Introduction

Nyctanthes arbor-tristis L. (family Oleaceae), commonly known as “night flowering jasmine,” is an important medicinal species. Although this plant is native to the Indian subcontinent, where it is distributed in the wild in sub-Himalayan regions, night flowering jasmine is also grown as an ornamental in subtropical gardens to cater to ritual and esthetic needs. This plant is a promising source of many therapeutic biomolecules, such as the iridoid glycosides arbortristosides, nyctanthoside, polyphenols, flavonoids, β-sitosterol, astragalin, nyctanthic acid, rengyolone, α-crocetin, and ursolic acid, among others (Tuntiwachwuttikul et al. 2003; Agrawal and Pal 2013; Khanapur et al. 2014; Saini et al. 2014). Plant-based extracts, derived either from leaves, flowers, seeds, roots, or bark of N. arbor-tristis, have been used in conventional medicine for the treatment of asthma, diuresis, cancer, rheumatism, sciatica, gout, malaria, filaria, liver dysfunction, skin diseases, and worm infection of intestine, because of their well-documented anti-arthritic, anti-malarial, anti-filarial, hepato-protective, anti-inflammatory, immuno-modulatory, anti-leishmanial, and antioxidant properties (Tuntiwachwuttikul et al. 2003; Rathee et al. 2007; Rani et al. 2012; Agarwal et al. 2013; Agrawal and Pal 2013; Saini et al. 2014; Mishra et al. 2016). Furthermore, some notable herbal preparations using plant parts of N. arbor-tristis are being sold today.

To date, the demand for N. arbor-tristis plant parts for pharmaceutical, esthetic, and ritual activities is met by the natural plant population, but such unrestricted utilization of the species has resulted in the unchecked exploitation of a vulnerable resource and threatens to bring this species in the near future to the brink of extinction in its native habitat. This situation is further aggravated because seed-based propagation of night flowering jasmine is constrained by low seed viability, poor seed germinating ability, and delayed root system growth at the sapling stage (Rout et al. 2008; Jahan et al. 2011). Thus, there is a growing need to optimize in vitro propagation protocols for rapid multiplication and conservation of this multipotent medicinal plant (Phillips 2004).

During the last couple of decades, several attempts to optimize the few existing protocols for in vitro organogenesis of N. arbor-tristis have been made (Rout et al. 2008; Jahan et al. 2011; Sahu et al. 2012). Additionally, several non-purine phenyl urea derivatives with cytokinin activity, such as thidiazuron (TDZ), N-phenyl-N′-benzothiazol-6-yl-urea (PBU), and N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU), have been used to promote either adventitious organogenesis or somatic embryogenesis in different species (Ricci and Bertoletti 2008; Rolli et al. 2011; Li et al. 2015). Jahan et al. (2011) reported that preconditioning axillary buds of N. arbor-tristis with TDZ for 8 d and subsequent implantation in Murashige and Skoog (MS) medium (Murashige and Skoog 1962) enhanced the rate of shoot multiplication in vitro, and similar reports have also been made for several related species. Alternatively, synthetic derivatives, such as PBU, have been tested in many species for in vitro propagation either via organogenesis or somatic embryogenesis (Torelli et al. 2006; Huang et al. 2010; Rolli et al. 2011; Carra et al. 2012; Li et al. 2015).

Considering the reported responses of plant cultures to PBU during organogenesis and somatic embryogenesis, in the present study, its application in the tissue culture of night flowering jasmine was investigated for an alternative propagation method, with the eventual goals of conservation of native N. arbor-tristis populations, while permitting production of bioactive metabolites on a commercial scale from propagated plants.

Furthermore, because genetic variation can arise during in vitro multiplication via organogenesis, whether as an expression of epigenetic imprints or due to the modifications in the genetic makeup induced by culture environments (Larkin and Scowcroft 1981), the genetic homogeneity of any resulting plantlets needed to be evaluated. The clonal fidelity of regenerated plantlets of N. arbor-tristis has not been assessed in any of the earlier studies, neither at the genetic level, nor in terms of the plant’s bioactive, therapeutic molecules and phytochemical content.

In other plant species, various DNA markers have been used, either individually or in conjunction with profiling of bioactive molecules, for the assessment of genetic homogeneity in plantlets regenerated in vitro (Ghaderi and Jafari 2014; Adeniran et al. 2018). Among these, start codon targeted polymorphism (SCoT; Collard and Mackill 2009) markers have been used successfully in recent days for the evaluation of genetic homogeneity of in vitro-derived plantlets in Dendrobium nobile Lindl. (Bhattacharyya et al. 2014), Alhagi maurorum Medik. (Agarwal et al. 2015), Helicteres isora L. (Muthukumar et al. 2016), and Abutilon indicum (L.) Sweet (Seth et al. 2017). These SCoT markers are preferred, because the primers are designed for short conserved regions around the ATG start codon and its targeting sequences, and are therefore most likely part of a gene (Collard and Mackill 2009; Bhattacharyya et al. 2014). Obviously, from a pharmacological viewpoint, consistency in terms of quality and content of bioactive metabolites of in vitro-regenerated medicinal plants is crucial (Dörnenburg and Knorr 1995).

The present report describes an alternative protocol for the in vitro propagation N. arbor-tristis from pretreated nodal explants and provides an assessment of the genetic fidelity of the in vitro-regenerated plantlets using SCoT marker profiling, along with a comparative evaluation of their bioactive molecules, antioxidants, and radical scavenging activities.

Materials and Methods

Explant preparation, media, chemicals, and culture conditions

Young and fresh apical stems of approximately1.5 cm length were obtained from 12-mo-old N. arbor-tristis (accession NAET-08) and were washed with water for 5 to 10 min, followed by 1% (v/v) Extran liquid detergent and 1% Bavistin® fungicide for 10 min each. Subsequently, truncated nodal explants were surface sterilized with 0.01% (w/v) HgCl2 for 3 to 4 min, followed by three rinses with sterile double-distilled water under a laminar flow hood. Basal MS (Murashige and Skoog 1962) medium, containing various combinations of 0.0, 2.5, 5.0, 7.5, 10.0, or 15.0 μM α-naphthaleneacetic acid (NAA), 6-benzyladenine (BA), or kinetin (KIN), and growth additives (0, 7.5, 15.0, 22.5, or 30.0 μM glutamine, 0.0, 5.0, 10.0, 15.0, or 20.0 μM proline, and 0.0, 15.0, 30.0, 45.0, or 60.0 μM adenine sulfate), or 25.0, 50.0, 75.0, 100.0, or 125.0 μM PBU were further supplemented with 3% (w/v) sucrose; the pH was adjusted to 5.8 ± 0.1 using 1 N HCl before 0.8% (w/v) agar-agar was added, and the medium was autoclaved at 1.06 kg cm−2 for 15 to 30 min. All the cultures were maintained in culture room at 25 ± 2°C, 60–70% relative humidity and a 16-h photoperiod, under approximate photosynthetic photon flux of 40 μmol m−2 s−1 using fluorescent tubes (Crompton Greaves, Mumbai, India).

Shoot bud induction and proliferation of shoots in vitro

The sterilized nodal explants were incubated on MS medium containing various combinations of 2.5, 5.0, 7.5, 10.0, or 15.0 μM BA, 2.5, 5.0, 7.5, 10.0, or 15.0 μM KIN, and 2.5, 5.0, 7.5, or 10.0 μM NAA, and the best responding plant growth regulator (PGR) combination, in terms of number of shoots per node vis-à-vis shoot length, was identified. This best combination of PGRs was further enriched with different concentrations and combinations of 15.0, 30.0, 45.0, or 60.0 μM adenine sulfate, 5.0, 10.0, 15.0, or 20.0 μM proline, and 7.5, 15.0, 22.5, or 30.0 μM glutamine, and their influence on shoot numbers per nodal explant was assessed.

In another set of experiments the nodal explants were pretreated with liquid MS medium containing either optimized concentrations of BA, KIN, NAA, and growth additives, or 25, 50, 75, 100, or 125 μM PBU for 2, 4, 6, and 8 d on a rotary shaker (Rivotek-50082001, Riviera Glass Private Limited, Mumbai, India) at 75 rpm, before being embedded in PGR-free solidified MS medium. The effects of different combinations of PGRs, growth additives, and pretreatments on regeneration response, number of shoots, and length of shoots after 8 wk of culture were recorded.

Rooting in vitro and acclimatization

In vitro-derived shoots of about 4 to 5 cm in length were excised from the clumps of shoots, pretreated on half-strength MS medium supplemented with 2.0, 4.0, 6.0, 8.0, or 10.0 μM NAA or indole-3-butyric acid (IBA), individually, for 1 wk, before transfer to PGR-free half-strength MS medium. Data on rooting response, number of roots, and average root length were recorded after 3 wk. Well-rooted plantlets were washed with sterile double-distilled water and transferred to plastic pots (3 in. diameter, 350 ml total volume; OEM Ltd., Bhubaneswar, India) containing 1:1:1 (v/v/v) garden soil, soil-rite mix (Keltech Energies Ltd., Bengaluru, India), and vermi-compost. These potted plantlets were retained in the culture room at 25 ± 2°C for 1 wk and successively transferred to a net house and the experimental field, following the routine protocol of acclimatization (Sahu et al. 2012). All the plant growth regulators, growth additives and chemicals used for the media preparation were obtained from Himedia Laboratories Pvt. Ltd., Mumbai, India.

Genetic fidelity analysis using SCoT markers

Along with their ex vitro donor plant, 39 in vitro-regenerated plantlets were randomly selected from the best-responding culture combinations for the assessment of genetic uniformity. Fresh and young leaves (approximately 1.5 g) were ground in liquid nitrogen and genomic DNA was extracted using the modified cetyl trimethyl ammonium bromide (CTAB) method described by Mishra et al. (2013). The crude DNA was purified using RNAse and proteinase K (B. Genei, Bangalore, India) treatment, followed by successive washes of 25:24:1 (v/v/v) phenol:chloroform:isoamyl alcohol (Himedia Laboratories Pvt. Ltd., Mumbai, India), and 24:1 (v/v) chloroform:isoamyl alcohol (Himedia Laboratories Pvt. Ltd., Mumbai, India), as described previously by Seth et al. (2017). The thus purified DNA was precipitated by addition of chilled ethanol, pelleted, and dried under vacuum. Subsequently, the DNA sample of each plant was dissolved in 10 mM tris (hydroxymethyl) aminomethane (Tris): 1 mM ethylene-diamine tetraacetic acid (EDTA) buffer and equilibrated to a concentration of 20 ng μL−1.

The polymerase chain reaction (PCR) mix of 25 μL total volume and containing 40 ng DNA, 2 mM MgCl2, 10 mM dNTP mix, 2.5 μL of 10× assay buffer (100 mM Tris-Cl, pH 8.3; 0.5 M KCl; 0.1% (w/v) gelatin), 1 U Taq DNA polymerase (B. Genei, Bangalore, India), and 20 ng of SCoT primer(s) was amplified in a MyCycler thermal cycler (BioRad, Hercules, CA) programmed for 35 cycles, as described in Seth et al. (2017). The amplified products were electrophoretically separated on 1.4% (w/v) agarose gel using TAE (40 mM Tris acetate; 2 mM EDTA) buffer at a constant 50 V, visualized with 0.5 μg mL−1 ethidium bromide staining, and recorded using the FireReader (UVITEC, Cambridge, UK) gel documentation system. The size of each amplified fragment was estimated by loading 250 bp step-up DNA ladder (B. Genei, Bangalore, India) as standard. To test the reproducibility, the amplifications and electrophoresis were repeated twice.

Preparation of extracts and determination of antioxidant and free radical scavenging activities

Leaves and flowers were obtained from the ex vitro- and in vitro-derived N. arbor-tristis NAET-08 plants, air dried under shade, then crushed to granular particles using grinder (K-10, Bajaj Electrical Ltd., Mumbai, India) and kept in an air-tight plastic jar at 25°C. To obtain aqueous extracts, 10 g of dried powder of each plant part were soaked in 1000 mL of double-distilled (dd) H2O at 60°C for 36 h, under agitation in a water bath. These decoctions were centrifuged at 12000 x g for 15 min, and the supernatants were collected in amber bottles and dried at 45°C, using a rotary vacuum evaporator (RV-10; IKA® WERKE GmbH & Co. KG, Staufen, Germany). To obtain methanolic and ethyl acetate extracts, each crushed sample (20 g) was extracted with 2000 mL of methanol or ethyl acetate, respectively, using a Soxhlet extractor. The extracts were dried as explained above and kept in amber bottles at 4°C. The yield percentage (w/w) was calculated following Mishra et al. (2016). From each of these dried extract preparations, 25 mg mL−1 stock solutions were prepared and used for the determination of polyphenol and flavonoid content, total antioxidant activity (TAA) and ferric ion reducing antioxidant power (FRAP), following protocols of Gul et al. (2011). The total polyphenols and flavonoids were expressed as gallic acid equivalents (μg GAE mg−1 dry weight) and quercetin equivalents (μg QE mg−1 dry weight) per mg dry weight, respectively. Similarly, TAA and FRAP were expressed as ascorbic acid equivalents (μg AAE mg−1 dry weight) per mg dry weight.

The free radical scavenging activities of the extracts were also assessed for 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydrogen peroxide (H2O2), and superoxide radicals (Kakkar et al. 1984; Braca et al. 2002; Gul et al. 2011) and the radical scavenging activity of each extract was measured as inhibition percentage, by using the formula:

Radical scavenging activity (%) = [(A0 − Ae)/A0] × 100, where A0 and Ae were absorbance measured at 517 nm (A517) of the control and extract(s), respectively.

Estimation of bioactive metabolites

Ursolic acid. The dried methanolic extract (10 g) was suspended in Milli-Q water and partitioned successively with ethyl acetate, n-butanol, and water (aqueous extract). The ethyl acetate fraction (2.3 g) was chromatographed over silica gel using a step gradient of petroleum ether (1000 ml; fraction-I), petroleum ether: Chloroform 1:1 (v/v; 2000 ml fraction-II), Chloroform (2000 ml; fraction-III), chloroform: methanol 9:1 (v/v; 2000 ml; fraction-IV) as described by Saini et al. (2014), and four different fractions were eluted. Fraction-IV was separated through 1515 Isocratic high-performance liquid chromatography (HPLC) (Waters Corporation, Milford, MA), using Whatman® 0.45 μm membrane (Merck KGaA, Darmstadt, Germany) and 20 μL of injected volume was analyzed using a C-18, 4.6 × 2 0 mm, 5 μm particle size XTerra™ RP column (Waters Corporation, Milford, MA). The entire HPLC analysis was carried out at 25 ± 2°C, the eluate was observed at 210 nm, and ursolic acid content (w/w) was estimated by standard curve method.

Rengyolone and nyctanthoside. The dried methanolic extracts (9.2 g) were chromatographed on a (70 to 230 mesh) silica gel column and fractionated successively as described by Tuntiwachwuttikul et al. (2003), and fraction I (198 to 224 mg) was purified over silica gel by step gradient elution, which gave rise to the expected colorless oil (453 to 512 mg) identified as rengyolone. Fractions II and III were also purified, eluted, and chromatographed as described previously by Tuntiwachwuttikul et al. (2003), and 92 to 108 mg of a resin-like substance was obtained, which was identified as nyctanthoside. The content of rengyolone and nyctanthoside (w/w) was measured for each sample.

Arbortristoside-A. The dried methanolic extract (~ 10 g) of each sample was dissolved in ddH2O and fractionated with diethyl ether, ethyl acetate, and n-butanol. The n-butanol fraction [yield: 45.42 to 48.75% (w/w)] of each sample was dried, ground with acetone, washed with 2 N HCl, and subsequently with hot ddH2O at 60°C. Finally, the fraction crystallized using a solvent system of 1:1 chloroform:methanol, and a light yellow-colored crystalline powder (308 to 344 mg) was obtained, and validated as arbortristoside-A using spectral analysis (Mendham et al. 2003), and its solubility in dimethyl sulfoxide (DMSO).

Statistical analysis

Visual observations were taken with respect to the effects of plant growth regulators, as well as pretreatment conditions, on the frequency of regeneration, number of shoots per explants, shoot length, percentage of rooting, and root length. All the experiments were repeated twice, with five replicas per experiment, in randomized methods, and the data were represented as mean ± standard error (SE). The data were subjected to one-way analysis of variance (ANOVA) and the arithmetic means were compared (P = 0.05) using Duncan’s multiple range test (Harter 1960) and the statistical software package SPSS® version 20.0 (IBM, Armonk, NY). The data on bioactive metabolite content, antioxidant activity, and radical scavenging activities were also statistically analyzed in the same way, also using SPPS® version 20.0 (IBM, Armonk, NY). The half concentration of inhibition (IC50) values were calculated from linear regression analysis using EXCEL add-in program ED50 plus version 1.0 (http://www.softlookup.com/display.asp? id=2972; Vargas 2000).

Results and Discussion

Effects of PGRs and growth additives on in vitro induction and multiplication of shoots

In the present study, the nodal explants of N. arbor-tristis cultured on MS medium fortified with different combinations of BA, KIN, and NAA exhibited morphogenic responses after 2 to 4 wk of culture. The nodal explants embedded in MS medium supplemented with 10.0 μM BA and 2.5 μM NAA responded well, as evidenced by the emergence of multiple shoot buds around the preexisting lateral meristem of the explants by the end of the second week (Fig. 1a), followed by the proliferation of multiple apical shoots after 4 wk of culture (Fig. 1b). These shoot buds proliferated to give rise 6 to 8 shoots (average of 6.60 ± 0.75; Table 1) per explant at the end of the eighth week, and the average shoot length for the PGR combination of 10.0 μM BA and 2.5 μM NAA was 3.04 ± 0.10 cm (Table 1). This response might be due to the coupled effects of low concentration of auxin along with high concentration of cytokinin on in vitro shoot multiplication, as reported in Rhinacanthus nasutus (L.) Kurz (Cheruvathur et al. 2012), Sida cordifolia L. (Sivanesan and Jeong 2007), and Trichodesma indicum (L.) Lehm. (Mahesh and Jeyachandran 2013). This exogenous application of auxin in low concentration might be involved in the asymmetric distribution of auxins necessary for the acquirement of organogenic competence, subsequent entry of active meristems and its adjoining cells into the cell cycle, canalized cell division, and de novo initiation of shoot buds under culture conditions, which then differentiate into multiple shoots later on (Sugiyama 1999; Phillips 2004; Zhao et al. 2008). In an attempt to improve the multiplication rate, as well as shoot elongation, MS medium with optimized combination of PGRs was further enriched with 45 μM adenine sulfate, 15 μM glutamine, and 10 μM proline, which increased the number of shoots per explant (Fig. 1c) to 8.60 ± 0.63 (Table 2), but no significant effect on shoot elongation (Fig. 1d) was observed. The additive effects of adenine sulfate, glutamine, and proline on shoot multiplication have previously been reported in Oryza sativa and Picrorhiza scrophularriflora (Bantawa et al. 2009; Shahsavari 2011; Pawar et al. 2015). The addition of adenine sulfate might be impeding the degradation of cytokinins, either by feed-back inhibition or by competing with the metabolites involved in cytokinin anabolism (Van Staden et al. 2008), whereas the addition of glutamine and proline to the medium might have provided alternative nitrogen sources for maintaining a high shoot bud induction rate (Shahsavari 2011; Pawar et al. 2015).

Figure 1.
figure 1

In vitro propagation in Nyctanthes arbor-tristis. (a) Emergence of shoot buds from the nodal explant on MS (Murashige and Skoog 1962) medium fortified with 10.0 μM 6-benzyladenine (BA) and 2.5 μM 1-naphthaleneacetic acid (NAA) at the end of 2 wk. (b) Proliferation of shoot buds showing multiple apical shoots at the end of 4 wk. (c) Proliferation of multiple shoots from the explant on MS medium fortified with 10.0 μM BA, 2.5 μM NAA, 45 μM adenine sulfate, 15 μM glutamine, and 10 μM proline at the end of 8 wk. (d) Elongation of shoots on the same medium after 8 wk. (e) Proliferation of multiple shoots from nodes pretreated with 100 μM N-phenyl-N′-benzothiazol-6-yl-urea (PBU) for 4 d and moved to on plant growth regulator (PGR) free MS medium at the end of 8 wk. (f) Emergence of roots from pretreated shoots on half strength MS medium without any PGR. (g) Acclimatization of shoots on plastic pots containing soil mix. (h) Genetic fidelity analysis of plantlets regenerated from PBU pretreated nodal explants using SCoT marker profiling (SCoT-1 and ScoT-2; Lane-M: 250 bp step-up ladder, MP-Donor plant; C1-C8: in vitro raised plantlets).

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Table 1. Effects of 6-benzyladenine (BA), kinetin (KIN), and 1-naphthaleneacetic acid (NAA) on the shoot multiplication of Nyctanthes arbor-tristis L. cultured on MS (Murashige and Skoog 1962) medium for 8 wk
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Table 2. Effect of adenine sulfate, glutamine, and proline on multiple shoot bud regeneration from nodal explants of Nyctanthes arbor-tristis L. cultured on optimized MS (Murashige and Skoog 1962) medium containing 10.0 μM 6-benzyladenine and 2.5 μM 1-naphthaleneacetic acid after 8 wk
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To improve the efficiency of shoot multiplication, preconditioning of explants with non-purine phenyl urea derivatives with cytokinin activity, such as TDZ, CPPU, and PBU, have been utilized in many species. Pretreatment of axillary buds of N. arbor-tristis with 75 μM TDZ for 8 d and subsequent implantation in MS medium showed a twofold increase in the rate of shoot multiplication (Jahan et al. 2011). Considering the structural similarity of PBU with TDZ, in the present study the nodal explants were pretreated for 2 to 8 d either with liquid MS medium containing either 25 to 125 μM PBU, or with the previously optimized combination of PGRs and growth additives, 10.0 μM BA + 2.5 μM NAA + 45 μM adenine sulfate + 15 μM glutamine + 10 μM proline, serving as the control before implantation in PGR-free MS medium (Fig. 2). Pretreatment of nodal explants in liquid MS medium with PBU had a significant effect on morphogenic differentiation and shoot bud induction, which culminated in increased number of shoots per explant (Table 3; Fig. 2). Specifically, pretreatment of nodal explants in liquid MS medium with 100 μM PBU for 4 d elevated the shoot proliferation rate by producing the highest number shoots per explant (17.40 ± 1.02) with an average shoot length of 5.96 ± 0.08 cm (Table 3; Fig. 1e) which was an average increment of 7 additional shoots per explant as compared to the control (Fig. 2). This increased rate of shoot proliferation might be attributed to PBU-mediated altered cytokinin metabolism by inhibiting the activity of cytokinin oxidase, and accumulation of adenine cytokinins in the explant, as has been observed with TDZ (Jahan et al. 2011; Kumari et al. 2018). Similar effects of PBU were also reported in Eucalyptus urophylla S. T. Blake (Huang et al. 2010; Li et al. 2015), Hyssopus officinalis L. (Rolli et al. 2011), and Capparis spinosa L. (Carra et al. 2012). On comparison between the effect of preconditioning with 100 μm PBU for 4 d and 75 μM TDZ for 8 d (Jahan et al. 2011), the effect of TDZ on the rate of shoot proliferation was slightly superior in term of the number of shoots per explant by producing an average of 20.0 ± 1.15 shoots per explant. This difference might be attributed to either the differential response of different explants or different genotypes or different non-purine phenyl urea compound with cytokinin activity used in both the studies. Although these non-purine phenyl urea derivatives have been reported to have a positive effect on shoot proliferation (Malik et al. 2010; Rolli et al. 2011), in many instances of deleterious effects on shoot elongation, fasciation of shoots, and poor rooting have also been noticed (Huetteman and Preece 1993; Guo et al. 2011). In turn, these negative effects might be due to PBU-accumulation in the plant tissues, because of its inefficient degradation by cytokinin oxidase (Zatloukal et al. 2008; Podwyszyńska et al. 2014). However, in the present study, no such adverse effects of PBU pretreatment had been noticed among the in vitro-regenerated N. arbor-tristis plantlets.

Figure 2.
figure 2

Graphical representation showing the effect of N-phenyl-N′-benzothiazol-6-yl-urea (PBU) pretreatment (dose and duration) on average number of shoots per nodal explant in Nyctanthes arbor-tristis.

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Table 3. Effect of preconditioning Nyctanthes arbor-tristis L. node explants with different durations and concentrations of PBU and optimized growth regulator combination on multiple shoot bud regeneration after 8 wk culture
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In vitro-rooting and acclimatization

Well-elongated shoots, about 4 to 5 cm long, were separated from the originating clump and implanted in rooting medium containing half-strength MS salts fortified with either IBA or NAA (Table 4). No root induction was observed from the shoots implanted in the rooting medium, even after 2 wk of incubation. Thus, these preincubated shoots were transferred to half-strength MS medium without extraneous auxin, where after 3 to 4 d roots were induced from the cut ends, and in some cases from 1 cm above the cut end (Fig. 1f). The duration for complete root development was almost 3 to 4 wk. Preincubation of shoots with rooting medium containing 6 μM IBA for 1 wk, followed by implantation on auxin-free half-strength MS medium, showed the best response (93.00 ± 0.89%) for rooting, with an average development of 6.20 ± 0.49 roots per shoot, and an average root length of 1.62 ± 0.05 cm (Table 4).

Table 4. Effect of auxin type and concentration on rooting percentage, root number, and root length of Nyctanthes arbor-tristis L. cultured on half-strength MS (Murashige and Skoog 1962) medium for 3 wk
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Between the two auxins tested, preincubation with IBA showed its superiority in terms of root induction and root growth, compared to that of NAA (Table 4), and this might be due to IBA’s presence in conjugated form, its stability, consistent weak auxin activity, and to IBA’s insensitivity to auxin-degrading enzymes, as reported in studies with Arabidopsis thaliana and Abutilon indicum (Ludwig-Müller et al. 2005; Seth and Panigrahi 2018). However, the average root length varied from 0.66 to 1.62 cm, depending upon the type and concentration of auxins added to the medium. This might be due in part to the effect of IBA on the development of the root system by regulating primary root elongation and lateral root formation (Marquez et al. 2016). In the present study, almost 78% of in vitro regenerated plants had survived at the end of acclimatization (Fig. 1g), and this plant-to-soil establishment process took almost 4 wk. These in vitro-regenerated plantlets were morphologically similar to their respective ex vitro-grown donor plant, and produced flowers after 8 mo of plant establishment in soil.

Genetic fidelity of in vitro-regenerated plantlets

Genetic variability is often observed during in vitro-propagation of many plant species and is attributed to organogenic induction driven by PGRs, culture environment, and epigenetic influences resulting in gene and ploidy mutations (Larkin and Scowcroft 1981; Kaeppler et al. 2000; Ramirez-Mosqueda and Iglesia-Andreau 2015). Use of PBU has been shown to induce 3.7% somaclonal variation during in vitro-propagation of Citrus madurensis Lour. via somatic embryogenesis (Siragusa et al. 2007). In light of these facts, genetic stability of in vitro-regenerated plantlets of N. arbor-tristis needed to be assessed, using DNA markers. Among different DNA markers, the SCoT marker analysis is quite simple and cost-effective, similar to random DNA markers; however, SCoT marker analysis exhibits increased stability and reliability due to the use of longer primers designed around the initiating codon (Collard and Mackill 2009; Agarwal et al. 2015). Moreover, these SCoT markers target the sequence flanking the start codon (Collard and Mackill 2009), which can provide further correlation with functional genes. In the present study, SCoT marker analysis using 10 primers produced consistently 33 amplified fragments (320 to 2405 bp; Table 5) among 39 regenerated plantlets and their donor plant (Fig. 1h; Table 5). This monomorphic banding pattern (Fig. 1h) demonstrated the lack of genetic variation among these in vitro-regenerated plantlets and their donor plant of N. arbor-tristis, in consonance with their morphology, and offered additional authentication supporting the stability of this in vitro propagation protocol. Furthermore, SCoT marker analysis has been effectively used for the genetic homogeneity assessment of in vitro-regenerated plantlets in H. isora, A.indicum, and A. maurorum (Agarwal et al. 2015; Muthukumar et al. 2016; Seth et al. 2017).

Table 5. Genetic fidelity assessment of Nyctanthes arbor-tristis L. in vitro-raised plantlets grown from 75 μM N-phenyl-N′-benzothiazol-6-yl-urea-pretreated nodal explants and their donor plant, using start codon targeted (SCoT) markers
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Comparative evaluation of bioactive phytochemicals and antioxidant activity among leaf and flower extracts of ex vitro- and in vitro-grown plantlets

N. arbor-tristis plant parts are the source of many important bioactive metabolites and have a wide range of biological and pharmacological activities (Rani et al. 2012; Agrawal and Pal 2013). The homogeneity and consistency of bioactive metabolite composition and accumulation in the target tissues of in vitro-raised plantlets should be similar to their ex vitro-grown donor plants as these plantlets will be the source of raw material for the isolation several therapeutic compounds. In view of these facts, the in vitro-regenerated plantlets and their plant parts produced in the current study were assessed for content of four important bioactive metabolites (ursolic acid, rengyolone, arbortristoside-A, and nyctanthoside), mostly used for the treatment of filaria and malaria (Tuntiwachwuttikul et al. 2003; Agarwal et al. 2013; Saini et al. 2014). The dried extracts under different solvents were assessed for yield percentage (w/w), which was 32.58% for methanolic, 20.36% for ethyl acetate, and 36.48% for aqueous extract, respectively. Ursolic acid was obtained only from leaf tissues of both the ex vitro- and in vitro-derived samples (Fig. 3a), whereas the remaining three phytochemicals were isolated and quantified from both leaves and flowers (Fig. 3b). Leaves obtained from in vitro-propagated plantlets contained 0.79 ± 0.03 μg mg−1 ursolic acid, 1.95 ± 0.05 μg mg−1 rengyolone, 1.69 ± 0.03 μg mg−1 arbortristoside-A, and 1.08 ± 0.02 μg mg−1 nyctanthoside, whereas leaves of ex vitro-grown donor plants contained 0.78 ± 0.02 μg mg−1 ursolic acid, 1.84 ± 0.05 μg mg−1 rengyolone, 1.68 ± 0.04 μg mg−1 arbortristoside-A, and 1.13 ± 0.06 μg mg−1 nyctanthoside (Fig. 3a). Akin to leaf tissues, the flowers of in vitro-derived plantlets contained 2.63 ± 0.12 μg mg−1 rengyolone, 1.90 ± 0.01 μg mg−1 arbortristoside-A, and 1.43 ± 0.03 μg mg−1 nyctanthoside, whereas flowers of ex vitro-grown plants contained 2.51 ± 0.07 μg mg−1 rengyolone, 1.87 ± 0.05 μg mg−1 arbortristoside-A, and 1.41 ± 0.02 μg mg−1 nyctanthoside (Fig. 3b). By comparison, the respective bioactive metabolites accumulated in leaves and flowers of in vitro-regenerated plantlets were nearly equal to those of the ex vitro-grown donor plants for ursolic acid, arbortristoside-A, and nyctanthoside content (Fig. 3a, b). However, the rengyolone content in the leaves and flowers of in vitro-regenerated plantlets was noticeably higher, compared to donor plants (Fig. 3a). This variation might be attributed to the influences of the PGRs used, tissue composition of explants, and culture environment, which probably necessitated the production and accumulation of more rengyolone, as has been reported for different bioactive metabolites in several medicinal species including Hypericum hirsutum L, H. maculatum Crantz., Agastache rugosa O. Kuntze, Musa accuminata L., and Aloe arborescens L. (Coste et al. 2011; Zielinska et al. 2011; Adeyemi et al. 2012; Amoo and Van Staden 2013).

Figure 3.
figure 3

Comparison of four bioactive metabolites content in the leaves (a) and flowers (b) of Nyctanthes arbor-tristis plants raised in vitro as well as ex vitro.

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Antioxidant activity

The harmful effects of free radicals and reactive oxygen species can be alleviated by antioxidant substances, preferably from natural sources, to prevent toxicity (Li et al. 2014). Polyphenols and flavonoids have been reported to be associated with such antioxidant activities (Gul et al. 2011; Riaz et al. 2014). A wide array of phytochemicals with proven antioxidant activities (Dasgupta and De 2007; Rathee et al. 2007; Khanapur et al. 2014; Mishra et al. 2016) has been identified in N. arbor-tristis plant parts. Thus, in the present study, three different extracts of leaves and flowers from ex vitro- and in vitro-grown plants were compared for total polyphenols and flavonoids content, as well as TAA, FRAP, and radical scavenging activities. As reported previously, both flower and leaf extracts of N. arbor-tristis show quite promising amounts of natural bioactive metabolites contributing to antioxidant and radical scavenging activities (Dasgupta and De 2007; Rathee et al. 2007; Mishra et al. 2016). Results obtained in the present study showed that total antioxidant activity of leaf extracts was higher than those of corresponding flower extracts (Fig. 4c), and a similar trend was noticed for polyphenols content (Fig. 4a), flavonoids content (Fig. 4b), and ferric ion reducing antioxidant power (Fig. 4d) among the extracts (Table 6). Similar canonical relationships between total polyphenol content and TAA have been established in a number of other medicinal plants including Abelmoschus moschatus Medik. L. and Fraxinus rhynchophylla Hance (Li et al. 2008; Gul et al. 2011). The antioxidant activity of different plant-based extracts is usually correlated directly to its reducing capacity, and the FRAP assays (Fig. 4d) conducted here substantiated prominent antioxidant activity (Fig. 4c) of leaf and flower extracts of N. arbor-tristis during this study (Table 6). Similar FRAP assays have been performed to evaluate antioxidant activity of flavonoid-rich tissue extracts in different species (Luximon-Ramma et al. 2002; Gul et al. 2011). In the present study, the leaf extracts showed positive correlation between flavonoids content (Fig. 4b) and ferric ion reducing power (Fig. 4d); when compared to the flower extracts, the leaf extracts showed higher flavonoids content, as well as elevated ferric ion reducing ability (Table 6; Fig. 4b, d). In the comparisons between in vitro- and ex vitro-raised plant parts shown in Table 6, the methanolic and ethyl acetate leaf extracts of in vitro-regenerated plants showed higher flavonoid content (149.00 ± 2.31 QE μg mg−1; Fig. 4b) and higher TAA (35.33 ± 0.88 AAE μg mg−1; Fig. 4c), respectively. Similarly, methanolic flower extracts of in vitro-raised plants also showed little higher content of polyphenols (156.33 ± 4.33 GAE μg mg−1) and TAA (25.0 ± 1.16 AAE μg mg−1), when compared to their ex vitro-raised counterpart (Table 6; Fig. 4). The superiority of the oxidative potential of methanolic and ethyl acetate extract of leave and flower, of in vitro-regenerated plant tissues might be due to the influence of PBU on the endogenous PGRs, as well as induced expression of genes involved in the biosynthesis of antioxidant-rich secondary metabolites and phytochemicals (Amoo and Van Staden 2013; Amoo et al. 2013). On the other hand, aqueous leaf and flower extracts of ex vitro-grown donor plant showed marginally higher, if statistically similar, FRAP and TAA activity (Table 6; Fig. 4c, d). This observed variation in total antioxidant activities among different extracts might also be attributed to solvents influencing either tissue-specific solubility of phytomolecules or environmental stresses encountered during ex vitro-growth (Masondo et al. 2015).

Figure 4.
figure 4

Comparative evaluation of antioxidant rich phytochemicals [(a) total phenol content and (b) total flavonoids content] and antioxidant potential [(c) total antioxidant activity and (d) ferric ion reducing power] of different extracts obtained from Nyctanthes arbor-tristis plants raised in vitro as well as ex vitro.

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Table 6. Comparative evaluation of polyphenols, flavonoids, total antioxidant activity, and ferric ion reducing antioxidant power in Nyctanthes arbor-tristis L. leaves and flowers obtained from in vitro and ex vitro-grown plantlets
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Radical scavenging activity

The leaf and flower extracts of N. arbor-tristis were evaluated for their ability to scavenge DPPH, H2O2, and superoxide radicals in a concentration-dependent manner (Table 7; Fig. 5) in the current study, recognizing that observed variation depends upon the type of tissues and solvents, as reported earlier (Dasgupta and De 2007; Rathee et al. 2007; Mishra et al. 2016). Similar to content of bioactive metabolites and antioxidant potential, radical scavenging activity of the extracts obtained from in vitro-regenerated plants were on a par with their respective counterpart from the donor plant, barring a few exceptions (Table 7; Fig. 5): the DPPH radical scavenging activity of analogous extracts of ex vitro- and in vitro-grown leaves and flowers were almost identical in terms of IC50 value, barring ethyl acetate and methanolic leaf extract (Fig. 5a). Ethyl acetate flower extracts showed superior DPPH radical quenching activity (as evidenced by lower IC50 values) in comparison to that of leaf extracts, regardless of plant origin (Fig. 5a), whereas aqueous leaf extracts revealed better DPPH scavenging activity than flower extracts (Fig. 5a). Contrasting to this, leaf extracts showed better H2O2 (Fig. 5b) and superoxide radical scavenging (Fig. 5c) activity than flower extracts (Table 7), for all categories, except for H2O2-scavanging activity of ethyl acetate-extracted leaves. In most cases both the in vitro and ex vitro leaf extracts showed equivalent radical scavenging activities in terms of their IC50 values, while in vitro flower extracts showed superiority over ex vitro flower extracts (Fig. 5). However, these variations might also have resulted due to either varying distribution of active components in different tissues, age of in vitro-regenerated plantlets grown in an ex vitro-environment, and fractionation during extraction, or even stresses levied during their growth, of the plants grown either in vitro or ex vitro (Malik et al. 2010; Riaz et al. 2014; Masondo et al. 2015).

Table 7. Comparative evaluation of half concentration of inhibition (IC50) values* of ethyl acetate, methanolic, and aqueous extracts of Nyctanthes arbor-tristis L. leaves and flowers obtained from in vitro and ex vitro-grown plantlets on different free radicals tested.
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Figure 5.
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Comparative evaluation of free radicals [(a) 1,1-diphenyl-2-picrylhydrazyl (DPPH), (b) hydrogen peroxide (H2O2), (c) superoxide] scavenging ability (in term of IC50 value) of leaf and flower extracts obtained from Nyctanthes arbor-tristis plants raised in vitro as well as ex vitro.

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Conclusion

The present study established an effective and alternative protocol for the micropropagation of N. arbor-tristis plants from PBU-treated nodal explants via organogenesis, where genetic stability of regenerated plantlets was affirmed by SCoT marker profiling, and by homogenous content of four sampled therapeutical metabolites, antioxidant potential, and equivalent radical scavenging activities. The presented propagation procedure should be of immense use for large-scale, clonal multiplication of N. arbor-tristis in vitro to meet the raw material demands of both the herbal industries and for esthetic purposes, as well as for the ex-situ conservation of N. arbor-tristis genotypes.