Environmentally Friendly Surface Modification Treatment of Flax Fibers by Supercritical Carbon Dioxide.

The present work investigates the effects of an environmentally friendly treatment based on supercritical carbon dioxide (scCO2) on the interfacial adhesion of flax fibers with thermoset matrices. In particular, the influence of this green treatment on the mechanical (by single yarn tensile test), thermal (by TGA), and chemical (by FT-IR) properties of commercially available flax yarns was preliminary addressed. Results showed that scCO2 can significantly modify the biochemical composition of flax fibers, by selectively removing lignin and hemicellulose, without altering their thermal stability and, most importantly, their mechanical properties. Single yarn fragmentation test results highlighted an increased interfacial adhesion after scCO2 treatment, especially for the vinylester matrix, in terms of reduced debonding and critical fragment length values compared to the untreated yarns by 18.9% and 15.1%, respectively. The treatment was less effective for epoxy matrix, for which debonding and critical fragment length values were reduced to a lesser extent, by 3.4% and 3.7%, respectively.

Ectopic lignification in the flax lignified bast fiber1 mutant stem is associated with tissue-specific modifications in gene expression and cell wall composition.

Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply.

[Genetic transformation of flax (Linum usitatissimum L.) with chimeric GFP-TUA6 gene for visualisation of microtubules].

The data of Agrobacterium-mediated transformation of some Linum usitatissimum cultivars zoned on the territories of Belarus and Ukraine with the plasmid carrying chimeric GFP-TUA6 gene and nptII gene as selectable marker conferring resistance to kanamycin are presented in this study. Transformation was affected by a number of factors including optical density (OD600), time of inoculation of explants with Agrobacterium and co-culture conditions. Transgenic nature of obtained lines was confirmed by PCR analysis. Expression of GFP-TUA6 gene was detected with confocal laser scanning microscopy. The obtained transgenic lines can be used for further functional studies the role of microtubules in the processes of building the flax fibres and resistance to wind.

Development of cellulosic secondary walls in flax fibers requires beta-galactosidase.

Bast (phloem) fibers, tension wood fibers, and other cells with gelatinous-type secondary walls are rich in crystalline cellulose. In developing bast fibers of flax (Linum usitatissimum), a galactan-enriched matrix (Gn-layer) is gradually modified into a mature cellulosic gelatinous-layer (G-layer), which ultimately comprises most of the secondary cell wall. Previous studies have correlated this maturation process with expression of a putative ß-galactosidase. Here, we demonstrate that ß-galactosidase activity is in fact necessary for the dynamic remodeling of polysaccharides that occurs during normal secondary wall development in flax fibers. We found that developing stems of transgenic (LuBGAL-RNAi) flax with reduced ß-galactosidase activity had lower concentrations of free Gal and had significant reductions in the thickness of mature cellulosic G-layers compared with controls. Conversely, Gn-layers, labeled intensively by the galactan-specific LM5 antibody, were greatly expanded in LuBGAL-RNAi transgenic plants. Gross morphology and stem anatomy, including the thickness of bast fiber walls, were otherwise unaffected by silencing of ß-galactosidase transcripts. These results demonstrate a specific requirement for ß-galactosidase in hydrolysis of galactans during formation of cellulosic G-layers. Transgenic lines with reduced ß-galactosidase activity also had biochemical and spectroscopic properties consistent with a reduction in cellulose crystallinity. We further demonstrated that the tensile strength of normal flax stems is dependent on ß-galactosidase-mediated development of the phloem fiber G-layer. Thus, the mechanical strength that typifies flax stems is dependent on a thick, cellulosic G-layer, which itself depends on ß-galactosidase activity within the precursor Gn-layer. These observations demonstrate a novel role for matrix polysaccharides in cellulose deposition; the relevance of these observations to the development of cell walls in other species is also discussed.

Ontogeny of floral organs in flax (Linum usitatissimum; Linaceae).

PREMISE OF THE STUDY: Flax (Linum usitatissimum) is an important crop worldwide; however, a detailed study on flower development of this species is lacking. Here we describe the pattern of initiation and a program of key developmental events in flax flower ontogeny. This study provides important fundamental information for future research in various aspects of flax biology and biotechnology. METHODS: Floral buds and organs were measured throughout development and examined using scanning electron microscopy. KEY RESULTS: Floral organs were initiated in the following sequence: sepals, stamens and petals, gynoecium, and nectaries. The five sepals originated in a helical pattern, followed evidently by simultaneous initiation of five stamens and five petals, the former opposite of the sepals and the latter alternate to them. The gynoecium, with five carpels, was produced from the remaining, central region of the floral apex. Stamens at early stages were dominated by anther growth but filaments elongated rapidly shortly before anthesis. Early gynoecium development occurred predominantly in the ovary, and ovule initiation began prior to enclosure of carpels. A characteristic feature was the twisted growth of styles, accompanied by the differentiation of papillate stigmas. Petal growth lagged behind that of other floral organs, but petals eventually grew rapidly to enclose the inner whorls after style elongation. Flask-shaped nectaries bearing stomata developed on the external surface of the filament bases. CONCLUSIONS: This is the first detailed study on flax floral organ development and has established a key of 12 developmental stages, which should be useful to flax researchers.

[Genetic polymorphism of flax Linum usitatissimum based on use of molecular cytogenetic markers].

Using a set of approaches based on the use of molecular cytogenetic markers (DAPI/C-banding, estimation of the total area of DAPI-positive regions in prophase nuclei, FISH with 26S and 5S rDNA probes) and the microsatellite (SSR-PCR) assay, we studied genomic polymorphism in 15 flax (Linum usitatissimum L.) varieties from different geographic regions belonging to three directions of selection (oil, fiber, and intermediate flaxes) and in the k-37 x Viking hybrid. All individual chromosomes have been identified in the karyotypes of these varieties on the basis of the patterns of differential DAPI/C-banding and the distribution of 26S and 5S rDNA, and idiograms of the chromosomes have been generated. Unlike the oil flax varieties, the chromosomes in the karyotypes of the fiber flax varieties have, as a rule, pericentromeric and telomeric DAPI-positive bands of smaller size, but contain larger intercalary regions. Two chromosomal rearrangements (chromosome 3 inversions) were discovered in the variety Luna and in the k-37 x Viking hybrid. In both these forms, no colocalization of 26S rDNA and 5S rDNA on the satellite chromosome was detected. The SSR assay with the use of 20 polymorphic pairs of primers revealed 22 polymorphic loci. Based on the SSR data, we analyzed genetic similarity of the flax forms studied and constructed a genetic similarity dendrogram. The genotypes studied here form three clusters. The oil varieties comprise an independent cluster. The genetically related fiber flax varieties Vita and Luna, as well as the landrace Lipinska XIII belonging to the intermediate type, proved to be closer to the oil varieties than the remaining fiber flax varieties. The results of the molecular chromosomal analysis in the fiber and oil flaxes confirm their very close genetic similarity. In spite of this, the combined use of the chromosomal and molecular markers has opened up unique possibilities for describing the genotypes of flax varieties and creating their genetic passports.

Lessons on textile history and fibre durability from a 4,000-year-old Egyptian flax yarn.

Flax has a long and fascinating history. This plant was domesticated around 8,000 BCE1 in the Fertile Crescent area2, first for its seeds and then for its fibres1,3. Although its uses existed long before domestication, residues of flax yarn dated 30,000 years ago have been found in the Caucasus area4. However, Ancient Egypt laid the foundations for the cultivation of flax as a textile fibre crop5. Today flax fibres are used in high-value textiles and in natural actuators6 or reinforcements in composite materials7. Flax is therefore a bridge between ages and civilizations. For several decades, the development of non- or micro-destructive analysis techniques has led to numerous works on the conservation of ancient textiles. Non-destructive methods, such as optical microscopy8 or vibrational techniques9,10, have been largely used to investigate archaeological textiles, principally to evaluate their degradation mechanisms and state of conservation. Vibrational spectroscopy studies can now benefit from synchrotron radiation11 and X-ray diffraction measurement in the archaeometric study of historical textiles12,13. Conservation of mechanical performance and the ultrastructural differences between ancient and modern flax varieties have not been examined thus far. Here we examine the morphological, ultrastructural and mechanical characteristics of a yarn from an Egyptian mortuary linen dating from the early Middle Kingdom (Eleventh Dynasty, ca. 2033-1963 BCE) and compare them with a modern flax yarn to assess the quality and durability of ancient flax fibres and relate these to their processing methods. Advanced microscopy techniques, such as nano-tomography, multiphoton excitation microscopy and atomic force microscopy were used. Our findings reveal the cultural know-how of this ancient civilization in producing high-fineness fibres, as well as the exceptional durability of flax, which is sometimes questioned, demonstrating their potential as reinforcements in high-technology composites.

Integument-Specific Transcriptional Regulation in the Mid-Stage of Flax Seed Development Influences the Release of Mucilage and the Seed Oil Content.

Flax (Linum usitatissimum L.) seed oil, which accumulates in the embryo, and mucilage, which is synthesized in the seed coat, are of great economic importance for food, pharmaceutical as well as chemical industries. Theories on the link between oil and mucilage production in seeds consist in the spatio-temporal competition of both compounds for photosynthates during the very early stages of seed development. In this study, we demonstrate a positive relationship between seed oil production and seed coat mucilage extrusion in the agronomic model, flax. Three recombinant inbred lines were selected for low, medium and high mucilage and seed oil contents. Metabolite and transcript profiling (1H NMR and DNA oligo-microarrays) was performed on the seeds during seed development. These analyses showed main changes in the seed coat transcriptome during the mid-phase of seed development (25 Days Post-Anthesis), once the mucilage biosynthesis and modification processes are thought to be finished. These transcriptome changes comprised genes that are putatively involved in mucilage chemical modification and oil synthesis, as well as gibberellic acid (GA) metabolism. The results of this integrative biology approach suggest that transcriptional regulations of seed oil and fatty acid (FA) metabolism could occur in the seed coat during the mid-stage of seed development, once the seed coat carbon supplies have been used for mucilage biosynthesis and mechanochemical properties of the mucilage secretory cells.

Direct insights on flax fiber structure by focused ion beam microscopy.

In this article, it is shown that focused ion beam (FIB) systems can be used to study the inner structure of flax fibers, the use of which as a reinforcing material in polymer composites still draws much interest from multiple disciplines. This technique requires none of the specific preparations necessary for scanning electron microscopy or transmission electron microscopy studies. Irradiation experiments performed on FIB prepared cross sections with very low Ga+ ion beam currents revealed the softer material components of fibers. Thus, it confirmed the presence of pectin-rich layers at the interfaces between the fibers of a bundle, but also allowed the precise localization of such layers within the secondary cell wall. Furthermore, it suggested new insights on the transition modes between the sublayers of the secondary cell wall.

Microwave-vacuum drying of flax fiber for biocomposite production.

Flax fiber is one of the important bast fiber available in North America which has low density and good mechanical properties for reinforcing various polymers to develop industrial biocomposite. But the hydrophilic nature of the fiber leads to poor adhesion between the fiber and the polymer matrices which, in turn, leads to poor dimensional stability. To enhance the adhesion between polymer matrix and fiber, chemical treatments of the fiber are necessary. After these treatments, the fiber needs to be uniformly dried to minimum moisture content for better compounding of flax in polymer matrices. Drying by conventional dryers at higher temperature usually results in overall quality loss due to surface drying, and it is also time consuming and energy intensive. In recent years, the microwave has been effectively used for enhancing bulk drying of biomaterials especially when coupled with vacuum. Such a volumetric heat transfer mechanism coupled with drying in vacuum provides an ideal low-temperature drying technique resulting in better organoleptic quality. In this study, the drying characteristics of flax fiber were studied under microwave-vacuum condition. The drying experiments were conducted in Enwave Microwave-vacuum dryer using three different process variables viz., microwave power (750 W and 375 W), vacuum level (25, 10, and 0 in Hg) and time of drying (0 to 14 mins). In order to select the appropriate drying curve equation, the drying data was fitted in three different models viz., Page model, single exponential model and approximation of diffusion model.

Thiol functionalization of flaxseed mucilage: Preparation, characterization and evaluation as mucoadhesive polymer.

The aim of present study is to browse mucoadhesive potential of flaxseed mucilage after thiol functionalization. Thiol-derivatization of flaxseed mucilage (FSM) polysaccharide was obtained by esterification with thioglycolic acid. Thiolation of FSM was confirmed by -SH stretch in FTIR spectra at 2549.01 cm-1. Thiolated flaxseed mucilage (TFSM) was distinguished by XRD, DSC, NMR & SEM analysis. TFSM was found to contain 325.6 mM of thiol groups/g as determined by Ellman's method. The mucoadhesive property of drug loaded TFSM pellets, carried out by using chicken buccal pouch membrane, displayed greater ex-vivo bioadhesion time as compared to FSM. This improvement in mucoadhesion property of TFSM over FSM can be attributed to the formation of disulphide bond between mucus and thiolated mucilage. Further, the in-vitro dissolution study conducted in phosphate buffer (pH 6.8) provided release of diclofenac sodium for a prolonged period of 12 h for TFSM pellets by anomalous transport mechanism of drug release following zero order model of release kinetics.

[Dynamics of ultrastructure changes in sheet plate fiber flax with braking transport assimilate by nitrate-anion].

Changes in leaf mesophyll cell ultrastructure under nitrate feeding into the apoplast of common flax (Linum usitatissimum L.) in the form of 50 mM KNO3 solution were studied. In 30 min after the beginning of nitrate feeding through the transpiration water stream, swelling of mitochondrial and microbodies, clarification of their matrices, and curling of dictyosome discs into annular structures were observed. These events characterized symplastic domain formed by mesophyll, bundle sheath and phloem parenchyma cells, and were not found in companion cell-sieve element complex. Simultaneously, formation of large central vacuoles in companion cells was noted. Restoration of organelle structures in assimilating cells and phloem parenchyma in 1-2 h after treatment was accompanied by enhancement of morphological changes in phloem elements and companion cells and signs of plasmolysis in the mesophyll cells. It was supposed that the two-phase character of changes in leaf organelle ultrastructure and photosynthesis might reflect duality of leaf cell response to nitrate ion. The rapid alterations of the structure can be coupled with direct influence of the anion on cell metabolism and(or) with signal-regulatory functions of oxidized nitrogen forms, while the slower ones reflect the result of suppression of photoassimilate export from leaves by the anion.

Flax stems: from a specific architecture to an instructive model for bioinspired composite structures.

The present paper proposes to carefully study and describe the reinforcement mechanisms within a flax stem, which is an exceptional natural model of composite structure. Thanks to accurate microscopic investigations, with both optical and SEM method, we finely depicted the flax stem architecture, which can be view as a composite structure with an outer protection, a unidirectional ply on the periphery and a porous core; each component has a specific function, such as mechanical reinforcement for the unidirectional ply and the porous core. The significant mechanical role of fibres was underlined, as well as their local organisation in cohesive bundles, obtained because of an intrusive growth and evidenced in this work through nanomechanical AFM measurement and 3D reconstruction. Following a biomimetic approach, these data provide a source of inspiration for the composite materials of tomorrow.

Development of gravitropic response: unusual behavior of flax phloem G-fibers.

The major mechanism of gravitropism that is discussed for herbal plants is based on the nonuniform elongation of cells located on the opposite stem sides, occurring in the growing zone of an organ. However, gravitropic response of flax (Linum usitatissimum L.) is well-pronounced in the lower half of developing stem, which has ceased elongation long in advance of plant inclination. We have analyzed the stem curvature region by various approaches of microscopy and found the undescribed earlier significant modifications in primary phloem fibers that have constitutively developed G-layer. In fibers on the pulling stem side, cell portions were widened with formation of "bottlenecks" between them, leading to the "sausage-like" shape of a cell. Lumen diameter in fiber widening increased, while cell wall thickness decreased. Callose was deposited in proximity to bottlenecks and sometimes totally occluded their lumen. Structure of fiber cell wall changed considerably, with formation of breaks between G- and S-layers. Thick fibrillar structures that were revealed in fiber cell wall by light microscopy got oblique orientation instead of parallel to the fiber axis one in control plants. The described changes occurred at various combinations of gravitational and mechanical stimuli. Thus, phloem fibers with constitutively formed gelatinous cell wall, located in nonelongating parts of herbal plant, are involved in gravitropism and may become an important element in general understanding of the gravity effects on plants. We suggest flax phloem fibers as the model system to study the mechanism of plant position correction, including signal perception and transduction.

ESTs from the fibre-bearing stem tissues of flax (Linum usitatissimum L.): expression analyses of sequences related to cell wall development.

In order to learn more about the diversity of genes expressed during flax fibre cell wall formation, expressed sequence tags (ESTs) were obtained from a cDNA library derived from the outer fibre-bearing tissues of flax (Linum usitatissimum) stems (cv Hermes) harvested at the mid-flowering stage. After elimination of vector and unreadable sequences, 927 ESTs were grouped into 67 clusters and 754 singletons. The flax ESTs have been submitted to the dbEST and GenBank databases with the accession numbers 25939634 - 25940560 (dbEST) and CV478070 - CV478996 (GenBank). Functional analysis allowed the grouping of ESTs into 13 functional categories and revealed that 62 % of ESTs were similar to known sequences, while 12.4 % of ESTs presented no similarity to any known sequences and 25.6 % of ESTs corresponded to proteins of unknown function. The most highly expressed transcripts belonged to four functional categories: protein maturation and metabolism (31 ESTs), signalling (22 ESTs), the cell wall (21 ESTs) and photosynthesis (19 ESTs). 4.4 % (41) of the total ESTs were potentially related to cell wall formation and maturation. The most highly expressed cell wall EST (15 ESTs) corresponded to a beta-xylosidase gene--potentially involved in cell wall remodelling during growth and development. Other cell wall-related ESTs corresponded to cellulose synthase, xyloglucan endotranglucosylase/hydrolase (XTH), beta-galactosidases, and peroxidases. The expression patterns of different cell wall-related ESTs were determined at different developmental stages in flax plants grown under different field conditions. The potential roles of gene products associated with cell wall related ESTs in fibre cell wall development is discussed.

Enhanced Extraction of Oil from Flaxseed (Linum usitatissimum L.) Using Microwave Pre-treatment.

The effect of microwave (MW) pre-treatment on the extraction of flaxseed oil was investigated by hot extraction (HE). Nine MW pre-treatments were established, combining three MW radiation intensities (12, 18 and 24 W/g) and three MW radiation times of pre-treatment (90, 150 and 210 s). Extraction yield increased significantly with MW pre-treatments of flaxseed, and a max oil extraction yield (78.11%) can be obtained using MW pre-treatment at 18 W/g for 210 s. Scanning electronic microscopy showed that the microstructure of treated samples (18 W/g and 210 s) was modified compared with that of untreated samples. The fatty acid compositions (palmitic acid 5.85±0.01%, stearic acid 3.00±0.01%, oleic acid 17.64±0.07%, linoleic acid 16.16±0.06%, and linolenic acid 57.37±1.32%) of the oils extracted by the MW pre-treatments HE were similar with that of the conventional HE method. Results showed that fatty acid compositions of flaxseed oil were not affected by MW pre-treatments.

Microscopic studies on mature flax fibers embedded in LR white: immunogold localization of cell wall matrix polysaccharides.

Flax fibers have been the subject of many biochemical studies, which revealed that cellulose and pectins are the major constituents of their walls. In contrast, little is known about the location of these polymers within the walls of mature fibers by microscopic methods. This has been technically hampered by the very thick secondary wall of fibers, resulting in inadequate tissue preservation unsuitable for immunogold microscopy. In this study, we adapted the basic chemical fixation, dehydration and infiltration methods to achieve a good preservation of the cell structures of mature fibers and reduced damage to antigens. We were able to apply postembedding immunocytochemical techniques to map the location of various pectic epitopes within the walls of mature fibers. Our immunolabeling data show that homogalacturonans were exclusively found in the middle lamellae and the cell junctions but were not detectable in the secondary wall. In contrast, rhamnogalacturonan I (RG I)-associated epitopes, as well as galactan and arabinan epitopes, were abundantly distributed over the secondary wall of mature fibers.

Enzyme-retting of flax and characterization of processed fibers.

Enzyme-retting formulations consisting of Viscozyme L, a pectinase-rich commercial enzyme product, and ethylenediaminetetraacetic acid (EDTA) were tested on Ariane fiber flax and North Dakota seed flax straw residue. Flax stems that were crimped to disrupt the outer layers were soaked with various proportions of Viscozyme-EDTA solutions, retted, and then cleaned and cottonized with commercial processing equipment. Fiber properties were determined and crude test yarns were made of raw and Shirley cleaned flax fibers and cotton in various blend levels. Cleaned fibers were obtained from both seed and fiber flax types, but with variations due to treatment. Retting formulations produced fibers having different properties, with enzyme levels of 0.3% (v/v as supplied) giving finer but weaker fibers than 0.05% regardless of EDTA level. Experimental yarns of blended flax and cotton fibers varied in mass coefficient of variation, single end strength, and nep imperfections due to sample and formulation. With cost and fiber and yarn quality as criteria, results established a range in the amounts of components comprising retting formulations as a basis for further studies to optimize enzyme-retting formulations for flax. Under conditions examined herein, Viscozyme L at 0.3% (v/v) plus 25 mM EDTA produced the best test yarns and, therefore, established a base for future studies to develop commercial-grade, short staple flax fibers for use in textiles.

Influence of nonuniform magnetic fields on orientation of plant seedlings in microgravity conditions.

Experiments on the spatial behavior of the flax (Linum usitatissimum, L.) seedlings in a nonuniform magnetic field were conducted on the orbital space stations "Salut" and "Mir". This field can displace sensory organelles (statoliths) inside receptor cells and such displacement should cause a physiological reaction of the plant-tropistic curvature. Experiments were conducted in the custom-built "Magnetogravistat" facility, where seeds were germinated and grown for 3-4 days in a magnetic field with the dynamic factor grad (H2/2) approximately equal to 10(7) Oe2/cm, then fixed on orbit and returned to Earth for analysis. It was found, that 93% of the seedlings were oriented in the field consistently with curvature in response to displacement of statoliths along the field gradient by ponderomotive magnetic forces, while control seedlings grew in the direction of the initial orientation of the seed. This suggests, that gravity receptors of plants recognized magnetic forces on statoliths as gravity, and that gravity stimulus can be substituted for plants by a force of a different physical nature.

Characterization of alkali treated flax fibres by means of FT Raman spectroscopy and environmental scanning electron microscopy.

Flax fibres grown under well managed conditions were submitted to NaOH chemical treatments, so called Mercerization. The extent of the polymorphic transformation of cellulose I into cellulose II taking place within the crystalline domains of the fibre cellulose was dependent on the alkali concentration. FT Raman spectroscopy turned out to represent an ideal tool for detecting the polymorphic transformation of the cellulosic fine structure of the flax fibres in vivo. In addition to the differences of the FT Raman spectra in the frequency range below 1500 cm(-1), second derivatives of the spectra in the range of the CH stretching vibrations could also be used to distinguish the two polymorphic modifications. The intensity ratio R of the stretching modes v(s)COC and v(as)COC represents a spectral parameter characterising the molecular structure of the flax fibres. As a supplementary tool, Environmental scanning electron microscopy (ESEM) was used to visualize the microstructural fibre properties dependent on the alkali concentrations during the Mercerization.