Introduction

Polyphenolics like stilbenes possess a broad spectrum of pharmacological and therapeutic effects such as antioxidative, anti-cancer, anti-atherosclerosis activities, as well as having cardioprotective, hepatoprotective and neuroprotective effects (Nassiri-Asl and Hosseinzadeh 2009; Waffo-Teguo et al. 2008; Baur and Sinclair 2006; Delmas et al. 2006). Low incidence of atherosclerosis in red wine drinking society is often co-related with significant level of stilbenes in red wine (Renaud et al. 1998; Waffo-Teguo et al. 2008).

Plant secondary products are of immense use as potential drugs, chemopreventive agents, nutraceuticals and food additives (Ramawat et al. 2009; Ramawat and Goyal 2009). Due to their limited availability and complexity for chemical synthesis, an alternative route such as plant cell culture is receiving importance for large-scale production of desired compound (Savitha et al. 2006). Root cultures (untransformed and transformed) are an excellent system to produce plant secondary metabolites (Putalun et al. 2007; Verma et al. 2007).

The bottleneck during in vitro multiplication and production of value added phytochemicals are governed by factors such as explant selection, its age and plant growth regulators that control the growth and cellular differentiation and production of secondary metabolites (Suri and Ramawat 1995; Goyal and Ramawat 2007). Roots are well-structured plant organs involved in the absorption of mineral nutrients and water from the soil or in a liquid culture medium (hydroponics) and supplied with suitable carbon source from the aerial parts of the plant. It may therefore be predicted that they should easily and readily be grown in a defined medium. Although Agrobacterium rhizogenes mediated hairy roots have wide acceptance and potential for large-scale culture for secondary metabolite production and plant regeneration, however hairy roots usually produce opine like substances, which are lethal to mammalian cells (Paek et al. 2005). Therefore researchers are now paying attention toward exploring the potential of adventitious roots for in vitro manipulation.

Phytoalexins from the Vitaceae constitute a rather restricted group of polyphenolic secondary metabolites belonging to the stilbenes family (piceid, resveratrol, viniferin, ampelopsin). Stilbenes production is mainly studied in cell cultures of Vitis vinifera (Waffo-Teguo et al. 2008).

We have previously reported occurrence of stilbenes in callus cultures of Cayratia trifolia (L.) Domin (Vitaceae), a tropical lianas (Roat et al. 2008) and their production in cell cultures as influenced by plant growth regulators (Roat and Ramawat 2009). The present study accomplishes the development and growth of untransformed roots of C. trifolia as affected by elicitors, growth retardant and precursors for the high yield of stilbenes.

Materials and methods

Cultures initiation and experimental setup

Root cultures of C. trifolia were initiated by transferring 4 g friable callus into 250 ml Erlenmeyer flask containing 100 ml of solidified MS medium (Murashige and Skoog 1962) supplemented with 0.5 mg l−1 NAA, 0.1 mg l−1 kinetin, 3% (w/v) sucrose and 0.8% (w/v) agar. Roots initiated on this medium were separated and transferred into 250 ml flasks containing 60 ml liquid MS medium of same composition. Initially 1 g roots were used to subculture. The inoculum comprises four to five roots (~5 cm long with two to three laterals). The cultures were grown for three to four passages in the medium to establish them in the liquid system. These cultures were maintained at 25 ± 1°C on an orbital shaker at 60 rpm under diffuse light and 55–60% relative humidity. The roots produced were subcultured after every fourth week into fresh medium.

To improve upon the growth, various combinations of auxins with kinetin 0.1 mg l−1 were incorporated in the MS medium. Time course of growth and stilbenes production was determined up to 40 days in the cultures grown in MS medium containing 0.5 mg l−1 NAA and 0.1 mg l−1 kinetin and 3% sucrose. Experimentation with inoculum density of 1 g 60 ml−1 with vessels of different sizes was carried out to determine optimal conditions of root growth in large vessels. All the culture vessels were harvested at 30th day of growth.

After optimizing the growth conditions the experiments were designed to increase the stilbenes production in the roots. For the stilbenes production roots were grown in the MS medium containing 0.5 mg l−1 NAA and 0.1 mg l−1 kinetin, 6% sucrose and various effectors were added at 25th day of growth. These media were inoculated with 2 g fresh roots in 250 ml flasks containing 60 ml medium and harvested at 30th day to obtain sufficient biomass. Here the sucrose concentration and inoculum size was doubled to attain moderate growth along with production. The elicitors were prepared as stock solutions and added to the cultures at appropriate concentrations after filter sterilization at 25th day of growth. The three sets of experiments were:

  1. 1.

    Effect of various biotic [yeast extract (YE) 500 mg l−1] and abiotic elicitors [methyl jasmonate (MeJa) 50 μM, salicylic acid (SA) 50 μM, ethrel 500 μM)].

  2. 2.

    Addition of 1 mg l−1 alar in the medium containing elicitor.

  3. 3.

    Addition of 500 mg l−1 phenylalanine in the medium containing elicitor.

Optimal concentration of selected effectors was applied on the basis of preliminary experiments (data not shown). Growth index (GI) was determined by carefully removing the roots from the flask, weighed and then dried in an oven at 60°C for 24 h to a constant weight and dry mass (DM) was determined. GI was calculated as (Final DM − initial DM/initial DM).

Sample preparation

The root cultures were harvested washed with distilled water and filtered under mild vacuum. Dried homogenized roots (50 mg) were extracted in acetone–water 3:2 (v/v) for 12 h (room temperature) on a test tube rotator, centrifuged at 2,000 rpm for 15 min and then the supernatant was concentrated under vacuum at 40°C till the complete removal of acetone, the aqueous extract was partitioned twice with equal amount of ethyl acetate; finally the ethyl acetate phase was concentrated under vacuum till dryness. This extract was then used for the HPLC analysis. The spent medium was also analyzed for stilbenes released in the medium by the same method. All the extracts were redissolved in HPLC grade methanol and analyzed by using HPLC (pump L2130, auto sampler L-2200, FL detector L-2485, Merck-Hitachi). Separation was accomplished on a (LichroCART)® 250 × 4 mm Purospher® (5 μm) RP-18 column protected by a guard column of the same material. The solvent system used was: Solvent A—0.0025% trifluoroacetic acid in water; solvent B-80% acetonitrile (E. Merck, India) in solvent A. The mobile phase consisted of solvent (A) and (B). The step gradient programme of solvent B was as follows: 0–3 min: 14–18%; 3–12 min: 18–18%; 12–25 min: 18–22%; 25–30 min: 22–22%; 30–38 min: 22–40%; 38–43 min: 40–40%; 43–46 min: 40–60%; 46–48 min: 60–70%; 48–50 min: 70–70%; 50–52 min:70–80%; 52–54 min: 80–80%; 54–56 min: 80–85%; 56–58 min: 85–100%; 58–60: 100–100%; 60–62 min: 100–14%; 62–65 min: 14–14%. Separation was performed at a flow rate of 1.0 ml min−1 and chromatographic peaks were monitored at λ exc 300 nm and λ em 390 nm (Krisa et al. 1999).

Standard compound trans-resveratrol (3,5,4′-trihydroxystilbene) was purchased from Sigma Chemical Co. (St Louis, MO, USA) and trans-piceid (3,3,4,5′tetrahydroxystilbene), trans-ε-viniferin and ampelopsin A were kindly provided by Prof J. M. Merillon, France. These were dissolved in methanol to yield a final concentration of 1.0 mg l−1 and standard curve was prepared using resveratrol with concentrations ranging from 50 to 500 ng ml−1. The amount of the other compounds was calculated on the basis of trans-resveratrol.

Statistical analysis

All results are averaged over two separate analyses for stilbenes estimation and two consecutive experiments with six replicate flasks in each treatment for growth value determination. The results are expressed as μg g−1 cell DM.

Results and discussion

The root culture of C. trifolia has been established as a new source of bioactive stilbenes production. It has been demonstrated that these cultures can be grown in large vessels and high yields of stilbenes could be obtained by elicitation and growth retardant combinations. The HPLC analysis of the spent medium showed the presence of some unknown compounds. These compounds were very low in amount to be detected. The initial growth of roots in liquid medium was slow producing a few laterals. The roots were thread like, light to dark brown in color and devoid of root hairs.

Interaction of auxins and kinetin

The maximum growth (GI 2.4) was observed in the culture supplemented with 0.5 mg l−1 NAA and 0.1 mg l−1 kinetin (Table 1) amongst the treatments used. All the treatments of kinetin with an auxin or a combination of two auxins in the MS medium were supportive to growth. However, higher concentrations of auxin induced callusing in the roots while on lower concentrations; low biomass was produced because of thin roots (all data not presented).

Table 1 Effect of different combination of auxins on root biomass of Cayratia trifolia after 30 days culture in MS medium containing 0.1 mg/l kinetin and 3% sucrose
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Growth curve and stilbenes accumulation

The growth and stilbenes accumulation was measured in root cultures of C. trifolia grown up to 40 days (Figs. 1, 2). The maximum biomass (3.4 g l−1) and stilbenes yield (~770 μg l−1) was recorded at 30th day of growth. The initial growth of the cultures was slow perhaps due to low inoculum density. All of the identified stilbenes could be detected at 10th day of growth. Prolongation of stationary phase up to 40 days resulted in decrease in stilbenes content but stable growth with dark brown roots. It was observed that maximum viniferin and ampelopsin accumulated in between 20th and 30th day of culture. However, piceid and resveratrol were accumulated at 30th to 40th day of culture when the growth was in the stationary phase.

Fig. 1
figure 1

Individual stilbene content in root cultures of C. trifolia

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Fig. 2
figure 2

Growth curve and stilbenes yield in root cultures of C. trifolia (yield dark filled square, growth dark filled triangle)

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Scale-up in large flasks

The results obtained with 1 g 60 ml−1 inoculum, with vessels of varying sizes are presented in the Table 2. Maximum biomass in terms of fresh weight, dry weight and GI was produced in 2 l flask containing 0.8 l medium. Growth of roots and GI value slightly increased with vessel size perhaps due to less aeration in large vessels and thickening of roots.

Table 2 Scale-up of roots of Cayratia trifolia in shake flask after 30 days culture in MS medium containing 0.5 mg/l NAA, 0.1 mg/l kinetin and 3% sucrose
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Effect of elicitors

Elicitors added at 25th day of root culture, increased the stilbenes accumulation significantly. Maximum increase was recorded in the roots treated with SA (Fig. 3) followed by YE and ethrel (Table 3). MeJa was not only least effective, it reduced the content lower than that recorded in the control tissues. Piceid and resveratrol contents were also maximal in the roots treated with SA except ampleopsin and viniferin, which were maximal in the tissues treated with YE. Growth of the tissues was almost similar as roots were treated in the late stationary phase.

Fig. 3
figure 3

HPLC chromatogram showing stilbenes accumulation in C. trifolia roots treated with salicylic acid

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Table 3 Effect of elicitors on stilbenes production in Cayratia trifolia roots after 30 days culture in MS medium containing 0.5 mg/l NAA, 0.1 mg/l kinetin and 6% sucrose
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Effect of phenylalanine

The roots treated with elicitors along with phenylalanine at 25th day, accumulated high amount of stilbenes over the control (roots without phenylalanine; Table 4). The increase in stilbenes yield was maximal in roots treated with SA (1,011 μg g−1 DM) followed by YE and ethrel. The increase in stilbenes in the roots treated with ethrel and YE were higher than that recorded in roots treated with these elicitors only or elicitors with alar.

Table 4 Effect of elicitors in presence of phenylalanine (500 mg/l) on stilbenes production in Cayratia trifolia after 30 days culture in MS medium containing 0.5 mg/l NAA, 0.1 mg/l kinetin and 6% sucrose
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Effect of alar

Alar added along with elicitors on 25th day of root culture in MS medium further enhanced the stilbenes yield over the control (without alar) and in roots treated with SA or ethrel alone. Effective reduction in stilbenes yield was recorded in the roots treated with alar and MeJa (Table 5). Maximum piceid, resveratrol and ampelopsin accumulation was recorded in the roots treated with SA.

Table 5 Effect of elicitors in presence of alar (1 mg/l) on stilbenes production in Cayratia trifolia roots after 30 days culture in MS medium containing 0.5 mg/l NAA, 0.1 mg/l kinetin and 6% sucrose
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When phenylalanine (Table 4) or alar (Table 5) alone was incorporated in the medium slightly enhanced the stilbenes content over the untreated control (Table 3). When effect of individual elicitor was compared, maximum accumulation was recorded in roots treated with SA and in case of combinations it was SA with alar followed by SA with phenylalanine. The total stilbenes content (1,415 μg g−1) and yield (9,198 μg l−1) recorded in the roots treated with SA and alar was maximum amongst all the treatments of elicitors, alar and phenylalanine. An overall improvement of ~12-fold higher yield was recorded in root cultures over the initial growth in 3% sucrose containing medium.

The higher content and yield of stilbenes have been achieved in root cultures (12-fold) of C. trifolia by the combined treatment of elicitor and precursor or alar over control cultures as well as ~eightfold increase over natural roots (Table 3). This was possible by use of 6% sucrose along with these effectors. In absence of high sucrose, stilbenes yield in the roots was low (data not presented), which was lower then cell cultures (Roat and Ramawat 2009). Though MeJa has been demonstrated as an effective elicitor for stilbenes accumulation, MeJa failed to enhance stilbenes in the root cultures of C. trifolia. SA and MeJa are involved in signal transduction and different species respond differently to these molecules (Sudha and Ravishankar 2002). We have also recorded a marked increase in isoflavonoids, another class of polyphenolics, in cells of Pueraria tuberosa treated with SA, MeJa and ethrel (Goyal and Ramawat 2008). The untransformed root system necessitates a special mention because of the easy production of valuable pharmaceutical compounds without genetic modification of the plant genome (Martin et al. 2008).

Alar belongs to the group of succinic acid. It has various effects in plants including reduction in growth and biomass (El-Sheibany et al. 2007; Basra 1994). Viniferin is a dimmer of resveratrol while piceid is a glucoside. Phenylalanine, a direct precursor enhanced resveratrol content resulting in increased accumulation of piceid and viniferin. A combined effect of precursor, growth retardant and elicitor stimulated accumulation of stilbenes.