Cent. Eur. J. Biol.• 6(6) • 2011 • 946-957 DOI: 10.2478/s11535-011-0057-1

Central European Journal of Biology

Rosmarinic acid content in basil plants grown in vitro and in hydroponics

Research Article

Claudia Kiferle1,*, Mariella Lucchesini1, Anna Mensuali-Sodi2, Rita Maggini1, Andrea Raffaelli3, Alberto Pardossi1

1Department of Crop Biology, University of Pisa, 56124 Pisa, Italy 2Sant’ Anna School of Advanced Studies, 56100 Pisa, Italy 3Institute for the Chemistry of Organo Metallic Compounds (ICCOM), National Council of Research (CNR), 56126 Pisa, Italy Received 19 January 2011; Accepted 29 May 2011

Abstract: The accumulation of selected derivatives (CADs), in particular (RA), was investigated in different tissues (leaves, roots and plantlet shoots) of sweet basil (Ocimum basilicum L.) plants grown either in vitro or in hydroponic culture (floating system) under greenhouse conditions. Two cultivars with green leaves (Genovese and Superbo) and one with purple leaves (Dark Opal) were tested. The content of CADs in HCl-methanol extracts was determined by HPLC. LC-MS and LC-MS-MS were used to confirm the identification of the metabolites of interest. Apart from rosmarinic acid (RA) and a methylated formofthis substance, no other CADs were detected at significant level in any of the analyzed samples. The content of RA ranged approximately from 4 to 63 mg/g DW, depending on the growing system. The highest RA content was found during the in vitro multiplication, in the acclimatized plants and in the roots of hydroponically-grown seedlings at full bloom. In vitro, 6-benzyladenine reduced the accumulation of RA in purple-leaf Dark Opal cultivar, but an opposite effect of this growth regulator was observed in the green-leaf genotypes. Our findings suggest the possibility to scale-up RA production by means of in vitro or hydroponic culture of sweet basil.

Keywords: 6-benzylaminopurine • Cytokinin • Floating system • Leaves • Micropropagation phase • Ocimum basilicum • Physiological stage • Roots • Soilless culture. © Versita Sp. z o.o.

1. Introduction Medicinal and herbal plants, including basil, are typically cultivated in open fields, resulting in year-to- Sweet basil (Ocimum basilicum L.) in the Lamiaceae year variability in the biomass production as well as in family is one of the most important herbs and is widely the content of secondary metabolites, which are both cultivated worldwide [1]. Basil leaves are largely employed affected by factors such as weather, soil fertility, growing as a flavoring agent for food. For instance, sweet basil practices and the presence of pests and diseases [7]. is the main ingredient of the well-known ‘pesto’ sauce Therefore, there is an increasing interest for the artificial and in Italy the cultivation of this species increased cultivation of these crops, either in vitro or in vivo considerably in the last years due to the growing demand (i.e. greenhouse hydroponic culture), where growing from the food industry [2]. Along with other species in the conditions can be strictly controlled to consistently Ocimum genus, sweet basil is used for pharmaceutical produce high-standard plant material all-year round and cosmetic preparations due to the high content of [8,9]. Sweet basil can be grown hydroponically, which essential oils [1,3] and rosmarinic acid [4,5]. Rosmarinic offers several advantages over traditional soil culture, acid (RA) is a caffeic acid ester with several important such as higher yield per unit ground area and better biological properties, including antioxidant, antibacterial, quality standards of harvested biomass. Hydroponically- antiviral and anti-inflammatory activities [6]. grown plant material is clean and easy to process due

* E-mail: [email protected] 946 C. Kiferle et al.

to minimal contamination from pollutants, pests and The explants were reduced in size, washed in current pathogens [10]. Floating systems is a hydroponic water for 30 min, surface-sterilized by soaking them technique that is increasingly used for greenhouse under agitation in a 15% aqueous solution of NaOCl (8% production of fresh-cut leafy vegetables, including basil active chlorine) with a few drops of Tween 20TM (Sigma- [11], and has also been utilized for the cultivation of Aldrich, Milan, Italy) for 15 min and rinsed three times medicinal plants [12,13]. with sterile deionized water. The explants were then cut Cell and tissue culture, or in vitro approaches, in single 1-cm long nodal segments under a laminar flow constitute an alternative to conventional agriculture cabinet. Each explant was placed horizontally in 30-ml for the production of high-value plant metabolites, polycarbonate UryTM vials (PBI, Milano, Italy) with 5 ml of because they provide an opportunity to regulate plant MS (Murashige and Skoog) [24] with 30 g L-1 of sucrose, biosynthetic pathways in a controlled environment 300 mg L-1 of reduced gluthatione (GSH) and 7 g L-1 [9,14]. The possible application of plant tissue culture agar. 2-(N-morpholino) ethanesulfonic acid (MES) for the production of bioactive compounds has been was added (500 mg L-1) to stabilize the pH, which was demonstrated in several species [9,14], although at adjusted to 5.8 before autoclaving at 121°C for 15 min. present only very few substances are produced in vitro MES concentration (2.3 mmol L-1) was much lower than on a commercial scale [15]. Different basil species the levels that were found to be toxic to callus culture were found to accumulate larger quantities of RA in (10 mmol L-1) [25] or to affect secondary metabolism cell, callus, hairy roots and shoot cultures than in vivo in Catharanthus roseus hairy roots (50 mmol L-1) [26]. [16-19]. The total phenolic and RA levels were higher During shoot initiation, 0.25 mg L-1 6-benzylaminopurine in sweet basil grown hydroponically than in soil-grown (BA) was added to the basal medium and the explants plants [20] were placed in a climatic chamber at 25±1°C with a Compared to in vitro culture, hydroponics offers the 16 h photoperiod and an irradiance of 70 mmol s-1 m-2 advantages of a higher rate of biomass production per from cool fluorescent tubes. In the multiplication phase, unit area and less expensive growing structures [21,22], nodal explants were sub-cultured every four weeks although the production cost per unit weight of the using PCCV25TM boxes (TQPL Co., New Milton, United metabolites of interest is not necessarily lower in vivo Kingdom) with 50 mL of solid growth medium (eight than in vitro [23]. explants per box). Root formation in nodal explants In this work, the accumulation of caffeic acid (1 cm long, excised from shoots at the end of the derivatives (CADs), in particular of RA, was studied in multiplication phase) was induced in PCCV25TM boxes three cultivars of sweet basil, with green (Genovese and containing 100 mL of perlite soaked with 50 mL of half- Superbo) or purple (Dark Opal) leaves, grown either strength MS nutrient solution without growth regulators in vitro or in hydroponic culture. To our knowledge, (eight explants per box). no paper has been published on the influence of Finally, well rooted plantlets were acclimatized in micropropagation phase on the accumulation of these 4-cm height LinfaboxTM boxes (Micropoli, Milano, Italy) metabolites in basil. containing 200 mL of sterilized peat-perlite mixture (1:1, v:v); five plantlets were transplanted in each box. The containers were wrapped with plastic and incubated in a 2. Experimental Procedures growth chamber at 20±3°C at 16 h of photoperiod with 100 mmol s-1 m-2 PAR (from HPS lamps). Plastic covers 2.1 Plant material were partially opened after one week and completely The plants were originated from seeds purchased from removed the subsequent week. Acclimatization SAIS (Cesena, Italy) and grown at University of Pisa concluded after four weeks, when the surviving (ex vitro) either in vitro or in hydroponics (floating system). plants were transferred to a greenhouse. We conducted two experiments using this 2.2 In vitro experiments previously-described protocol. In the first experiment, An original protocol for the micropropagation was growth and tissue concentration of CADs and pigments developed previously. Nodal segments were excised, (anthocyanins and chlorophyll) were determined in at the time of flowering, from basil seedlings grown shoot explants of cv. Genovese at the end of each in floating systems under greenhouse conditions. To micropropagation phase. In the second experiment, we prevent pathogen contamination, the mother plants investigated the effect of BA concentration (0.1, 0.25, were sprayed weekly with Benomyl (1.0 g L−1; Du Pont 0.5 and 1 mg L-1) on the rate of shoot proliferation and Agricultural products, Wilmington, Delaware, USA) in the content of CADs and pigments in all genotypes at the last three weeks before the collection of explants. the end of the multiplication phase.

947 Rosmarinic acid content in basil plants grown in vitro and in hydroponics

2.3 Hydroponic culture 5000 rpm. Following centrifugation, the supernatant was Seeds were germinated in rockwool tray plugs in a collected in plastic tubes and stored overnight at -20°C. growth chamber (25±1°C; 250 mmol s-1 m-2 PAR; The pellet was extracted again with 5 ml of solvent and 12 h photoperiod) and seedlings were transferred at the two supernatants were combined for analysis. All the second-leaf stage to a glasshouse under natural extracts were filtered with Chromafil® 0.45 μm cellulose temperature and light conditions. Two weeks after mixed esters membrane, 25 mm diameter syringe filters sowing, the plants were transferred to 12 separate (Macherey-Nagel, Düren, Germany) prior to HPLC hydroponic systems, each consisting of a polystyrene separations. plug tray floating in a plastic tank with stagnant nutrient HPLC grade solvents and the following chemically- solution (300 L m-2), which was continuously aerated pure standards were used: , caffeic (oxygen content >6.0 mg L-1). Crop density was 40 acid, , t-, p-coumaric acid plants per tank, corresponding roughly to 160 plants m-2 (Sigma–Aldrich, Milano, Italy) and RA (Extrasynthese (ground area). The nutrient solution contained the S.A., Genay, France). HPLC analytical equipment following concentrations of macro- and micro-nutrients: included a Jasco (Tokyo, Japan) PU-2089 four-solvent -3 -3 -3 4.0 mol m N-NO3, 1.0 mol m N-NH4, 0.5 mol m low-pressure gradient pump and a UV-2077 UV/Vis -3 -3 -3 P-H2PO4, 2.5 mol m K, 3.0 mol m Ca, 1.0 mol m Mg, multichannel detector. Analyses were performed -3 -3 -3 ® 0.5 mol m S-SO4, 40.0 mmol m Fe, 40.0 mmol m B, using a Macherey–Nagel C18 250/4.6 Nucleosil 5.7 mmol m-3 Cu, 17.6 mmol m-3 Zn, 10.0 mmol m-3 Mn, 100-5 column, at a flow rate of 1 ml min-1, equipped 1.0 mmol m-3 Mo. Electrical conductivity (EC) of nutrient with a guard column, using acetonitrile (solvent A) and solution oscillated between 1.55-1.80 dS m-1 and pH was aqueous 0.1% phosphoric acid (solvent B) as elution maintained between 5.5 and 7.0 by frequent adjustment solvents. The gradient elution was programmed as with diluted sulphuric acid. follows: 0.0-0.4 min, B 95%; 0.4-0.5 min, B 95-85%; Climatic parameters were continuously monitored 0.5-10 min, B 85-80%; 10-20 min, B 80-60%; 20-21 min, by a weather station located inside the greenhouse. B 60-5%; 21-25 min, B 5%; 25-26 min, B 5-95%; The minimum and ventilation air temperature were 16 26-30 min, B 95%. Detection was made at four and 27°C, respectively; maximum temperature reached wavelengths: 325 nm, 280 nm, 300 nm and 350 nm. up to 30–32°C during daytime in early summer. Daily The injection volume was 20 μL and the analyses global radiation and mean air temperature averaged, were performed at room temperature (23–29°C). The respectively, 12.0 MJ m-2 and 25.1°C. substances of interest were identified by comparing Plants were sampled during the vegetative and their retention times with those of reference standards flowering stages (two and four weeks after transplanting, and quantified on the basis of the integrated peak area, respectively) for growth and chemical analysis. Plant as compared with a standard curve. height and the fresh and dry weight of leaves, stems The content of CADs was expressed per gram DW and roots were measured in each sample consisting of on the basis of the dry matter content determined in an one individual plant. All the roots and the leaves (apart aliquot of each sample after desiccation in a ventilated from a few basal leaves, if senescent) were sampled oven at 85°C (to reach a given weight). The detection and a representative aliquot was extracted for the limit of the analytical protocol was on the order of determination of CADs and pigments. 0.05 mg g-1 DW. Peak identification was accomplished by LC-MS and 2.4 Phytochemical analysis LC-MS-MS using a PE Sciex API 365 triple quadrupole Plant samples from in vitro or hydroponic cultures mass spectrometer LC-MS System (Concord, ON, were rapidly washed in tap water, rinsed in deionised Canada), equipped with a Turbospray source and water and gently dried with a towel. A sub-sample of coupled to a 200 Series HPLC system with quaternary approximately 0.5 g FW was frozen in liquid nitrogen pump and autosampler. The separations were carried and stored at -80°C before laboratory analysis, out with the same column used for HPLC analysis. In which was performed within a few weeks following order to improve the analytical sensitivity, phosphoric sampling. acid was replaced by formic acid because the former The concentration of CADs in each sample was produces a strong suppression effect under electrospray determined as reported by Maggini et al. [13]. Briefly, ionization conditions [27]; this modification did not frozen tissue was ground in a mortar with 5 ml alter the elution pattern. The identification of selected

extraction solvent (MeOH:H2O:HCl 70:29:1 v/v) and CADs was confirmed by comparison with authentic sieved into plastic tubes. Samples were shaken for 4 h standards showing the same retention times and on a magnetic stirrer and then centrifuged for 8 min at MS-MS fragmentation patterns.

948 C. Kiferle et al.

2.5 Pigments all samples, although a quantitative determination of this Total content of chlorophyll and anthocyanins was compound was not performed. determined spectrophotometrically and expressed on a DW basis. 3.2 In vitro culture Leaf samples (20 mg) were extracted with 2 mL In all tissue culture experiments, the plantlets ethanol 95% (v/v) overnight at 4°C in the dark. The developed normally and exhibited well-formed shoots extracts were centrifuged (3 min at 5000 rpm) and without hyperhydric symptoms in each phase of subsequently analyzed at 666.2 nm and 654.6 nm. micropropagation (Figure 2). All ex vitro plants survived Chlorophyll concentrations were calculated according and adapted well to greenhouse cultivation. to Lichtenthaler’s [28]. For anthocyanin analysis, Total dry mass of in vitro plantlets (on average, methanol 80% (v/v) containing 1.2 M HCl was used 50.34 mg each) and the length of newly-formed for extraction and the concentration of cyanidin-3- shoots (1.05 cm) were similar at the end of induction glucoside equivalents was determined by measuring the and multiplication phase, whereas shoot number was absorbance of the extracts at 535 nm. higher in the latter stage (Table 1). Dry mass and shoot formation were lower during rooting than in any 2.6 Statistical analysis other culture phase (Table 1). Acclimatized plantlets The experimental design was completely randomized. exhibited the highest dry biomass of all stages sampled. Data were subjected to a one-way or two-way analysis Rosmarinic acid was detected in in vitro plantlets during of variance (ANOVA) and the means were separated all phases of micropropagation (Figure 3). The highest using the Tukey’s test. Each experiment was repeated RA content (43.21 mg g-1 DW) was found in acclimatized two to three times with similar results and those from a plantlets, although the highest production of RA under representative run are reported in this paper. genuine in vitro conditions was detected in the explants at the multiplication phase. In the second experiment, which was carried out with 3. Results all three cultivars, we demonstrated that the proliferation rate of explants could be increased by modulating the BA 3.1 Quantification of CADs in basil tissues concentration in the medium. No clear effects of the tested Preliminary experiments were conducted in order to BA concentrations (0.10, 0.25, 0.50 and 1.00 mg L-1) validate the HPLC method adopted for this study and to were found on the number and the length of regenerated identify the major CADs contained in frozen, non-dried, or oven-dried (70°C) roots and leaves of sweet basil plants grown in hydroponic culture and/or in vitro. Among the six CADs of interest, only RA was found in considerable concentrations in all analyzed samples, while the other metabolites were present in trace quantities (e.g. ferulic and caffeic acid) or below the detection limits (0.05 mg g-1 DW). The typical chromatogram of methanolic extract of basil sample showed two main peaks, one of which was identified as RA by means of LC-MS analysis while the other corresponded to a methylated derivative of this substance (Figure 1). LC-MS-MS analysis suggested that methylation was present on the caffeic acid moiety. Frozen, non-dried samples of all types of tissues contained invariably much more RA and the methylated derivative compared to the dried ones (data not shown). Therefore, in the experiments with both in vitro and in vivo plants, only frozen, non-dried samples were analyzed. Only trace amounts of chlorogenic acid, caffeic acid, Figure 1. Typical HPLC chromatogram of pure standard of rosmarinic ferulic acid, t-cinnamic acid and p-coumaric acid were acid (RA, top) and HCl-methanolic extract of in vitro shoots of sweet basil (O. basilicum L.) (bottom). The peak marked detected in the analyzed samples, while the methylated with asterisk in the bottom chromatogram was identified form of RA was detected at significant concentrations in by LC-MS as a methylated form of RA.

949 Rosmarinic acid content in basil plants grown in vitro and in hydroponics

Figure 2. Micropropagation of sweet basil (O. basilicum L., cv. Genovese): in vivo (hydroponic) cultivation of mother plants (A); shoot bud induction on MS + 0.25 mg L-1BA (B); shoot proliferation on MS + 0.25 mg L-1BA (C); rooting of nodal segments on ½ MS (D); acclimatized plants (E).

Phase Shoot number Shoot length (cm) Plantlet height(cm) Fresh weight (g) Dry weight (mg)

Induction 1.88b 0.96bc 1.80c 0.598a 50.68b

Multiplication 2.57a 1.14ab 2.52b 0.602a 50.00b

Rooting 1.09c 1.41a 1.65c 0.169b 11.50c

Acclimatization 1.20c 0.55c 3.80a 0.742a 82.77a

Table 1. Growth parameters of sweet basil (O. basilicum L., cv. Genovese) in vitro plantlets at the end of each micropropagation phase. Each phase lasted four weeks. Mean values of 15 replicates, each consisting of one plantlet. One-way ANOVA was performed. Values followed by different letter differs significantly (P≤0.001).

shoots, and on the total dry weight of individual plantlets than in Dark Opal (on average, 8.59 mg g-1 DW) and, (data not shown), which averaged 2.14 shoot per in the latter cultivar, increased with BA concentration explants, 1.35 cm and 48.16 mg, respectively. However, (Figure 5). The chlorophyll content was not significantly a strong genotype-dependent effect of BA was observed affected by plant genotype nor BA level, averaging on RA production. In green-leaf cultivars, the highest 9.92 μg g-1 DW. RA contents were observed in the plantlets grown with 1.0 mg L-1 BA in the medium, while in red-leaf Dark Opal 3.3 Hydroponic culture the RA concentration decreased with increasing BA In the floating systems, plants grew vigorously and level (Figure 4). flowered abundantly within one month after planting The content of anthocyanins was much lower in (Figure 6). Fresh and/or dry matter (Table 2) accumulation the green-leaf cultivars (0.61 mg g-1 DW, on average) in shoots, leaves and roots of hydroponically-grown

950 C. Kiferle et al.

Figure 3. The content of rosmarinic acid (RA) in vitro plantlets of Figure 5. The effect of BA concentrations in the growing medium sweet basil (O. basilicum L. cv. Genovese) sampled at on the content of anthocyanins (Cy-3-Glc equivalents, the end of each micropropagation phase. Each phase measured spectrophotometrically) in the shoots of sweet lasted four weeks: Induction (I); Multiplication (M); basil (O. basilicum L. cv. Dark Opal) grown in vitro and Rooting (R); Acclimatization (A). Mean values of four sampled at the end of the multiplication phase, which lasted replicates, each consisting one single plantlet. One way four weeks. Mean values of four replicates, each consisting ANOVA was performed. Values followed by different of one single plantlet. The effects of BA concentration (A) letters differ significantly (P≤0.05). and genotype, and their interaction (A x B), were significant at P≤0.001 according to ANOVA. Values followed by different letters differ significantly (P≤0.05). RA content increased at the flowering stage in both leaves and roots of Genovese and Superbo plants compared to the vegetative stage, with levels reaching 28.88 mg g-1 DW (Figure 7). In contrast, no difference in RA levels between developmental stages were observed in Dark Opal plants, which on average contained much less RA than the other genotypes (4.87 vs. 13.60 mg g-1 DW in the leaves and 9.29 vs. 21.23 mg g-1 DW in the roots). A significant difference in RA level between Genovese and Superbo was found only in the leaves of blooming plants, which was considerably higher in the latter cultivar (Figure 7). The level of RA was higher in the roots (on average, 17.64 mg g-1 DW ) than in the leaves (10.69 mg g-1 DW) regardless of the cultivar and Figure 4. The effect of BA concentrations in the growing medium on the content of rosmarinic acid (RA) in different the physiological stage, with the exception of flowering cultivars (Genovese, Dark Opal and Superbo) of Superbo (Figure 7). There were no differences among sweet basil (O. basilicum L.) in vitro plantlets at the end of multiplication phase, which lasted four weeks. the cultivars in chlorophyll content, which averaged Mean values of four replicates, each consisting of one 16.65 μg g-1 DW. However, the leaves of Dark Opal single plantlet. The effects of BA concentration (A) and seedlings contained more anthocyanins than those of genotype, and their interaction (A x B), were significant -1 at P≤0.001 according to ANOVA. Values followed by other cultivars (on average, 13.15 mg g DW compared different letters differ significantly (P≤0.05). to 0.89 mg g-1 DW). plants was much higher at the flowering stage compared to the vegetative stage. Results indicate that four to five crops per year could be performed with an estimated 4. Discussion annual leaf fresh biomass production of 30 kg m-2. The root to shoot DW ratio increased approximately two- 4.1 CADs quantification fold at the flowering stage in all the cultivars. At the Many authors found several CADs in basil tissues vegetative stage, there were no significant differences although, in many cases, RA was the most abundant among the cultivars in terms of plant height and total [4,5]. In contrast, in our samples only RA was present dry mass. However, the red-leaf Dark Opal plants were at concentrations well above the detection limit of the much smaller than the green-leaf genotypes (Genovese analytical method used in this study; all other CADs and Superbo) when sampled at the flowering stage were found in trace quantities regardless of growing (Table 2). system and plant tissue. In addition, we observed a

951 Rosmarinic acid content in basil plants grown in vitro and in hydroponics

Figure 6. Hydroponic cultivation (floating system) of different cultivars (Genovese, Dark Opal and Superbo) of sweet basil (O. basilicum L.): plants at full bloom, which typically occurred four weeks after planting (A); the typical leaves of cv. Genovese (B), cv. Dark Opal (C), cv. Superbo (D); the typical inflorescences of the cultivars with purple leaves (cv. Dark Opal; E) or green leaves (cv. Genovese and Superbo; F).

Height Shoot FW Shoot DW Leaf FW Leaf DW Root DW Cultivar (cm) (g) (g) (g) (g) (g)

Vegetative stage (2 weeks after transplanting)

Genovese 25.80c 13.037b 0.787c 8,407b 0.517b 0.058d

Dark Opal 22.00c 9.918b 0.611c 6,373b 0.379b 0.024d

Superbo 27.70c 13.863b 0.862c 9,195b 0.537b 0.053d

Full bloom (4 weeks after transplanting)

Genovese 83.59a 67.138a 7.516a 41,752a 3.835 a 1.047a

Dark Opal 53.65b 60.667a 4.128b 39,446a 3.260a 0.362b

Superbo 80.60a 72.138a 8.158a 47,392a 4.365a 0.952a

Significance

Cultivar (A) *** * * NS NS ***

Developing stage (B) *** *** *** *** *** ***

A x B ** NS NS NS NS ***

Table 2. Growth parameters of different cultivars (Genovese, Dark Opal and Superbo) of sweet basil (O. basilicum L.) plants grown hydroponically and sampled during vegetative stage and at full bloom. The shoots included inflorescences when sampled at flowering stage. Mean value of 15 replicates, each consisting of one individual plant. Values followed by different letters differ significantly (P≤0.05). Two-way ANOVA was performed (*P≤0.05; **P≤0.01; ***P≤0.001; NS=not significant).

952 C. Kiferle et al.

and further work is in progress to more thoroughly characterize the methylated form of RA and to verify whether its production is a result of genuine biosynthesis or artifactual methylation during methanol extraction. The high levels of RA detected in our study (approximately, from 4 to 60 mg g-1 DW), were within those reported in the literature for Sweet basil tissues, which range from less than 0.1 mg g-1 DW [17,20] to nearly 100 mg g-1 DW [4]. This big variability is probably a consequence of differences of plant genotype, growing conditions as well as of the method used for the quantification. The observed difference in RA levels is likely a result of differences among plant genotypes, growing conditions, as well as of the method used for RA quantification. In our preliminary work, RA content was much lower in samples that had been desiccated at 70°C. Sample heating was found to reduce the root content of echinacoside in Echinacea angustifolia [33] and Echinacea pallida [34] due to volatilization or thermal decomposition. Consequently, only frozen, not dried, samples were processed and analyzed in our reported experiments.

Figure 7. The content of rosmarinic acid (RA) in leaves 4.2 In vitro culture and roots of different cultivars (Genovese, In experiment 1, the proliferation aptitude of explants, Dark Opal and Superbo) of sweet basil as indicated by the number and length of shoots at the (O. basilicum L.) plants grown hydroponically and sampled during vegetative stage and at full bloom. end of multiplication stage, was lower compared with Vegetative and flowering plants were sampled two and those found in similar studies with sweet basil [35,36]. four weeks after transplanting, respectively. Mean values of four replicates, each consisting of one single plant. Nevertheless, our protocol resulted in biomass production The effects of cultivar (A) and physiological stage (B), on the order of 50 mg DW per plantlet (Table 1), which and their interaction (A x B), were significant at P≤0.001 according to ANOVA. Values followed by different letters was nearly 50-fold higher than that observed by Kintzios differ significantly (P≤0.05). et al. [17] in nodal segments cultured in a bioreactor. The lowest shoot dry weight was found in rooted peak in all samples that LC-MS analysis attributed to a plantlets (Table 1). Root formation may have occurred methylated form of RA. In many samples, the peak area at the expense of shoot development, as found in other corresponding to this compound was even higher than plant species grown in vitro [e.g. 37]. that of RA (data not shown). Ex vitro plantlets acclimatized successfully, with Methyl rosmarinate (MeRA) has been detected considerable growth (1.56 mg per plantlet per day) in several plants species, particularly within the measured in the four weeks following the transfer out Lamiaceae [29,30]. However, to our knowledge no of in vitro vessels, although newly-formed lateral shoots paper has been published reporting the presence of were shorter compared to those developed in the in vitro MeRA in basil tissues. In the present study, LC-MS-MS phases (Table 1). Ex vitro conditions in combination analysis excluded the correspondence of the unknown with the absence of cytokinin application likely restored peak with MeRA because the methylation was present apical dominance, enhancing plant elongation and on the caffeic acid moiety. Similar to our work, a later- reducing the development of lateral shoots. eluting RA methylated derivative was found in basil The highest RA content (43.21 mg g-1 DW) cell suspensions by Strazzer et al. [31]. Since RA was found at the end of acclimatization (Figure 3). methylation can occur both in vivo and during extraction Increased RA accumulation at this stage was likely a with methanol, Strazzer et al. [31] quantified RA in response to the stress induced by ex vitro conditions samples by considering both the area of the RA peak [38] which stimulated secondary metabolism. It is well and that of its methylated form. known that the synthesis of CADs is enhanced by In vitro studies showed that the antioxidant capacity both biotic and abiotic stress [39].The accumulation of of RA alkyl derivatives could be higher than RA [32], considerable amounts of biomass (Table 1) and the high

953 Rosmarinic acid content in basil plants grown in vitro and in hydroponics

RA content (30.45 mg g-1 DW; Figure 3) indicates the 4.3 Hydroponic culture feasibility of in vitro shoot culture for the production of The floating system was found to be an efficient RA in sweet basil. approach for producing large amounts of plant material In experiment 2, we did not observe any significant that can be easily processed on an industrial scale effect of BA on shoot proliferation. In contrast, previous (Table 2) in agreement with previous findings [10,11]. studies testing BA concentrations similar to those used in One of the advantages of the hydroponic growth our study have demonstrated a strict correlation between system is that it facilitates harvesting of root tissue, shoot proliferation and the level of BA in the medium for which accounted for 10% of total dry mass (Table 2) and O. basilicum [36,40]. The influence of cytokines on shoot had significantly higher RA concentrations than shoot initiation in in vitro culture is affected by many factors, tissues (Figure 7). Previous studies on members of including the growing protocol and plant genotype [41], the Lamiaceae also reported higher RA content in the which may explain the differences between our findings roots compared to the leaves [19,47]. The antimicrobial and those reported by other authors. properties of RA may represent a constitutive defense The hormone composition of the growth medium of the plants against microbial infection, playing an may affect the synthesis of plant secondary important role in the root-pathogen interaction [19]. metabolites in vitro [14,42]. For instance, addition Shoot and root concentrations of RA ranged between of cytokines to the culture medium enhanced the 4 and 29 mg g-1 DW, and were similar to levels reported accumulation of alkamide and CADs in Echinacea in previous studies [4,43]. The highest levels of RA were angustifolia [42]. In this work, we observed the most detected at full bloom in both leaves and roots (Figure 7). significant differences in RA content among the three Juliani et al. [5] reported that leaf concentration of RA cultivars at the highest BA concentration (Figure 4). in basil increased during flowering with respect to the Previous research has also shown a large variability in vegetative stage, although to a lesser extent in Dark RA accumulation of different basil cultivars in culture Opal plants than in the green-leaf genotypes under [43]. Increasing BA concentrations resulted in an investigation. Similarly, the highest content and the best increase of RA content in green-leaf cultivars, which composition of essential oils are generally found in basil coincides with previous findings (Figure 4). Rady plants at full bloom [3]. In contrast, in other Lamiaceae, and Nazif [18] observed that RA content increased such as rosemary [48], spearmint and peppermint [49], in in vitro shoots of Ocimum americanum L. with BA leaf RA content was lower at the flowering stage than at concentrations up to 1 mg L-1. In contrast to green- the vegetative stage. leaf basil genotypes, RA content decreased in purple- Contrasting results are reported in the literature leaf Dark Opal at BA concentrations above 0.5 mg L-1. regarding RA content in basil cultivars with different leaf Anthocyanin accumulation increased in response to color. In our study, leaf and root concentrations of RA BA addition in Dark Opal plantlets (Figure 5), which in the purple-leaf cultivar Dark Opal were lower than in agrees with previous findings in other species such as the green-leaf cultivars (Figure 7), in agreement with Catharanthus roseus, Celosia argentea and Cordyline Javanmardi et al. [4]. Opposite results were reported by terminalis [44]. The accumulation of anthocyanins Juliani et al. [5] and Nguyen and Niemeyer [43], who was not affected by BA in green-leaf cultivars (data found higher RA levels in Dark Opal plants than in other not shown). cultivars with green leaves. Regardless of genotype, the concentration of RA in O. basilicum shoot tissues (40 to 60 mg g-1 DW) 4.4 In vitro vs. hydroponic culture exceeded the levels previously reported by Kintzios In vitro grown shoots, especially those of the Dark et al. [16,17] and by Moschopoulou and Kintzios Opal cultivar, contained much higher levels of RA [45], who used different in vitro systems (e.g. callus, (Figures 3, 4) than did the plants in hydroponic culture cell suspension and immobilized cells) and growth (Figure 7). In experiments with various Ocimum regulators (e.g. NAA, 2,4D ). These findings suggest species, tissues or cells cultured in vitro accumulated that the pattern of RA accumulation in vitro is not RA to levels that were 3- to 11-fold higher than in donor predictable. On the other hand, increased synthesis plants [16,50]. Secondary metabolism may be altered of secondary metabolites is often associated with by artificial environmental conditions provided byin vitro cell or tissue differentiation, such as aggregation of culture and by the composition of the growth medium cell suspensions or the formation of more complex [14,51]. structures such as adventitious roots or shoots [46], The lower content of both chlorophylls and which may explain the accumulation of high amounts anthocyanins in basil tissues grown in vitro compared of RA in cultured shoots in our study. to hydroponically-grown plants was likely a result of

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the presence of sugars in the medium as well as of found a positive correlation between the content of RA the reduced irradiance inside the cultivation vessels. and anthocyanins. Exogenous supply of sucrose reduced chlorophyll In conclusion, the highest RA concentrations content in Solanum tuberosum [52]. Anthocyanins are were observed during the in vitro multiplication, in the able to reduce photo-oxidative injury in leaves and their acclimatized plants and in the roots of hydroponically- concentration is generally reduced when the plants are grown seedlings at full bloom. In vitro, BA reduced the grown under low irradiance [53]. Moreover, the reduction accumulation of RA in purple-leaf Dark Opal cultivar, but of gas exchange in cultivation vessels could lead to an increased RA accumulation in the green-leaf genotypes. accumulation of ethylene and CO2, which stimulates the Our findings suggest RA production can be improved degradation of photosynthetic pigments [54]. by utilizing in vitro or hydroponic culture of sweet basil. Observing the RA content of plants cultured both The differences in RA and anthocyanin content in vivo and in vitro, it stands out a negative relation observed in vivo and in vitro suggest a negative relationship between the accumulation of the metabolite and between the accumulation of these two compounds. Work anthocyanins. When in vivo and in vitro plants, green is in progress to elucidate the possible interaction between and purple-leaf genotypes or the Dark Opal plantlets the synthesis of these metabolites in basil tissues as well cultured in vitro at different BA concentrations in the as the nature and the origin (genuine in vivo biosynthesis basal medium are compared in terms of RA and or artifact during the methanol extraction) of the methylated anthocyanins content, it emerges a negative relation form of RA detected in our samples. between the accumulation of these metabolites. A common substrate of RA and anthocyanins pathways is 4-coumaroyl-CoA [55] and a competition between Acknowledgements their individual biosynthesis could be hypothesized, as also suggested by the higher RA content in green- vs. This work was partially supported by 2008-PRIN project red- varieties of Perilla frutescens [56]. However, in titled “Plant cultures as source of endocrine interfering cell suspensions of Dark Opal basil, Strazzer et al. [31] metabolites” (scientific resp. A. Mensuali).

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