Euphytica (2006) 150: 339–345

DOI: 10.1007/s10681-006-9120-9 C© Springer 2006

Relative efficiency of anther culture and chromosome elimination techniquesfor haploid induction in triticale × wheat and triticale × triticale hybrids

Aditya Pratap1,2, Gurdeep Singh Sethi1,∗ & Harinder K. Chaudhary1

1Cytogenetics and Tissue Culture Lab., Department of Plant Breeding and Genetics, CSK Himachal PradeshAgricultural University, Palampur – 176062, India; 2Present address: Division of Genetics & Plant Breeding, SKUniversity of Agricultural Sciences & Technology, FOA, Main Campus, Chatha, Jammu – 180009, India(∗author for correspondence: e-mail: ctlhkc@hillagric.ernet.in)

Received 10 June 2004 ; accepted 5 February 2006

Key words: anther culture, chromosome elimination technique, haploids, triticale × triticale hybrids, triticale ×wheat hybrids, wheat × maize system

Summary

The study was undertaken to evaluate the relative efficiency of anther culture and chromosome elimination (bycrosses with maize) techniques of haploid induction in intergenotypic triticale and triticale × wheat hybrids. Forthis, 15 triticale × wheat and 8 triticale × triticale F1 hybrids were subjected to anther culture and were alsosimultaneously crossed with the ‘Madgran Local’ genotype of maize (Zea mays L.) to induce haploids through thechromosome elimination technique. The haploid embryo formation frequency through the chromosome eliminationtechnique was significantly higher in both, triticale × wheat (20.4%) and triticale × triticale (17.0%) F1 genotypes,as compared to the calli induction frequencies through anther culture (1.6 and 1.4%, respectively). Further, fourtriticale × wheat and three triticale × triticale F1 genotypes failed to respond to anther culture, whereas, all the F1

genotypes formed sufficient number of haploid embryos through the chromosome elimination technique with norecovery of albino plantlets. The haploid plantlet regeneration frequencies were also significantly higher through thelatter technique in both triticale × wheat (42.7%) and triticale × triticale (49.4%) F1s as compared to anther culture(8.2 and 4.0%, respectively), where the efficiency was drastically reduced by several constraints like, high genotypicspecificity, low regeneration frequency and albinism. The overall success rates of obtaining doubled haploids per100 pollinated florets/anthers cultured were also significantly higher through the chromosome elimination technique(1.1% in triticale × wheat and 1.5% in triticale × triticale hybrids), proving it to be a highly efficient and economicallymore viable technique of haploid induction as compared to anther culture, where the success rates were only 0.2%and 0.1%, respectively.

Introduction

In vitro haploid production followed by chromosomedoubling has played an important role in modern plantbreeding programmes as the systematic introductionand rapid fixation of alien chromosomes, chromosomesegments or even genes can be achieved in a singlestep. The haploid breeding approach increases the cor-rectness of selection of multiple crosses by offeringthe reliability of selection and the assurance that thegenes involved are completely homozygous and the

characters selected will not be lost due to residual het-erozygosity. Haploids may also find use in mutationbreeding as their hemizygous expression allows thedominant as well recessive mutations to be expresseddirectly among regenerants and deleterious mutationsare eliminated.

In a major portion of the world, wheat (Triticumaestivum L.) is grown under diverse agro-climatic andrain-fed conditions and faces constraints such as sus-ceptibility to diseases, drought and cold stresses. How-ever, it has a large assortment of relatives, among which

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rye (Secale cereale L.) is a rich source of genes for im-proving many characters in wheat (Sethi, 1989). Trans-fer of desirable genes into wheat can occur throughD- and R-genome chromosome substitution (Merker,1975; May & Appels, 1980; Sethi & Plaha, 1988),Robertsonian (whole-arm) translocations (Driscoll &Sears, 1965; May & Appels, 1978; Sethi & Plaha,1988) as well as cryptic translocations (Lukaszewski &Gustafson, 1983; Sethi, 1989). Therefore, by bringingtogether these diverse gene pools through hybridiza-tion followed by fixation of the promising recombi-nants through doubled-haploidy breeding, we can ob-tain in the shortest possible time the reconstituted ho-mozygous lines that are resistant to various biotic andabiotic stresses.

Anther or microspore culture (androgenesis) iswidely used in breeding wheat, triticale and any othercrops because of availability of a large number of mi-crospores within each anther that may produce haploidplants (Zhou, 1996). However, due to necessity for an-ther isolation, the anther culture method seems to bemore laborious (Pauk et al., 2000). Moreover, the stronggenotype dependence of androgenesis, low regenera-tion rate, and high frequency of albinism are the majorconstraints limiting its wider application at the currenttime. Therefore, alternative methods are sought for hap-

Table 1. Parentage and source of different genotypes of wheat and triticale

S. no. Genotype Parentage Source

Wheat

1. HPW42 VEE”S”/4/PVN”S”/CBB//CNO”S”/3/JAR/ORZ”S” CSK HPKV, Palampur

2. RL 14-1 RL 14/CPAN 1922 CSK HPKV, Palampur

3. HS396 PFAU/4/B6/Gu/GJ/F.30.;70//KAL/B6/5/Vee #7 IARI, RS, Shimla

4. PW565 – AICWIP, Karnal

5. Raj 3702 – AICWIP, Karnal

6. UP2418 HD2320/UP2263 GBPUAT, Pantnagar

7. VL 798 – VPKAS, Almora

8. VL 802 CPAN 3018/CPAN 3004/PBW65 VPKAS, Almora

Triticale

1. DT123 TR190/C306//TR239/3/TR170/4//TR169/PBW34//TR255 IARI, New Delhi

2. DT126 C306/WSP536//JNIT140/3/TR180/4/TR241 IARI, New Delhi

3. TL2900 JNIT 128/GP 288/TL 2729 AICWIP, Karnal

4. TL2908 TL 2614/JNIT 141 AICWIP, Karnal

5. TL2919 TL2734/3/MZACIN/JNIT128//TL2433 PAU, Ludhiana

6. TL2920 PBW189/WHITE RYE/JNIT128 PAU, Ludhiana

7. ITSN 105 #58 – ITSN, CIMMYT, Mexico

8. ITSN65 – ITSN, CIMMYT, Mexico

9. ITSN109 – ITSN, CIMMYT, Mexico

10. ITSN163 – ITSN, CIMMYT, Mexico

loid induction in wheat and triticale (× TriticosecaleWittm.). Hybridization of wheat with maize, followedby auxin treatment has become a well-establishedmethod of obtaining haploid wheat (Laurie et al., 1990)and triticale plants (Wedzony et al., 1998). This is pos-sible due to the insensitivity of maize to the action ofthe crossability inhibitor Kr genes (Laurie & Bennett,1987). However, very little work on these lines has beendone in triticales and triticale × wheat hybrids. Theobjective in the present study was to compare the effi-ciencies of these two techniques of haploid induction.

Materials and methods

Plant materials

Ten elite and diverse genotypes of triticale (AABBRR)viz., DT 123, DT 126, TL 2900, TL 2908, TL 2919,TL 2920, ITSN 105 #58, ITSN 65, ITSN 109 and ITSN163 were crossed with 8 different genotypes of wheat(AABBDD) viz., HPW 42, HS 396, PW 565, Raj 3702,RL 14-1, UP 2418, VL 798 and VL 802. These geno-types were selected on the basis of their high yield,grain quality (amber, bold and hard grains preferred),disease resistance and diverse parentage (Table 1).The

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15 triticale × wheat F1s and 8 triticale × triticale hy-brid combinations were raised in two replicates undernatural field conditions. In each replicate, 25 seeds percross were sown in single rows, 1.25 m long and 23 cmapart. One of the replicates was used for anther cultureand the other was used for the chromosome elimination(by crosses with maize) technique of haploid induction.

Anther culture

The main spikes of 15 plants in each cross were selectedat the mid- to late-uninucleate stage of the pollen devel-opment. The selected main tillers were cut at the base,then kept in darkness at 4 ◦C for 48 h cold treatmentbefore culturing. The anthers were excised asepticallyand cultured on Potato-II medium (Chuang et al., 1978)supplemented with 0.5 mg/l kinetin and 2.0 mg/l 2,4-D. The medium also contained 0.5 mg/l glutamine (deBuyser & Henry, 1980) and was solidified by adding6.0 g/l agarose (Henry et al., 1984), followed by auto-claving. In 150 × 20 mm Petri plate containing 25 mlmedium, 35–50 anthers were cultured with a total of525 anthers per F1 genotype. The Petri plates weresealed with parafilm and kept in darkness at 28 ± 1 ◦Ctemperature and 80 per cent relative humidity (RH) foreight days. After eight days, the physical environmentwas changed to a temperature of 25 ± 1 ◦C, 80 percent RH and a 10 h photoperiod for a period of 35–50days till callus initiation was observed. As soon as thecallus was formed, it was transferred to plant regener-ation medium with 0.5 mg/l of naphthalene acetic acid(Chuang et al., 1978). For profuse rooting, the callus-derived plantlets were transferred onto the liquid root-ing medium comprising half the strength of MS salts(Murashige & Skoog, 1962), 1 mg/l each of naphtha-lene acetic acid and indole-3-butyric acid and devoidof sucrose and agar.

Chromosome elimination

The triticale × wheat and triticale × triticale F1s werecrossed with maize. In each F1 genotype, 15 mainemasculated spikes were pollinated with fresh pollenfrom the ‘Madgran Local’ genotype of maize. 24 h af-ter pollination, the hollow of the uppermost internodeof the crossed spike was filled with 250 ppm 2,4-D so-lution by injecting at its base. The injections were re-peated twice on consecutive days (three injections overthree days) to promote in vivo embryo formation. Thecrossed spikes were harvested from the tiller base 18–20 days after pollination. The expanding ovules were

dissected from the ovaries and embryos removed. Theembryos were transferred under asceptic conditionsto MS medium (Murashige & Skoog, 1962) supple-mented with 0.5 mg/l kinetin, 150 mg/l glutamine and20 mg/l each of L- arginine, L- cysteine and L- leucine.The pH of the medium was 5.8 and it was autoclaved.The embryos were cultured in 150 × 20 mm culturetubes and were given cold treatment at 4 ◦C in darknessfor 24 h. Following the low temperature treatment, theywere incubated at 25±1 ◦C in 10 h light, 14 h dark pho-toperiod regime for regeneration. When the embryoid-derived plantlets reached 3–4 leaflet stage, they weretransferred to liquid rooting medium as described inanther culture for profuse rooting and subsequentlyto the potting mixture of soil. Somatic chromosomenumber was examined in the root tip cells of three ran-domly taken haploid plantlets following the procedureof Tsuchiya (1971).

The haploid plantlets obtained through both the an-ther culture and chromosome elimination techniqueswere treated with colchicine as described by Inagaki(1985). At the three- to five-tiller stage, the roots of thehaploid plants were cut back to 2–3 cm. The crown ofthe haploid plants was immersed in 0.1% colchicinesolution that was supplemented with 0.5% dimethylsulphoxide at 25 ± 1 ◦C for 5 h. The treated plantswere washed thoroughly in tap water, potted in soiland grown to maturity. Those plants which producedseeds were taken as doubled haploid plants.

Recording of observations

Observations were recorded of callus induction, plant-let regeneration and green haploid plantlet formationfor the anther culture experiment, and seed formation,embryo formation and regeneration frequencies in thechromosome elimination experiment.

Results

The embryo formation frequencies through the chro-mosome elimination technique were significantlyhigher (t-test, at the p = 0.001 level) in all the trit-icale × wheat and triticale × triticale F1 hybrids incomparison to the callus induction frequencies throughanther culture. As shown in Tables 2 and 3,callus in-duction in anther culture technique ranged between 0to 5.1 per cent in the triticale × wheat F1s and 0 to14.2 per cent in triticale × triticale F1 genotypes. Onthe other hand, embryo formation in the chromosome

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Table 2. Relative efficiency of anther culture and chromosome elimination techniques for the production of haploids and doubled-haploids in

triticale × wheat hybrids

Parameters of anther culture technique (%)∗ Parameters of chromosome elimination technique (%)∗∗

S. no. Name of the F1 genotype ci r a gp(h) dh sr f sf ef r dh sr

1. TL 2900 × RL 14-1 1.7 (9) 33.3 (3) 11.1 (1) 22.2 (2) 22.2 (2) 0.4 480 33.7 (162) 9.9 (16) 25.0 (4) 25.0 (4) 0.8

2. DT 126 × Raj 3702 0.6 (3) 33.3 (1) 0 33.3 (1) 33.3 (1) 0.2 544 29.2 (159) 9.4 (15) 6.7 (1) 6.7 (1) 0.2

3. DT 126 × HS 396 2.7 (14) 7.1 (1) 0 7.1 (1) 7.1 (1) 0.2 460 5.6 (26) 65.4 (17) 52.9 (9) 47.1 (8) 1.74

4. ITSN 105 #58 × VL 802 0 – – – – – 512 18.0 (92) 20.6 (19) 10.5 (2) 10.5 (2) 0.4

5. ITSN 105 #58 × UP 2418 0.6 (3) 0 – – – – 503 7.2 (36) 47.2 (17) 82.3 (14) 70.6 (12) 2.4

6. ITSN 105 #58 × Raj 3702 0.8 (4) 50.0 (2) 100.0 (2) 0 – – 498 9.2 (46) 21.7 (10) 20.0 (2) 20.0 (2) 0.4

7. ITSN 105 #58 × VL 798 0 – – – – – 528 53.8 (284) 3.9 (11) 100.0 (11) 90.9 (10) 1.9

8. TL 2908 × VL 802 4.2 (22) 22.7 (5) 9.1 (2) 13.6 (3) 13.6 (3) 0.6 580 42.4 (246) 19.5 (48) 25.0 (12) 25.0 (12) 2.1

9. TL 2919 × Raj 3702 3.6 (19) 15.8 (3) 5.3 (1) 10.5 (2) 10.5 (2) 0.4 468 25.6 (120) 9.2 (11) 54.5 (6) 36.4 (4) 0.8

10. TL 2920 × HPW 42 0 – – – – – 521 31.9 (166) 7.8 (13) 61.5 (8) 38.5 (5) 1.0

11. TL 2920 × PW 565 5.1 (27) 25.9 (7) 14.8 (4) 11.1 (3) 11.1 (3) 0.6 610 9.5 (58) 36.2 (21) 28.6 (6) 28.6 (6) 1.0

12. DT 123 × VL 798 0 – – – – – 607 50.4 (306) 1.6 (5) 40.0 (2) 40.0 (2) 0.3

Mean 1.6 15.7 11.7 8.2 8.2 0.2 26.4 20.4 42.3 36.6 1.1

∗ci = callus induction, r = regeneration, a = albino plantlets, gp(h) = green haploid plantlets, dh = doubled haploids, sr = success rate.∗∗f = florets pollinated with maize pollen, sf = seed formation, ef = embryo formation, r = regeneration, dh = doubled haploids.

Figures in parentheses represent the numbers obtained.

Table 3. Relative efficiency of anther culture and chromosome elimination techniques for the production of haploids and doubled-haploids in

triticale × triticale hybrids

Parameters of anther culture technique (%)∗ Parameters of chromosome elimination technique (%)∗∗

S. no. Name of the F1 genotype ci r a gp(h) dh sr f sf ef r dh sr

1. TL 2900 × TL 2920 0 – – – – – 623 27.6 (172) 4.1 (7) 57.1 (4) 57.1 (4) 0.6

2. ITSN 105 #58 × ITSN 65 4.2 (22) 13.6 (3) 9.1 (2) 4.5 (1) 4.5 (1) 0.2 596 35.8 (209) 10.5 (22) 36.4 (8) 27.3 (6) 1.0

3. TL 2908 × ITSN 105 #58 3.2 (17) 29.4 (5) 11.8 (2) 17.6 (3) 17.6 (3) 0.6 544 1.8 (10) 80.0 (8) 87.5 (7) 75.0 (6) 0.1

4. TL 2920 × DT 123 0 – – – – – 499 68.7 (343) 21.0 (72) 41.7 (30) 30.6 (22) 4.4

5. ITSN 109 × ITSN 65 1.7 (9) 44.4 (4) 0 – – – 507 61.7 (313) 5.1 (16) 75.0 (12) 75.0 (12) 2.4

6. ITSN 65 × ITSN 163 0.3 (2) 0 – – – – 485 86.6 (428) 10.2 (43) 39.5 (17) 37.2 (16) 3.3

7. ITSN 163 × DT 123 0 – – – – – 640 19.8 (127) 3.1 (4) 25.0 (1) 25.0 (1) 0.2

8. DT 126 × ITSN 105 #58 1.9 (10) 20.0 (2) 10.0 (1) 10.0 (1) 10.0 (1) 0.2 619 27.5 (170) 1.7 (3) 33.3 (1) 33.3 (1) 0.2

Mean 2.7 13.4 3.9 4.0 4.0 0.1 41.1 17.0 49.4 45.1 1.6

∗ci = callus induction, r = regeneration, a = albino plantlets, gp(h) = green haploid plantlets, dh = doubled haploids, sr = success rate.∗∗f = florets pollinated with maize pollen, sf = seed formation, ef = embryo formation, r = regeneration, dh = doubled haploids.

Figures in parentheses represent the numbers obtained.

elimination technique ranged between 1.6 to 65.04 percent and 1.8 to 80.0 per cent respectively, in the triticale× wheat and triticale × triticale F1 hybrids. Averagedover the genotypes, the embryo formation frequencieswere many times higher in comparison to the callusinduction frequencies in both triticale × wheat (26.4%vs. 1.6%) and triticale × triticale (41.7% vs. 2.7%) F1

hybrids. Also the genotypic effect was very strong inanther culture, where 4 triticale × wheat F1s (ITSN

105 #58 × VL 802, ITSN 105 #58 × VL 798, TL 2920× HPW 42 and DT 123 × VL 798) and 3 triticale ×triticale F1 hybrids (TL 2900 × TL 2920, TL 2920 ×DT 123 and ITSN 163 × DT 123) did not show callusformation. On the contrary, the chromosome elimina-tion technique was free from the genotypic effect whereall the F1 genotypes formed a good number of haploidembryos, many of which were subsequently regener-ated into haploid plantlets.

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The recovery of green haploid plantlets was dras-tically reduced by albinism in the anther culturetechnique. The average green haploid plantlet regen-eration frequencies through this technique were only8.2 and 4.0 per cent, respectively in the triticale ×wheat and triticale × triticale F1 genotypes. On theother hand, these were much higher through the chro-mosome elimination technique (42.3% and 49.4%).The F1 genotypes, TL 2920 × HPW 42 among thetriticale × wheat F1s and ITSN 109 × ITSN 65amongst triticale × triticale F1s, which formed callusin the anther culture experiment, could not regeneratehaploid plantlets. However, all the genotypes, whichrecorded embryo formation in the chromosome elim-ination experiment, exhibited green haploid plantletregeneration.

The haploid plantlets obtained through both thetechniques were successfully converted into doubledhaploids after colchicine treatment. On an average, thedoubled haploid plants recovered through anther cul-ture were 8.2 and 4.0 per cent, respectively in triticale ×wheat and triticale × triticale F1 hybrids. Through thechromosome elimination technique these frequencieswere significantly higher (36.6% and 45.1%). The over-all success rates of obtaining haploids through antherculture were observed to be only 0.2 and 0.1 per cent ofthe anthers cultured in case of triticale × wheat and trit-icale × triticale F1 hybrids, respectively. On the otherhand, through the chromosome elimination technique,the overall success rates were observed to be signifi-cantly higher and these were 1.1 and 1.6 per cent of thetotal florets pollinated in triticale × wheat and triticale× triticale F1s, respectively. The highest success rate incase of triticale × wheat F1s through anther culture wasrecorded in crosses TL 2908 × VL 802 and TL 2920 ×PW 565 (0.6% each), followed by TL 2900 × RL 14-1and TL 2919 × Raj 3702 (0.4% each). Six F1 geno-types did not record any success. In case of triticale ×triticale F1s, the highest success rate was observed inTL 2908 × ITSN 105 #58 (0.6%), followed by ITSN105 #58 × ITSN 65 and DT 126 × ITSN 105 #58 (0.2%each) and five F1 genotypes did not exhibit any suc-cess. On the other hand, through the chromosome elim-ination technique, ITSN 105 #58 × UP 2418 recordedthe highest success rate (2.4%) among the triticale ×wheat hybrids, followed by TL 2908 × VL 802 (2.1%)and ITSN 105 #58 × VL 798 (1.9%). Among the trit-icale × triticale hybrids, the highest success rate wasobserved in TL 2920 × DT 123 (4.4%), followed byITSN 65 × ITSN 163 (3.3%) and ITSN 109 × ITSN 65(2.4%).

Discussion

It is evident from the results that the chromosome elim-ination technique outperformed the anther culture tech-nique in both triticale × wheat and triticale × triticaleF1 genotypes with respect to all the haploid inductionparameters under study. Callus induction through an-ther culture was much lower than embryo formationthrough the chromosome elimination technique. Fur-ther, callus induction did not occur in 4 triticale×wheatand 3 triticale × triticale F1 genotypes. This could beattributed to high genotypic specificity, which is a char-acteristic of the androgenetic haploid system and haslimited the exploitation of this method in commercialwheat breeding. Differential androgenetic response intriticale × wheat F1s has also been reported earlier byHu et al. (1988), Tao and Hu (1990) and Wang et al.(1993), who derived a number of wheat lines throughanther culture with different rye constitutions using thetriticale × wheat F1s. However, Tuvesson (2000) ob-served that the triticale responded better to anther cul-ture and wheat responded in varying degrees. The lowresponse of triticale × wheat F1s to anther culture mayalso be attributed to the large extent of pollen sterilityin such plants because of meiotic irregularities, whichare caused by the lack of pairing between the R- andD- genomes (Wang et al., 1993). In general, in the caseof both triticale × wheat and triticale × triticale F1

hybrids, the influence of genotypes appears to be themajor disadvantage in the large recovery of haploidplantlets. On the other hand, the high response of allthe F1s to the chromosome elimination technique withrespect to embryo formation was mainly due to geno-typic non-specificity, which is because of the insen-sitivity of the maize pollen to the action of Kr1 andKr2 genes, which express in the style of many wheatvarieties (Laurie & Bennett, 1987). Results regardingthe high response of the chromosome elimination tech-nique have earlier been reported by Inagaki and Tahir(1990) in wheat and Rogalska and Mikulski (1995) andRogalska et al. (1996) in triticale.

The haploid plantlet regeneration frequencies werealso significantly higher through the chromosome elim-ination technique in the case of both triticale × wheatand triticale × triticale F1 genotypes. These results con-firm the earlier reports of Kisana et al. (1993) andGury et al. (1993) who found regeneration of hap-loid plants in the chomosome elimination technique(by crosses with maize) to be better in comparison tothat in the anther culture technique. The calli obtainedin the case of anther culture needed to be proliferated

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and differentiated in vitro, requiring many sub-cultures,which might have resulted in injury and subsequent lossof regenerability in many of the calli. Further, genemutations, chromosomal aberrations and aneuploidsarising during the cell divisions in vitro might havealso led to the production of gametoclonal variants. Onthe other hand, high haploid regeneration frequenciesthrough the chromosome elimination technique wereobtained due to the well-developed haploid embryoswith healthy root and shoot primordia at the time of res-cue on culture medium. Further, no albino plants wereobtained through the latter technique, which otherwisewas a major limitation in recovery of a large numberof haploid plantlets through anther culture in both trit-icale × wheat and triticale × triticale F1 hybrids. Highrates of albino plantlets through anther culture in triti-cale and wheat have also been reported by Schumann(1988) and Pauk et al. (2000). Studies of Karsai et al.(1994) suggested that the incubation medium contain-ing agar and Ficoll 400 could significantly increasethe efficiency of anther culture in wheat. Also, a highconcentration of maltose (0.25 M) increased embryoinduction in both wheat and triticale, though highergreen plantlet regeneration frequency was observed incase of wheat only. Many times higher frequencies ofhaploids and doubled haploids in both triticale × trit-icale and triticale × wheat F1 genotypes through thechromosome elimination technique confirm its superi-ority over anther culture for the purpose. Recent stud-ies have suggested some new Gramineae genera whichcould replace maize for the purpose of haploid induc-tion through the chromosome elimination technique,out of which Imperata cylindrica has been found to beas efficient as maize (Chaudhary et al., 2005 and Pratapet al., 2005).

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