PLANT TISSUE CULTURE

Cardenolide estimation in callus-mediated regenerantsof Digitalis lamarckii Ivanina (dwarf foxglove)

Buhara Yücesan & Frieder Müller-Uri & Wolfgang Kreis &

Ekrem Gürel

Received: 25 February 2013 /Accepted: 29 July 2013 / Editor: J. Forster# The Society for In Vitro Biology 2013

Abstract Digitalis cardenolides can regulate heart rhythmsand are effective agents in cancer chemotherapy, in particular,for treating prostate and breast cancer. In this study, an opti-mized and efficient plant tissue culture protocol was establishedusing callus cultures of Digitalis lamarckii Ivanina, commonlyknown as dwarf foxglove. Lamina explants developed calluswhen cultured on Linsmaier and Skoog (LS) medium contain-ing different concentrations of 6-benzyladenine (BA; 4.4, 13.3,or 22.2 μM) and α-naphthalene acetic acid (NAA; 2.7, 5.4, or10.8 μM). The highest incidence of callus formation (100%)was achieved on LS medium containing 13.3 μM BA and10.8 μMNAA. Indirect shoot regeneration was achieved whenthe callus explants were cultured on LS medium supplementedwith varying concentrations of BA (0.4, 1.1, or 2.2 μM) and/orgibberellic acid (0.7 or 1.4μM) for 8 wk. Following the rootingof shoots on LS medium supplemented with either indole-3-acetic acid (ranging from 1.4 to 5.7 μM) or NAA (1.3 to5.2μM), lamina and petiole tissues of the 4-mo-old regeneratedplants were compared for their cardenolide contents. Laminaextracts showed nearly three times higher cardenolide accumu-lation than petiole extracts. Of the cardenolides analyzed byreverse-phase high-performance liquid chromatography, neo-odorobioside G and glucogitoroside were abundant in laminaextracts (170.3 and 143.9 mg/kg dry weight, respectively). Theregeneration protocol described in this study can be used for thein vitro production of certain cardenolides from D. lamarckii .

Keywords Digitalis lamarckii . Cardenolides . Calluscultures . In vitro regeneration

Introduction

After disintegration of the family Scrophulariaceae, the genusDigitalis was accepted into the Plantaginaceae family(Olmstead et al. 2001; Bräuchler et al. 2004).Digitalis lamarckiiIvanina, commonly known as dwarf foxglove, is classified as aleast-concern plant species endemic to the rangelands of Turkeyin the Black Sea region (Davis 1978; Ekim et al. 2000).Members of the genus Digitalis are medicinally and economi-cally important as they contain cardenolides (cardiac glyco-sides), which can increase the force of systolic contractionsand regulate heart rhythms (Baytop 1999). In addition to thecardiotonic effects, they are effective anticancer agents, particu-larly targeting prostate and breast cancer cells (Lopez-Lazaro2007; Newman et al. 2008). Furthermore, Baytop (1999) report-ed that D. lamarckii was one of the most poisonous foxgloves,with a lethal dose of ∼0.025 g/kg (for cats) from dried leaves,and Benli et al. (2009) reported antibactericidal effects of leafand flower extracts of D. lamarckii .

The European cardiovascular drug market was estimated tobe around $36 billion in 2012 (Clemente et al. 2011), indicatinga significant interest in commercially important cardenolides.Plant tissue culture techniques have been employed for theconservation of many medicinal plants, for plant genetic andbiochemical improvement studies, and for molecular engineer-ing for secondary metabolite production (Collin 2001; Butiuc-Keul et al. 2012). Therefore, protocols for efficient plant tissueculture and cardenolide profiling of in vitro regenerated planttissues could be useful for propagating the genus Digitalis andmake an important contribution toward large-scale productionof cardenolides.

Plant regeneration via somatic embryogenesis and organ-ogenesis has been reported for several Digitalis species, in-cluding Digitalis lanata , Digitalis minor, Digitalis purpurea ,Digitalis thapsi , Digitalis trojana , and Digitalis obscura(Clemente et al. 2011). In this study, we aimed (1) to establishcallus cultures of D. lamarckii employing alternative tissue

B. Yücesan (*) : E. GürelDepartment of Biology, Abant Izzet Baysal University, 14280 Bolu,Turkeye-mail: ibuhara@yahoo.com

F. Müller-Uri :W. KreisDepartment of Biology, Friedrich-Alexander-University,Staudtstraße 5, 91058 Erlangen, Germany

In Vitro Cell.Dev.Biol.—PlantDOI 10.1007/s11627-013-9549-1

culture protocols to the earlier investigations in severalDigitalis species and (2) to estimate and compare the amountsof major cardenolides in the lamina and petiole tissues of basalleaves excised from 4-mo-old plantlets developed underin vitro conditions.

Materials and Methods

Preparation of plant material and germination. Seeds of D.lamarckii Ivanina were collected in September 2010 fromnatural populations growing in Bolu, southwest of the BlackSea region, Turkey (altitude 1,510 m, N 40°37.709′ E032°26.265′). Voucher specimens of all collected plants(voucher no. EKER-1973 ) were deposited in the Abant IzzetBaysal University Herbarium. Seeds were surface sterilized bywashing in 50mL of 20% (v/v) commercial bleach (Domestos®)with a few drops of Tween-20® in a 250-mL beaker and stirringat 250 rpm on a magnetic stirrer plate for 10 min, followed byrinsing with sterile distilled water several times. For germination,an average of 20–25 seeds were aseptically cultured in Petridishes (90×15 mm) containing 30 mL of Linsmaier and Skoog(LS) medium (Linsmaier and Skoog 1965) supplemented with3% (w /v) sucrose. The medium was solidified with 0.8% (w/v)agar and autoclaved at 121°C and 1.06 kg/cm2 pressure for15 min after adjusting the pH to 5.8 with 0.1 N HCl or 0.1 NKOH. The cultures (except rooting stage) were grown in dispos-able Petri dishes (90×15 mm) and kept at 23±1°C under a 16-hlight photoperiod provided by cool-white fluorescent light withan irradiance of ∼35 μmol−2 s−1 at a relative humidity of 60%.

Callus induction, adventitious shoot regeneration, androoting. Leaf lamina explants (ca. 35–40 mm2) were isolatedfrom 4-wk-old germinated seedlings and transferred to differ-ent callus induction media supplemented with LS salts, vari-ous concentrations and combinations of 6-benzyladenine(BA; 4.4, 13.2, or 22.2 μM), and α-naphthalene acetic acid(NAA; 2.7, 5.4, or 10.8 μM) for 6 wk, using five laminaexplants per treatment with three repeats (a total of 15 explantsper treatment). Green and nodular callus which developed onleaf lamina explants was transferred to LS medium containinglower concentrations of BA (0.4, 1.1, or 2.2 μM) and/orgibberellic acid (GA3; 1.4 or 2.8 μM) for shoot regenerationfor 8 wk, using four callus clumps (ca. 150–200 mg) pertreatment with three repeats (a total of 12 callus clumps pertreatment). Regenerated shoots were rooted in Magenta® ves-sels (77×77×97 mm) containing 25 mL of LS mediumsupplemented with various concentrations of indole-3-aceticacid (IAA) (1.4, 2.8, or 5.7 μM) or NAA (1.3, 2.6, or 5.2 μM)and cultured for 8 wk. Each rooting treatment used ten shoots,and the experiment was repeated three times (a total of 30explants per treatment). The rooted shoots were transferred topots containing an autoclaved mixture of soil, manure, moss,

and sand at a ratio of 1:2:2:1 and were kept under greenhouseconditions at 20–22°C with low humidity (25–35%). After therooting stage, some of the randomly selected plantlets,irrespective of medium composition, were used for cardenolideanalysis.

Quantitative analysis of cardenolides. Cardenolides wereextracted as described previously by Wiegrebe and Wichtl(1993) with slight modifications. In this study, leaf materials(leaf and petiole) were dried at 40°C in an oven for 2 d. Driedmaterials were ground finely using a mortar and pestle. Forextraction, 50 mg powdered dry material was transferred to amicrocentrifuge tube containing 1 mL of 70% (v /v ) methanol.After 30min of treatment in an ultrasonic bath at 65–70°C, theextract was rapidly cooled on ice for 3 min and then centri-fuged for 10 min at 13,000 rpm in a microcentrifuge at roomtemperature. The supernatant was thoroughly mixed with0.25 mL of 15% (w /v ) lead acetate solution and centrifugedagain. After removing the lead acetate residue, 0.5 mL of 4%(w /v ) monosodium phosphate was added and centrifuged.The supernatant was transferred to 2-mL centrifuge tubes,diluted to a final volume of 2 mL with water, and centrifugedat room temperature (RT) for 8 min at 13,000 rpm. Thesupernatant was thoroughly mixed with 0.5 mL chloroform/isopropanol (3:2) and centrifuged for 5 min as before. Thelower phase was transferred to 2-mL microcentrifuge tubes asa first extraction. The remaining methanolic solution was usedfor the second extraction by adding chloroform/isopropanolmixture and centrifuged at RT for 5 min at 13,000 rpm. Bothextractions were combined prior to evaporation under an air-flow chamber for 3 h and then dissolved in 500 μL methanol(HPLC grade). For the qualitative analysis of the compoundsprior to HPLC, thin-layer chromatography (TLC) wasperformed on TLC plates (10×20 cm silica gel 60 W, Merck,Darmstadt, Germany) using 50 μL of each extract. The TLCplates were developed in a glass chamber containing mobilephase as follows chloroform/methanol/water (80:18:2). After10–25 min, the plates were removed from the chamber andsprayed with Jensen–Kny's reagent to identify the cardenolidegroups depending on coloration of the bands at 120°C for10 min (Jork et al. 1990). The spots appeared on the chromato-gram at 366 nm UV light as yellow, green, or blue bandsreferred to as A-, B-, or C-type cardenolides, respectively.For the determination of pregnane structures, the mixture ofreference substances, including lanatosides A, B, and C, wastreated with or without acid hydrolysis on TLC plates.

HPLC determination of the cardenolides. A published proto-col (Wiegrebe and Wichtl 1993) was modified for reverse-phase (RP)-HPLC determination and was carried out using aflow rate of 1.2 mL/min achieved using a binary pump solventdelivery system, a dual λ absorbance UV detector operating at220 and 350 nm, and an autosampler (Waters Autosampler

YÜCESAN ETAL.

717 Plus) injecting 20 μL of each sample. For the column, aReproSil-Pur® C18 AQ 5 μm, 250×4 mm (Dr. Maisch,GmbH, Ammerbuch, Germany), for RP-HPLC was used.Cardenolides were eluted with acetonitrile (A) and water (B)gradients as follows: 0–20 min 20% (A), 80% (B); 20–27 min32% (A), 68% (B); 27–35 min 58% (A), 42% (B); 35–40 min60% (A), 40% (B); 40–60 min 0% (A), 100% (B); and 60–65 min 20% (A), 80% (B). HPLC calibration was performedusing different internal standard (IS; β-methyl-digitoxin) con-centrations (ranging from 1 to 100 ppm dissolved in HPLC-grade methanol). Cardenolides as reference substances(glucogitoroside, strospeside, neo-digitalinum verum, neo-glucodigifucoside, neo-odorobioside G, purpurea glycosideB, gluco-evatromonode, digitoxin, lanatoside C, and digoxin)were obtained from the FAU, Pharmaceutical BiologyDepartment, Erlangen, Germany.

Statistical analysis. Data obtained from tissue culture exper-iments were statistically analyzed using SPSS, version 17.0(SPSS Inc., Chicago, IL). Analysis of variance (ANOVA) was

used to calculate statistical significance, and means ± standarderror (SE) differing significantly were determined usingDuncan's multiple range test at P <0.05 level.

Results

Callus formation and regeneration. Green and nodular calli,obtained from leaf lamina explants cultured for 8 wk on LSmedium containing various concentrations of BA (ranging from4.4 to 22.2 μM) and NAA (from 2.7 to 10.8 μM; Table 1;Fig. 1A, B), were transferred to LS medium containing lowerconcentrations of BA (0.4, 1.1, or 2.2 μM) and/or GA3 (0.7 or1.4 μM) for shoot regeneration. A combination of 13.2 μMBAand 10.8 μM NAA induced the highest incidence (100%) ofcallus formation. Callus development was significantly lowerwhen 4.4 μM BA was combined with any concentration ofNAA (Table 1). For example, 4.4 μM BA combined withNAA produced the lowest incidence of explants developingcallus (65%). No callus was observed when explants were

Figure 1. Stages of shootregeneration from callus obtainedfrom leaf explants ofD. lamarckiicultured on medium containing13.2 μM BA and 10.8 μM NAA:scanning electron micrograph (a)and light micrograph (b) of callusafter 6 wk of cultivation; shootdevelopment from calluscultivated on 1.1 μM BA alone(c) or in combination with1.4 μM GA3 (d), rootedregenerated plants used forcardenolide extraction (e), andregenerated plants being hardenedunder growth chamber conditions(f).

IN VITRO D. LAMARCKII AND CARDENOLIDES

cultured on media without plant growth regulators (PGR), andthese explants eventually died (data not shown).

After a further 8 wk of culture, the green nodular callusdeveloped dark green shoots over the entire surface of tissue,producing a mean of 6.2, 4.1, and 2.8 shoots per callusexplant, when the culture medium, respectively, contained0.4, 1.1, and 2.2 μM BA alone (Table 2; Fig. 1C). A similarpattern was observed when these concentrations of BA werecombined with 0.7 μM GA3 (Table 2). Using either 0.7 or1.4 μM GA3 alone was not effective for enhancing shootinduction. However, an increased shoot production wasobtained when 1.4 μM GA3 was combined with higher con-centrations (1.1 and 2.2 μM) of BA. As seen in Table 2,combinations of 1.1 or 2.2 μM BA with 0.7 μM GA3 pro-duced means of 4.3 and 2.5 shoots per callus explant, respec-tively, while the same combinations of BAwith 1.4 μM GA3

produced means of 6.6 and 6.0 shoots per callus explant,respectively (see Fig. 1D).

For rooting the regenerated shoots, two types of auxin, IAAand NAA, were tested at three different concentrations (1.4,2.8, and 5.6 μM IAA; 1.3, 2.6, and 5.2 μMNAA; Table 3). Itwas clear that IAAwas significantly more effective than NAAfor root induction; IAA induced means of 4.2, 4.4, and 3.2roots per shoot, respectively, compared to means of 2.3, 2.1,and 2.5 roots per shoot induced by the respective three con-centrations of NAA (Table 3). Mean root length was alsogreater when culture medium contained IAA (8.6, 7.8, and7.1 cm, respectively). On the other hand, mean root lengthdecreased from 8.6 to 7.1 cm when the IAA concentrationincreased from 1.4 to 5.7 μM. About 95% of the regeneratedplantlets generated from all treatments were successfully ac-climatized in a mixture of soil, manure, moss, and sand at aratio of 1:2:2:1 (1:1; v /v ) under nonaxenic conditions in thegreenhouse (Fig. 1F).

Cardenolide estimation in regenerated plants. Cardenolideextraction was successfully achieved with respect to the recov-ery rate of the reference substances. Regression (R2) was de-termined as 0.97, and method recovery with spike method (ISadded to plant material) was calculated to be 82% (n =10,100 mg/L IS; Table 4). Lamina and petiole tissues from 4-mo-old regenerated plants rooted in Magenta vessels (Fig. 1E)were used for cardenolide extraction. Overall cardenolide ac-cumulation was higher (nearly 3-fold) in lamina compared topetiole tissues (Table 4). Of the cardenolides analyzed, bothneo-odorobioside G and glucogitoroside were detected at muchhigher concentrations compared to strospeside, neo-digitalinumverum, neo-glucodigifucoside, and glucoevatromonosidein both lamina (170.3 and 143.9 mg/kg dry weight [DW], re-spectively) and petiole (56.4 and 53.4 mg/kg DW, respectively).

Table 1. Incidence (%) of leaf lamina explants developing green nodularcallus following 6 wk culture on LS medium supplemented with variousconcentrations of BA and NAA

PGRs (μM) Explants developing callus (%)x

Control 0

4.4 BA+2.7 NAA 83

13.2 BA+2.7 NAA 91

22.2 BA+2.7 NAA 95

4.4 BA+5.4 NAA 65

13.2 BA+5.4 NAA 75

22.2 BA+5.4 NAA 82

4.4 BA+10.8 NAA 79

13.2 BA+10.8 NAA 100

22.2 BA+10.8 NAA 66

x A total of 15 explants were used per treatment

Table 2. Shoot regeneration from callus of D. lamarckii cultured on LSmedium containing BA alone or in combination with GA3 after culturefor 8 wk

PGRs (μM) Frequency (%) ofcallus developing shootsx

No. of shoots percallus (mean ± SE)

Control 0 0

0.4 BA 83 6.2±2.1ab

1.1 BA 83 4.1±1.0b

2.2 BA 50 2.8±0.2c

0.7 GA3 41 2.3±0.1c

0.7 GA3+0.4 BA 91 6.1±2.0ab

0.7 GA3+1.1 BA 58 4.3±1.1b

0.7 GA3+2.2 BA 80 2.5±0.9bc

1.4 GA3 50 3.1±0.9bc

1.4 GA3+0.4 BA 75 5.7±1.5ab

1.4 GA3+1.1 BA 91 6.6±1.6ab

1.4 GA3+2.2 BA 83 6.0±1.5ab

Means (±SE) with the same letter are not significantly different at P=0.05xA total of 12 callus clumps were used per treatment

Table 3. Effects of IAA and NAA on rooting of the regenerated shootsof D. lamarckii after 8 wk of culture

PGRs (μM) Shoots withrootsx (%)

No. of roots/shoot(mean±SE)

Root length(mean±SE)

Control 66 2.1±0.4c 4.0±0.5d

1.4 IAA 85 4.2±1.1a 8.6±0.9a

2.8 IAA 100 4.4±1.0a 7.8±0.6ab

5.7 IAA 95 3.2±0.6ab 7.1±0.4b

1.3 NAA 66 2.3±0.4c 5.1±0.6c

2.6 NAA 75 2.1±0.1c 5.0±0.6c

5.2 NAA 60 2.5±0.2bc 4.7±0.4cd

Means (±SE) with the same letter within the same column are notsignificantly different at P= 0.05xAverage basal leaf length was 12–14 cm

YÜCESAN ETAL.

In contrast to the major compounds, purpurea glycoside B,digoxin, lanatoside C, and digitoxin were not detected in thesein vitro-derived tissues.

Discussion

In vitro culture of medicinal plants makes it possible tocultivate aerial parts (shoots) for the production of targetedmetabolites through multiplication of elite clones and long-term cultivation of plants that, under natural growth condi-tions, would otherwise die at the end of the vegetation period.In this study,D. lamarckii is shown to be a suitable species forrapid in vitro regeneration, employing various tissue culturetechniques for the production of plantlets with basal leaves inan average length of 12–14 cm within 20–22 wk compared tothe traditional plant breeding techniques. The callus tissuewith organogenic potential, as described in this study, can bea useful tool for shoot regeneration. Callus tissues have beenused mainly for somatic embryogenesis and/or indirect shootorganogenesis in several Digitalis species (Reinbothe et al.1990; Fatima et al. 2009; Corduk and Aki 2010; Gurel et al.2011; Verma et al. 2012). Although Verma et al. (2011a, b)reported in vitro regeneration studies based on somatic em-bryogenesis and direct shoot organogenesis from cotyledon-ary leaves of D. lamarckii , methods for propagation and sec-ondary metabolite estimation remained a major challenge. Inthis study, an efficient and straightforward tissue culture proto-col forD. lamarckii was established from lamina explants, withthe incidence of callus formation ranging between 65% and100%, depending onBA andNAA concentrations (Table 1). Ofthe callus induction media tested, almost all combinations ofBA and NAA were found to be effective, but control groups

without PGR produced no callus. Synergistic effects of BA andNAA at various concentrations have been demonstrated previ-ously in some Digitalis species (Perez-Bermudez et al. 1983;Cacho et al. 1991; Corduk and Aki 2010; Verma et al. 2012).BA concentrations higher than 22.2 μM are not recommendedfor the callus production as such high BA concentrations havebeen implicated in chromosome number changes (Bairu et al.2011). In this study, callus developed from lamina explants andproduced shoots after 8 wk of incubation when transferred toLS medium supplemented with various concentrations of GA3

and/or BA (Table 2). The mean number of shoots produced percallus explant on LS medium containing 0.4 μM BA alone orBA in combination with GA3 (0.7 or 1.4 μM) was more than6.0. Therefore, our study shows that callus production and itssubsequent prolonged subcultivation might provide a new ap-proach for a continuous plant production without depletingnatural populations. In this respect, we have been able tomaintain 2-yr-old callus clumps with organogenic potential,which are ready for plantlet formation once transferred intoshoot regeneration media (data not shown).

There have been few tissue culture studies in whichgibberellic acid was tested inDigitalis spp. To our knowledge,there has been only one report on effects of GA3 on morpho-genesis inD. obscura (a Spanish endemic foxglove), in whichgibberellic acid alone did not induce morphogenesis, butmodified responses promoted by auxins and/or cytokinins(Gavidia et al. 1993). Our results demonstrated that GA3 alone(0.7 or 1.4 μM) was effective in promoting 2.3 or 3.1 shootsper callus explant, while nearly a 3-fold higher response (6.6shoots per callus with a regeneration frequency of 91%) wasobtained in the presence of BA (1.4μMGA3 plus 1.1μMBA;Table 2).

Following the shoot regeneration stage, individual shootswere transferred to Magenta vessels containing LS mediumsupplemented with or without auxin for rooting. Of the testedauxins, IAAwas found to be most effective over 8 wk whenused at 1.4 or 2.8 μM, producing 4.2 and 4.4 roots per shoot,respectively (Table 3). This finding was also consistent withthe reports of D. davisiana (Gurel et al. 2011) and D. trojana(Verma et al. 2012) in which IAAwas claimed as an effectivePGR for rooting. As for the hardening stage, upon transfer tosmall plastic pots containing mixture of soil, manure, moss,and sand, about 95% of the rooted regenerated plantsdisplayed normal morphological characteristics under green-house conditions.

The indirect organogenesis system described here might beapplicable to other Digitalis species for large-scale plantproduction. To test the efficiency of the system, cardenolideestimation was carried out in two different leaf tissues, lamina,and petiole. Higher cardenolide accumulation occurred inlamina tissue, which might be attributed to the transportationor localization of the cardenolide in the leaf mesophyll cells(Kreis et al. 1998). Of the analyzed cardenolides, for example

Table 4. Comparison of cardenolide content (in milligrams per kilogramDW) in petiole and lamina extracts from 4-mo-old regenerated plantlets

Cardenolide content (mg/kg DW)

Cardenolides Lamina Petiole

Glucogitoroside 143.9±21.6 53.4±13.5

Strospeside 47.9±7.2 17.6±4.1

Neo-digitalinum verum 45.2±6.2 14.3±3.9

Neo-glucodigifucoside 79.4±11.7 28.1±8.0

Neo-odorobioside G 170.3±26.5 56.4±11.7

Purpurea glycoside B nd nd

Glucoevatromonoside 61.0±8.8 10.6±1.5

Digitoxin nd nd

Lanatoside C nd nd

Digoxin nd nd

Values represent means ± SE. n =3 for each in vitro plant sampleharvested

nd not detected

IN VITRO D. LAMARCKII AND CARDENOLIDES

neo-odorobioside G, the content in the lamina was almostthree times higher than that of petiole samples. Furthermore,the highest difference (nearly 6-fold) was observed withglucoevatromonoside (compare 61.0 in lamina to 10.6 mg/kg DW in petiole samples; Table 4). Production of gluco-evatromonoside in vitro might be feasible, since this com-pound was recently reported to have the highest antiviralactivity against Herpes virus (Bertol et al. 2011). Althoughdigitoxin and lanatoside C were reported by Wichtl andHuesmann (1982) in natural populations ofD. lamarckii , thesecompounds were not detected in in vitro -derived samples. Itmay be that occurrence of such compounds is due to thephysiological ages of plants and the lack of enzymatic regula-tion in the formation of cardenolides for cultured samples.

Conclusion

The protocol described here could contribute to the establish-ment of a large-scale production system using tissue culture,which could be applied to D. lamarckii and perhaps othermedicinal herbs for commercial cultivation and cardenolideproduction. This is the first well-documented study on thecardenolide content of any Digitalis leaf material producedin vitro . This method could be also used to accelerate eliteclone production and facilitate genetic transformation andmetabolic engineering studies on cardenolide metabolism indwarf foxglove. In this manner, production of certain metab-olites in D. lamarckii , such as glucoevatromonoside, viatissue culture techniques might be plausible and also avoidsexcessive harvesting from limited natural sources.

Acknowledgments The authors are grateful to the Scientific and Tech-nological Research Council of Turkey (TÜBİTAK) for a research grantfor the international doctoral research fellowship program (2214) to BY.

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