Virus-Induced Gene Silencing (VIGS) in Flax (Linum usitatissimum L.) Seed Coat: Description of an Effective Procedure Using the transparent testa 2 Gene as a Selectable Marker

  • Christophe HanoEmail author
  • Samantha Drouet
  • Eric Lainé
Part of the Methods in Molecular Biology book series (MIMB, volume 2172)


Virus-induced gene silencing (VIGS) has been successfully applied for functional analysis of genes in many plant species. Many protocols have been established but mainly for gene expression study in vegetative tissue. Here, we present the critical steps of an optimized procedure of VIGS in flax (Linum usitatissimum L.) seed coat using the transparent testa 2 gene as a selectable marker. The present protocol may serve as an effective tool for functional characterization of genes involved in seed coat formation and/or biological functions.

Key words

Flax Flavonoids Functional genetic Seed coat Virus-induced gene silencing 



This work was supported by Ligue contre le Cancer and Loire Valley Regional Council.


  1. 1.
    Francoz E, Lepiniec L, North HM (2018) Seed coats as an alternative molecular factory: thinking outside the box. Plant Reprod 31:327–342CrossRefGoogle Scholar
  2. 2.
    Fliniaux O, Corbin C, Ramsay A et al (2014) Microwave-assisted extraction of herbacetin diglucoside from flax (Linum usitatissimum L.) seed cakes and its quantification using an RP-HPLC-UV system. Molecules 19:3025–3037CrossRefGoogle Scholar
  3. 3.
    Corbin C, Fidel T, Leclerc EA et al (2015) Development and validation of an efficient ultrasound assisted extraction of phenolic compounds from flax (Linum usitatissimum L.) seeds. Ultrason Sonochem 26:176–185CrossRefGoogle Scholar
  4. 4.
    Renouard S, Corbin C, Lopez T et al (2012) Abscisic acid regulates pinoresinol-lariciresinol reductase gene expression and secoisolariciresinol accumulation in developing flax (Linum usitatissimum L.) seeds. Planta 235:85–98CrossRefGoogle Scholar
  5. 5.
    Garros L, Drouet S, Corbin C et al (2018) Insight into the influence of cultivar type, cultivation year, and site on the lignans and related phenolic profiles, and the health-promoting antioxidant potential of flax (Linum usitatissimum L.) seeds. Molecules 23:2636CrossRefGoogle Scholar
  6. 6.
    Lamblin F, Aimé A, Hano C et al (2007) The use of the phosphomannose isomerase gene as alternative selectable marker for Agrobacterium-mediated transformation of flax (Linum usitatissimum). Plant Cell Rep 26:765–772CrossRefGoogle Scholar
  7. 7.
    Lacomme C (2015) Strategies for alternative plant traits using virus-induced gene silencing technologies. In: Mysore KS, Senthil-Kumar M (eds) Plant gene silencing: methods and protocols, vol 1287. Springer Sciences+Business Media, New YorkCrossRefGoogle Scholar
  8. 8.
    Chantreau M, Chabbert B, Billiard S et al (2015) Functional analyses of cellulose synthase genes in flax (Linum usitatissimum) by virus-induced gene silencing. Plant Biotechnol J 13:1312–1324CrossRefGoogle Scholar
  9. 9.
    Lepiniec L, Debeaujon I, Routaboul J et al (2006) Genetics and biochemistry of seed flavonoids. Annu Rev Plant Biol 57:405–430CrossRefGoogle Scholar
  10. 10.
    Corbin C, Drouet S, Mateljak I et al (2017) Functional characterization of the pinoresinol–lariciresinol reductase-2 gene reveals its roles in yatein biosynthesis and flax defense response. Planta 246:405–420CrossRefGoogle Scholar
  11. 11.
    Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158CrossRefGoogle Scholar
  12. 12.
    Morel G, Wetmore RH (1951) Fern callus tissue culture. Am J Bot 38:141–143CrossRefGoogle Scholar
  13. 13.
    Yoshida K, Ma D, Constabel CP (2015) The MYB182 protein down-regulates proanthocyanidin and anthocyanin biosynthesis in poplar by repressing both structural and regulatory flavonoid genes. Plant Physiol 167:693–710CrossRefGoogle Scholar
  14. 14.
    von Lintig J, Kreusch D, Schroder J (1994) Opine-regulated promoters and LysR-type regulators in the nopaline (noc) and octopine (occ) catabolic regions of Ti plasmids of Agrobacterium tumefaciens. J Bacteriol 176:495–503CrossRefGoogle Scholar
  15. 15.
    Hano C, Martin I, Fliniaux O et al (2006) Pinoresinol-lariciresinol reductase gene expression and secoisolariciresinol diglucoside accumulation in developing flax (Linum usitatissimum) seeds. Planta 224:1291–1301CrossRefGoogle Scholar
  16. 16.
    Liu Y, Schiff M, Dinesh-Kumar SP et al (2002) Virus-induced gene silencing in tomato. Plant J 31:777–786CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC INRA USC1328)Université d’Orléans—Pôle Universitaire d’Eure et LoirChartresFrance

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