Andressa Reis

Stéphanie Boutet-Mercey

Sophie Massot

Pascal Ratet
email: pascal.ratet@ips2.universite-paris-saclay.fr

José Angelo Silveira Zuanazzi

Section of the Journal -Plant Cell Technology Isoflavone production in hairy root cultures and plantlets of Trifolium pratense

Keywords: Elicitors, plantlets, red clover, salicylic acid, sucrose

Objectives

The aim of this study was to develop a T. pratense hairy root (HR) production protocol and select HR lines with high isoflavone yield following elicitor treatments.

Results

We obtained 13 independent HR lines, producing approximately 3 times more isoflavonoids than seedlings (3.3 mg/g dry weight) and in which 27 isoflavonoids were detected. Each HR line had its own isoflavonoid profile. These lines produced as major components daidzein, genistein, formononetin and biochanin A. Sucrose, salicylic acid (SA), yeast extract (YE) and flagellin 22 (flg22) were tested as elicitors. Using SA 140 mg/L, allowed the maximum isoflavonoid production in plantlets (11.9 mg/g dry weight) but reduced root growth, possibly as a result of its toxicity. The highest isoflavone production in HR (27.9 mg/g dry weight) was obtained using sucrose 60 g /L, for 3.5 days.

Conclusion

This work reports the high production of various isoflavonoids with T. pratense elicited HR cultures.

Introduction

Red clover (Trifolium pratense L.) is a forage legume producing as main isoflavones formononetin, biochanin A, daidzein and genistein (Fig. 1-A to D) [START_REF] Saviranta | Red clover (Trifolium pratense L.) isoflavones: determination of concentrations by plant stage, flower colour, plant part and cultivar[END_REF]).

These molecules present several biological activities related to their phytoestrogenic-like activy, due to some similarities with the structure of 17-estradiol (Fig. 1-e) [START_REF] Monteiro | Impact of microbiota on the use and effects of isoflavones in the relief of climacteric symptoms in menopausal women -A review[END_REF]).

Insert Fig. 1 These economically interesting plant secondary metabolites generally accumulate in roots [START_REF] Saviranta | Red clover (Trifolium pratense L.) isoflavones: root phenolic compounds affected by biotic and abiotic stress factors[END_REF]. To avoid destroying the plant, the Agrobacterium rhizogenes transformed hairy root (HR) cultures represent a good alternative for biotechnological applications [START_REF] Cosson | Hairy root research: recent scenario and exciting prospects[END_REF][START_REF] Putalun | Improvement of artemisinin production by chitosan in hairy root cultures of Artemisia annua L[END_REF].

HR cultures are cytologically stable, exhibit continued growth without any hormonal supplement, have negative gravitropism and produce opines. This stability allows biochemical and genetic manipulations (Webb et al. 1990) and the development of controlled cultures [START_REF] Spagnuolo | Isoflavone content and estrogenic activity of different batches of red clover (Trifolium pratense L.) extracts: an in vitro study in MCF-7 cells[END_REF]). To date, two studies have used red clover HR [START_REF] Beach | In vitro culture of legume root tissue transformed by Agrobacterium rhizogenes[END_REF]Webb et al. 1990), but none of them focused on the secondary metabolites production.

Plants produce various natural products as part of their development or in response to elicitors that can be used to increase secondary metabolite production [START_REF] Shinde | Impact of nutrient components on production of the phytoestrogens daidzein and genistein by hairy roots of Psoralea corylifolia[END_REF][START_REF] Udomsuk | Improved isoflavonoid production in Pueraria candollei hairy root cultures using elicitation[END_REF]. Yeast extracts (YE, Saccharomyces cerevisiae) and salicylic acid (SA) can be used as elicitor in HR cultures [START_REF] Ramirez-Estrada | Elicitation, an effective strategy for the biotechnological production of bioactive high-added value compounds in plant cell factories[END_REF]Shinde et al. 2009b;[START_REF] Udomsuk | Improved isoflavonoid production in Pueraria candollei hairy root cultures using elicitation[END_REF]. The bacterial flagellin or part of it (flg22 peptide) is also a potent elicitor, eliciting basal plant defenses in plants like Arabidopsis [START_REF] Maffei | Natural elicitors, effectors and modulators of plant responses[END_REF]De Coninck et al. 2015). Sucrose has important signaling functions throughout all stages of the plant life cycle [START_REF] Smeekens | Sugar-induced signal transduction in plants[END_REF][START_REF] Sujatha | Establishment of fast-growing in vitro root culture system in Artemisia vulgaris[END_REF]. At high concentrations it can trigger reactive oxygen species production and also polyphenol, including anthocyanin, accumulation in grape (Vitis vinifera) [START_REF] Ramakrishna | Influence of abiotic stress signals on secondary metabolites in plants[END_REF] and also flavonoids and anthocyanins production in A. thaliana [START_REF] Solfanelli | Sucrose-specific induction of the anthocyanin biosynthetic pathway in Arabidopsis[END_REF] .

The aim of our study was to develop a T. pratense hairy root (HR) production protocol and select HR lines with high isoflavone yield following elicitor treatments. We report a medium term (five months) study for the production of daidzein, genistein, formononetin and biochanin A in HR culture.

Experimental

Plant material

Seeds of T. pratense var. URS-BRS Mesclador were donated by Dr. Miguel Dall'Agnol (UFRGS). The specimen was deposited under the registration code HAS 87114, number 4291 (10/14/1986) (Museum of Natural Sciences -Zoobotanic Foundation of Rio Grande do Sul). The harvest point is (latitude: -30.0331/ longitude: -51.23 [err: ± 29946 WGS84]).

Seed disinfection and germination

In order to initiate the in vitro germination process, T. pratense seeds were scarified with fine sandpaper and the disinfection process started with sodium hypochlorite (2% active chlorine) plus tween 20 (two drops/ 100 mL) under stirring conditions for 30 minutes, followed by triple washes with sterile water and inoculation in agar/ water medium (agar 8 g /L). The seeds were maintained for 48 hours at 4 °C before their transfer to 24 °C (2 days) in the dark. The seedlings have started to grow at this point ( 1.5 cm in length) and were shifted to a growth chamber with 16 h photoperiod (200 μE/s/m 2 ) in ½ MS medium [START_REF] Murashige | A revised medium for rapid growth and bio assays with tobacco tissue cultures[END_REF] plus sucrose 10 g /L and agar 7 g /L (Supplementary material) .

Screening for elicitor effects on the roots of T. pratense plantlets

In order to test the elicitor effects on T. pratense development, the experiment was initiated with 5 days-old T. pratense seedlings that were cultivated in the same conditions as described above. The elicitation effect was measured at 0, 12, 24, 36, 48 and 72, 96, 120 or 168 h after inoculation.

Elicitor preparation and treatment

Elicitors treatment was tested using the Flagellin 22 peptide QRLSTGSRINSAKDDAAGLQIA (flg22, Proteogenix, Schiltigheim France), yeast extract (BD Biosciences), salicylic acid (Sigma Aldrich) and sucrose. The elicitor action was estimated as the percentage of root growth inhibition after treatment. Seedlings were grown on agar 7 g /L or Phytagel TM 2 g /L as gelling agents. Elicitors were dissolved in distilled sterile water and added to the medium in the following concentrations. Flg22 was used in the concentrations of 0; 0.25; 0.5; 1 and 2.5 µM [START_REF] Millet | Innate Immune Responses Activated in Arabidopsis Roots by Microbe-Associated Molecular Patterns[END_REF]. The YE (Supplementary material) was tested at 0.5; 1; 2; 3 and 4 g /L of medium. YE solution was filter sterilized and added after the medium sterilization [START_REF] Sivesind | Effects of foliar application of elicitors on red clover isoflavone content[END_REF]. SA was dissolved in distilled water and autoclaved at 121 •C for 15 min. After sterilization, SA was added at concentrations of 0, 1.4, 14, 70 and 140 mg /L. Sucrose (Sigma Aldrich) was employed from 10, 30, 60, 90 until 180 g /L and was autoclaved with the medium.

Hairy root culture a)

Agrobacterium culture and tissue infection

The A. rhizogenes strain A4TC24 [START_REF] Petit | Further extension of the opine concept: Plasmids in Agrobacterium rhizogenes cooperate for opine degradation[END_REF] was cultured in the flask containing 4 ml of YEB liquid medium + rifampicin 100 µg /mL. The incubation was done at 30°C, in a rotary shaker at 200 rpm. One day before the transformation, 100 µL of the liquid culture was plated on a YEB agar plate with appropriated antibiotics. The culture was incubated overnight at 30°C to obtain a layer of A. rhizogenes on the plate to proceed with the transformation.

The 7 days-old seedlings were infected aseptically by cutting the root meristem and dipping it into the Agrobacterium layer or by wounding the principal root using Agrobacterium-filled syringes. Inoculated seedlings were kept for two days in ½ MS, storing the plate with an angle of 45° and the root covered with opaque black plastic film. After this time, the infected seedlings were transferred to ½ MS + 400 µg /mL Amoxicillin Na and clavulanate K 5:1 + 1% sucrose for 15-21 days (photon flux of 40 µmol m -2 s -1 ), 25 ± 2 ºC, with the roots covered to remain in the dark (Wright and Wang 2015).

When the new growing HR reached the size of 10-15 cm, they were cut from the plant and transferred to new medium, with the same components and sub cultured each thirty days.

Roots growing efficiently after two subcultures independently of the plant were considered as true HR. These HR were transferred to Erlenmeyer flasks containing 50 mL of ½ MS liquid medium and maintained at 20 °C on a shaker (100 rpm) in the dark. These were also sub cultured every 30 days.

Evaluation of the isoflavone content in seedlings and HR cultures

The seedlings were grown for seven days using the media including the elicitors (sucrose + phytagel, sucrose + agar, SA, YE and Flg22), as cited above (Elicitor preparation and treatment section) and harvested for analysis. Thirteen HR lines with good growth capacities were isolated and partly harvested each 30 days of culture. The material was stored at -80 C

for posterior flavonoids extraction and control of isoflavone content during the first 5 months of the experiment.

The flavonoids analysis by Ultra Performance in Liquid Chromatography (UPLC) was adapted from the literature (Galland et al. 2014) and the samples were compared to standards of the commercially acquired isoflavones daidzein, genistein, formononetin and biochanin A (Sigma-Aldrich). The hairy root cultures were lyophilized, and the extraction made using around 100 mg of T. pratense material reduced to powder using liquid nitrogen, mortar and pestle. To this powder, 1.5 mL of extractive solution (methanol/ water/ acetone/ TFA, v/v 40/ 32/ 28/ 0.05 %) was added and the membranes were further disrupted by placing the samples in an ultrasonic bath for 20 min at 25 kHz at 4 °C. These samples were then centrifuged at 20 000 g for 20 min. The process of extraction was performed twice with the same sample and the two resulting supernatants were pooled and dried in a rotary evaporator and freeze dryer. Quantitative analyzes were performed using commercial isoflavone standards to verify the UV absorption, retention time and the linearity. Each curve was made with six different concentrations. For daidzein the concentration range used was 1.5 -5.5 μg /mL, for genistein 4.3 -216.4 μg /mL, for formononetin 76.3 -225 μg /mL and for biochanin A 5.9 -357.2 μg /mL. The linear equation and the determination coefficient (R²) were calculated for each isoflavone standard curve.

b) Isoflavone identification

The following flavonoid extraction protocol was adapted from the literature (Galland et al. 2014). HR flavonoids were extracted from 100 mg of 13 different HR T. pratense lines cultivated for 60 days, after grounding in liquid nitrogen using mortar and pestle. Then, 1 ml of methanol 80% (v/v) was added to the HR powder samples and membranes were further ruptured by placing the samples in an ultrasonic bath for 20 min at 25 kHz at 4 °C and later on a rotating wheel for 1 hour.

After centrifugation during 10 min at 20000g at 4 °C, the pellet was extracted with 1 ml of methanol 80% (v/v) overnight at 4°C on a rotating wheel. The two resulting supernatants were pooled, filtered at 0.45 µm (Millipore) and dried on a SpeedVac for 8 h.

The dry pellet was dissolved in methanol/ water/ acetone/ trifluoroacetic acid (40/32/28/0.05, v/v). At this stage, 20 ng of rhamnetin (Extrasynthese) were added as internal standard.

Finally, the extract was filtered again. The extracts were then analyzed by HPLCelectrospray-MSMS (HPLC-ESI-MS/MS).

The compounds were introduced in the ESI source using a Waters UPLC Acquity I-Class (Waters) equipped with a Waters PDA detector. Separation was achieved on a reversephase column (Uptisphere C18 ODB, 150*2.1 mm, Interchim) using a flow rate of 0.40 mL /min and a binary gradient: (A) acetic acid 0.1% (v/v) and (B) acetic acid 0.1% (v/v) in acetonitrile. The solvent gradient was programmed as following: 0-4 min 10% B, 4-17 min 60% B, 17-23 min 100% B, 23-25 min 14% B [START_REF] Kerhoas | Structural characterization of the major flavonoid glycosides from Arabidopsis thaliana seeds[END_REF]).

Analyses were performed on a Waters Xevo TQ-S (Waters) MS triple quadripole mass spectrometer operating in MS full scan for the relative quantification and identification of the compounds, ESI positive and negative modes and In Source-Collision Induced Dissociation (IS-CID) positive modes. Relevant instrumental parameters were set as following: cone voltage 20 V and 80 V for usual and IS-CID modes respectively, capillary 2.70 kV, source block and desolvation gas temperatures 150°C and 600°C, respectively. Nitrogen gas was used to assist nebulization and desolvation (7 bar and 1000 L/h, respectively). When MS/MS modes were used for the confirmation of the identification (Product ion scanning and Precursor ion scanning 20 eV was used as collision energy in the collision cell).

Hairy root elicitation

Elicitation was done on 5 months stabilized cultures. For this, approximately 7 days after subculture, hairy root cultures were transferred to ½ MS medium containing: sucrose 10 g/L (control) or sucrose 60 g/L or sucrose 10 g/L plus SA 10 or 30 mg/L as elicitors. The first measurements were done after 3 days (72 h) of culture and at 84, 96, 108 and 120 h (3.5, 4, 4.5 and 5 days, respectively). For control (sucrose 10 g/L), the measurements were done until the 14 th day in culture. The HR cultures were collected, lyophilized, grounded and extracted for total isoflavonoids measurement. The extraction and chemical analysis of isoflavones was performed according to the technique described in UPLC isoflavones quantitative analysis section.

Statistical analysis

All culture experiments were conducted in triplicate. The root length experiments were composed of different elicitor treatments at different concentrations. For each treatment 5 replicates of three plates were used, considering each plate as an experimental unit. HR cultures were run in quadruplicates for each line and the replicates of each line were combined in two, prior to isoflavones extraction and analysis. Elicitation of the HR cultures was performed in triplicates. Isoflavone concentrations were determined by means of the standard curves for each one of the isoflavones analyzed in the samples, as well as the standard deviation of the samples under study and Analysis of Variance (ANOVA) and Tukey test at 5% significance level [START_REF] Machado | Programa estatístico WinStat Sistema de Análise Estatístico para Windows[END_REF].

Results and discussion

Effect of elicitor on roots length and isoflavone content in T. pratense plantlets

In order to estimate the effects of different elicitors on T. pratense, we tested their influence on seedling root growth and isoflavones content. Seedling root growth inhibition is easier to test than HR growth inhibition, because it is fast and easy to measure on many conditions. Compared to the control condition (sucrose 10 g/L) the different treatments either stimulated or inhibited root growth. We postulated that the elicitor action will mimic defense reactions and will inhibit root growth in these experiments. This was observed for high sucrose (90 and 120 g/L) and SA (140 mg/L) treatments. Root growth stimulation rather reflect a positive nutrition effect and was obtained in the treatments using YE 4 mg/L (106.4 mm), which did not differ statistically from SA 1.4 mg/L (105.6 mm), YE 1 mg/L (94 mm) and sucrose 60 g/L + agar (88.9 mm) (Table S1-supplementary material).

These experiments were done using agar and phytagel as gelling agents. Roots were growing slightly better on phytagel with sucrose at 10 g/L (Fig. 2-A), and similarly at 30 and 60 g/L. At sucrose concentration of 90 and 120 g/L the root growth inhibition was less pronounced using agar as compared to phytagel.

Insert Fig. 2 Similarly, in Arabidopsis thaliana seedlings, sugar promoted growth at low concentrations, although, at high concentrations, it acts synergistically to abscisic acid to inhibit growth [START_REF] León | Sugar and hormone connections[END_REF]. Agar is routinely used as a gelling agent for tissue culture; however, it is known that depending on the sources from which it comes, it may contain impurities interfering with the experiments [START_REF] Cosson | Hairy root research: recent scenario and exciting prospects[END_REF]. Phytagel TM , a selected grade of gelrite, is a very clean gelling agent, very effective in different applications [START_REF] Veramendi | Gelrite as an alternative to agar for micropropagation and microtuberization of Solanum tuberosum L. cv. Baraka[END_REF]. Despite that gelling agents should be inert constituents of the culture media [START_REF] Arregui | Effect of gelling agents on in vitro tuberization of six potato cultivars[END_REF], in our red clover seedlings root length experiment phytagel and agar were similar at low sugar concentration (until 60 g/L) but plants grew better on agar (Figure 3-A) at higher sugar concentration.

Insert Fig. 3 This experiment (Fig. 3 -A, Table S1) shows that total T. pratense isoflavones content could be enhanced at high concentrations of sucrose in combination with agar, with a production reaching 11.3 mg/g dry weight using 120 g/L of sucrose when it is only 3.2 mg/g dry weight using 10g/L sucrose. Using phytagel the best isoflavones production (6 mg/g dry weight) was obtained with 90 g/L of sucrose. Although the high sucrose and agar concentrations were the most effective, they were extremely damaging to the seedlings root growth (Fig. 2-A). Plantlets developed on phytagel showed a colour change at 90 g/L of sucrose, with the formation of magenta colour near the cotyledons which correlated with the colour of the flavonoid extracts obtained from them (Fig. S1). Thus, our study also indicates that in T. pratense seedlings sucrose induces phenylpropanoid production with both an increase in anthocyanin and isoflavonoid production (Fig. 3-A and Fig. S1).

SA at low concentration (1.4 mg /mL; Fig. 2-B) increases root growth. At 14 mg /mL there is no change to control and at higher concentrations, root growth is inhibited. SA treatment did not change isoflavonoid production until the concentration reaches 140 mg/L (Fig. 3-B) by which the flavonoid amount almost tripled (11.9 mg/g dry weight). Similar results were described with cell cultures and hairy root cultures of P. corylifolia (Shinde et al. 2009a) and for Phaseolus vulgaris cotyledons (Durango et al. 2013).

Contrary to sucrose and SA, YE (Fig. 2-C) had a rather positive effect on T. pratense seedling root growth. The isoflavonoids production in these YE treated roots decreased for 0.5 and 1 mg/L (Fig. 3-C), was similar to the control for 2 mg/L and increased slightly above the control for 4 mg/L YE. In studies with P. candollei HR cultures, YE was the most efficient elicitor for flavonoid production [START_REF] Udomsuk | Improved isoflavonoid production in Pueraria candollei hairy root cultures using elicitation[END_REF]) and in P. corylifolia cell cultures and HR, YE was the best elicitor, inducing higher levels of daidzein and genistein in both studies (Shinde et al. 2009a;Shinde et al. 2009b). Thus, in contrast to previous studies, at the concentration used YE did not behave as an elicitor but rather promoted growth and was not used further in our experiments.

Flagellin 22 (flg22) is a conserved 22-amino acid-long N-terminal part (QRLSTGSRINSAKDDAAGLQIA) of the main bacterial flagellum protein that can act as a potent biotic elicitor in plants. For T. pratense seedlings, the different concentrations tested did not change root growth significantly (Fig. 2-D) but increased slightly isoflavonoids production, using 0.5 M/L (Fig. 3-D). As for YE, flagellin was not used further in our experiment due to its poor elicitor effect on T. pratense.

All together isoflavonoid production was inversely related to the root growth inhibition (elicitor effect), validating the elicitor testing protocol, and the best effects were obtained with SA and sucrose that multiplied isoflavonoid production by three.

Establishment of the Hairy Root (HR) cultures

Hairy roots are induced by the transfer to the plant recipient genome of the A.

rhizogenes T-DNA sequence containing plant oncogenes [START_REF] Petit | Further extension of the opine concept: Plasmids in Agrobacterium rhizogenes cooperate for opine degradation[END_REF]). These oncogenes allow formation of root cultures that also produce opines. The transformed nature of the roots is attested by the high HR growth rate in absence of hormones, their negative gravitropism and the constancy in metabolite yield due to their genetic stability [START_REF] Chandra | Engineering secondary metabolite production in hairy roots[END_REF].

T. pratense seedlings were infected with the A. rhizogenes strain A4TC24 five days after germination. They showed visible adventitious root development 10 days after inoculation for about 90% of them, with root formation at the site of infection (Fig. 4-A). The putatively transformed roots were cut and transferred to solid medium (Fig. 4-B) where they showed rapid growth and loss of the response to gravity (negative gravitropism; Fig. 4-C).

Rapid elongation is a phenotypic characteristic of such transformed roots, and the rapid lateral branching is responsible for the increase in mass and accumulation of these roots (Eskandari-Samet et al. 2012). When properly developed, the cultures were shifted to liquid culture medium (Fig. 4-D) where the multiplication rate was exponential (Fig. 4-E to H). All these characteristics demonstrate that the root cultures obtained are true HR. Using this protocol, thirteen T. pratense HR cultures were established and further studied.

Insert Fig. 4 a) Isoflavone identification

The thirteen HR cultures were harvested after the second month of culture to analyze their total isoflavonoids content by U-HPLC-ESI-MS/MS. According to these analyses 27 compounds were found in the chromatograms of the extracts obtained from these cultures (Table 1, compounds 1 to 27). Among these compounds it was possible to identify both aglycones and conjugated forms such as glycoside and glucoside malonates (Table 1 and Fig. 5), in agreement with studies done using Trifolium roots [START_REF] Saviranta | Red clover (Trifolium pratense L.) isoflavones: root phenolic compounds affected by biotic and abiotic stress factors[END_REF]).

Insert Fig. 5 Fig. 5 shows that all the cultures were isoflavone producers, with the biochanin A (4) isomers as major compound representing 26.3 to 58.0 % of the total isoflavonoid content. The second most abundant molecule was the formononetin aglycone ( 14), representing 10.1 to 36.5 % of the compounds, followed by other isoflavonoids, such as irilone/ afrormosinemalonyl hexose (9; 4.4 to 17.7 %) and biochanin isomers (6; 6.7 to 13.5%). Other molecules were produced in minor quantities and some of the cultures were unique in having certain molecules. For example, the HR10 culture was the only one to produce compounds 12 and 3, and the HR5 culture the only one producing compound 16.

The HR cultures could be differentiated by their growth capacity and morphology, but mainly by their biochemical production, demonstrating a high variability in isoflavonoids types and content. These dissimilarities can result from genetic differences between the seedlings used to start the HR cultures. Indeed T. pratense is an allogamous diploid plant and individuals are genetically different.

Another important source of variation can be related to the effect of the A. rhizogenes rol genes that can have different levels of expression between the different HR lines. This can result in different auxins and cytokinins contents (or sensitivity) in each line (Fu et al. 2005).

These variations can produce differences in the accumulation patterns of secondary metabolites as well as variations in growth rate and productivity between the clones. Previous studies also showed that, even if the cultures come from a single root, they may present a certain heterogeneity [START_REF] Hu | Hairy Root and Its Application in Plant Genetic Engineering[END_REF].

b)

Isoflavone content in the first five months of culture

In order to study the stability of the red clover root cultures isoflavone profiles during the first months after the transformation, four HR cultures (1HR, 2HR, 4HR and 6HR) were selected based on their good multiplication rate and stability. At the time of liquid culture subculture (each four weeks) part of the cultures were harvested and stored to analyze their isoflavonoids content. Lines 1HR, 2HR, 4HR and 6HR were then evaluated for their daidzein, genistein, formononetin and biochanin A content (Fig. 6-A to D and Table S2-Supplementary material). Fig. 6-E shows the variations in the total isoflavone content of these molecules over time for these four HR cultures.

Insert Fig. 6 The most abundant isoflavones described in red clover are biochanin A (25) and formononetin ( 22), with the second being more present in roots [START_REF] Saviranta | Red clover (Trifolium pratense L.) isoflavones: determination of concentrations by plant stage, flower colour, plant part and cultivar[END_REF]. In contrast, the transformed root cultures analyzed here presented different most abundant metabolites. Line 4HR was the richest in daidzein (27, 1.73 mg /g) at the end of the 150 days (Fig. 6-A, Table S2). Genistein was produced in large quantities in line 2HR (1.01 mg /g) at 120 days of culture (Fig. 6-B, Table S2) and formononetin ( 22) in line 6HR (20.79 mg /g) after 3 months of culture (Fig. 6-C, Table S2). Biochanin A (25) was the main compound detected in line 4HR (13.90 mg /g) after 4 months in liquid medium (Fig. 6-D, Table S2).

The total isoflavones content was also estimated in these four lines. At the first point measured (30 days, Table S2) the total isoflavonoid content of the four HR cultures was three time higher than the seedling one (9.4 to 11.6 mg/g dry roots versus 3.2 mg/g dry plant, Table S1, sucrose 10 g/l and Table S2). The isoflavone content even reached 27 mg per g of dry root in some cultures. Isoflavone production was stable for 1HR and 2HR over the 5 months of the experiment. For 6HR an increase production was observed at month 3 and for line 4HR production increased months 4 and 5 (Fig. 6-E and Table S2). Previous studies did not address isoflavone production over several months. However, they have shown that HR lines can maintain genotypes and stability for extended periods, only loosing this ability with drastic changes of the media or the addition of plant hormones that induce metabolic modifications and cause disorganization in the cultures, as with Datura and Nicotiana cultures (Doran 1997; Anil [START_REF] Kumar | Enhanced production of pharmaceutically important isoflavones from hairy root rhizoclones of Trifolium pratense L[END_REF]).

c) Elicitation of the isoflavonoid production

After the characterization, two lines, 4HR and 8HR, were selected to conduct elicitation tests with sucrose 10 (control) and 60 g/L, as well as with salicylic acid 10 and 30 mg/L. After the treatment, the samples were harvested, stored and extracted for their evaluation in daidzein, genistein, formononetin, biochanin A and the total content of these four molecules using UPLC for a period of 3 to 14 days, depending on the sample analyzed.

In line 4HR grown in presence of sucrose 10 g/L (Fig. 7-A) the isoflavone total concentration decreased during the culture from 11.21 mg /g at 3 days, down to 2.53 mg /g of dry mass in the end of 14 days in culture. The highest production for daidzein (27), formononetin ( 22), biochanin A (25) and total isoflavones was observed 3 days after treatment (Fig. 7-A) and decreased slowly during the experiment. In the case of genistein it was 8 days after treatment.

Insert Fig. 7 For the line 8HR (Fig. 7-B) the total isoflavones content at 3 days (9.77 mg /g) increased slightly at 4 days and decreased to 6.81 mg /g after 8 days. The time determined as best for the highest production was 4 days. The most abundant molecule for this control culture was biochanin A (25). The isoflavone production decreased after 4 days.

Based on these results, isoflavonoid production following elicitation was only studied for 4.5 days.

The use of 60 g/L sucrose in line 4HR (Fig. 7-C) increased the total isoflavones production after 3.5 days of treatment to 27.88 mg /g. This is 2.5-fold the highest concentration (11.209 mg /g) measured for the control experiment and nearly 10 time more than in seedlings. Formononetin (22) increased 4 times, from 4.05 mg /g in control to 17.01 mg /g but genistein production remained low. At 3.5 and 4 days of treatment daidzein (27) was produced in large amounts but at 4.5 days formononetin ( 22) was again the main compound.

Using sucrose at 60 g/L for line 8HR (Fig. 7-D) resulted in a total isoflavones production peaking after 3.5 days of treatment to 21.21 mg /g. Similarly, to what was observed in line 4HR, the isoflavonoid production was different to the one observed in the control culture. Formononetin ( 22) instead of biochanin A (25) was the most abundant compound (14.76 mg /g) after 3.5 days of culture.

Different results were described in Pueraria phaseoloides transformed roots, where 30 g/L sucrose promoted better accumulation of isoflavones after 16 days of treatment [START_REF] Liang | Effect of sucrose concentration on the growth and production of secondary metabolites in Pueraria phaseoloides hairy roots[END_REF]). In Hypericum perforatum L. root cultures, high quantities of sucrose were shown to stimulate the production of total phenols and flavonoids (Cui et al. 2010), similar to what was found in P. corylifolia root cultures, for which high concentrations, like 50 g/L, prolonged the isoflavonoid biosynthesis and induced the accumulation even after more than 30 days of culture [START_REF] Shinde | Impact of nutrient components on production of the phytoestrogens daidzein and genistein by hairy roots of Psoralea corylifolia[END_REF].

T. pratense hairy root cultures were tested using two SA concentrations (10 and 30 mg/L). The 4HR line was relatively insensitive to the 10 mg/L treatment (SA 10 treatment, Fig. 7-E) with the best isoflavone production at 3 days (11.64 mg /g) similar to the control one. In contrast, using 30 mg/L (SA 30 treatment, Fig. 7 -G) the flavonoid content (5.24 mg /g) at 3 days of culture was low as compared to control culture and declined close to zero at the end of 4.5 days. In both cases the treatment did not change drastically the relative content of the different molecules.

The 8HR line produced more isoflavonoid (17.17 mg /g at 3 days) after the 10 mg/L treatment than the control (Fig. 7-F) but this production decreased rapidly to control level in the next sampling time. In the 30 mg/L treatment the production was high (12.20 mg /g) at 3.5 days but reduced importantly later. Note that the isoflavonoids profiles were different from the control experiment. In the SA 10 experiment the main difference to control in the isoflavonoid composition was an increase in daidzein ( 27) content and the absence of genistein. Formononetin (22, 4.67 mg /g) and biochanin A (25, 5.74 mg /g) were the most abundant, at 3.5 days with SA 30 after elicitation. Their production decreased toward the end of the experiment.

Our experiments thus show that the isoflavonoid production can be stimulated in the T. pratense HR cultures by addition of sucrose 60 g/L (Table S3-Supplementary material), mainly for the 4HR line, increasing daidzein (27), formononetin ( 22) and the total isoflavones content well above the values obtained with the seedlings. Note that genistein production was the highest using control sucrose condition (10 g/L). In contrast, SA treatment had rather limited and short-term effects on isoflavonoid production, increasing only biochanin A (25) production 3 days after the treatment. Higher SA concentration had rather negative effects. This is in contrast to the P. corylifolia HR study for which isoflavonoid production was independent of the SA concentration and of the length of the treatment [START_REF] Udomsuk | Improved isoflavonoid production in Pueraria candollei hairy root cultures using elicitation[END_REF].

Similar results for the negative effect of the SA treatments were reported in another study (Shinde et al. 2009b).

Conclusion

In this work, we have produced A. rhizogenes-mediated HR culture lines of T.

pratense and studied their isoflavone content. Both the total or main aglycone isoflavones present in seedlings and HR cultures were determined and their production in the presence of elicitors tested. Seedlings had better root growth in the presence of salicylic acid 1.4 mg/L and yeast extract 4 mg/L, but higher elicitor concentrations inhibited growth. The highest accumulation of total isoflavones in seedlings (11.9 mg/g dry weight) was found using SA 140 mg/L. The independent hairy root cultures showed fast growth rates during the five months of the experiment and increased isoflavonoid production as compared to the seedling.

Interestingly these cultures exhibited different isoflavonoid profiles and these profiles changed in the presence of elicitors, with high-level isoflavone production (27.9 mg/g dry weight) when sucrose was added at 60 g/L in the two lines analyzed.

Thus, these experiments showed that the use of A. rhizogenes strain A4TC24 mediated hairy root cultures in the presence of sucrose 60 g/L could be used to produce different T.

pratense isoflavonoids. The rather simple procedure described here can now be used as the starting point to establish large-scale cultures in bioreactors using amplification of cultures as described in [START_REF] Mehrotra | Bioreactor Technology for Hairy Roots Cultivation[END_REF] or for the manipulation of these cultures by expressing specific genes allowing overproduction of specific molecules.

Notes 4 .

 4 49 g Murashige and Skoog medium basal salt mixture including vitamins and microelements (Duchefa,), 30 g sucrose pH 5.8, 1% agar (Difco,) for solid medium. extract (Difco), 10 g Tryptone (Difco), 10 g NaCl, 10 g agar (Difco) for solid medium, pH 7.0. Sterilized by autoclaving. b) YEB medium: 5 g Beef extract (Difco), 1 g Yeast extract (Difco), 5 g Peptone (Difco), 5 g Sucrose, 2 mM MgSO 4 , pH 7.2. Sterilized by autoclaving. transformation using leaf explants, pp115-27 in Methods in Molecular Biology, Agrobacterium protocols, Kan Wang editor, 343, Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512. Cui X, Chakrabarty D, Lee E, Paek K (2010) Production of adventitious roots and secondary metabolites by Hypericum perforatum L. in a bioreactor. Bioresour Technol 101:4708-4716. doi: 10.1016/j.biortech.2010.01.115 De Coninck B, Timmermans P, Vos C, et al (2015) What lies beneath: Belowground defense strategies in plants. Trends Plant Sci 20:91-101. doi: 10.1016/j.tplants.2014.09.007 Doran PM (1997) Hairy roots. CRC Press Du H, Huang Y, Tang Y (2010) Genetic and metabolic engineering of isoflavonoid biosynthesis. Appl Microbiol Biotechnol 86:1293-1312. DOI: 10.1007/s00253-010-2512-8 Durango D, Pulgarin N, Echeverri F, et al (2013) Effect of salicylic acid and structurally related compounds in the accumulation of phytoalexins in cotyledons of common bean A, Piri K, Kayhanfar M, Hasanloo T (2012) Enhancement of tropane alkaloids production among several clones and explants types of hairy root of Atropa belladona L. J Med Plants By-products 1:35-42. Fu C, Zhao D, Xue X, et al (2005) Transformation of Saussurea involucrata by Agrobacterium rhizogenes: Hairy root induction and syringin production. 40:3789-3794. DOI: 10.1016/j.procbio.2005.03.063 Galland M, Boutet-Mercey S, Lounifi I, et al (2014) Compartmentation and dynamics of flavone metabolism in dry and germinated rice seeds. Plant Cell Physiol 55:1646-1659. DOI: 10.1093/pcp/pcu095 Webb JK, Jones S, Robbins MP, Minchin FR (1990) Characterization of transgenic root cultures of Trifolium repens, Trifolium pratense and Lotus corniculatus and transgenic plants of Lotus corniculatus. Plant Sci 70:243-254. Wright E, Wang Z-Y (2015) Medicago trunculata transformation using cotyledonary explants. In: Wang K (ed) Agrobacterium protocols. Springer New York, New York,

Fig. 1 Fig. 2 Fig. 3 Fig. 5 Fig. 6 Fig. 7

 123567 Figure Captions:

Table S1 .

 S1 Root growth (mm) and isoflavone content (mg /g dry plants) on T. pratense seedlings grown in medium with elicitors at different concentrations during the first seven days after germination*. Results are the mean of three replicates ± SD and ANOVA followed by Tukey test at 5% of probability. Different letters for the same treatment are significantly different at P< 0.05.

	Elicitor	Dosage	Total isoflavone Root lenght (mm) Standard deviation
		10.0	3.20 D	67.9 EFG	0.163
		30.0	5.75 CD	84.8 CDE	0.227
	Sucrose +
		60.0	5.76 CD	88.9 ABCD	0.310
	agar (g/L)
		90.0	3.91 CD	47.7 G	0.334
		120.0	11.34 AB	21.9 H	0.676
		10.0	3.24 D	76.6 CDEF	0.440
	Sucrose +	30.0	2.35 D	85.4 BCDE	0.088
	phytagel	60.0	2.3 D	82.9 CDE	0.149
	(g/L)	90.0	6.26 BCD	20.7 H	0.205
		120.0	3.09 D	12.2 H	0.337
		1.4	3.42 D	105.6 AB	0.144
	Salicylic
		14.0	3.28 D	60.3 FG	0.191
	acid
		70.0	4.65 CD	16.7 H	0.095
	(mg/L)
		140.0	11.90 A	12.6 H	0.289
		0.25	4.13 CD	83.8 CDE	0.089
	Flagellin	0.50	5.59 CD	69.3 DEF	0.112
	22 (µM/ L)	1.0	2.33 D	70.8 DEF	0.118
		2.0	4.26 CD	85.4 BCDE	0.203
		0.5	2.59 D	77.0 CDEF	0.088
	Yeast
		1.0	2.51 D	94.0 ABC	0.074
	extract
		2.0	3.35 D	82.9 CDE	0.151
	(mg/L)
		4.0	4.28 CD	106.4 A	0.137

*

Table S2 .

 S2 Isoflavones and total isoflavones content of the T. pratense HR lines 1HR, 2HR, 4HR and 6HR during the first 5 months of culture. Results are the mean of three replicates ± SD and different letters in the same isoflavone detected are significantly different at P< 0.05, using Tukey test.

					29
		1HR	9.90 ± 0.58 BCD	2.80 ± 0.27 CD	2.51 ± 0.35 CD	9.27 ± 0.28 BCD	5.76 ± 0.29 CD
	Total isoflavone (mg/	2HR	11.65 ± 0.41 BC	5.41 ± 0.12 CD	5.31 ± 0.08 CD	11.30 ± 0.23 BC	6.45 ± 0.29 CD
	Analysis	Culture	30 days	60 days	90 days	120 days	150 days
	g dry roots)	4HR	10.70 ± 0.14 BCD	7.04 ± 0.02 CD	5.62 ± 0.12 CD	27.01 ± 0.28 A	18.79 ± 0.09 AB
		1HR	0.48 ± 0.22 C	0.90 ± 0.11 B	0.09 ± 0.003 DE	0.00 E	0.21 ± 0.053 DE
		6HR	9.36 ± 0.22 BCD	7.49 ± 0.11 BCD	26.96 ± 0.08 A	2.49 ± 0.05 CD	0.13 ± 0.002 D
	Daidzein (mg/ g dry	2HR	0.00 E	0.43 ± 0.02 C	0.00 E	0.00 E	0.71 ± 0.116 B
	roots)	4HR	0.43 ± 0.09 C	0.00 E	0.00 E	0.00 E	1.73 ± 0.01 A
		6HR	0.15 ± 0.06 DE	0.27 ± 0.01 CD	0.00 E	0.00 E	0.13 ± 0.007 DE
		1HR	0.19 ± 0.07 EF	0.19 ± 0.05 EF	0.19 ± 0.06 EF	0.49 ± 0.06 C	0.24 ± 0.04 DE
	Genistein (mg/ g dry	2HR	0.52 ± 0.04 B	0.00 G	0.00 G	1.01 ± 0.001 A	0.27 ± 0.03 DE
	roots)	4HR	0.13 ± 0.06 F	0.48 ± 0.03 C	0.48 ± 0.05 C	0.48 ± 0.05 C	0.00 G
		6HR	0.32 ± 0.04 D	0.23 ± 0.01 DEF	0.69 ± 0.01 B	0.17 ± 0.01 EF	0.00 G
		1HR	4.81 ± 0.90 EF	1.16 ± 0.24 JK	1.98 ± 0.62 IJ	3.44 ± 0.46 GH	3.04 ± 0.35 HI
	Formononetin (mg/	2HR	5.70 ± 0.48 DE	5.48 ± 0.18 E	2.68 ± 0.18 HI	5.64 ± 0.51 DE	5.35 ± 0.73 EF
	g dry roots)	4HR	6.78 ± 0.06 D	4.27 ± 0.03 FG	2.18 ± 0.20 IJ	12.62 ± 0.60 C	13.87 ± 0.11 B
		6HR	5.63 ± 0.43 DE	5.12 ± 0.32 EF	20.79 ± 0.18 A	1.14 ± 0.08 JK	0.00 K
		1HR	5.67 ± 1.14 B	1.47 ± 0.67 E	1.43 ± 0.71 EF	5.34 ± 0.61 B	2.97 ± 0.73 CD
	Biochanin A (mg/ g	2HR	5.36 ± 1.12 B	1.61 ± 0.29 DE	2.63 ± 0.14 CDE	5.16 ± 0.43 B	2.78 ± 0.27 CDE
	dry roots)	4HR	3.36 ± 0.36 C	2.29 ± 0.002 CDE	3.22 ± 0.28 C	13.90 ± 0.46 A	3.19 ± 0.26 C
		6HR	5.00 ± 0.35 B	2.12 ± 0.11 CDE	5.48 ± 0.14 B	1.56 ± 0.09 DE	0.00 F

*

Table S3 .

 S3 Effect of elicitors on the accumulation of major and total isoflavones (mg/ g dry plant) in two T. pratense hairy roots line. *

				4 12.465 ± 1.720 A		0.620 ± 0.061 DEFG	2.843 ± 0.432 CD 0.147 ± 0.076 LM 3',7-Di-O-Methylorobol (5Hydroxy-3-(4-hydroxy-3-methoxyphenyl)-7-methoxy-4H-16.075 ± 0.572 BCDE
	11		11.71	315	315	-
	Elicitor HR line Days 4.5 1.895 ± 0.031 EFGHI Daidzein	Genistein 0.513 ± 0.031 FGHIJ 15.184 ± 5.289 A 4.217 ± 0.673 CD Formononetin Biochanin A chromen-4-one)	Total isoflavones 21.808 ± 1.561 ABC
	12		11.94	3 4.975 ± 0.159 BCD 3 4.184 ± 0.164 BCDE 269 517	0.159 ± 0.002 K 0.687 ± 0.014 DEF 248	4.049 ± 0.180 CD 1.846 ± 0.121 FGHIJK 0.604 ± 0.088 D 0.157 ± 0.055 LM Formononetine -Malonyl Hexose	11.029 ± 0.116 DEFGHIJ 5.461 ± 0.080 GHIJKL
	13 14		12.05 HR 12.33	3.5 2.942 ± 0.059 BCDEFGH 0.110 ± 0.001 K 3.5 1.937 ± 0.298 EFGHI 0.395 ± 0.009 FGHIJK 14.763 ± 0.879 A 4.114 ± 0.302 CD 2.053 ± 0.026 CD 0.708 ± 0.013 JKLM 283 531 248 Pseudobaptigenin-Malonyl Hexose 4 3.850 ± 0.001 BCDEF 0.167 ± 0.005 JK 2.294 ± 0.049 CD 0.882 ± 0.003 IJKLM 4 5.397 ± 0.271 B 0.220 ± 0.028 HIJK 7.973 ± 1.031 BC 1.803 ± 0.239 FGHIJK 285 285 -Biochanin/calycosin/maackiain/Glycitein/Prunetin/Texasin 21.209 ± 0.372 ABC 5.814 ± 0.025 GHIJKL 7.192 ± 0.014 FGHIJKL 14.943 ± 0.392 BCDEF
	15		12.41	4.5 1.906 ± 0.052 EFGHI 4.5 5.002 ± 1.103 BCD 269 517	0.625 ± 0.001 DEFG 0.612 ± 0.150 DEFG 248	2.263 ± 0.010 CD 1.388 ± 0.027 GHIJKLM 6.182 ± 0.022 FGHIJKL 2.054 ± 0.467 CD 0.418 ± 0.085 KLM 8.086 ± 0.451 EFGHIJKL Formononetin -Malonyl Hexose
	16		4 HR 13.26	5 1.360 ± 0.010 FGHI 3 5.171 ± 0.010 BC 285	533	0.448 ± 0.004 FGHIJK 1.268 ± 0.008 D 0.301 ± 0.028 GHIJK 3.526 ± 0.118 CD 2.642 ± 0.092 EFG 1.109 ± 0.012 HIJKLM 4.185 ± 0.008 HIJKL 11.639 ± 0.062 DEFGHI 248 Biochanin/calycosin/maackiain/Glycitein/Prunetin/Texasin -Malonyl Hexose
	Sucrose 10 g/L 17 10 18 mg/L 19 acid 20 Salicylic		13.52 HR 13.54 14.41 14.58 HR	8 2.583 ± 0.036 CDEFGHI 0.869 ± 0.012 CDE 3.5 4.099 ± 0.064 BCDE 0.208 ± 0.008 HIJK 299 547 162 11 0.559 ± 0.002 HI 0.533 ± 0.003 EFGHI 0.400 ± 0.006 D 0.547 ± 0.003 D 1.752 ± 0.033 CD 1.503 ± 0.112 GHIJKL Irilone/Afrormosine-Malonyl Hexose 0.937 ± 0.063 HIJKLM 4.937 ± 0.029 HIJKL 7.563 ± 0.054 EFGHIJKL 1.707 ± 0.144 GHIJK 3.199 ± 0.039 IJKL 14 0.313 ± 0.027 HI 0.356 ± 0.004 FGHIJK 0.548 ± 0.001 D 4 5.229 ± 1.089 BC 0.239 ± 0.011 HIJK 2.243 ± 0.355 CD 1.276 ± 0.183 GHIJKLM 8.987 ± 0.410 DEFGHIJKL 301 549 248 Pratensein/ 3 O Methylorobol (5,7-dihydroxy-3-(4-hydroxy-3-methoxyphenyl)-4Hchromen-4.5 4.167 ± 0.153 BCDE 1.153 ± 0.019 BC 1.652 ± 0.090 CD 1.513 ± 0.076 GHIJKL 4one) Malonyl Hexose 8.485 ± 0.084 DEFGHIJKL 1.312 ± 0.119 GHIJKLM 2.529 ± 0.038 JKL 3 0.988 ± 0.004 GHI 0.924 ± 0.003 CD 3.521 ± 0.024 CD 4.341 ± 0.026 BCD 9.774 ± 0.014 DEFGHIJK 3.5 1.206 ± 0.007 FGHI 1.326 ± 0.005 B 3.015 ± 0.001 CD 4.373 ± 0.003 BCD 9.921 ± 0.004 DEFGHIJ 285 533 248 Biochanin/calycosin/maackiain/Glycitein/Prunetin/Texasin -Malonyl Hexose 3 3.618 ± 0.617 BCDEFG 0.410 ± 0.061 FGHIJK 5.867 ± 0.866 CD 7.279 ± 1.347 A 17.174 ± 0.723 BCD 3.5 1.384 ± 0.003 FGHI 0.202 ± 0.002 HIJK 2.444 ± 0.044 CD 3.463 ± 0.002 DE 7.492 ± 0.013 EFGHIJKL 301 301 -Pratensein/ 3 O Methylorobol (5,7-dihydroxy-3-(4-hydroxy-3-methoxyphenyl)-4Hchromen-4one)
	21 22		15.51 8 HR 15.92	4 2.479 ± 0.011 DEFGHI 4 2.208 ± 0.073 EFGHI 283 283 4.5 1.558 ± 0.015 EFGHI 4.5 2.327 ± 0.561 DEFGHI 269 269	2.525 ± 0.045 A 0.123 ± 0.005 K -1.380 ± 0.010 B 0.548 ± 0.355 EFGH -	3.938 ± 0.004 CD 4.995 ± 0.034 BC 1.556 ± 0.016 D 2.036 ± 0.034 EFGHIJ Pseudobaptigenin 0.499 ± 0.028 D 0.795 ± 0.008 JKLM 1.220 ± 0.133 D 1.335 ± 0.170 GHIJKLM 5.181 ± 0.306 GHIJKL 5.923 ± 0.032 GHIJKL 13.938 ± 0.024 CDEFG 4.232 ± 0.015 HIJKL Formononetin
	mg/L 23		17.29	5 1.057 ± 0.024 GHI 3 1.935 ± 0.077 EFGHI 299 299	1.165 ± 0.010 BC 0.599 ± 0.030 DEFG	1.507 ± 0.055 D 1.379 ± 0.085 D Irilone/Afrormosine	2.247 ± 0.043 EFGHI 1.324 ± 0.086 GHIJKLM 5.237 ± 0.070 GHIJKL 5.840 ± 0.033 GHIJKL
	Sucrose 60 24 30 25 Salicylic acid 26	g/L	4 HR HR 18.29 18.51 19.36	8 1.128 ± 0.003 GHI 3 1.536 ± 0.305 EFGHI 3.5 12.774 ± 1.332 A 3.5 1.318 ± 0.032 FGHI 4 0.375 ± 0.012 HI 307 365 285 285 4.5 0.172 ± 0.016 I 269 372	1.114 ± 0.025 BC 0.404 ± 0.029 FGHIJK 17.011 ± 5.510 A 4.354 ± 0.740 BCD 1.397 ± 0.006 D 3.175 ± 0.023 DEF 0.652 ± 0.066 DEF 13.383 ± 1.156 AB 1.075 ± 0.199 HIJKLM 27.883 ± 0.688 A 6.815 ± 0.014 FGHIJKL 23.306 ± 1.646 AB 0.167 ± 0.008 JK 0.886 ± 0.022 D 0.815 ± 0.019 IJKLM 3.186 ± 0.020 IJKL 0.397 ± 0.003 FGHIJK 0.013 ± 0.006 D 0.171 ± 0.0002 LM 0.956 ± 0.005 KL g -Biochanin A 0.155 ± 0.001 K 0.178 ± 0.007 D 0.042 ± 0.005 M 0.369 ± 0.007 L 103 Formononetine Malonyl

Acknowledgements

Thanks to the Coordination for the Improvement of Higher Education Personnel and National Council for Scientific and Technological Development/ Brazil for the financial assistance provided through PhD and Scientific Initiation scholarships. J.A.S.Z. thanks CNPq for the researcher fellowship, Programa Iberoamericano CYTED -proyecto BIFRENES 416RT0511 and the National Institute of Science and Technology -INCT BioNat -grant # 465637/2014-0. Thanks also to Dr. Sandra Beatriz Rech, Dr. Miguel Dall'Agnol, Dr. Amelia Henriques and Marí Castro for the collaboration. Thanks to Dr. Marie Garmier for careful reading and editing of the manuscript. The Project benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS).

Compliance with ethical standards

Conflict of Interest: The authors declare that they have no conflict of interest.

Supplementary material

Media

All media were calculated for a volume of 1L and were sterilized by autoclaving for 20 min at 120 C. Antibiotics, hormones or other components, were added after autoclaving the media.


Yoan Bourlier

Bruno Bérini

Mathieu Frégnaux

Arnaud Fouchet

Damien Aureau

Yves Dumont

Transfer of Epitaxial SrTiO 3 Nanothick Layers Using Water-Soluble Sacrificial Perovskite Oxide

Keywords: SrVO 3, SrTiO 3 pseudo-substrate, sacrificial layer, silicon, water soluble

Integration of functional thin film materials with adapted properties is essential for the development of new paradigms in information technology. Among them, complex oxides with perovskite structures have huge potential based on the particularly large diversity of physical properties. Here we demonstrate the possibility of transferring perovskite oxide materials like SrTiO 3 onto silicon substrate using an environmentally friendly process at nanoscale, by means of a water-soluble perovskite sacrificial layer: SrVO 3 . Based on in situ monitoring atomic force microscopy and photoemission studies, we reveal that dissolution is initiated from a strontium rich phase at the extreme surface of SrVO 3 . The transferred nanothick SrTiO 3 layer on silicon presents an effective morphology and monocrystalline quality, providing a proof of concept for the integration and development of an all perovskite oxide electronics or "oxitronics" onto any Si-based substrates.

MANUSCRIPT

INTRODUCTION

Thin films technology, particularly from ABO 3 perovskite oxides, has always been considered as strong impactful research for science and technology in a perspective beyond silicon electronics. [1][2][3] Tremendous optical and electrical responses can now be induced at nanoscale, where significant results have been demonstrated, for example the presently famous and extensively studied LaAlO 3 /SrTiO 3 heterostructure. [4][5][6][7][8][9][10][11][12] However, the growth of functional perovskite oxides on silicon or amorphous substrate like glass remains a long-standing challenge due to mechanical and chemical incompatibilities between the supports and the crystalline structure of the film. Indeed, a SiO 2 layer at the surface avoids a direct epitaxy and the perovskite oxide films can be grown only at the cost of high temperature (in order to crystallize) and only in polycrystalline form, which generally decreases the intensity of the targeted functional properties. This is due to the inherent anisotropies in pristine or distorted perovskite oxide structures. Different approaches have been developed recently to break through this critical technological barrier. First, special care can be used to remove this SiO 2 protective layer and stabilize the Si surface. This approach has been developed by Mc Kee et al. [13] in order to stabilize a buffer layer of SrTiO 3 (STO). Furthermore, significant efforts have been made to develop STO buffer layer on silicon using molecular beam epitaxy (MBE) [14][15][16][17][18][19][20][21] or other associated techniques such as hybrid MBE (hMBE) giving the latest great improvements. [22] However, these buffer layers are deposited at high temperature, and are barely scalable at industry level. Furthermore, this STO/Si interface remains compressively strained with defects at interface. [23] Recent promising work shows the possibility of growing STO on Si using only pulsed laser deposition (PLD), but the resulting films are oxygen-deficient due to the use of a low oxygen pressure to preserve an intimate interface. [24] An alternative for the growth of perovskites on amorphous substrate is the use of nanosheets acting as seed layers. These nanosheets are deposited onto the substrates by Langmuir-like deposition using soft chemistry and the most common one is Ca 2 Nb 3 O 10 -. [25][26][27][28][29][30][31][32][33] These nanosheets are 2D materials, with a thickness of 1 to 2 nm and in-plane length from 500 nm to a few micrometers. The in-plane lattice parameter is 0.386 nm, close to the targeted perovskite used.

These nanosheets are nevertheless randomly deposited on the substrate. Consequently, the final film is textured out of plane and presents in most of the cases a random orientation in plane.

Another similar work using 2D materials was reported of the growth of STO using PLD on graphene coated Si substrate; but even when a high (00l)-oriented crystalline STO is reported, other crystalline orientations are also observed. [34] Another approach is the use of a sacrificial layer (SL) which preserves the in plane orientation and retains the structural quality of the transferred film. This process implies the deposition of the targeted functional monocrystalline film on a SL which can be dissolved by chemical treatment depending on its nature. The resulting freestanding functional monocrystalline film is then released onto another substrate. This process was first initiated to transfer metallic layers with NaCl crystals acting as SL and dissolving in water. [35] After several years, the process evolved to transfer much more complicated materials such as graphene layers, using hBN flakes, [36,37] or by dissolving Cu or Ni foils in FeCl 3 and HCl chemical baths. [38][39][40][41][42][43] Transfer of perovskite oxide crystalline layers such as PbZr 0.2 Ti 0.8 O 3 (PZT) or SrRuO 3 (SRO) using SL was investigated later, using either La 0.7 Sr 0.3 MnO 3 , [44] or directly STO substrate, [45] as SL with their dissolution in HCl or HF respectively. Nevertheless, the transferred film must be strongly chemically stable to those acids, which limits the choice of the materials to be transferred. Alternatively, another phase of Sr 3 Al 2 O 6 , is water soluble and can also be used as SL. [46,47] Even if this material is interesting, special care has to be considered for the growth of the transferable heterostructure, because of the possible change of crystal structure rendering Sr 3 Al 2 O 6 insoluble. [48] Ideal SL materials for functional perovskite oxides should be of the same family of materials, with a close lattice parameter, and present water solubility. In the present study, we demonstrate that strontium vanadate perovskite oxide: SrVO 3 , noted SVO, is a good candidate for perovskite SL as it fulfilled all these conditions. Bulk SrVO 3 presents a cubic crystal structure with lattice parameter of 0.384 nm, [49] and is well adapted for the growth of all perovskites.

Recent research on SVO has shown that this material is a promising transparent conductive oxide for integration of new devices in microelectronics industry. [50][51][52] Furthermore the surface of this material can be adjusted under specific growth conditions in order to obtain either a very smooth surface with a global roughness (in root mean square or RMS) of 0.5 ± 0.1 nm or a surface governed by auto-organized nanostructures. These nanostructures have already been identified as crystallites of Sr-rich phases of Sr 3 V 2 O 8 . [53,54] In our previous study, a selective etching of these Sr-rich nanostructures was evidenced using water. Moreover all Sr-rich phases were found to be removed from the surface by the action of water. [55] In this paper, we expose the process of SVO dissolution in water, proving that SVO can be used as a SL for perovskite oxides transfer. Through AFM and XPS analyses, we show that strontium rich phases exist at the surface of SVO layers. Such phases are obtained at the end of the growth, as already seen from STO surfaces where SrO surface segregation takes place after annealing, [56][57][58] and are essential to initiate the SVO solubilization by preferential etching. At first, two different surfaces, a smooth one and another presenting the Sr-rich nanostructures, were considered. Finally, we are showing the transfer of STO thin films. This latter compound is widely used as a substrate for epitaxial growth of a wide range of perovskite oxides with numerous different functionalities. Transferring well-conformed thin films of STO onto Si is a first step towards the intriguing challenge to make efficient epitaxial regrowth and developing Si-integrated technologies with based-oxide electronic devices for a new dawn of "oxitronics".

RESULTS

A. In situ monitoring of the SrVO 3 layer dissolution in water

Study of the SVO layer dissolution in water can be observed in figure 1 for two types of surface morphology: the smooth one and the nanostructured one. First, a dynamic imaging concerning a 40 nm-thick film with nanostructured surface is investigated by in situ atomic force probe (AFM) monitoring, in liquid mode (see the layout in figure 1a). From the numerous AFM micrographs taken in liquid mode analysis, an editing video on the in situ dissolution of the nanostructured SVO thin film through the action of water was produced and is available as supplementary information (SI) S1 (see the attached MPEG file to the web version of this article) with an associated graph of roughness evolution (root mean square or RMS) through acquisition time.

Then, three AFM images collected from the video are shown in figure 1b, one before water contact, one after 7 min, and one after 2 h 04 min. We observe a relatively fast dissolution of the nanostructures in the first minutes in water. These nanostructures were already analyzed by transmission electron microscopy (TEM) as Sr 3 V 2 O 8 phase, [53] and also described as half-buried through the surface, [54,55] where the dissolution in water first leaves their imprints on the surface. Thus, a preferential etching of these Sr-rich nanostructures is evidenced using water. Then, for longer times in water (from 3 min to 52 min), the roughness progressively increases and SVO layer dissolution is initiated around the nanostructures imprints. After more than 50 min, the nanoimprints are no longer discernable, SVO layer dissolution increases and the entire film starts to decompose in larger aggregates until it almost totally disappears after 2 hours.

In comparison to the SVO nanostructured thin film, the dissolution of the SVO smooth thin film is presented in figure 1c. This film is 40 nm-thick and presents a low roughness of 0.6 nm (root mean square or RMS), characterized by AFM. In this experiment, the middle of the smooth SVO layer is covered by a protective polymer resin, leaving unprotected both sides of the sample. After one day in water at room temperature and without stirring, the SVO uncovered zones are completely dissolved, with respect to film center which remains intact. The protective polymer resin can be removed in acetone in order to access the pristine SVO layer. Indeed, SVO smooth thin films are water soluble. Since these thin films are proven to be Sr-rich on their surfaces, [55] we can affirm that the dissolution of SVO is initiated by them. Furthermore, SVO thin films containing nanostructures appear readily soluble compared to the smooth thin films where dissolution takes longer. Thus, the kinetics of the SVO thin film dissolution seems to be accelerated by the effective roughness resulting from the imprint of the nanostructures.

B. Study of SrVO 3 and SrTiO 3 nanothick layers during transfer

In the light of these observations, we have shown that SVO layer can be used as a watersoluble SL for transferring a thin film of perovskite oxide such as STO onto silicon (Si) substrate. Figure 2a presents the outline of the transfer process, similar to other works. [42,46] First, a 20 nm-thick SVO film was grown onto a STO substrate, followed by a 90 nm-thick STO film, using pulsed laser deposition (PLD) technique. The STO thin film was deposited through a mechanical mask, [59] leaving a smaller area of the STO film (2.5×2.5 mm²) compared to the SVO film deposited on the entire STO substrate (10×10 mm²). This difference in size will ease the future transfer of the STO layer.

Within the transfer process, the surface of the sample is first covered with a silicone type film serving as a stamp (see step 1 in figure 2a). The success for transferring the totality of the STO layer relies on the use of commercial "screen protector" (SP) film. Such SP film are basically constituted of a silicone coating on back side (applying in front of the STO thin film, and of a poly-ethylene-terephtalate (PET) film on its front side for the rigidity of the whole film.

The use of SP film has been particularly chosen compared to other silicon type resin film such as (poly-dimethyl siloxane) PDMS film. Indeed, when PDMS film was used, we encountered difficulties for transferring the entire STO layer. Supporting information S2 shows a STO layer initially of 3.9×3.9 mm² square-sized onto Si substrate where less than 20 % of the area is finally transferred by the use of PDMS film as intermediate support. According to the work of X.-D.

Chen et al. [42] about transfer of graphene films, the transfer efficiency and integrity is strongly dependent o, the surface tension of each layer. Contrary to SP film which has the ability to easily transfer the supported film, transfer using PDMS film is very sensitive to pressure and release time. [60] This can be explained by a smaller work of adhesion at the SP/STO layer interface than the PDMS/STO layer interface.

Figure 2b

illustrates the XPS mapping analysis on the corner of the square-shaped STO/SVO stack before transfer. STO area is highlighted by the presence of Ti2p (at 459.0 eV) and the Sr3d (at 134.0 eV) signals. Corresponding high-resolution energy spectra for associated Ti2p and Sr3d spectral windows are generated from the "STO area" zone (see S3 in SI). XPS chemical composition of the STO surface gives a cationic ratio "Sr/Ti" of 1.03 ± 0.05. The "SVO area" is also clearly delimited by the V2p (at 517.1 eV) and the Sr3d (at 132.4 eV) spectral regions. The generated spectra of Sr3d in the "STO area" and "SVO area" are definitely distinct. The different binding energies indicate two different chemical environments. The XPS chemical composition of the "SVO area" reveals a cationic ratio "Sr/V" of 1.44 ± 0.05. This Srrich ratio from the SVO layer is in good agreement with a previous study evidencing that Sr-rich phases exist at the extreme surface of these SVO thin films. [55] As Sr-rich phases were found to be readily soluble in water, this will be an asset for its use as a SL. Figure 2c represents AFM analyses of the STO and SVO surfaces. We first observe a very smooth and homogeneous STO surface with a very low RMS of 0.56 nm. However, SVO is characterized by the presence of nanostructures identified as Sr 3 V 2 O 8 crystalline structure as previously discussed. [53] This fact is due to the reaction between the masked part of SVO and O 2 partial pressure during the growth of STO layer. The presence of nanostructures in the SVO area reinforces the fact that the surface is composed of Sr-rich phases.

After applying the SP film at the surface of the STO layer, the sample is immersed in water at 50°C during about 5 days, under stirring, until all the SVO underneath layer is completely dissolved. The relatively long time of dissolution of the SVO SL can be explained by the efficient sealing of the surface with SP film. However, the process can be accelerated by adding a surfactant to lower the surface tension of water. Furthermore, we observed that a slightly alkaline aqueous media at pH near 8 (by adding KOH salt for instance) help to reduce the dissolution time of the SL. When the entire SVO layer is dissolved, the sample is carefully released from the water and SP is removed by slowly peeling off the film from the substrate (step 2 in figure 2a). At this step, as observed in figure 3a, STO thin film is released from the initial substrate to the SP film. Thus, we can analyze the back-side of the STO layer on SP film, which was in contact with SVO. Figure 3b shows the XPS survey spectrum of the STO thin film standing on the SP stamp. It indicates the presence of the chemical elements Si, Sr, C, Ti and O by their main spectral signatures of Si2p, Sr3d, C1s, Ti2p, and O1s, at their respective binding energies of 101 eV, 132 eV, 284 eV, 460 eV, and 532 eV. In addition to the peaks associated with the STO, silicon is detected. As we can see in figure 3a, the free standing STO film is partially cracked. Therefore, XPS analysis also detects the SP film related to the Si spectral signature. Figure 3c shows the XPS high resolution energy spectrum of the O1s-V2p region of interest. The O1s is clearly divided in two different components, one at a binding energy of 531.5 eV, "O1s (Si-O),(C-O)" attributed to SP film spectral signature and organic contamination, and one at a binding energy of 528.5 eV, "O1s (M-O)" attributed to the STO transferred layer.

However, no V2p signature was found on this spectrum, which means that no vanadium was detected or its amount is below the XPS sensitivity threshold (0.1 at.%). Therefore, the SVO layer is fully dissolved by our process. Furthermore, the STO layer standing on SP is analyzed by XRD analysis (figure 3d). A preferential orientation of STO perpendicular to the [001] direction is observed with STO (001) and STO (002) Bragg peaks at 2θ angles of 22.68°, and 46.18°, respectively. The broad peak at 25.88° is related to SP template (amorphous SiO 2 lattice). These analyses confirm that the entire SVO layer is dissolved, leaving a free-standing crystalline STO film with the pristine orientation.

C. Description of the final SrTiO 3 nanothick layer transferred onto Si substrate

The final step is then to transfer the STO standing on the SP film onto Si substrate by making a physical contact (step 3 in figure 2a). An annealing at 70°C for 10 min is also performed to dry the layer before gently peeling off the SP film. In figure 4a, an optical image of the STO layer on Si is shown. We observe that the entire STO layer is transferred on Si substrate.

Even if some cracks remained, the STO transferred layer is a well-define 2.5×2.5 mm² squareshaped characterized by domains larger than 200 µm. To further improve the quality of the perovskite transferred layer, two main optimizations can be considered. STO upper layer could be protected before conducted the SL process, using for example a photosensitive resin in order to lower the stress during transfer. We can also transfer a perovskite oxide layer with a bulk lattice parameter closer to the one of SVO (a = 0.3840 nm in theoretical values), such as LaAlO 3 (a = 0.3790 nm) compared to STO (a = 0.3905 nm) to reduce strain at interface.

Figure 4c

represents the XPS mapping of the STO transferred layer onto Si substrate. The chemical cartography of the transferred layer is revealed by the presence of the main spectral signatures of Ti2p (458.0 eV), Sr3d (133.5 eV), and O1s (529.0 eV) inside the "square" of STO, shaped by the spectral signature of Si2p (99.0 eV). It proves that strontium and titanium are effectively transferred inside this area. These chemical maps are used to generate an averaged spectrum of each spectral region of interest where the associated spectra are visible in supplementary material (S4 in SI). The resulting XPS chemical composition is characterized by a cationic ratio "Sr/Ti" of 0.96 ± 0.05 which is relevant compared to the analyzed "STO area" from the sample before processing the transfer (figure 2b). The O1s spectra is fitted with the previously introduced approach assuming two different O1s chemical environments: "O1s (Si-O),(C-O)" and "O1s (M-O)". Due to XPS mapping information, the oxygen stoichiometry of the STO layer can be also evaluated. "(Sr+Ti)/O" oxygen ratio is then extracted, through quantification of Sr3d, Ti2p and O1s (M-O) contributions, and was estimated to "0.71± 0.05" which is slightly higher than a STO oxygen stoichiometry of 0.67 for the theoretical "(Sr+Ti)/O" ratio. This definitely evidences the conservation of the STO layer chemical composition after transfer onto Si.

In addition, figure 5a shows a 2x2 µm² AFM image of the STO transferred layers, selected from an area free of cracks. A well-preserved smooth and homogeneous surface is observed, characterized by a low roughness of RMS = 0.66 nm, similarly to the one measured on STO surface before transfer (RMS = 0.56 nm, see figure 2c). The conservation of the surface roughness proves the good quality of the transferred layer. The AFM height scan analysis in figure 5b is taken at the side of the reported STO layer. It reveals an averaged thickness of 88 nm, which is in good agreement with the original thickness of STO layer (90 nm-thick STO layer assumed by the PLD process). It confirms that only SVO was dissolved and that the entire STO thickness is transferred.

Most importantly, XRD analysis of the STO layer on Si substrate is performed (figure 5c).

The Bragg peaks at 2θ angles of 32.99°, and 69.13° are associated to (002) and (004) planes of the Si substrate. STO Bragg peaks corresponding to (001), (002), and (004) indexes, are visible at 2θ angles of 22.66°, 46.25°, and 103.48°, respectively. It proves that the STO transferred layer is well crystallized with its original preferential orientation preserved, following the (00l) crystalline planes. Moreover, the Laue oscillations around STO (002) peak is measured and simulated (represented in figure 5d), giving a lattice parameter of c = 0.392 nm and a number of 230 unit-cells. The resulting thickness estimation of 90 nm is in perfect agreement with the ones calculated from AFM height scan and estimated from PLD process conditions. The same observation was done on thinner STO layer using a 20 nm-thick square of 3.9×3.9 mm² (see S5, S6, and S7 in SI, for microscopy images, AFM analyses, and XRD analyses respectively). These well-resolved Laue oscillations up to high order confirm the atomic flatness of the STO in the rear face and the good quality of an intimate STO/Si interface. All these measurements demonstrate that the initial crystalline structure, thickness, and roughness on both sides of the STO thin film are conserved upon transfer onto Si substrate.

Such featured results conclusively show that SVO layer can be used as a SL to report STO thin film onto Si substrate, encouraging a future vertical integration onto Si substrate of other perovskite oxides and/or associated heterostructures.

CONCLUSION

In this paper, we demonstrate the feasibility of transferring SrTiO 3 (STO) perovskite oxide thin films onto Si using SrVO 3 (SVO) as sacrificial layer (SL). The presented process is soft, low-cost, and environmentally friendly with only water as solvent. The process of SVO dissolution was studied by in situ AFM monitoring. We show that existence of Sr-rich phases at the extreme surface of SVO thin film is a clue to initiate its dissolution in water. The STO transferred layer onto Si exhibits a similar roughness and thickness than the ones before transfer, and most importantly, the initial stoichiometry and monocrystalline structure following the (00l) oriented plans are preserved. Transferring homogeneous and well-conform STO oxide layers using a SL of perovskite SrVO 3 is opening a new way for perovskite oxides integration onto Si substrate and other supports such as glass or flexible substrates. This proof of concept remains the crucial step for direct integration of such oxides in microelectronic and the development of a new electronics based on functional perovskite oxides.

Experimental Section

Epitaxial thin films growth conditions: SrVO 3 (SVO) and SrTiO 3 (STO) targets for pulsed laser deposition were prepared in the laboratory from high purity binary oxides powders (SrO/V 2 O 5 and SrO/TiO 2 ) by standard ceramic processing method. The pressed pellet was sintered in air with additional oxygen flow. The "Sr/V = 1" and "Sr/Ti = 1" target metallic ratios were controlled by chemical titration. Depositions of both layers were performed in an ultra-high vacuum chamber with base pressure of 5×10 -7 Pa. Targets were ablated using a KrF laser with a wavelength of 248 nm and 20 ns of pulse duration. Single crystal (SurfaceNet GmbH) STO (100) substrates (10×10×0.5 mm 3 ) were used. The laser fluency was about 1.85 J/cm -3 for SVO and STO. The distance between target and substrate was kept to 5 cm. The temperature and oxygen pressure for SrVO 3 layer was maintained to 760°C and 1.2×10 -4 Pa, respectively. [53] In this conditions, SVO film show an excellent conductivity with a R300/R2=2.4 at the state of the art for PLD growth onto STO (100). [61] The thickness was estimated to 20 nm from RHEED measurements. Then, a shadow mask was pressed to create four STO square per sample of 2.5×2.5 mm² each. A shadow mask of 3.9×3.9 mm² was also used to create larger square-shaped areas. Temperature was rapidly decreased to 720 °C (approximately 4 min) and pressure was kept constant during the beginning of STO growth to minimize the appearance of nanostructures on the underlying layer and progressively increased to 7.5×10 -6 Pa in order to obtain a better oxygenation. 90 nm-thick layer of STO was deposited. Samples were cooled directly after the growth with the same pressure in 2 hours. A 20 nm-thick layer of STO was also deposited in the same growth conditions with exception that the shadow mask was applied for both SVO and STO layers. A longer time to dissolve the entire SVO layer was observed probably due to the absence of the step between the two layers. The corresponding STO layer transferred onto Si substrate is shown in supporting information (relative to figures S5, S6 and S7).

Thin films transfer method: All SrTiO 3 (STO) layers were transferred using commercial "screen protector" (SP) sheets (PET/silicone film). SP film was adhered to STO surface sample and introduced into water at a temperature of 50°C with stirring for 5 days, in order to dissolve the entire SVO layer. After the complete etching of SVO, the SP film supporting the STO layer was applied onto Si substrate. The SP film was then released after a short annealing at 70°C for 30 min to dry the film. A single STO/SVO thin film sample was partially transferred onto Si substrate by using a poly-dimethyl siloxane (PDMS) resin film (see figure S3 in supporting information). The synthesis of PDMS film was conducted using a Dow Corning ® 184 Silicone Elastomer.

Surface characterizations details:

A Dimension ICON (Bruker) Atomic Force Microscope was employed in PeakForce Tapping mode to control the morphology of both SVO and STO thin films (see figure 2c and figure 5a) using commercial silicon tips on a nitride lever with 70 kHz resonant frequency and 0.4 N.m -1 spring constant (ScanAsyst-AIR Bruker). The scanning speed was kept constant at 2 µm.sec -1 in order to compare RMS for images of different size.

More specifically, a sharpened ScanAsyst-Fluid + tip (Bruker) was used in a fluid cell mounted on a 90 µm × 90 µm × 14 µm scanner to follow by in situ monitoring the dissolution of the SVO layer in deionized water as function of time (see figures 1a andb). In order to pursue this in situ AFM analysis, an image was first acquired in air to obtain the initial state. Then, a water droplet was placed on the tip by using a syringe, and another droplet deposited on the sample, giving the time t = 0 of the experiment. The scanner is lowered until the droplet on the cantilever joins and formed a meniscus with the droplet on the sample (see figure 1a). Consequently, the scan area is not exactly the same than the first image in air. For faster acquisition, the scan size range was fixed to 2 µm with an aspect ratio 3:1. For this experiment 117 images were collected, the first image of the SrVO 3 surface in water was obtained after 2 minutes 20 seconds and the last image after 124 minutes and 11 seconds. The scanning frequency was set to 2 Hz, with 256 samples per line for images n°1 to n°78, allowing an acquisition time of 50 seconds, and with 512 samples per line for images n°79 to n°117 for a resulting acquisition time of approximatively 100 seconds. Images were analyzed with the Nanoscope Analysis 1.8 software (Bruker) and gathered in Windows Movie Maker v2.6 to make a movie of the SVO dissolution in water (see the supporting information S1). We used one-in-two images for images n°1 to n°78 and all images from n°79 to n°117 to reach a similar interval of time for the video. Regular X-ray Photoelectron Spectroscopy (XPS) measurements were carried out using a Thermofisher Scientific ESCALab 250 Xi spectrometer with a monochromatic Al-Kα X-ray source (hν = 1486.6 eV). The detection was performed perpendicular to the sample surface using a constant analyzer energy (CAE) mode (pass energy 20 eV), and spectra were recorded with a 0.1 eV energy step. The survey spectrum in figure 3b was collected with a pass energy of 100 eV and an energy step of 1 eV. The use of a low-energy electron and ion flood gun was necessary to perform the analysis. Quantification was performed on the C1s, O1s, Sr3d, V2p 3/2 peak areas after a Shirley type background subtraction using the Thermofisher Scientific Avantage © software. XPS Imaging of the reported STO layers on top of Si substrate was performed in parallel imaging acquisition mode using a 900 µm spot, i.e. photoelectrons from the whole of the field of view (1 mm) are detected simultaneously. Electrons of a given kinetic energy are focused on the channel plate detector to produce a direct image of the sample without scanning. Integrating images from consecutive energies, an average spectrum of the selected area can be generated. Maps were recorded in the energy range of O1s, Sr3d and V2p, using a 20 eV pass energy and 0.1 eV energy step between each acquisition. XPS imaging of the SVO-STO stack was recorded with a Thermofisher Scientific Nexsa spectrometer with a monochromatic Al-Kα X-ray source (hν = 1486.6 eV) in the "SnapMap" mode. In such imaging mode, the micro-focused beam was rastered across the sample by rapid stage movements, yielding an elementary map (O1s, Sr3d, Ti2p and V2p) with a field of view of 3 mm². Full spectra were acquired at each pixel with pass energy of 150 eV.

The crystal structure of the STO layers were characterized by X-ray diffraction (XRD) analyses using a Bruker D8 Discover diffractometer equipped with a Cu Kα1 radiation source (λ = 0.15406 nm).

ASSOCIATED CONTENT

Supporting Information

The following files are available free of charge on the ACS Publications website at DOI:

FIGURESFigure 1 :

 1 FIGURES

Figure 2 :

 2 Figure 2: SVO used as a sacrificial layer for perovskite thin film transfer onto Si substrate,

Figure 3 :

 3 Figure 3: Analysis of the STO layer supported by the SP film, (a) photography of the

Figure 4 :

 4 Figure 4: STO thin film transferred onto Si substrate, (a) optical micrograph reconstruction

Figure 5 :

 5 Figure 5: STO transferred layer onto Si substrate with physical properties conserved, (a)

S6: AFM analyses of the STO transferred layer related to figure S5. S7: XRD analyses of the STO transferred layer related to figure S5.

AUTHOR INFORMATION

Corresponding Author

*E-mail: yoan.bourlier@uvsq.fr.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT