Isolation and characterisation of RNA from flax (Linum usitatissimum L.)

A method for isolation of flax RNA is described; properties of the isolated RNA are given. RNA degradation was held to a minimum through a high pH (9.5) extraction buffer, diethylpyrocarbonate (4%) as a nuclease inhibitor, a high concentration (1.5%) of sodium dodecylsulphate, 2 mm Mg²⁺, and separation of the RNA from contaminating materials on Sephadex G-50.     

Integrated consensus genetic and physical maps of flax (Linum usitatissimum L.)

Three linkage maps of flax (Linum usitatissimum L.) were constructed from populations CDC Bethune/Macbeth, E1747/Viking and SP2047/UGG5-5 containing between 385 and 469 mapped markers each. The first consensus map of flax was constructed incorporating 770 markers based on 371 shared markers including 114 that were shared by all three populations and 257 shared between any two populations. The 15 linkage group map corresponds to the haploid number of chromosomes of this species. The marker order of the consensus map was largely collinear in all three individual maps but a few local inversions and marker rearrangements spanning short intervals were observed. Segregation distortion was present in all linkage groups which contained 1–52 markers displaying non-Mendelian segregation. The total length of the consensus genetic map is 1,551?cM with a mean marker density of 2.0?cM. A total of 670 markers were anchored to 204 of the 416 fingerprinted contigs of the physical map corresponding to ~274?Mb or 74?% of the estimated flax genome size of 370?Mb. This high resolution consensus map will be a resource for comparative genomics, genome organization, evolution studies and anchoring of the whole genome shotgun sequence.     

RAPD analysis of the flax (Linum usitatissimum L.) varieties and hybrids of various productivity

Genetic polymorphism in varieties and hybrids of cultivated flax (Linum usitatissimum L.) has been investigated by RAPD-PCR. Analysis with 15 primers has revealed varietal specificity and hybrid inheritance of RAPD alleles. This allows genetic certification of the original varieties and their hybrids for breeding purposes. Polymorphic amplification products were obtained in RAPD analysis of DNA from two cultivated flax varieties with the use of 10-11 nucleotide primers.     

A lipoxygenase-divinyl ether synthase pathway in flax (Linum usitatissimum L.) leaves

Incubation of linoleic acid with an enzyme preparation from leaves of flax (Linum usitatissimum L.) led to the formation of a divinyl ether fatty acid, i.e. (9Z,11E,1'Z)-12-(1'-hexenyloxy)-9,11-dodecadienoic [(omega5Z)-etheroleic] acid, as well as smaller amounts of 13-hydroxy-9(Z),11(E)-octadecadienoic acid. The 13-hydroperoxide of linoleic acid afforded the same set of products, whereas incubations of alpha-linolenic acid and its 13-hydroperoxide afforded the divinyl ether (9Z,11E,1'Z,3'Z)-12-(1',3'-hexadienyloxy)-9,11-dodecadienoic [(omega5Z)-etherolenic] as the main product. Identification of both divinyl ethers was substantiated by their UV, mass-, (1)H NMR and COSY spectral data. In addition to the 13-lipoxygenase and divinyl ether synthase activities demonstrated by these results, flax leaves also contained allene oxide synthase activity as judged by the presence of endogenously formed (15Z)-cis-12-oxo-10,15-phytodienoic acid in all incubations.     

Development of specific and degenerate primers for CesA genes encoding cellulose synthase in flax (Linum usitatissimum L.)

Representatives of the CesA multigene family that control the synthesis of the catalytic subunits of the cellulose synthase complex were described for a number of higher plants. It has been established that the HVR2 region of these genes is class-specific and determines the involvement of the gene product in the synthesis of either the primary or secondary cell wall. The purpose of the current research was to develop degenerate and specific primers for parts of the CesA genes to allow the construction of molecular markers for the class-specific HVR2 region. Two pairs of specific primers for the CesA-1 and CesA-6 genes as well as a pair of degenerate primers for the HVR2 region of all flax CesA genes were developed, based on analysis of the CesA ESTs as well as the full-length cDNA sequences of the CesA genes in Arabidopsis, poplar, maize, and cotton that are available in the GenBank. Fragments of the expected size were amplified using flax cDNA as a template (201 bp for CesA-1, 300 bp for CesA-6, and 600 bp for HVR2). The markers developed in this research can be used for CesA gene cloning and sequencing, analysis of gene copy numbers as well as characterization of tissue- and development specific gene expression.     

SSR and morphological trait based population structure analysis of 130 diverse flax (Linum usitatissimum L.) accessions

A total of 130 flax accessions of diverse morphotypes and worldwide origin were assessed for genetic diversity and population structure using 11 morphological traits and microsatellite markers (15 gSSRs and 7 EST–SSRs). Analysis performed after classifying these accessions on the basis of plant height, branching pattern, seed size, Indian/foreign origin into six categories called sub-populations viz. fibre type exotic, fibre type indigenous, intermediate type exotic, intermediate type indigenous, linseed type exotic and linseed type indigenous. The study assessed different diversity indices, AMOVA, population structure and included a principal coordinate analysis based on different marker systems. The highest diversity was exhibited by gSSR markers (SI = 0.46; He = 0.31; P = 85.11). AMOVA based on all markers explained significant difference among fibre type, intermediate type and linseed type populations of flax. In terms of variation explained by different markers, EST-SSR markers (12%) better differentiated flax populations compared to morphological (9%) and gSSR (6%) markers at P = 0.01. The maximum Nei's unbiased genetic distance (D = 0.11) was observed between fibre type and linseed type exotic sub-populations based on EST-SSR markers. The combined structure analysis by using all markers grouped Indian fibre type accessions (63.4%) in a separate cluster along with the Indian intermediate type (48.7%), whereas Indian accessions (82.16%) of linseed type constituted an independent cluster. These findings were supported by the results of the principal coordinate analysis. Morphological markers employed in the study found complementary with microsatellite based markers in deciphering genetic diversity and population structure of the flax germplasm.     

Cellulose synthase genes that control the fiber formation of flax (Linum usitatissimum L.)

Four cellulose synthase genes were identified by analysis of their class-specific regions (CSRII) in plants of fiber flax during the “rapid growth” stage. These genes were designated as LusCesA1, LusCesA4, LusCesA7 and LusCesA9. LusCesA4, LusCesA7, and LusCesA9 genes were expressed in the stem; LusCesA1 and LusCesA4 genes were expressed in the apex part of plants; and the LusCesA4 gene was expressed in the leaves of fiber flax. The expression of the LusCesA7 and LusCesA9 genes was specific to the stems of fiber flax. These genes may influence the quality of the flax fiber.     

Genetic diversity of cultivated flax (Linum usitatissimum L.) germplasm assessed by retrotransposon-based markers

Retrotransposon segments were characterized and inter-retrotransposon amplified polymorphism (IRAP) markers developed for cultivated flax (Linum usitatissimum L.) and the Linum genus. Over 75 distinct long terminal repeat retrotransposon segments were cloned, the first set for Linum, and specific primers designed for them. IRAP was then used to evaluate genetic diversity among 708 accessions of cultivated flax comprising 143 landraces, 387 varieties, and 178 breeding lines. These included both traditional and modern, oil (86), fiber (351), and combined-use (271) accessions, originating from 36 countries, and 10 wild Linum species. The set of 10 most polymorphic primers yielded 141 reproducible informative data points per accession, with 52% polymorphism and a 0.34 Shannon diversity index. The maximal genetic diversity was detected among wild Linum species (100% IRAP polymorphism and 0.57 Jaccard similarity), while diversity within cultivated germplasm decreased from landraces (58%, 0.63) to breeding lines (48%, 0.85) and cultivars (50%, 0.81). Application of Bayesian methods for clustering resulted in the robust identification of 20 clusters of accessions, which were unstratified according to origin or user type. This indicates an overlap in genetic diversity despite disruptive selection for fiber versus oil types. Nevertheless, eight clusters contained high proportions (70?C100%) of commercial cultivars, whereas two clusters were rich (60%) in landraces. These findings provide a basis for better flax germplasm management, core collection establishment, and exploration of diversity in breeding, as well as for exploration of the role of retrotransposons in flax genome dynamics.     

Notes on Thysanoptera found on flax (Linum usitatissimum L.) in the British Isles

The following eighteen species of Thysanoptera Terebrantia have been found on flax in the British Isles: Melanthrips fuscus (Sulzer), Aeolothrips fasciatus (L.), Anaphothrips obscurus (Müler), Aptinothrips rufus (Gmelin), Chirothrips manicatus Hal., Limothrips cerealium Hal., L. denticornis Hal., Stenothrips graminum Uzel, Taeniothrips atratus (Hal.), T. vulgatissimus (Hal.), Thrips angusticeps Uzel, T. discolor Hal., T. flavus Schrank, T. fuscipennis Hal., T. major Uzel, T. minutissimus L., T. physapus L., T. tabaci Lindeman. Each species is described briefly with notes on habits of adults and larvae, place of pupation, number of generations in the year, hibernation, time of occurrence on plants, plants and objects on which found, host plants of larvae and adults, importance to flax, record of locality and collector on flax, distribution, including altitudes, in the British Isles. More species occur in the south than in the north of Great Britain, and species common to both regions usually occur in greater numbers in the south. The insects breed on certain species of crop plants, weeds or trees of arable land. No damage of economic importance to flax by Thysanoptera has been proven in the British Isles, and the flax thrips, Thrips lini Ladureau, has not been found. Taeniothrips vulgatissimus (Hal.) may breed on flax and its adults, and those of T. atratus (Hal.) may cause superficial damage to petals of flowers. Thrips angusticeps Uzel and T. tabaci Lindeman will probably breed on flax.     

Histological study of embryo-like structures initiated from hypocotyl segments of flax (Linum usitatissimum L.)

Cultivation of flax hypocotyl segments on MS medium supplemented with auxin (2,4-d, NAA) and combination of auxin (NAA) and cytokinin (BAP, zeatin) resulted in production of callus on the cut ends of segments and prolonged cultivation in globular structures resembling early stages of somatic embryos. Embryo-like structures protruded on the surface directly from the subepidermal layers of hypocotyl segments. Despite these globular structures closely resembling somatic embryos, histological observations did not reveal their embryogenic character–organogenesis was the predominant developmental morphogenic pathway. Based on our experiments, as well as on critical revision of existing reports on flax somatic embryogenesis, we conclude, that there has not yet been convincing histological proof of somatic embyogenesis from flax hypocotyl segments.     

Purification of several pectin methyltransferases from cell suspension cultures of flax (Linum usitatissimum L.)

Three pectin methyltransferases (PMT5, PMT7, PMT18; EC 2.1.1.6.x) were solubilized from the endo-membrane complex of flax cells, with 0.05% Triton X-100. After a 3 step-chromatography procedure, PMT7 and PMT5 were purified to apparent homogeneity. PMT5 and PMT7 differed regarding their optimum pH (5 or 7), the methyl acceptor (low or highly methylesterified pectin), their focusing pH range (6-7 or 8-9) and relative molecular mass (40 +/- 5 or 110 +/- 10 kDa). SDS-PAGE of PMT5 and PMT7 did not reveal bands at 40 or 110 kDa but only a silver stained band of about 18 kDa. Two independent methods (photo labelling and enzymatic activity) showed that this silverstained band corresponded to a methyltransferase with affinity for pectins. This polypeptide was of the same size as the enzyme designed PMT18 (18 +/- 3 kDa; pl 4-4.5) recovered during size exclusion chromatography of either PMT7 or PMT5, suggesting that PMT18 bears the catalytic site of PMT5 and PMT7.     

Sensitivity of different flax (Linum usitatissimum L.) genotypes to salinity determined by GE biplot

Plants respond differently to salt stress depending on their genetic structure and the severity of the stress. Salinity reduces seed germination, delays plant emergence, and inhibits seedling growth. The selection of the tolerant genotypes, however, plays a vital role in increasing agricultural output since various genotypes greatly vary for their tolerance to salinity. Therefore, this study determined the impact of five different NaCl levels (i.e., 0, 50, 100, 150 and 200 mM) on seed germination and growth attributes of 10 flax (Linum usitatissimum L.) genotypes. The germination and growth characteristics of the genotypes under study were examined using the biplot approach at varied salt levels. The results indicated that individual and interactive effects of genotypes and salinity levels significantly (p ≤ 0.01 or p ≤ 0.05) affected several seed germination traits. The relations of genotype × germination traits indicated that ‘G4′ and ‘G6′ were the most stable genotypes with the highest performance regarding seed germination characteristics. The genotype ‘G2′ was associated with shoot length, while ‘G7′ was linked with salinity tolerance index. The biplot divided the germination characteristics into five different groups according to sector analysis. Most of the germination parameters had higher values under 100 mM, while some of the parameters had better values under 0, 50 and 200 mM NaCl levels. The tested genotypes varied for their seed germination and growth response depending on the NaCl levels. The genotypes ‘G4′, ‘G5′ and ‘G6′ proved more tolerant to high NaCl levels. Therefore, these genotypes can be used to improve flax productivity under saline soils.     

Identification of microspore-derived plants in anther culture of flax (Linum usitatissimum L.) using molecular markers

The microspore origin of anther-culture-derived plants of flax was determined using inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) markers. Polymorphic fragments between the two parents of the F₁ donor plants were identified and their segregation patterns in anther-culture-derived plants were used to elucidate the origin of those plants and to determine the degree of independence of plants regenerated from the same callus. Using one ISSR primer (UBC 889) and two RAPD primers (UBC 556 and 561), 12 out of 16 plants were unequivocally identified as being derived from microspores. Plants derived from the same callus had identical PCR patterns at five polymorphic loci and thus were likely derived from the same microspore. Therefore, it is proposed that the number of calli forming shoots be used to describe the anther culture efficiency in flax. Received: 3 February 1998 / Revision received: 8 June 1998 / Accepted: 8 July 1998     

Technologically indicative properties of straw fractions of flax, linseed (Linum usitatissimum L.) and fibre hemp (Cannabis sativa L.)

In this study of the behaviour of the fractions of unretted and frost-retted fibre straws in damp air, a production scale method to separate fibre and shive from fibre plants was introduced and tested on bast fibre plants (Linum usitatissimum L. and Cannabis sativa L.). The method consists of optional drying of stalks, unloading bales, milling the straws with a hammer mill, separating the fractions from air stream with a cyclone and finally separating fibres from shives with a screening drum. Fractions were characterized focusing on technologically indicative properties such as equilibrium moisture content, ash and microbiological quality. Unretted fractions of the bast fibre plant stem reached higher equilibrium moisture contents than the retted fractions, and hemp fibres absorbed more moisture from air than did the Linum fibres. In very humid air, all fractions began to lose weight due to moulding. The weight decrease during the first week was lower in frost-retted than in unretted fractions. The frost-retted fractions appeared to be more resistant to humidity in the short term. The total number of microbes and especially the numbers of yeasts and moulds can be used as a criterion of hygienic level. For green fractions, the mould level was similar in fibres and in shives, but frost-retted shives contained more moulds than the unretted shives. The mould content of a fraction had no direct correlation with the moulding tendency of the fraction. The ash contents of fibres were somewhat higher than those of shives, due to a probable soil contamination. Ash content did not have significant correlation with microbiological quality, although ash is a possible risk factor for hygienic quality. According to the results of this study it is highly important to study the quality of the production chain of bast fibre plants to ensure the quality of industrial products. From the producer's point of view, raw material with defined quality can be directed to the most suitable application. The behaviour of fractions in various ambient atmospheres, and other quality aspects such as hygienic level can be used as criteria for defining the most appropriate product applications.     

Xylem changes in the correlative inhibition of lateral bud growth in flax (Linum usitatissimum L.)

Xylem or tracheary changes at the base of the cotyledonary buds of flax seedlings (Linum usitatissimum L.), released from inhibition by decapitation of the main apex were studied. The differentiation of xylem strands and/or tracheary elements was correlated with the growth in length of the lateral buds, especially 48–72 hr after the removal of the main apex. The xylem strands, connected to the hypocotylary stele or not, and the tracheary elements increased with age within and outside the strands of both non-decapitated and decapitated seedlings. In the latter, the differentiation of these structures, however, occurred much earlier and in greater abundance in the same regions. The early growth in length of lateral buds, 1 or 2 hr after decapitation, was correlated with the early development of tracheary perforations in the xylem strands. The xylary strands with perforated elements are known to be more efficient than those without them. Therefore, it is suggested that the inhibition of lateral-bud growth was due, in fact, to a lack of appropriate tracheary perforations in the bud xylem strands that were connected with the hypocotylary stele of flax seedlings.