In vitro regeneration of a rare antidiabetic plant Epaltes divaricata L.

In vitro regeneration of a rare antidiabetic plant Epaltes divaricata L.Barathi Kannan K, Paul Agastian *

Research Department of Plant Biology and Biotechnology, Loyola College, Chennai - 600 034, India

*Corresponding authorDr. P. AgastianE-mail: [email protected] / [email protected]: +91 9444433117, Fax: +9144 28175566

Artilce History: Received 15 March 2015Revised 21 May 2015Accepted 2 June 2015

Keywords: MS mediumMultiple shootsRegenerationHardeningEpaltes divaricata L.

Manuscript Details Abstract

This study was conducted to develop an efficient protocol for in vitro direct and indirect re-generation of Epaltes divaricata L. MS (Murashige and Skoog) medium was supplemented with different growth hormones both individually and in combinations. MS medium con-taining BAP (1.0 mg/L) and TDZ (0.1 mg/L) in combination proved to be most effective in inducing maximum callus with multiple shoot formation using shoot tip from the explants producing maximum of 10.77±0.4 shootlets / explants with mean length of 4.30±0.3 cm from leaf explants. Regenerated multiple shoot induction using nodal explants supplemented BAP (1.0 mg/l) with NAA (0.1 mg/L) produced 1.86±1.05 average number of shoots / explants, with mean length of shoots 3.00±0.64 cm whereas BAP (1.0 mg/L) with KN (0.1 mg/L) pro-duced 4.16±0.36 average number of shoots with mean length of shoot 3.08±0.33 cm. The leaf explants produced good callus in the combination of growth regulator BAP (0.5 mg/L) and NAA (0.1 mg/L) 80% response and number of shoot per callus is 3.38 ± 0.21. In vitro rooting of micro propagated shoot response in MS media with IBA (0.1 mg/L) producing more roots (13.31±0.24 numbers) with root length 7.16±0.51 cm followed by NAA (0.1 mg/L) producing 8.21 ±0.26 numbers with root length 5.22 ±0.41 cm. Regenerated of Epaltes divaricata L. plant-lets were hardened in organic manure with sand showed acclimatization percentage is 73.33 % after 2 months in the field.

South Indian Journal of Biological Sciences 2015 (1) 52-59

1. Introduction

Epaltes divaricata L. (Family: Compositae), a plant used in traditional ayurvedic medicine, is found in Sri Lan-ka, India, Myanmar, Java and China. It is used to alle-viate jaundice, urethral discharges and acute dyspep-sia, and is also regarded as a diaphoretic, diuretic and a stimulating expectorant. Currently, there has been a marked rise in scientific interest in the pharmacologi-cal activities of medicinal plants, particularly in relation to folklore medicine. The plant and tissue cultures have been enabled to increase the knowledge in many areas including differentiation, cell division, cell nutrition and cell preservation but now, cells are cultivated in vitro in bulk or as clone from single cells to grow whole plants from isolated meristem, then induce callus and develop

complete plantlets by organogenesis or by embryogen-esis. The research needs are based on the elements of scientific progress and development of new techniques, which either enables more critical experiment to be un-dertaken, or rendering easy accessibility to complicated problems through experimental studies (Mantell and Smith 1983; Evans et al., 1983). Since the world population is increasing rapidly, there is an extreme pressure on the available cultivatable land to produce food and fulfill the needs. Therefore, for other uses such as production of pharmaceuticals and chemicals from plants, the available land should be used effectively. The development of micro propagation methods for a number of medicinal plant species has been already reported and needs to be adopted (Naik 1998). Plants are valuable source of a wide range of sec-

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ondary metabolites which are used as pharmaceuticals, agro-chemicals, flavors, fragrances, colors, biopesticides and food additives. Over 80% of the approximately 30,000 known natural products like camptothecin, taxol (anticancer), gymnemic acid, chirantin (antidiabetic) are of plant origin (Phillipson 1990; Balandrin and Klocke 1988; Gloribai et al., 2015). Through plant tissue culture, the totipotent characteristics of plant can be used for the in vitro regeneration of plant. The great enthusiasms of biotechnologists are seen in the potential use of cell cul-ture in the production of valuable secondary products. Plant tissue culture is a noble approach to obtain their substances in large scale. In the present scenario, it is an effective and efficientprocedure for converting less me-dicinally important plant metabolites to a valuable prod-uct using biotransformation. Many companies in India and abroad are showing interest in this direction. Tissue culture has been applied as a potent method for multi-plication and conservation of various plant species. As compared to conventional propagation, micro-propaga-tion benefits from the advantage of allowing rapid prop-agation within limited time and space. Tissue culture method has been proved to be a promising technique for conservation, and large-scale micropropagation-with rapid multiplication of several forest rare species (Balaraju et al., 2011). The selected plants are found only during win-ter seasons and start disappearing during early summer itself. Hence the plant materials are non-available for plant tissue culture during rest of the year. But till date, no report has been found for any kind of plant tissue cul-ture work of Epaltes divaricata. The present study was under taken with the view to standardize the plant tissue culture protocol for mass propagation of this important medicinal plant through in vitro culture and ensuring production of genetically identical plantlets for further field culture as well as conservation.

2. Materials and methods2.1. Collection and identificationEpaltes divaricata L. plants were collected from the banks of Oragadamlake near Chennai, TN, India. Plants were raised in Botanical garden of Loyola College. Epal-tes divaricata L. was identified and authenticated by Dr. J. Jeyajothi, Taxonomist from the Department of Plant Biology and Biotechnology, Loyola College, Chennai, India. A voucher specimen was deposited at the Depart-mental Herbarium. Healthy plants were collected from the plants raised in the Botanical Garden, Loyola Col-lege. Explants were excised in the morning in an asep-tic container. Depending upon the experiments, the explants were surface sterilized and inoculated into the appropriate medium supplemented with respective test hormones.

2.2. Surface sterilization of explantsThe surface sterilization of the plants was done by fol-lowing the method of Balaraju et al., (2011). Briefly, Apical meristem / nodal explants were collected from in vivo grown plants from the Botanical garden of Loyola

College, Chennai, India, cut as per desired size, and then washed in running tap water for 10 min. The explants were washed with detergent (Tween 20) for 5 min. and rinsed 2 – 3 times with sterile distilled water and then soaked in fungicide (Bavistin 1%) for 5 min followed by rinsing with sterile distilled water. Thereafter, the ex-plants were surface disinfected with 70 % ethanol for 30 seconds and rinsed 2 – 3 times with sterile distilled wa-ter, then treated with 0.1% aqueous mercuric chloride (HgCl2) for 3 minutes and thoroughly washed 5 times with sterile double distilled water under aseptic condi-tion. The surface sterilized explants were used for inoc-ulation on MS medium with various concentrations of hormones.

2.3. Culture medium and growth conditionsThe medium and growth conditions were followed as reported by Balaraju et al., (2011). Briefly, MS medium with 2% (w/v) sucrose was used in the present study. The medium was further amended with 0.5, 1.0, 1.5 2.0 mg/L BAP, KN and TDZ individually, BAP was also checked in combinations with 0.1 mg/L KN, 0.1 mg/L NAA and 0.1 mg/L TDZ. The pH of the medium was adjusted to 5.8 before gelling with 0.8% agar. Molten medium (10 ml) was dispensed into 50 ml test tubes (Borosil) and plugged with non-absorbent cotton plugs. The culture tubes containing the media were autoclaved at 121 °C for 15 min. The surface sterilized explants were inoculated 1-2 per test tube in the Laminar Air Flow Chamber. All the cultures were maintained at 22 ±2 °C under a 16 h photoperiod at a photosynthetic flux of 35-50 µmol / m-2

s-1), provided by cool day light fluorescent lamps.

2.4. Multiple shoot induction and elongation me-diumThe multiple shoot induction was carried out by follow-ing the procedure of Balaraju et al., (2011). Briefly, The surface sterilized shoot tips / nodal explants were inocu-lated onto the MS medium supplemented with BAP, KN (0.5, 1.0, 1.5, 2.0 mg/L) and TDZ (0.5, 1.0, 1.5, 2.0 mg/L) individually and BAP in combinations with KN (0.1 mg/L), TDZ (0.1 mg/L) and NAA (0.1 mg/L) for shoot proliferation. Subsequent sub culturing was done once in 4 weeks. After one or two sub culturing in the same concentration, callus was transferred to the medium with reduced concentration of the above said hormones for enhancing the proliferation of shoots. Finally shoots were sub cultured in half-strength and full-strength MS medium with 2% (w/v) sucrose containing BAP or KN individually for shoot elongation.

2.5. Rooting mediumFor in vitro root induction, the shoots (3-4 cm long) with 4-5 leaves were transferred to half-strength and full-strength MS medium with 2% (w/v) sucrose and 3% (w/v) bacto-agar. The medium was further supplement-ed with IBA and NAA (0.05, 0.1 and 0.2 mg/L) individu-ally. After 4 weeks, the rooted shoots were transferred to the above medium devoid of growth regulators for root elongation.

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2.6. Acclimatization of plantletsFor easy acclimatization, the test tubes with plantlets (fully expanded leaflets with a height of 5-6 cm) were kept open for a week after removing the cotton plugs in the culture room itself. Then the plantlets were removed from the culture medium and washed with distilled wa-ter and transferred to plastic cups containing autoclaved organic manure, garden soil and sand in ratio 1:1, 1:2 and 1:3. The potted plantlets were maintained inside the plant tissue culture room at 22±2 °C under a 16 h pho-toperiod provided by cool day light fluorescent lamps. The relative humidity was reduced gradually and after 30 days, the plantlets were transplanted to earthenware pots (8 cm diameter) containing garden soil and maintained in the green house.

2.7. Statistical analysis Statistical parameters like percentage, mean, standard error were estimated by using the Microsoft Office Excel.

3. Results 3.1. Shoot proliferation using shoot tip and nodal explantsShoot tip explants of E. divaricata L produced multi-ple shoots on MS medium supplemented with different plant growth regulators individually and in combina-tions. Shoot tip explants took nearly 35 days to initiate shoots. The type and the concentration of cytokinin in-fluenced the average number of shoots produced per ex-plants as well as mean length of the shoots. Of the three cytokinins tested BAP with TDZ responded more effec-tive in inducing bud break and multiple shoot formation from the explants producing maximum of 10.77±0.4 shootlets / explants (Table 1). BAP+NAA moderately induced average number of shoots / explants as well as mean length of the shoots. In case of BAP with KN grad-ually reduced the number of shoots / explants as well as mean length of shoots. With reference to the multiple shoot induction, BAP with NAA (1.00 +0.1 mg/L) sig-nificantly increased the production of average number of shoots per nodal explants (4.16±0.36) also increase in mean length of shoots (3.08 cm) (Table 2). BAP+KN at (1.00+0.1) mg/L also found to increase the responses on number of shoots with mean length of shoots. Stunt-ed growth and less number of shoots were observed at higher concentrations of the tested hormones. Generally an increasing BAP concentrations with constant KN or NAA concentrations responded decrease in number of shoots / explants as well mean length of shoots (Fig.1 and 2).

3.2. Shoot elongationShoot initiation and growth in the two sets of experi-ments showed a similar pattern. The number of shoots produced per explants was higher on the medium con-taining either 1.0 or 1.5 mg/L of BAP in combination with 0.1 mg/L TDZ. Basal callus was observed when the explants were kept for a prolonged period on the medi-um. In our present investigation, BAP in combination TDZ played an important role as a plant growth regu-

lator and had a significant effect on the average number of shoots initiated per shoot tip explants. The percentage of shoot formation and the number of shoots per node was increased during the first two culture passages; af-terwards a gradual decline in the rate of multiplication was observed (Table 1 and Table 2). Effect of growth regulators on callus induction and shoot induction was tested with 2,4-D, NAA and BAP. Among which, 2, 4-D and NAA produced callus with light yellow colour and semi friable nature (0.20 – 1.50 mg/L). At lowest concen-trations, friable calli were produced. Increasing concen-trations of auxins reduced the callus formation (Fig.3). BAP with NAA (0.50+0.1 mg/L) showed 80% callusing followed by shoot induction after 30 days of incubation. 2,4-D or NAA alone does not responded for shoot in-duction (Table-3).

3.3. In vitro root inductionRooting occurred from regenerated shoots cultured on medium containing auxins. Excised shoots failed to pro-duce roots when cultured on media containing half or full strength MS medium without any growth regulator even after 40 d of culture. However, before root forma-tion, basal callus was observed from the explants in re-sponse to the presence of auxin in the medium for long duration. After 25 d of culture, basal callus was excised with a scalpel and the shootlets were sub cultured into a fresh medium containing the same concentrations of auxin. There was no significant difference in the mor-phology of average number of roots produced per shoot in the treatment containing IBA or NAA. Most of the shoots produced only one or two roots in all the con-centrations of auxins tested. The best rooting in terms of rooting percentage (13.31±0.24) and root length (7.16±0.51) was obtained using IBA as compared with NAA. A maximum frequency of root formation (55% with 5.16 cm length) was achieved on MS medium sup-plemented with IBA (0.1 mg/L) after 25 d (Table 4).

3.4. Hardening and acclimatizationAfter 30-40 d of root induction, regenerated plantlets with fully expanded leaflets and well developed roots were washed several times with water to remove all ad-hering culture medium. The plantlets were hardened inside an environmentally controlled room for 25-30 d. Three planting substrates used were; organic manure and sand, vermicomposting with sand and garden oil with sand. Of the three different types of substrates exam-ined, survival rate of the plantlets was highest (73.33%) in organic manure with sand (Table 5). After 45 d, when the plant shows sign of acclimatization with new shoot growth after a month, they were transferred to green house and exposed to natural air condition (Fig. 2).

4. DiscussionMass propagation of plant species through in vitro cul-tures is one of the best and most successful examples of commercial applications of plant tissue culture technol-ogy. Recently there has been much progress in this tech-nology for conservation of genetic resources and clonal

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A

E

C

B

D

Fig.1. In vitro studies of Epaltes divaricata L. using shoot tip culture

A & B) Regeneration of shoot, C) Elongation of shoots D. Plantlet showing shooting and rooting, E) Plantlet after 75 d.

Fig.2. In vitro studies of Epaltes divaricata L using nodal explants

A1

D2 C2

B

A1, A2. Nodal explants induce shooting, B. Sub-culture of shoots induced by explants. C1, C2. Induction of multiple shoots, D1, D2. Hardening of in vitro raised plantlets

C1

55

A

C

B

D D

Fig.3. Shows the in vitro studies of Epaltes divaricata L. using leaf explants

A. Leaf explants induce callus, B. Sub-culture of callus, C. 30 – days old callus, D. induction of shoot from callus.

Table 1. Effect of growth regulators in combination on multiple shoot induction using shoot tip explants of Epaltes divaricata.Hormone Concentration (mg/L)

% of explants responded Average number Mean length

BAP+ KN0.50+0.11.00+0.11.50+0.12.00+0.1BAP + NAA0.50+0.11.00+0.11.50+0.12.00+0.1BAP + TDZ00.50+0.11.00+0.11.50+0.12.00+0.1

67.159.458.457.3

68.174.475.466.3

67.184.483.476.3

1.52±1.11.77±0.41.48±0.51.36±0.4

2.54±1.13.77±0.42.52±0.52.36±0.4

1.84±1.110.77±0.48.52±0.55.36±0.4

1.52±0.22.90±0.31.90±0.51.78±0.4

2.30±0.31.97±0.51.20±0.4

2.73±0.24.30±0.32.97±0.52.20±0.4

The values are (mean ± SE) collected after 40 days from two experiments each with 10 replicates per treatment.

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Table 2. Effect of growth regulators in combination on multiple shoot induction using nodal explants of Epaltes divaricata L. Hormone Concentration (mg/L)

% of explants responded Average number of shoots/ explants*

Mean length of shoots (mm)*

BAP + KN 0.50+0.11.00+0.11.50+0.12.00+0.12.50+0.1BAP+NAA0.50+0.11.00+0.11.50+0.12.00+0.12.50+0.1

66.658.3757560

66.675

66.663.355

1.41±1.141.86±1.051.41±0.691.34±0.230.58±0.65

2.16±0.524.16±0.363.83±0.322.25±0.771.58±0.65

1.11±0.453.00±0.642.00±0.661.66±0.181.16±0.20

1.66±0.373.08±0.332.61±0.202.41±0.411.16±0.20

The values are (mean ± SE) collected after 40 days from two experiments each with 10 replicates per treatment.

Table 3. Effect of growth regulators either individually or in combination on callus induction using leaf explants of Epaltes divaricata L. Growth Regulators (mg/L) % of response Nature of the callus No. of Shoots / callus2,4-D0.20.511.5NAA0.20.511.5BAP+NAA0.20+0.10.50+0.11.00+0.11.50+0.1

6070906060

70605070

806060

CL YL YL YL GL Y

L YL YL YL G

L GL GL G

TFFS FS FS F

S FS FS FS F

S FS FS F

--------

--------

2.53±0.133.38±0.212.03±0.121.91±0.10

The values are (mean ±SE) collected after 35 days from experiments each with 12 replicates per treatment. C – colour; T – Texture; L Y – callus with Light Yellow,L G – callus with Light Green; S F - callus with Semi Fria-ble; F callus friable. – No response.

Table 4. Effect of IBA and NAA and strength of MS medium on in vitro rooting of micropropagated shoots of Epates divaricata L.Hormone Used mg/L Medium used Rooting response (%) No. of roots/shoot * Root length (cm)00IBA 0.05IBA 0.1IBA 0.2IBA 0.05IBA 0.1 IBA 0.2

NAA 0.05NAA 0.1NAA 0.2NAA 0.05NAA 0.1NAA 0.2

½ MSMS

½ MS½ MS½ MS MS MS MS

½ MS½ MS½ MS MS MS MS

-----3545--455535---

3045504535

------

1.55±0.112.43±0.04

---4.16±0.22

13.31±0.243.81±0.16

---

2.13±0.411.67±0.234.52±0.138.21±0.262.62±0.33

------

3.01±0.122.53±0.02

---3.10±0.147.16±0.514.11±0.23

----

2.55±0.222.22±0.144.62±0.135.22±0.413.12±0.24

The values are (mean ± SE) collected after 40 days from two experiments each with 10 replicates per treatment. --- indicates no response.

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improvement (Barz et al., 1977). Rapid shoot regenera-tion has been achieved with a wide range of species with initial explants being taken from normal aerial shoots of field grown herbaceous medicinal plant species (Rai 2002; Hall and Camper 2002). The germination was poor in freshly harvested seeds as well as seasonal availability of plants are found to be difficult for in vitro studies in the test plant Epal-tes divaricata L, which is found to be highly valuable in ayurveda and siddha for developing antidiabetic drugs. Therefore the test plant was chosen for developing the protocol using tissue culture methods using shoot tip explants and nodal explants for continuous availability. The use of BAP over other cytokinins,is well reported in woody tree species including Swartzia mabegascar-iensies, Sterculia urens, Dalbergia sissoo and Sesbania rostrapa (Berger and schaffner 1995; Purohit and Dave 1996; Pradhan et al.,1998; Jha et al., 2004) the results cor-roborate with earlier findings of several workers,where the addition of low level of auxin with cytokinin promot-ed shoot proliferation in Lagerstromia parviflora (Tiwari et al., 2002). An inhibitory effect of higher concentration of BAP on shoot formation has also been reported in P. marsupium (Anis et al., 2005). BAP was found to be more effective than KN for bud break and shoot growth.MS medium with cytokinin and auxin was selected as optimum medium and used for further multiplication. Multiple shoot formation (3.8) was observed in 68 % of basal nodes cultured on medium with optimum concentration of 4.43 mM BA and0.54 mM NAA after 8 weeks. Terminal nodes were not suitable for inducing multiple shoots. Irrespective of the orientation vertical/horizontal), all shoot tip explants responded with a sin-gle shoot in all the combinations of plant growth regula-tor (Sudha et al., 1998). The role of auxins incorporated in the medium in combination with cytokinins for shoot multiplication has been reported in number of cases and higher concentrations of auxins (2.5 and 5.0 µM) sup-pressed the shoot regeneration but induced basal callus. IBA was more effective for rooting when compared with other auxins as reported previously in Pterocarpus mar-supium and other plants species. The incorporation of an auxin in the medium generally promotes rooting, but in the present study auxin alone was found to be effective for rooting. Addition of activated charcoal with IBA for root initiation has been demonstrated in Hagenia abys-sinica (Feyissa et al., 2005), but in our study root induc-tion occurred without the addition of activated charcoal. Lychnophora pinaster, known as arnica, is a medicinal plant of the Cerrado ecosystem in Brazil. The best me-dium for germination of arnica embryos and plantlet growth was a quarter strength semisolid Murashige and Skoog medium (MS/4) containing 0.75% (w/v) sucrose. For shoot induction, the best results were obtained on MS/4 with 2.76 mM of benzylaminopurine. Maximum shoot elongation before rooting occurred in the presence of 8.67 mM of gibberellic acid for 19 d. In vitro plantlets were successfully rooted in the presence of 10.7 mM of naphthalene acetic acid for 15 d. The rooted plantlets were acclimatized in a greenhouse for 20 d, the survival

rate was 100% when planted in a soil from the area of occurrence of the species, whereas 0 % survived when planted in Plant max. Similarly a Asteraceae member Eclipta alba (L.) Hassk, an important medicinal plant, was standardized for reproducible protocol for mass propagation of by culturing shoot tips and nodal seg-ments taken from in vitro raised plants. Maximum shoot proliferation occurred when the explants were cultured on Murashige and Skoog (MS) medium supplemented with 1 mg/ L benzylaminopurine (BAP). The shoot buds formed were further multiplied and maintained on me-dium containing BAP (0.5 mg/ L) and gibberellic acid (0.5 mg/L). Rooting was best on MS medium supple-mented with 1mg/ L indole-3-butyric acid. Dong et al. (2003) reported that explants derived from adventitious buds, rhizomes, stems, and leaves of a medicinal plant, Polygonatum cyrtonema, were studied for plantlet re-generation, and only adventitious bud explants were able to be regenerated into plantlets. Regeneration was also accompanied by the formation of rhizome-like tissue, the medicinal part of the plant.The optimum hormone combination for plantlet regeneration was 4.44 mM ben-zyladenine plus 2.26 mM 2,4- dichlorophenoxyacetic acid, at which new adventitious buds were obtained from 96.6 % of the adventitious bud explants,with an av-erage of 5.2 buds per explant. The best medium for root induction was half-strength Murashige and Skoog medi-um with 4.57 mM α-naphthaleneacetic acid, as 92 % of regenerated buds were rooted. Jiang et al. (2012) stated that Dysosma versipel-lis (Hance) M. Cheng, when the explants of rhizome buds were cultured on Murashige and Skoog’s (MS) me-dium with 6-benzyladenine (BA) (1.0 mg/ L), gibberellic acid (GA3) (0.5 mg/ L) and zeatin (0.5 mg/ L), multiple buds were regenerated directly onthe explants without callusing within 6 weeks. Callus wasinduced from the leaf segment cultures on MS basal medium supplement-ed with 2,4-dichlorophenoxyaceticacid (2,4-D) (0.5 mg/ L) and BA (0.2 mg/ L) within 4 weeks. The adventitious buds were differentiated when thecalli were subcul-tured on MS medium supplemented with BA (1.0 mg/ L) and thidiazuron (TDZ) (0.2 mg/ L) within 6 weeks. The adventitious buds obtained from callusand the rhi-zome-buds rooted with a frequency of 100% on half strength MS medium fortified with indole-3-butyricacid (IBA) 0.5 mg/ L and activated charcoal (AC) 0.5 g/ L for 4 weeks.

5. ConclusionIn vitro propagation can become an important alterna-tive to conventional propagation and breeding proce-dures for E. divaricata L which is both an industrially and medicinally important herb. The above studies showed the potential for use in tissue culture methods to get a rapid mass propagation of E. divaricata L. The explants - and plant growth regulators levels have significant im-pact on accelerated micro-propagation of E. divaricata L to regenerate, genetically true to the type propagates. In most cases, growth regulators like BAP and NAA were found to be essential for growth and multiple shoot for-

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mation. Tissue culture protocols have been developed for this test plant which is overexploited in pharmaceu-tical industries and need conservation.

Conflict of interest statement We declare that we have no conflict of interest.

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