/L IBA.Thesurvival percentage of transplanting plantlets was 82.22%in soil:vermiculite:perlite(5:3:1).Our study is the firstto establish an effective organogenesis protocol for T.amurensis using mature zygotic embryos.

Sulfated Sterols Isolated from Starfish Asterias amurensis Lutken

Aim To study the chemical constituents of starfish Asterias amurensis. Methods The constituents were separated and purified by different chromatographic methods, and their structures were elucidated by MS and NMR. Results Six compounds were isolated from Asterias amurensis Lutken. Their structures were identified as 3β-O-sulfated-cholest-5-en sodium salt （1）, 3β-O-sulfated-6α-ol- pregn-9（ 11 ） -en-20-one sodium salt （ 2 ）, 3β-O-sulfated-6α-ol-cholest-9 （ 11 ） -en-23-one sodium salt （3）, 3β-O-sulfated-6α, 20β-diol-cholest-9 （ 11 ）-en-23-one sodium salt （ 4 ）, 3β-O-sulfated-6α-ol- cholesta-9 （ 11 ）, 20 （ 22 ） -dien-23-one sodium salt （ 5 ）, and 3β-O-sulfated-6ct-ol-ergost-9 （ 11 ） -en-23- one sodium salt （6）. Conclusion Compounds 1 - 6 were obtained from this species for the first time.

Optimal ultrasonic extraction condition and determination of polysaccharides in Tilia amurensis flowers

Seven extracting temperatures （25, 35, 45, 55, 65, 75, 85℃） and extracting time （30, 40, 50, 60, 70, 80, 90 min） were designed for selecting the optimal extracting time and extracting temperature for ultrasonic extraction of T. amurensis flowers. Polysaccharides from T. amurensis flowers were isolated and determined by spectrophotometry. Results show that the optimal ultrasonic temperature was 75℃ and ex tracting time 52 min. The content of polysaccharides in T. amurensis flowers measured by anthrone--H2SO4 colorimetry under 580 nm, was 9.74% with 0.47% of relative standard deviation （RSD, n=3）. This study demonstrated that ultrasonic extraction method was simple, and the color of the treated samples was stable in 4 h. The average recovery value for the polysaccharides measured was 99.48%±1.01%, with 0.112% of RSD （n=3）.

土壤理化性质对暴马丁香(Syringa reticulata var.amurensis)天然林林分因子的影响

通过对处于不同坡位的暴马丁香（Syringa reticulata var.amurensis）天然林的土壤理化性质及林分因子的研究,探讨了暴马丁香天然林的林分特征及不同土壤理化性质对林分因子的影响。认为：在不同坡位样地中,处于坡中位置的暴马丁香林分因子占优势地位;土壤全磷含量、全钾含量、速效钾含量及有效磷含量对暴马丁香各林分因子有显著相关性。

黑龙江草蜥(Takydromus amurensis)消化道嗜银细胞的研究

为了研究黑龙江草蜥(Takydromus amurensis)消化道嗜银细胞,试验采用Grimelius银染法,对黑龙江草蜥消化道嗜银细胞的分布、密度及形态进行观察。结果表明:嗜银细胞分布于从食管至直肠的消化道全长,细胞形态多样,有圆形、椭圆形、锥体形等,分布于消化道黏膜上皮细胞基部、上皮细胞之间以及腺泡上皮细胞之间,密度分布高峰位于胃体及幽门,根据其形态认为黑龙江草蜥消化道嗜银细胞具有内分泌、外分泌功能。

Comparative metabolic profiling of Vitis amurensis and Vitis vinifera during cold acclimation

Vitis amurensis is a wild Vitis plant that can withstand extreme cold temperatures.However,the accumulation of metabolites during cold acclimation(CA)in V.amurensis remains largely unknown.In this study,plantlets of V.amurensis and V.vinifera cv.Muscat of Hamburg were treated at 4℃ for 24 and 72 h,and changes of metabolites in leaves were detected by gas chromatography coupled with time-of-flight mass spectrometry.Most of the identified metabolites,including carbohydrates,amino acids,and organic acids,accumulated in the two types of grape after CA.Galactinol,raffinose,fructose,mannose,glycine,and ascorbate were continuously induced by cold in V.amurensis,but not in Muscat of Hamburg.Twelve metabolites,including isoleucine,valine,proline,2-oxoglutarate,and putrescine,increased in V.amurensis during CA.More galactinol,ascorbate,2-oxoglutarate,and putrescine,accumulated in V.amurensis,but not in Muscat of Hamburg,during CA,which may be responsible for the excellent cold tolerance in V.amurensis.The expression levels of the genes encodingβ-amylase(BAMY),galactinol synthase(GolS),and raffinose synthase(RafS)were evaluated by quantitative reverse transcription-PCR.The expression BAMY(VIT_02s0012 g00170)and RafS(VIT_05s0077 g00840)were primarily responsible for the accumulation of maltose and raffinose,respectively.The accumulation of galactinol was attributed to different members of GolS in the two grapes.In conclusion,these results show the inherent differences in metabolites between V.amurensis and V.vinifera under CA.

Ectomycorrhizal communities associated with Tilia amurensis trees in natural versus urban forests of Heilongjiang in northeast China

The deciduous linden tree （Tilia amurensis Rupr.） is protected at National Level II in China as a species of ecological and economic importance. The objective of this study was to assess and compare the ectomycorrhizal communities associated with T. amurensis in natural versus urban forests of central Heilongjiang Province. The percentage of T. amurensis colonisation by ectomycorrhiza was more than 60 % in urban forests, compared to 34-49 % in natural forests. Use of a combi- nation of morphological and molecular methods docu- mented 18 ECM （ectomycorrhizal） types among three sites; 12-13 ECM species were identified in the natural sites versus 9 species in the urban site. Four ECM species （Boletus sp., Tuber sp., Inocybe sp.2, Leccinum sp.1） were the dominant mycorrhizal symbionts, and Cenococcum geophilum and Russula sp. were found only in the natural forests.

Synthesis of Amurensin H, a New Resveratrol Dimer from the Roots of Vitis Amurensis

Amurensin H(1) is a new resveratrol dimer isolated from the roots of Vitis amurensis Rupr. Its structure was determined by spectroscopic methods. II was synthesized from resveratrol with an oxidative coupling reaction as a key step.

Comparison of Lipids in Organs of the Starfish Asterias amurensis Associated with Different Treatments

Lipids were extracted from organs of the starfish Asterias amurens＆ associated with different treatments （raw-control, boiling and heating）, and then analyzed for lipid content, lipid oxidation index, lipid classes and fatty acid composition. Results showed that boiling softened the hard starfish shells, thus facilitating the collection of starfish organs. As compared with raw organs, the boiled organs had lower water content and higher lipid content, possibly due to the loss of water-holding capacity caused by pro- tein denaturation. Both boiling and heating increased the peroxide value （PV）, thiobarbituric acid （TBA） value and carbon value （CV） of lipids. Despite slight increases in the content of complex lipids, associated lipid composition had no substantial variations upon boiling and heating. For simple lipids, the content of 1, 2-diglyceride decreased in boiled and heated organs, with free fatty acids observed on thin layer chromatography （TLC）. However, neither boiling nor heating significantly changed the fatty acid composi- tions of simple or complex lipids in starfish organs, suggesting that these two treatments had no significant effects on complex lipids in starfish organs. Together, our results indicated that boiling of starfish soon after capture facilitated the handling and extraction of useful complex lipids consisting of abundant glucosylceramide and eicosapentaenoic acid （EPA）-bounded phospholipids.

Expression of structural genes related to anthocyanin biosynthesis of Vitis amurensis

This research was designed to assess the changes in anthocyanin content in grape skins of Vitis amurensis and to explore m RNA transcriptions of 11 structural genes（PAL,CHS3, CHI1, F3H2, F30 H, F3050 H, DFR, LDOX, UFGT,OMT and GST） related to anthocyanin biosynthesis during grape berry development, by the use of HPLC-MS/MS and real-time Q-PCR analysis. Accumulation of anthocyanins began at veraison, continued throughout the later berry development and reached a peak at maturity. Veraison is the time when the berries turn from green to purple. Expression of PAL, CHI1, and LDOX were up-regulated from 2 to4 weeks after flowering（WAF）, down-regulated from6 WAF to veraison, whereas DFR was up-regulated at8 WAF, and then up-regulated from veraison to maturity.CHS3, F3050 H, UFGT, GST, and OMT were down-regulated from 2 WAF to veraison, and then up-regulated from veraison to maturity. The transcriptional expressions of the11 structural genes also showed positive correlations with the anthocyanin content from veraison to maturity. Positive correlations were also observed between OMT transcriptional level and the content of methoxyl-anthocyanins, and between F3050 H transcriptional level and the content of delphinidin anthocyanins. F3H2 and F30 H expression was up-regulated at 2 WAF. F3H2 expression was down-regulated from 4 WAF to veraison and then up-regulated again from veraison to maturity. F30 H expression was down-regulated at 4 WAF and then up-regulated again from 6 WAF to maturity. F30 H transcriptional level was correlated positively with the cyanidin anthocyanin concentration from veraison to maturity. These results indicate that the onset of anthocyanin synthesis during berry development coincides with a coordinated increase in the expression of a number of genes in the anthocyanin biosynthetic pathway.

A New Oligostilbene from the Roots of Vitis Amurensis

A new resveratrol trimer. amurensin G (1), was isolated from the roots of Vitis amurensis Rupr. Its structure and relative configuration were established on the basis of spectral evidence. especially on HMBC spectrum and NOE difference experiments.

Expressed sequence tags (ESTs) analysis of the ripening Vitis amurensis cv. Shuang Hong berry skins

Vitis amurensis is a valuable resource for wine production. Ripening of the grape berry is the key phase which determines the com- position of wine. To better understand the gene expression that manifest in V. amurensis berry skins during the ripening, cDNA library of V. amurensis berry skins was constructed. A total of 935 high quality ex- pressed sequence tags （ESTs） were obtained from the library. These ESTs represent 636 unigenes, including 108 contigs and 528 singletons. The EST analysis was performed and genes were assigned to functional categories according to their primary BLAST match. Of these 25.35% were involved with metabolism, 6.27% with cell rescue and defense, 6.84% energy, 11.68% protein synthesis, 18.8% protein activity regula- tion, 11.11% cell structure, 7.98% transport, 6.27% transcription and the remaining 5.7% were signal transduction. The generated ESTs were characterized by the gene ontology analysis and were categorized ac- cording to its cellular component, molecular function and biological process. In the cDNA library, some genes are relevant to the biosynthesis of anthocyanins, while some genes are related to grape berry maturation.

Isolation and expression analysis of CBF4 from <i>Vitis amurensis</i>associated with stress

The C-repeat Binding Factor/Dehydration Responsive Element Binding factor (CBF/DREB) responsive pathway is involved in plant response to abiotic stress. In this study, we report the isolation of VaCBF4 (a complete cDNA) and its promoter from Vitis amurensis through rapid amplification of cDNA ends and genome walking techniques, respectively. The CBF4 transcript accumulation of V. amurensis increased under cold, salinity, and abscisic acid (ABA) and salicylic acid (SA) treatments, whereas that of Vitis vinifera showed a different change. The transcript levels of VaCBF4 inthe roots, stems, leaves, and petioles under cold, salinity, and ABA and SA treatments were up-regulated in a timedependent manner. The presence of the cis-elements MBC, ABRE, and as-2-box in the VaCBF4 promoter further confirmed that this promoter is a component of the CBF transduction pathway, which is involved in plant response to multistress.

Isolation and expression characterization of CBF2 in <i>vitis amurensis</i>with stress

The transcription factor VaCBF2, which interacts with C-repeat/DRE and its promoter, was isolated from Vitis amurensis. The VaCBF2 amino acid sequence contained a conserved AP2 domain of 56 amino acids and a potential nuclear localization sequence. The sequence of VaCBF2 showed a high level of homology with other CBF2 family members. Phylogenetic analysis showed that the amino acid sequences may be CBF2 proteins with evolutionary relationship. Quantitative reverse-transcription polymerase chain reaction analysis indicated that the expression of VaCBF2 gene in tissues (roots, stems, leaves, and petioles) was induced by low temperature, high salinity, and application of abscisic acid and salicylic acid in a time-dependent manner but to different extents in the cold-hardy V. amurensis and the less cold-hardy Vitis vinifera. The presence of cis-elements such as MYC and ABRE in VaCBF2 promoter further confirmed that this promoter was a component of the CBF transduction pathway involved in plant response to multiple stresses.

The development of the region between the preexisting and nascent membranes during the first cleavage of Rana amurensis eggs

The region between the preexisting and nascentmembranes of a cleaving Rana egg is a dense protrusionregion of nearly 40μm wide at 180°stage.Later,thisregion differentiate,into an upper part,a strip withlong, branched and randomly distributed protrusionswhich are derived largely from the preexistingmembrane,and a lower part,a band with microvilli.During 4-and 8-cel1 stages,the strip is almost vanishedand microvilli in the band is shortened.The nascentmembrane is smooth at the 180°stage,then a rough areaappears below the smooth region and quickly expands.Wheat germ agglutinin which can bind to preexistingmembrane interrupts the development of the regionbetween the preexisting and nascent membranes.Detergent,Brij,having the property to increase the areaof nascent membrane,does not interrupt the developmentof the region between the preexisting and nascentmembranes.

Biomass allocation and assimilation efficiency of natural Tilia amurensis samplings in response to different light regimes

Biomass allocation and assimilation efficiency of natural Amour linden (Tilia amurensis) samplings in different light regimes were analyzed in the paper. The results showed that shoot increment of samplings in gap was the highest and that of samplings under canopy was the least. Samplings in gap expressed apical dominance strongly but samplings in full sun and under canopy behaved intensive branching. Lateral competition or moderate shading was favored to bole construction. The patters of biomass allocation of samplings in different light environment were rather similar. The biomass translocated to stem was more than that to other organs, and about one half of photosynthate was used to support leaf turn over. On the contrary, photosynthates of samplings in full sun were mostly consumed in leaves bearing and energy balancing. The carbon assimilation for leaves of samplings in gap was the most efficient, and more carbons were fixed and translocated to non-photosynthetic organs, especially to stemwood.

Laser scanning fluorescence microscopic measurement of the movement of cleaving egg surface of Rana Amurensis

By laser scanning fluorescence microscopy for quan-titative measurement of fluorescence intensity changes on egg surface stained with fluorescein isothiocyanate duxing cleavage furrow extending forward, it was found that in area of presumptive cleavage furrow the scanning curve became ∨ shape, indicating dark stripe appeared in that place. Then the fluorescence intensity increased at the place where the botton of ∨ shape had located, and the scanning curve tuxned to ∧ shape, indicating single stripe was formed. While enhanced fluorescence appeared on the borders of ∧ shape, an M shape curve was found, show-ing double stripe occurred. During the distance between two borders of M shape incresing from 50 μm to 100μm,a fluorescence peak came to sight in the middle of the M shape, which being the cleavge furrow bottom. The two lateral sides of furrow bottom with decreasing fluorescence were nascent membrane. At that time the curve became W shape. By the sides of cleavage furrow the the stress folds became conspicous after double stripe stage, showing the stretching of the egg surface being increased. With our[31, 33] and others[32] reports that polylysine could induce the appearance of nascent membrane and phyto-hemagglutinins could decrease or prevent the appearance of nascent membrane, we believed the idea of Schroeder[25] that increasing mechanical stress could initiate nascent membrane formation and thought that the stress lay to the outsides of cleavage furrow.

Prehibernation Energy Storage in Heilongjiang Brown Frogs(Rana amurensis) from Five Populations in North China

Energy storage is an important component in the life history of species that directly inlfuences survival and reproduction. The energetic demands of amphibian reproduction can differ between the sexes, with environmental conditions, reproductive pattern or process of the species, and depending upon the timing of breeding, and the reproductive season for a species. Surprisingly, comparative studies of pre-hibernation energy storage for anuran populations from different latitudes are relatively few in Asia, especially in China. Here we investigated the patterns of pre-hibernation energy storage of Heilongjiang brown frogsRana amurensis, based on ifve populations along a ifnely latitudinal gradient in north China （40.7–43.7°N）. We found that pre-hibernation energy storage of the frogs did not show a clear latitudinal cline, but differed strongly between the sexes, with males depositing more energy reserves into the muscle and liver, whereas females accumulate more energy in the gonads. The sexual differences in energy storage may result from differential timing of energy allocation for reproduction.

Genetic diversity and population structure of the sea star Asterias amurensis in the northern coast of China

The sea star Asterias amurensis is widely viewed as a severe“marine pest”because of its broad feeding habits.Over the past few decades,A.amurensis undergoes massive and sporadic population outbreaks worldwide,causing extensive economic and ecological losses to the local aquaculture industry and marine ecosystem.Understanding the genetic diversity and population structure of A.amurensis can provide vital information for resource management.By analyzing the polymorphism of the mitochondrial cytochrome C oxidase subunit I(COI)gene and ten simple sequence repeat(SSR)microsatellites markers,the genetic diversity and population structure of A.amurensis of four populations along the northern coast of China was uncovered.A total of 36 haplotypes were identified,and a main haplotype was found in four populations.The Qingdao(QD)population displayed the highest genetic diversity among all the populations.The AMOVA and pairwise F_(st)showed that there was small but statistically significant population differentiation among the four populations,especially between QD and Weihai(WH).Moreover,the principal component analysis(PCA)and admixture analysis showed that several individuals in Yantai(YT)and Dalian(DL)had little genetic association with other individuals.Overall,this study provided useful information of the genetic diversity and population structure of A.amurensis and will contribute to the resource management of A.amurensis in China.

Fallen wood decomposition of Pinus koraiensis and Tilia amurensis

Fallen wood decomposition rate ofPinus koraiensis andTilia amurensis in broadleaved Korean pine forest was studied in this paper. The result showed that decomposition rate of fallen wood was different from that of little diameter wood and coarse woody debris for the same tree species. Fallen wood decomposition was generally rotten from outside to inside. And decomposition speed of fallen woods was different according to tree species and site, and it was also related to diameter of fallen woods. Decomposition depth ofTilia amurensis fallen wood for 17 years was 14 cm, but that ofPinus koraiensis in the same condition was less than 7 cm.Tilia amurensis was decomposed faster thanPinus koraiensis. For same tree species, if the diameter was small, the decomposition speed was quick.