Development and testing of Flax (Linum Ussitatisimum L.

) TILLING Population as a Resource for the Plant Genetic Improvement

Flax (Linum Ussitatisimum L., Linaceae) fibre is pale yellow, rich in cellulose (70%), luxurious, elegant, comfortable, thermo regulating, non-allergenic, antistatic and antibacterial but less flexible and stronger than cotton. The fibre has various applications especially in textile industry. In India, due to lack of high yielding varieties with quality fibre, flax cultivation is yet to take off substantially despite of having conducive agro-climatic conditions in different regions. The cultivation of fibre flax to meet the domestic demand (around 800 MT of flax fibre), India spend valuable foreign currency equivalent of 400 Million Rupees in order to import raw flax fibre from Belgium and Poland. Though, CRIJAF has taken the initiative and developed high fibre yielding breeding lines (JRF-2, JRF-1) even than the quality of flax fibres is not at par to the European countries. In this regard improvement of fibre quality in flax is of utmost importance. Since, narrow genetic diversity in flax germplasm is a serious limiting factor in flax fibre quality improvement programme. Hence, there is a need to effectively expand and spot the desirable genetic variability for the target traits by inducing EMS mediated mutation in flax. Moreover, identification of dlpf (deficient lignified phloem fibre) mutant of C. olitorius having low lignin supports possibility of mutation for the development of better quality flax mutants. The reverse genetic strategy like, TILLING (Targeting Induced Local Lesion IN Genome) identify lesions in specific genes prior to any phenotypic characterisation. This takes advantage of growing knowledge of genomic sequence and hypotheses for gene functions based on research in model organism and multiple sequence alignments. Moreover, Plants obtained through the TILLING method are not regarded as genetically modified, are not subjected to special regulations and may escape the political context of controversial plants like BT-cotton and brinjal. Against this background, there are enough justification to initiate research on TILLING of EMS induced mutants of Flax by employing growing knowledge of genomics resources and tools for improving fibre yield and quality in Flax. The proposed research focuses on the generation of mutations in flax using chemical mutagen, determination of mutation in the targeted genes and the utilization of the useful mutations in the flax improvement programs. Objectives: 1. 2. 3. 4. To optimize mutagenesis and develop TILLING population of Flax To produce a phenotypic data base and detailed characterisation of selected Flax fibre mutants To set-up a functional flax TILLING panel To undertake TILLING for improving bast fibre quality traits in terms of lignin and hemicelluloses contents

Budget: Head Non-recurring equipment Civil works recurring Total 25 25 50 50 50 30 130 30 30 25 25 25 25 75 50 135 260 2012-13 2013-14 2014-15 2015-16 2016-17 total

In order to be competitive in the world market, changes in flax fibre lignin content including gross architecture (texture and cell wall constituents) through a gain of control over key metabolites in the lignin metabolic grid (phenylpropanoid pathway) is imperative.
Lignification of flax fibers, although weak, causes a decrease in their commercial quality. due to difference in type of linkage, fiber lignin mainly consists of guaiacyl (G) units in contrast to the mixed guaiacyl-syringyl (G-S) lignin type occurring in xylem fibers. The type of linkages within a lignin polymer depends on the methylation of either the 3-hydroxyl groups or both 3OH and 5-OH groups, which is controlled by two enzymes: caffeate 3-O-methyltransferase (COMT) and caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT).

Reverse genetic

approaches TILLING (Targeting Induced Local Lesion IN Genome) which identify lesions in specific genes prior to any phenotypic characterisation are effective strategies for developing lines with improved fibre qualities in terms of fibre chemistry (such as low lignin content). . This takes advantage of growing knowledge of genomic sequence and hypotheses for gene functions based on research in model organism and multiple sequence alignments. Moreover, Plants obtained through the TILLING method are not regarded as genetically modified, are not subjected to special regulations. TILLING for candidate genes, such as COMT and CCoAOMT in the lignin metabolic grid, will be an effective strategy for developing lowlignin flax, qualitatively superior in terms of fibre fineness and tensile strength.

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT, EC 2.1.1.104) down-regulated-flax (Linum usitatissimum) plants were generated using an antisense strategy and functionally characterized. Chemical analyses (acetyl bromide and thioacidolysis) revealed that the lignin quantity was reduced and that the Syringyl/Guaacyl (S/G) lignin monomer ratio was modified in the non-condensed lignin fraction of two independent down-regulated lines. These modifications were associated with altered xylem organization (both lines), reduced cell-wall thickness (one line) and the appearance of an irregular xylem (irx) phenotype (both lines). In addition UV microspectroscopy also indicated that CCoAOMT down-regulation induced changes in xylem cell-wall structure and the lignin fractions. Microscopic examination also suggested that CCoAOMT down-regulation could influence individual xylem cell size and identity. As a first step towards investigating the cellular mechanisms responsible for the unusual structure of flax lignin (G-rich, condensed), recombinant flax CCoAOMT protein was produced and its affinity for different potential substrates evaluated. Results indicated that the preferred substrate was caffeoyl coenzyme A, followed by 5-hydroxyconiferaldehyde suggesting that flax CCoAOMT possesses a small, but probably significant 5' methylating activity, in addition to a more usual 3' methylating activity.

Caffeoyl-coenzyme A 3-O-methyltransferase enzyme activity, protein and transcript accumulation in flax (Linum usitatissimum) stem during development.
Day A, Dehorter B, Neutelings G, Czeszak X, Chabbert B, Belingheri L, David H.

Source
Laboratoire de Physiologie des Parois Vgtales UPRES 2702, Universit des Sciences et Technologies de Lille, Bat SN2, F-59655 Villeneuve d'Ascq cedex, France Laboratoire de Glycobiologie Structurale et Fonctionnelle UMR-CNRS 8576, Universit des Sciences et Technologies de Lille, Bat C9, F-59655 Villeneuve d'Ascq cedex, France Equipe Parois et matriaux fibreux, UMR-INRA FARE, Esplanade R Garros BP224, F-51686 Reims cedex 2, France.

Abstract
Flax (Linum usitatissimum) is a commercially important fiber crop in Europe. Lignification of its phloem fibers, although weak, causes a decrease in their commercial quality. In flax, fiber lignin mainly consists of guaiacyl (G) units in contrast to the mixed guaiacyl-syringyl (G-S) lignin type occurring in xylem fibers. G lignins are reported as more condensed polymers due to a higher frequency of 5-5 linkages, whereas the deposition of syringyl end groups in lignins increases the proportion of alky-aryl ether linkages as beta-O-4-bonds. The type of linkages within a lignin polymer depends on the methylation of either the 3-hydroxyl groups or both 3-OH and 5-OH groups, which is controlled by two enzymes: caffeate 3-O-methyltransferase (COMT) and caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT). First, we measured the in vitro activity of both OMTs in the flax stem tissues during stem development. CCoAOMT activity varied in the same way as COMT, i.e. increased gradually with stem maturity, from the top to the bottom of the stem, was maximum at the flowering stage and was lower, but still scorable, in the outer fiber-bearing tissues than in the xylem cells. In a second step, we focused our studies on the characterization of CCoAOMT in order to understand the implication of this enzyme in the lignification of flax fiber cells. CCoAOMT activity appeared to be associated with the accumulation of an acidic 33-kDa polypeptide identified as a CCoAOMT after immunological crossreactivity with a poplar CCoAOMT and microsequencing. The differential accumulation of the CCoAOMT protein was confirmed by immunolocalization on tissue prints and correlated with that of the transcripts, suggesting a transcriptional regulation of CCoAOMT in the flax stem.