An efficient plant regeneration protocol of an industrially important plant, Hedychium coronarium J. Koenig and establishment of genetic & biochemical fidelity of the regenerants

Industrial Crops & Products 126 (2018) 58–68

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An efficient plant regeneration protocol of an industrially important plant, Hedychium coronarium J. Koenig and establishment of genetic & biochemical fidelity of the regenerants

T

Shashikanta Beheraa, Pradeep K. Kamilab, Kedar K. Routc, Durga P. Barika, Pratap C. Pandab, ⁎ Soumendra K. Naika, a b c

Department of Botany, Ravenshaw University, Cuttack, 753003, Odisha, India Plant Taxonomy and Conservation Division, Regional Plant Resource Centre, Bhubaneswar, 751015, Odisha, India Siddhi Binayak Science College, Angul, 759122, Odisha, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Hedychium coronarium Medicinal plant Micropropagation ISSR HPTLC Coronarin D

Hedychium coronarium is a valuable medicinal plant which is also known for its uses in cosmetics and perfumes industries. Due to high demand and unsustainable harvesting, this plant is becoming threatened in different regions of India. Tissue culture mediated plant regeneration is an alternative platform for large scale production of such plants to meet the commercial demand of the plant and plant materials in a sustainable manner. Here an efficient and improved in vitro plant regeneration protocol was established for H. coronarium along with the genetic and biochemical fidelity analysis of the regenerants. MS medium augmented with different types, concentrations, and combinations of plant growth regulators were tested in this study. The highest number of shoot regeneration was observed from rhizome segments on Murashige and Skoog’s (1962) (MS) basal medium supplemented with 0.8 mg/L thidiazuron (TDZ) (shoot induction medium) followed by their sub-culture on MS containing 1.0 mg/L gibberellic acid (GA3) (shoot elongation medium). Up-scaling of shoots was carried out using axenic stem segments, isolated from primary in vitro shoots, on a fresh medium of the same composition. Roots developed simultaneously during shoot multiplication and thus could eliminate the requirement of an additional step of rooting. Using this protocol ca. 540 plantlets were produced starting from single explant within 14 weeks. About 95% of these plantlets were successfully acclimatized and eventually established in the field. The micropropagated plants were morphologically similar to the mother plant. Genetic fidelity analysis of the field established micropropagated plants with that of the mother plant were carried out by using Inter Simple Sequence Repeat (ISSR) and monomorphic banding profile confirmed the genetic uniformity of the regenerants. Biochemical fidelity of the tissue culture raised plants was also validated by quantitative phytochemical analysis and High Performance Thin Layer Chromatography (HPTLC). This efficient plant regeneration protocol could be useful for commercial propagation and conservation of H. coronarium.

1. Introduction Hedychium coronarium J. Koenig (White butterfly lily) is a rhizomatous herb, belonging to the family of Zingiberaceae. The plant is popular in India and China for its use in traditional systems of medicine (Kunnumakkara et al., 2008; Chan and Wong, 2015). The rhizome of the plant has been used for the treatment of inflammation, headache, diabetes, and rheumatic pain (Jain et al., 1995; Suresh et al., 2010; Chan and Wong, 2015). In addition rhizome has also been known to be used for remedies of fever, throat related problems, diarrhoea, and skin diseases (Behera et al., 2018). The leaves are reported to be useful for

⁎

treatment of indigestion, hypertension and, stiff and sore joints (Ribeiro et al., 1986; Behera et al., 2018). Recent studies revealed various biological activities of H. coronarium such as antimicrobial, anti-inflammatory, antioxidant, cytotoxicity, and analgesic (Hartati et al., 2014; Chan and Wong, 2015). The biological activities of this important medicinal plant are probably due to the presence of different labdane diterpenes and sesquiterpenes type of compounds (Suresh et al., 2010; Kiem et al., 2011). Hedychium coronarium is a rich source of coronarin D (a labdane diterpenes) which has been isolated, identified and studied for its various biological activities (Kunnumakkara et al., 2008; Kiem et al., 2011; Ray et al., 2017a). The plant is also of horticultural

Corresponding author. E-mail address: [email protected] (S.K. Naik).

https://doi.org/10.1016/j.indcrop.2018.09.058 Received 27 June 2018; Received in revised form 23 August 2018; Accepted 30 September 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

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importance due to its beautiful (butter fly shape) and fragrant flower and thus, often used for ornamental purposes in the gardens of tropical regions (Yue et al., 2014; 2015). The plant has also commercial and industrial importance due to presence of volatile compounds in its flower which exhibit more fragrant than species like rose and snapdragon (Yue et al., 2014) and thus useful for perfumery (Chadha, 2005). Not only flowers but the rhizomes of the plant are also good source of essential/volatile oil and used for making perfumes (Chan and Wong, 2015; Yue et al., 2015). The stem of the plant is potential source of raw materials in paper making industries as it contain about 42–48% cellulose (Chadha, 2005). Rhizomes, flowers, and tender shoots of this plant are also used as food and consumed as raw vegetables in different parts of globes including India, Japan, and Hawaii (Devi et al., 2010; Misra et al., 2013; Chan and Wong, 2015). Due to multifarious use and high demand for medicinal uses, H. coronarium has already been overexploited in their natural habitat and rightly reported as a medicinal plant of conservation concerns of different states of India including Madhya Pradesh, Odisha, Kerala, and Karnataka (Ved et al., 2008; http://envis.frlht.org/mpcc-species). Conventional methods of plant propagation are not always adequate to produce large pool of plants to be used for germplasm conservation through reintroduction as well as in various pharmaceutical and cosmetic industries. Biotechnological approach using plant tissue culture to circumvent these problems of producing large number of genetically and biochemically clonal plants are already evident by now. But possibilities of variations in plants regenerated through tissue culture cannot be ruled out as in vitro culture environments at times induce changes at phenotypic as well as genetic level (Bairu et al., 2011; Krishna et al., 2016). Thus along with morphological similarity, the assessment of genetic and biochemical fidelity of the tissue culture regenerants is imperative. Keeping all the above points of view, present study was carried out to develop an improved in vitro plant regeneration protocol of H. coronarium. Most importantly the genetic and biochemical fidelity of the regenerants were also established with their mother plant.

(0.25–1.5 mg/L), and a growth adjuvant, Adenine sulphate (ADS) (25–100 mg/L). The culture medium was supplemented with 3% (w/v) sucrose and gelled with 0.7% (w/v) agar. The pH of the medium was adjusted to 5.8 ± 0.1 prior to autoclave. Shoots failed to elongate on MS augmented with TDZ medium. Thus after 3 weeks the explants (with multiple shoot buds) were transferred to either plant growth regulator free MS or MS supplemented with 1.0 mg/L Gibberellic acid (GA3) medium for further elongation of shoots. With the aim to upscale the shoot number within a short period of time, the in vitro primary shoots regenerated from the explants (rhizomatic bud segments) were harvested. The primary shoots were cut into small segments and inoculated as explants on MS medium augmented with 0.8 mg/L TDZ, which was optimized as best medium for shoot regeneration/one step plant regeneration. After harvest of primary shoots the original mother explants were also repeatedly sub-cultured on medium with same composition i.e. MS + 0.8 mg/L TDZ for further shoot multiplication. After initiation of multiple shoots from the shoot segment explants as well as original mother explants, the cultures were transferred to MS medium containing 1.0 mg/L GA3 for further elongation of shoots. All the cultures were maintained at 25 ± 1 °C with 16 h photoperiod and 50 μmol m−2 s−1 light intensity provided with cool white fluorescent tubes (Philips, India). 2.3. Acclimatization of plantlets The clump of in vitro plantlets was carefully removed from the culture vessel followed by thorough washing of the roots to remove the adhered media. The in vitro plantlets (shoot with few roots formed simultaneously) were separated carefully from the clump of culture. The plantlets (one shoot with minimum shoot length of 4.0 cm having few roots with at least 3.5 cm length) were transferred to small pots (6.5 × 7.5 cm) containing either autoclaved garden soil, or mixture of garden soil and sand (1:1) and the substrates were moistened with tap water. The potted plantlets were covered with polyethylene bags to ensure humidity and kept in the culture room under same controlled environmental conditions as described earlier. The plantlets were inspected at regular interval and watered as per the requirement. After one week from the date of planting, the polyethylene bags were removed and plantlets were maintained in the same condition for another one week then taken out from culture room and kept in shade for one week and watered at two days interval. Acclimatized plants were transferred to clay pots (13 × 17 cm) containing the respective substrate and kept out door under shade for one week prior to transferring under full sun for three weeks. Finally these plants were transfer to field conditions.

2. Materials and methods 2.1. Collection, sterilization, and preparation of explants Young, sprouting rhizomatic axillary buds (10–15 cm) were collected from two years old H. coronarium plants, having average height of 1.15 m, maintained in the garden of Department of Botany, Ravenshaw University, Cuttack, Odisha, India and brought to the laboratory. The rhizomatic buds were washed carefully in running tap water to remove soil and sand adhering to it and subsequently roots and outer scale were removed. They were washed thoroughly with 5% (v/v) aqueous solution of liquid detergent ‘Teepol’ (Reckitt Benckiser Ltd., India) and then treated with 2% (w/v) ‘Bavistin’ (fungicide) (BASF, Mumbai, India) for 10 min followed by 3–5 times rinsed with double distilled water. The explants were surface sterilized with mercuric chloride [0.1% (w/v) HgCl2; Hi-Media, India] for 12 min followed by 3–5 times washing with sterile double distilled water. The surface sterilized rhizomatic buds were transversely cut into pieces (0.5–1.0 cm thickness) each having eye bud, followed by removal of leaf sheath and ultimately used as starting plant materials (Fig. 1A).

2.4. Morphological characteristic study and estimation of total chlorophyll content in leaf The morphological characters such as plant height, tiller number/ plant, leaf number/shoot, and leaf size of both the types i.e. conventionally grown and micropropagated plants of one and half year old were compared. Fresh leaf of mother and micropropagated plants of H. coronarium were used to estimate the total chlorophyll content using acetone. Fresh leaves (1.0 g) were crushed and homogenized using 10 mL distilled water. From this sample 500 μL was taken and volume was made up to 5 mL using 80% acetone. The sample was centrifuged at 10,000 rpm for 5 min and the supernatant was collected and the optical density (OD) was measured at 663 and 645 nm. The total chlorophyll content of both types of leaves was calculated using the following formula (Arnon, 1949; Sudhakar et al., 2016).

2.2. Shoot proliferation and plantlet formation Surface sterilized rhizomatic segments (with eye bud) were cultured either on Murashige and Skoog’s (1962) (MS) medium alone or augmented with different concentrations and combinations of plant growth regulators such as N6-Benzyladenine (BA) (1.0–5.0 mg/L), Kinetin (KIN) (1.0–5.0 mg/L), Zeatin (Z) (1.0–5.0 mg/L), meta-Topolin (mT) (1.0–5.0 mg/L), Thidiazuron (TDZ) (0.25–2.0 mg/L), α-Naphthaleneacetic acid (NAA) (0.25–1.5 mg/L), Indole-3-butyric acid (IBA)

mg total chlorophyll/g tissue = [20.2 (A645) + 8.02 (A663)] × V/ (1000 × W) Where, 59

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(caption on next page)

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Fig. 1. (A) Rhizomatic segments having eye bud; (B) One step plant regeneration on growth regulator free-MS medium; (C) Multiple shoot bud initiation from rhizomatic segment on MS + 3.0 mg/L BA; (D) Simultaneous proliferation of multiple shoot and root on MS + 3.0 mg/L BA; (E) One step formation of multiple shoot and root on MS + 3.0 mg/L mT; (F) Multiple shoot bud initiation from rhizomatic segment on MS + 0.8 mg/L TDZ within 3 weeks of culture; (G) Simultaneous proliferation of multiple shoot and root on MS + 1.0 mg/L GA3 followed by transfer from MS + 0.8 mg/L TDZ; (H) Multiple shoot initiated on MS + 0.8 mg/L TDZ after 5 weeks of culture failed to elongate on MS + 1.0 mg/L GA3; (I) Production of shoot and root from axenic stem explant on MS + 1.0 mg/L GA3 medium followed by subculture from MS + 0.8 mg/L TDZ; (J, K) Multiple shoot proliferation from mother rhizomatic explant during fourth passage of culture on MS + 1.0 mg/L GA3 medium at 2 and 4 weeks of culture respectively after transfer from MS + 0.8 mg/L TDZ; (L) In vitro shoots having roots (plantlets) after separation from the cluster of shoots, prior to acclimatization; (M) Acclimatized plants in small pots containing autoclaved garden soil and sand (1:1); (N) Acclimatized plants in the larger clay pots. Bars: 1 cm; EB: eye bud.

methanol for overnight at room temperature. The samples were filtered in Whatman filter paper no. 42. The filtrates were kept in a water bath and allowed to evaporate until it achieved a constant weight. The quantities of flavonoids were calculated and represented as mg/g dry weight of sample (Bankole et al., 2017).

A = absorbance at specific wavelengths V = final volume of chlorophyll extract W = fresh weight of tissue extracted 2.5. Genetic fidelity study using ISSR markers In this study genetic fidelity of the micropropagated plants was checked using inter simple sequence repeats (ISSR) markers. Genomic DNA was isolated from fresh tender leaves of mother plant as well as ten randomly selected field established micropropagated plants using the method as described by Doyle and Doyle (1990) with minor modification. The quality as well as the quantity of DNA was checked on 0.8% agarose gel using uncut λ DNA as standard. The DNA samples were diluted with T10E1 buffer to a working concentration of 25 ng/μL for PCR analysis. Ten ISSR primers were randomly selected to check the genetic fidelity of micropropagated plants and out of these, five primers were used for final analysis on basis of their reproducible and clear banding pattern. The polymerase chain reaction (PCR) amplification mixture of 25 μL contained 1.0 μL of 25 ng template DNA, 2.5 μL of 10 × PCR buffer (100 mM Tris HCl pH 8.3, 500 mM KCl and 0.1% gelatin), 1.5 mM MgCl2, 200 μM each dNTPs, 15 ng primer and 0.5 U Taq DNA polymerase (Bangalore Genei, India) and volume was adjusted by adding molecular grade water (Hi-Media, India). The PCR was programmed as: initial denaturation at 94 °C for 5 min followed by 42 cycles of 1 min denaturation of template DNA at 94 °C, 1 min annealing of primers at 45 °C–66.1 °C (depending upon the primer used), 2 min primer extension at 72 °C and a final extension cycle at 72 °C for 7 min in a thermal cycler (Applied Biosystem, Model 9700). The amplified products were separated in 1.5% agarose gel in 1 × TAE buffer (pH 8.0). The gels were visualized and photographed using gel documentation system (Bio-Rad, USA). The sizes of the amplicons were estimated comparing with a DNA ladder (Medium Range GeneRuler [5 kb], Genei-Merck, India or GeneRuler 100 bp Plus DNA Ladder [3 kb], Thermo Scientific, USA). Only reproducible bands with same migration were considered homologous bands and scored for analysis irrespective of their intensity.

2.6.3. Estimation of saponin content Saponin content was estimated using the method described by Bankole et al. (2017) with slight modification. Five gram of rhizome, leaf, and root powder of both mother and micropropagated plants were taken and 50 mL of 20% aqueous ethanol was added in it. The sample was constantly stirred for 4 h at 55 °C on a hot plate magnetic stirrer. The mixture was collected by filtering and the residue was re-extracted by adding 50 mL of 20% aqueous ethanol. The combined filtrates (extract) were reduced to about 20 mL over water bath at 90 °C. The concentrated sample extract was transferred into 60 mL separating funnel and 10 mL of diethyl ether was added to it and shaken vigorously. The aqueous layer was collected and ether layer was discarded. This purification process was repeated twice. To the combined aqueous, 10 mL of n-butanol was added. The combined n-butanol extracts was washed twice with 10 mL of 5% aqueous NaCl. The remaining solution was allowed to heat in a water bath. After evaporation, the concentrated sample was dried in dry bath to a constant weight and saponin content was calculated as mg/g in dry weight.

2.6.4. Estimation of phenolic content Samples (5 g each) were taken separately and percolates in 50 mL of double distilled water for overnight at room temperature. The extracts were filtered and taken for phenolic assay. Phenolic content was determined by Folin Ciocalteu’s method. One mL of sample and gallic acid as standard (10, 20, 40, 60, 80, 100 μg/mL) were taken separately in each test tube, 5 mL of double distilled water and 0.5 mL Folin Ciocalteu’s reagent were added to it. The mixture was gently shaken and allowed to incubate for 5 min. The volume was adjusted to 10 mL followed by adding 1.5 mL of 20% sodium carbonate and allowed to incubate for 2 h at room temperature and absorbance was measured at 750 nm using UV–vis spectrophotometer. The amount of total phenolics was calculated as mg of gallic acid equivalent weight (GAE)/g of dry weight (Kamtekar et al., 2014).

2.6. Phytochemical analysis 2.6.1. Preparation of samples Fresh rhizome, leaf, and root samples of both mother and micropropagated plants of H. coronarium were collected from one and half year old plants grown in the departmental garden. They were thoroughly washed with tap water followed by double distilled water, and cut into small pieces. Each sample was kept separately in room temperature and shade dried to a constant weight. They were then powdered and stored in air tight container for the different phytochemical analysis. All types of samples (rhizome, leaf, and root) were used for estimation of different phytochemicals such as flavonoids, saponins, phenolics, and tannins, whereas only rhizome sample was used for HPTLC analysis.

2.6.5. Estimation of tannin content Sample powders (5 g each) were dissolved in 50 mL of double distilled water and incubated overnight at room temperature. After filtration the samples were used for estimation of tannin content by Folin Ciocalteu method (Tambe and Bhambar, 2014). One mL of extract from each sample and tannic acid as standard (100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 μg/mL) were taken separately in each volumetric flask containing 7.5 mL distilled water, 0.5 mL of Folin Ciocalteu phenol reagent and 1 mL of 35% sodium carbonate solution. The mixture was shaken properly and incubated for 30 min at room temperature. Absorbance for test and standard solution were measured against the blank at 700 nm using UV–vis spectrophotometer. The tannin content was expressed in terms of mg of tannic acid equivalent weight (TAE)/g of dry weight.

2.6.2. Estimation of flavonoid content Five gram each of rhizome, leaf, and root powder of both mother and micropropagated plants were dissolved separately in 50 mL of 80% 61

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To achieve large scale shoot multiplication the primary in vitro shoots were cut into small pieces (on an average 3 explants were obtained from one shoot thus ca. 45–47 explants were found from an average of 15.8 shoots) and inoculated on the MS medium supplemented with 0.8 mg/L TDZ (shoot induction medium). About 90% of these explants showed multiple shoot induction within 3 weeks. Finally an average of 12.0 shoots with shoot length 5.0 cm and 27 roots with root length 4.8 cm were recorded per explant on MS + 1.0 mg/L GA3 (shoot elongation medium) within four weeks of subculture (Fig. 1I). Therefore, in a period of 14 weeks, ca. 540 plantlets (shoot with roots) were obtained starting from a single rhizomatic segment. Elimination of an additional step of in vitro rooting, as described here, reduces the time and cost of micropropagation. After harvesting of the in vitro shoots the original mother explants were also sub-culture repeatedly on shoot induction medium followed by shoot elongation medium to evaluate their shoot regeneration ability. No significant reduction of the shoot number from the mother explants was observed till 5th sub-culture passage (Fig. 2). But a number of regenerated shoots during the 5th sub-culture passages were comparatively thin and weak. Highest shoot number (23.6 shoots/explant) was recorded during the 4th sub-culture passage, where all the shoots were healthy (Fig. 1J, K).

2.6.6. HPTLC analysis for coronarin D HPTLC analysis for coronarin D was carried out by following the protocol of Ray et al. (2017b). A HPTLC system (Camag, Switzerland) equipped with an automatic sample applicator Linomat 5 (100-μL syringe), TLC scanner 3 and integrated Win CATS software (version 1.4.2) was used in this study. The sample solution for HPTLC analysis was prepared by dissolving 20 mg of dry acetone extract of rhizome in 1 mL of acetone. A 6 μL sample solution was applied on HPTLC plate by using Linomat 5 applicator as 6 mm band. The chromatography was carried out by using n-hexane- ethyl acetate (8:2) as mobile phase and densitometry scanning was performed at 231 nm. The presence of coronarin D was detected at RF value of 0.20 as reported by Ray et al. (2017b). 2.7. Data recording and statistical analysis All the experiments were repeated three times. The tissue culture experiments were having fifteen replica (culture vessels) and each culture vessel consisted of one explant. The data was recorded at regular interval and final data on mean number of shoots per explant, mean number of roots per explant, mean length of shoot, and root in centimetre (cm) was obtained after seven weeks of culture. Data were analysed using analysis of variance (ANOVA) for a completely randomized design (CRD). Duncan’s multiple range tests (DMRT; Gomez and Gomez, 1984) was used to separate the mean values for significant effect. Data for quantitative phytochemical analysis and morphological studies was analysed using t-test.

3.2. Acclimatization of in vitro generated plantlets From the clump of in vitro regenerated shoots having roots (Fig. 1I), individual plantlets (shoot with few roots; Fig. 1L) were carefully separated and used for acclimatization. About 95% of the plantlets survived in small pots containing a mixture of garden soil and sand (1:1) after three weeks from the start of hardening process (Fig. 1M). Plant survival in garden soil was comparatively less than the sand and soil mixture (data not shown). On subsequent transfer of these plants to larger clay pots having garden soil and sand (1:1) zero mortality was observed (Fig. 1N) and all the plants were subsequently established in the field.

3. Results 3.1. Multiple shoot proliferation and in vitro plant regeneration An improved micropropagation protocol has been established for Hedychium coronarium. Shoot bud initiation from rhizomatic segment explants was observed within one week of culture, irrespective of media tested. The explants responded differently to various media but a common interesting event i.e. simultaneous formation of both shoot and root was observed in all the culture media including plant growth regulator free MS medium (Table 1). MS medium devoid of any plant growth regulators exhibited shoot proliferation from 55.6% of the explants. On this medium regeneration of only one shoot with simultaneous rooting was observed (Fig. 1B). Maximum explant response (91.1%) as well as highest number of shoot regeneration was observed on MS augmented with TDZ (0.8 mg/L) (Table 1). BA, one of the most popular cytokinins, was not effective for shoot regeneration. BA at an optimum concentration of 3.0 mg/L exhibited 4.9 shoots/explant with average shoot length of 5.0 cm and 16.5 roots/explant (Fig. 1C, D). About 86.7% explants responded to the optimum concentration of mT (3.0 mg/L) and about 11.0 shoots and 25.0 roots were recorded per explant (Fig. 1E). When rhizomatic segments were inoculated on MS augmented with 0.25–2.0 mg/L TDZ, maximum shoot buds were observed on 0.8 mg/L TDZ containing MS medium (shoot induction medium) in three weeks but they failed to elongate on the same medium (Fig. 1F). Therefore, the explants with induced shoot buds were transferred to either MS basal medium alone or MS supplemented with 1.0 mg/L GA3. Maximum shoots (15.8 shoots/explant) with average shoot length of 5.5 cm and about 45.5 roots/explant were observed simultaneously from the explants sub-cultured on MS + 1.0 mg/ L GA3 (shoot elongation medium) after four weeks of sub-culture (Fig 1 G; Table 1). The number of shoot bud regeneration was found to be increased when there was an increase in the culture period from three weeks to five weeks on MS with 0.8 mg/L TDZ. But unfortunately these multiple shoot buds failed to grow and elongate even after transfer to elongation medium i.e. MS + 1.0 mg/L GA3 (Fig.1H). Thus initial culture of explants on MS + 0.8 mg/L TDZ for three weeks followed by their subculture on MS + 1.0 mg/L GA3 for four weeks was optimized for in vitro plant regeneration.

3.3. Morphological characteristic and chlorophyll content of micropropagated plants Analysis of morphological features of both micropropagated plant and the mother plant (one and half year old) grown under field condition was carried out. Both types of plants did not show any kind of variations among themselves. The morphological features including plant height, tiller number/plant, leaf number/shoot, and leaf size of the tissue culture regenerated plants resembled the mother plant (Table 2). The total chlorophyll content of both types of plants was also estimated and found to be almost similar (37.8 mg/g FW: micropropagated plant and 36.9 mg/g FW: mother plant). 3.4. Clonal fidelity analysis by ISSR markers Clonal fidelity analysis of the micropropagated plants with that of the mother plant was carried out using ISSR markers. Out of the ten primers used for initial screening, five primers produced clear and reproducible bands. A total of 25 bands were produced by these five primers, with an average of 5.0 bands per primer. The number of scorable bands amplified by ISSR primers varied from 2 [Oligo 4(b)] to 9 (PCP-3), whereas the size of these bands ranged from 400 to 3000 bp. All the primers produced unique and monomorphic banding profile (Table 3). Similar types of bands for both in vitro regenerated plants and mother plant meant no variation among them and which indicated that the regenerants are genetically similar to the mother (Fig. 3A, B). 3.5. Phytochemical analysis Phytochemical analysis of flavonoids, saponins, phenolics, and 62

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Table 1 Effect of plant growth regulators and growth adjuvant on in vitro shoot and one step plant regeneration from rhizome bud segments of H. coronarium. MS basal medium + growth regulators (mg/L) a

BA

KIN

Z

mT

TDZ

NAA

IBA

ADS

0 1.0 2.0 3.0 4.0 5.0 – – – – – – – – – – – – – – – – – – – – – 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0

0 – – – – – 1.0 2.0 3.0 4.0 5.0 – – – – – – – – – – – – – – – – – – – – – – – – – – – –

0 – – – – – – – – – – 1.0 2.0 3.0 4.0 5.0 – – – – – – – – – – – – – – – – – – – – – – –

0 – – – – – – – – – – – – – – – 1.0 2.0 3.0 4.0 5.0 – – – – – – – – – – – – – – – – – –

0 – – – – – – – – – – – – – – – – – – – – 0.25 0.5 0.8 1.0 1.5 2.0 – – – – – – – – – – – –

0 – – – – – – – – – – – – – – – – – – – – – – – – – – 0.25 0.5 1.0 1.5 – – – – – – – –

0 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 0.25 0.5 1.0 1.5 – – – –

0 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 25 50 75 100

% response

Shoots/explant

Roots/explant

Shoot length (cm)

55.6 ± 1.0 j 73.3 ± 1.7 g 82.2 ± 2.0 d 88.9 ± 1.0 ab 86.7 ± 1.0 bc 77.8 ± 1.0 ef 62.2 ± 1.0 i 66.7 ± 1.0 h 77.8 ± 0.0 ef 77.8 ± 1.0 ef 73.3 ± 2.6 g 73.3 ± 1.7 g 82.2 ± 0.0 d 86.7 ± 1.0 bc 77.8 ± 3.0 ef 77.8 ± 1.0 ef 73.3 ± 0.9 g 86.7 ± 1.0 bc 86.7 ± 0.9 bc 77.8 ± 1.7 ef 66.7 ± 3.5 h 77.8 ± 3.0 ef 86.7 ± 4.0 bc 91.1 ± 1.9 a 88.9 ± 2.0 ab 80.0 ± 2.6 de 73.3 ± 1.0 g 91.1 ± 1.8 a 91.1 ± 2.8 a 88.9 ± 2.0 ab 88.9 ± 2.6 ab 88.9 ± 3.0 ab 86.7 ± 1.7 bc 86.7 ± 1.7 bc 77.8 ± 1.0 ef 91.1 ± 1.8 a 91.1 ± 2.8 a 91.1 ± 2.8 a 88.9 ± 1.0 ab

1.0 ± 0.0 v–x 2.6 ± 0.3 o–t 3.3 ± 0.3 m–q 4.9 ± 0.4 h–l 2.3 ± 0.0 p–u 2.0 ± 0.2 r–v 1.0 ± 0.0 v–x 1.5 ± 0.0 s–w 2.0 ± 0.5 r–v 3.0 ± 0.5 n–r 2.0 ± 0.0 r–v 1.5 ± 0.0 s–w 3.0 ± 0.5 n–r 3.5 ± 0.4 m–p 3.0 ± 0.5 n–r 2.7 ± 0.1o–s 3.8 ± 0.1k–o 7.0 ± 0.9 ef 11.0 ± 2.1b 6.5 ± 0.5 fg 4.0 ± 0.2 j–n 5.0 ± 0.3 h–k 9.6 ± 0.2 cd 15.8 ± 1.6 a 10.9 ± 0.8 bc 8.0 ± 2.0 e 6.0 ± 0.0 f–h 4.9 ± 0.4 h–l 5.0 ± 0.7 h–k 3.5 ± 0.2 m–p 3.5 ± 0.3 m–p 5.2 ± 0.3 g–j 5.9 ± 0.1f–i 3.5 ± 0.1 m–p 3.0 ± 0.5 n–r 5.9 ± 0.4 f–i 6.0 ± 0.8 f–h 5.2 ± 0.2 g–j 4.4 ± 0.9 j–m

2.3 ± 0.3 xy 7.0 ± 1.0 q–t 13.5 ± 0.9 jk 16.5 ± 0.6 gh 9.3 ± 0.5 m–q 7.5 ± 0.5 p–s 4.0 ± 0.7 u–x 5.5 ± 0.9 s–u 10.0 ± 2.6 l–o 12.0 ± 2.0 kl 7.0 ± 1.7 q–t 4.5 ± 0.5 u–w 7.5 ± 0.5 p–s 10.3 ± 0.5 l–n 9.7 ± 0.1l–p 5.0 ± 0.5 t–v 7.0 ± 1.0 q–t 19.0 ± 2.0 ef 25.0 ± 1.7 bc 9.3 ± 0.9 m–q 9.0 ± 1.0 m–r 16.0 ± 1.0 g–i 26.5 ± 1.3 b 45.5 ± 2.3 a 23.3 ± 0.3 cd 15.5 ± 0.1h–j 13.5 ± 1.0 jk 18.0 ± 1.0 e–g 19.5 ± 2.3 e 18.0 ± 0.5 e–g 18.0 ± 2.0 e–g 15.5 ± 0.9 h–j 18.0 ± 1.0 e–g 10.0 ± 1.8 l–o 7.0 ± 1.7 q–t 9.7 ± 1.0 l–p 15.5 ± 0.9 h–j 13.5 ± 0.5 jk 11.2 ± 1.3 k–m

3.0 3.3 3.7 5.0 4.5 3.5 3.0 3.5 4.8 5.0 4.5 3.5 3.8 4.0 4.0 3.0 3.8 3.2 4.8 4.5 3.0 5.0 5.3 5.5 5.3 3.7 2.3 5.1 6.0 5.0 4.8 4.2 5.0 4.6 4.2 4.7 5.5 5.2 4.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.4 o–s 0.3 m–q 0.2 l–o 0.5 b–f 0.3 d–k 0.0 l–p 0.2 o–s 0.0 l–p 0.5 b–h 0.9 b–f 0.5 d–k 0.3 l–p 0.3 k–n 0.3 i–m 0.8 i–m 0.5 o–s 0.1k–n 0.3 n–r 0.5 b–h 0.5 d–k 0.0 o–s 0.4 b–f 0.3 a–c 0.5 ab 0.3 a–c 0.7 l–o 0.5 t 0.6 b–e 1.0 a 0.5 b–f 0.4 b–h 0.2 g–l 0.0 b–f 0.6 c–j 0.2 g–l 0.0 c–i 0.3 ab 0.2 b–d 0.4 b–g

Root length (cm)

1.5 ± 0.5 rs 2.0 ± 0.2 o–r 3.4 ± 0.2 i–k 4.2 ± 0.3 c–f 3.9 ± 0.4 d–i 3.7 ± 0.3 f–j 2.5 ± 0.5 m–o 2.5 ± 0.0 m–o 3.4 ± 0.2 i–k 3.2 ± 0.2 j–l 3.0 ± 0.0 k–m 2.0 ± 0.0 o–r 2.3 ± 0.3 n–p 3.4 ± 0.4 i–k 3.0 ± 0.5 k–m 2.5 ± 0.3 m–o 2.0 ± 0.0 o–r 2.0 ± 0.9 o–r 2.8 ± 0.2 l–n 2.0 ± 0.0 o–r 1.5 ± 0.5 rs 3.9 ± 0.0 d–i 4.8 ± 0.1ab 5.2 ± 0.2 a 3.4 ± 0.1i–k 3.2 ± 0.1 j–l 2.1 ± 0.1 o–q 4.3 ± 0.2 b–e 4.5 ± 0.5 bc 4.1 ± 0.2 c–g 4.0 ± 0.3 c–h 2.8 ± 0.3 l–n 3.0 ± 0.3 k–m 3.0 ± 0.5 k–m 2.8 ± 0.3 l–n 3.4 ± 0.2 i–k 4.4 ± 0.1 b–d 4.2 ± 0.3 c–f 3.7 ± 0.2 f–j

Values represent means ± standard deviation (SD). Different letters in superscripts in a column specify statistically significant difference between the means (P ≤ 0.05; Duncan’s multiple range test). BA: N6-benzyladenine, KIN: kinetin, Z: zeatin, mT: meta-Topolin, TDZ: thidiazuron, NAA: α-naphthaleneacetic acid, IBA: indole-3-butyric acid, ADS: adenine sulphate. a Data (shoots/explant, roots/explant, shoot length, root length) were recorded after sub-culture on MS + 1.0 mg/L GA3. Table 2 Comparison of morphological features of one and half year old field grown mother and micropropagated plants. Morphological features

Mother plant

Micropropagated plant

Plant height (cm) Tiller number/plant Leaf number/shoot Leaf length (cm) Leaf width (cm) Total Chlorophyll (mg/g FW)

115.0 ± 1.7b 8.0 ± 1.0b 12.0 ± 1.0a 32.2 ± 1.7a 5.0 ± 0.8a 36.9 ± 1.0a

121.0 ± 1.7a 11.0 ± 1.0a 13.0 ± 1.0a 34.3 ± 1.1a 5.8 ± 0.5a 37.8 ± 1.0a

Values represent means ± standard deviation (SD) of 30 replicates (plants). In a row, different letters in superscripts followed by mean value designate statistically significant difference between the means (P ≤ 0.05; t- test). FW: fresh weight.

Fig. 2. Shoot regeneration potential of rhizome bud segments at various culture passages. Bars with different letters specify statistically significant difference between the means (P ≤ 0.5; Duncan’s multiple range test).

flavonoids in the rhizome of micropropagated plants (10.0 mg/g dry wt.) was significantly higher compared with the conventionally grown mother plant (5.4 mg/g dry wt). In contrast, the quantities of saponins and tannins in the rhizome of mother plant were significantly higher than the micropropagated plants. However, the flavonoids, saponins, and tannins content in the root of mother plant were found to be

tannins indicated their presence in both the types of plant i.e. mother plant and in vitro regenerated plants. Most importantly almost similar amounts of these phytochemicals were present in different plant parts of both plant types with few variations (Table 4). The quantity of 63

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Table 3 ISSR primers used for evaluation of genetic fidelity of the micropropagated plants of H. coronarium, their sequence, melting temperature, annealing temperature, number of bands and size range of amplified products. No.

Primer code

Primer sequences (5’–3’)

Melting temperature (ºC)

Annealing temperature (ºC)

No. of scorable bands

Approximate range of amplifications (bp)

1. 2. 3. 4. 5. Total

Oligo4(a) Oligo4(b) Oligo-5(a) Oligo-5(b) PCP-3

(GACA)4T T(GACA)4 G(GA CA)4 (GACA)4G (GTGC)4

50 50 52 52 71.1

45 45 47 47 66.1

4 2 6 4 9 25

750–1450 500–1050 500–3000 850–1700 400–2300

alternative method for the year round production of clonal plants, both biochemically as well as genetically, in a large scale. The organised meristematic tissue is usually preferred by workers for micropropagation since such plant regeneration system is less prone to genetic variation and chances of production of genetically cloned plant is more (Shenoy and Vasil, 1992; Bhatia et al., 2011). Thus, in the present study, meristematic rhizomatic segment was chosen and used as explants for micropropagation of H. coronarium. Plant parts associated with rhizome have also been used for the micropropagation of H. coronarium (Mohanty et al., 2013; Parida et al., 2013; Verma and Bansal, 2013, 2014). Type and concentration of plant growth regulators have considerable influence on the regeneration of shoots and roots, ultimately the process of micropropagation. In this present study, TDZ (0.8 mg/L) supplemented MS was found to be most effective for shoot initiation compared with all the other cytokinins as well as cytokinin and auxin or ADS combinations. TDZ has also been reported to be more efficient than BA or KIN for multiple shoot proliferation in number of plant species (Murthy et al., 1998; Faisal et al., 2018). However, in this study shoot elongation was a problem in TDZ supplemented medium

comparatively higher than the micropropagated plants (Table 4). 3.6. HPTLC analysis for coronarin D The HPTLC analysis for coronarin D content in acetone extracts of rhizome of mother as well as micropropagated plant of H. coronarium was carried out and their level was compared on the basis of their peak area. The coronarin D content was found higher in rhizome of the micropropagated plant than the mother plant (Fig. 4A, B). The result clearly suggested that the plant regenerated by the presently described method does not impart any adverse influence on the production of coronarin D, which is one of the most important secondary metabolites present in H. coronarium. 4. Discussion 4.1. Multiple shoot proliferation and in vitro plant regeneration Plant tissue culture technique has already been proven as an

Fig. 3. ISSR banding profiles of mother and micropropagated plants of H. coronarium using ISSR primers (A) Oligo 4a and (B) PCP 3; Lane 1 mother plant, Lane 2–11 micropropagated plants. M is the marker (5 kb). 64

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this study was in agreement with the earlier study of Verma and Bansal (2014) in H. coronarium as well as other medicinal plant species of Zingiberaceae family including Curcuma longa (Parthanturarug et al., 2003), Curcuma soloensis (Zhang et al., 2011), and Hedychium spicatum (Giri and Tatma, 2011) where TDZ was reported to be effective for shoot proliferation. In this study, shoot number could be up-scaled by culturing the axenic stem segments isolated from the primary in vitro shoots regenerated from rhizome explants. An average of 12 shoots (with simultaneous rootlets) per stem explant were obtained, thus about 540 plantlets could be regenerated within 14 weeks.

Table 4 Comparative quantitative phytochemical profiling of mother and micropropagated plant of H. coronarium. Phytochemicals

Mother plant

Micropropagated plant

Flavonoids (mg/g DW) Saponins (mg/g DW) Tannins (mg TAE/g DW) Phenolics (mg GAE/g DW)

Rhizome 5.4 ± 0.4b 29.0 ± 0.8a 169.6 ± 2.0a 12.1 ± 0.5a

10.0 ± 1.0a 24.4 ± 1.0b 140.0 ± 0.8b 12.3 ± 0.1a

Flavonoids (mg/g DW) Saponins (mg/g DW) Tannins (mg TAE/g DW) Phenolics (mg GAE/g DW)

Root 9.2 ± 0.6a 21.7 ± 1.0a 123.2 ± 1.3a 5.4 ± 0.4a

6.0 ± 0.4b 19.9 ± 0.6b 115.5 ± 1.5b 5.1 ± 0.3a

Flavonoids (mg/g DW) Saponins (mg/g DW) Tannins (mg TAE/g DW) Phenolics (mg GAE/g DW)

Leaf 5.5 ± 0.5a 22.3 ± 1.1a 201.0 ± 1.0a 20.9 ± 0.4a

4.9 ± 0.4a 24.6 ± 1.5a 202.4 ± 1.2a 20.3 ± 0.5a

4.2. Acclimatization of plantlets In this study, ninety five percent of in vitro regenerated plantlets were successfully established in the field after step wise acclimatization. This was in contrast to previous report (Mohanty et al., 2013) where, cent-percent acclimatization of plantlets was reported. This difference was may be due to the difference in the robustness of the plantlets obtained in the respective protocols. Number of plantlets regenerated in the current protocol was quite high (more than 500) in compare to the earlier report of Mohanty et al. (2013) but may be the plantlets produced in this study were relatively less robust. Thus normal step wise acclimatization process was carried out in this study, the success of acclimatization (95%) was at par with previous plant regeneration protocols of H. coronarium by Verma and Bansal (2012) (95%) or better than Parida et al. (2013) (80%). This protocol yielded significantly higher plantlets than the earlier reports in H. coronarium (Bisht et al., 2012; Mohanty et al., 2013; Parida et al., 2013; Verma and Bansal, 2013, 2014).

Values represent means ± standard deviation (SD). In a row, different letters in superscripts followed by mean value designate statistically significant difference between the means (P ≤ 0.05; t-test). DW: dry weight, GAE: gallic acid equivalent, TAE: tannic acid equivalent.

and it was overcome by transferring the cultures to MS containing 1.0 mg/L GA3 medium after three weeks of culture on TDZ supplemented MS. Thus there was a requirement of two separate media, one for shoot induction (MS + 0.8 mg/L TDZ) and another for shoot elongation (MS + 1.0 mg/L GA3), for this micropropagation system. This system yielded highest shoots (15.8) which was significantly more compare to other media. Interestingly, in this study both shoot and root were produced simultaneously on a single culture medium and thus, additional treatment or step was not necessary for induction of roots. In fact, this type of unique feature is characteristic in the members of Zingiberaceae family (Alpinia galanga: Singh et al., 2014; Curcuma caesia: Mohanty et al., 2010; Zingiber officinale: Jain et al., 2018; Zinger zerumbet: Faridah et al., 2011). Simultaneous production of roots along with shoots in the plants of Zingiberaceae family is probably due to the presence of ‘intrinsic’ root inducing factors in their rhizome (Agretious et al., 1996), which is influenced by the type and concentrations of plant growth regulators. Similar event has also been previously reported in H. coronarium (Mohanty et al., 2013). TDZ differs from other natural or synthetic plant growth regulators due to their ability to stimulate shoot regeneration even after a short duration of exposure to the explants (Huetteman and Preece, 1993; Khalafalla and Hattori, 1999; Murthy et al., 1998; Alatar, 2015). In this study, initial culture duration of explants on a TDZ supplemented medium was found to influence the regeneration of shoot. In contrast to three weeks, the cultures incubated for five weeks on TDZ (0.8 mg/L) medium showed higher production of shoots, but unfortunately they failed to elongate further even after transfer to plant growth regulator free or GA3 supplemented media. The result of this study was in agreement with the earlier reports where problems of shoot proliferation and poor elongation of shoots on medium containing TDZ has been reported to be influenced by the exposure duration and concentration of TDZ (Liu et al., 2003; Tang and Newton, 2005; Tao et al., 2011; Bhattacharyya et al., 2014; Alatar, 2015). In contrast to the present study, a combination of cytokinin and auxin was used for optimum production of multiple shoots in H. coronarium i.e. 2.0 mg/L BA and 0.5 mg/L NAA (Mohanty et al., 2013). Similarly a combination of three plant growth regulators (3.0 mg/L BA + 3.0 mg/L KIN + 0.2 mg/L TDZ) was reported to be effective for shoot proliferation from axillary bud of rhizome of H. coronarium (Parida et al., 2013, 2015). Verma and Bansal (2014) reported yielding of highest number of shoots (14.21) from rhizome bud on MS augmented with TDZ (1.0 mg/L) followed by sub-culture to 1.0 mg/L BA supplemented MS medium. The result of

4.3. Morphological and genetic fidelity study Upholding of genetic stability of the regenerants is one of the important requirements of any micropropagation protocol. Use of organised meristematic explants also at times failed to produce true-to-type plants (Devarumath et al., 2002) as during culture different factors including light, temperature, and plant growth regulators, especially synthetic ones may induce stress leading to genetic variation in micropropagated plants (Krishna et al., 2016). Besides, prolong culture even at optimal culture conditions may also often responsible for variations (Saha et al., 2016). Therefore, assessment of clonal fidelity of in vitro regenerated plant is essential for the success of micropropagation. In this study, no detectable morphological variations were observed among the micropropagated plants and mother plant. But plant comparison in terms of morphology has certain limitations as the morphological characters are usually influenced by environmental factors and not necessarily represent the genetic makeup of the plants (Mandal et al., 2001; Mallaya and Ravishankar, 2013). At the same time, molecular markers are independent of the influence of environmental factors, thus in this study assessment the genetic stability of the regenerated plants was carried out using molecular markers (ISSR). Although a number of micropropagation protocols have been reported for H. coronarium, only single study was on molecular marker based assessment of the regenerants (Parida et al., 2013). No difference in banding pattern of all micropropagated plants for a particular primer with respect to the mother plant was observed during the ISSR analysis, which confirmed the genetic stability of the plants produced by this protocol. The other advantages of using the ISSR markers are their costeffectiveness, simplicity, quickness, and reliability (Lakshmanan et al., 2007). Use ISSR markers for molecular analysis of micropropagated plants for authentication of their fidelity has already been documented in a number of plant species including Alpinia galanga (Baradwaj et al., 2017), Globba marantina (Parida et al., 2018), Nothapodytes nimmoniana (Prakash et al., 2016), and Tylophora indica (Sharma et al., 2014). 65

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Fig. 4. Chromatogram of coronarin D; (A) rhizome of mother plant, (B) rhizome of field established micropropagated plant.

4.4. Biochemical fidelity study

(Morikawa et al., 2002), antifungal (Kaomongkolgit et al., 2012), and antimicrobial activity (Reuk-ngam et al., 2014). Keeping all these in mind, in this study phytochemicals such as phenolics, saponins, flavonoids, and tannins were estimated and their content in the micropropagated plants was compared with that of the mother plant. Presence of all these phytochemicals was observed in both the types of plants with not much variation in their quantities. HPTLC analysis of coronarin D further established the biochemical fidelity of the micropropagated plants with that of the mother plant. To be best of knowledge, here, quantitative estimation of phytochemicals such as phenolics, saponins, flavonoids, and tannins as well as HPTLC analysis of coronarin D in the mother plant and micropropagated plants was reported for the first time.

Assessment of biochemical uniformity among the micropropagated plants and mother plant is imperative for the utility of any suggested tissue culture protocol. The medicinal properties of any plants are attributed to the secondary metabolites or phytochemicals present therein. Usually the secondary metabolites show an array of pharmacological properties and responsible for providing remedy for a number of diseases including cancer and diabetes (Wink, 2015). Phenolics, saponins, flavonoids, and tannins are some of the important plant secondary metabolites in general. At the same time, a number of labdanetype diterpenes have been isolated and identified from the rhizome of H. coronarium (Chan and Wong, 2015). Many of these compounds have antimicrobial, anti-inflammatory, and cytotoxicity activities (Suresh et al., 2010; Kiem et al., 2011; Chan and Wong, 2015; Behera et al., 2018). Hedychium coronarium rhizome derived coronarin D, a labdane diterpene, possesses anticancer (Kunnumakkara et al., 2008; Chen et al., 2017), anti-inflammatory (Kiem et al., 2011), anti-allergic

5. Conclusion In the present study, an improved and efficient plant regeneration protocol is established for H. coronarium. An initial culture of rhizome 66

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segments on MS medium augmented with 0.8 mg/L TDZ for three weeks was best for induction of shoots. Subsequent sub-culture of these explants on MS medium containing 1.0 mg/L GA3 for four weeks was optimized for in vitro plant regeneration i.e. shoot elongation with simultaneous rooting. Further, axenic stem segments derived from primary in vitro shoots were also used for up-scaling of plantlets. So, using this protocol ca. 540 plantlets were obtained starting from single explant within 14 weeks. Genetic fidelity using ISSR markers confirmed the true-to-type of the field established tissue culture raised plants. Further, HPTLC analysis of coronarin D and quantitative analysis of other phytochemicals also ascertained the biochemical fidelity of these micropropagated plants, probably for the first time in H. coronarium. In conclusion, this improved protocol has the ability to produce a large number of genetically and biochemically clonal plants within a short period of time. Thus the protocol presented herein could be useful for conservation purposes of H. coronarium and also in pharmaceutical industries for obtaining valuable secondary metabolites.

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