In vitro plant regeneration, phenolic compound production and pharmacological activities of Coleonema pulchellum

Effects of plant growth regulators (PGRs) and organic elicitors (OEs) on Coleonema pulchellum in vitro micropropagation, secondary product production and pharmacological activities were evaluated. In vitro , ex vitro and parental plants of C. pulchellum were investigated for their potential to produce phenolic and pharma- cological compounds. Different morphogenic characteristics of shoots were obtained with PGRs- and OEs-containingmedia.Ahighernumberofnormalshootswereachievedwithalowconcentrationofthidiazuron (TDZ: 4.5 μ M). Lesser numbers were found with combinations of TDZ (13.6 μ M) + indole-3-acetic acid (IAA: 2.9 μ M); haemoglobin (HB: 300 mg l − 1 ) or glutamine (GM: 40 μ M) + benzyladenine (BA: 8.8 μ M). Shoots were rooted in vitro and successfully acclimatized. Plant growth regulators and OEs had a signi ﬁ cant effect on the synthesis and accumulation of phenolic compounds and ﬂ avonoids. In particular, casein hydrolysate (CH) aswellasacombinationofGMandBAinducedhighlevelsoftotalphenolicsand ﬂ avonoidsduring invitro culture. CytokininsandOEshadasigni ﬁ canteffectonDPPHradicalscavengingandantibacterialactivitiesof C.pulchellum extracts. Acclimatized C. pulchellum plants can be used as substitute alternative to natural populations.


Introduction
Coleonema pulchellum Williams (Rutaceae) is an evergreen, erect and dense shrub, which occurs from the western to the eastern Cape in South Africa. It is an ideal aromatic garden plant which remains beautiful with pink flowers throughout the year. The plant contains phenylpropenes, phenylpropanoids and terpenoids which exhibit antimicrobial properties (Brader et al., 1997).
In vitro plant cell cultures have been carried out in the recent past for biosynthesis of compounds with pharmaceutical properties (Palacio et al., 2008). Plant tissue culture techniques offer a viable tool for in vitro regeneration in the conservation and commercial propagation of medicinal plants (Pattnaik and Chand, 1996). The capacity for plant cell, tissue and organ cultures to produce and accumulate many of the same valuable chemical compounds as the parent plant in nature has been recognized since the inception of in vitro technology. At the present time, many valuable compounds are produced by in vitro plant cell cultures (Baskaran et al., 2012). However, in other cases production requires more differentiated organ cultures such as shoots and roots (Karuppusamy, 2009). No studies on in vitro propagation of C. pulchellum have been reported.
The aim of this study was to investigate the potential of C. pulchellum in vitro cultures to synthesize phenolic compounds and to evaluate the changes in phenolic content due to plant growth regulators and organic elicitors used in the propagation process. The antioxidant and antibacterial activities were also evaluated in in vitro regenerated plants in comparison with parental (wild) plants.
Aseptic shoot-tip explants (10 mm in length) were cultured on MS medium (Murashige and Skoog, 1962) supplemented with 30 g l −1 sucrose, 8 g l −1 agar and different plant growth regulators (PGRs) and organic elicitors (OEs) alone or in combination for high-frequency rapid shoot multiplication. The concentration and combinations of PGRs are indicated in Tables 1 and 2. The pH of the medium was adjusted to 5.8 with 0.1 N NaOH and/or 0.1 N HCl before gelling with 8 g l −1 agar and autoclaved at 121°C for 20 min. The cultures were maintained at 25 ± 2°C under a 16 h photoperiod and PPF of 40 μmol m −2 s −1 provided by cool white fluorescent light (Osram L 58W/640, Germany).

Rooting of shoots and acclimatization
Proliferating shoots (at least 3.0 cm in length) were obtained from shoot-tip explants on MS medium containing 4.4 μM BA, and transferred to half-and full-strength MS media supplemented with different concentrations of auxins (IAA, IBA and NAA) and 8 g l −1 agar. In addition, halfstrength MS medium supplemented with 5.4 μM NAA, 8 g l −1 agar and different concentrations (10-40 g l −1 ) of sucrose was also tested. The optimized root induction medium (half-strength MS basal medium plus 5.4 μM NAA, 30 g l −1 sucrose and 8 g l −1 agar) was used in all treated shoots for root induction. Media devoid of PGRs were used as controls. The pH of the media was adjusted to 5.8 with 0.1 N NaOH and/or 0.1 N HCl prior to addition of agar and autoclaving at 121°C for 20 min. All cultures were maintained at 25 ± 2°C under cool-white fluorescent light at an intensity of 40 μmol m −2 s −1 with a 16 h photoperiod.
All rooted shoots were removed gently from the rooting medium after 8 weeks, and transferred to terracotta pots (95 × 120 mm, 500 ml) containing a 1:1 (v/v) vermiculite:sand mixture and irrigated with tap water every second day. These plantlets were maintained in the greenhouse (25 ± 2°C under natural photoperiod conditions and a midday PPF of 950 ± 50 μmol m −2 s −1 ) for acclimatization. Plant survival (%) was recorded after 10 weeks.

Sample preparation
Ten-week-old in vitro-grown shoots, one-year-old ex vitro-grown plants (grown on MS + 4.5 μM TDZ) and parental plant tissues (leaves, stem and roots) of C. pulchellum were oven dried at 50°C to constant weight. The dried materials were ground into fine powders using a grinder (IKA®, USA). The finely ground samples (0.1 g) were extracted with 10 ml 50% methanol in a sonication bath (Branson model 5210, Branson Ultrasonics B.V., Soest, Netherlands) for 20 min on ice. The methanolic extracts were then filtered under vacuum through Whatman No. 1 filter paper and the filtrates were immediately used for the determination of total phenolics and flavonoids.
Dried and finely ground plant extracts for antibacterial and antioxidant assays were extracted in a sonication bath on ice for 1 h (1.0 g extract in 40 ml of 70% acetone), and concentrated in a rotary vacuum evaporator (Büchi, Switzerland) at 30°C. The concentrated extracts were then dried over a stream of cold air at room temperature, redissolved in 50% DMSO to known concentrations and immediately used for the determination of antibacterial and DPPH radical scavenging antioxidant activities using appropriate solvent controls.

Phytochemical analysis
The Folin & Ciocalteu assay (Singleton and Rossi, 1965) was used for the determination of total phenolic compounds. As control a blank that contained 50% aqueous methanol was used instead of sample extracts. A standard curve of gallic acid was used to quantify the total phenolic content in the extracts. Total phenolic compounds were expressed in mg gallic acid equivalents (GAE) per g dry weight (DW).
The aluminium chloride (AlCl 3 ) method was used to quantify total flavonoid content (Zhishen et al., 1999). Suitable aliquots of catechin were used to derive a standard calibration curve. Total flavonoid content was expressed in mg catechin equivalents (CE) per g DW.
2.6. DPPH (2,2-diphenyl-1-picryl hydrazyl) radical scavenging activity The DPPH assay for the determination of free radical scavenging activity was done as outlined by Moyo et al. (2010). Methanolic DPPH (100 μM) was freshly prepared before the assay. Decrease in the purple colouration of the reaction mixtures was read at 517 nm in a Cary 50 UV-visible spectrophotometer. Ascorbic acid was used as a standard antioxidant. The assay was done using four replicates. The free radical scavenging activity (RSA) was calculated according to the formula: where A E is the absorbance of the reaction mixture containing the sample extract or standard antioxidant, and A D is the absorbance of the negative control.

Antibacterial microdilution assay
The minimum inhibition concentration (MIC) of the extracts was evaluated using the serial micro-dilution assay (Eloff, 1998). Four bacterial strains Gram-positive (Enterococcus faecalis ATCC 19433 and Staphylococcus aureus ATCC 12600) and Gram-negative (Escherichia coli ATCC 11775 and Pseudomonas aeruginosa ATCC 10145) were used. Overnight bacterial cultures were diluted with sterile Mueller-Hinton (MH) broth to a final inoculum concentration of 10 6 colony forming units (CFU)/ml. Plant extracts were re-suspended in 50% DMSO to a concentration of 25 mg/ml. A positive control, neomycin (100 μg/ml) was used against each bacterial strain. Sterile distilled water, 50% DMSO, bacteria-free MH broth, and each bacterial strain, with no extract added completed the set of controls. The assay was done in triplicate. INT (0.2 mg/ml) dissolved in distilled water was used as the indicator of bacterial growth after incubation at 37°C for 1 h.

Statistical analysis
Data were collected after 10 weeks of culture for shoot multiplication and 8 weeks for rooting experiments. All PTC experiments were repeated three times with 50 explants per treatment. The data were subjected to one-way analysis of variance (ANOVA) using SPSS version 18.0 for Windows (Chicago, USA). Significantly different means were separated using Duncan's multiple range test (p = 0.05).

Influence of PGRs on shoot regeneration
Shoot-tip explants of C. pulchellum were cultured on MS medium with different cytokinins [benzyladenine (BA), meta-topolin (mT) and thidiazuron (TDZ)] at various concentrations (4.2-22.7 μM) to evaluate their potency on shoot multiplication. Different morphogenic characteristics were noticed in these in vitro cultures (Table 1). The shoot regeneration rate varied significantly between the control and PGR-containing treatments after 10 weeks of culture (Table 1). TDZ (13.6 μM) produced significantly higher shoot numbers (37.0 shoots per explant) after 10 weeks of culture (Table 1; Fig. 1A). However, these shoots exhibited shoot-tip necrosis symptoms (Table 1). On the other hand, BA, mT and lower concentrations of TDZ (4.5 μM) produced normal shoots (Table 1; Fig. 1B). The present study indicates that shoot-tip necrosis, a physiological disorder commonly observed in in vitro cultures affecting a wide range of plants, depends on the type and concentration of cytokinin in the culture medium. It is a phenomenon whereby the apical shoot initially becomes brown and later dies (Lakshmi and Raghava, 1993;Bairu et al., 2009). Benzyladenine was successfully used to control shoot-tip necrosis in chestnut cultures (Piagnani et al., 1996). Excessive yellowing of leaves and necrosis was observed at higher concentrations of 2-isopentenyladenine (2iP), kinetin or TDZ in Mexican redbud plants (Mackay et al., 1995). In the present study, various combinations and concentrations of PGRs were used to develop high-frequency rapid normal shoot multiplication from shoot-tip explants. A combination of TDZ (13.6 μM) and IAA (2.9 μM) significantly increased normal shoot production compared to other treatments (Table 1; Fig. 1C), indicating cytokinin-auxin synergistic effects in shoot proliferation. These cultures induced different types of basal callus after 10 weeks of culture (Table 1). A low auxin concentration in combination with high cytokinin concentration promoted shoot proliferation in other species of Rutaceae (Bohidar et al., 2008;Vennel Raj and Basavaraju, 2012).

Influence of organic elicitors and PGRs on shoot regeneration
The importance of organic elicitors (OEs: casein hydrolysate, haemoglobin, glutamine and mebendazole) alone and in combination with cytokinin (TDZ and BA) was examined for rapid shoot proliferation (Table 2). Organic elicitors showed significant rapid shoot proliferation in C. pulchellum. The promotional effects of elicitors on shoot regeneration have been reported in other plant species (Baskaran and Jayabalan, 2005;Baskaran and Van Staden, 2011). In this study, GM and HB treatments increased the number of shoots after 10 weeks of culture (Table 2; Fig. 1D). The HB treatment produced normal shoots as well as green compact basal callus (GCBC) after 10 weeks of culture. Shoots produced in the CH treatment exhibited shoot-tip necrosis symptoms, root growth and green friable basal callus (Table 2; Fig. 1E). A combination of TDZ and GM significantly increased shoot number (Table 2), but the shoots were also necrotic after 10 weeks of culture (Table 2; Fig. 1F). Combinations of CH, MBZ or HB with TDZ produced hyperhydric shoots as well as green compact basal callus (Table 2; Fig. 1G). Hyperhydricity is characterized by a glassy or swollen appearance to the tissue, usually resulting in reduced multiplication rates, poor quality shoots and tissue necrosis (Ziv, 1991). Their organs are translucent, in some cases less green and easily breakable (Ziv, 1991). In this study, combinations of HB or GM and BA produced normal shoots with a significantly higher shoot length (Table 2; Fig. 1H). These organic elicitors can be considered to mimic PGRs in the culture medium. Hyperhydricity has been reported to be influenced by cytokinin type and concentration (Kadota and Niimi, 2003), which is in agreement with our present findings.
The current investigation showed that OE and PGR combinations in the MS medium enhanced adventitious shoot multiplication.

Effect of medium strength, sucrose concentration and auxins on in vitro rooting and acclimatization
Excised shoots from MS medium containing 4.4 μM BA were rooted using both half-and full-strength MS media with different types of auxins (Fig. 1I). Both half-and full-strength MS media containing 5.4 μM NAA induced higher numbers of roots after 8 weeks of culture (Table 3). Root length was significantly higher in full-strength MS medium containing 5.4 μM NAA after 8 weeks of culture (Table 3). These cultures produced normal shoots and roots as well as white friable basal callus (Table 3). In this study, concentrations of sucrose (10-40 g l −1 ) were also tested in half-strength MS medium with 5.4 μM NAA. Different rates of root growth were observed after 8 weeks of culture (Table 3). The medium containing 30 g l −1 sucrose was more effective in induction of root growth compared to other sucrose concentrations (Table 3). The present study revealed the importance of sucrose concentration on root induction in C. pulchellum. A similar trend was also observed in Persian walnut (Vahdati et al., 2004). Half-strength MS medium supplemented with 5.4 μM NAA was selected as the standard growth medium for root induction in subsequent experiments. All the PGR and OE-treated shoots produced roots with different morphogenic characteristics (Tables 1 and 2). Root induction rate varied significantly between the shooting media after 8 weeks of culture (Tables 1  and 2). GM-produced shoots were the most amenable to root induction, and produced significantly higher root numbers (12.0 roots per shoot) and root lengths (13.24 cm) after 8 weeks of culture (Table 2; Fig. 1J). These cultures induced green roots and white friable basal callus Fig. 1. In vitro propagation from shoot-tip explants of C. pulchellum. (A) Multiple shoots developing with shoot-tip necrosis from shoot-tip explants on MS medium with 13.6 μM TDZ after 10 week of culture (Bar, 10 mm), (B) Shoot multiplication on MS medium with 4.5 μM TDZ (Bar, 10 mm), (C) Shoot multiplication with green compact basal callus on MS medium with 13.6 μM TDZ and 2.9 μM IAA (Bar, 10 mm), (D) Adventitious shoot regeneration with green compact basal callus on MS medium containing 300 mg l −1 HB (Bar, 10 mm), (E) Shoot multiplication with shoot-tip necrosis, root growth and green friable basal callus on MS medium containing 300 mg l −1 CH (Bar, 10 mm), (F) Shoot multiplication plus shoot-tip necrosis on MS medium containing a combination of 40 μM GM and 4.5 μM TDZ (Bar, 10 mm), (G) Shoot multiplication with hyperhydric shoots and green compact basal callus on MS medium with 300 mg l −1 CH and 4.5 μM TDZ (Bar, 10 mm), (H) Production of adventitious shoots on MS medium with 300 mg l −1 HB and 8.8 μM BA (Bar, 10 mm), (I) Excised shoots from shooting medium and rooting of shoots (Bar, 10 mm), (J) Rooting of shoots derived from GM treatment (Bar, 10 mm), (K) Acclimatized plants with ex vitro flowering after 10 months. ( Table 2). The present study indicates that beneficial root growth depends on type as well as concentration of PGRs and OEs in the media (Tables 1 and 2). The in vitro-rooted shoots were removed from the culture media after 8 weeks and successfully acclimatized (100%) in a vermiculite:soil mixture (1:1 v/v) in the greenhouse (25 ± 2°C) under natural photoperiod conditions with a midday PPF of 950 ± 50 μmol m −2 s −1 (Fig. 1K).

Phytochemical and antioxidant properties of C. pulchellum extracts
The results on the effect of cytokinins and OEs are presented in Table 4. Total phenolic and flavonoid contents were higher in the leaves and stems compared to the roots for both acclimatized and natural plants. Shoot cultures provide a viable option for the production of secondary metabolites, especially where the compounds are synthesized and/or stored in the aerial plant parts (Amoo et al., 2012). Cytokinin type and OEs had a significant effect on total phenolic and flavonoid contents. In particular, casein hydrolysate significantly upregulated the levels of total phenolic compounds and flavonoids (Table 4). Total phenolic and flavonoid contents were also upregulated in in vitro C. pulchellum cultures treated with BA. Different types and concentrations of PGRs have been reported to regulate developmental processes and modify the concentration of secondary metabolites in plants differently (Palacio et al., 2008;Baskaran et al., 2012). A combination of GM and BA significantly increased the in vitro accumulation of phenolics and flavonoids, compared to when the compounds were used singly. The results show that the concentration of secondary metabolites in in vitro cultures is influenced by the composition of the medium (Abbasi et al., 2010;Baskaran et al., 2012). Natural phenolic compounds and flavonoids are widely known for a range of biological activities such as antimicrobial, anti-cancer and cardiovascular regulatory effects (Brader et al., 1997;Palacio et al., 2008;Moyo et al., 2010).
All the treatments exhibited a dose-dependent increase in DPPH radical scavenging activity (Table 4). In vitro treatment using cytokinins and organic elicitors had a significant effect on DPPH radical scavenging activity, in particular BA, TDZ and CH as well as their respective combinations. Natural antioxidants play a crucial role in ameliorating the detrimental effects of oxidative stress caused by reactive oxygen species as well as being food additives that inhibit the oxidation of nutrients in foodstuffs. There is a strong correlation between disease pathogenesis and oxidative stress levels, hence the use of natural antioxidant compounds is considered an effective therapeutic approach against several diseases (Moyo et al., 2010). The potential use of in vitro technology for the synthesis of antioxidant compounds from medicinal plants has been widely reported (Matkowski, 2008;Abbasi et al., 2010;Amoo et al., 2012;Khateeb et al., 2012).

Antibacterial activity of C. pulchellum extracts
The antibacterial activity of C. pulchellum extracts is presented in Table 5. The tested extracts had better activity against Gram-positive Enterococcus faecalis. In vitro extracts derived from mT and CH-treated plants exhibited high antibacterial activity against E. faecalis with MIC values of 0.781 and 1.041 mg/ml, respectively. Furthermore, CH-treated C. pulchellum plants had a low MIC (1.56 mg/ml) against the Gramnegative bacteria E. coli. Isolated phenylpropenes from C. pulchellum had good antimicrobial activity (Brader et al., 1997). The authors attributed the observed antimicrobial activity to the reactivity of the phenolic hydroxyl group (Brader et al., 1997). In a similar study, Khateeb et al. Table 4 Effect of plant growth regulators and organic elicitors on secondary metabolite production and radical scavenging activity of in vitro shoots, acclimatized and natural-grown plants of Coleonema pulchellum I. Williams. S + Lstem and leaves; Rroots.
Values are mean ± standard error (SE). Means followed by same letters in each column are not significantly different (p = 0.05) using Duncan's multiple range test. (2012) reported the antimicrobial activity of in vitro-derived secondary metabolites of Cichorium pumilum. The present study shows that C. pulchellum contains biologically potent therapeutic phytochemicals with high antibacterial and antioxidant activities.

Conclusions
The present study demonstrated that C. pulchellum shoot-tip explants are a good starting material for in vitro culture establishment. A combination of TDZ and IAA produced a significant number of normal shoots while lower TDZ concentrations favourably affected the induction of normal shoots. The best rooting was achieved with halfstrength MS medium containing 30 g l −1 sucrose and NAA. In vitro and ex vitro-grown C. pulchellum treated with combinations of OEs and PGRs exhibited high antioxidant and antibacterial activities. This study showed that in vitro regenerated C. pulchellum plant tissues and organs contain the active phytochemical constituents, which have medicinal properties. The present micropropagation system can be recommended for the mass propagation of C. pulchellum plants. The acclimatized in vitro-derived plants can be used as an alternative to natural populations of C. pulchellum.