BASE Biotechnol. Agron. Soc. Environ.201418(1),32-36 Short note

Studyoftheimpactofgrowthsubstancetreatmentandmaize(Zea maysL.)varietyinspelt(Triticum speltaL.)haplodiploidizationEmmanuelleEscarnot(1),CharlotteThibaut(2),PaulForgeois(2)(1)CentrewallondeRecherchesagronomiques(CRA-W).DépartementSciencesduVivant.UnitéAméliorationdesEspècesetBiodiversité.BâtimentÉmileMarchal.RuedeLiroux,4.B-5030Gembloux(Belgium).E-mail:e.escarnot@cra.wallonie.be(2)InstitutdeGenech.RuedelaLibération.F-59242Genech(France).

ReceivedonJanuary21,2013;acceptedonJanuary13,2014.

Sofarasweknow,thisisthefirststudyonspelt(Triticum speltaL.)haplodiploidization.Thetechniqueusedinvolvedaninter-genericcrosswithmaize(Zea maysL.).Therateofembryos/100pollinatedfloretswas16.1.Thespeltbreedinglinehadnosignificanteffectonembryoproduction,butthemaizevarietydid,andaninteractionbetweenspeltbreedinglineandmaizevarietywasfound.Thebestratewasobtainedwithamaizevarietyofthepopcorntype.Therateofcaryopsisformationwaslow(66.2caryopses/100pollinatedflorets).Therateofplantletregenerationwasverylow(38plantlets/100embryos),withthemaizevarietyhavinganimpact,butnotthespeltline.Keywords.Triticum spelta,Zea mays,haplomethods,intergenerichybridization.

Étude de l’influence du traitement hormonal et de la variété de maïs (Zea mays L.) dans l’haplodiploïdisation de l’épeautre (Triticum spelta L.). D’après nos connaissances, il s’agit de la première étude sur l’haplodiploïdisation del’épeautre(Triticum speltaL.).Latechniqueutiliséeimpliqueuncroisementintergénériqueaveclemaïs(Zea maysL.).Letauxd’embryons/100fleurspolliniséesétaitde16,1.La lignéed’épeautren’avaitpasd’effet significatif sur laproductiond’embryon,aucontrairedelavariétédemaïsetuneinteractionentrelalignéed’épeautreetlavariétédemaïsaétéobservée.Lemeilleur taux a été obtenu avec une variété demaïs de type popcorn. Le taux de formation de nouaison était faible(66,2nouaisons/100fleurs pollinisées).Le taux de régénération de plantules était très faible (38plantules/100embryons),avecunimpactdelavariétédemaïs,maispasdelalignéed’épeautre.Mots-clés.Triticum spelta,Zea mays,haplométhode,hybridationintergénérique.

1. INTRODUCTION

TheworksofZenkteleretal.(1984),Laurieetal.(1986,1987,1988)andSuenagaetal.(1989)enabledtosetthetechniquesofhaplodiploidizationonwheatthroughcrossing with maize. Since then, haplodiploidizationhasbecomeanimportanttoolinplantbreedingandisincreasinglyusedinpure-lineprograms.Spelt(Triticum spelta L.) isanancientsubspeciesofcommonwheat(Triticum aestivum L.) mainly cultivated in Europe.So far asweknow, noworkhas beendoneon spelthaploidiploidization. The objective of the study wasto evaluate the impact of themaize genotype on thehybridization with spelt; and of growth substancetreatmentontheproductionofcaryopses(C).

2. MATERIALS AND METHODS

2.1. Materials

TenF1breedinglinesfromCRA-Wwereselectedonthediversityof theirgenitors (22).Twosweetmaizevarieties(GBandTS)andonepopcornvariety(MPC)werechosen.

2.2. Methods

Twenty-sevengrainsofthespeltlineweresownin4Lpots.Thesubstratewashalfblondeandhalfbrownpeat.Onemonthaftersowing,theplantswerevernalizedat4°Cover8weeks.Theyweretransplantedandplaced

Growthsubstancetreatmentandmaizevarietyinspelthaplodiploidization 33

in a greenhouse at 16-18°C, with a photoperiod of16hoflightand8hofdark.Thesodium-vaporlampprovided107µE.m-2.s-1.Fifteengrainsofmaizeweresown in10Lpotswith the samesubstrateas for thespelt. About 19-20weeks later, some mature pollenwasavailable.Theplantsweretransplantedandplacedinagreenhouseat20-35°C;photoperiodandlightingwerethesameasforspelt.

Whenspeltheadingoccurredandbeforetheanthersdehisced,onlytheprimaryandsecondaryfloretsonthecentralportionofthespikewereretained.Emasculationwas donemanually 1-4days before anthesis (Sarrafiet al.,1994; Inagakietal.,1998).Whenspelt stigmawasfeatheryandfluffy(Campbelletal.,1998),1-4daysafter emasculation, fresh maize pollen was placedon theflorets.Two solutions of 2,4-dicholorphenoxyacetic acid (2,4-D) and gibberellic acid (GA3) bothat100mg.l-1werestoredat4°C.The2,4-DactsasagrowthsubstanceandGA3playsaroleinthegrowthofthecaryopsis(C).Twenty-fourhoursafterpollination,spikewaspulverised.

Fourteendaysafterpollination,Cswere removedfromthespikesanddisinfectedunderlaminarflow.TheCswere rinsed three times over 5minwith distilledwaterandtheCswerethendissectedtocheckforthepresenceofanembryo(E).TheEswerecontrolledtobehaploids(H)withtheaspectoftheC:aCfromaninterspecificcrossinghasanaqueousendospermwhilea C from self-pollination has a milky endosperm.H Eswere removed under amicroscope LeicaM26(4x10)andsetincultureinaPetridishwithculturemediaB5(Gamborgetal.,1968).ThePetridishwasclosedandplacedindarknessinanincubatorat28°Cuntilgerminationandtheappearanceofthefirstroots.ThePetridishwasmovedtoacultureroomat20°C,with a photoperiod of 16h light and 8h dark under28.5µE.m-2.s-1. After 14days, plantlets (P) weretransplanted into a 55mm diameter container withculturemediaB5.

In the first experiment, the spelt breeding linesGE10,12,21,28,33and43werechosen.Tennon-emasculatedspikesperbreeding linewerepollinatedwith eachmaizevariety.This represented180spikesfortheexperiment:5,404pollinatedflorets(PFs).Onlythe2,4-Dsolutionwaspulverised.

Inthesecondexperiment, thespeltbreedinglinesGE 9, 22, 26 and 38were selected. Fourteen spikesperbreedinglinewerepollinated(sevenemasculatedspikes and seven non-emasculated spikes) and twotreatmentswereappliedtobothsetsofspikes:– 2,4-Dalone24hafterpollination;– 2,4-D 24h after pollination plus GA3 48h after pollination.

In total, 28spikes were worked per breeding line:112spikesor3,374PFs.NumberofCs,EsandPswas

collected.Theresultswerereportedonaper-PForper-Ebasis.TheproportionsofEsper100PFs(E/100PF),PperE(P/E),CsperPF(C/PF)andPperPF(P/PF)were calculated. An analysis of variance (ANOVA)evaluated the impact of the treatments, based on thegeneral linearmodel, using the software Statbox 7.1(Grimmersoft,Issy-les-Moulineaux,France).

3. RESULTS AND DISCUSSION

3.1. Impact of maize variety on hybridization with spelt

In the first experiment, 3,484Cs, 872Es and 280Pswereobtained.

Production of embryos.Forthesixbreedinglinesandthethreemaizevarieties,16.1E/100PFwereobtainedonaverage,withthehighestbeing18.5±6.8E/100PFfor MPC (Table 1). The rates of embryo formationreportedinliteraturewere:14.4-28.0%(Laurieetal.,1987),20.6%(Laurieetal.,1988);8.3-21.1%(Inagakiet al., 1992); 15.1% (Verma et al., 1999); 22.5%(Martins-Lopes et al., 2001); 13.9% (Sharma et al.,2002);and3.8%E/PF(Torresetal.,2010).

In thepresentstudy, theANOVAshowedthat themaize variety had a significant effect on the successofembryoproduction(p=0.049)whichisconsistentwith literature (Suenaga et al., 1989; Matzk et al.,1994;Zhanget al.,1996)and thebestmaizevarietywas the popcorn type, which is consistent with theworkofVermaetal.(1999).

Herehowever,theANOVAshowedthatthebreedinglinehadnoeffectonthesuccessofembryoproduction.In the literature, on the one hand, a considerablevarietaldifferenceinefficiencyamongwheatvarietieshasbeenobservedinembryoproduction(Bitschetal.,1998;Martins-Lopesetal.,2001;Torresetal.,2010)oronC/PFandE/Crates(Sharmaetal.,2002).Whileon theotherhand, severalauthorshave reported thatwheat genotypes do not affect embryo production(Suenagaetal.,1989;Matzketal.,1994;Zhangetal.,1996). In the present study, a significant interactionbetween spelt breeding line and maize variety wasobserved(p=0.004).Bitschetal.(1998)andLefebvreet al. (1996) reported that interactionwas significantfor HP formation and for the number of P/100PF,respectively.

Regeneration of embryos to plantlets. Inthepresentstudy,thenumberofPobtainedfromthenumberofEsplacedinculturewas38.0%(Table 2).Theliteraturereports rates of 68.1% (Laurie et al., 1988), 85%(Laurieetal.,1991),43.1%(Inagakietal.,1992),43%(Sharmaetal.,2002)and26.1%(Torresetal.,2010).

34 Biotechnol. Agron. Soc. Environ. 201418(1),32-36 EscarnotE.,ThibautCh.&ForgeoisP.

Lefebvreetal.(1996)reportedarateofHPproductionof 9.1 (4.4-14.7) per 100florets, comparedwith 4.8-8.6P/100PFinthepresentstudy,whichislower.

TheANOVAshowedaneffectofthemaizevariety(p=0.02),butnoeffectofthespeltbreedingline,noran interactionbetween the two factorson the rateofregeneration.ThehighestrateforMPCwas46.5%;itwas34.6%forGBand32.8%forTS.Eventhebest

rate obtained in the present studywas not as good as those obtainedbyLaurie,butitwasclosetoothersfromliterature.

3.2. Impact of the growth substance treatment

Inthissecondexperiment,2,099Csand574Eswereobtained.

Formation of caryopses. ThenumberofCsobtainedisattributedto the application of growthsubstances. The ANOVA showedthat pulverisation had a significanteffect (p=0.002). The numberof C/100PF was: 66.2 with onepulverisation of 2,4-D 24h afterpollination;and57.4C/100PFwithone pulverisation of 2,4-D after24h and one of GA3 48h afterpollination.Thesimpleuseof2,4-D24hafterpollinationthereforegavethe highest number of Cs. Theserates were low compared withthose reported in the literature forwheat, 87% (Sharma et al., 2002)and69.4%(Torresetal.,2010).Theresults obtained from other studies(Laurie et al., 1991; Inagaki et al.,1995) agreed with the finding ofthe present study that 2,4-D aloneperformsbetterthan2,4-DplusGA3.The C/PF increased significantly(p=0.019) with emasculation,from 58.6 to 65.1%. Emasculationprobably enabled the glumes to beopenedsothathormonaltreatmentswould have better access to thestigmaandbetterpenetration.

Production of embryos. TheANOVA showed that neither thespelt breeding line nor the natureof growth substance influencedthe rate of production of Es. Theemasculation factor, however,

playedasignificantrole(p=0.004).Whenspikeswereemasculated 1-4 days prior to anthesis, 19.3E wereobtainedfor100PFwhereasonly14.6wereobtainedwhen theywere not.Emasculationhas beendone asearlyas4(Sarrafietal.,1994)to5daysbeforeanthesis(Suenagaetal.,1989),2-3daysbeforeanthesis(Zhanget al., 1996; Suenaga et al., 1997), 1-2days beforeanthesis(Matzketal.,1994;Campbelletal.,1998)and

Table 1.Meannumbersofcaryopses,embryosandhaploidplantletsproducedin six spelt breeding lines, depending onmaize variety—Nombre moyen de nouaisons, embryons et plantules haploïdes produites chez six lignées d’épeautre selon la variété de maïs.Spelt lines Evaluated traits Maize varieties*

GB MPC TSGE10 Pollinatedspikes 10 10 10

C/100Fs 71.3 71.8 68.7E/100Fs 14.4 20.4 21.7HPs/100Es 48.4 33.8 38.8

GE12 Pollinatedspikes 10 10 10C/100Fs 50.4 62.6 56.6E/100Fs 10.8 25.0 11.2HPs/100Es 13.7 60.5 34.8

GE21 Pollinatedspikes 10 10 10C/100Fs 66.4 72.1 59.2E/100Fs 15.8 17.6 20.5HPs/100Es 40.8 37.1 27.3

GE28 Pollinatedspikes 10 10 10C/100Fs 62.1 71.2 58.9E/100Fs 12.8 20.0 8.9HPs/100Es 46.3 55.7 30.4

GE33 Pollinatedspikes 10 10 10C/100Fs 46.7 63.5 62.4E/100Fs 16.0 5.7 16.9HPs/100Es 30.0 33.3 19.2

GE43 Pollinatedspikes 10 10 10C/100Fs 67.5 70.9 70.8E/100Fs 13.3 22.4 16.8HPs/100Es 28.8 58.9 46.3

Generalaverage Pollinatedspikes 60 60 60C/100Fs 60.7b 68.7a 62.8b

E/100Fs 13.9±2.0b 18.5±6.8a 16±5.1a,b

HPs/100Es 34.6±13.1b 46.5±13.1a 32.8±9.4b

*:valuesinthesamerowsfollowedbydifferentlettersdiffersignificantlyatP=0.05—les valeurs sur les mêmes lignes suivies de lettres différentes diffèrent significativement à la valeur P = 0,05.

Growthsubstancetreatmentandmaizevarietyinspelthaplodiploidization 35

1daybeforeanthesis(Inagakietal.,1998).Accordingto Laurie (1989) however, intact glumes result inhigher embryoproduction thanwhenglumes are cutbackforemasculation,butSuenagaetal.(1997)foundthattherewasnodifference.Theresultsobtainedinthepresentstudycontradictthosereportedintheliteraturebutemasculationinthecaseofspeltseemedpromising.

4. CONCLUSION

For thefirst experimenton spelt haplodiploidization,therateofEproductionwassatisfying.TherateofPregenerationwasweakoverallexceptforonevarietyofmaize;thissuggeststhatabetterratecouldbeobtainedand integrated into a breeding program. The rate ofCformationwasweak,although in thepresent studyitdidnothaveanimpactontherateofEproduction.Deeper investigations and new procedures need tobe further experimented in order to propose newprotocolsforspelthaplodiploidizationtothescientificcommunity.

Abbreviations

C:caryopse2,4-D:2,4-dicholorphenoxyaceticacidE:embryoGA3:gibberellicacidHP:haploidplantP:plantletPF:pollinatedfloret

Acknowledgements

WewishtothanktheInstitutdeGenechteam–ChristopheBrame, Sophie Fouchet, Marion Levaux, and MélanieWostyn–andtheCentrewallondeRecherchesagronomiquesteam–LucWatelet–fortheirtechnicalassistance.Theauthorsdeclarethattheyhavenoconflictofinterest.

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Table 2.Meannumbersofcaryopsesandembryosproduced in fourspeltbreeding lines,dependingonauxin treatmentandemasculation—Nombre moyen de nouaisons et embryons produit chez quatre lignées d’épeautre selon le traitement hormonal et la castration.Spelt lines Evaluated traits Auxin treatment/emasculation*

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