est destinée au dépôt

Let us specify here that core temperature (set to 470 K) is taken precisely into account in two steps that will be especially detailed in the next part. Basically, the first step is the proper homogenization of the core material (from volumic proportions of UO 2 , H 2 O and SiO 2 , as described in [START_REF] Bentridi | Criticality of Oklo Natural Reactors : Realistic Model of Reaction Zone 9[END_REF]), involving the temperature-dependent density of water. The second step concerns the nuclear code of each isotope (.03c for data at 300 K) combined with the use of Serpent 2's "tmp" instruction allowing to extrapolate these data up to the desired temperature (provided it is below the next available value of 600 K).

All Monte Carlo runs are performed by Serpent 2 [START_REF] Leppänen | User manual of the Monte Carlo code Serpent 2[END_REF] and before that generated by SMURE [START_REF] Méplan | SMURE, a Serpent-MCNP Utility for Reactor Evolution[END_REF] to both compute this core's material evolution and estimate its lifetime. Natural Gd and Sm probably played the important role of Burnable Poisons (BP) to help Oklo Reaction Zones staying critical over a few tens of thousands of years at least. Their common massic proportion within UO 2 is set to 10 ppm and core volumic power to 100 W/m 3 (10 6 times less than in a standard PWR core), which gives a total power of 230 W. In this part, the bare core evolution (described above) at constant power is given as a control case for the following. No reactivity control is made and k eff is let free, increasing from 1 (0.998) at BOC (Beginning Of Cycle) during the first 5 ky and then decreasing (cf. Fig. 1). Fig. 1. Evolution of the bare core keff with initial poisons (10 ppm of Sm and Gd in UO2), while core temperature and power are kept constant. According to this first model, reactivity reserve allows a lifetime of about 20 ky only. EOC (End Of Cycle) is considered attained when k eff goes back to 1, about 20 ky later. Of course, this is a rough estimation and part II will aim at making a more realistic evolution calculation, for a more precise estimation of the lifetime of this same core. The difference between the two estimations will help us to feel how much the new method with variable temperature (and power) is necessary.

One advantage of this first control case is to make obvious the temporary excess reactivity due to the consumption of initial BP. Just to give an idea of the initial rate of this insertion, at run 1 (i.e. 500 y "only" after BOC) k eff has increased from 0.998 to 1.001 (to reach about 1.004 at run 2, i.e. at t = 1 ky). Fig. 2 below shows the evolution of main BP capture rates. While Gd isotopes (157 first and mainly, then 155) clearly bring reactivity through their consumption along the lifetime of 20 ky, 149 Sm has no similar behavior. Much more produced by fission (with an equivalent yield of 1%, 100 times higher than Gd's), its capture rate (close to its equilibrium value in this case) remains relatively constant.

II. Description of all methods within our simple model for core temperature evolution

The main goal is, following inspiration from early thoughts on Oklo ways of reactivity management [START_REF] Naudet | Oklo : des réacteurs nucléaires fossiles (étude physique)[END_REF], to regularly change the core temperature so that criticality is maintained. In reality, reactivity from BP makes power increase first, then temperature. The trick in our model is simply to change temperature first (by means of a pre-evaluated thermal feedback coefficient), and to update power accordingly (by means of a simple lumped thermal model). Such a simple method makes us find the most recent models for Oklo core evolution calculations way too complicated i.e. too rich in unessential hypotheses like cyclic water boiling within the core [START_REF] Ibekwe | Modeling the short-term and long-term behaviour of the Oklo natural nuclear reactor phenomenon[END_REF]. Such a postulated boiling is against most of experimental evidence [START_REF] Naudet | Oklo : des réacteurs nucléaires fossiles (étude physique)[END_REF]. And more generally, it is against a progressive approach of the problem.

For all these reasons, we take the opposite view and suppose that typical temperature increases have been sufficiently limited in Oklo cores so that they can be explained without systematic boiling of their water content. This way, we can use SMURE (with Serpent 2) and simple methods (embedded in the KNACK toolbox described in [START_REF] Nuttin | Study of D2O/H2O-cooled thorium-fueled PWR-like SMR cores using the KNACK toolbox: conversion and safety assessment[END_REF]) to check if such a simple long-term model is compatible or not with some of the most known features of a typical Oklo core. Before describing its main results on our bare core, we detail the complete set of necessary methods in the consistent order of included effects (first water dilatation, then global thermal feedback, temperature update and finally power update).

II.A. Water Dilatation vs. core temperature and re-definition of core material accordingly

Like in [START_REF] Bentridi | Criticality of Oklo Natural Reactors : Realistic Model of Reaction Zone 9[END_REF], volumic proportions and initial densities (of 10.6, 0.88 and 2.65 g/cm 3 for UO 2 , H 2 O and SiO 2 respectively) gives to core material its density (of about 2.52 g/cm 3 ) as well as weight fractions of each element. Water is the only of the three homogenized core parts to see its density significantly change with temperature (at constant pressure). Each time temperature will be updated, the new value of water density will be linearly interpolated (cf. Fig. 3). Then the core material will be re-homogenized, with a new density used together with the new temperature by SMURE for the new Serpent run. Fig. 3. Evolution of water density at 200 bars vs. temperature in K according to NIST [START_REF]Thermophysical Properties of Fluid Systems[END_REF]. The [470 K, 570 K] range is used for linear interpolation (from 420 K to 600 K and above, albeit we will check that 600 K is never reached).

II.B. Constant Global Temperature Coefficient (GTC, pcm/K) for core thermal feedback

Just after BOC, due to BP consumption, k eff is going to increase and will be set close to 1.000 (± a few hundreds of pcm) again by increasing core temperature. Such periodic temperature adjustments (first increases, then decreases towards EOC) will use a Global Temperature Coefficient (GTC) accounting for both Water Dilatation and Uranium Absorption Effects. For our bare core case, without material removal or geometric change, this GTC can be supposed (and will be checked) to remain constant.

GTC is computed as the reactivity difference at BOC between one MC run at nominal conditions and a second one 100 K hotter, divided by this temperature increase. 800 active cycles of 32000 neutrons per cycle give a statistical precision on GTC of ±0.4 pcm/K (which will be rounded to ±1 pcm/K for systematic errors within our very simple homogeneous model). Water Dilatation Effect, occurring in the H 2 O part of the real core, is taken into account by a complete re-homogenization of core material (new density and isotopic proportions) at 470+100 = 570 K (starting from a water density of 0.74 g/cm 3 , as detailed by Fig. 3). Uranium Absorption Effect, occurring in the UO 2 part, is due to the reduction of resonance self-shielding through Doppler Effect. It is added via Serpent's "tmp" instruction (made available in SMURE) which extrapolates parameters for resonant absorption from the temperature of the used extension code (here .03c for 300 K) up to the temperature wanted (here 570 K), provided it is below the next temperature in the "xsdata" file (here 600 K). Beginning of core material definition in Serpent is thus changed from "mat m1 -2.5165 tmp 470" at BOC to "mat m1 -2.4745 tmp 570" in its hot version, giving a GTC of -34 pcm/K. Such a high value is typical of homogeneous well-moderated cores and is worth noticing, since it directly explains why temperature increase remains quite limited.

II.C. Algorithm based on GTC for conversion of (excess or missing) reactivity into temperature

Fig. 4. Principle of temperature adjustment for criticality, every 2 runs (every ky) via a constant GTC of -34 pcm/K. Like in the control case at constant power, one Serpent run is performed every 500 years to update average cross-sections. The difference here is that k eff is kept as close as possible to unity, by the only change of core temperature. During a first phase, reactivity from BP makes temperature increase (thus converting initial reactivity reserve based on Sm and Gd into a "thermal" form). In the second and last phase, temperature decrease allows to keep the core critical until EOC when T ext is reached. Fig. 4 describes how temperature is updated just after BOC. The two first runs (0 and 1) are used to extrapolate what would be k eff at t = 1 ky without temperature update. Excess reactivity ∆ρ (400 pcm) is then cancelled out by adding, just before run 2, ∆T = [0 (goal) -∆ρ (start)]/GTC = -∆ρ/GTC to core temperature. New water density asks for complete core material re-homogenization, with new density and isotopic proportions. Like for GTC, 800 active cycles of 32000 neutrons per cycle are used, with a standard deviation on k eff of 25 pcm. This causes adjusted k eff to fluctuate within a few hundreds of pcm around 1, which is sufficient (with about one week on 32 processors for a 100-ky evolution).

II.D. Constant global core-to-exterior conductance G (W.K -1 ) for associated power update

Core power has to be adjusted too, consistently with temperature. For that purpose, a simple lumped thermal coupling model adapted from [START_REF] Nuttin | Study of D2O/H2O-cooled thorium-fueled PWR-like SMR cores using the KNACK toolbox: conversion and safety assessment[END_REF] and described by equation ( 1) is used. In this simple thermal balance of the core in W, d is its density (in kg.m -3

), V its volume (2.3 m 3 here) and C its heat capacity (in J.kg

-1 .K -1
). Main trick of this model is the global conductance G (in W.K -1

), which is supposed (like GTC, for same reason) constant and allows to make a quasi-static power correction according to each temperature update. At BOC, core power P is 230 W. Together with initial T core of 470 K and constant T ext of 420 K, it allows to set G = 230 / (470 -420) = 4.6 W.K -1

. For comparison, typical value for G in a PWR-like core (where heat transfer to coolant is optimized) is higher by 4 orders of magnitude [START_REF] Nuttin | Study of D2O/H2O-cooled thorium-fueled PWR-like SMR cores using the KNACK toolbox: conversion and safety assessment[END_REF].

𝑑 𝑐𝑜𝑟𝑒 𝑉 𝑐𝑜𝑟𝑒 𝐶 𝑐𝑜𝑟𝑒 𝑑𝑇 𝑐𝑜𝑟𝑒 (𝑡) 𝑑𝑡 = 𝑃 𝑐𝑜𝑟𝑒 (𝑡) -𝐺[𝑇 𝑐𝑜𝑟𝑒 (𝑡) -𝑇 𝑒𝑥𝑡 ] (1)
Equation ( 1) will not be solved continuously but used every ky under quasi-static form of equation [START_REF] Leppänen | User manual of the Monte Carlo code Serpent 2[END_REF].

In this formulation at equilibrium, "2n" stands for any even number of MC run (like run 2 on Fig. 4) just before which temperature is adjusted and power adapted accordingly.

𝑃 𝑐𝑜𝑟𝑒 𝑄𝑆 (𝑡 2𝑛 𝑀𝐶 ) = 𝐺[𝑇 𝑐𝑜𝑟𝑒 𝑄𝑆 (𝑡 2𝑛 𝑀𝐶 ) -𝑇 𝑒𝑥𝑡 ] (2)
In order to directly relate power variation ∆P to temperature variation ∆T, the difference between two successive even MC run times can be computed for each term, giving equation [START_REF] Méplan | SMURE, a Serpent-MCNP Utility for Reactor Evolution[END_REF].

∆𝑃 𝑐𝑜𝑟𝑒 𝑄𝑆 (𝑡 2𝑛 𝑀𝐶 ) = 𝐺 ∆𝑇 𝑐𝑜𝑟𝑒 𝑄𝑆 (𝑡 2𝑛 𝑀𝐶 ) (3)
Let us note that such a power update will have drastic consequences on evolution calculations. As temperature will first increase, power will follow. So will do the average flux level, speeding up uranium consumption and all other reaction rates. During the second phase (of temperature decrease down to T ext ), power will progressively go down to zero according to equation ( 3). This time will give us EOC, i.e. an estimation of core lifetime under the hypothesis of such a simple operation based on BP only.

III. Main results, including thermal evolution, obtained for our bare core test case

In part I, the studied bare core has been evaluated to last only 22 ky when operated at constant power. Corresponding final burnup is 1.7 GWd/t (of metal U, whose initial inventory is 1.1 tons). Main goal of this part is, using methods described in part II, to see how core lifetime and burnup are extended when considering its T/P history. Main results are first given. Then associated power and flux curves are considered, before analyzing evolution dynamics based on BP by means of a simple neutron balance. Main inventories, related to neutron balance, are finally detailed to complete these results.

III.A. Maintained criticality, lifetime estimation and temperature curve

Fig. 5 shows that k eff can be maintained to 1.000 until t = 60 ky, before clearly decreasing (despite its approximative adjustment due to the limited MC precision and especially to large 500-y MC steps). Corresponding final burnup is 3.1 GWd/t (thus almost doubled compared to control case). Let us note that, compared to Fig. 1's increase of almost 1000 pcm from BOC in control case, the jagged k eff evolution is sufficiently flat though to give the quite acceptable temperature curve of Fig. 6.

Another remark about Fig. 5 is that reactivity drop after EOC is much slower here (about -10 pcm/ky) than for control case (about -60 pcm/ky), because of the power drop. Hence almost only the decay of 239 Pu (more reactive than 235 U, i.e. with higher νΣ fis ) plays a role on k eff decrease afterwards.

Fig. 6. Evolution of core temperature from Tinit (470 K) at BOC down to Text (420 K) at EOC (reached at t = 60 ky).

Fig. 6 shows that a maximal temperature of 495 K (25 K above T init ) is reached at t = 5 ky. It then takes about 55 ky to use the full "thermal" reactivity reserve -75 K x -34 pcm/K = 2550 pcm). At t = 5 ky, water and core material densities are minimal, slightly below 0.85 and 2.506 g/cm 3 respectively.

III.B. Associated power and flux evolutions, with remarks on such a quasi-static approach

According to equation ( 3) and as shown by Fig. 7, power is directly adjusted from temperature updates. It can be disturbing for a reactor physicist to see power evolving so much while k eff has just been said to be maintained equal to 1. The explanation is that (super-or sub-critical) power changes occur over very short time intervals in reality, which are implicitely accounted for in our quasi-static approach. Like in standard core evolution calculations, the average flux within each evolving cell (unique in our simple case here) is normalized by the total power. Hence, flux on Fig. 8 follows (with possible slight differences due to small spectrum variations) the same trend as power on Fig. 7. Let us recall about power that, in reality, transients start with a reactivity insertion that makes power increase first. Then, after a thermal delay, power triggers temperature increase which finally counter-acts on reactivity via thermal feedback. In our method, power and temperature interventions are inverted. Temperature is updated according to GTC first, then power is corrected consistently via the simple thermal model.

III.C. Main reaction rates, simplified neutron balance and analysis of BP roles in core dynamics

Fig. 9 and 10 show the evolution of the main reaction (capture or fission) rates, which are sufficient to sketch the neutron balance of our reference bare core, at any time. For instance, at t = 5 ky (T and P peak time), 235 U fission rate is about 10 THz (in the following, "THz" is used for 10 12 s -1 as an ugly but convenient unit). Multiplied by an average number of neutrons produced per fission of 2.5, it gives a total disappareance rate for neutrons of about 25 THz at t = 5 ky. Hence, establishing the (critical) neutron balance at this peak time simply consists in detailing main disappearance rates (including leakage) whose sum is equal to 25 THz. This is done (by means of Fig. 9 and 10) in Tab. 1. Tab. 1 is shown here for peak time only as an example to explain the principle of such a simplified neutron balance. Within Light Nuclei (whose peak contribution is evaluated to about 0.3+1.2+2.5 THz according to Fig. 10), the share of Fission Products is rather low (0.3 THz) and relatively constant until a few tens of ky at least. As expected, leakage rate is high from this bare core with a probability of 4/25 = 16%, checked (in Serpent outputs) to remain constant over the whole cycle.

In order to specify BP contribution to initial reactivity insertion (and associated temperature increase), we show below in Tab. 2 another neutron balance which details more precisely BOC. With total power of 230 W (against 340 W at peak time), the total disappearance rate is logically (and proportionally) lower and amounts to 16.9 THz. Capture rates by Sm and Gd isotopes (cf. Fig. 11), normalized by the total disappearance rate at BOC, will give us their effective reactivity worths and finally (via GTC) their respective contributions to the temperature increase from BOC to peak time.

Fig. 2 .

 2 Fig. 2. Detail of reaction rates for initial Sm and Gd most capturing isotopes, showing their chronology in reactivity management (first and mainly 157 Gd, then 155 Gd). In this control case at constant power, 149 Sm is no real BP.

Fig. 5 .

 5 Fig. 5. Evolution of keff maintained to unity (at ± 200 pcm) by temperature adjustment, until EOC around t = 60 ky.

Fig. 7 .

 7 Fig. 7. Evolution of core power, consistently with temperature, from Pinit (230 W) at BOC down to zero at EOC.

Fig. 8 .

 8 Fig. 8. Evolution of the core-averaged flux (max. of 9.3 108 cm -2 .s -1 at t = 5 ky), shaped by the variable power level.

Fig. 9 .

 9 Fig. 9. Evolution of the main reaction rates of actinides ( 235 U fission is largely dominating, while 239 Pu is negligible).

Fig. 10 .Tab. 1 .

 101 Fig. 10. Evolution of the main capture rates of Light Nuclei. So-called Fission Products include initial Sm and Gd.

UTab. 2 .

 2 Main reaction rates for neutron loss (values in THz) at BOC, for BP contributions to temperature increase.

Fig. 11 .

 11 Fig. 11. Reaction rates of Sm and Gd most capturing isotopes, with only contribution before peak being 157 Gd's.

Fig. 11

 11 Fig. 11 shows that, like in control case (with same initial concentrations of 10 ppm for natural Sm and Gd in UO 2 ), 157 Gd plays alone an effective role of BP during first phase of temperature increase (until peak at t = 5 ky). During second phase, decreases of

Gd's capture rate and especially of 149 Sm's are nothing but simple consequences of power (and flux) decrease already described (cf. III.B). While 155 Gd's contribution is delayed and remains negligible because of its lower cross-section (of 7500 barn against 32000 for 157 Gd), 149 Sm (with intermediary average of 17000 barn) more than compensates its capture rate by a high equivalent fission yield of about 1% (100 times higher than Gd's).

At BOC, 157 Gd represents a potential reactivity of about 850 pcm (0.144/16.9), which is quite quickly delivered over the first 5 ky. This is consistent, via GTC of -34 pcm/K, with the computed temperature increase of 25 K (-850/-34). Of course, BP respective efficiencies depend on their initial concentrations (which should be compared to their equilibrium values to identify those which are too low at BOC to play a role). Yet 157 Gd remains the main contributor in all studied cases, like in these two extremes: -In the case of an initial concentration as low as 5 ppm of Gd and Sm in UO 2 , with core radius and height reduced to 88 cm, temperature rise is maximal at same peak time of t = 5 ky and reaches 10 K only. This is consistent with initial reactivity from 157 Gd of about 400 pcm (and GTC of -35 pcm/K).

-A high initial BP concentration of 20 ppm in UO 2 , with core radius and height of 96 cm, gives at t = 4 ky a maximal temperature rise of 80 K. Let us note that even in this case, maximal temperature (of 550 K) still remains well below the boiling limit of 640 K. Logically, 157 Gd's reactivity is doubled compared to reference case and amounts to 1700 pcm. Via GTC of -33 pcm/K, this gives only about 50 K of temperature rise. Indeed at such concentrations, other isotopes are high enough to start above their equilibrium concentrations at BOC and thus to be consumed before peak time, contributing to the remaining 900 pcm (and 30 K) with about one third only coming from 149 Sm.

Let us go back to our reference case (10 ppm of BP at BOC) for second phase of temperature drop, from its maximal value of 495 K down to T ext (420 K). This corresponds to a reactivity reserve of about 2600 pcm (-75 K x -34 pcm/K), progressively used to compensate the core reactivity swing estimated at constant power, for the control case, to about -60 pcm/ky (cf. Fig. 1). Here, the power drop reduces impact on neutron balance of capture rates from nuclei with non negligible decay rates. This lowers the average reactivity loss to slightly less than -50 pcm/ky, making second phase lasts about 55 ky.

It should be noted that the lengths of both phases (and thus core lifetime) do not depend on the initial BP concentration. For first phase, peak time is close to 5 ky in all cases since it is determined by the complete consumption of the "fastest" BP isotope (which is 157 Gd and whose kinetics does not change with BP concentration). As for second phase duration (55 ky), it is related to criticality features like core size and fissile content (and thus common to all BP cases). Only maximal temperature attained depends on BP concentration (via associated reactivity reserve at BOC), with 480 K, 495 K and 550 K obtained for initial concentration of 5, 10 and 20 ppm of natural Gd (and Sm) in UO 2 respectively. Among other inventories, 235 U's one on Fig. 13 is a good indicator of how power and flux are reduced until core final "shutdown". Beyond the easily interpreted inflection point at t = 5 ky, it is interesting to notice that its minimal inventory is attained before EOC (around t = 45 ky), when its production by 239 Pu decay starts to outweigh its consumption. Fig. 14 shows the impact of 239 Pu decay on its own inventory after about one half-life (of 24 ky). Like 236 U, Fission Products show a quite unusual build-up (due once again to variable power), progressively slowing down until final equilibrium at BOC.

III.D. Main core inventories related to neutron balance and reactivity swing

IV. Conclusions and perspectives (towards more realistic "long-term only" models)

Previous evolution calculations, performed at constant power, had already shown the important role of Burnable Poisons (BP) played by Rare Earths (RE) initially present in uranium ore. Let us note that some other natural RE, like Dy or Er, could be considered to complete this study. Explained here, our last developments allow to semi-continuously convert BP reactivity into temperature T, first increasing (over a rather short period of a few ky) and then decreasing (over a longer period of a few tens of ky) down to the "ambient" value T ext . At this time, core shutdown is imposed by our simple thermal lumped model that couples directly power to T-T ext . Such a simple methodology, applied to a simple case for the sake of principle description, confirms that the "usual boiling" hypothesis is not mandatory at all. In our opinion, a good safeguard in the gigantic work of reconstituting operational schemes of Oklo's reaction zones is to add complexity only when it is absolutely necessary, and to convoke short-term phenomena (like in [START_REF] Ibekwe | Modeling the short-term and long-term behaviour of the Oklo natural nuclear reactor phenomenon[END_REF]) only when all likely long-term effects have been considered and used.

Additional effects (involving geology, geochemistry, tectonics, etc) could probably extend lifetime by a few tens of ky. Most promising one could be Quartz Dissolution (QD), resulting in the semi-continuous reduction of SiO 2 volumic proportion within the core. According to [START_REF] Naudet | Oklo : des réacteurs nucléaires fossiles (étude physique)[END_REF], QD could have taken place just after criticality and operated at a constant rate (of typically a few vol% evacuated every ky). This would lead to a progressive reduction of core volume (e.g. of its height), which could be over-compensated by uranium concentration in core material (thus adding some extra reactivity reserve to BP's). Due to such tremendous changes, GTC will probably have to be regularly re-evaluated. Extra axial cells being necessary to model QD effects on core geometry with SMURE, reflected cores will be considered.


Thi Dao Vu

Cyril C Jousse
email: cyril.jousse@uca.fr

Nathalie Pawlicki-Jullian

Séverine Schiltz
email: severine.schiltz@u-picardie.fr

Thi Kieu

Oanh Nguyen

Thi Le

Minh Tran

Laure-Anne Bouquet
email: laureanne.bouquet@gmail.com

Alain Hehn
email: alain.hehn@univ-lorraine.fr

Michèle Boitel-Conti

Julie Moussaron
email: jmoussaron@gmail.com

Thi Le Minh Tran

Flore Biteau
email: florebiteau@yahoo.fr

Corinne Assaf-Ducrocq
email: corinne.assaf@u-picardie.fr

Christophe Robin
email: christophe.robin@univ-lorraine.fr

Frédéric Bourgaud

Armand Guckert
email: armand.guckert@univ-lorraine.fr

Eric Gontier
email: eric.gontier@u-picardie.fr

Datura innoxia plants hydroponically-inoculated with Agrobacterium rhizogenes display an enhanced growth and alkaloid metabolism

Keywords: Agrobacterium rhizogenes, Datura innoxia, hydroponics, tropane alkaloids, chimeric plant, semi-composite plant

come   L'archive ouverte pluridisciplinaire

Introduction

Higher plants can synthesize a wide range of biologically active specialized metabolites used as pharmaceuticals of generally high value (e.g. morphine, codeine, atropine, scopolamine, quinine). These structurally complex plant products are currently isolated from wild or cultivated plants, or from specific plant organs since their chemical synthesis is not economically profitable [START_REF] Nguyen | From Bioreactor to Entire Plants: Development of Production Systems for Secondary Metabolites[END_REF]. Many medicinal plants require a special climate for growth [START_REF] Ha | Hairy root cultures of Panax vietnamensis, a promising approach for the production of ocotilloltype ginsenosides[END_REF]. For naturally grown plants, there is a risk of over-collecting endangered species [START_REF] Nguyen | From Bioreactor to Entire Plants: Development of Production Systems for Secondary Metabolites[END_REF][START_REF] Ha | Hairy root cultures of Panax vietnamensis, a promising approach for the production of ocotilloltype ginsenosides[END_REF][START_REF] Ha | Study on obtaining of hairy root of Panax vietnamensis by using rol gene transformation via Agrobacterium rhizogenes[END_REF]. Alternative methods for producing these plant-derived drugs are therefore desirable. Biotechnological production of specialized metabolites in plant cell cultures provides obvious advantages: biomass production is generally quicker than for the whole plant; plant growth conditions and plant nutrient requirements can be easily controlled; over-collection of endangered species is avoided; geopolitical and import price fluctuations can be eliminated [START_REF] Oksman-Caldentey | Transgenic crops for improved pharmaceutical products[END_REF].

Callus and suspension cultures of undifferentiated cells are one of the possible biotechnological methods used to produce pharmaceutically active compounds. Despite significant effort over nearly 30 years, many limitations remain and only a few compounds produced in this way have been commercialized [START_REF] Mccoy | Natural products from plant cell cultures[END_REF][START_REF] Weathers | Bench to batch: advances in plant cell culture for producing useful products[END_REF]. The major constraint with cell cultures is that they are metabolically unstable: cultured cells tend to produce low yields of specialized metabolites, which generally vary and decrease over months or years of culture. An alternative method of producing biologically active compounds involves the use of differentiated cultures of shoots [START_REF] Gontier | Development and validation of an efficient low cost bioreactor for furanocoumarin production with Ruta graveolens shoot cultures[END_REF] or roots [START_REF] Boitel-Conti | Permeabilization of Datura innoxia hairy roots for release of stored tropane alkaloids[END_REF][START_REF] Boitel-Conti | Influence of feeding precursors on tropane alkaloid production during an abiotic stress in Datura innoxia transformed roots[END_REF]. The behavior of plant organ cultures is more predictable, and the concentrations of such products are often found to be comparable to, or higher than, those of whole plants [START_REF] Massot | Optimized culture conditions for the production of furanocoumarins by micropropagated shoots of Ruta gravolens[END_REF][START_REF] Milesi | a promising species for the production of furanocoumarins[END_REF]. Many studies have shown that transformed root cultures offer better possibilities of producing specialized metabolites [START_REF] Sevon | Agrobacterium rhizogenes-mediated transformation: root cultures as a source of alkaloids[END_REF][START_REF] Srivastava | Hairy root culture for mass-production of high-value secondary metabolites[END_REF]. Hairy roots, transformed with the natural vector Agrobacterium rhizogenes (A.r.; also renamed Rhizobium rhizogenes [START_REF] Young | A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter[END_REF]), the causative agent of hairy root disease in plants, have been found to be suitable to produce specialized metabolites because of their stable and high productivity in hormone-free culture conditions [START_REF] Thiruvengadam | Induction of hairy roots by Agrobacterium rhizogenesmediated transformation of spine gourd (Momordica dioica Roxb.ex.willd) for the assessment of phenolic compounds and biological activities[END_REF]. The possibility of genetic manipulations to increase biosynthetic capacity has initiated considerable interest in them as both a fundamental research tool and a way for producing valuable products [START_REF] Mishra | Growth of hairy-root cultures in various bioreactors for the production of secondary metabolites[END_REF].

A. r. has been widely described as leading to large modifications in growth and metabolism in the case of hairy root cultures as well as for other uses [START_REF] Boitel-Conti | Permeabilization of Datura innoxia hairy roots for release of stored tropane alkaloids[END_REF][START_REF] Veena | Agrobacterium rhizogenes: recent developments and promising applications[END_REF]. As such, it has been considered as a biological tool for improving plant rooting [START_REF] Kavino | Biological hardening -A new approach to enhance resistance against biotic and abiotic stresses in micropropagated plants[END_REF], especially for recalcitrant [START_REF] Devi | Agrobacterium rhizogenes induced rooting of in vitro regenerated shoots of the hybrid Helianthus annuus X Helianthus tuberosus[END_REF][START_REF] Felker | A survey of environmental and biological factors (Azospirillum spp, Agrobacterium rhizogenes, Pseudomonas aurantiaca) for their influence in rooting cuttings of Prosopis alba clones[END_REF] woody plants [START_REF] Strobel | Composition comprising Agrobacterium rhizogenes and Agrobacterium radiobacter and process for the development of plant roots[END_REF][START_REF] Strobel | Agrobacterium rhizogenes promotes the initial growth of bare root stock almond[END_REF][START_REF] Strobel | Improvements in the growth and yield of olive trees by transformation with the Ri plasmid of Agrobacterium rhizogenes[END_REF][START_REF] Caboni | Root induction by Agrobacterium rhizogenes in walnut[END_REF][START_REF] Häggman | Agrobacterium rhizogenes for rooting recalcitrant woody plants[END_REF]. However, no very large-scale application in agriculture or forestry has emerged.

Since the late 1990s, we study the potential of entire plants for producing specialized metabolites in soilless conditions, such as hydroponics or aeroponics, conducted in classic "non-axenic" greenhouses [START_REF] Gontier | Method for producing metabolites from plants cultivated in soil-less medium[END_REF][START_REF] Gontier | Hydroponic combined with natural or forced root permeabilization: a promising technique for plant secondary metabolite production[END_REF]. We made evidence that the root tropane alkaloid profile was different between hydroponic cultures of Datura innoxia conducted in sterile and non-sterile conditions [START_REF] Jousse | Tropane alkaloid profiling of hydroponic Datura innoxia Mill. plants inoculated with Agrobacterium rhizogenes[END_REF]. We also showed that hyoscyamine and scopolamine contents could be up to seven times higher in the hydroponic whole plant when it is co-cultivated with A.r.. The interaction of plant roots and A.r. has been studied and deciphered through a metabolomic approach including correlation network analysis [START_REF] Nguyen | Unravelling the architecture and dynamics of tropane alkaloid biosynthesis pathways using metabolite correlation networks[END_REF]. Regulation points in the tropane alkaloid pathway have been identified in control roots and in plant roots after co-culture with A.r.. We showed that interaction between roots of whole plants and A.r. led to a significant growth improvement and metabolic changes in Datura roots and shoots. However, the mechanisms involved in the plant response were not clearly established. We assumed that a DNA horizontal transfer occurred from the wild type bacterial plasmid to the genome of the plant cell roots, but this remained to be proved.

Because hydroponic or aeroponic culture of medicinal plants can be performed at industrial scale to produce specialized metabolites, and because the co-culture of such plants with A.r. (added in the nutrient solution) can enhance growth and specialized metabolite biosynthesis, it may be possible to recommend the co-culture of plants with A.r. on an industrial scale. A prerequisite is to clarify the way in which A.r. impacts the plant. It is thus necessary to choose a biological system as a model. Our choice was Datura belonging to the Solanaceae family. It is composed of many species including innoxia, metel and stramonium. Such plants produce tropane alkaloids, and particularly scopolamine and hyoscyamine that are used as anticholinergic drugs [START_REF] Renner | Pharmacokinetics and pharmacodynamics in clinical use of scopolamine[END_REF]. For decades, D. innoxia has been used as a model for medicinal plants, especially for studies devoted to biotechnological research on hairy root cultures. Many studies were thus carried out to establish and optimize plant growth conditions and phytochemical production [START_REF] Amdoun | Influence of minerals and elicitation on Datura stramonium L. tropane alkaloid production: Modelization of the in vitro biochemical response[END_REF][START_REF] Amdoun | Optimization of the Culture Medium Composition to Improve the Production of Hyoscyamine in Elicited Datura stramonium L. Hairy Roots Using the Response Surface Methodology (RSM)[END_REF][START_REF] Pinol | Influence of calcium ion-concentration in the medium on tropane alkaloid accumulation in Datura stramonium hairy roots[END_REF]. The effects of feeding precursors [START_REF] Boitel-Conti | Influence of feeding precursors on tropane alkaloid production during an abiotic stress in Datura innoxia transformed roots[END_REF], elicitors [START_REF] Amdoun | Influence of minerals and elicitation on Datura stramonium L. tropane alkaloid production: Modelization of the in vitro biochemical response[END_REF] and heavy metals [START_REF] Vernay | Effect of chromium species on phytochemical and physiological parameters in Datura innoxia[END_REF] were also tested. The effect of the biotic environment on tropane alkaloids was less studied, particularly in whole plants.

In the present work, our first objective was to assess the effects of four different A.r. strains on the development, growth, and hyoscyamine and scopolamine production of hydroponic D. innoxia plants. The second objective was to study the best bacterial strain and to determine if gene transfer really occurred during the early co-culture. Finally, the aim was to build a model of plant root-bacteria interaction that could be applied as a generic tool for specialized metabolite production in whole plants cultivated in greenhouses (i.e. in semi-controlled conditions).

Materials and Methods

Biological material

Seeds of Datura innoxia Mill., initially obtained from Genbank (Gatersleben, Germany) were germinated under greenhouse conditions. Agrobacterium rhizogenes TR7, TR107, 11325 and 15834 wild-type strains (BCCM TM /LMG Bacteria Collection, University of Gent) and the derived A.r. TR7-egfp and TR7-gus were grown at 28°C in liquid YMB medium [START_REF] Hooykaas | Transfer to the Agrobacterium tumefaciens Ti plasmid to avirulent Agrobacteria and Rhizobium ex planta[END_REF] for 24 h. Cells were harvested by centrifugation (3 min, 1100 g), resuspended in distilled water and adjusted to the desired concentration before being used as inoculum. For the construction of the two targeted strains, TR7-gus and TR7-egfp, plasmid pTDE4 (kindly provided by the group of Prof. Van Montagu, Gent) bearing the GUS reporter gene, and plasmid pPE113 (kindly provided by Prof. F. Guérineau, University of Picardie Jules Verne) coding for a modified Green Fluorescent Protein (EGFP), were introduced, respectively, by electroporation into A.r. TR7. The pTDE4 T-DNA contained i) the GUS gene under the control of CaMV 35S promoter and followed by the nos polyadenylation signal, and, ii) the nptII gene between the nos (nopaline synthase) promoter and the ocs (octopine synthase) polyadenylation signal. The pPE113 T-DNA contained i) the 2X35S promoter, the egfp gene and the CaMV polyA sequence and, ii) a nos-nptII-nos fusion.

Non-axenic hydroponic cultures

Seeds of Datura innoxia Mill. were sown in the greenhouse in a 1:1 (v/v) mixture of sand/humus and pots were watered daily. After four to five weeks, plants were transferred into hydroponic culture under greenhouse conditions at a day/night temperature of 27/23°C with a photoperiod of 16 h. An amount of 160 µmol of photons.m -2 .s -1 was provided to the plants by sodium lamps (Philips Son-T Agro 400). The hydroponic system consisted of an extruded polystyrene plate, drilled, floating on a 40-L container (L 56 x W 36 x H 23 cm) with ~30 L of mineral MS nutrient solution [START_REF] Murashige | A revised medium for rapid growth and bioassays with tobacco cultures[END_REF] as previously described [START_REF] Gontier | Hydroponic combined with natural or forced root permeabilization: a promising technique for plant secondary metabolite production[END_REF]. Young Datura innoxia plants were inserted into the holes of plates and roots were kept in the dark. The nutrient solution was inoculated at 3.10 5 CFU.ml -1 with an A.r. wild-type solution and was continuously aerated by a capillary placed at the bottom of the tray [START_REF] Gontier | Hydroponic combined with natural or forced root permeabilization: a promising technique for plant secondary metabolite production[END_REF]. Plant density was 90 plants.m -2 , i.e. each tray contained 18 plants. To study growth and development parameters, Datura innoxia plants were cultivated in eight trays (2 per modality), each containing a different A.r. wild-type strain (TR7, TR107, 11325 and 15834) for five weeks. Plants cultivated in the same conditions without bacteria were used as a control (2 more trays). The total weight of the raft was measured over time on one of the trays. At day 21, 3 plants were used for morphological analysis on the replicate 1 of each tray (root morphology analysis -using WinRHIZO-was not possible later because of root growth was too extensive). For the replicate 2 of each tray, at days 2, 9, 17, 23 and 35, 3 plants were sacrificed for each experimental measurement (fresh and dry weight, alkaloid levels).

Axenic hydroponic cultures of Datura innoxia plants

Seeds of Datura innoxia Mill. were surface-sterilized and germinated in glass jars (10 x 5 cm diameter) containing MS/2 medium (22°C, 60% relative humidity, 16 h photoperiod, irradiance of 160 µmol m -2 s -1 ). After eight weeks, plantlets with three to four leaves were transferred, under sterile conditions, to hydroponic bottles containing 1 L MS solution with one plant per bottle. Each sterile (autoclaved) culture system was composed of a bottle, a tubing with a 0.2 µm filter for 0.1 VVM air bubbling into the nutrient solution and a 44 X 20.5 cm polypropylene cover bag (Sunbag, Sigma, France). Plants were grown in the growth chamber (60-75% relative humidity, 16 h photoperiod, 25°C day vs. 20°C night, irradiance of 180 µmol m -2 s -1 ). In order to check that the culture had been conducted in sterile conditions, a sample of the nutrient solution was used for microbial testing. The sterile hydroponic solution was then inoculated with one of the different A. rhizogenes strains -the wild-type TR7, the tagged TR7-egfp or the TR7-gus -with ~0.7 10 5 CFU.ml -1 . In these conditions, no microorganism other than the added A.r. can lead to a physiological response of the plant.

In vitro isolation of root lines from the roots of axenic plants inoculated by A.r.

After 25 days of culture, roots of the different inoculated D. innoxia plants were excised, carefully rinsed to eliminate a maximum of bacteria and cultured individually on solid half-strength MS medium (MS/2) supplemented with 30 g.L -1 sucrose and 300 mg.L -1 cefotaxime. These explants were subcultured 6 more times in the same conditions. As a bacteriostatic, cefotaxime was used in order to eliminate the bacteria gradually throughout the subcultures. The roots were then transferred into liquid B5 medium [START_REF] Gamborg | Nutrients requirements of suspension culture of soybean root cells[END_REF] supplemented with 30 g.L -1 sucrose and kept in a rotary shaker (200 rpm) at 25°C in the dark. These cultures were performed without cefotaxime in order to check that no viable A.r. remained.

Measurement of plant growth and root architecture

Three plants per treatment were harvested in order to measure plant growth. Measurements were typically made once a week, for five weeks. Shoots and roots were separated, weighed (fresh biomass), oven dried at 80°C for 72 h, weighed again (dried biomass) and ground for further analysis. After three weeks of culture, leaves were removed from three other plants and scanned at 200 dpi. Leaf area was then determined by the software WinFOLIA (version 2002a). Root morphology of the same three plants was analyzed by the software WinRHIZO (versions 2002b and 2002c, Regent, Canada). Roots were spread in a tray containing water, scanned and analyzed using the parameters recommended by Bouma et al. [START_REF] Bouma | Sample preparation and scanning protocol for computerised analysis of root length and diameter[END_REF]. Number of root apices, total root length and root diameters were measured by WinRHIZO. Root area and volume were then calculated. It was not technically possible to analyze root architecture at dates later than 3 weeks (impossible to spread the roots in the tray because of their too extensive growth).

Sample extraction and HPLC analysis

After five weeks of culture, three plants per treatment were harvested. Shoots and roots were separated and immediately frozen at -18°C. Ground freeze-dried tissues (30 ± 5 mg) were extracted with 1.5 mL of an 80/20 (v/v) 0.1 M HCl-95% EtOH mixture. After 30 min, with 3 × 1 min stirring at ambient temperature, the samples were centrifuged at 12000 g for 10 min. The supernatant was directly injected into the HPLC apparatus after filtration through a 0.2 μm filter (Minisart RC15, Sartorius, Aubagne, France). As previously described [START_REF] Lanoue | Occurrence of circadian rhythms in hairy root cultures under controlled conditions[END_REF][START_REF] Dechaux | A strategy for an overaccumulation of scopolamine in Datura innoxia hairy roots cultures[END_REF], hyoscyamine and scopolamine levels were measured with an HPLC system consisting of: a TSP P100 pump coupled to a TSP AS100 automatic injector (ThermoElectron, France); a UV WAT 484 detector (Waters, France) used at 204 nm; an AZUR® V3.0 software (Datalys, France); and a 4.6 mm i.d. × 250 mm, 5 μm, 120Å YMC ODS-AQ reverse phase column (AIT, France). The mobile phase (flow: 0.8 mL.min -1 ) was composed of 0.05% formic acid in an 86/14 (v/v) water/acetonitrile mixture.

Evaluation of the transgenic state of plant roots

The histochemical GUS assay was carried out by incubating root fragments in GUS assay buffer at 37°C overnight and decolorizing with 95% ethanol prior to observation [START_REF] Jefferson | GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[END_REF]. Imaging of EGFP fluorescence was performed using a fluorescence microscope (Nikon Eclipse E-800) with an excitation filter (450-490 nm). Fluorescent images were recorded using a digital camera.

Total DNA was isolated from fresh roots (500 mg) using the "DNeasy Plant Mini Kit" (Qiagen). For the PCR analysis, the following PCR mixture was used: 200 ng of total DNA, 12.5 µM of each primer, 200 µM d NTPs, 1.5 u Taq DNA polymerase (Q-Biogene), and buffer supplied by the enzyme manufacturer in a total volume of 25 µL. The rolA specific primers used (rolA DIR cagaatggaattagccggacta / rolA REV cgtattaatcccgtaggtttgttt; Sevon et al., 1997) generated a 310 bp PCR product; the virA primers (VirAAr85D ccatcttgtgagcggcaacga / VirAAr230R tactcagcgcacgagtgaagctg; [START_REF] Sevon | Characterization of transgenic plants derived from hairy root of Hyoscyamus muticus[END_REF]) generated a 145 bp PCR product; the egfp primers (egfp222D ctaccccgaccacatgaagcag / egfp395R ttgaagtcgatgcccttcagctc) generated a 173 bp product and the gus primers (35SDIR100 cactatccttcgcaagacccttcctctatataaggaa / gus284R ccgtaatgagtgaccgcatcgaa) generated a 384 bp fragment. PCR amplifications were performed under the following conditions: initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 50°C (for the rolA gene) or 55°C (for the gus gene) or 62°C (for the virA and egfp primers) for 30 s, and extension at 72°C for 1 min, with a final extension at 72°C for 10 min. The PCR reaction mixtures (25 µL) were loaded directly onto a 1.5% agarose gel for electrophoretic analysis. A DNA ladder (Eurogentec) was used as a molecular weight marker for the PCR-amplified double-stranded DNA fragment.

Statistical analysis

All data were analyzed using the Statbox Pro variance procedure. Means were classified according to the Newman-Keuls test. Data followed by different letters are significantly different (p<0.05).

Results

Inoculation of the nutrient solution of hydroponic Datura innoxia plants by different strains of

Agrobacterium rhizogenes

Growth and development of D. innoxia plants

Shoot and root dry matter of D. innoxia plants grown in the greenhouse under non-axenic hydroponic conditions was determined on three plants per condition at days 2, 9, 17, 23 and 35 (Fig. 1). These plants were grown with or without one of the four A.r. strains. Leaf area per plant, root length, root area, root volume and root apex number were already higher after three weeks in plants with A.r. than in controls, regardless of the strain (Fig. 2). Because the root growth was too extensive, it was not possible to spread, scan and analyze the root morphology with WinRHIZO at later dates.

After 35 days, the presence of A.r. had increased dry matter accumulation in shoots and roots compared to control plants (Fig. 1). TR7 strain led to the highest dry matter accumulation in whole plants (17.8 gDW for shoots and 4.7 gDW for roots). The mean plant biomass per plant was doubled in TR7 compared to control plants (22.5/11.5 gDW per plant). Based on the culture density of 90 plants per square meter, this corresponds to a biomass production of 2.025 kg DW.m -2 for D. innoxia hydroponic culture inoculated with TR7, and only 1.035 kg DW.m -2 for the control plants after 35 days of culture.

Tropane alkaloid levels in D. innoxia plants

Hyoscyamine and scopolamine contents were also measured in shoots and roots after 35 days of culture (Fig. 3). A.r. TR7 strain significantly increased the hyoscyamine content in shoots compared to controls (Fig. 3). TR7 and 11325 had the same positive effect for hyoscyamine in the roots of inoculated plants. The scopolamine concentration was enhanced in the shoots when TR7, 11325 and 15834 strains were added to the nutrient solution. It was significantly enhanced in the roots only with the 11325 strain. The inoculation with TR7 bacterial strain enhanced the hyoscyamine and scopolamine levels from 2 to 8 in roots and shoots. This led to an estimated total accumulation of 2.5 g of alkaloids per square meter after a culture of 35 days. The total productivity could thus be determined as 71 mg of hyoscyamine and scopolamine produced per day and per square meter of hydroponic culture.

To validate these results at a larger scale, the same kind of experiment was carried out at a pilot scale (Fig. S1.1 to S1.9). Two parallel hydroponic cultures of 252 plants were conducted in two 3 m 2 [L: 5 m x W: 0.6 m x H: 0.1 m; 200 L of nutrient solution] containers under the same greenhouse conditions. One was inoculated by A.r. TR7, the other was the control. This assay confirmed the previous ones and demonstrated the value of inoculating D. innoxia with A.r. TR7 strain: increased plant growth (Fig. S1.1 to S1.7) and higher hyoscyamine and scopolamine contents in shoots and roots (Fig. S1.8). Compared to the control, the initial addition of A.r. TR7 into the nutrient solution of the pilot-scale hydroponic system did not lead to evident modification of the total microflora that grew spontaneously in the nutrient solution (Fig. S1.9).

Evaluation of the transgenic state of roots

Culture of Datura in axenic hydroponic conditions and inoculation of the nutrient solution with modified A.r. TR7 containing the egfp or gus reporter gene

Because the inoculation of the nutrient solution by dead A.r. TR7 (boiled twice in a microwave oven) did not lead to growth (Fig. S2.1) or alkaloid level (Fig. S2.2 and S2.3) improvement, we hypothesized that the positive effect of A.r. TR7 could be related to gene transfer events. To check this hypothesis, two genetically modified A.r. strains called TR7-gus and TR7-egfp were constructed by transfection with the two plasmids pTDE4 and pPE113, respectively. These two strains have two available plasmids: pRi and either pTDE4 or pPE113. The vir proteins can transfer one or both T-DNAs into the plant genome and can therefore generate transformed tissues with the rol genes and/or the reporter genes. Axenic cultures of hydroponic Datura were performed (Fig. S3.1 and S3.2) and used for the co-culture with the different TR7 strains. After 25 days, the different root systems of D. innoxia inoculated with A.r. TR7 wild-type, TR7-egfp and TR7-gus were more developed than that of the control. To determine the presence of EGFP protein, the roots were observed with UV light under a microscope. Control roots (Fig. 4a) never showed any kind of yellow-green fluorescence. In contrast, some of the TR7-egfp roots showed the typical yellow-green fluorescence due to the presence of EGFP protein. The observation of pieces of TR7egfp roots clearly indicated that EGFP accumulated only in some parts of the root system; for example, in the tips (Fig. 4b) and the proximal parts of some roots (Fig. 4c). Likewise, histochemical analyses of the roots of TR7-gus-inoculated hydroponic plants showed some gus activity after 25 days (Fig. 5a) in contrast to controls (Fig. 5b). However, this staining was not homogeneous (Fig. 5a and5c) and showed a chimeric distribution in the roots (Fig. 5d). Only some parts of the root system thus appeared to be gus-positive.

Isolation of roots from A.r. TR7 inoculated Datura and in vitro characterization of their hairy root-like phenotype after eradication of the bacteria

To determine whether gene transfer occurred during the plant-root interaction, some root apices from the different hydroponic plants were isolated and transferred to Petri dishes on MS/2 medium without hormone but with cefotaxime at 300 mg/L (Fig. 6a to 6d). After 18 days of culture (Fig. 6e to 6h), differences were noted in the root growth and phenotypes. The roots from control plants did not grow, even after a long time (Fig. 6e). In comparison, some of the roots from the A.r. co-cultured plants presented a fast and significant growth with numerous ramifications like hairy roots (examples in Fig. 6f to 6h). Some others were thick with a positive geotropism. In some cases, some of the root apices did not develop, like control roots (not visible on these Petri dishes). The isolated roots were then subcultured six more times on solid agar medium without phytohormone but with cefotaxime 300 mg/L in order to remove any remaining agrobacteria. Some explants were transferred onto solid medium without cefotaxime to check that no living bacteria could grow again. In addition, small explants (1.5 -2 cm) of the rapidly well-developed roots were isolated and cultured in liquid B5 medium without phytohormones and without cefotaxime. No bacterial contaminant occurred in the cultures. The roots quickly developed and presented all the attributes of hairy roots (Fig. S4.1) with a two hundred-fold increase in the fresh biomass (from 0.02gFW to values of 4 to 4.3g for TR7/TR7-egfp/TR7-gus; Fig. S4.2). Compared to the control roots, the alkaloid contents were six to seven times larger (Fig. 7).

Molecular proof of gene transfer occurrence

To establish that transformation events had definitely occurred, we checked the integration of the different foreign genes in the in vitro isolated root lines that grew without additional phytohormone in liquid medium. These cultures contained no viable Agrobacterium cells: no bacteria grew during the microbiological tests performed on the root media and tissues. Using PCR, no virA gene could be detected in all these root cultures. Furthermore, the positive controls (A.r. TR7) were all positive, confirming that the molecular test was efficient. Thus, it could be concluded that no more -even dead-Agrobacterium remained in the flasks (Fig. 8a to 8c; see A.r. TR7).

The occurrence of (i) the rol A gene from the plasmid pRi TR7 in the wild-type TR7 (Fig. 8a to 8c), (ii) the egfp gene from the plasmid pPE113 in TR7-egfp (Fig. 8b) and (iii) the gus gene from the plasmid pTDE4 in TR7-gus (Fig. 8c) was confirmed by PCR analysis in root lines from each inoculation experiment. The systematic presence of the rolA gene thus proved that a gene transfer had systematically occurred; i.e. a genetic transformation by A.r.. A difference in the intensity of amplified genes could also be observed. This may be explained by the still chimeric state of the root biomass in the Erlenmeyer flasks (some roots may have integrated more than one DNA copy). The egfp gene was recovered in 4 of the 6 "TR7-egfp" tested root lines (Fig. 8b). Of the seven tested root lines, two were positive for the gus gene after amplification (Fig. 8c). The combined presence of these genes (rolA + [egfp or gus]) proved that a double transformation event had occurred. The rolA gene was systematically present and lead to the hairy root phenotype whereas the egfp or the gus gene are present in some independent transformation.

Discussion

Hairy root (HR) cultures have been widely studied as a model of choice for the characterization of Specialized Metabolic (SM) pathways and for the development of the biotechnological production of plant metabolites [START_REF] Ha | Hairy root cultures of Panax vietnamensis, a promising approach for the production of ocotilloltype ginsenosides[END_REF][START_REF] Jaziri | Genetic transformation of Atropa belladonna[END_REF][START_REF] Bonhomme | Tropane alkaloid production by hairy root of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only[END_REF][START_REF] Tran | Synthèse et accumulation d'alcaloïdes tropaniques chez Datura innoxia Mill. cultivé en hydroponie : analyse des effets de l'environnement biotique et abiotique ; essais de mise en place d'une nouvelle technologie de production[END_REF][START_REF] Pinol | Effects of Ri T-DNA from Agrobacterium rhizogenes on growth and hyoscyamine production in Datura stramonium roots[END_REF][START_REF] Palazon | Relation between the amount of rolC gene product and indole alkaloid accumulation in Catharanthus roseus transformed root cultures[END_REF][START_REF] Bulgakov | The impact of plant rol C oncogene on ginsenoside production by ginseng hairy root cultures[END_REF][START_REF] Diop | Hairy root and tissue cultures of Leucojum aestivum L. -relationships to galanthamine content[END_REF]. Typically, HRs are cultured in vitro, in Erlenmeyer flasks and in different types of higher scale bioreactors. The initiation of such HRs generally starts by the inoculation of plant material (seedlings, plant part) with Agrobacterium rhizogenes (A.r.). It is well known that such a bacterium can transfer some plasmidic DNA (ri-tDNA) into the genome of a plant cell. The transformed cell display a modified phytohormone balance and sensitivity [START_REF] Lütken | Genetic engineering and sustainable production of ornamentals: current status and future directions[END_REF] and tend to divide and differentiate into root tissues that can be multiplied in vitro [START_REF] Guillon | Hairy root research: recent scenario and exciting prospects[END_REF][START_REF] Chaudhuri | Spontaneous plant regeneration in transformed roots and calli from Tylophora indica: changes in morphological phenotype and tylophorine accumulation associated with transformation by Agrobacterium rhizogenes[END_REF][START_REF] Makhzoum | Hairy Roots: An ideal platform for transgenic plant production and other promising applications[END_REF]. Such in vitro plant material is of very great interest because it generally has a relatively high growth rate and the levels of Specialized Metabolites are comparable to or higher than those of the roots in entire plants. These characteristics are stable throughout the subcultures. Nevertheless, with the aim of the industrial production of plant specialized metabolites, it is necessary to overcome the difficulty of both i) scaling up and ii) maintaining the axenic characteristic (i.e. sterility) of the culture. In our strategic perspective, we chose here to use entire plants cultivated in open air conditions (i.e. nonsterile) and with the possibility of an easy scale-up of the cultures carried out in greenhouses. Soilless culture, like hydroponics (or aeroponics), opens up such perspectives. It also enables a better control of plant growth parameters (nutrient solution, temperature, humidity, light, etc.). Plant biomass containing large amounts of Specialized Metabolites of interest can be produced by optimizing the process. We demonstrate here that inoculation of the nutrient solution of hydroponic D. innoxia with chosen A.r. strains led to improved growth and alkaloid production. This work also reveals the mechanisms by which the plant-bacteria interaction acts on the productivity of the system. Due to A.r., the novel plants now harbor a chimeric root system composed of "normal roots" and "transformed roots". The latter, like in vitro HRs, probably grow faster and synthesize more alkaloids. Furthermore, they most probably induce an increase in growth and secondary metabolism in neighboring "normal roots". These modifications would influence the aerial parts, which may grow more and accumulate more alkaloids.

In addition, in our experiments the aerial parts of the plants were normal. They did not have the phenotype observed in Datura plants regenerated from hairy roots. In an unpublished experiment, we obtained these kinds of plants regenerated from a more than ten-year-old D. innoxia hairy root strain cultivated in vitro. The regenerated plants were smaller than controls (dwarf phenotype as described by Casanova et al. [START_REF] Casanova | Influence of rol genes in floriculture[END_REF]). Their internodes were shorter, and their leaves were wrinkled. Flowering occurred but no fruit production was observed. In the case of the Datura plants described in this article, the shoots were absolutely comparable to those of control plants. No modification of architecture or fertility was observed.

Cultivating medicinal plants in hydroponics (or aeroponics), optimizing culture condition parameters and inoculating the nutrient solution with chosen A.r. to produce semi-composite plants could be a strategy of generic interest to produce plant secondary metabolites. Bacterization of the nutrient solutions with a synthetic inoculum composed of different A. rhizogenes strains could be suggested in greenhouse-controlled conditions [START_REF] Oksman-Caldentey | Transgenic crops for improved pharmaceutical products[END_REF][START_REF] Van Der Salm | Prospects for applications of rol genes for crop improvement[END_REF] or for the seedlings before the greenhouse culture.

Consequently, other points must be clarified. The first one is would such semi-composite (chimeric) plants obtained with a wild strain of A.r. be considered genetically modified organisms? The answer is no, based on legal requirements in European countries. Also, is there a risk of using a pathogenic bacterial (A.r.) species in a greenhouse? Based on the recent review of Bosman et al. [START_REF] Bosmans | Rhizogenic agrobacteria in hydroponic crops: epidemics, diagnostics and control[END_REF]), the answer is yes. Some biovars from Agrobacterium species are causative agents of pathology, the hairy root disease, particularly in the hydroponic cultivation of tomato, cucumber and eggplant. However, some other Agrobacteria are not pathogenic. Lastly, in the case of problems within the greenhouse, some methods are available to eradicate or at least minimize the presence and/or the effects of the pathogenic bacterium [START_REF] Strobel | Composition comprising Agrobacterium rhizogenes and Agrobacterium radiobacter and process for the development of plant roots[END_REF].

In this work, A.r. was used to improve the production of plant roots (and subsequently aerial parts) which then produce more metabolites of interest. In Datura, roots are known to be the main biosynthetic site of tropane alkaloid synthesis prior to further translocation into shoots and preferentially the youngest leaves [START_REF] Gontier | Tropane alkaloid levels in the leaves of micropropagated Datura innoxia Mill. plants[END_REF]. The increase in the root biomass and number of root apices could have directly led to a higher biosynthetic potential of the root system. Therefore, the whole plant contained more tropane alkaloids after partitioning and remobilization from roots to leaves.

As a conclusion, we suggest here producing plant secondary metabolites from medicinal plants through the soilless culture of entire plants and with an optimized inoculation of the nutrient solution containing a chosen strain of A.r.. The final plant biomass should be greater and may contain higher levels of phytochemicals than non-inoculated control plants. (c) TR7-gus.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Dry matter accumulation per D. innoxia plant in shoots (a) and roots (b) in the presence or not of four different Agrobacterium rhizogenes strains. The values are means of 3 plants ± standard error; for day 35, significant ANOVA at p< 0.05 was followed by a Student Newman Keuls test at p< 0.05 (different letters for significant differences).

Figure 4 :

 4 Figure 4: Fluorescence in a root of a hydroponic plant cultivated with A. rhizogenes TR7-egfp. Root apex from a control plant (a; no fluorescence), and fluorescent root apex from a co-cultured plant (b). Distribution of fluorescent signal in the root, above the apex (c).

Figure 5 :Figure 6 :Figure 7 :

 567 Figure 5: Blue color from the metabolization of X-gluc substrate in roots of axenic hydroponic plants cocultured with A. rhizogenes TR7-gus: (a) non-uniform blue color in roots, (b) root of a control plant, (c) emergence of a lateral blue root from a blue-colored root. (d) Root system of a 21-day-old A.r. TR7-gus plant showing a chimeric distribution of blue color that reveals x-gluc activity and thus the distribution of potential sites of transformation with the gus gene. Control plants never showed any coloration.

Figure 8 :

 8 Figure 8: PCR analysis of the virA, rolA, egfp and gus genes in the different root lines. (a) TR7, (b) TR7-egfp;

Acknowledgments:

We thank the "Conseil Regional de Picardie" for the financial support of part of this work (program MTV: Metabo-Typage Végétal). We also thank Dr. Michael Deyholos (UBC, Canada) who spent valuable time on the reading and improvement of this manuscript. We also want to thank Dr. A. Pühler and C.W.W. Sensen and their scientific collaborators for their valued advice, which enabled a better presentation of this work

Appendix A: Supplementary data 1; Effects of Agrobacterium rhizogenes TR7 on growth and tropane alkaloid content of Datura innoxia cultivated in a large-scale hydroponic system; including Fig. S1. 1-9. Appendix B: Supplementary data 2; Effects of microwaved boiled Agrobacterium rhizogenes TR7 initial inoculation on the growth and tropane alkaloid levels of hydroponic Datura innoxia plants; including Fig.

S2.1-3.

Appendix C: Supplementary data 3; Inoculation of modified Agrobacterium rhizogenes TR7 strains in the nutrient solution of axenic-hydroponic cultures of Datura innoxia; including Fig. S3.1-2.

Appendix D: Supplementary data 4; Characterization of growth and tropane alkaloid levels in the axenic and stabilized liquid cultures of roots isolated from axenic hydroponic Datura innoxia plants in which the nutrient solution had been inoculated with Agrobacterium rhizogenes TR7, TR7-egfp or TR7-gus strain; including Fig. S4. 1-3.