Rosmarinic Acid Content in Basil Plants Grown in Vitro and in Hydroponics
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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 caffeic acid derivatives (CADs), in particular rosmarinic acid (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 form of this 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: chlorogenic acid, caffeic (oxygen content >6.0 mg L-1).