Isolation and General Characterization of Polysaccharides from Tansy Tanacetum vulgare L.

Using extraction with 0.75% aqueous ammonium oxalate, the following polysaccharide fractions were isolated: tanacetans TVF, TVS, and TVR from floscules, sprouts, and roots, respectively, of Tanacetum vulgare L., spread throughout the European North of Russia. The sugar chain of tanacetan TVF consists of D-galacturonic acid (61.4%), arabinose (14.7%), galactose (10.2%), and rhamnose (3.7%) as the main constituents as well as xylose, glucose, mannose, apiose, and 2-O-methylxylose in trace amounts. Tanacetans TVS and TVR were shown to differ in the sugar quantitative composition. They contain 67 and 28% galacturonic acid, respectively. A partial acid hydrolysis of the tanacetan TVF gave a polysaccharide fragment TVF1, -1,4-D-galacturonan (GalA 98.2%). Digestion with pectinase (-1,4-D-polygalacturonase) resulted in fragment TVF3, containing residues of arabinose (27.1%) and galactose (17.3%). NMR spectroscopy allowed detection of the terminal residues of -Araf and -Galp as well as of the residues of -Arap substituted in 3,5- and 5-positions. Thus, tanacetan TVF was proved to be a pectic polysaccharide.     

Structural studies of the pectic polysaccharide from duckweed Lemna minor L

The pectic polysaccharide of duckweed Lemna minor L. termed lemnan (LM) was shown to contain the ramified, "hairy" region. Using partial acid hydrolysis and Smith degradation followed by NMR spectroscopy of the fragments obtained, some structural features of the hairy region of LM were elucidated. Partial acid hydrolysis of LM afforded the crude polysaccharide fraction LMH that was separated into two polysaccharide fractions: LMH-1 and LMH-2. In addition, the oligosaccharide fraction LMH-3 contained 97% D-apiose was obtained from the supernatant. A further more rigorous acidic hydrolysis of LMH led to the crude polysaccharide fraction LMHR which was separated in to two fractions: LMHR-1 and LMHR-2. Smith degradation of LMH afforded the polysaccharide fragment LMHS differed in low contents of apiose residues. Unfortunately, NMR-spectroscopy failed to provide significant evidence concerning the structure of LMH-1 due to the complexity of the macromolecule. The structure of the 1H/13C-NMR spectroscopy including the correlation 2D NMR spectroscopy. As a result, alpha-1,4-D-galactopyranosyluronan was confirmed to be the main constituent of the LM backbone. In addition, the ramified, "hairy" region of the macromolecule appeared to contain segments consisting of residues of terminal and beta-1,5-linked apiofuranose, terminal and alpha-1,5-linked arabinofuranose, terminal and beta-1,3- and beta-1,4- linked galactopyranose, the terminal and beta-1,4-linked xylopyranose, and beta-1,4-linked 2-mono-O-methyl xylopyranose. Analytical and NMR-spectral data of LMHS confirmed the presence of considerable amounts of the non-oxidized of 1,4-linked D-galactopyranosyl uronic acid residues. Thus, some side chains of the ramified region of lemnan appeared to attach to D-galactopyranosyl uronic acid residues of the backbone.     

Structural Studies on Pectin from Marsh Cinquefoil <Emphasis Type="Italic">Comarum palustre</Emphasis> L.

Pectin with [alpha]D(20) +192 degrees (c 0.1; water), named comaruman, was isolated from marsh cinquefoil Comarum palustre L., which is widespread in the European North. The sugar chain of comaruman contains residues of D-galacturonic acid (64%), D-galactose (13%), L-rhamnose (12%), L-arabinose (6%), and trace amounts of xylose and glucose. Partial acid hydrolysis and digestion with pectinase demonstrated that comaruman composed of the backbone comprised regions of linear alpha-1,4-D-galactopyranosyl uronan interconnected by numerous residues of alpha-1,2-L-rhamnopyranose. In addition to the backbone (core of the macromolecule), ramified regions are involved in comaruman and comprise alpha-2,4-L-rhamno-alpha-4-D-galacturonan with side chains consisting mainly of beta-1,4-linked residues of D-galactopyranose. The ramified region contains additionally residues of 5-O-substituted arabinofuranose and 3- and 6-O-substituted galactopyranose. The present 3,4- and 4,6-di-O-substituted residues of galactopyranose appear to be branching points of the side chains. Some galactopyranose residues were found to occupy the terminal positions of the side chains or appeared to be single sugar residues attached to the side chains. Methylation analysis data indicated that comaruman contains residues of terminal, 3- and 3,4-di-O-substituted galactopyranosyl uronic acid, which appeared to be constituents of the side chains, and the latter represented additionally branching points of the backbone.     

Structural studies and physiological activity of lemnan, a pectin fromLemna minor L

A pectic polysaccharide, lemnan, was isolated from freshly collected duckweedLemna minor L. Its sugar chain was shown to be mainly composed of the residues ofD-galacturonic acid (64%), galactose, arabinose, xylose, andD-apiose, a branched chain sugar. The high content ofD-apiose (25%) indicated that lemnan is an apiogalacturonan type pectin similar to zosteran, a pectic polysaccharide from a sea phanerogam of the Zosteraceae family. The results of partial acidic hydrolysis, pectinase digestion, and NMR studies of lemnan demonstrated that its macromolecule contains regions of the linear α-1,4-D-galacturonan and branched apiogalacturonan. The side chains of apiogalacturonan were found to be formed of single and 1,5-linked residues ofD-apiofuranose attached to 2- and 3-positions of theD-galacturonic acid residues of the apiogalacturonan backbone. Lemnan was shown to exhibit an immunomodulatory effect activating the system of phagocytosis.     

Branching of the galacturonan backbone of comaruman, a pectin from the marsh cinquefoil Comarum palustre L.

Galacturonan, the main constituent of the backbone (core) of the comaruman macromolecule, a pectin from the marsh cinquefoil Comarum palustre L., was obtained on partial acid hydrolysis of the pectin. Using atomic force microscopy and methylation analysis of the galacturonan, the backbone of the comaruman macromolecule was shown to contain branches as side chains consisting of α-1,4-linked residues of D-galactopyranosyl uronic acid attached to the 2-and 3-positions of the galacturonic acid residues of the core, in addition to linear regions of α-1,4-D-galacturonan. A few side chains appear to attach to 2,3-positions of the D-galacturonic acid residues. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 5, pp. 666–671.     

Alterations in growth of tissue-cultured tansy (Tanacetum vulgare L.) treated with antibiotics

Effects of three antibiotics (cefotaxime, rifampicin and gentamicin) were tested on in vitro shoots cultures of tansy (Tanacetum vulgare L.)- These antibiotics were selected because endophytic bacteria isolated from the in vitro shoot cultures of tansy showed that all bacteria were Gram-negative and their growth was reduced by these three antibiotics. Five isolates were Enterobacteriaceae, three were fluorescent Pseudomonas, and two were aerobic bacteria. Increased concentrations of antibiotics caused usually linear or quadratic changes on the initiation of shoot growth, shoot number, growth rate, and shoot height. These changes and changes in pH of the culture media were tansy genotype-dependent following treatments with gentamicin. Also, the treatment with rifampicin or cefotaxime showed a genotype-dependent effect, because they resulted in significantly higher percentage of rooted plants in one of the three tansy genotypes tested. The growth rate and length of shoots were reduced in the media containing both gentamicin and rifampicin, but less so than in media containing both gentamicin and cefotaxime.     

Chemical and genetic characterization of calli derived from somatic hybridization between tansy (Tanacetum vulgare L.) and pyrethrum (Tanacetum cinerariifolium (Trevir.) Schultz-Bip.)

 Pyrethrum (Tanacetum cinerariifolium (Trevir.) Schultz-Bip.) produces environmentally benign pesticides, the pyrethrins, and tansy (Tanacetum vulgare L.) lower terpenes of variable biological effectiveness. As an approach to improve the oil content and composition of tansy for enhanced biological activity, a somatic hybridization technique between tansy and pyrethrum was established. About 1×10⁶ of leaf-mesophyll protoplasts of both species were mixed and fused with a solution containing 15% polyethylene glycol. Light-green and yellowish calli developed from the fusion experiments. The fusion-derived calli grew vigorously on MS medium supplemented with 6.4 mg l^-1 of BAP, 0.8 mg l^-1 of NAA, and 30–40 g l^-1 of glucose. Nuclear DNA content, RAPD patterns, and volatile compounds were analyzed to determine the hybridity of the calli. The nuclear DNA content of the tansy and pyrethrum genotypes, and the protoplast-derived calli of tansy were 6.41, 7.39, 13.84, and 8.11 pg, respectively. The nuclear DNA content of individual calli derived from the protoplast fusion between tansy +tansy ranged from 8.84 (F43A) to 19.59 pg (F43C) while those of the tansy+pyrethrum fusions were 10.66 (F46A) and 31.87 pg (F46B). Using four 10-mer primers a total of 56 RAPD-PCR fragments were produced. The distance matrices of fragments were calculated by average linkage cluster analysis. Two visually separated clusters were observed. One cluster consisted of the two tansy genotypes and the fusion-derived callus F43A; the other consisted of pyrethrum and fusion-derived calli F46B and F46C. Volatile compounds, such as decadienal, artedouglasia oxide, heptadecane, syringaldehyde and coniferyl alcohol, analyzed by gas chromatography mass spectrometry, were found only in the protoplast fusion-derived calli F43A and F46B. Several less volatile compounds were also detected only in fusion calli. Hexadecanoic and linoleic acids were common to fusion-derived calli and tansy, and one unknown compound to fusion-derived calli and pyrethrum. Pyrethrins I and II were detected from pyrethrum, but not from the fusion-derived calli. The additive nuclear DNA content of protoplast fusion-derived calli and the results of the RAPDs suggest that interspecific fusions had occurred. The small number of volatile compounds detected from both the fusion calli and from the donor species indicates that the unorganized callus tissue is unable to produce tissue-specific volatile compounds. Received: 4 August 1998 / Accepted: 30 September 1998     

Genetic and morphological diversity of Finnish tansy (Tanacetum vulgare L., Asteraceae)

 Morphological and genetical differences of twenty Finnish tansy (Tanacetum vulgare) genotypes were studied. The genotypes were distinguishable by morphology, and number of flower heads, which correlated positively with height of the plant and height of the corymb. The mean nuclear DNA content in the leaves of tissue-cultured tansy plantlets was 8.86 pg, and variation between genotypes was 27%. The genotypes were also distinguishable on the basis of their RAPD patterns. The RAPD and morphological data were subjected to analysis of principal components, according to which the genotypes were separated into two main groups. Group I included 7 genotypes from southern Finland and 2 genotypes from the eastern lake district. They started to flower later, had more flower heads and nodes per stem, and the corymb was longer than in the other genotypes that originated in the western and central part of Finland and formed group II. Our data are supported by previous studies and suggest that group I may represent native tansy populations in Finland, whereas group II may represent tansy genotypes that have been transported to other parts of Finland through agriculture and attempts of domestication. Received: 19 November 1997 / Accepted: 25 November 1997     

Ecdysteroids from Polypodium vulgare L

Three new compounds (3, 7, and 11) together with eight known phytoecdysteroids (1, 2, 4-6, and 8-10) were isolated from the rhizomes of common polypody, Polypodium vulgare L. The structures of compounds were elucidated by spectroscopic methods including 1D and 2D NMR measurements. The ¹H and ¹³C NMR assignments of compounds 1, 6, 9 and 10 are included.     

Production of gynogenic haploids of Hordeum vulgare L.

Haploid plants were regenerated from cultured unfertilized ovaries of Hordeum vulgare L. (barley). Optimal response was obtained by the addition of 0.6 M 4-chloro-2-methylphenoxyacetic acid (MCPA), 2.8 M indole-3-acetic acid (IAA) and 4.4 M 6-benzyladenine (BA) in the N₆ medium. Further increase in the rate of callus formation and the number of green plants produced was possible with the addition of 90 g/l sucrose and 100 g/l coconut water. The stage of development of the ovaries at the time of culture was critical; the largest number of plants being produced by ovaries from flowers at the trinucleate stage of pollen.Abbreviations (BA) 6-benzyladenine - (MCPA) 4-chloro-2-methylphenoxyaceticacid - (2,4-D) 2,4-dichlorophenoxyaceticacid - (GA₃) gibberellic acid - (IAA) indole-3-acetic acid     

Primary callus as source of totipotent barley (Hordeum vulgare L.) protoplasts

Summary Primary callus of barley (Hordeum vulgare L.) derived from scutella (cv. Dissa) and anthers (cv. Igri) was used for protoplast isolation and plant regeneration. The protoplasts were embedded in agarose and cultured with nurse cells. The plating efficiency varied from 0.1% to 0.7%. Shoots regenerated from the developing callus. Plantlets were transferred to soil and cultivated in the greenhouse three to five months after protoplast isolation. All plants were normal in morphology, and most of them flowered and set seeds.     

Monosomic and double monosomic substitutions of Hordeum bulbosum L. chromosomes into H. vulgare L.

Summary One of the aims of the interspecific crossing programme between Hordeum vulgare L. and H. bulbosum L. has been to introgress desirable genes into barley from the wild species. However, despite their close taxonomic relationship there have been few reports of achieving this objective using amphidiploid hybrids. In order to broaden the range of available hybrids, partially fertile triploids between H. vulgare (2n = 2x = 14) and H. bulbosum (2n = 4x = 28) were developed and crossed with H. vulgare as female parent. From 580 progeny which were screened, eight putative single monosomic chromosome substitution lines and one double monosomic substitution were identified by cytological analysis. These involved the substitution of H. vulgare chromosome 1 (two lines), 6 (four lines), 6L (one line), 7 (one line) and 1 + 4 (one line) by their respective H. bulbosum homoeologues. The H. bulbosum chromosome was frequently eliminated during plant development, but it was observed regularly in pollen mother cells of two lines. However, pairing between the H. bulbosum chromosome and its H. vulgare homoeologue was low. Several of the lines were more resistant than their H. vulgare parents to powdery mildew (Erysiphe graminis DC. f.sp. hordei Em. Marchai), net blotch (Drechslera teres Sacc.) and scald (Rhynchosporium secalis (Oudem.) Davis). Apart from their use in studying genome relationships, their value to plant breeders will depend on the ease of inducing translocations between the parental chromosomes.     

Chromosomal variation in dividing protoplasts derived from cell suspensions of barley (Hordeum vulgare L.)

Summary Numerical and structural chromosome variation was analysed in dividing protoplasts isolated from suspension cells of barley. Five cell lines exhibited distribution patterns in chromosome number with different peaks and ranges. Embryogenic/morphogenic cell lines showed a peak at 2n = 14 (ca. 50%) after 6–7 months in culture, while older non-embryogenic cell lines had peaks at aneuploid or polyploid chromosome numbers. Culture duration had a clear effect on numerical and structural chromosome variation in embryogenic cell lines. With ageing of the cultures chromosome variation accumulated and the proportion of 2n = 14 cells decreased. The effect of protoplast isolation and culture on chromosome variation was examined; more cells with normal chromosome sets (12%) were maintained in protoplast-derived colonies than in source suspension cells (4%) of the same culture age.Abbreviations DC Dicentric - F fragment - T telocentric     

Biosynthesis of the irregular monoterpene artemisia ketone, the sesquiterpene germacrene D and other isoprenoids in Tanacetum vulgare L. (Asteraceae)

The incorporation of [1-13C]-labeled glucose into the irregular monoterpene artemisia ketone, the regular monoterpenes camphor and beta-thujone, the sesquiterpene germacrene D, the diterpene trans-phytol and beta-sitosterol and isofucosterol has been studied in axenic cultures of Tanacetum vulgare L. (Asteraceae). Quantitative 13C NMR spectroscopic analysis of the resulting labeling patterns showed that the isoprene units of the monoterpenes and the diterpene are formed via the methylerythritol phosphate (MEP) pathway, whereas the isoprene building blocks of the sesquiterpene and the sterols originate from the mevalonic acid (MVA) pathway.     

Structure of Silenan, a Pectic Polysaccharide from Campion Silene vulgaris (Moench) Garcke

A pectic polysaccharide named silenan, [alpha]D20 +148.6 degrees (c 0.1; H2O), was isolated earlier from the aerial part of campion, Silene vulgaris (Moench) Garcke. Silenan has been shown to contain homogalacturonan segments as "smooth regions" and rhamnogalacturonan fragments as "hairy regions". The present study reveals a generalization of structural features of silenan. Silenan was subjected to enzymic digestion with pectinase, to Smith degradation, and to lithium-degradation to determine the conforming poly- and oligosaccharide fragments of "hairy regions" of silenan. The NMR-spectral data and mass-spectrometry confirmed that the core of the ramified region of silenan consisted of residues of alpha-rhamnopyranose 2-O-glycosylated with the residues of alpha-1,4-D-galactopyranosyl uronic acid. The part of the alpha-rhamnopyranose residues of the backbone are branched at O-4. On the basis of the data, the hairy regions of silenan proved to contain mainly linear chains of beta-1,3-, beta-1,4-, and beta-1,6-galactopyranan and alpha-1,5-arabinofuranan. The side chains of the ramified region were shown to have branching points represented 2,3-, 3,6-, 4,6-di-O-substituted beta-galactopyranose residues.     

Optimizing somatic embryogenesis and particle bombardment of barley (Hordeum vulgare L.) immature embryos

Summary A highly regenerable target tissue and a high-frequency DNA delivery system are required for the routine production of transgenic barley. This project separately optimized tissue culture and particle bombardment parameters. Immature zygotic embryos (0.7 to 1.2 mm) were excised and culture on B5L solid medium. Klages and H930-36 cultivars regenerated significantly more green plants than Sabarlis and Bruce. The regeneration pathway shifted from organogenesis to somatic embryogenesis when maltose was used as the medium carbohydrate source instead of sucrose. More somatic embryos were induced on 5 mg/liter 2,4-dichlorophenoxyacetic acid than 2 mg/liter. Gene delivery was optimized using anthocyanin regulatory genes as a transient marker. A 3-mm rupture disc-to-macrocarrier gap distance, a 1-day prebombardment embryo culture period, and a maltose carbohydrate source were each significantly better than other treatments. Double bombardments per plate, a 6-mm macrocarrier fly distance, and 650-psi rupture discs each had the highest number of transiently expressing cells in individual experiments, although the results were not statistically significant compared to the other treatments. Using the optimized parameters, over 200 cells routinely expressed anthocyanin in a bombarded immature embryo. In tissue culture experiments, 350 to 400 green plants regenerated per 100 immature embryos. The improvement of green plant regeneration and gene delivery forms a strong basis to develop a practical barley transformation system.     

Non-host plant odor (Tanacetum vulgare; Asteracea) affects the reproductive behavior ofLobesia botrana Den. et Schiff (Lepidoptera: Tortricidae)

Females ofLobesia botrana Den. et Schiff. (Lepidoptera Tortricidae) are attracted in natural conditions by volatiles released by a nonhost plant: tansy (Tanacetum vulgare L.; Asteracea). We have shown that both tansy flowers and their odor inhibit oviposition behavior and mating behavior and reduce adult longevity. The mean number of eggs laid per female isolated with tansy flowers was reduced by up to 50% every 2 days during the 6 days of exposure. This reduction was maintained after the tansy was removed. In the presence of tansy essential oil, the egg-laying reduction ranged from about 30 to 80% according to the odor concentration. The number of spermatophores found in females isolated with tansy flowers was also reduced twofold compared to the control treatment, indicating that the presence of tansy reduced mating activity. This mating activity is strongly reduced, by two-thirds, when adults face the highest dose of essential oil compared to controls. The number of eggs laid by the controls cannot be explained by the number of spermatophores. Therefore, the reduction in oviposition has been attributed to the presence of tansy flowers or to the tansy odor. Tansy flowers and tansy odor increased male mortality during the exposure (10% in the control, 50% in the tansy treatment, and up to 98% in the odor treatment). The highest rates of male mortality occurred during the 4- to 6-day period of exposure to flowers or odor. Repellence resulting in sustained locomotor activity is a possible cause of such a mortality. Female mortality was increased only in response to the highest dose of odor. This increase might be due to egg retention, and not directly to a plant effect. We discuss the effects of tansy flower odor on different patterns relative to the reproductive behavior ofL. botrana and, especially, on oviposition behavior in the ecological context of plant selection and polyphagy.     

Characterization of a gibberellin sensitive dwarf mutant of barley (Hordeum vulgare L.,Mut. Dornburg 576)

The radiation (³²P) induced dwarf mutant of spring barley,Hordeum vulgare L., Mut. Dornburg 576, was genetically studied by crosses with the mother variety and characterized by seedling assays and investigations on development and yield formation. In comparison to the normal mother variety (Hordeum vulgare L., cv. Saale) mutant plants exhibit drastically reduced culm length, intensive tillering, a prolonged life cycle and a smaller biomass and grain yield formation. These characters are controlled by one gene in a pleiotropic way. The mutant responds with normal growth and development to the application of gibberellins.