Production of high value compounds from plants using the hairy roots method

1. Genetic transformation of Gentiana lutea with Agrobacterium rhizogenes: production and quantitative determination of secoiridoid glucoside in transformed hairy root (HR) cultures.

The aim is to produce high value plant’s compounds using the hairy root (HR) method. This method is based on the infection of plants by Agrobacterium rhizogenes which induces rapid growth of adventive roots (figure 1). From the transformed roots it is possible to extract and purify targeted high value molecules. This method present many advantages :

ü No hormones are neededü Simpler and shorter than field culturesü Provides constant quality end productsü Sustainable and respectuful towards the environment

Infection of the explants with Agrobacterium rhizogene

Set up of the HRs culture

Scaling up into mist bioreactors

ExtractionCaracterization and purification

Production of high value compounds

The hairy roots method

IntroductionGentianaceae is a family of flowering plants which are traditionally used in European medicine for their beneficial gastro-intestinal properties. The roots of Gentiana lutea are remarkable for the intensely bitter properties due to the high content of "secoiridoid glucoside" biomolecules. It is a slow-growing plant and takes a lot of time before the roots are able to be harvested. Because of the uncontrolled use of roots for glucoside extraction and for liquor manufacturing, G. lutea is close to extinction in its natural habitats.Hairy roots technologies applied to this G. lutea could be a sustainable and easier alternative to agricultural processes or wild crop production for producing valuable biomolecules.The aim of my study was to transform G. lutea with Agrobacterium rhizogenesand to determine the capacity of G. lutea HR to produce "secoiridoid glucosides".

MVApathway

GPP

Sweroside

-Gluc.

Swertiamarin

-Gluc.

Gentiopicroside

-Gluc.

Amarogentin

MEPpathway

Fig 2: Schematic representation of the secoiridoids pathway (adapted from Padhan et al. (2014))1

Sweroside which is synthesized from geranyl diphosphate (GPP) was the common intermediate of the secoiridoid glucosides.Swertiamarin, gentiopicroside and amarogentin take the mevalonate (MVA) or the non mevalonate (MEP) pathway of terpene biosynthesis. In addition, amarogentin contains the biphenylcarboxylic acid (BCA) moiety which was presumed to be biosynthesized by the shikimate pathway.

shikimate pathway

HR ?

BCA

0

20

40

60

80

100

120

Shoots from in vitro culture

Shoots from transformed plants

Roots from transformed plants

Gentiopicrodide

Sweroside

Swertiamarin

Transformed plants

Fig 4: Variation in content of gentiopicroside, sweroside and swertiamarin in three different cultures of Centaurium erythraea (adapted from Piatczak et al. (2006)2

The roots from transformed plants contain the lowest level of sweroside and swertiamarin but the highest level of gentiopicroside. These results suggest a higher activity of putative enzyme(s) involved in the conversion of swertiamarin to gentiopicroside in HR.

HR

References(1) Padhan, J.K., Kumar, V., Sood, H., Singh, T.R., and Chauhan, R.S. (2015). Contents of therapeutic metabolites in

Swertia chirayita correlate with the expression profiles of multiple genes in corresponding biosynthesis pathways. Phytochemistry 116, 38–47.

(2) Piatczak, E., Krolicka, A., and Wysokinska, H. (2006). Genetic transformation of Centaurium erythraea Rafn by Agrobacterium rhizogenes and the production of secoiridoids. Plant Cell Reports 25, 1308–1315.

(3) Bensaddek, L., Villarreal, M.L., and Fliniaux, M.-A. (2008). Induction and growth of hairy roots for the production of medicinal compounds. Electronic Journal of Integrative Biosciences 3, 2–9.

The first step consists on generation of axenic plant from G. lutea seeds. Usually the seeds of G. lutea require a long cold stratification period. To break down (to by-pass) the dormancy and reduce the time before germination we will use "pretreated seeds” purchased from Jelitto®.

The second step consists on transformation of G. lutea with A. rhizogenes.The leaf explants were incubated with A. rhizogenes.The putatively transformed roots are checked using PCR to confirm T-DNA integration into the plant.

HPLC

HPTLC

PCR

Extraction and phytochemical analysis of secoiridoids biomolecules from G. lutea are performed by using HPLC and HTPLC. The goal of this step is to compare qualitative and quantitative bitter compounds content to wild, cultured and transformed G. lutea roots.

1st Step

2nd Step

3nd Step

Fig 3: From seed to interest biomolecules

ConclusionPiatczak et al. (2006) shows that the transformed plants of Centaurium erythraea, a member of gentianaceae family, are able to produce more secoiridoid than non transformed plants of C. erythraea .The transformation of G. lutea represents a key step . According to Bensaddek et Al (2008)3, the success of the transformation depends on various parameters such as the nature of of the A. rhizogenes strain, the infection conditions (culture medium) and the species of the plant.Scaling up G. lutea HR to industrial levels represents a great challenge. The purpose of this preliminary study was to determine if G. lutea HR may represent a valuable source of secoiridooids.

Results and discussion

Material and Methods

-Gluc.-

Fig 1: Representation of the hairy roots method

Christophe Loth, Dounia Dhaou, Arthur Muller, Master’s students, M1 Biologie et Valorisation des Plantes – Valorisation des Ressources Végétales, Université de Strasbourg, France

Supervisor: Laurence Gondet, Rozenn Menard, Pascaline Ullmann

mg.g-1DW