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1007/s00299-005-0022-3
Chenna Reddy Aswath Sung Youn Mo Doo Hwan Kim S. Won Park
Agrobacterium and biolistic transformation of onion using non-antibiotic selection marker phosphomannose isomerase
Received: 20 November 2003 / Revised: 25 January 2005 / Accepted: 26 January 2005 / Published online: 7 October 2005 C Springer-Verlag 2005
Abstract A new selection system for onion transformation that does not require the use of antibiotics or herbicides was developed. The selection system used the Escherichia coli gene that encodes phosphomannose isomerase (pmi). Transgenic plants carrying the manA gene that codes for pmi can detoxify mannose-6-phosphate by conversion to fructose-6-phosphate, an intermediate of glycolysis, via the pmi activity. Six-week-old embryogenic callus initiated from seedling radicle was used for transformation. Transgenic plants were produced efciently with transformation rates of 27 and 23% using Agrobacterium and biolistic system, respectively. Untransformed shoots were eliminated by a stepwise increase from 10 g l1 sucrose with 10 g l1 mannose in the rst selection to only10 g l1 mannose in the second selection. Integrative transformation was conrmed by PCR, RT-PCR and Southern hybridization. Keywords Onion . Transformation . Phosphomannoseisomerase . Positive selection Abbreviations pmi: Phosphomannose isomerase . 2,4-D: 2,4-Dichlorophenoxy acetic acid . ABA: Abscisic acid
Introduction Onion (Allium cepa L.) is one of the most widely planted vegetable crops in the world with a global production of
Communicated by I. S. Chung C. R. Aswath Indian Institute of Horticultural Research, Bangalore, 560089, India e-mail: aswath@iihr.ernet.in S. Y. Mo D. H. Kim S. W. Park ( ) Department of Horticultural Science, Konkuk University, Seoul, South Korea e-mail: sewapark@konkuk.ac.kr e-mail: mosy0708@hanmail.net e-mail: kimdh@konkuk.ac.kr
about 105 billion pounds in 6.7 million ha. In previous studies, callus induction, regeneration and transformation of A. cepa by various explants was reported. Among them, some reported direct DNA delivery (Klein et al. 1987; Eady et al. 1996; Barandiaran et al. 1998; Scott et al. 1999), while some others reported Agrobacterium transformation using mature (Joubert et al. 1995; Eady et al. 1996; Zheng et al. 2001) and immature embryos (Eady et al. 2000). However the use of mature embryos is tedious, while immature embryo leads to contamination. Hence seedling radicle was used as an explant in this study. The presence of antibiotic marker genes, seen as an unpredictable hazard to the ecosystem and human health, can be solved by removing the selectable antibiotic marker gene. Recently, a number of markers for positive selection for transformation have been developed (Joersbo and Okkels 1996; Joersbo 2001; Zhang et al. 2000). Among them the Escherichia coli gene-encoding phosphomannose isomerase (pmi) using mannose as the selection indices is useful in the transformation of many plant species (Joersbo et al. 1998; Negrotto et al. 2000; Wang et al. 2000; Lucca et al. 2001). Mannose in plant cells converts into mannose-6-phosphate, an inhibitor of glycolysis, thereby inhibiting germination and development. The pmi activity converts mannose-6-phosphate to fructose-6-phosphate, an intermediate of glycolysis that positively supports growth of transformed cells. Transformed cells are able to utilize mannose as a carbon source and grow either in the absence of or with the addition of only small amounts of other carbon sources such as glucose or sucrose. Cells genetically transformed to express pmi acquire a growth advantage (positive selection) on mannose-containing media, which makes it a useful selection agent for generation of transgenic plants. Since onion is consumed on a large scale, there is a need for the elimination of controversial antibiotic markers. Hence, the study was taken up to evaluate the use of pmi as a selectable marker for the recovery of transgenic onion bulbs following Agrobacterium and biolistic-mediated transformation of embryogenic calli initiated from seedling radicle.
93 Fig. 1 Flow diagram showing the different steps of transformation using callus initiated from seedling radicle in onion
Materials and methods Plant material Seeds of onion inbred line KU-31 were rinsed in 70% ethanol for 30 s, washed for 1 min, soaked in detergent for 5 min, rinsed and surface disinfected with 4% sodium hypochlorite for 30 min by agitation. They were then washed in sterile water 5 times, and stored in sterile water at 4 C for 16 h. The surface was disinfected for the second time with sodium hypochlorite for 10 min followed by rinsing in sterile water. The seeds were germinated aseptically and the seedling radicle of 1 cm in length growing from the shoot apex was cut (Fig. 3a) and cultured in a 9 cm petridish containing MS basal medium supplemented
with 5 M 2,4-dichlorophenoxy acetic acid (2,4-D; the best medium from Zheng et al. 1998). Twelve explants were cultured per petridish and incubated at 25 1 C in dark for 4 weeks; later, the callus was subcultured in the same conditions for another 2 weeks (Fig. 1). Six-week-old proliferated callus was then used for transformation. Determination of mannose concentration for selection Six-week-old compact and nodular embryogenic callus was cultured on MS basal medium supplemented with 5 M 2,4-D, varying concentrations of sucrose (020 g l1 ) and mannose (020 g l1 ) to establish mannose lethality. To test whether mannose could substitute sucrose in sustaining growth, sucrose was not added to the mannose containing
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media in a few treatments. Each treatment contained three replications; each replication (one petridish, 9 mm diameter) contained nine clumps each of 3 mm2 (0.25 0.05 g) callus. The callus weight was measured after 4 weeks of selection culture. Increase in weight was calculated by subtracting the initial weight of 0.25 g from the nal weight of the callus. Agrobacterium-mediated transformation Plasmid pNOV2819 conferring streptomycin resistance harbored in Agrobacterium tumefaciens strain LBA4404 was obtained from seed company (Syngenta Seeds AG, www.syngenta.com). It carries pmi gene with constitutive promoter CMPS (Cestrum yellow leaf curling virus Promoter-Shorter version), followed by the NOS terminator sequences. A single colony of bacteria was inoculated into a liquid YEP medium containing 50 mg l1 spectinomycin and incubated for more than 24 h at 28 C with reciprocal shaking (150 cycles min1 ). Cultured bacterial cells were collected by centrifugation (2,000g,10 min) and suspended to a nal OD 600 of 20 ml liquid MS medium with 5 M 2,4D, 20 g l1 sucrose, and 100 M acetosyringone (Sigma Aldrich, www.sigmaaldrich.com). Six-week-old callus was segmented into small pieces of 3 mm2 , 20 clumps were immersed in bacterial suspension for 5 min every time and blotted on sterile lter papers. Twenty explants were placed onto 25 ml aliquots of above medium with 7 g l1 agar in 9-cm petridishes; then the plates were sealed with serene wrap and incubated at 25 C in dark for 3 days. Biolistic transformation A fragment preparation of construct pNOV2820 containing the E. coli derived manA gene encoding pmi driven by CMPS promoter and NOS terminator sequences was used for transformation. DNA was extracted using the Qiagen mega preparation kit. Typically, 250 g of plasmid DNA was digested in a nal volume of 500 l and then run on 0.8% agarose gel in TBE buffer. The band containing the fragment DNA was cut from the gel and eluted using kit (Takara, www.takaratoys.co.jp). The DNA was then precipitated and resuspended in 100 l of TE buffer to a nal concentration of 1 g l1 . The target plate was composed of six-week-old actively growing embryogenic callus of nine clumps of 3 mm2 arranged in concentric circles of 2.5-cm diameter in the center of the plate. Fragment DNA was precipitated on to gold micro carriers (<1 m) as described in manual. Five microgram DNA was used in each six-shot micro carrier preparation. Genes were delivered to the target tissue cells using the PDS-1000HE BiolisticsTM device. The setting on the device were as follows: 6 mm between the rupture disk and macro carrier, 10 mm between the macro carrier and the stopping screen and 6 cm between the stopping screen and the target. The target callus were shot twice with 1 g DNA
using 1,100-psi rupture disks (Eady et al. 1996). After the gene delivery, tissues were incubated in MS basal medium supplemented with 5 M 2,4-D and 20 g l1 sucrose in the dark at 23 C. Transformation efciency After 3 days of co-culture with Agrobacterium or bombardment, the callus was transferred to MS basal medium supplemented with 5 M 2,4-D, 10 g l1 mannose, and 10 g l1 sucrose (with 300 mg l1 cefotaxime in case of Agrobacterium) (Fig. 1). The transformation efciency was recorded at the end of 7th week as the number of callus clumps survived without browning. The callus was then transferred to only mannose medium without any sucrose in order to reduce the number of escaped untransformed callus and improve the selection efciency. At the end of 9th week transformation efciency was counted as the number of callus clumps proliferated. The proliferated sectors from resistant callus were then transferred to the regeneration medium containing MS medium with 5 M Kinetin, 1 M abscisic acid (ABA) and 10 g l1 mannose for induction of somatic embryos. At the end of the 15th week the transformation efciency was counted as number of somatic embryos formed in each callus clumps. The developed somatic embryos were then transferred to MS basal medium supplemented with 10 g l1 mannose. At the end of the 19th week the transformation efciency was counted as number of plantlets formed from well-developed somatic embryos and then transferred to half-strength MS medium with 10 g l1 mannose. At the end of the 23rd week, the rooted plantlets were transferred to soil. For negative control, the callus was inoculated with an Agrobacterium strain free of any plant selectable marker, in case of biolistic experiments, the callus was bombarded without DNA. For positive control, the callus without co-culture or bombardment was transferred to respective growth hormone medium with 20 g l1 sucrose. Analysis of transformants Total genomic DNA was isolated from leaf tissues of the control and putatively transformed plants regenerated from mannose resistant calli (Wettasinghe and Pefey 1998). For PCR analysis of manA gene, the primer set 5 ACAGCCACTCTCCATTCA 3 and 5 GTTTGCCATCACTTCCAG 3 , which amplies 514 bp, was used. PCR amplication reaction contained 20 ng of template DNA, 0.4 M of each primer, 0.5 l dNTP mixture (2.5 mM), 1 Taq DNA polymerase reaction buffer and one unit Taq DNA polymerase (Takara, www.takaratoys.co.jp) in a 20 l nal volume. PCR (Perkin Elmer, www.perkinelmer.com) conditions were performed as follows: one cycle at 94 C for 5 min followed by 30 cycles at 94 C for 1 min, 60 C for 30 s, and 72 C for 30 s. Amplied products were resolved in 1% (w/v) agarose gel.
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For RT-PCR, total RNA was conducted according to Manuel et al. (1999). RT-PCR was performed using Reverse-iTTM one step RT-PCR Kit, ReddyMixTM (Tamar, www.tamar.co.il) according to the manufacturers recommendations. To ensure that the amplied bands were not from trace genomic DNA in the RNA solution, the RNA solutions were used without reverse transcriptase. ManA gene was amplied using the above indicated primers according to the following cyclic conditions: one cycle at 47 C for 30 min and 94 C for 5 min followed by 30 cycles at 94 C for 1 min, 60 C for 30 s, and 72 C for 30 s. Amplied products were resolved in 1% (w/v) agarose gel. For Southern blotting, genomic DNA (about 10 g) was restricted with EcoRI, separated on a 0.8% agarose gel and transferred onto Hybond-N nylon membranes as per manufacturers instructions (Amersham Biosciences, www1.amershambiosciences.com). Both plasmids were restricted with HindIII and Kpn1 and isolated using the QIAquick Gel Extraction Kit. Filters were hybridized with probe as template using Alkphos Direct Labeling Reagent (Amersham Biosciences, www1.amershambiosciences.com). Hybridization, washing and detection were performed according to the instruction manual of Alkphos Direct Labeling and Detection System with CDP-Star (Amersham Biosciences, www1.amershambiosciences.com). Selection for pmi positive plants Mannose, like phosphinothricin, is an effective selection agent for plants grown in soil under nonsterile conditions. Therefore, mannose selection on soil-grown onion was investigated. For selection of transformants, the transgenics and control plants were transferred to pots during the 24th week; later they were hardened in green house for 1 week. Different concentration of mannose (2, 5, 10, and 50 mM) were dissolved in water and sprayed daily for 25 days, after which necrosis and mortality were recorded. Results Response of callus to mannose concentration A mannose dose response curve revealed that, even in the presence of sucrose, the callus growth decreases as the percentage of mannose increases (Fig. 2). Ten grams per liter mannose was found to starve the tissue within the rst 2 weeks of exposure. Ten grams per liter mannose, the osmotic equivalent of 20 g l1 sucrose, was the minimum amount of carbohydrate used in the experiments for the maintenance of onion cultures. The mix of 10 g l1 mannose with 10 g l1 sucrose decreased the tissue growth by 42% as compared to normal 20 g l1 sucrose. The toxic effect of mannose decreases with increasing sucrose concentrations in the medium.
Fig. 2 The effect of mannose concentration (g l1 ) (, 0; , 5; ,10; , 15; , 20) on average gram fresh weight (gFW) when cultured with different sucrose concentration. Value represents meansSE
Callus multiplication and regeneration Callus formation from seedling radicle could be observed after 45 days of culture. Three morphologically different callus types could be easily distinguished: a compact type, a white and nodular type with no apparent structures and a watery and transparent type. Compact callus was prominent at the site of the apex whereas friable callus was abundantly produced at the other end. In further subcultures, callus became swollen and enlarged considerably. For transformation, only compact and friable callus were used. As early as 1 week after the transfer of callus to regeneration medium, multiple embryos were formed (Fig. 3b). Though many of these embryos failed to form somatic embryos, they turned green and formed roots. In some callus clumps, somatic embryos and embryo-like structures were formed (Fig. 3c). These somatic embryos germinated and developed into plantlets, which readily formed roots upon transfer to half-strength MS medium without growth regulators (Fig. 3d). Plants continued to grow after transfer to the glasshouse under short-day conditions. Upon transfer to long-day conditions, the regenerated plants produced bulbs. During initial selection at the 7th week (Table 1), untransformed callus clumps were starved and did not develop further (Fig. 3e). There was no signicant difference between Agrobacterium and biolistic transformation, where both recorded 2327% positive callus for the mannose selection. At the end of the 9th week, since most of the untransformed callus was ltered earlier, proliferation was 33% in Agrobacterium and 27% in biolistic transformation. After the 15th week, the percentage of callus forming primaries was more in biolistic than Agrobacterium (Fig. 3f). At the end of 19th week, as compared to the control, the percentage somatic embryos forming plantlets was much less with no signicant difference between Agrobacterium and biolistic.
96 Table 1 Effect of Agrobacterium and biolistic-mediated transformation on survival, proliferation and regeneration of onion callus under different periods of selections Callus survived at the end of 7th week (%) Agrobacterium Biolistic Negative control (Agrobacterium) Negative control (biolistic) Positive control 27b 23b 3.7c 4.5c 95.5a Callus proliferated Callus forming at the end of 9th somatic embryos week (%) (SEs) at the end 15th week (%) 33b 27b 2.1c 2.2c 90.5a 17.4b 23.4b 0.0c 0.0c 45.5a SEs forming plantlets at the end of 19th week (%) 22.5b 23.7b 0.0c 0.0c 65.5a
Note. For Agrobacterium and biolistic transformation, 20 and 9 callus clumps were taken each time, respectively. The experiment was repeated 20 times. The percentage was calculated based on total number of callus clumps. Mean separation in column by Duncans multiple range tests. Within the column gure followed by same letter do not differ signicantly at P0.05 Fig. 3 Plant regeneration via somatic embryogenesis in onion. a Germinated seed showing radicle, hatched area represents the explant. b Compact callus derived from seedling radicle used for transformation. c Somatic embryos under MS with 5 M kinetin, 1 M ABA and 10 g l1 mannose. d Plantlets developed from somatic embryos. e Co-cultured callus at the end of the 7th week. Note the untransformed callus starving. f Selection at the end of the 15th week under MS with 5 M 2,4-D and 10 g l1 mannose
In positive control, it was frequently observed that as the structures enlarged, additional shoot meristem regions were produced raising the number of shoots than would be expected from the initial number of embryogenic structures within the culture (data not shown). An average of 3.47 shoots per callus clump was observed in both transformed
and non-transformed treatments. Plantlets could be readily induced to form roots by transfer to half-strength MS medium without growth regulators. In the negative controls of both Agrobacterium and biolistic transformation, 34% callus proliferated at the end of the 7th week, while in the 9th week 12% of the untransformed callus starved but
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ever 50 mM of mannose effectively killed non-transformed plants within 10 days of spray, while the transformed plants were completely resistant. Discussion A reproducible and stable transformation system for onion has been developed using Agrobacterium tumefaciens and biolistic approach with 6-week-old callus induced from seedling radical. This is the rst report on the use of mannose as a selection index in onion, whereas previously only kanamycin and hygromycin were used. A common feature among transformation studies in onion was the use of immature embryos as target explants due to their excellent morphogenetic competence (Eady et al. 1996, 2000). However, they reported that the contamination rate, which probably arose from infected embryos, ranged from 40 to 100%. Zheng et al. (2001) used mature embryos, which is tedious to remove and requires stereomicroscope to identify the shoot apex portion. Klein et al. (1987) and Scott et al. (1999) used epidermal tissues in onion with high velocity microprojectiles resulting in transient expression of chloromphenicol acetyl transferase and green uorescent protein gene, respectively. However contamination rate was also high in epidermis. We have used seedling radicle, which is in no way inferior to other explants with an added advantage of easy extraction and zero contamination. The most common selective protocols for plant transformation are the use of kanamycin, hygromycin, and phosphinothricin. In onion Eady and Lister (1998) demonstrated that kanamycin was ineffective up to 200 mg l1 and used 50100 mg l1 geneticin. Kanamycin is not recommended for spray, as it is very expensive (Altmann et al. 1992). Though hygromycin is moderately expensive and works well both in media and soil it is not recommended because of its high toxicity to humans (Altmann et al. 1992), even 2050 mg l1 hygromycin in onion resulted in few escapes (Eady and Lister 1998). Phosphinothricin is an excellent marker for soil-grown plants. Selection on plates requires puried and expensive phosphinothricin. Additionally, a large number of escapes were noticed during selection on media containing 1030 mg l1 phosphinothricin (Eady et al. 2000). Unlike most selectable markers, pmi confers a positive advantage to plants grown in tissue culture using mannose, which is a carbon source. In addition it does not lead to necrotic cells, which may release toxic compounds and is non-toxic to humans. Mannose selection of resistant plants works equally well in soil and in plates, and is very cheap, perhaps the least expensive among all agents (Todd and Tague 2001). When onion callus was exposed to 10 g l1 mannose without sucrose, the starving of tissue was observed within 2 weeks. Callus growth continued when mannose was added with sucrose at higher concentrations, which is consistent with the ndings of Wright et al. (2001) and Negrotto et al. (2000). However, it was in contrast with Kim et al. (2002) who observed reduction in shoot regeneration capacity in
Fig. 4 Molecular analysis of transgenics. a PCR analysis of transgenics for pmi gene. Lane P1 plasmid from pNOV 2819 used for Agrobacterium transformation, lane P2 plasmid pNOV2820 used for biolistic transformation, lanes 112 transformed plants through Agrobacterium (lanes 16) and biolistic (lanes 712) mediated transformation. b RT-PCR analysis for the leaf tissue of transgenic onion plants for pmi gene. Lane C untransformed control, lanes 15 transformed plants through Agrobacterium (lanes 13) and biolistic (lanes 45) mediated transformation, lane N negative control without reverse transcriptase. c Southern hybridization analysis of genomic DNA derived from putative transgenic plants. Lane C control plant, lanes 16 transformed plants through Agrobacterium (lanes 13) and biolistic (lanes 46) mediated transformation
still survived. In the rooting test (data not shown) rooting of wild type shoots was totally inhibited by 10 g l1 mannose, whereas the transgenic pmi-expressing plants developed a normal root system. Consequently, 10 g l1 mannose was used to screen for transgenic plants which can root normally, while control shoots failed to produce any roots. Conrmation of transformation PCR amplication of manA gene showed that all the plants except untransformed control produced bands of an expected size of 514 bp for the pmi fragment at the same position as the binary vector positive control (Fig. 4a). RT-PCR transgenic plants yielded the expected 514-bp band while untransformed control did not have the band (Fig. 4b). No band was produced in the negative control (lane N in Fig. 4b). Southern hybridization following EcoRI digestion produced a single band for DNA from the transformed plantlets and none for the control (Fig. 4c). Selection for pmi-positive onion in soil We observed negligible effect with the spray of the lowest concentrations (2, 5, and 10 mM) of mannose. How-
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pepper with the addition of sucrose to mannose at higher concentration. In our studies, the prolonged exposure of escaped calli to high mannose without any sucrose for 2 weeks, further exposure of 8 weeks in regeneration media, and continuing of the mannose during rooting might have caused distortion of untransformed cells and virtually eliminated all escapes. Further spray of 50 mM mannose to plants grown in pots perfected the selection without any escapes. Wright et al. (2001) utilized only mannose (10 g l1 ) and a mannose (7 g l1 ) +sucrose (3 g l1 ) combination during callus selection regeneration in maize and wheat transformation, respectively. Negrotto et al. (2000) set a combination of 5 g l1 sucrose and 10 g l1 mannose as the selection index for maize transformation, while Kim et al. (2002) recommended 20 g l1 sucrose and 15 g l1 or more of mannose (up to 50 g l1 ) in pepper transformation. The mannose concentration of 20 g l1 but not 30 g l1 , still allowed a low frequency of organogenesis in cassava (Zhang and Puonti-Kaerlas 2000), while 3 g l1 inhibited regeneration in sugar beet totally (Joersbo et al. 1998). The average transformation frequency was 25.0%, which was lower than that reported for non-transformed calli using similar plant growth regulators (95.0%). Shoot regeneration from resistance calli initiated from mature embryo of onion following Agrobacterium-mediated transformation occurred at a lower frequency of 1.95% using hygromycin (Zheng et al. 2001) and 2.5% using immature embryo with selection agent geneticin (Eady et al. 2000). The lower frequency by earlier workers was also due to large contamination of explants. Negrotto et al. (2000) recovered frequency of 30% in maize plants using Agrobacterium transformation. Wright et al. (2001) also observed mean frequencies of 45% for maize and 20% for wheat using biolistic transformation, which is a 10-fold higher than the herbicidal selection of previous workers. In sugar beet, the mannose selection system using Agrobacterium was reported to result in 10-fold higher transformation frequencies as compared to kanamycin selection (Joersbo et al. 1998); however in cassava, the efciency of hygromycin selection was about 2-fold higher than that of mannose selection-using PEGmediated particle bombardment (Zhang and Puonti-Kaerlas 2000). This indicated that transformation frequency on mannose selection varies with each crop. Southern blot analysis of EcoRI restriction digests of genomic DNA from transgenic plants using pmi probe revealed one band in the transgenic lines, indicating the integration of one copy of the pmi gene. Agrobacteriummediated transformed plants (plants 1, 2, and 3 of Fig. 4c) showed integration of gene at one locus while polymorphism of hybridization pattern among plants bombarded by gene gun (plants 4, 5, and 6) indicated random integration. RT-PCR transgenic plants yielded the expected 514-bp fragment while untransformed control did not have the fragment. No fragment was produced in the negative control (lane N in Fig. 4b), where RNA solutions was used without reverse transcriptase indicating that the RT-PCR bands from transgenic lines were produced from the mRNA of pmi.
Even though bombarded callus recorded better transformation than Agrobacterium there was no statistical difference between the two methods. Hence for onion transformation both approaches can be used. Conclusion Callus induction using MS basal medium supplemented with 5 M 2,4-D from seedling radicle, and shoot regeneration via somatic embryogenesis using MS medium with 5 M Kinetin and 1 M ABA has been demonstrated to be a very effective procedure for both micro propagation and transformation studies. Initial selection of callus in MS medium with 10 g l1 mannose and 10 g l1 sucrose and the subsequent selection with only 10 g l1 mannose prevented the production of escapes. The results indicated that, if the right gene constructs are delivered into the right tissue and selected with a non-controversial selection marker like mannose, it should be possible to produce safer transgenic onions.
Acknowledgements We thank the Korean Federation of Science and Technology, Republic of Korea, for the nancial support under Brain pool project and ICAR, Government of India, for the study leave for C. Aswath. We also thank Syngenta seeds, Switzerland for sparing the pmi construct. We would like to acknowledge Dr Tim Conner for critically reviewing the manuscript.
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