i C?2o Q\7- C

Synthesis andfertilit y of xBrassicoraphanus andway s of transferring Raphanus characters to Brassica

O.Dolst i

NN0B201.912 O. Dolstra

Synthesis andfertilit y ofxBrassicoraphanu s andway so ftransferrin g Raphanus characters toBrassic a

Proefschrift terverkrijgin gva nd egraa dva n doctori nd elandbouwwetenschappen , op gezagva nd erecto rmagnificus , dr.C.C .Oosterlee , hoogleraari nd eveeteeltwetenschap , inhe topenbaa rt everdedige n opvrijda g2 9oktobe r198 2 desnamiddag s tevie ruu ri nd eaul a vand eLandbouwhogeschoo l teWageningen .

Centre for Agricultural Publishing and Documentation

Wageningen - 1982 Abstract

Dolstra,0. ,1982 .Synthesi san d fertility ofxBrassicoraphanu s andway s oftransfer ­ ringRaphanu s characters toBrassica .Agric .Res .Rep . (Versl.landbouwk .Onderz. ) 917,ISB N90-220-0805-3 ,(viii )+ 9 0p. ,3 8tables ,1 4figs ,11 4refs ,Eng .an dDutc h summaries. Also:Doctora lthesis ,Wageningen .

About25 0intergeneri chybrid swit hth egenom e constitutionAR ,AAR Ro rAR Rwer eob ­ tained fromove r 1500 0crosse sbetwee nBrassic a campestris (AAo rAAAA ,femal eparent ) andRaphanu s sativus (RRo rRRRR) .Th epoo rcrossabilit ywa s shownt ob ea consequenc e ofvariou sbreedin gbarriers .Th ediploi d andtetraploi dhybrid swer e studied inform , fertility,chromosom eassociatio na tmeiosi san d crossabilitybot hamon gth ehybrid s and incrosse swit hth eparenta l speciesan dBrassic a napus•XBrassicoraphanu s (AARR) wasbackcrosse d twicewit hB .campestri s (AA),an dwa sals ocrosse d andbackcrosse d withB .napu s (AACC).For man d chromosomeassociatio na tmeiosi s inAA Ran dAAC Rhy ­ bridswer estudied . Twopopulation so fXBrassicoraphanu s (AARR)wer epropagate d and studied inmor ede ­ tail,partl y forpossibl esuitabilit ya sa ne wcrop .Mos tplant swer ehighl ysteril e becauseo fbreedin gbarriers ,whic hwer esimila rt othos ei nth eorigina l intergeneric crosses.Th eincreas ei nfertilit yan dchromosoma l stabilitywer e studied inth efirs t threegeneration s ofth epopulations .

Freedescriptors :Brassic a campestris,Raphanu s sativus,incongruity ,breedin gbar ­ riers,embry oculture ,intergeneri chybrids ,XBrassicoraphanus ,fertility ,matin g system,introgression ,Brassic anapus ,allosyndesis ,forag ecrop .

Thisthesi swil l alsob epublishe d asAgricultura l Research Reports917 .

© Centre forAgricultura l Publishing andDocumentation ,Wageningen ,1982 .

Nopar t ofthi sboo kma yb e reproduced orpublishe d in any form,b yprint , photoprint,microfil m oran y othermean swithou twritte npermissio n from thepublishers . Stellingen

1.Selecti e in Brassica campestris en Raphanus sativus opee ngoed ekruis - baarheid tussen deze soorten,i nhe tbijzonde r volgens demethod eva n de reciproke récurrente selectie,za lnie t alleen leiden tot eenverbeterin g vand ekruisbaarheid , maar ook totee nverhoogd e fertiliteitva nd e allo- tetraploïde geslachtshybriden.

Ditproefschrift .

2.Mannelij k sterielehomozygot e lijnenva nkoolzaa d enbladkoo l zijnver ­ krijgbaar uitheterozygoo tmannelij k steriel uitgangsmateriaal doorher ­ haalde inductieva n diploïdeparthénogenès e (matromorfie)vi abestuivin ­ genme t xBrassicoraphanus.

Ditproefschrift .

3.Polyploïdisati e doorvalentiekruisinge n (4xx 2 xkruisingen )i soo kbi j generatiefvermeerderd e gewassent everkieze nbove nmitotisch everdubbe ­ lingva nhe tchromosoomaantal .

4.He t iswenselijk , datsynthetisch e amphi(di)ploïde vormen diebruik ­ baar kunnenzij nbi j deveredelin g van cultuurgewassen wordenbewaar d in genenbanken, ookwannee rhe tnu thierva nnie tdirec tduidelij kis .

5.Te nonrecht ebeschouw tRöbbele n allopolyploïdie alsee n doodlopende weg ind eevoluti eva nplanten .

G.Röbbelen ,1979 .Proc .Conf .Broadenin gGenet .Bas eCrops .Wageningen ,1978 . Pudoc,Wageningen ,p .249-255 .

6.D everedelin g vanmai s zou zeergebaa tzij nbi j openbaarheid van de formulesva nhybrid e rassen enbi jee nregelin g diehe tmogelij k maakt datd eouderlijne nva nhybrid e rassenbeschikbaa r komenvoo rwetenschap ­ pelijk onderzoek en kruisingsdoeleinden.

7.He t isnie twaarschijnlij k dati nNederlan d de teeltva nmai svoo r 'corn-cob-mix'ee ngrot evluch t zalnemen . 8. De plantenveredeling zou verrijkt worden met een aantrekkelijke methode voor het verbeteren van waardevolle genotypen, indien somatische recombi- natie in een relatief hoge frequentie geïnduceerd zou kunnen worden in cultures van protoplasten en cellen.

9. De 'recombinant-DNA'-technieken vormen weliswaar een belangwekkende nieuwe technologische ontwikkeling, maar de betekenis ervan voor de plantenveredeling wordt erg overtrokken.

R. Keuning et al., 1980. Chemisch Magazine. Bijvoegsel Chemisch Weekblad 10: 156-161. J. Ingle, 1982. Span 25: 50-53.

Proefschrift van 0. Dolstra characters' £££* °f ^-^^or^^s and ways of transferring Ra^hanus Wageningen,2 9oktobe r198 2 Woord vooraf

Hetbeschreve n onderzoekwer dverrich t aan de Stichtingvoo rPlanten ­ veredeling (SVP)t eWageningen , inhe tkade rva nhe tprojec t 'Cytogene- tischonderzoe k aansoort -e ngeslachtsbastaarde nva nlandbouwcruciferen' . Hetbestuu r end edirecti eva n de SVPhebbe nmi j in staatgestel d dit onderzoek teverrichte n end e resultaten tebewerke n totee nproef ­ schrift. Ikbe nhe n zeer erkentelijk voor hetvertrouwe n datz e daarbij inmi jgestel dhebben . Ditproefschrif t isto tstan dgekome nn a intensief overlegme tprof . dr. ir.J.G.Th . Hermsen enprof .dr . ir.J . Sybenga.Hu n opbouwende com­ mentaren hebben eenduidelijk e invloed gehad op de eindversie van dit proefschrift. Ikbe nhu nhiervoo rvee l dank verschuldigd. Eenpromotie-onderzoe k isnooi tee n eenmansactie.Vee l SVP-ers hebben hieraan eenzee rgewaardeerd e bijdrage geleverd. Enkelenwi l ikhie r met name noemen. Ir.H .Toxopeu s heeftee ngrot e inbreng gehad bijhe t formu­ lerenva nbovengenoem d project enveelvuldi g alsvraagbaa k gefungeerd. Zijnenthousiasm evoo r de landbouwcruciferen werktebijzonde r stimulerend. Verderwer dvee l steunverkrege nva n ing.G.J . Speckmann en ind e laatste jarenvoora lva n dr.ir .W . Lange.D e heerJ.H.M .Zuidgees theef t ind e eerste jarenva nhe tonderzoe k opbekwam e en actievewijz e assistentie verleend. Ind e slotfaseva nhe tonderzoe k hebbend ehere n J.H. Lubberts enH .Dijkstr a mettoewijdin g informatie verzameld over de fertiliteit van xBrassicoraphanus. Kortetij d zijnvoort s de STOVA-stagiaires Marianne van derBlom ,Aleid ava n deKraat s enGerar d Bremer actiefbi jhe tonder ­ zoekbetrokke ngeweest . Hetvel e typewerk iso p de SVPuitgevoerd , waarbij inhe tbijzonde r mw.A .Meereboe r zichverdienstelij k heeftgemaakt .Vee l foto's in hetproefschrif tzij ngemaak tdoo r fotografenva nhe tBG D teWageningen . De grafiekenwerde ngeteken d door dehee rG . Post (SVP).D e correctie van deEngels e tekste nd euitgav eva nhe tproefschrif twerde n respectieve­ lijkverzorg d door dehee r J.C. Rigg B.Sc. en ing.J . Casteleinva n het PUDOCt eWageningen . Curriculum vitae

De auteurwer d op2 6 januari 1946 te Steenwijkerwold geboren. In196 5 behaalde hij aanhe tJ.H . TrompMeesterslyceu m te Steenwijk het diploma HBS-B.N ahe tvervulle nva nzij nmilitair e dienstplicht,bego n hij in 1967ee n studie aand eLandbouwhogeschoo l teWageningen ,waa rhi j inja ­ nuari 1975he tdoctoraaldiplom a behaalde ind eplantenveredelin g met als keuzevakken erfelijkheidsleer (verzwaard)e n algemene planteziektenkunde. Sinds 1 september 1974i shi j indiens tva nd e Stichtingvoo r Plantenver­ edeling teWageningen ,waa r sindsdien hetbeschreve n promotie-onderzoek werdverricht . Ind eperiod e 1974-1977wa shi j tevensverantwoordelij k voor het cytogenetisch onderzoek aangranen . Sinds eind 1977 is de auteur belastme the tveredelingsonderzoe k aansnijmais . Contents

1 General introduction 1 1.1 Rationale 1 1.2 Speciesan dallopolyploi dhybrid s 1 1.3 Scopeo fth eresearc hprogramm e 2

2 Crosses between Brassica campestris and Raphanus sativus 5 2.1 Introduction 5 2.2 Materialan dmethod s 6 2.3 Results 9 2.3.1 Settingo fsiliqua ean dseed s 9 2.3.2 Seedsiz e 9 2.3.3 Productiono fhybrid san dmatromorph s 10 2.3.4 Useo fgeneti cmale-sterilit y 13 2.3.5 Cultureo fembryo s 14 2.3.6 Genomeconstitutio nan dnumbe ro fchromosome si n theprimar yhybrid s 15 2.4 Discussion 17

3 Nature of breeding barriers 19 3.1 Introduction 19 3.2 Materialan dmethod s 19 3.3 Results 20 3.4 Discussion 22

4 Primary allodiploid ARhybrids and their progeny 24 4.1 Introduction 24 4.2 Materialan dmethod s 25 4.3 Results 26 4.3.1 Formo fA Rhybrid s 26 4.3.2 Chromosomeassociatio na tmeiosi s 28 4.3.3 Malean dsee dfertilit y 30 4.3.4 Crossabilityo fA Rhybrid swit hvariou sspecie s 31 4.3.5 Somatican dmeioti cdoublin go fchromosom enumbe r 33 4.3.6 Progenyo fcrosse sbetwee nA Rhybrid s 33 4.4 Discussion 34 5 xBrassicoraphanus: form, fertility and crossability with various species 37 5.1 Introduction 37 5.2 Material andmethod s 38 5.3 Results 38 5.3.1 Form and fertilityo fprimar y hybrids 38 5.3.2 Crossabilitywit h Brassica campestris, Raphanus sativus and B. napus 40 5.4 Discussion 42

6 xBrassicoraphanus: sterility factors and mating system 44 6.1 Introduction 44 6.2 Material andmethod s 45 6.3 Results 45 6.3.1 Meiosis 45 6.3.2 Barriersi nth eprogami c phase 49 6.3.3 Barriers after fertilization 52 6.3.4 Mating system 54 6.4 Discussion 55

7 Improvementin fertility of xBrassicoraphanus 57 7.1 Introduction 57 7.2 Material andmethod s 57 7.3 Results 59 7.3.1 Chromosome numbers 59 7.3.2 Changesi nfou r fertility characteristics 60 7.3.3 Effectso fchromosom e numbero nfertilit y 64 7.4 Discussion 64

8 AAR hybrids and introduction of alien chromosomes into Brassica campestris 66 8.1 Introduction 66 8.2 Material andmethod s 66 8.3 Results 67 8.3.1 Form and chromosome association inAA R hybrids 67 8.3.2 Attemptst ointroduc e alien chromosomes 69 8.4 Discussion 71

9 Ways of transferring Raphanus characteristics to Brassica napus 72 9.1 Introduction 72 9.2 Material andmethod s 72

9.3 Results 73 9.3.1 Hybridsfro mcrosse sbetwee n xBrassicoraphanus and Brassica napus 73 9.3.2 Chromosomeassociatio ni nAAC Rhybrid s 75 9.3.3 Firstbackcros sgeneratio n 76 9.4 Discussion 76

10 Prospects 78 10.1 Resistancet obee teelwor m 78 10.2 xBrassicoraphanus 78 10.3 Introgression 79

Summary 80

Samenvatting 83

References 86 1 General introduction

1.1 RATIONALE

Likemos tothe rcruciferou scrops , Brassica spp.ar ehost st oth ebee t eelworm (Heterodera schachtii Schm.)(de nOuden ,1954 ;Steele ,1965) , whichi sdistribute dal love rth eworl d (Stelter,1973 )an di sth emos t seriousnematod eo fsugar-bee t (Beta vulgaris L.)i nEurop e(Heijbroek , 1979).I nth eNetherlands ,fo rexample ,a quarte ro fth esugar-bee tare a hasbee ninfecte db ythi snematode ,especiall yi nth esouth-wes tan d south-east (Heijbroek,1979) .Problem saris emainl yfro mto onarro wcro p rotations.Th egrowin go fsugar-bee ti seconomicall yattractiv et oth e farmers,an dthe ywil ltr yt ocontinu ewit hsuc hrotations ,despit edis ­ advantagesfro ma phytosanitar ypoin to fview . Underpresen tcircumstances ,n oplac ei slef tfo r Brassica cropsi n rotationswit hbeets ,unles sresistan tvarietie sar ebre dwit hpreferabl y theabilit yt oreduc eth epopulatio ndensit yo fth eeelworm .I nth egenu s Brassica, however,n oresistanc eha sbee nfoun dsofa r(Baukloh ,1976 ; Lubberts& Toxopeus ,1982) .I nth e1960s ,report sappeare di nGerma n farmingjournal so nresistanc ei n Raphanus sativus L.var . oleiformis, theoil-see dradish .Th eearl ycontradictor yreport swer ereviewe db y Hirling (1976).Wit hoil-see dradis hth eeelwor musuall ymultiplie dslow ­ ly,bu ti na fe wreport srathe rquickly .I nrecen tyears ,th epresenc e ofresistanc ei n Raphanus hasbee nconfirme db yBauklo h (1976)an dToxo ­ peus& Lubbert s (1979).Bauklo h(1976 )studie dth einheritanc ean dassum ­ eda singl edominan tgen efo rresistance . Thegenu s Brassica includesmajo rcrop sfo rfodder ,oil-see dproduc ­ tion,an dgree nmanure ,som eo fwhic hmigh tfi tquit ewel lint ocro pro ­ tationswit hsugar-beet .S o aprogramm ewa sstarte do nintergeneri chy ­ bridizationbetwee n Brassica spp.an dnematode-resistan tform so f R. sa­ tivus withth eultimat egoa lo ftransferrin gresistanc et o Brassica. Asecon dargumen tfo rthi sprogramm ewa sth epresenc eo fresistanc et o club-root (Plasmodiophora brassicae Woron.)i noil-see dradish .

1.2 SPECIESAN DALLOPOLYPLOI DHYBRID S

Theprogramm eo nintergeneri chybridizatio ninclude sth especie s R. sativus (2w= 18 ,RR) ,B. campestris (2n= 20 ,AA )an d B. napus (2n= 38 , AACC).Thei rgenom econstitution san dinterrelationship sar eoutline di n Figure1 (Mizushima ,1980) . B. napus isa natura lallotetraploi dan di s composedo ftw ogenome so f B. campestris andtw oo f B. oleracea (2n =18 , CC).Tabl e1 enumerate sth esubspecie so rbotanica lvarietie sdistin ­ guishedi nthes especies .Accordin gt oHel m (1957), R. sativus comprises fivebotanica lvarieties ,whic hinclud evariou svegetable san dagricul ­ turalcrops .Th ebes tknow ni nEurop ear eradish ,gian tradish ,blac k radish,an doil-see do rfodde rradish .Th elatte ri showeve rmainl yuse d asgree nmanure .Mougri-radis hi sgrow ni nsouth-eas tAsia ,especiall y forit sleave san dyoun gpod s (Banga,1976) .Fo r B. campestris, theclas ­ sificationo fOlsso n(1954 )ha sbee nfollowed .H edistinguishe dvariou s subspecies,whic hwer eclassifie db yothe rtaxonomist sa sseparat espe ­ cies. B. campestris containsman yorienta lleaf yvegetables ,an dsevera l root,oilsee dan dfodde rcrops . B. napus alsoinclude smajo rroot ,oil ­ seedan dfodde rcrops ,althoug hit sdomesticatio nstarte donl ya fe whun ­ dredyear sag o(Toxopeus ,1974a ;McNaughton ,1976) .Sinc eth ediscover y ofth egenom econstitutio no f B. napus (U,1935) ,thi sspecie sha softe n beenresynthesize d fromit sdiploi dancestors .Thi sha sincrease dgeneti c variationi nth eexistin gcrop san deve nresulte di na headin gtyp ecal ­ led 'Hakuran'(Nishi ,1980) .Mor edetaile dinformatio no nth efou rspe ­ ciesi nth epresen tstud yi sfoun di nBoswel l (1949),Herklot s (1972), Simmonds(1976 )an dNish i (1980). Intergenerichybrid sbetwee n R. sativus and Brassica specieshav ea longan dwel lknow nhistory .A tpresen tvariou smeioticall ystabl eal ­ lopolyploidsar eknow n(Fig .1) .Plant swit hth egenom econstitutio n RRCCwer ealread yobtaine db yKarpechenk o (1927;1928 )an dname d Raphanobrassica. Someyear slater ,Terasaw a (1932)wa sth efirs tt opro ­ duceallotetraploid swit hth egenom econstitutio nAARR ,whic hh ecalle d Brassicoraphanus. Inthi s study,th ename sx Raphanobrassica and x-Brassi- coraphanus areuse dfo rplant swit hth egenom econstitutio nRRC Can d AARR,respectively ,irrespectiv eo fth efemal eparen ti nth eorigina l crosses.Accordin gt oth einternationa lrule sfo rnomenclature ,thos e namesshoul db eprecede db ymultiplicatio nsig n(Internationa lBurea u forPlan tTaxonom yan dNomenclature ,1980) .A les sknow ntyp ei sth e allohexaploidwit hth econstitutio nAACCRR ,whic hwa srecentl yproduce d byClaus s (1978).

1.3SCOP EO FTH ERESEARC HPROGRAMM E

Thepresenc eo fresistanc et oth ebee teelwor mi n R. sativus wasth e mainreaso nfo rstartin ga programm eo nintergeneri chybridizatio ni n ordert otransfe rthi strai tt oth egenu s Brassica, especiallyt o B. campestris and B. napus. Thosespecie sinclud emos to fth emajo rarabl e Brassica cropsgrow ni nth eNetherlands .Figur e1 indicate stha tvariou s waysfo rtransferrin ggene sfro m Raphanus to Brassica mayb efeasible . Table 1.Lati nan d commonEnglis hname so fsubspecie s andbotanica lvarietie s in Raphanussativu sL. ,Brassic a campestrisL .an dBrassic anapu sL .

Species Subspecies/ Commonname(s ) botanicalvariet y

R.sativu sL . var.gayanu sWebb , wild radish var.oleiformi sPers . oil-seed orfodde rradis h var.mougr iHel m Mougri-radisho rrat-taile d radish var.nige r (Miller) blackradish ,gian tradish , Pers.sens ulat . Japaneseradis h var. sativusHel m radish

B.campestri sL . ssp.eu-campestri sOlsso n wildtyp e ssp.oleifer a (Metzg.)Sinsk . turnip-rape ssp.rapifer a (Metzg.)Sinsk . turnip ssp.chinensi s (L.)Makin o Chinesemustar d ssp.pekinensi s (Lour.)Olsso n Pe-tsai,Chines ecabbag e ssp.narinos a (Bailey)Olsso n ssp.nipposinic a (Bailey)Olsso n ssp.dichotom a (Roxb.)Olsso n toria ssp.perviridi sBaile y tendergreen ssp.triloculari s (Roxb.)Olsso n yellow-seeded sarson

var.napobrassic a (L.)Peterm . swede ssp.oleifer a (Metzg.)Sinsk . oil-seed and fodder rape

R. sativus

xBrassicoraphanu s xRaphanobrassic a

B.campestri s B.napu s B.oleracea Figure 1.Genom edesignations ,chromosom enumber san dinterrelationship s ofRaphanu s sativus,som eBrassic a speciesan dallopolyploi dhybrids . Directintrogressio nint o B. napus bycrossin gthi sspecie swit h R. sa- tivus wasapparentl ydifficult ,althoug hsom ehybrid shav ebee nobtaine d (seefo rinstanc eYarnell ,1956) .A mor epromisin gfirs tste pwa sth e cross B. campestris x R. sativus, because (1)th ecrossabilit yi sbette r (Yarnell,1956 )an d(2 )th eresultin gallotetraploi dhybrid sma yb ea bridgebetwee n R. sativus and B. napus. Soal leffort swer econcentra ­ tedo nthi sparticula rcros s(Chapte r2) . Mostinitia lhybrid sgre wvigorousl yan dsuggeste dtha tth eallote ­ traploidhybrid s (= xBrassicoraphanus) mightb esuitabl ea sa fodde r crop,combinin grapi dgrowt han dbee teelwor man dclub-roo tresistanc e ofoil-see dradis hwit hth ebette rpalatabilit yo fth e Brassica parent. Aprerequisit efo rsuccessfu lselectio nwithi nsuc ha 'newspecies 'i s theavailabilit yo fa broa dgeneti cvariation ,i.e .t oproduc ehybrid s involvingman ygenotype so fth eparenta lspecies .S oth enatur eo fbreed ­ ingbarrier san dway so fimprovin gcrossabilit ybetwee n B. campestris and R. sativus werestudie d(Chapte r2 an d3) . Severalpropertie so f.th eprimar yhybrids ,e.g .form ,fertility , crossabilityan dmeiosi sar edescribe di nChapte r4 an d5 .Possibl e causeso fsterilit yan dimprovemen to ffertilit yi n xBrassicoraphanus aregive ni nChapte r6 an d7 . Wetrie dt otransfe r Raphanus genest o Brassica, intw oways .Th e firstapproac hwa st oproduc ei n B. campestris monosomicadditio nline s carryinga nextr a Raphanus chromosome.Th eintentio nwa st otes tsuc h linesfo rresistanc et ovariou sdisease san dafterward st otransfe rth e resistancegene sfro mseparat e Raphanus chromosomesi na mor edirecte d attemptint oth e B. campestris genome (Chapter 8). Thesecon dapproac h wasa backcros sprogramm eo f xBrassicoraphanus with B. napus asth ere ­ currentparen t(Chapte r 9). 2 Crosses between Brassica campestris and Raphanus sativus

2.1 INTRODUCTION

Sinceth e1920 ssevera lresearc hworkers ,especiall yfro mJapa nhav e reportedth ehybridizatio no f B. campestris and R. sativus (Table 2). Terasawa& Shimotoma i (1928)obtaine dth efirs tdiploi dhybrids .Mor e reportso nsuccessfu lhybridizatio nfollowed .Initiall yth ecrosse swer e .carriedou ta tth ediploi dleve lonl ybu tlate rautotetraploi d formso f eitheron eo rbot hspecie shav ebee nuse da swell ,especiall ydurin gth e lasttw odecade s(Tabl e 2). Crossabilitywa spoo ri nal lcombinations . Thebes tresult swer enevertheles sobtaine dfro mcrosse sbetwee ndiploi d forms,usin g B. campestris asth efemal eparent .I nthos ecrosses ,ther e wasmarke dvariatio ni nth enumbe ro fhybrid sproduce dpe rpollinatio n (Table 2). Asfa ra sknown ,th eprimar yhybrid sreporte db yvariou sauthor s (Table2 )ha dnearl yalway sth eexpecte dgenom econstitution ,bu tsom e deviantplant swer efoun di n2 xx 2 xcrosse swit h R. sativus asth efe ­ maleparent .U e tal . (1937),Morri s& Richhari a (1937)an dMizushim a (1950a)obtaine damon gthei rhybrid sfiv eallotriploi dplant scomprisin g twogenome so f Raphanus andon eo f Brassica. Theseplant sprobabl yaros e froma fertilize d2 neg gcell . Apartfor ma ninteres ti nth eintergeneri chybrid sa ssuch ,th e crossesmentione di nTabl e2 hav ebee nmad emainl yfo rtw oreason s:t o studyth ecause so fmatromorph y (Nishie tal. ,1964 ;Tokumasu ,1965 ; 1970a;Open a& Lo ,1978 )an dt oge tinformatio no ninterspecifi cincom ­ patibility (Kakizaki,1925 ;Becker ,1951 ;Hinat ae tal. ,1974) .Usuall y progenyo fcrosse sbetwee n B. campestris and R. sativus consist partly ofmatromorphi cplants .Tokumas u (1965;1970a )showe dtha tsuc hplant s wereno tcompletel yhomozygou sa sNish ie tal . (1964)assumed .Matromor ­ phyi nCrucifera ei sa kin do pdiploi dparthenogenesi san dha sbee nob ­ servedi nman yinterspecifi can dintergeneri ccrosse swithi nthi sfamil y (Eenink,1974) . Thischapte rpresent sresult so fintergeneri ccrosse sbetwee ndiploi d andtetraploi dform so f B. campestris and R. sativus. Thelatte rspecie s wasalway suse da sth epolle ndonor ,becaus ei nthi sdirectio nth ecross - abilitywa smuc hbette rtha ni nth ereciproca l (Table 2).Man yaccession s of B. campestris wereuse di norde rt ominimiz eth echanc eo fusin gpoor ­ lycrossabl egenotype sonly .Th emal eparent svarie dless ,bein gconcen - Table 2. List of reported crosses between diploid and tetraploid forms of Brassica campestris (AA and AAAA) and Raphanus sativus (RR and RRRR).

Cross Numbero f Numbero f Numberfract ­ Source po:llination s hybrids ionhybrid s to pollinat- ions(% )

AAx R R 33 0 0 Kakizaki (1925) 90 6 6. 7 Terasawa& Shimotoma i (1928) 428 12 2.8 Morris& Richhari a (1937) 217 0 0 Becker (1951) 903 167 18.5 Mizushima (1952) 8 003 263 3.3 Nishie tal . (1964) 286 31 10. 8 Tokumasu (1970b) 82 0 0 Hinatae tal . (1974) 13 287 44 0.3 Opena& l o (1978) 2 111 13 0.6 Namai (1980) 24 0 0 Takeshitae tal . (1980)

RRx A A 39 0 0 Kakizaki (1925) 30 0 0 Terasawa& Shimotoma i (1928) ? >2 0.2 Ue tal . (1937) 413 2 0.5 Morris& Richhari a (1937) 328 0 0 Becker (1951) 4 775 2 0.0 4 Nishie tal . (1964) 417 0 0 Tokumasu (1965) 1 111 0 0 Namai (1980) 19 0 0 Takeshitae tal . (1980)

AAx RRR R ? 1 2 Ellerström (pers.commun. )

RRRRx A A ? •> 0 Ellerstrom (pers.commun. )

AAAAx R R 481 0 0 Nishie tal .(1964 )

RRx AAA A 471 0 0 Nishie tal .(1964 )

AAAAX RRR R 58 0 0 Tokumasu (1970a)

RRRRx AAA A 132 0 0 Tokumasu (1970a)

Remark: Intergeneric hybrids were also reported by Morris (1936), Nishiyama (1946), Hosoda (1946; 1947) Mizushima (1950a), Clauss (1978), McNaughton &Ros s (1978), Ellerström (1978), Olsson &Ellerströ m (1980), Namai et al. (1980).

trated to fodder radish because of the requirement of combined resistance to beet eelworm and club-root. Some attempts were made to improve the re­ sults of these intergeneric crosses by culture of immature embryos in vitro.

2.2 MATERIAL AND METHODS

The diploid and tetraploid genotypes of B. campestris and R. sativus used for intergeneric hybridization are listed in Table 3. The fodder radishes 'Siletta', 'Levana', 'Palet' and RS14 were chosen because they were partially resistant to beet eelworm. In most tests for resistance Table3 .Description ,ploid yan dorigi no fBrassic aan dRaphanu saccession suse d forintergeneri ccrosses .

Designation Species/subspecies Description Ploidy Origin orbotanica l variety.

B.campestris Al ssp. rapifera inbred lineBC18 3 2x 1 A2 ssp. rapifera inbred lineBC18 2 2x 1 A3 ssp. rapifera inbred lineBC17 1 2x , 1 A4 ssp. rapifera inbred lineBC17 2 2x 1 A5 ssp. rapifera inbred lineBC18 1 2x 1 A6 ssp. rapifera inbred lineBC17 6 2x 1 A7. ssp. rapifera inbredlin eBC18 4 2x 1 A8 ssp. rapifera cv.Trofe e 2x 2 A9 ssp. oleifera boterzaad 2x 1 A10 ssp. pekinensis 2x 1 All ssp. chinensis Shan-Jue-Man-Tsien-Tsaj 2x 3 A12 ssp. chinensis 2x 1 A13 ssp. chinensis S linefro mA1 2 2x AA1 ssp. rapifera cv.Novita s 4x 4 AA2 ssp. rapifera cv.Tarond a 4x 5 AA3 ssp. rapifera cv.Tigr a 4x 6 AA4 ssp. pekinensis 2243 4x 7 AA5 ssp. nipposinica 2245 4x 7 AA6 ssp. perviridis 2240 4x 7 AA7 ssp. narinosa 2260 4x 7 AA8 ssp. chinensis 2242 4x 7

R.sativus Rl var. oleiformis cv.Silett a 2x 8 R2 var. oleiformis cv.Levan a 2x 8 R3 var. mougri Rs4.0 1 2x 1 R4 var. niger Ra74/6 5Chin a1956:Kanto n 352 x 3 R5 var. oleiformis Rs6.0 4 2x 1 R6 var. niger Ra 113/64Japanes ewhit e NewDelh i 2x 3 RR1 var. oleiformis cv.Pale t 4x 8 RR2 var. oleiformis RS14 4x 9

*] 1.Stichtin gvoo rPlantenveredelin g (SVP),Wageningen ,Th eNetherlands . 2.Zwaa n& D eWiljes ,Zaadteel te nZaadhande lBV ,Scheemda ,Th eNetherlands . 3.Zentralinstitu tfü rGeneti kun dKulturpflanzenforschung ,Gatersleben , DDR. 4.Cebeco-Handelsraad ,Rotterdam ,Th eNetherlands . 5.Zelde rBV ,Ottersum ,Th eNetherlands . 6.Kon .Zaadteel te nZaadhande lSlui se nGroo tBV ,Enkhuizen ,Th eNetherlands . 7.Swedis hSee dAssociation ,Svalöv .Sweden . 8.Kon .Kweekbedrij fe nZaadhande lD.J .va nde rHav eBV ,Kappelle , TheNetherlands . 9.Scottis hPlan tBreedin gStation ,Pentlandfield ,Unite dKingdom .

usually somemultiplicatio n ofthi snematod e occurred,bu tt o arelati ­ vely low degree (Toxopeus,pers . commun.). Fodder-radish varieties often have ahig h degree ofresistanc e to club-root (Crutee tal. , 1980). The Germanvariet y Silettawa s resistant toman y isolates of club-root (Toxopeus, 1974b). The fodder radishes 'Levana' and 'Palet',ar e direct derivatives from 'Siletta'an dhav eprobabl y the same genes forresis ­ tance. Mosto fth eplan tmateria lwa s grown inrathe rwel l conditioned green­ houses attemperature svaryin g from 15t o 20 °Can d adaylengt h between 14an d1 7h .Th eplant swer e growni npot s ofcapacit y about 2\ litres. Theintergeneri ccrosse swer emad eb yhan d inth eperio d from Januaryt o Mayo f197 5an d 1976.Ope n flowers andyoun g flowerbud swer e removed from the inflorescences and subsequently theres to fth ebud swa semas ­ culated.Th e inflorescences,wit h an average ofabou t1 5bud swer e bagged.The ywer epollinate d between 13:00 and 15:00 immediately after emasculation in197 5 andtw ot othre eday s after emasculation in197 6b y putting asmal l amounto fa polle nmixtur e onto the stigmaswit h asmal l softbrush . Suchmixture swer e composed of almostequa l amounts of fresh pollencollecte d from2 5t o4 0plant spe r accession.A t themomen t of pollination in1976 ,th eoldes tbu dwa smarke d andbud s andope n flowers ofeac h inflorescence were counted.Thre e to fourweek s later thedevel ­ oped siliquaewer e counted. Atharvest ,th etota l number ofsiliqua e and thenumbe r ofseed swer e counted foreac h inflorescence.Th e largest diameter ofeac h seedwa sde ­ termined asa measur e ofsee dsize .Larg e seedswer egerminate d inPetr i dishes onwe t filterpape r at2 3 °Ci nth edark . Small and shrivelled seedswer e firstdisinfecte d for2 0min .i n aqueous sodium hypochlorite ofmas s concentration 3 g/1, followedb y thorough rinsing in sterilized water.Th e disinfected seedswer e thenals o germinated at2 3 °Ci nth e darkbu ti n smallPetr i dishes on asoli d nutrientmediu m ofth ecompo ­ sitiongive nb yGuh a& Maheswar i (1964). Harberd'smetho d ofembry o culture (1969;1971)wa s used.Th edevelop ­ mental stageso fth eculture d embryoswer e established according to the drawings ofWilma r& Hellendoor n (1968)fo r B. oleracea. Initially all developed ovuleswer e cultured,whethe r anembry owa spresen t ornot , butonl yovule swit h avisibl e embryo havebee n cultured since1976 . Thedistinctio nbetwee nhybrid s andmatromorphi cplant swa s feasible onth ebasi s ofplan tmorphology , aswa s confirmed by countingchromo ­ somes inroot-ti p ormeioti c cells.Th e root-tipswer e treated and squashed forcountin g thechromosome s according to themetho d of Jochemsen& M^ynie c (1974). For counting inmeioti c cells youngbud s were fixed ina solutio n ofthre epart sb yvolum e of aqueous ethanol (volume fraction ofethano l 0.96) and ofon epar tglacia l acetic acid andplace d at4 °Cfo r afe wdays .Th ebud swer e thentransferre d to aqueous ethanol (volume fractiono fethano l 0.7) and kepti nstorag e at 4 °Cfo ra fe wweeks .Th ebud swer e subsequently stainedb y Snow'smeth ­ od (1963)fo rabou t 14h at6 0 °C. Ina smal lexperimen ti n197 7th epossibilit ywa steste d ofusin gge ­ neticallymale-steril e B. campestris plants for intergeneric hybridiza­ tion. Fourmale-steril e F2plant s from acros sbetwee n amale-steril e plant from accessionA 5 and aplan to fAl lwer e transplanted intoiso - lationcage swit h floweringplant so fR. sativus. Honey-bees were used forpollination .

2.3 RESULTS

2.3.1 Setting of siliquae andseeds

Settingo fsiliqua ewa s assessed foral lth e crossesi n197 6 threet o fourweek s afterpollinatio nan da tharvesting .Th e results for2 xx 2x , 2xx 4x , 4xx 2 xan d4 xx4 xar e summarizedi nTabl e4 fo reac h Brassica accession, irrespectiveo fpollinator .Afte r 21-28 days the Brassica ac­ cessions differed verymuc hi nsettin go fsiliquae .Th eploid yo fth e pollinatorsha d apparentlyn olarg eeffec t (Table4) . A lowproportio no fsiliqua e shrivelled away after initial development andwer eno tharveste d (Table 4). Theremainde r onlypartl y contained seeds.Thos e seeds gave sometimes riset omatromorphs , especiallyi n crossesi nwhic hth enumbe r ratioo fdevelope d siliquaet opollinate d flowerswa s comparatively low,a so nA8 ,A9 ,AA2 ,AA 3an dAA4 .

2.3.2 Seed size

Nearly all seeds obtained from thecrosse si n197 5 and 1976wer e

Table4 .Numbe r fractiono fsiliqua ean do fsiliqua ewit hseed st opollinations , and number ratioo fseed spe rpollinatio ni nth ecrosse smad ei n197 6betwee ndiploi dan d tetraploid formso fBrassic a campestrisan dRaphanu s sativus (Tables5 ,6 an d 7).Th e settingo fsiliqua ewa sassesse d21-2 8day safte rpollinatio nan da tharvest .Th ecolum n subheadings indicate thegenom econstitutio no fth epollinator .

Female Numberfractio no fsiliqua e Numberrati oo f Number ratioo f parent topollination s(% ) siliquaewit hseed s seedst o topollination s(% ) pollinations after21-2 8day s at harvest

RR RRRR RR RRRR RR RRRR RR RRRR

B.campestri s (2x) A8 29 20 18 i9 10 16 0.18* 0.35* A9 22 26 22 23 10 23 0.16* 0.45* A13 80 69 72 61 27 13 0.37 0.22

B.campestri s (4x) AA2 9 13 9 13 0.08* 0.12* AA3 2 3 2 3 2 0.02* 0.02 AA4 5 6 5 1 0 0.03* 0 AA5 49 45 15 25 5 0 0.06 AA6 48 53 31 47 17 0.04 0.20 AA7 51 48 16 14 1 0.03* 0.01* AA8 43 40 33 33 3 0.01 0.05

*]Seed sgav emostl yris et omatromorphs . Cumulative frequency

100 ?' y' 90

80 1 ' 70 f I 60 1 I y / ' 50 p / / ƒ

1 40 1 // / f Hybrids 30 - / / ' / o o 2x x 2i / / ' ' û -à 4x x 4x 20 - / / ' ' Matromorphs / / A : 0----O 2x x 2x 10 1/ '' * û—•& 4x x 4x 0 -V^ =32^_i <^_, • 0.5 1-0 1.5 2.0 2.5 3.0 Seed size(mm ) Figure2 .Cumulativ efrequenc ydistributio no fsee d sizeo fhybri dan draatromorphic seedsobtaine dfro m2 xx 2 xan d4 xx 4 xcrosse sbetwee nBrassic a campestrisan d Raphanussativus .

measured.Th e seedswer emostl ywell-fille d andwer e ovoid-spherical, although somewer e shrivelled. The seed size ofhybri d andmatromorphi c seedsobtaine d from2 xx 2 x and4 xx 4 x ispresente d inFigur e 2.Th e hybrid seedswer e onaverag e smaller than seedsyieldin g matromorphic plants,bu tther ewa s tosom eexten t anoverla p in seed size ofhybrid s andmatromorphs ,especiall y for2 xx 2xcrosses .Matromorphi c seeds from 4xx 2xan d2 xx 4 xcrosse swer e similar insiz e to thato f4 xx 4 x and 2xx 2xcrosses ,respectively . The fewhybri d seeds from those crosses were also smaller andwer e atmos t 1-2 mm indiameter . The germinability ofth e seedswa s related to seed size.Th e seeds notgerminatin g orwhos e seedlings diedbefor e the cotyledon stagewer e on average smaller thanth e germinated hybrid seeds andwer e probably mainly hybrid seeds.Th e respective ratios of seeds notgerminatin g to hybrid seeds in2 xx 2x ,4 xx 2x,2 xx 4x and4 xx .4 xwer e approximately 1,7 ,1 8 and 1,indicatin g thatespeciall y in4 x x 2x and 2xx 4 x the germinability ofhybri d seedswa spoor .

2.3.3 Production of hybrids and matromorphs

Theresult s of all intergeneric crossesmad e in 1975 and 1976 are giveni nTabl e 5, 6an d 7.Tabl e 5list s thecrosse sbetwee ndiploids . In197 5nearl y all availableplant s frommos t Brassica accessions were crossed as femaleswit hth e Raphanus accessions,R l and R3. Usually each

10 Table5 .Result so fcrosse sbetwee ndiploi dform so fBrassic a campestris (9)an d Raphanus sativus in197 5 and1976 .

Cross Numbero f Number of Numbero f Numbero f Numberrati oo f hybrids pollinations female plants hybrids matromorphs topollination s (%)

tested yielding average maximum hybrids

1975 Al x Rl 488 18 4 12 21 2.5 17.1 R3 267 13 4 8 17 3.0 11.9 A2 x Rl 207 8 0 0 27 0 0 R3 205 8 0 0 6 0 0 A3 x Rl 429 17 3 6 25 1.4 7.4 R3 462 17 2 6 48 1.3 8.0 A4 x Rl 249 10 0 0 8 0 0 R3 283 10 2 2 11 0.7 3.3 A5 x Rl 443 18 0 0 12 0 0 R3 400 17 2 3 11 0.8 7.4 A6 x Rl 364 15 2 2 3 0.5 6.3 R3 344 14 3 4 3 1.2 15.0 A7 x Rl 423 18 2 4 7 0.9 10.0 R3 432 17 3 11 21 2.5 36.0 A10x Rl 44 .2 0 0 0 0 0 Allx Rl 346 14 3 3 20 0.9 5.6 R3 295 13 0 0 30 0 0 A12x Rl 102 1 1 4 0 3.9 3.9

1976 A8 x Rl 239 10 0 0 41 0 0 A9 x Rl 105 7 1 1 16 1.0 5.0 A13x Rl 182 11 10 44 8 24.2 43.8 R2 129 4 3 18 1 14.0 122.2

Total 6438 128 336 2.0 122.2

plantwa s pollinated withbot hpollinator s onth e same day and an almost equal number ofpollination swa smad e foreac hpollinator .Th e number ratio ofhybrid spe rpollinatio nwa s low inbot hyear s andrange d from 0 to24. 2% betwee ncrosses .A largevariatio n inhybri dproductio nwa s also foundwithi n the Brassica accessions (Table 5).Usuall y alo wpro ­ portion ofth e femaleplant s actually gave riset ohybrid s (Table 5).I n 1975, no striking differenceswer e observed between the two pollinators inproductio n ofhybrid s from crosseswit hvariou s Brassica accessions (Table 5). Crosses ofAl 3gav e riset o asubstantia l number ofhybrids ; nearly allplant s from that accessionproduce d some hybrids in crosses withth eaccession s Rl aridR2 . A13wa s anS 1 lineobtaine db y selfinga particular A12plant ,whic h in 1975 showed agoo d fruit-set andgoo d pro­ ductiono fhybrids ,i.e .numbe r ratioo fhybrid st opollination s (cross- ability)3. 9 % incrosse swit h Rl.Th e resultswit hA1 3 suggest thatse ­ lectionwithi n B. campestris may giveris e toa considerabl e increase in crossability. Ifw e exclude the results ofcrosse swit hA13 ,th emea n crossablility over the twoyear swa s about1 % .Th e crossability ofth e

11 Table6 .Result so fcrosse sbetwee nautotetraploi d formso fBrassic a campestris (ç)an d Raphanus satlvusi n197 5an d1976 .

Cross Numbero f Numbero f Numbero f Numbero f Number ratioo fhybrid s pollinations femaleplant s hybrids matromorphs topollination s(% )

tested yielding average maximum hybrids

1975 AA1x RR2 629 29 0 0 1 0 0

1976 AA2x RR1 138 8 0 0 12 0 0 RR2 133 8 0 0 18 0 0 AA3x RR1 73 6 0 0 0 0 0 RR2 128 7 1 1 1 0.8 5.0 AA4x RR1 121 7 0 0 0 0 0 RR2 26 2 0 0 0 0 0 AA5x RR1 39 6 3 3 1 7.7 25.0 RR2 107 10 1 1 0 0.9 9.1 AA6x RR1 455 18 13 39 5 8.6 53.3 RR2 74 6 3 6 1 8.1 30.0 AA7x RR1 158 8 0 0 3 0 0 RR2 52 6 0 0 0 0 0 AA8x RR1 398 18 2 12 2 3.0 57.9 RR2 68 6 0 0 0 0 0

Total 2599 62 44 2.4 57.9

bestA1 3plan twa s evenmor e than10 0% (Table5) . Table 6list s crossesbetwee n tetraploids.Th emea n crossability was slightlybette r thanbetwee n diploids;o n average 2.4 %wit h arang e 0-8.6 %.Fou rtetraploi d accessions of B. campestris gaven ohybrid s at all;o fthos eaccession s thatproduce d hybrids,usuall y only a few plants did so,AA 6bein g awelcom e exception.Femal e plantswit h good crossability were found inAA5 ,AA 8 and especiallyAA6 . Crossesbetwee n diploid formso f B. campestris and tetraploid R. sativus gave riset o 5hybrid s from313 2pollination s on about 130 Brassica plants (Table 7). Themea ncrossabilit ywa s only 0.16 %. Incrosse swit htetraploi d formso f B. campestris as femaleparent s anddiploi d formso f R. sativus aspollinator , crossability waspoo r too (Table 7). Intotal ,141 1pollination s on several Brassica plants gave riset oon ehybri d only,whic h amounts to acrossabilit y of0.0 7 %. Matromorphswer e frequently found in2 xx 2 x and2 xx 4 x crosses and

less frequently in4 xx 2xan d4 xx 4 X crosses (Table 5,6 an d 7). The respective number ratioso fmatromorph s topollination s were 5.2,8.1 , 1.6 and 1.7 %. Sodiploi d formso f B. campestris gave riset omatro ­ morphsmor e easily thantetraploi d forms.

12 Table 7. Results of 2x x 4x and 4x x 2x crosses between Brassica campestrls (p) and 175an d 1976.

Cross Year Number of Number of Numbero f Numbero f pollingition s female plants hybrids matromorphs

tested yielding hybrids

2x x4 x Al xRR 2 1975 441 17 0 0 52 A2 xRR 2 1975 213 8 0 0 12 A3 xRR 2 1975 413 17 0 0 44 A4 xRR 2 1975 189 8 0 0 3 A5 xRR 2 1975 464 18 0 0 12 A6 xRR 2 1975 353 15 2 2 3 A7 xRR 2 1975 437 18 0 0 21 A8 xRR 1 1976 69 4 0 0 15 RR2 1976 99 7 0 0 40 A9 xRR 1 1976 31 2 0 0 14 All xRR 2 1975 319 14 0 0 30 A13 xRR 1 1976 38 4 0 0 1 RR2 1976 66 7 2 3 7

Total 3132 254 4xx 2 x AA1x R 3 1975 727 29 0 7 AA2x R i 1976 97 7 0 8 AA3x R 2 1976 161 7 0 3 AA4x R i 1976 58 5 0 2 AA5x R l 1976 9 1 0 0 R2 1976 32 6 0 0 AA6x R i 1976 107 12 ] 0 AA7x R i 1976 10 1 0 0 R2 1976 27 4 0 1 AA8x R i 1976 114 12 0 1 R2 1976 69 6 0 0

Total 1411 22

2.3.4 Use of genetic male-sterility

To avoid time-consuming hand-pollination, use of genetically male- sterile plants of B. campestris was tested in 1977, though on a limited scale. Four male-sterile plants grown in four isolation cages together with a flowering diploid or tetraploid accession of Raphanus gave rise to a considerable number of small seeds (Table 8). Unfortunately these cages were not completely pollen proof and some undesired cross-pollina­ tion occurred from flowering plants of B. carinata (BBCC) growing quite near to the cages. Therefore deviant plants with the genome constitution ABC were recovered as well as the expected intergeneric hybrids and matro­ morphs .

13 Table8 .Result so fcrosse sbetwee nmale-steril eplant so fBrassic a campestrls (2x)an d diploido rtetraploi d formso fRaphanu ssativu s (1977).Plant swer egrow ni nisolatio n cagesan dwer epollinate db ybees .

ParentN o Pollinator Numbero fseed s Numbero f

obtained sown plants hybrids matromorphs

true false*

R4 156 15 12 10 R5 2 2 0 0 R6 70 15 9 0 RR1 15 15 5 3

*]Numbe ro fhybrid swit hth egenom e constitutionAB Cobtaine db yillegitimat e pollination.

Table9 .Settin go fsiliquae ,ovule san dhybri dan dmatromorphi c embryos incrosse s in197 6betwee ndiploi dan dtetraploi d formso fBrassic a campestris (AAan dAAAA )an d Raphanus sativus (RRan dRRRR) ,a swel la sth edevelopmenta l stageo fth ehybri d embryos3- 4week safte rpollination .

Female Numbero f Embryostag eo fhybri dembryos* " Numberrati o parent ofhybri d polli- siliquaedevelope d embryos* ? G H LHE TT LTW S M embryost o nations ovules pollinations M H (%) AAX R R A8 61 18 24 14 7 3 4 11 A9 109 24 37 21 8 2 1 2 3 7 A13 62 54 88 7 46 4 4 2 1 2 4 29 74 AAx RRR R A8 54 10 21 16 2 1 1 A9 84 32 64 64 0 A13 25 18 8 0 2 1 AAAAx R R AA5 126 50 10 1 5 1 3 4 AA6 164 78 59 2 29 2 10 7 3 2 3 18 AA7 14 10 1 0 0 0 AA8 106 57 41 0 18 1 6 9 17 AAAAX RRR R AA5 310 137 57 3 29 11115 3 2 15 9 AA6 251 131 187 14 119 2 12 23 19 18 12 2 5 '26 47 AA7 97 94 42 120 33235 2 2 21 AA8 175 81 128 6 61 1 15 25 7 3 4 1 1 4 35

*] Mmatromorphi c embryo; H hybrid embryo. **] ? unidentified; G globular; H heart + early heart; LH late heart; ET early torpedo; T torpedo; LT late torpedo; WS walking stick; Mmatur e I + II (Wilmar & Hellendoorn, 1968).

2.3.5 Culture of embryos

Embryo culture depends first on the availability of sufficient hybrid embryos. Table 9 summarizes data from intergeneric crosses made in 1976

14 forembry o culture.Ther ewer e largedifference sbetwee n femaleparent s innumbe r ratio ofdevelope d ovules topollinations ,an dnumbe r ratios of matromorphic andhybri d embryos topollinations .Siliqua e nearly always contained one ormor e developed ovules,whic h ingenera lwer e rather small and oftenmilk y oreve nbrow n and shrivelled. Embryos obtained from such ovuleswer e classified ashybri d embryos.However ,larg eovule swer e found too,especiall y incrosse swit hA 8 andA9 .Thes e ovules contained large embryos,whic hwer e classed asmatromorphic .A s fara splant swer e raised from these ovules,th e largeone s gaveexclusivel ymatromorph s andth e small oneshybrids .No t all developed ovulesha d adevelope dembryo . Success ofculturin g embryos invitr o further depends onth e size and condition ofth e embryos.Th ehybri d embryos cultured in 1976 differed verymuc h indevelopmenta l stage (Table 9). Classification ofth e embryos by the scheme ofWilma r& Hellendoor n (1968)wa s usuallypossibl e except fora fewembryo s of irregular shape.Th e developmental stage ranged fromglobula r tomatur e I+ II.Th e greatmajorit y ofth eembryo sha d already reached their final stage,a sjudge d fromth econditio n ofth e ovules.Thi smean s thatth edevelopmen tusuall y stoppedbefor e embryo maturity. The results ofcultur e ofembryo s fromcrosse sbetwee n B. campestris and R. sativus invitr o are summarized inTabl e 10.I n197 5th etechni ­ quewa s tested onembryo s fromcrosse s ofA1 0 andA1 2wit h 'Siletta'. Thedevelopmenta l stages ofthos e embryos ranged from globular toma ­ ture.Th eproportio n ofplant s raised fromembryo swa s about3 1% ,in ­ cludinghybrid s andmatromorphs . In1976 ,al l embryosmentione d inTa ­ ble 9wer e cultured invitro .Hybrid s andmatromorph swer e raised from 2xx 2x and4 xx 4 xcrosse s and onlymatromorph s from2 xx 4 x crosses (Table 10).Th ematromorph s were obtained fromcomparativel y largeem ­ bryos,o n average, 11% ofal lembryo s cultured in197 6 gaveris e toa plant.Th enumbe r ratio ofhybrid s topollination s wasunaccountabl y low incompariso n to thehybri dproductio nwithou tembry o culture (Table5 , 6 and 7). Initiallymos tembryo s grew fairlywel l inth e liquidmedium , but afterwards many turned yellow anddied .

2.3.6 Genome constitution and number of chromosomes in the primary hy­ brids

Thechromosom e number of14 0primar yhybrid swa s determined (Table11) . Intergeneric crossesbetwee ndiploid s gave onlyplant swit h 19chromoso ­ mes, although severalhybrid s hadroot-ti p cellswit hdouble d number of chromosomes.Apparentl y allembryo s from these crosses hadth e expected genome constitutionAR . Crossesbetwee ntetraploid s gave riset ohybrids ,whos e chromosome number ranged from 35t o40 .Abou t4 8% ha d 38chromosomes ,th e expected

15 Table10 .Summar yo fth eresult so fcultur eo fembryo sobtaine d from crossesbetwee n diploidan dtetraploi d formso fBrassic a campestris (AAan dAAAA )an dRaphanu s sativus (BRan dRRRR) .Th eembryo swer eisolate d21-2 8day safte rpollination .

Cross Year Numbero f Numbero f Numbero fNumbe r ofNumbe r ratioo f pollina- hybrids matro- hybridst o embryos ovules morphs pollinations(% ) cultured cultured*

AAx R R AIOX R I 1975 69 1 39 0 0 A12x R i 1975 191 132 76 38 19.9 A8 xR I 1976 61 21 0 0 A9 xR I 1976 109 29 0 0 A13x R i 1976 36 36 7 19.4 R2 1976 26 17 3 11.5 Total 492 236 48 9.8 AAX RRR R A8 xRR l 1976 37 0 RR2 1976 17 10 1 A9 xRR 2 1976 84 63 19 A13x RR l 1976 8 1 0 RR2 1976 17 1 0 Total 163 83 20

AAAAx R R AA5x R i 1976 6 1 R2 1976 120 5 AA6x R i 1976 164 31 AA7x R 2 1976 14 0 AA8x R i 1976 106 18 Total 410 55

AAAAX RRR R AA5x RR l 1976 143 20 0 0 RR2 1976 167 12 2 1.2 AA6x RR l 1976 207 108 4 1.9 RR2 1976 44 15 2 4.5 AA7x RR l 1976 34 10 0 0 RR2 1976 63 11 0 0 AA8x RR l 1976 175 67 2 1.1 Total 833 243 10 1.2

'] Ovuleswit hn ovisibl eembryo .

Table 11.Chromosom enumber san dprobabl egenom econstitution s ofprimar y hybridsfro mcrosse sbetwee ndiploi dan dtetraploi d formso fBrassic a campestris (AAan dAAAA )an dRaphanu s sativus (RRan d RRRR).

Cross Numbero f Genomeconstitutio nan d chromosomenumbe r hybrids classified AR ARR AARR

19 28 29 35 36 37 38 39 40

AA xR R 85 85 AA xRRR R 6 3 1 2 AAAAX R R 1 1 AAAAx RRR R 48 1 1 12 23 10 1 Total 140 85 3 1 1 1 13 25 10 1

16 euploid chromosome number ofplant swit h thegenom e constitutionAARR . Chromosome numbers deviating from 38migh tresul t from fusiono faneu - ploid gametes derived fromeithe r ofth e twoparents ,especiall y thefe ­ maleparent . The 2xx 4xcrosse s resulted intw o tetraploid and fourtriploi d hy­ bridswit hprobabl e genome constitutions AARR andARR , respectively. One ofth e triploids washyperploi d (2n= 29) .Th etw o allotetraploids werepresumabl y derived from afertilize d 2n egg cell. The4 xx 2xcrosse s gaveris e toon ehybri dwit h 37chromosomes .A plausible explanation forthi sunexpecte d chromosome number is fusion of ananeuploi d egg-cell and a2 nmal egamete .

2.4 DISCUSSION

Inthi s study, the crossabilitybetwee ndiploi d formso f B. campes­ tris and R. sativus was intermediate totha t foundb y otherworker s (Table 2). Themea n crossability atth etetraploi d level,however ,wa s better than inpreviou s reports.Th eresult s of2 xx 4 xan d4 xx 2 x crosseswer epoo r incompariso n tothos eo fcrosse s atth ediploi d and tetraploid level.S other e isapparentl y aneffec to fploid y oncross - ability inthi s intergeneric cross.Suc heffect shav ebee ndescribe d in various interspecific and intraspecific crosses among Cruciferae (e.g. Howard, 1942b). Inth e crosses studied, theoptimu m ratioo f thegenom e number inembry o and endosperm isapparentl y 2t o3 .Th eoccurrenc e of anunexpecte d highproportio n of allotetraploids amongth ehybrid s from 2xx 4 x and4 xx 2x supports thisview . Theviabl e seed from intergeneric crossesconsiste d ofhybri d andma - tromorphic ones.Usuall y small seedsgav e riset ohybrids ,a swa sear ­ lier reported byNish i et al. (1964)an dTokumas u (1970b).Plum p seeds resulted inmatromorphs . Manymor ematromorph swer e obtainedpe rpollinatio n incrosse s ondi ­ ploid forms of B. campestris thano ntetraploids .Althoug h illegitimate self-pollination and cross-pollination cannotb eprecluded , the diploid forms seemed tohav e abette rparthenogeneti c ability of2 n eggs thanth e tetraploids afterpollinatio nwit h Raphanus. Clear-cut differences between andwithi n B. campestris accessionsex ­ isted incrossabilit y with Raphanus. Only asmal lproportio n of thefe ­ male plants used inthi s studyproduce d hybrids.Variatio n incrossabil ­ itycoul db e causedb yvariatio n inenvironmenta l conditions.Howeve r as the femaleplant swer e grownan dtreate d ina simila rway ,ther emus tb e genotypicvariatio n incrossability .Th e finding thatnearl y allplant s from an Sj^lin e (A13),whic hha dbee nobtaine db y selfinga plan twit h good crossability, alsoha d agoo d crossability, supportsthi svie w and proves that selection amongth e femaleparent s forimprove d crossability

17 couldb eeffective .Th eeffec to fpollinato r genotype on crossability, was lesspronounced .Possibl e genotypicvariatio nwithi nth emal eacces ­ sions,however ,ma yhav ebee nmaske db y theus e ofbulke d pollen. Theproductio no flarg enumber s ofhybrid s istime-consuming . Effi­ ciency couldb e improvedb y theus e ofgeneticall y male-sterile plants of B. campestris. To facilitate large-scale production ofhybrid s it mightb ewort htryin gt ocombin emale-sterilit y with agoo d genetically determined crossability. Another interesting approach isembry o culture.Developmen t of hybrid embryosmostl y stopsbefor e embryomaturity .Th e results of embryo cul­ ture inthi s study,however ,wer e disappointing, although in 1975 itcon ­ siderably increased thenumbe r ofhybrid s raisedpe rpollination . 3 Nature of breeding barriers

3.1 INTRODUCTION

Only a fewbrie f reports existo nbreedin gbarrier s inth e progamic phase of crossesbetwee n B. campestris and R. sativus. Oelke (1957)ob ­ served thatpolle n of severalunname d cruciferous specieswa s inhibited onradis h stigmas.On e ofthos e specieswa s B. campestris spp. rapifera (Sampson, 1962). Itspolle n germinatedbu t didno tpenetrat e the radish stigma.Reciproca l crossesbetwee nth e two specieswer e studied atth e ScottishPlan tBreedin g Stationb y fluorescence microscopy. 'Pollenger ­ mination and growthwer e fairly goodbu tver y fewovule s starteddevel ­ opment' (ScottishPlan tBreedin g Station,1971 ,p .28) . Self-incomptability in B. campestris and R. sativus isgoverne d by the sporophytic action ofon eS-alleli cserie s (reviewedb yHinat a &Nishio , 1980). Incontras t to Lewis &Crow e (1958), Sampson (1962)observe d that mostcrosse sbetwee n self-incompatible species inCrucifera ewer e incom­ patible, except inth ebu d stageo f flowers.Oelk e (1957)ha d also found thatbu dpollinatio n had abeneficia l effecti ncrosse sbetwee n kohlrabi (B. oleracea var. gongyloides L.) an dradish ,usin g the former aspis ­ tillateparent .Thu sbu dpollinatio n seemed anattractiv emetho d for overcoming interspecific breedingbarriers . Thepresen t studywa s intended toclarif y thenatur eo fbreedin gbar ­ riers during theprogami cphas e of fertilization incrosse sbetwee ndi ­ ploid and tetraploid formso f B. campestris (9)an d R. sativus andt o test the significance ofbud-pollination .

3.2 MATERIAL AND METHODS

Parto fth e 1976 crossingprogramm e was anexperimen to nbreedin gbar ­ riers.Plan tmaterial ,growin g conditions andcrossin gmethod swer e as described inChapte r 2, flowersbein gpollinate d 2-3 days afteremascu ­ lationwhe n some flowerbud s had reached theopen-flowe r stage and the restwer e still inth ebu d stage.Th e oldestbu d ofeac h inflorescence wasmarke d andthre e days later the thirdpisti lbelo w and above the markedpisti lwer e fixed inCarno y solution (6volume s of aqueousetha ­ nol (volume fraction 0.96), 3volume s ofchlorofor m and 1volum e ofgla ­ cial acetic acid)an d stored ina refrigerator .Th e resto fth e pistils of each inflorescence were lefto nth eplant s anduse d forembry o cul-

19 tureafterwards .Th eresult s ofthes e cultures are reported inTabl e10 . The fixedpistil swer e subsequently softened inNaO H solutiono f sub­ stanceconcentratio n 1mol/ 1 for 1h at6 0 °Can d stained in an aqueous solutionwit hmas s concentration ofanilin eblu e 2g/l and K3P04 20 g/1. Thepistil swer egentl y squashed ina dro p ofglycero l and studied under a fluorescencemicroscope . Inal lpistils ,th eproportion s ofgerminate d pollen and thepropor ­ tionso fpolle ngrain swit h apolle n tube thatha dpenetrate d the stigma werecounte d among arando m group of about 50polle n grainspe r stigma. Althoughusuall y over 1000polle ngrain swer e applied oneac hstigma , mostly afe whundre d grainswer e stillpresen t and sometimes even less than50 .Th ereductio n innumbe r ofpolle ngrain swa sprobably , at least inpart ,cause db yth e losso fungerminate d pollen during fixation,ma ­ ceration andstaining . Inal lpistils ,th epolle n tubeswer e counted in thetransitiona l areabetwee n style and ovary and the ovules fertilized, i.e.th eovule swit h apolle ntub e inthei rmicropyle ,wer e counted for eachovary .

3.3 RESULTS

Intergeneric incompatibility was studied in2 xx 2x,2 xx 4x,4 xx 2x and4 xx 4 xcrosse sbetwee n B. campestris and R. sativus. Inal lcrosses , typical incompatibility reactions occurred onth e stigmas similar toin ­ compatibility reactions after self-pollination.Usuall ymor e than8 0% of thepolle n stillpresen t ona stigm aha d germinated. Germination was somewhatbette r on stigmaspollinate d inth eopen-flowe r stage than in thebu d stage.Th epolle ntube swer emostl y short and strongly fluores­ cent,becaus e ofcallos e formation (Fig.3A) .Callos e depositswer eusu ­ ally alsoobserve d inman y stigmapapillae .Suc h reactions apparently prevented thepenetratio n ofpolle n tubes into the style.Howeve r acer ­ taindegre e ofpenetratio nwa s usually observed afterbu d and open-flower pollination. Some abnormalities occasionally occurred within stigma papillae, such asbranchin g ofpolle n tubes and inhibition ofpolle n tubegrowt h (Fig. 3B). Nobarrie r seemed toexis t inth e style,an d one ormor epolle n tubes entered theovar y innearl y allpistils ,irrespectiv e ofth emod e of pol­ lination (Table 12).A hig hproportio n ofpistil s in allcrosse sha d more thante npolle n tubespe r style.Bu dpollinatio n didno t increase thenum ­ bero fpolle ntube spe r style.Th eresult s forth evariou s crosses indi­ catetha tther ewa sn omarke d effecto fploid y onpenetratio n ofpolle n tubes into the style. Inth eovaries ,mos tpolle n tubes usually grew fairly straight to the bottom,wher e growthbecam e erratic.Usuall y only a fewpolle n tubes grew inth edirectio no f anovul e andpenetrate d through themicropyl e (Table13) .

20 MPPil 1 1 «fFff i*.'V^.v-. ; îf*":*^-.--..*'.,'>.•*-."- • :%- -.'. ;r. •\->*:'-ï'~~.-y'-; '-".-'. .

- • -"••»f'. ^' »• - - *

'-i • *^

"?•*•-

Figure3 .Incompatibilit y reactionsi nth eprogami cphas eo fcrosse sbetwee nBrassic a campestrisan dRaphanu ssativu s (ultravioletfluorescence) . A.Inhibitio no fgrowt ho fpolle ntub eo na stigm apapill a (x490). B.Inhibitio no fgrowt ho fpolle ntub ewithi na stigm apapill a (x490).

Table 12.Numbe ro fpolle ntube senterin gth eovar yafte rbu dan dopen-flowe r pollinationi ncrosse sbetwee ndiploi dan dtetraploi d formso fBrassic acampestri s (AAan dAAAA )an dRaphanu s sativus (RRan dRRRR) . The data arebase d on observations inth esam epistil sa si n Table13 .

Cross Modeo f Numbero f Number of pistils pollinati on pistils withn o with1 - •9 withÔ1 0 tubes tubes tubes

AA XR R Bud 13 1 5 7 Flower 13 1 6 6

AA XRRR R Bud 12* 0 3 7 Flower 13 1 4 7

AAAAx R R Bud 18 1 5 13 Flower 17 1 5 11

AAAAX RRR R Bud 44 4 12 28 Flower 44 2 9 33

•] Data fromtw opistil smissing .

21 Table13 .Numbe ro ffertilize dovule san dth enumbe ro fpistil s inwhic hfertilizatio n wasobserve di ncrosse sbetwee ndiploi dan dtetraploi d formso fBrassic a campestris (AAan dAAAA )an dRaphanu ssativu s (RRan dRRRR) .

Cross Numbero f Numbero f Fertilized Pistils withovule s pistils ovules ovules fertilized* * observed observed* number number proportion(%) AA xR R A8 xR l 9 203 11 5.4 3 A9 xR l 33 17 362 17 4.7 8 47 AA xRRR R A8 xRR1/RR 2 12 285 9 3.2 5 42 A9 xRR 1 13 278 12 4.3 8 62 AAAA xR R AA6 xR l 19 424 14 3.3 10 53 AA8 XR l 16 276 17 6.2 7 44 AAAA XRRR R AA4 xRR 1 9 141 2 1.4 AA5 XRR 1 2 22 5 108 1 0.9 AA6 xRR 1 1 20 27 637 30 4.7 15 56 AA6 xRR 2 6 130 5 3.8 3 50 AA7 xRR1/RR 2 12 248 23 9.3 9 75 AA8 xRR 1 29 512 17 3.3 11 38 *]Onl yeasil yvisibl eovule si neac hovar ywer eassessed . **]Fertilizatio ni sunderestimate d because someovu l eswer eno tvisible .

Occasionally erraticgrowt h ofpolle n tubeswa s also observed near theen ­ trancet oth emicropyle .Th eproportio n ofovule s fertilized was low, 0.9 to9. 3 %. Inman ypistils ,n o fertilizationwa s observed, and thenumbe r offertilize d ovulespe rpisti l averaged less than two in thebes t cross (Table13) .

3.4 DISCUSSION

Inth eprogami cphas e ofth e intergeneric crosses studied, therewer e obviousbarrier s tobreeding :poo rpenetratio n ofpolle n tubes into the stigmas anderrati c growth ofth epolle n tubewithi n the ovaries.Th e firstbarrie rwa sno t fully effective and inmos t crosses a considerable number ofpolle n tubes reached the ovaries.Thes e observations agree quitewel lwit h those atth e Scottish PlantBreedin g Station (1971). The poor development ofovule s observed in Scotland and inthi s study (Ta­ ble 9)seem st ob e causedprimaril y by scanty fertilization. Apparently theovule s failed to attractpolle n tubes.Suc h abreedin gbarrie r was alsoobserve d ininterspecifi c crosses in Arabidopsis (Berger, 1968). A ratherhig hproportio n of fertilized ovules apparently gave rise tode ­ veloped ovules inwhic h anembry o frequently waspresen t (Table 9).Se ­ lectionwithi n B. campestris for improved crossability mightb e achieved

22 by selection forgenotype swit h arelativel y good fertilization asde ­ tectedb yultraviole tmicroscop y or forgenotype swit h ahig hproportio n ofdevelope d ovules or embryospe rpollination . Inth eprogami c phase,ther ewer en o striking differences between 2x x 2x,2 xx 4x ,4 xx 2 x and4 xx 4 xcrosse s forth enumbe r ofpolle ntu ­ bes observed inth e style and success of fertilization. The effecto f ploidy oncrossabilit y (Section2.4 ) apparently came after fertilization andno tbefor eit . Budpollinatio nwa s certainly not aneffectiv emetho d ofcircumven ­ ting thebreedin gbarrier s onth e stigma,a swoul db e expected from the results of Sampson (1962),becaus e therewa sn omarke d differencebe ­ tweenbu d andopen-flowe rpollinatio n indegre eo fth e incompatibility reactions onth e stigma andpenetratio n ofpolle n tubes into the stigma. A role of theS-locu s ininterspecifi c incompatibility orincongru ­ ity asdefine d byHogenboo m (1973;1975 )wa s assumed, forinstanc eb y Sampson (1962), but shouldb e queried,becaus e the activities oftha t locus are found instigma s ofope n flowers and olderbuds .Perhap s the buds studied were too old andth eS-locu swa s already operative.Howeve r the results ofNama i (1980),wh o recently studied the effecto f ageo f buds incrosse sbetwee n B. campestris and R. sativus, indicate that crossability ofyounge rbud s iseve n less.

23 4 Primary allodiploid AR hybrids and their progeny

4.1 INTRODUCTION

Several authors (Table2 )hav e obtained AR hybrids from crossesbe ­ tweendiploi d formso f B. campestris and R. sativus. Terasawa &Shimo - tomai (1928),Tokumas u (1970b)an dOpefi a& Lo (1978)hav e described such hybrids fromcrosse sbetwee norienta l vegetable forms of Brassica and commonradis ho rJapanes e radish.Th eplant swer e intermediate between theparenta l species inman y respectswit h alea frosett e inth evege ­ tative stagean dhardl y show anythickenin g ofth e roots,i f at all.Th e whitean dpurpl e flower of R. sativus proved tob e dominant over the yellowo f Brassica. Clauss (1978), however, obtained onlyyellow-flower ­ edhybrid s from similarcrosses .Th e siliquae ofth ehybrid s consisted oftw opart s asi n Brassica, i.e. avalvat e part and arathe r large in- dehiscentpart ,th ebeak .Bot hpart s contained ovules (Terasawa 5=Shi - motomai,1928 ;Tokumasu , 1970b). Tokumasu (1970b)observe d also thatth e hybrids grewvigorousl y inth e later stages ofdevelopment . Various authors studiedmeioti c chromosome behaviour insuc h hybrids (Terasawa& Shimotomai ,1928 ;Richharia , 1937;U et al., 1937;Mizushi - ma, 1944b;1950a ;Tokumasu , 1970b). The datapresente d vary considera­ bly.Accordin g toU et al. (1937), Terasawa found amaximu m of fivebi ­ valentspe r cell, m theirow nhybrids ,U et al. (1937)counte d nomor e thanthre ebivalent s andRichhari a (1937)eve n found amaximu m of eight bivalentspe r cell.Tokumas u (1970b)observe d sixbivalent s per cell at mostan d also found alo w frequency oftrivalent s and quadrivalents. The causes ofth edifference s indegre e ofchromosom e association areun ­ known,bu tmigh tb e inpar tgenetic . Another intriguing aspecto f allodiploid hybrids isthei r fertility. Morris (1936)an dTokumas u (1970b)studie dmal e fertility.Morri s found thatal lhybrid s studiedproduce d aconsiderabl e amount ofdiploi d ger- minablepollen .Polle n stainability varied considerably from day to day andprobabl y fromplan tt oplant .Tokumas u (1970b)als onotice d arathe r highproportio n ofstainabl epollen ,varyin g from4 to3 0% with amea n of 11% .Howeve r seed fertility ofth ehybrids ,a sdetermine d by inter- mating,wa sver ypoor .Terasaw a & Shimotomai (1928)obtaine d someF 0 plants,whic hdiffere d in form and flower colour.Als oMorri s (1936)an d

Mizushima (1952)obtaine d F2plants .Howeve rTokumas u (1970b)di d not

succeed m obtaining aprogen y from Fx hybrids.

24 Thecrossabilit y ofth e diploid hybridswit hth e twoparenta l species, B. campestris and R. sativus was equallypoo r (Terasawa & Shimotomai,1928 ; Tokumasu, 1970b). OnlyTerasaw a& Shimotomai (1928)obtaine d a fewcom ­ pletely sterileplant s frombackcrossin g F..hybrid s asfemale swit h the Brassica parent.Backcrosse swit h R. sativus failed. This chapterpresent s observations onvariou s aspects ofth e diploid Fjhybrid s obtained inthi s study, such as form,meioti c association of chromosomes,polle n and seed fertility.Als o reported arechromosom e num­

ber and fertility ofF 2 plants,attempt s todoubl e thechromosom e number

ofF 1 hybrids and crosses ofF .hybrid swit h B. campestris, R. sativus and B. napus respectively.

4.2 MATERIALAN DMETHOD S

All hybrids studiedwer e obtained from the crossesbetwee nth edip ­ loid accessions of B. campestris and R. sativus. Allhybri dplants, a s far aswa s known,ha d 19chromosome s andth e genome constitution was probably AR.Th eplant swil lb ereferre d to asA Rhybrid s or rapifera hybrids, for instance,referrin g toth e Brassica parent inth e original intergeneric crosses.Th e hybridswer egrow n inth e samewa y asthei r parents, andunde r similar conditions,bu tmos twer etransplante d into soilo f aheate d greenhouse justbefor e flowering.Non-boltin g plants were firstplace d ina col d greenhouse for about4 0day s inorde r to induce flowering. Pollen stainabilitywa s assessed on freshpolle ncollecte d fromva ­ rious flowersb y ametho d of Sass (1964)wit h arando m sample of about 200polle ngrain spe rplant .Som e slideswer e alsouse d formeasurin g pollen grain size.Th emethod s used forstudyin gmitosi s andmeiosi s havebee n described in Section2.2 .Al l data onth econstitutio n of sporad cellswer e obtained fromanther s squashed ina dro p ofpropiono - iron-alum haematoxylin (Henderson& Lu , 1968). SeveralA R hybridswer evegetativel y propagated from cuttings, cul­ ture invitr o of stem andpetiol e explants (Karthae t al., 1974)o ro f axillary buds. Propagationb ycuttings ,whic hwer e treatedwit h atalcu m powder containing indolebutyric acid (IBA)a ta substanc e content of 59/kg, gaveusuall y rise towea k and smallplants .Th etw omethod s of culture invitr o resulted inwel l developed plants.Th e overall results of propagation through stem andpetiol e expiants,however ,wer epoor ; some shoot formationoccurre d ona cultur emediu m ofMurashig e & Skoog (1962)containin g 6-benzylaminopurin e (BA)an d1-naphthaleneaceti c acid (NAA)a tsubstanc e concentrations of1 0an d 0,5 mmol/m3, respectively. Culture conditions andmediu m for invitr o culture of axillarybud s were the same as inKarth a et al. (1974).Th emetho d gaverelativel y goodre ­ sults on amediu m containingB A at5 mmol/m 3 andNA A at1 mmol/m 3.

25 Cuttingso fvariou s ARhybrid swer e transplanted inisolatio n cages (2.5m x 2. 5 m)place d outside and intercrossed with thehel p ofhoney ­ bees toobtai na nF ,generation . Subsequently, theF 2plant s weremulti ­ plied ina simila rwa y incage s ina greenhouse . Allaxillar ybud s ofroote d cuttings of several AR-hybrids were treatedwit hcolchicin e in four substance concentrations ranging from2 to6 g/1 .Th ecolchicin e treatmentswer e carried out according to the traganth-slimemetho d ofSchwanit z (1949)a tabou t 18 °C andwit hhig h airhumidity .

4.3 RESULTS

4.3.1 Form of AR hybrids

Intergeneric crossesbetwee n B. campestris and R. sativus gave rise tohybrid s andmatromorphs .Th edistinctio nbetwee n the two types proved tob eeas y andwa susuall ypossibl e ina nearl y developmental stage.Fo r example,hybrid susuall y had awhitis h hypocotyl and their firstlea f waspubescent . Incontrast ,matromorph s fromcrosse s between B. campes­ tris ssp. chinensis and fodder radish, for instance,ha d plain green and morerobus thypocotyl s andglabrou s leaves.Th ehybrid s subsequentlyde ­ veloped aleaf-rosett e with several slightlypubescen t leaves (Fig.4A) . Theshap eo fth eleave swa s intermediate,bu tver yvariable ,rangin g from simplet opinnate .Hybrid susuall y had slightlypubescen tpetioles ,whic h were sometimes slightly red from anthocyanins. Somehybrid s from crosses with turnip showed aswolle n turnip-like root.O n thewhole ,th e hybrids provedt ob evigorous ,althoug h afe wwer e stunted oreve n deformed. Thetransitio n fromvegetativ e togenerativ e stage didno t require cold treatment,excep t forsom ehybrid s originating from crosses with stubble turnip,a for m of B. campestris ssp. rapifera thatneed s cold for flowerinduction .Th e flowering time ofA R hybridswa s intermediate betweentha to fth eparenta lspecies . Inth egenerativ e stage,hybrid susuall y had anerec tmai n stemwit ha terminal inflorescence (Fig.4B) .Afterward s many lateral branches with secondary inflorescences developed and theplant s reached aheigh t of 1.3-1.7m .Th eindeterminat e flowering observed inA R hybrids,probabl y causedb y ahig h degree ofsterility , resulted finally in awil d dense buncho fbranches .O nth e stem and lateralbranches , asligh t pubescence and some anthocyanin colorationwa s occasionallypresent . The inflorescences ofA R hybridswer emor e similart o B. campestris thant o R. sativus innumbe r of flowers,flowe r size and the position of flowers and flowerbud s (Fig.4C) .Th ebud swer eusuall y slightly pubescent.Mos tA R hybrids hadwhit e orpurpl e flowers with clear-cut veins, like Raphanus, but someplant sha dplai nwhit e flowers oreve n

26 -5 t •

'< \, [ . ' , '• 1

1 1 Î \ t > •

' ft',-: )

Figure4 .Hybrid swit hth egenom econstitutio nA Rfro mth ecros sBrassic acampestri s ssp.chinensi sx Raphanu s sativus cv.Siletta . A.Vegetativ estage . B.Generativ estage . C.Inflorescence .

27 yellow flowers,lik e Brassica. Intotal ,abou t 11% of all flowering AR hybridswer eyellow-flowered .Th e flowersmostl y had strikingveins ,bu t a fewha dplai nyello w flowers. Ina fe whybrid swit h white or purple flowers,sectoria l chimaeras for flowercolou rwer e observed; usuallya small sectoro fa peta lbein gyellow . SomeA Rhybrid s showed some setting of smallbivalve d siliquae witha largebeak .

4.3.2 Chromosome association at meiosis

Earlymeioti c stages inA Rhybrid swer e quite regularbu t indiaki - nesisan dmetaphas e I,frequen tunivalen t formation disturbed meiosis (Table 14). Datapresente d inth eTabl e 14shoul db e interpreted with caution.Analysi s ofchromosom e association in suchhybrid s wasdifficult , because thehig h frequency ofunivalent s sometimes hindered recognition ofmeioti c stages (metaphase Ian d anaphase I, for example). Itals o pro­ ved difficultt odistinguis hbetwee n truebivalent s and secondary asso­ ciationso funivalents . Nochromosom e associationwa s observed in 67% of allpolle nmothe r cellsstudie d insi xA R hybrids.Cell swit h one or twobivalent s occur­ red in2 1% and8 % o fth epolle nmothe r cells,respectively , andtri - valents in2. 6 % ofth e cells.Th emaximu m number ofboun d chromosomes waseigh ti na cel lwit h fourbivalents .Mos tbivalent s were rod-shaped (Fig.5A ,B) ,an d alltrivalent swer e chains.Th e differences inchromo -

Table14 .Distribution so fmeioti cconfiguration si ndiakinesi so rmetaphas eI i nsi x ARhybrid sfro mcrosse sbetwee nBrassic acampestri sssp .chinensi san dRaphanu ssativu s cv.Siletta .

Configurations* Numberfraction s(% ) ofname dhybrid s Numbero fcell s EC119 EC186 EC209 EC274 EC3H 75.1040-9 Observed Expected*

- - 65 ? 62 8 361 6 "Ï + 1II i5 7 26 4 îï°n III - - 33 16I + 1 m 15-7 1(\M, 24-° 13-8 21.2 24.4 115 158 IS T+ •>T T 1-1 2.0 1.7 7 HHunnn 5-9 5-6 t! «•? "•! " Ä 13 I + 3 II i L 1-7 5\ t 1 1 6 12I + 2H + H„ "J - ^ c 3* 111+411 ' 1.1 °-6 \] 1 Totalnumbe ro fcell s 51 72 50 94 99 172 538 *]I ,I Ian dII Iar esymbol sfo runivalents ,bivalent san dtrivalents ,respectively . **]Expectatio nbase do na Poisso ndistributio nfo ra mea nnumbe ro f0.4 7bivalent s percell .

28 B

•

;

.« /*

D

f\ sociation,dya d formationan dstainabl epolle ni nA Rhybrid s Figure5 .Chromosom eass c

A.~Pollen'mothercel la tmetaphas eI wit h1 5I + 2 I I ^^40). B-Polle nmothe r cella tmetaphas e Iwit h 17I + 1 H l*^^- C.Spora d stagewit hdyad san don etetra d (x550). D-Stainabl epolle n (X550) Table 15. Frequency distribution of pollen stainability in ARhybrid s from crosses between Brassica campestris and Raphanus sativus. Data from 1975.

Pollen stainability (%) Number of plants Number fraction of plants (%) 0 53 62 1-10 16 19 11-20 8 9 21-30 6 7 31-40 2 2 Total 85

some association between the AR hybrids were only small. The mean number of bivalents per cell in the six AR hybrids was 0.47 (one trivalent calculated as two bivalents). The frequency distribution of the number of bivalents observed per cell deviates significantly from

a Poisson distribution (P < 0.01) for M = 0.47, because of an evident excess of cells with exclusively univalents and cells with one trivalent or two bivalents. Chromosome association is apparently not simply a mat­ ter of chance; some chromosomes probably pair more easily than others.

4.3.3 Male and seed fertility

The majority of AR hybrids were completely male-sterile, but 38 %o f the plants studied produced some stainable pollen grains (Table 15, Fig. 5D). All AR hybrids from crosses with male-sterile turnips, for in­ stance, were partly male-fertile. The density function of proportion of pollen grains stained showed that two plants produced over 30 % staina­ ble pollen. Stainable pollen was usually ovoid-spherical and had a dia­ meter of 35.2 pm (mean for 5 AR hybrids). Cytokinesis in AR hybrids was irregular, resulting in various types of sporad : besides normal tetrads, monads, dyads, triads, pentads and hexads (Table 16). The size of the cells in a sporad usually varied con­ siderably, but dyads usually contained two cells of equal size (Fig. 5C). Relatively high frequencies of dyads occurred in several chinensis hy­ brids and in nearly all rapifera hybrids. Most of these plants also had a relatively high proportion of stainable pollen. So dyad formation is hvbrT\T? bUt n0t a gUarSntee f°r POllen sta-ability. One rapifera hybrid, 75.1283-1, also produced and exceptionally high number of monads (47 out of 98). The high proportion of dyads in many AR hybrids can be explained by the occurrence of restitution nuclei at interkinesis, as such nuclei 11117 ln PlantS With S high dyad polleponen Üstainabilityr H" . Production or a high The well filled and stainable pollen of AR hybrids were germinable.

30 Table 16.Distributio n of sporads with different number of cells per sporad in some AR hybrids originating from crosses between Brassica campestris ssp. chinensis or ssp. rapifera and Raphanus sativus, cv. Sile tta or var . mougri.

Plants Total Numt1e ro f sporads Number fract- Pollen stainab- number ion of dyads ility(% ) of sporads 1 2 3 4 5 6 (%)

chinensis hyb rids EC119 87 0 15 10 52 10 0 17 0 EC138 58 0 1 6 48 1 2 2 1 EC186 129 2 32 8 84 2 1 25 6 EC189 74 2 58 7 7 0 0 78 24 EC200 65 2 12 0 48 3 0 18 0 EC263 137 0 43 21 71 2 0 31 7 EC274 63 0 0 8 44 11 0 0 0 EC277 69 0 4 5 52 7 1 6 0 EC282 68 1 25 2 40 0 0 37 32 EC305 57 0 20 7 30 0 0 35 11 EC314 55 3 23 2 27 0 0 42 17 75.1040-9 72 0 5 6 60 1 0 7 0 75.1088-1 94 2 6 2 81 3 0 6 0

rapifera hybrids 75.1283-1 98 47 49 1 1 0 0 50 2 75.1308-2 66 0 34 9 23 0 0 52 8 75.1310-2 72 1 37 11 22 1 0 51 6 75.1427-3* 79 0 51 8 20 0 0 65 20 75.1671-1 56 0 1 1 53 1 0 2 0 75.1682-5* 62 0 41 1 20 0 0 66 25

Total 1461 60 457 115 783 42 4 31 8

"]Th e marked AR hybrids originated from R. sativus var . mougri.

Incrosse s ofA R hybrids asmal eparent swit h B. campestris ssp. chinen­ sis and B. napus, for example,germinate d pollen couldb e demonstrated byultraviole tmicroscopy . Two days afterpollination ,polle ntube swer e oftenobserve d inth e style and someo fthe mha d evenreache d themicro - Pyleo fa novule . Seed fertility ofA R hybrids was poor.Man y attemptswer emad e topro ­ pagate suchhybrid s by intercrossing, evenwit hcultur e ofimmatur eem ­ bryos invitr o (Table 17).Th e considerable effortswit h intercrossing of ARhybrid s resulted inonl y threeplants .However ,propagatio n inisola ­ tioncage s of someA R hybrids orcutting s of them still gave rise tosev ­ eralmor e seeds (Table 18).I ntotal ,3 6% ofth ehybrid sproduce d seeds inthi sway .

4.3.4 Crossabilitu of AR hybrids with various species

The results of crossesbetwee nA R hybrids and B. campestris, B. napus and R. sativus, respectively werepoo r (Table 17). Fromcrosse s ofA R hybrids (2n = 19)wit h B. campestris (2n =20 )a spollinator ,onl yon e hybridwa s obtained with4 7chromosomes .Th ereciproca l crossgav eonl y

31 Table17 .Number so fseed san dembryo sobtaine db yintercrossin gA Rhybrid san db y crossingA Rhybrid swit hBrassic acampestri s (AA),Raphanu ssativu s (RR),Brassic a napus (AACC)an dAAR Rhybrids .Al lembryo swer eculture d invitro .

Cross Numbero f Numbero f Numbero f Number ofplant s pollinat­ ions seeds plants embryos plants hybrids matromorphs obtained cultured obtained

AR XA R ca250 0 5 2 11 1

AR X AA ca 350 1 1 2 0 1 0 AR X AACC 120 4 2 3 1 3 0

AA X AR ca 900 13 4 26 4 0 8 RR X AR 66 0 0 1 0 0 0 AACC X AR 811 105 84 15 4 1 87

AARR X AR ca 1000 12 6 2 0 6 0 AR X AARR ca 250 3 0 0 0 0 0

matromorphs,som eo fwhic hwer e raisedb y embryo culture. Pollinationo fA Rhybrid sb y B. napus gave threever y similarhybrids , allderive d from oneparticula r rapifera hybrid.Th eplant s had aste m liketha to fforag erap e and leaves andpetiole s werepubescent . Inth e generative stage,thes eplant sproduce d only afe w flowers,whic hwer e completelywhite .Unfortunatel y the chromosomes ofthes e hybrids were notcounted .Th ereciproca l crosses gave animpressiv e number ofplants , partly raisedb yembry o culture.Onl y oneplan twa s atru ehybri d with 2n= 55 ;th eother swer ematromorphs .Th ehybri dwa s obtained from a small seed; itgre wirregularly , hadwhit e flowers and evenproduce d stainablepollen . Crosses of fodderradis h (?)an dA R hybridswer emad e on a small scale, resulting inonl yon eembry o (Table 17).Cultur e ofthi s embryo invitr o failed.

Table18 .See dproductio ni nth eF. .an dF _generatio no ftw opopulation so f mAR\hybridsuyurias .Thin eF 1 ananddF .t'plant plats fromeac hpopulatio nwer epropagate d in cagesan dpollinate db yBees .

Origin Gene­ Number of plants Numbero fseed s ration tested producing seeds rapiferahybrid s 21 9 45 24 10 chinensishybrid s 53 15* 4 23 15 10 441

32 4.3.5 Somatic and meiotic doubling of chromosome number

Various methods were used to double the chromosome number of AR hy­ brids. The first method, colchicine treatment of axillary buds of 90 rooted cuttings from 28 AR hybrids, resulted in doubling in 32 cuttings. Recognition of such doubled sectors proved rather easy, because flowers were larger and pollen was usually more stainable. A treatment with col­ chicine solution of mass concentration 4 g/1 (in traganth slime) gave the best results, but differences from other colchicine concentrations were small. Another approach for doubling the chromosome numbers of AR hybrids was to intercross AR and AARR hybrids. Doubling was expected in this way by meiotic nuclear restitution in the AR hybrids. Table 17 shows that no plants were obtained if AR hybrids were used as female parent. The reciprocal cross gave rise to six vigorous hybrids. Two of these plants had 38 chromosomes and one plant had 36; the chromosomes of the others were not counted. Chromosome doubling occurred by chance in a stem expiant of one AR hybrid cultured in vitro.

4.3.6 Progeny of crosses between AR hybrids

Seeds from crosses between AR hybrids were produced by hand-pollina­ tion (Table 17) or insect pollination (Table 18). Of the seeds, about 6* %germinate d and most of those gave rise to viable plants. The off­ spring of such crosses varied widely in chromosome number (Table 19). However, a remarkably high proportion of the plants had about 57 chromo­ somes, especially if obtained from crosses between chinensis hybrids. Such hexaploids probably had the genome constitution AAARRR. The remai­ ning plants mostly had aneuploid chromosome numbers and were genetically less balanced than the hexaploid plants. The progeny of crosses between AR hybrids also varied widely in form, not surprisingly in view of the variation in chromosome number. Some

Table 19. Somatic chromosome numbers in progeny of AR hybrids produced by intercrossing Sinensis hybrids and rapifera hybrids respectively.

PoDulati „, , Number of r Ll™ Chromosome number niants 29 31 35 36 37 38 39 40 46 52 54 55 56 57 58 gassi-

£S£i|era hybrids 21121231 1 \ l ?° SlüSÊSsis hybrids 2 1 1 1 2 ^ , 11 Total 4 1 1 2 1 2 3 1 1 1 1 1 2 U 1J5!

33 v^.yyi..wiffLjrvm frmwminm. 'g? psampmpppnm

I t t '>C i rV •&

, ^ •-•—•? /•* ^ /4ïr . ;- r-

•1 M MIJ" 1

Figure6 .Tw ohexaploi dplant swit hprobabl egenom econstitutio nAAARR Robtaine db y intercrossingA Rhybrid sderive d fromcrosse sbetwee nstubbl eturni p (2x)an dRaphanu s sativus (2x).

plants showed seriousgrowt hirregularities ,bu tother s grewvigorously , especiallyth ehexaploid s (Fig.6) .Th ehexaploi dplant s usuallyha d plump flowerbuds , largewhit eo rpurpl e flowers,an dproduce d some stainablepollen . Forpropagation ,mos toffsprin go f rapifera and chinensis hybridswa s transplanted intothre eisolatio ncage swit hbee s forpollinatio n (Table 18). Over5 0% o fth eoffsprin gproduce d some seed.Th esee d production perplan twa s2. 2an d29. 4i nth eoffsprin go f rapifera and chinensis hybrids,respectively .Mos to fth eseed-producin g plants were hexaploids with about5 7chromosomes .

4.4 DISCUSSION

The formo fA Rhybrid s obtainedb yothe r authors (Table2 )an do fth e onesi nth epresen t studywer e similar. Someo fth epresen t hybrids, however,ha dyello w flowers,a sreporte db yClaus s (1978). Inhybrid s withwhit ean dpurpl e flowers,th esynthesi s offlavonoid swa sapparentl y suppressed, asi n R. sativus (Bonnet, 1978). This suppressor systemi s perhaps inactivated inth eyellow-flowere dhybrid sb yon eo rmor e genes

34 fromth eA genome. Interactionbetwee n genes ofth e twogener a alsooccur ­ redi npartiall y male-fertile hybrids,whic horiginate d fromcrosse sbe ­ tweenmale-steril e turnips and R. sativus. Pollen sterility inturni p provedt ob e inherited in a simplemonofactoria l and recessivewa y (Dolstra,unpublished) . Sooccurrenc e ofpolle n fertility inA Rhybrid s ofmale-steril e turnips indicated thatth eeffec to fth egen e forpolle n sterility inthes e hybrids is compensated bygene(s )o f Raphanus. Only alo w degree of chromosome associationwa s found atmeiosi si n ARhybrids . Incompariso nwit h theresult s ofRichhari a (1937)an d Tokumasu (1970b), the frequency ofcell swit honl yunivalent swa shig h andth emaximu m number of associated chromosomes percel llow .Genotypi c andenvironmenta l variationma y account forsuc hdifferences . Inexten ­ sivestudie s onchromosom e association ininterspecifi c hybridsan d haploids,Mizushim a (1980)foun d amaximu m oftw o autosyndeticchromo ­ somepair s inth eA andR genome together. Iftha testimat e iscorrect , themaximu mnumbe r of allosyndetic chromosomepair snotice d inth epre ­ sentA Rhybrid s is two.Highe rvalue shav ebee n found (Mizushima, 1980). Aremarkabl e phenomenon is thata hig hproportio n ofth eA Rhybrid s produced aconsiderabl e proportion ofstainabl epollen .Th emai ncaus e isth eoccurrenc e of dyads inth e sporad stage,probabl yb yth eforma ­ tiono frestitutio n nuclei in interkinesis,a ssuggeste db yMorris , (1936).However ,Tokumas u (1970b)observe d low frequencies ofdyad si n threehybrid swit h arathe r goodpolle n stainability.Th emechanis m of formationo frestitutio n nuclei is still obscure.A possibl e causemigh t bea poorl y developed spindle as aconsequenc e ofscant ypairin go fchro ­ mosomes.However ,variatio n inth e degree ofdya d formationwa sno trela ­ tedt oth edegre e of chromosome association. Intercrossing ofA R hybrids gave aver ypoo r seed set.Th eonl yfea ­ siblemetho d of obtaining seedwa s togro wth ehybrid s inisolatio nan d tous ebee s forpollination .A remarkable number ofF 1plant sproduce d inthi swa y had ahig hnumbe r of chromosomes comparedwit h theresult s ofTerasaw a & Shimotomai (1928). Suchplant swit h about5 7chromosome s were frequently found andpresumabl y had thegenom e constitutionAAARRR . Fusiono f4 n egg cells andunreduce d male gametesma ygiv eris e tosuc h Plants, A hybrid with 47 chromosomes from acros sbetwee n anA Rhybri d <2«= 19 )an d B. campestris (2n= 20 )possibl y also originatedb y fusion °fa hypoploi d 4n gamete and anormall y reduced Brassica gamete. InCru - ciferae, the occurrence of4 n gameteswa s reported earlier forhaploid s of B. napus (Heyn,1974) . Chromosome doubling by colchicine treatmentwit h thetraganth-slim e methodo fSchwanit z (1949)wa spossible ,bu tha d thedisadvantag e that thedouble d sectorsmostl ywer e rather small. Asprimar yAAR R hybrids have apoo r fertility (Section 5.3.1), seedproductio no nsuc hdouble d sectorsofte n failed because of ashortag e offlowers .

35 Theproductio no fviabl eunreduce d pollen byman yA R hybrids makes ittemptin gt odoubl e thechromosom e numbers of these hybridsmeiotical - lyb yusin gthe m aspollinato r incrosse swit hAAR R hybrids.However , suchcrosse sgav erathe rpoo r results,probabl ybecaus e ofth e poorfer ­ tilityo fth eAAR Rhybrid sused .Th eus eo fmor e fertile xBrassicorapha- nus may improveth eseed-se t considerably.

36 5 xBrassicoraphanus: form,fertilit y andcrossabilit y with various species

5.1 INTRODUCTION

Nopublishe d datawer e available onth e agriculturalvalu e of xBrassi­ coraphanus, although such allotetraploid hybrids havebee nproduce d in variousways .Mizushim a (1968)expecte d thatsynthesi s of xBrassicora­ phanus by crossing turnips and radishcoul db e apromisin gwa yo fobtain ­ inga ne wroo tcrop . Allotetraploids with the genome constitutionAAR Rwer e firstfoun di n

anF 4generatio n of someA R hybrids (Terasawa, 1932). Thecommo nmethod s ofsynthesi swer e doubling ofth echromosom e number ofA Rhybrid sb ycol ­ chicinetreatmen t (Mizushima, 1950b;Tokumasu , 1976;Sectio n4.3.5 )an d crossingo ftetraploi d forms of thetw oparenta l species (Nishiyama,1946 ; Tokumasu,1970b ;Olsso n& Ellerström, 1980;McNaughto n& Ross , 1978;Sec ­ tion2.3) . Less attractive methods are2 xx 4 xcrosse sbetwee n B. campes- tris and R. sativus, although itwa spossibl e toobtai nhybrid s inthi s way (Section 2.3.6). McNaughton (ScottishPlan tBreedin g Station,1976 , P-16 )trie d toproduc e xBrassicoraphanus indirectlyb ycrossin g B. napo- campestris, an artificial hexaploidwit h thegenom econstitutio nAAAACC , withautotetraploi d forms of R. sativus (RRRR). Inth e subsequentgenera ­ tionso fth eAARR C hybrids, agradua l loss ofth eC genom ewa sexpected . Manyhundred s ofpollination s gave only oneseed . Thefor m of theAAR R hybrids waspoorl y described. Inman yrespect s suchhybrid swer e known tob e intermediate between theparenta l species andsimila r to the corresponding AR hybrids (Terasawa, 1932).A nundesi - redtrai to f suchplant s was thepoo r fertility (Mizushima,1950b ; Tokumasu,1976 ;Olsso n & Ellerström, 1980). Thecrossabilit y of xBrassicoraphanus asa femal eparen twit hth e diploidparenta l specieswa s ratherpoor ,bu tseed swer eobtaine d (Tera­ sawa,1933 ;Nishiyam a & Inomata, 1966;Kat o& Tokumas u 1979;1980) .Th e reciprocal crosses gave no seeds atal l (Nishiyama & Inomata, 1966).Th e failureo fsuc h crosses was due either to thedeat ho fhybri d embryoso r toabnorma l development of the endosperm and inhibitiono fembry odevel ­ opment(Nishiyam a & Inomata, 1966). Kato &Tokumas u (1979)reporte d that XBrassicoraphanus withwhit e flowers showedmuc hmor e cross-affinitywit h white-fiowered R. sativus thanwit h diploidyellow-flowere d B. japomca Sieb- (= B. campestris ssp. nipposinica). Therevers ewa s found foryel - !ow-flowered xBrassicoraphanus. Pollination ofwhit e flowered xBrassico-

37 raphanus withtetraploi d R. sativus gave rise to 1.1 seeds perpollina ­ tion,wherea spollinatio nwit h tetraploid B. japonica gave no seed at all (Kato& Tokumasu , 1978). Terasawa (1933)teste d thecrossabilit y of xBrassicoraphanus with B. napus (AACC), B. cernua (2n =36 ,AABB )an d B. oleracea (CC),usin g the Brassica spp. aspollinators .A n exceptionally high seed-setwa sobtai ­ ned fromcrosse swit h B. napus, arathe r good seed-setwit h B. cernua andn o seedwit h B. oleracea. The form and fertility ofprimar y allotetraploid hybrids was studied andth ecrossabilit ywit hvariou s specieswa s tested.

5.2 MATERIALAN DMETHOD S

Theprimar y allotetraploid hybrids described below arose directly from theintergeneri c crossesmentione d inTable s 6an d 10.Th e data presented on seed-seto fprimar yAAR R hybrids,however , also include crosses with 'doubled'A Rhybrid s (Section 4.3.5). Theparenta l material of xBrassi­ coraphanus used incrosse swit hvariou s species consisted in 1976 and 1977o fprimar y hybrids and in197 8o fplant s from the second generation. Sometimesth evariou shybrid s arebelo w called, forinstance , chinensis or perviridis hybrid, referring to the Brassica parent in the original intergeneric cross. Most accessions ofth evariou s species crossedwit h xBrassicoraphanus werementione d inTabl e 3,excep t fortw ovarietie s of spring oil-seed rape. Seedo f 'Zephyr', aCanadia nvariety ,wa s kindly supplied byDr . G.R. Stringam (Research StationCanad aDepartmen t ofAgriculture , Saska­ toon, Canada)an d seed of 'Tantal', aFrenc hvariety ,b y Zelder BV (Ottersum,Netherlands) . Plantmateria l wastreate d normally, excepttha tth eAAR R plants were transplanted before flowering into the soil ofa heate d greenhouse.Cross ­ ingtechniqu e andothe rmethod swer e the same as inpreviou s experiments. In1976 ,flowe rbud swer emostl ypollinate d immediately after emasculation but, since 1977,tw oo rthre eday s afteremasculation ,whe n some buds hadopened .Th ereaso n forthi s change oftechniqu e was the questionable significanceo fbu dpollinatio n in intergeneric hybridization.

5.3 RESULTS

5.3.1 Form and fertility of primary hybrids

Growthhabi to f Fx AARRhybrid swa s similar totha to fA R hybrids.I n thevegetativ e stage,al lha d aleaf-rosette , irrespective of the Bras­ sica parentuse d (Fig.7) .Lea fshap ewa s intermediatebu tvarie d widely. Leaves andpetiole swer e lesspubescen t thano f Raphanus. Hairinesspro -

38 ^ *

^^•1 \

"*«

1 r **#•<* .* I t "i

r ~-« ."•-•• ' '1 II*. , ^ -' --•*

J

l

"i

Figure7 .Allotetraploi dhybrid s fromcrosse so fa tetraploi d fodderradish ,RS14 . A.Wit ha diploi d inbred lineo fturni p(^) . B.Wit htetraploi d turnip cv.Tigr a ($>). C.Wit ha tetraploi d formo fBrassic a campestris ssp.perviridi s (oj. ved tob e anexcellen tmarke r inearl y stages ofdevelopmen tt odistin ­ guishhybrid s frommatromorph s incrosse sbetwee norienta l vegetable forms of B. campestris and R. sativus, because the formerwer eglabrous . Similarity ingrowt hhabi tt oA Rhybrid swa s alsoremarkabl e inth e generative stage.Al l hybrids had amai n flower stalk,whic h laterpro ­ ducedman y lateral branches.Th e flowerswer e somewhatlarge rtha ni n tetraploid B. campestris andwer ewhit e orpurple .However ,tw ohybrids , bothwit h 39 chromosomes,ha d yellow flowers.Mos t flowers,whateve r the colour,ha d petalswit h strikingly darkveins ,althoug hplant swit h Plainwhit e orplai nyello w flowers occurred too.Variatio n inth eex ­ tento f floweringwa s frequently observed. Someplant sproduce donl ya

39 B

Tv .1

fe' 'i Î i :

f- V Cl

•> f .:>

Figure8 .Siliqua ean dseed so fF hybridswit hgenom econstitutio nAARR . A.Siliqua eo fa hybri d fromth ecros sBrassic a campestrisssp .chinensi s is (4x)an d fodderradis hcv .Palet . B.Seed so fB .campestri s ssp.perviridi s (left row),a nAAR Rhybri d (middlerow )an d Raphanussativu scv .Pale t (rightrow) .

fewflowers ,i fa tall ;wherea s the flowerbud s generally dried and droppedprematurily ,other s flowered abundantly. Siliquae of xBrassico- raphanus were intermediate between those ofth eparent s (Fig.8A) .Eac h consisted ofa dehiscen tbivalve d partwit h aseptu m and arathe r large indéhiscentpart ,th ebea k or rostrum.Th emea nnumbe r of ovules per pistilwa s about 19,wit h some inth ebeak .

The fertility of F± AARR hybridswil lb e described inmor e detail in Section 7.3.2. Insummary , thehybrid s showed alarg evariatio n in pollen stainability andvarie dwidel y insettin g of siliquae.Th e seed- set,o nth e otherhand ,wa sver ypoo r incrosse sbetwee nAAR Rhybrids ; onaverag e0.00 8see dpe rpollination .Usually , these seedswer e rather well-filled andwer e similar indiamete r and 1000-grainweigh t to B. campestris (Fig. 8B).

5.3.2 Crossability with Brassica campestris, Raphanus sativus and Brassi­ ca napus

xBrassicoraphanus wasuse d extensively incrosse swit h the parental species and B. napus (Table 20). The crosses inwhic h diploid forms of

40 Table 20. Results of crosses of XBrassicoraphanus (AARR) with Brassica campestris (AA and AAAA), Raphanus sativus (RRRR) and Brassica napus (AACC).

Cross YearNumbe ro f female seed polli- sili- seeds hy- matro- plants produ-nat - quae bridsmorph s cing ions obta-sow n plants ined

AARR x AA chinensis hybrids x B.c.ssp. chinensis 1976 4 1 ca 85 34 19 19 9 1977 4 0 90 21 0 0 0 chinensis hybrids x B.c.ssp. oleifera 1976 1 1 ca 6C 7 5 5 3 perviridis hybrids x B.c.ssp. chinensis 1977 6 1 112 6 4 4 3 1976 1 0 7 0 0 0 0 rapifera hybrids x B.c.ssp. chinensis 1976 3 0 16 2 0 0 0 rapifera hybrids x B.c.ssp. oleifera 1976 1 0 ca11 0 0 0 0 0

AARR X AAAA 0 0 perviridis hybrids x B.c.ssp. chinensis 1977 1 0 19 6 0 chinensis hybrids x B.c.ssp. chinensis 1977 2 0 19 0 0 0 0 perviridis hybrids x B.c.ssp. perviridis 1978 6 0 113 0 0 0 0 AARR X RRRR 1 1 1 perviridis hybrids x 'Palet' 1976 1 1 12 6 1978 8 0 98 25 0 0 0

AARR_x_AACC 20 10 9 EÜHSSsis hybrids x 'Zephyr' 1977 5 3 51 13 1 27 1 1 0 0 'Tantal' 1977 3 39 6 185 52 115 48 Bgrviridis hybrids x 'Zephyr' 1977 12 0 'Tantal' 1977 6 1 48 7 7 0 19 1 1 0 0 îâ£lfera hybrids x 'Zephyr' 1977 2 1 0 'Tantal' 1977 4 0 83 0 0 0

MAAJ<_AARR 94 50 0 0 0 ?-£-ssp. perviridis x perviridis hybrids 1978 4 0 RRRR x AARR 1 0 0 0 'Palet' X perviridis hybrids 1Q7R fi 0 70

MCCj^AARR |Zephyr'x perviridi s hybrids 1977 8 ZePhyr'x chinensi s hybrids 1977 8

B. campestris were used as pollinators gave an average of 0.06 seed an 0.03 hybrid per pollination. In 1976, only one chinensis hybrid produced seed in crosses with B. campestris ssp. chinensis and ssp. oleifera. This plant originated from an AR hybrid, whose chromosome number had doubled during regeneration of plantlets in vitro from stem expiants addltl n total, this plant gave rise to 12 backcross hybrids. In ° ;.^nsis hybrids from crosses between this plant and B. campestris ssp. c ^_ (2x) were obtained by culture of 43 embryos in vitro (from 13 po roduced e tions only). In 1977, only one perviridis hybrid P " fMBS of s. Crosses between XBrassicoraphanus (female) and tetraploi o ,_ . see*>Ad/Tahl (Table20)e 20) .Tn .Th ere ree - campestris (male)gav e very few siliguae andn o

41 ciprocalcrosses ,however ,resulte d in amuc hbette r setting ofsiliquae , thoughthe ywer e seedless.Crosse sbetwee n xBrassicoraphanus (female) andtetraploi d formso f R. sativus (male)gav e good setting of siliquae andon eseed ,bu tth ereciproca l crosses yielded only one siliqua andn o seed. Inbot h setso freciproca lbackcrosses ,th e reciprocals differ dis­ tinctly insettin go fsiliquae ,bu t inopposit e directions. Incrosse sbetwee n xBrassicoraphanus (female)an d B. napus, especial­ ly 'Zephyr',a muc hbette r seed-setwa s obtained; on average about 0.35 seedpe rpollination . Several females gave seed, although therewa swid e variationbetwee n females inth e setting of siliquae and seeds.Th emea n seedproductio no fth ebes t female incrosse swit h rapewa s 3.4 seedpe r pollination.Th eseed s fromthes e crosseswer e larger than from inter­ crossing XBrassicoraphanus andwer e on average 2.2 mm indiameter .Th e reciprocal cross,AAC Cx AARR ,gav e riset o evenmor e seedpe rpollina ­ tion,o naverag e 1.3.Howeve rnearl y all 220plant s raised from these seedsturne d outt ob ematromorphs . Only fourplant s were hybrids and arose from small seedswit h adiamete r less than 2.0 mm.

5.4 DISCUSSION

Theprimar yAAR Rhybrid smostl y had awel lbalanced , rather vigorous andregula r growth.Th egrowin g conditions,however , dono tpermi t far- reachingconclusion s aboutth e agronomicvalu e of xBrassicoraphanus, al­ though thevigou r ofth ehybrid s indicatedpotentia l as a forage crop. Thelesse rhairines s than fodderradis hwoul dmak e themmor e palatable forcattle .Morphologica l characteristics, like abundant flowering anda ratherhig hpotentia l seedproductio n per siliqua indicate that xBras­ sicoraphanus couldyiel d amuc h larger number of seed than fodder radish butth esee d size ismuc h smaller thano f fodder radish.Threshin gdiffi ­ culties, asi n R. sativus, canb e expected to some extent in xBrassico­ raphanus, because someovule s are inth e indéhiscentbea k ofth esiliquae . Giventh eappearanc e ofth ehybrid s and the large diversity in B. campes- tris and R. sativus, thecreatio n ofattractiv e andvariabl e sourcemate ­ rial forth eproductio n of ane w forage crop, xBrassicoraphanus, ispossi ­ ble.Th emai nhindranc e in achieving this goal isth ever y poor fertility ofnewl y synthesizedhybrids . Mostcrosse sbetwee n xBrassicoraphanus (female)an ddiploi d forms of B. campestris failed to setseed .Onl y twowhite-flowere dhybrid s produ­ cedseed ;on eplan teve nha d anexcellen t setting of siliquae andseeds . Thedifference s observed incrossabilit ywer ecertainl y notrelate d to theflowe rcolou r of xBrassicoraphanus assuch , as suggested by Kato & Tokumasu (1979).Th etw oyellow-flowere d plants set fruitpoorl y and gave no seed atall .

Reciprocal crosses of xBrassicoraphanus with tetraploid forms ofS .

42 campestris and of R. sativus were nearly all unsuccessful. The differ­ ences in setting of siliguae were remarkable; crosses with R. sativus (4x) gave the best results with xBrassicoraphanus as female parent. The reverse held true for crosses with B. campestris (4x). These contrasts in fruit setting might reflect the effect of selection for better cross- able genotypes in B. campestris and R. sativus, which probably occurred during the synthesis of xBrassicoraphanus. Setting of siliguae and of hybrid seed were quite good in crosses be­ tween xBrassicoraphanus (female) and B. napus. The reciprocal crosses also resulted in a good seed-set, but most seeds gave rise to matromorphs. Although some matromorphs might have originated from illegitimate sel- fings, matromorphy must occur frequently in the rape variety 'Zephyr'. The occurrence of hexaploid hybrids in offspring of these crosses further supports this view (Table 36). Strikingly large numbers of matromorphs were also obtained in crosses between B. napus and AR hybrids (Table 17). So AR and AARR hybrids apparently have an excellent ability to induce parthenogenesis in B. napus. R. sativus probably has a similar ability in rape (McNaughton & Ross, 1978). The reciprocal difference in production of hybrid seed in crosses be­ tween xBrassicoraphanus and B. napus was strikingly similar to a classic example of unilateral incompatibility, namely the cross between B. olera- cea and R. sativus. In both combinations, crosses are more difficult with species having a C genome for female parent; B. napus (AACC) and B. olera- cea (CC), respectively. The good seed-set of xBrassicoraphanus plants after pollination by B. napus showed that the poor fertility of xBrassicoraphanus was not caused bY a disturbed megasporogenesis.

43 6 xBrassicoraphanus: sterility factors and mating system

6.1 INTRODUCTION

Poor fertilityi sa norma l phenomenon inearl y generations of xBrassi­ coraphanus (Section 7.3.2). Small-scale cytogenetic studies have shown thatmeiosi si nthi shybri d ismostl y regular:usuall y onlybivalent s were founda tmetaphas eI (Terasawa , 1932;Mizushima , 1944b, 1950b;Nishiyam a& Inomata,1966 ; Tokumasu, 1976). Tokumasu (1976), forexample , found only bivalents in6 8% o fth epolle nmothe r cells examined; theothe r cells con­ tained someunivalent so rmultivalents .H econclude d thatth efrequenc yo f meiotic irregularitieswa sto olo wt ocaus e suchpoo r fertility. Nishiyama & Inomata (1966)showe d thatmeioti c irregularities might reduce fertility m latergenerations .The y examined meiosis intw ostrain s originating from theamphidiploid sproduce db yTerasaw a (1932)an dfoun d thaton estrai n withhighl y fertilepolle nan dhig h seed-setha dregula r meiosis andth e other strainwit hpolle no flo wfertilit yha dslightl y irregular meiosis (afe wunivalents , sometimes fragments). Ingeneral ,meiosi s in xBrassi­ coraphanus gave riset obalance d gametes,whic h were oftenviable .Fo r example,th eprimar yAAR R hybrids usually produced functional andnormal ­ lyreduce deg gcells ,a sindicate db yth egoo d crossability with B. napus (Section 5.3.2).

Sosterilit yi n xBrassicoraphanus mustb ecause db ybarrier s inth e progamicphas ean dafte r fertilization. Kato (1971)showe d that5 0t o 70% o fal lembry o sacs (ina fe wplant so fprobabl y anadvance d genera­ tion)ha dbee n fertilized fiveday s afterpollination . Fifteen days after pollination,th eembryo sha dreache dth eglobula ro rearl y heartstage . Subsequently,th edevelopmen to fembryo swa sretarde d incompariso nt o nested Par6ntal Species and many «»bryosstoppe dgrowin gan ddege -

Tokumasu (1976)an dKat o& Tokumasu (1976)observe da sudde nincreas e IJ* ^^ ^ aSSOGiation with *chang ei nflowe rcolou rfro mwhit et o TollZ 7 T0rS COnClUded that a Sterllity gene «»trollingth edeve - S <0r qeneand°t hTr . ^^ "** <*—* ^ toth ewhite-flowe r W C&USed tweena B ""*""* ** ^^ " ^ ™ interchangebe - sultedin7r?- and.a RaPhanUS Chr°—• ™* interchangefinall yre - ;l L wel! ;rtl0n'defiCienCy tyPe °fPlant < lackin*th esterilit y white n T Whlte~fl— *»«. However,th esterilit y ofth e white-flowered plantsma yals ob ecause db ya discordanc e betweenth e

44 cytoplasmo f Brassica andth ewhite-flowe r gene (orrathe rgene s linked toit )fro m Raphanus. Recently thesam e authors suggested thatther e mustb estil l other genes affecting fertility (Tokumasu& Kato ,1980) . Sterilityi sa seriou s andpoorl y understood problem.S ovariou sas ­ pectso fi twer e studied: thechromosoma l behaviouri nmeiosis ;th e occurrenceo fbreedin g barriers;th epresenc e andsignificanc eo fself - incompatibility.

6.2 MATERIALAN DMETHOD S

Meiosiswa sexamine d inG l andG 2 plants fromth eP Han dC Hpopulatio n of xBrassicoraphanus (Section 7.2).Th ecytologica l techniques werede ­ scribedi nSectio n2.2 . Breedingbarrier s andexten to fself-incompatibilit y in xBrassico­ raphanus were tested ina nexperimen twit h sevenP Hplant san dfiv eC H

plantso fG 2.Abou t five buds offou r inflorescences oneac ho fthes e Plantswer e emasculated andbagged . Three days later,tw oinflorescence s ofeac hP Han dC Hplan t were pollinated withbulke dpolle n fromal l availableP Ho rC Hplants , respectively.A tth esam e time,th etw oremai ­ ninginflorescence s were selfed.Th epistil so fthos e inflorescences fixedtw oday s after pollination andlate r studiedb yultraviole tmicros ­ copyfo rpolle n germination, penetration ofpolle n tubes intoth est ye ^ thenumbe ro fpolle n tubespe rstyl e andth enumbe ro fovule swit ha P lentub ei nth emicropyl e (Section 3.2).Th ewhol e testwa srepeat e

threeweek s later. „ u_

SixG 2plant s ofth eP Hpopulatio n andthre eG 2plant so fth eC po^ plant P lationwer e tested separately forsettin go fsiligua ean d ^ Qf deVel Pme variationi nnumbe r ofovule s developedpe rsiliqu aan d ° "uina_ embryos,A totalo f6 0t o8 0emasculate d flowerspe rplan twer ep o ^^ tedwit hbulke d pollen from severalP Ho rC Hplants ,respectiv ey ^ ^^ d *yslater ,th edevelope d siliguae andovule swer e counted an ^^ Q£ lopmentalstag eo fal lembryo swa sdetermine d accordingt o e Wilmar& Hellendoor n (1968).

6-3 RESULTS

6-3-l Meiosis

Meiosisi n xBrassicoraphanus wassimila rt otha t inth e ip ^ ^ entalspecie san dshowe d almost exclusively bivalents (Fig. . ^ randomsampl eo feuploi d G,plant s fromth eP Han dC Hpopula t ^

talents onlyi n 79an d8 7% o fth epolle nmothe r cells,r eP and f°rG 2 piants these figures were 91an d9 4% (Table 21).^ ^ ^ Themai n irregularity inmeiosi s ofeuploi d plants ( n 45 B

^\ ^ ' a ill

c ••

4 • *

* ^ ^ • • ft

••"•••• . *

Figure 9. Meiosis in XBrassicoraphanus (AARR hybrids). A., B. Pollen mother cells at metaphase I with 19 II (xl540). C. Pollen mother cell at anaphase I (xi090). occurrence of univalents. Two univalents were found in about 13 % of all

ZmvalTed Snd f°Ur ln °nly °ne Cel1- ^ °nly °th- irregularity, had JIT' rCUrred SP°radiCall*<- «** I-* %« f all cells ex^inel had a quadnvalent, usually of chain configuration otic i3

^xzi:tTPT;T~ 7) " vaient and "is Mvaient—— s in abou t e2 % of the ceiis ;ab : 2-2 Mo : :rtr . five„niv » , „\ • he remainlng cellsha d three or al n ds. H^loi: Plal; ; lentS WerS °bSerVed ^"^ally, as inth e euploi IZIZI hut Zsl V 39> Sh°WetrlValentd lSn ab °U2t %5 °**f • -lis c ne ttree or even five univaients we" : 'b : i ;; :e ™/ ' -

aients (Table 23). Quadnvalents were notnoticed .

46 Table 21.Chromosom e association at diakinesis and metaphase I in pollen mother cells of euploid G and G plants (2n = 38) from the PH and CH population of xBrassicoraphanus•

Plant Numbero f Number of cells with * cellsex ­ amined 1911 1811+ 2 1171 1+ 4 1171 1+ 1I V 1IV+ 161 1+2 1

PHpopulation/G. . PH3 50 42 6 PH5 25 22 2 PH6 25 22 3 PH9 25 22 3 PH18 25 22 3 PH10 16 5 11 PH12 9 1 PH15 25 18 76.2079-2 7 6 76.2257-4 25 21 3 75.2326-1 25 19 5 76.2256-1A 25 22 2 76.2256-1B 25 22 3 Total 307 244 56

PH population/G. PH2-1 z. 25 22 2 PH3-1 22 18 4 PH5-1 25 23 2 PH7-1 25 24 1 PH7-3 25 22 3 PH9-6 25 25 Total 147 134 12

ÇHpopulation/G, CHI — 1 25 24 1 CH2 25 21 4 CH3 25 20 3 76.2104-2 25 22 3 Total 100 87 11

£?LE°pulation/G„ CH3-4 z 25 22 CH4-3 25 25 CH4-6 50 47 Total 100 94

*]I , II,II I and IV are symbols for univalents,bivalents , trivalents and quadrivalents, respectively.

Atanaphas e I,th ehomologou s chromosomes separated synchronously and wereusuall y regularly distributed toth epole s (Fig.9C) .Laggard san d misdivisionso f chromosomes were observed, though rarely (Table24) .Abou t 88% o fal lpolle nmothe r cells inth eeuploi dplant s showed anequa l chromosome distribution (19/19). Liketh e firstmeioti cdivision ,th e secondmeioti c divisionwa s quite regularwit hoccasiona l laggardsan d "»isdivisions. Thehig h frequency ofpolle nmothe r cellswit hunivalent s inth eaneu - Ploid,probabl y monosomic and trisomie,plant s resulted inmor e halfchro - Table22 .Chromosom eassociatio na tdiakinesi san dmetaphas eI inpolle nmothe rcell so fhypoploi d G andG plants (2n= 37 ) fromth eP Han dC Hpopulatio no fxBrassicoraphanus .

Plant Number of Number of cells; wit h* cells examined 1811+ 11 1711 +3 1 1611 +5 1othe r* *

PHpopulation/ G PHI 25 24 1 PH4 25 24 1 PH14 25 23 1 1 76.2147-2 13 7 4 1 1 76.2229-1 25 25 76.2229-12 4 1 3 76.2237-7 25 2 14 9 76.2251-2 25 23 2 Total 167 129 26 10 2

CHpopulation/ G CH-5 25 20 5 76.2104-7 25 23 1 1 76.2202-2 25 25 Total 75 68 6 1

CHpopulation/ G CH2-1 l 14 12

*]I ,II ,II Ian dI Var esymbol sfo runivalents ,bivalents ,trivalent s andquadrivalents ,respectively . **]Othe rchromosom econfigurations ,i.e . 1II I+ 1 5I I+ 4 I ,1 I V+ 1 6I I+ 1 I ,o r1 II I+ 1 6I I+ 2 I

Table23 .Chromosom eassociatio na tdiakinesi san dmetaphas e Ii npolle n mothercell so fhyperploi dG andG plants (2n= 39 )fro mth eP Han dC H populationo fXBrassicoraphanus •

Plant Numbero f Number ofc e ill; swith * cells examined 1811 + IUI 1911+ 11 1811 +3 1 1711+ 5 1

PHpopulation/ G PH8 1 26 2 16 6 2 PH11 25 6 18 1 76.2154-5 25 8 14 3 76.2237-11 25 2 9 12 2 Total 101 18 57 22 4 CHpopulation/ G CH7 * 25 5 20 76.2104-3 2 1 1 Total 27 6 21 CHpopulation/G „ 2 CH1-1 25 10 14 1 CH4-5 18 7 8 3 Total 33 17 22 4

*]I ,II ,II Iar esymbol sfo runivalents ,bivalents ,trivalent s respectively.

48 Table24 .Chromosom edistributio na tlat eanaphas eI an dmetaphas e IIi npolle n

Plantswit h; 2n= 38 Plantsw i th 2n =3 7 Plantsw i th 2n =3 9

distribution numbero f distribut ion numbero f distribut ion numbero f cells cells cells

19 :1 9 169 19 :1 8 64 19 :2 0 29 18: 2 0 16 20 :1 7 1 19%:19 % 8 19: 18* 4 18 :18 * 9 19 :19 * 3 17: 2 1 1 18%:18 % 17 Other 1 Other 2 Other 13

Totalnumbe r ofcell s 192 104 41 examined

Numbero f laggardspe r 0.021 0.13 0.07 cell

Numbero f misdivisions per cell 0.016 0.26 0.20

*]plu son elaggar d

mosomes and laggards per cell inth e subsequentmeioti c stages thani n euploidplants .Th eprobabl e cause ofhal fchromosome swa s centromere misdivision inth eunivalents . Inth e euploids,rathe rhig h frequencies ofunbalance d gameteswer eproduced ,becaus e ofth e occurrence ofuni ­ valents.Mos tunivalent s were included inon eo fth e anaphase groups of chromosomes. Univalent formationwa s apparently under genetic control,becaus emos t

Gx plantswit hman y univalents were derived from the same Brassica plant, i.e.PH1 0 andPH1 2 inTabl e 21,plan t 76.2237-7 inTabl e 22 andplan t 76.2237-11 inTabl e 23.No t surprisingly, thehalf-si b families derived from theseplant s contained ahig h frequency ofaneuploi dplants .

6.3.2 Barriers in the progamic phase

Stigmas ofth e cross-pollinated flowers ofG 2plant s from theC Han d PHpopulatio n always showed serious incompatibility reactions twoday s afterpollination . Strongultraviole t fluorescence duet ocallos edepo ­ sitsoccurre d inman y stigmapapilla e (Fig.10A) ,an dpolle ngrain smost ­ lyfaile d to germinate orproduce d ashor tultraviolet-fluorescen t pollen tube,onl y few tubes actuallypenetrate d the stigma.Th e finalresul to f these incompatibility reactions was apoo r penetration ofpolle n tubes into style and ovary.Th enumbe r oftube s counted per stylevarie d from 0 to2 2an dwa s on an average about10 .I twa snearl y always lesstha n

49 WW-MSW«W?I

y '•VV'-" :

r w i v

* ^ r •"{• -H i ,, >.* *

Î :!" t *• 4 ; \f t

•' Ï

•tf•*j 4 3 ^ •?'" . \ i i:. •''''t--Vv '• '.. »„l '*'''""?"v.. -, ..•..-.-*• J . , •*;-•

?:. '' .,'i.-'' .. . • ' "' •i i iteadJalaAaiààata-^»/.':!.!-n.*,- ^- '•.-.

Figure10 .Ultraviole tfluorescenc ephotograph so fth eprogami cphas ei n .' \\\ \" xBrassicoraphanus2 day safte rpollination . A.Callos ei nstigm apapilla eafte rcross-pollinatio n (xlOO). B.Callos ei nstigm apalilla eafte rself-pollinatio n (X24). C.Fertilize dovul e (xlOO). D.Bundl eo fpolle ntube si nth estyl eafte rself-pollinatio n (XlOO).

50 the number of ovules in an ovary (about 19). Significant differences were observed between individual plants (Table 25). In a few plants, how­ ever, there was rather large variation between the test dates, for in­ stance plant PH9-4. The pollen tubes that penetrated apparently grew unhindered through the style and nearly all reached the ovary. Many tubes subsequently en­ tered an ovule through the micropyle. Such ovules with a tube in their micropyle were classed as fertilized, although it was unknown whether fertilization had occurred (Fig. IOC). There were large and significant differences in the number of fertilizated ovules per pistil between PH plants but not between the CH plants. Number of fertilized ovules per pistil ranged from 1.2 in PH7-2 to 14.9 in PH3-2. Except for PH9-4, this number was nearly equal on the two evaluation dates. The low number for PH9-4 in the second test was obviously associated with the few pollen tubes in the style at that date; it accounted completely for the diffe­ rence between the overall means of 6.3 and 4.9 fertilized ovules per ova­ ry (against 5.5 and 5.3 respectively if the values for PH9-4 were ex­ cluded ). Table 25 shows a large variation in the ratio of number of fertilized ovules and number of pollen tubes in the style, i.e. the fertilization

Table 25. Results of ultraviolet-fluorescence studies on breeding barriers in the progamic phase of fertilization after cross-pollination in the PH and CH population of xBrassicoraphanus. These tests were repeated after three weeks. Each value represents the mean of 4 or 5 flowers.

nt Number of pollen Number of fertilized Fertilization tubes per style ovules per ratio (B/A) (A) ovary (B)*

Test 1 Test 2 Test 1 Test 2 Test 1 Test 2

PH population . „, „ n1 PH3^2 14.60 17.50 14.00 15.75 0.96 0.91 PH5-1 4.75 11-00 3.25 5.00 0.80 0.42 PH7-1 9.80 6.25 6.00 3.00 0.57 0.50 PH7-2 11.80 11.60 1.40 1.00 0.12 0.08 PH7-3 12.20 11.75 11.00 10.00 0.89 0.83 PH9-2 5.00 8.00 2.00 2.40 0.47 0.31 PH9-4 17.40 2.80 17.00 1.40 0.98 0.3b

ÇHpopulation „ EO n -in CHÎ^f 11.00 17.75 5.60 5.50 0.52 0.30 CH4-3 6.50 12.40 3.00 3.60 0.64 0.37 CH4-4 5.60 12.25 3.60 3.25 0.78 0.60 CH6-1 6.80 5.60 5.20 3.00 0.39 CH7-1 9.75 - 3.75 0.63 0.47 Mean 9.60 10.63 6-32 4.90

Leastsignifican t __ _ „, , 270 .26 difference (0.05) 3.36 5.70 2.80 2.96 0- fertilized ovulewa sdefine d asovul ewit hpolle ntub ei nit smicropyle .

51 ratio.Thi svariatio ndi dno tmerel y reflectvariatio n in thenumbe r of pollentube s inth e stylebecaus e theplan tmean s for these traits were notsignificantl y correlated (r= 0.18).Th e fertilization ratio andth e number ofpolle ntube s inth e stylewer e apparently two independent causes ofvariatio n innumbe r of fertilized ovulespe r ovary.Thi s agrees withth eobservatio n thati nplant swit hpoo r fertilization theorienta ­ tiono fpolle n tubegrowt hwithi n theovar ywa s often disturbed asi n theorigina l intergenericcrosses .

6.3.3 Barriers after fertilization

Ina sampl e of xBrassicoraphanus plants,th e setting of siliquae and thedevelopmen to fovule s and embryoswer e assessed thirty days after cross-pollination. Bythen ,nearl y allembryo sha d reached the final size andstage .Th esettin g ofsiliqua e ranged from 38% inCH4-4 t o 97% in PH7-3 (Table 26). Theplant s also differed inmea n length and outerap ­ pearance ofth e siliquae (Table 26).Smal l siliquaewer e often yellow greenan d sometimes slightly shrivelled. Other fruits,mainl y the larger ones,wer enorma l green and often swollen.A positiv e correlation of 0.65 was found forplant sbetwee n setting and average length ofsiliquae . Thevariatio n inbot h characteristics probably reflected variation in thenumbe r of fertilized ovules,becaus e both traitswer e significantly correlatedwit h thenumbe r ofdevelope d ovulespe r siliqua (r= 0.93 and r = 0.65, respectively).

Table26 .Developmen to fsiliquae ,ovule san dembryo so nG plantsfro mth e PHan dC Hpopulatio n3 0day safte rpollinatio nwit hbulke dpolle nmixtur e fromvariou splant so fth eP Ho rC Hpopulation ,respectively .

Plant Numbero f Numbero f Mean Numbero f Numbero f Numbero f polli­ siliquae length ovules ovules embryos nations obtained ofsili ­ developed collapsed persiliqu a quae (cm) persili ­ after qua initital development PHpopulatio n PH3-2 82 77 4.3 6.6 5.2 1.3 PH4-4 62 44 3.7 2.0 1.2 0.6 PH7-2 62 37 1.7 0 0 PH7-3 63 61 2.5 1.7 0.6 0.9 PH9-4 62 51 2.3 0.7 0.6 0.04 PH12-2 61 54 4.3 7.5 1.9 3.8 CHpopulatio n CH4-4 61 23 1.7 0.4 0.4 0 CH4-6 64 59 5.1 8.0 7.6 0.3 CH6-1 86 46 2.8 1.2 1.2 0.02 Mean 3.2 3.1 2.3 0.77

52 The traitdevelope d ovulespe r siliqua (Table26 )include d all ini­ tially developed ovules.Ther ewa s enormousplant-to-plan tvariatio n in it.O n thewhole ,th enumbe r ofovule s developed per siliquawa s lower thanth enumbe r ofovule swit h apolle n tube inthei rmicropyl e (Table 25). A highproportio n of the initially developed ovules later collapsed and shrivelled (Table26) . Mostunshrivelle d ovules contained anembryo ,bu t about2 6% ofthe m contained no embryo.Th e number ofembryo spe r siliquawa sonl y 0.77, whichwa s far lesstha n thenumbe r ofinitiall y developed ovules.How ­ ever individual plants differed widely;PH12- 2produce d 3.8 embryospe r siliqua,wherea s inothe rplant s not asingl e embryo couldb e detected. The developmental stage ofal lembryo swa s classified according to the system ofWilma r &Hellendoor n (1968).Althoug h this systemwa sde ­ vised forBrussel s sprouts,i tcoul db e readily applied to xBrassico- raphanus. The number ofembryo s isolatedpe rplan tdiffere d greatly,bu t nearly allplant s had an almostcontinuou s frequency distribution ofth e embryos over thevariou s developmental stages,indicatin g thatther ei s nota specifi c stage atwhic hgrowt h and development stopped.Th ediffe ­ rences in frequency distribution betweenplant s suggesttha tsom evaria ­ tionexiste d inth edegre e of inhibition ofgrowt h anddevelopmen t (con­ trastPH7- 3 and PH12-2). The condition ofth eovule s indicated thatmos t embryosha d already stopped developing or completed development.Unde r normal conditions,onl y the ovuleswit hembryo s inth ewalkin g sticko r mature I+ II stagewoul dprobabl y give riset oviabl e seed (Table27) .

Table27 .Classificatio nb yth eschem eo fWilma r& Hellendoor n (1968) ofal lembryo sobtaine d fromth ecrosse smentione d inTabl e25 .

1!c Plant Numbero f Developmental stage embryos G H ET T LT WS M

PHpopulatio n 3 2 PH3-2 97 4 46 25 16 1 PH4-4 26 6 7 4 5 3 1 0 PH7-3 55 2 8 13 16 6 6 4 PH9-4 2 1 0 0 1 U 0 0 PH12-2 203 17 90 62 26 b 1 1

CHpopulatio n 5 0 3 CH4-6 18 0 0 7 3 CH6-1 1 0 0 0 1 0 0 0 21 11 10 Total 402 30 151 111 68 --] Gglobula rstage ;H hear t+ earl yhear tstage ;E Tearl ytorped o stage;T torped ostage ;L Tlat etorped ostage ;W Swalkin gstic k stage;M matur eI + II .

SI 6.3.4. Hating system

The parental species of xBrassicoraphanus are predominantly outbree- ders, because of their self-incompatibility systems accompanied by insect pollination. xBrassicoraphanus plants also attract insects, but there was no published data on whether they are self-incompatible. To study this critical aspect of fertilization, two series of selfings were made on the same plants as used for tests on breeding barriers in the proga­ mic phase of fertilization. The mean number of ovules fertilized per ova­ ry with selfing and cross-pollination were presented in Table 28. Four plants were completely self-incompatible (Fig. 10B), and three, PH3-2, PH7-2 and CH4-3, had a much smaller number of pollen tubes per style after selfing than after cross-pollination. Absence of pollen tubes in the style could be caused by a poor pollen germinability. However, pollen stainabil- ity was not related to self-incompatibility and germinated pollen grains were observed on selfed stigmas of all plants examined by ultraviolet microscopy. So such plants were probably truly self-incompatible, as would be expected for allotetraploids derived from two species with a sporophytic incompatibility system. In contrast, five plants were fully self-compatible. In these plants, the number of pollen tubes per style after selfing was even higher than that after cross-pollination (Fig. 10D). However, the progamic barrier on the stigma (Section 6.3.2) was observed in all stig-

Table 28. Comparison of xBrassicoraphanus plants from the PH and CH popula­ tions for number of pollen tubes per style and number of ovules fertilized per ovary after self-pollination and after cross-pollination. Each value represents the mean of 8-10 flowers.

Plant Pollen Numbero f pollen Numbero f ovules stainability tubespe r style fertilize!i perovar y (%) crossed* selfed crossed* selfed

PHpopulatio n PH3-2 74 16.05 0.20 14.88 0.00 PH5-1 66 7.88 0.00 4.13 0.00 PH7-1 39 8.03 8.10 4.50 6.20 PH7-2 64 11.70 1.75 1.20 0.00 PH7-3 61 11.98 0.00 10.50 0.00 PH9-2 28 6.50 0.00 2.20 0.00 PH9-4 62 10.10 13.90 9.20 10.10

CHpopulatio n CH1-1 33 14.38 15.45 5.55 5.70 CH4-3 63 9.45 1.50 3.30 1.00 CH4-4 37 8.93 9.45 3.43 3.35 CH6-1 28 6.20 0.00 4.10 0.00 CH7-1 55 9.75 10.10 3.75 2.50

Mean 51 10.08 5.04 5.56 2.40 *] Data from Table 25.

54 mas, whether self-o r cross-pollinated. So self-pollination ofself-in ­ compatibleplant s only further reducedpenetratio n ofpolle ntube s into thestyle .

6.4 DISCUSSION

Inth epopulation s studied, sterilityha d several causes.Th emai n factors were incompatibility reactions onth e stigma,disorientatio n of pollen-tube growthwithi n theovary ,poo r settingo rgrowt h ofsiliguae , andpoo r growth and arrested development of embryos.Al l these factors dono toperat e equally in allplant s orhav e the same impacto nfertil ­ ity.A disorientation ofpollen-tub e growth, forexample ,wa s absenti n someplants .Th emajo r cause of sterilitywa s undoubtedly poor growth and arrested development ofembryos ,probabl y bymalfunctionin g ofendo ­ sperm, asconclude d by Kato &Tokumas u (1976). The sterility factorswer e quite similar toth ebreedin gbarrier sob ­ served inth e original intergeneric crosses. Interm s ofth etheor y of incongruity (Hogenboom 1973;1975) , thismean s thatth ebarrie r andpene ­ tration capacity of xBrassicoraphanus matched poorly. Soresidua l fac­ torso fth e incongruity system of B. campestris and R. sativus were at leastpartiall y responsible forsterility .Th e situation insuc hnewl y established allotetraploids isprobabl y like adoo rwit h adoubl e lock: thebarrie r capacities of B. campestris and R. sativus. Fertilization only occurs if the key, the combined penetration capacities ofthos e specieswithi n xBrassicoraphanus, fitsbot h locks.A consequence ofthi s hypothesis is that selection foreasie r crossable genotypeswithi nth e parental species of xBrassicoraphanus may alsohav e apositiv e effecto n fertility of allotetraploids derived from suchgenotypes . Chromosome association inmeiosi s agreed quitewel lwit htha tdescri ­ bedb y Terasawa (1932), Mizushima (1950b)an dNishiyam a & Inomata (1966). Theonl y irregularities were acertai ndegre e ofuni -an d multivalent formation.Tokumas u (1976), incontrast , foundman ymor e multivalents perpolle nmothe r cell. Since one ofhi sF 1 hybridswa s amixoploi d

(2x/4x)wit h adiploi d sectorproducin g stainablepollen ,hi sF 2 plants may have originated from a4 x x 2xcross .Suc h anorigi nwoul d givea more satisfying explanation forth e remarkably high frequency ofmulti ­ valents. The degree ofunivalenc e in xBrassicoraphanus was farles s than inoctoploi d andhexaploi d triticale (Scoles& Kaltsikes , 1974), sotha t aneuploidy in xBrassicoraphanus seems ales s importantproblem .Ther e was some genetic variation inthi s characteristic of xBrassicoraphanus and so selection againstplant swit h acomparativel y high degree ofde - synapsis should reduce the frequency ofaneuploids . Fertility problems inth enewl y established populations areprobabl y under genetic control and atleas t somegeneti cvariatio n seemst o exist

55 inal lcomponent s of sterility.A simplebreedin gprogramm e toaccumul ­ atefertilit y factors should improve fertility, asmus t havehappene d in theextensiv ebreedin gprogramme s of xRaphanobrassica (McNaughton,1973 ; Ellerstróm& Zagorscheva ,1977 ;Iwas a& Ellerström , 1981). The question remainswhethe r thepopulation s contain sufficient genetic variation for all sterility factors.I fnot ,structura l changes inth e karyotype (e.g. losso fa chromosom e segmentcarryin g asterilit y gene)coul db ebenefi ­ cial (Tokumasu, 1976). However, aseriou s disadvantage of such changes isth epossibl e losso fusefu l genetic information. For instance,intro ­ duction into thepresen tpopulation s of xBrassicoraphanus of the defi­ ciency thatwa s describedb y Tokumasu (1976)t o improve the fertility wouldprobabl y soonresul t incompletel y homozygous populations forthi s deficiency,becaus e ofth e selective advantage ofremova l of a sterility factor. Itsgeneti c load,however ,i sunknow n and so introduction into thebreedin gprogramm e shouldb e avoided. Mizushima (1950b)reporte d that xBrassicoraphanus was self-sterile. Thepresen tresults ,however , showtha t such allotetraploids could be self-compatible,despit e thepresume d presence of aone-locu s sporo- phytic self-incompatibility system inth eparenta l species of xBrassi­ coraphanus. Theoccurrenc e of self-compatible plants has also beenob ­ served inothe r artificial cruciferous allotetraploids derived from self-incompatible diploid species,e.g . in xRaphanobrassica (Howard, 1942a). The occurrence ofself-compatibl e plants inou rpopulation s indi­ cates thatth eself-incompatibilit y systems ofth eparenta l species were sometimes entirely orpartiall y eliminated. Thepresenc e of self-incom­ patibleplant sdoe sno tnecessaril y imply that self-incompatibility mech­ anisms ofth eparenta l specieswer e actively involved;poorl y matching barrier andpenetratio n capacities couldb e responsible aswell . Ifso , improvement inmatchin gbetwee nbarrie r andpenetratio n capacities should increase theproportio n ofself-fertil eplants .

56 7 Improvement in fertility of xBrassicoraphanus

7.1 INTRODUCTION

Primary allotetraploidhybrid s derived from crossesbetwee n B. campes- tris and R. sativus werepoorl y fertile orproduce d no seeds atal l (Hosoda,1946 ,1947 ;Mizushima , 1950b,1952 ;Tokumasu , 1976;Olsso n & Ellerström, 1980). Inm y hybrids, forexample ,th emea n seed-setpe r pol­ linationwa s only about 0.008.Mizushim a (1950b)eve nfaile d to obtain anyseed s atall .I nth e subsequent generations ofnewl y established xBrassicoraphanus, Olsson &Ellerströ m (1980)observe d deterioration and extinctionb y the fifth generation.Othe r authors,however ,obtaine d rath­ er fertile strains of xBrassicoraphanus (Terasawa,1932 ;Tokumasu ,1976 ; Tokumasu &Kato , 1980).

Terasawa (1932)reporte d the occurrence of allotetraploids inth eF 4 ofa cros sbetwee n B. campestris ssp. chinensis (2x)an d R. sativus (2x). Mosto fthes eplant swer e completely fertile. In1966 , two strains of Terasawa'smateria l still existed (Nishiyama & Inomata, 1966). One of them (2n= 38 )ha dhighl y fertilepolle n and ahig h seed-set.Anothe r fertile strain of xBrassicoraphanus produced byTokumas u (1976)showe d a

sudden increase in fertility inF 3 derived fromtw o F± hybrids ofwhic h thechromosom e numberwa s doubledwit h colchicine.Th e fertility inthi s strain gradually improved even further inth e subsequent generations (Kato& Tokumasu , 1976). Recently thesam e authorsobtaine d ane w rather fertile strain from an allodiploid ofa cros sbetwee n atriploi d B. japon- ica (AAA)an d diploid R. sativus (RR).On eplan tobtaine db y open-pollina­ tion (possiblywit h the fertile strainproduce d earlier asth epolle n donor)wa s an allotetraploid andproduce d aboutthre e seedspe r siliqua. All existing strains of xBrassicoraphanus with arathe r goodfertil ­ ityhav e anarro w geneticbasis . Incontrast ,th ehybrid s obtained in thepresen t studyhav e amuc hbroade r origin. Improvement in fertility isprerequisit e forexploitatio n of any agriculturalvalu eo f xBrassico­ raphanus. So fertility improvement and chromosomal stabilitywer e stud­ ied inearl y generations oftw opopulation s composed ofprimar yhybrids .

7.2 MATERIAL ANDMETHOD S

The two xBrassicoraphanus populations coded PHan dCH ,wer e composed ofprimar y allotetraploids.Th eP Hpopulatio n consisted of4 8 perviridis

57 Table29 .Siz eo fth eP Han dC Hpopulatio n inth ethre e generationsstudie dan dth enumbe ro fisolatio ncage suse d ineac hgeneration .

Generation Population Number of Numbero f isolati on plants* cages

PH 48 Gl 3 CH 1 14 PH 3 82(-17) G2 CH 1 30(-9) PH 6 180 G3 CH 1 25

*]I nbracket sth enumbe ro fplant seliminate dbecaus eo f highlydevian tchromosom enumbers .

hybrids from crossesbetwee n B. campestris ssp. perviridis (4x)an d the tetraploid fodderradis h accessions 'Palet' andRS1 4 (Tables 6an d10) . TheC Hpopulatio nwa s composed of1 4 chinensis hybrids derived from crossesbetwee n B. campestris ssp. chinensis (4x)an d 'Palet' (Tables6 and10) . Bothpopulation s werepropagate d forthre e generations in isolation cages (Table 29). Ineac hcage ,rando mmatin gwa s stimulatedb yhoney ­

bees foralmos tth ewhol e floweringperiod .Th egeneration s G2 andG 3 of thetw opopulation s included allhalf-si b families obtained inth epre ­ vious generation.T o avoid inbreeding asmuc h aspossible , the samenum ­ ber ofseedling swa sraise d foreac hhalf-si b family, ifsufficien t seeds were available,an d forP Hequall y distributed over the cages.S oP H in

Ov G2 andG 3 consisted of3 ,3 and 6subpopulations , respectively,where ­ asC Hwa spropagate d as onepopulation . Someplant s with exceptionally

higho rlo w chromosome numberswer e excluded fromG 2 andmultiplie d sepa­ rately (Table 29). ifpossible ,thes eplant swer e replacedb y otherplant s from thesam ehalf-si b families. Theisolatio ncage suse d forpropagatio nwer e2. 5 m-x 2.5 m or 3m* 3 m andwer emounte d ina heate d greenhouse (18 °C)fo rG andG ,an d outdoors forG3 .Th eplant swer e raised inpot s of about2, 5 litres and weretransplante d into thecage sbefor e flowering.Th emai n flowering

periodwa s fromFebruar y toJun e in Gx andG 2 from June toAugus t in G3. Four fertility traits,i.e .polle n stainability,numbe r ofsiliqua e perplant ,numbe r ofseed spe rplan t and seedproductio npe r siliqua, were studied inth e firstthre egeneration s ofth epopulations .Polle n stainability wasusuall y assessed twice ata tim e interval of several weekswit h lactophenol acid fuchsinb yth emetho d ofSas s (1964). Pollen preparations fromon e flowerwer e used for G.; about50 0polle n grains wereexamine d per flower. InG 2 and G3, arando m sample of20 0 grains was examined inbulke dpolle n from about five flowers

58 The remaining fertility traitswer emonitore d atharvest ,agai n for eachplan t separately. InG - andG_ ,al l siliquae developedwer eharves ­ ted,counte d and shelled byhand .Al l seedswer e thencounte d andth e number ofseed spe r siliquawa s calculated. The sameprocedur ewa suse d

forG 3 when thenumbe r of siliquaepe rplan twa s less than100 . Ifmor e than 100 siliquae werepresen t inG_ ,al l siliquaewer eweighte d andsee d productionpe r siliquawa s estimated in arando m sample of10 0siliquae , whichwa s alsoweighte d inorde r toestimat e thetota lnumbe r ofsiliquae . Chromosomes were counted atmeiosi s ormitosi s ofG .an dG _plants .

7.3 RESULTS

7.3.1 Chromosome numbers

The chromosomes of4 6G. .plant s and 118G plants fromth eP H andC H populations were counted (Table 30).I nG, , thechromosom e numbers ranged

from3 5 to3 9 and amongG ,plant s from 34t o 58.Bot hpopulation s inG 2, had aremarkabl ebimoda l frequency distribution forchromosom e number withmaxim a at3 8 and 57.Mor e than 12% ofG ,plant s classified had over 50chromosomes . InG~ ,th e frequency ofeuploi dplant s (2n= 3 8o r 57)

washighe r than inG 1 but theproportio n ofplant swit h 38 chromosomes hadhardl y increased. The number ofhypoploi d plants,especiall y inth e PHpopulation ,wa smuc hhighe r thanth enumbe r ofhyperploi d plants,es ­

pecially inG 2. The frequency ofchromosom enumber s inoffsprin g ofal lseed-produc ­

ingG 1 plants fromth e twopopulation s studied isliste d inTabl e 31.G 1 plantswit h 37chromosome s gavepredominantl y euploidplant s (2n= 38), like the euploidG- .plants .Nearl y allhypoploi dplant s inth e half-sib progenies ofeuploi dG. .plant swer e derived fromPH1 0 andPH1 2 (Table 31). Thechromosom e number inoffsprin go f G^^ plantswit h3 9 chromosomes was quitevariable .Mos t aberrantP Hplant swer e derived from thehybri d PHll,whic h originated from acros s onth e same Brassica plant asPH1 0 andPH12 ,tw o hybrids thatshowe d acomparativel y high frequency ofuni ­ valents.

Table30 .Frequenc yo fchromosom enumber s inG jan dG 2o fth eP Han dC Hpopulation .

Genera- Popula- Numbero f Chromosomenumbe r tion tion plants 34 35 36 37 38 39 40 41 43 45 52 54 56 57 58

G! CH 10 3 5 2 PH 36 1 1 11 16 7 G2 CH 32 1 2 15 4 112 42 PH 86 1 5 9 12 47 1 2 1115 1

59 Table31 .Frequenc yo fchromosom enumber si nhalf-si bfamilie so fG plants withvariou snumber so fchromosome si nth eP Han dC Hpopulation .

Seedparent s (G plants) Chromosomenumbe rG plants* Numbero f G plants Chromo-Popula -Numbe r 34 35 36 37 38 39 40 41 43 45 52 54 56 57 58 some tion of number plants

37 CH 1 1 1 2 PH 3 2 3 11 16

38 CH 4 19 3 15 PH 11 4 6 3 28 1 3 46 (3)(6 )(3 )(1 ) (1) (1)

39 CH 1 3 1 1 1 3 1 10 PH 2 1114 7 1 1 2 1 20 (1)(1 )(1 )(1 )(2 ) (1) (2) (1) unknown CH 1 1 2 1 1 5 PH 2 2 1 1 4

*]I nbracket sth enumbe ro fplant s fromPH10 ,PH1 1an dPH12 .

Ina fe whalf-si b families,G 2 plantswit h 52-58 chromosomes were found.O fth e2 5 families raised, only 7gav eris e to such aberrant plants. Theoccurrenc e ofhexaploi d andhighl y aneuploid plants inG ,seeme d a serious threatt oth echromosoma l stability ofth eP H and CHpopula ­ tion.Therefore ,al lG 2plant swit hmor e than3 9 and less than 37chro ­ mosomeswer e excluded from thetw opopulations .Som e aneuploids,however , were noteliminate d because their chromosome numberwa s not established properlybefor e transplantation intoth e isolationcages .

InG 3 ofth eP H andC Hpopulations ,n ohexaploid s were detected:mor ­ phological observations,measurement s ofpolle n size and checks ofchro ­ mosomenumbe r ina fe wsuspect s revealed nohexaploids .

7.3.2 Changes in four fertility traits

Changes inpolle n stainability, setting of siliquae,see d production and seed-setpe r siliquawer e studied in the firstthre e generations of theP Hpopulatio n (Fig.11 )an d CHpopulatio n (Fig.12) .Th e results were expressed ascumulative-frequenc ydistributions .Th e actual number ofplants, o nwhic hth egraph s arebased ,ma y differ slightly from each otherbecaus e ofmissin g data andbecaus e a fewplant s died prematurely and soprovide d nodata .Th ethre e generations ofth etw o populations were successively studied ina perio d ofthre eyears .S odifference sbe ­ tweengeneration s canb e attributed to some extentt oyea r influences. Theplant-to-plan tvariatio n inpolle n stainability wasver y large

60 Cumulative frequency Cumulative frequency CAO CAO 100 100

G1(N=47 _ _ G2(N=S4) D—O G3CN=178)

100 1000 O 10 20 30 40 50 60 70 80 90 100 Number of siliquae per plant pollen stamabilityi0/»)

Cumulative frequency Cumulative frequency CAO C/o) 100 100 ID

10 10° . Number of seeds per 100 s.l.quae

Figure 11. emulative frequency ^^Zlll^^^^^ three generations of the PH population of xBrassico^ A. Pollen stainability (%)• B. Number of siliquae per plant. C Number of seeds per 100 siliquae. D. Number of seeds per plant. _ number of plants in the gener The population means are marked with an arrow.

ts of the PH population pro- (Fig. HA and 12A). About 35 % of G P ^ ^.^ had various causes, duced no stainable pollen at all. mis» ^ flowers in a few plants, such as the absence of pollen shedding or even o plants grad­ in the subsequent generations, the proportion of male 61 Cumulative frequency Cumulative frequency CAO (°A0 100 100

G1 (N=14) G2(N=21) I63(N=25)

l(log) 20 30 40 50 60 70 80 90 100 10 100 1000 10000 Pollen sta inability CAO Number of siliquae per plant Cumulative frequency Cumulative frequency CAO 100 (%) 10O r

G1 (N=9 ) G1 (N= 1 4 ) 'G2 ( N=16) G2( N=21 ) G3( N=23) G OG3(N =25 )

klog) , , . . IciogJ 10 100 250 10 100 1000 10000 Number of seeds per 100 siliquae Number of seeds per plant

thfeeeJn;^rUlatirire^enCy distributio^ of various fertility traits in the first three generations of the CH populations of xßrassicoraphanus. A. Pollen stainability (%). * B. Number of siliquae per plant. C. Number of seeds per 100 siliquae. D. Number of seeds per plant. The population means are marked with an arrow. N = number of plants in the generation.

ually decreased and the mean pollen stainability of the PH population im­

proved from 24 % in Gl to 49 % in G3. The CH population had a higher pollen staxnability initially, mainly because of fewer male-sterile plants; there

was no improvement in subsequent generations; the G2 even showed a decline.

62 Onth ewhole ,polle n stainability was highly variable. Ina separat etest , acoefficien t ofvariatio n ofabou t3 0% wa s found. Sodat amus tb einter ­ preted cautiously. TheP Hpopulatio n also showed an increase inth e three successivege ­ nerations inth enumbe r of siliguaeharveste dpe rplan t (Fig. IIB). The proportion ofplant swithou t any siliquae atal ldecrease d considerably.

The samewa s true forth eC Hpopulatio n (Fig.12B) .Th e G±plant s ofth e PHpopulatio n produced only fewsiliquae .Howeve r astrikin g improvement occurred inth enex ttw o generationsb y areductio n inth eproportio n of plantswithou t siliquae andplant swit h fewsiliquae .Th emaximu mproduc ­ tion inth e respective generationswa s 1482,138 6 and 1947 siliquaepe r plant.Th e data onth e CHpopulatio n areles s indicative because fewer plantswer e available ineac hgeneration .However ,G 2 showed aseriou s drop inproductio n of siliquaepe rplant ,thoug hrecoverin gt o levelo f G followed inth enex tgeneration . Themos tcrucia l trait isundoubtedl y seedproduction .Th eP H andC H populations hadver y low fertility inG x andproduce d only 6.5 and 5.6 seedspe rplant ,respectivel y (Fig.11 D and 12D,respectively) . Inth e following generations of theP Hpopulation , aconsiderabl e improvement occurred, theG 3 plantsproducin g about7 2 seedspe rplant .Th e frequen­ cydistribution s for seedproductio n inth eP Hpopulatio n showed a striking reduction in theproportio n ofcompletel y seedlessplant s in successive generations.Th e lastgeneratio n showed amarke d shiftto ­ wards ahighe rproductivity . TheC Hpopulation ,o nth e otherhand ,ha da setback in seed production inG 2paralle l to thedecreas e inproductio n ofsiliquae ,bu tth eresult s ofG 3 weremuc hbetter ,eve ntha nG^^ . The frequency distributions of seed-setpe r siliqua differed only slightlybetwee nG andG 2 ofth eP Hpopulation ; about0.0 5 seedwer e obtained inbot h generations (Fig.11C) .Th e G3, however,showe d a striking improvement. Someplant s evenha d aproductio n ofove r one seed per siliqua, and the frequency ofplant swit honl y seedless siliquae further decreased. The range of seed-setpe r siliqua inth ethre esucces ­

sive generations ofth e CHpopulatio n was aboutth e same asi n G±o fth e

PHpopulation .Afte r asetbac k in seed-seti nG 2 by increase inth enumbe r ofplant swit h seedless siliquae,th e seed-seti nG 3greatl y improved. Insummary , seedproductio n inth eP Hpopulatio n improvedwit ha nin ­ crease insiliqu aproductio n and seed-setpe r siliqua.Th e twotrait s were only slightly correlated (r= 0.17 in G3), butwer ebot hpositivel y correlated with seedproductio n (r= 0.53 and r= 0.7 0 inG3 , respective­ ly). Similar relationships were found inG 3o fth eC Hpopulation .Th eim ­ provement in fertility ofthi spopulatio nwa sbrough t aboutentirel yb y animprove d seed-setpe r siliqua.Th e fourth fertility trait pollen stainability, was independento f seedproductio n (r- 0.23 andr

inG 3 ofth e CH andP Hpopulations ,respectively) .

63 7.3.3 Effects of chromosome number on fertility

Chromosomenumbe rvarie d in the G± andG 2 ofth e twopopulation s of xBrassicoraphanus studied.Th e impacto fthi s variation onvariou s fer­ tility traits issummarize d inTabl e 32.Euploi dplant s had, onaverage , more stainablepolle n andproduce d considerably more siliquae and seeds thananeuploids .A nexceptio nwa s the class ofplant s with 36 chromosomes. Sixo fthos eplants ,however ,wer e derived from the sameG plant,whic h had acomparativel y good fertility. Soth erathe r good fertility ofthi s classwa sprobabl y notcause d by adirec teffec to f chromosome numbero n fertility. Inconclusion ,aneuploid y had asmal l negative effect on all fertility traits. Soselectio n for fertility favours euploid xBrassicoraphanus plants. Cytological selection foreuploid y seems not necessary.

7.4 DISCUSSION

Thepopulation s responded fairly strongly tonatura l selection forim ­ proved fertility.Th eP Hpopulatio n responded with agradua l improvement m allth etraits ;th e appearance ofth eplant s improved too, resulting

m G3 ofgreate runiformity , abundant flowering, and only a fewplant s of abnormal form.Th eC Hpopulation , onth e other hand, showed less obvious

trends. InG 2, anobviou s loss ofvigou rwa s accompanied by a serious setback inal l fertility characteristics.Th epopulation , however,reco ­ vered tosom e extent inG_ . A largevariatio n in all fertility traits studied was still present

m G3 ofbot hpopulations .Polle n stainability and fruit setting varied from zero toalmos t10 0% . m contrast, seed-setpe r siligua was always farbelo wpotential . InG3 , forexample ,th ebes t seed-set observed was about1 an d 0.3 seedpe r siligua inth eP H and CHpopulation , respect- In"6an dG ff:ftthefx^r°mOSOmer* " °nVariatio n in "me fertility traits 1 M ^20 f the xBrassxcoraphanuspopulation s PHan dCH . Characteristic Chromosomenumbe r

35 36 37 38 39 40 41 45 Numbero fplants * 1 7 27(2) 80(2) 14 1 1 1 Meanpolle nstainabilit y 15 57 Numbero fsiliqua e 28 41 32 13 0 19 perplan t 3 284 Numbero fseed spe rplan t 75 159 120 0 4 13 0 9.7 Numberfractio no fplant s 2.5 13.4 8. 1 0 0 0 withsiliqua e(% ) 100 86 Numbero fseed spe r 56 81 57 0 100 100 100siliqua e 0 4.1 3.6 5.8 5. 7 0 0 0 'plants , which diedb e forematuri t V-

64 ively,wherea s thenumbe r ofovule spe r siliquawa s about 19.Th e large variation in setting of siliguae and of seeds per siliqua inG . suggested thatsucceedin g generations would furtherimprove . Prerequisite forcommercia l use of xBrassicoraphanus isa stabl echro ­

mosome number. In G± andG 2,mos tplant s had about3 8 chromosomes,bu t several plants had an aberrant number.Th emos t striking phenomenonwa s theoccurrenc e ofhexaploi d plants with about 57chromosome s in someG , families fromboth 7populations. Suchplant sprobabl y arose from fusion ofa nunreduce d and areduce d gamete,resultin g inplant swit h the genome constitutionAAARRR . The occurrence ofunreduce d gametes indicates that xBrassicoraphanus may form unreduced diplosporic or aposporic embryosacs as xRaphanobrassica (Ellerström& Zagorscheva, 1977). The hexaploid

G2 plants usually showed only rather small differences in appearance compared toth e tetraploid counterparts,suc h as larger flowerbud s and flowers, andusuall yha d somewhat larger stainablepollen . InG~ ,n o hexaploids were detected byvisua l screening andb ymeasurin gpolle nsize . The occurrence of suchhexaploid s couldbadl y affectchromosoma l stabil­ ity innewl y createdpopulations .Th e deterioration and subsequentextinc ­ tion inEllerström' smateria l (Olsson& Ellerström , 1980)migh thav ebee n caused by non-reduction. The other important aspecto fchromosoma l stability, the aneuploidy, was alreadypresen t in G1. Intha tgeneration , theoccurrenc e of aneuploid plants was largely enhanced by theus eo f autotetraploid formso fth epar ­ ental species inth eproductio n of xBrassicoraphanus. InG 2,mos tplant s derived frommonosomi c and trisomieG ,plant s turned outt ob e euploid, indicating thatbalance d gametes and zygoteswer e fitter andha d aselec ­ tive advantage.Howeve r several aneuploids were still found inG 2,proba ­ blybecaus e of formation ofunivalent s inmeiosis . Insummary , sterility inearl y generations of xBrassicoraphanus ispre ­ dominantly caused byvariou s breedingbarriers ,whic hwer e geneticallyde ­ termined.Meioti c irregularities likenon-reductio n and desynapsisproba ­ blyha d no effect on fertility assuch ,bu tma y giveris et o aneuploid offspringwit h reduced fertility.Howeve r non-reduction and desynapsis seem of little consequence.

65 8 AAR hybrids and introduction of alien chromosomes into Brassica campestris

8.1 INTRODUCTION

Introgressiono f Raphanus genesint o B. campestris hasno tye tbee n accomplished.Th eai mo fth epresen tprogramme ,wa st oproduc emonosomi c additionsi n B. campestris, eachplan thavin gth enorma lcomplemen to f chromosomesplu sa nextr achromosom efro m R. sativus. Theexpectatio n wastha tsuc hplant swoul dserv ea ssteppin gstone sfo rtransfe ro fpar ­ ticular Raphanus genes,e.g .resistanc et obee teelwor mo rt oclub-root . Themetho dchose nfo rproductio no fsuc haddition sinclude dtw osteps : productiono fAA Rhybrid san dbackcrossin gwit h B. campestris (AA)a s recurrentparent .Thi schapte rreport sattempt st oachiev eth eai man d discussesth eproblems ,tha tma yhampe rsuccessfu ltransfe ro f Raphanus genes into B. campestris.

8.2 MATERIALAN DMETHOD S

Theattempt st oproduc eAA Rhybrid swer esummarize di nTabl e20 .I n total,1 9firs tbackcros shybrid s(BC^ )includin g4 plant sproduce db y embryocultur ewer eobtaine dfro mcrosse so ftw oprimar yAAR Rhybrids , 75.1040-9(2 n= 38 )an d76.2229-1 3 (2n= 39 )wit h B. campestris (2x). Theallotetraploid ,75.1040-9 ,originate dfro mdoublin gi nvitr oo fth e chromosomenumbe ro fa diploi dhybri dfro ma cros sbetwee n B. campestris ssp. chinensis (2x)an dcv .Silett a (2x).Th eothe rallotetraploid , 76.2229-13,wa sobtaine dfro ma cros sbetwee n B. campestris ssp. perviri- dis (4x)an dcv .Palet .Th eAA Rhybrid swer estudie dfo rform ,chromosom e number,fertilit yi ncrosse swit hB . campestris ssp. chinensis (2x)an d inparticula rfo rchromosom eassociatio ni nmeiosis . TheAA Rhybrid san d B. campestris ssp. chinensis (2x)wer ecrosse di n thewinter s1976/7 7an d1977/7 8i na heate dgreenhouse ,i nwhic hal l parentplant swer egrow ntogether .I nth efirs twinter ,newl yopene d flowerso fth eAA Rhybrid swer ehand-pollinate dwit hbulke dpolle nfro m therecurren tparent ,S . campestris, twoo rthre etime sa wee kove ra periodo fabou tthre emonths .I nth esecon dwinter ,th eparent swer e growntogethe ri na nisolatio ncag ewit hbee sfo rpollination .

Thesecon dbackcros sgeneratio n(BC 2)wa sgrow ni na simila rwa yt o theB Clgeneratio ni n1977/78 ;i na nisolatio ncag ewit hth erecurren t parentan dwit hbee sfo rpollination .

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s fi

j* 'YB£ « ULtîttP # ^b..j--*-^Ji^^|1 j^|f-,iulw,M|rfiijäB^^^ .i

Figure 13.Backcros sderivative so fa 'doubled 'F hybrid fromth ecros sBrassic a campestris ssp.chinensi s (2x)x Raphanu s sativuscv .Silett a (2x)an dB .campestri s ssp. chinensis (2x)a sth erecurren tparent . A.Firs tbackcros s derivative (AAR,2 n=29 )i nth evegetativ estage . B. Firstbackcros sderivativ e (AAR,2 n=29 )i nth egenerativ estage . C Secondbackcros sderivativ e (2n=2 5plu s fragment).

8• 3 RESULTS

8.3.1 Form and chromosome association in AAR hybrids

Of1 2BC , plants,te nplant sha d2 9chromosome s andwer e apparently true allotriploids withth egenom e constitution AAR.Th etw oremainin g plantsha d2 8chromosome s plusa fragmen tan d3 1chromosomes ,respec ­ tively.Th elatte r occurred inth eprogen yo fplan t76.2229-13 .

67 TheBC ^plant s derived from crossesbetwee n 75.1040-9 (4x)an d B. campestris ssp. chinensis orssp . oleifera grew quitevigorousl y and were inman y respects intermediatebetwee nth eparents .Al l plants formed aleaf-rosett e inth evegetativ e stage andha d shallowly lobed and slightly pubescentleave s (Fig.13A) .Th e incisions ofth e leaves weremor epronounce d inplant s fromcrosse swit h B. campestris ssp.

oleiferea. Inth egenerativ e stage,th eBC 1 plants initially had an erectmai nste mwit ha termina l inflorescence (Fig. 13B)an d reached a heighto fabou t1.5m .Afterward s many secondarybranche s were formed, resulting ina dens ebush yplant .Th ehybrid s derived from plant 75.1040-9 (4x)flowere d abundantlywit hplai nwhit e flowers like those ofplan t75.1040-9 . Incontrast ,th ethre eBC ^plant s originating fromplan t 76.2229-13 (4x)showe ddisturbe d growthwit h irregularly shaped leaves.Onl y oneo f theseplant s flowered;th e flowerswer eveine d andpurple . Noneo fth eAA R hybridsproduce d stainable pollen, the anthers were already shrivelledbefor epolle n shed.Th edevelopmen t of floralpart s was apparently quitenormal ,althoug hpoorl y developed ovuleswer eob ­ served ina fe whybrids .Afte rpollinatio nwit hpolle n from B. campes­ tris, afe wsiliqua edeveloped ;the ywer ebivalve dwit h awel l developed beak andha d amea nlengt ho fabou t2. 6 cm. Chromosome association atmetaphas e Io fmeiosi swa s studied inthre e closelyrelate d allotriploidplant s (Table 33). Thepresenc e of atriva ­ lenti na fe wpolle nmothe r cells and the largeproportio n ofpolle n mothercell s (86% )wit h1 0bivalent s and 9univalent s (Fig.14A )indi ­ cate thathomologou spairin g occurred almostexclusively .Howeve r the occurrence ofpolle nmothe rcell swit heleve nbivalent s shows thatsom e autosyndeticpairin gbetwee nR-chromosome s occurred (Fig.14C) .Th epres ­ enceo fa fe wchain-trivalent s further suggested that allosyndetic pairingbetwee nA andR chromosomes waspossibl e (Fig.14B ;Tabl e33) .

Table33 .Chromosom eassociatio na tmetaphas eI i nthre e AARhybrid s (2n= 29 )fro mcrosse sbetwee na homozygou s xBrassicoraphanusplan t (75.1040-9 (4x))an dBrassic a campestrisssp .chinensi s(2x) .

Chromosome Numbero fcell sobserve d configurations* Hybrid 1 Hybrid 2 Hybrid3

11I + 9I I 3 1 0 9 I+ 1 0I I 57 34 29 8 I+ 9I I+ 1 II I 1 2 0 7I + 1 1I I 7 3 2 Total 68 40 31

*]I ,I Ian dII Iar esymbol sfo runivalents ,biva ­ lentsan dtrivalents ,respectively .

68 if- B

•: *

• A

» lï' * * N» « *

Figure 14.Chromosom eassociatio na tmetaphas eI i nAA Rhybrid sfro ma cros sbetwee n a 'doubled'A Rhybrid ,76.1040- 9 (4x),an dBrassic a campestrisssp .chinensi s (2x). A.1 0I I+ 9 I (X1540) . B. 1II I+ 9 I I+ 8 I (X1540) . C 11I I+ 7 I (X1090) .

Thedifference s inchromosom e associationbetwee nth ethre eAA R hybrids were small.On e reasonma yb e thatth eplant sha d an identical seto f R andA chromosomes.

8.3.2 Attempts to introduce alien chromosomes

In197 7 and 1978,thousand s of flowers ofth eAA R hybridswer epollin ­ atedb y hand orb ybee swit hpolle n from B. campestris (2x).Th e results ofthes e crosses,i.e . BO,, are summarized inTabl e 34.Mostl yn osiliqua e

69 Table34 .Seed-se to nsevera lAA Rhybrid susin gBrassic acampestri s ssp.chinensi s (AA)a s pollinator.

Origino f Year Numt1e r of Totalnumbe r AARhybrid s AAR hyb rids ofseed s

75.1040-9 (4x)x 1977 11 5 B.c.ssp .chinensi s 1978 2 0

75.1040-9 (4x)x B.c.ssp .oleifer a 1978 3 27

76.2229-13 (4x)x B.c.ssp .chinensi s 1978 3 0

oronl y afe wdevelope d oneac hplant ,excep to non eAA R hybrid derived from acros sbetwee n 75.1040-9 (4x)an d B. campestris ssp. oleifera. De­ spiteconsiderabl e efforts,onl y3 2 seedswer eproduce d from six ofth e plantsonly .

TheBC 2 generation consisted ofeleve nplants ,o fwhic h three died beforematurity . In1978 ,anothe r three BC2 plantswer e raised by cul­ tureo f2 6embryo s invitro .Th e chromosomes ofmos t full-grown BC_ plantswer ecounte d (Table 35). Mostha d a few chromosomes less thanth e originalAA Rhybrids ,bu ttw oplant sha d considerablymor echromosomes .

Son omonosomi c additionswer eobtained .Th e BC2 plants were usually weak and floweredpoorly .Al lplant swer emale-steril e and had white flowers,excep ttw oplant swit hpal eyello w flowers.Figur e 13C shows theonl yBC 2 plantwit h 25 chromosomes (plus a fragment). Thisplan tha d ratherirregula rgrowth , andmos t Raphanus characteristics were apparent­ lylost .

All BC2 plantswit h2 9o r fewer chromosomes werebackcrosse d againwit h B. campestris ssp. chinensis (2x). Despite large-scale pollinations,non e of these plants produced seeds.

Table35 .Frequenc yo fchromosom enumber si nfull-grow nplant s obtained fromcrosse sbetwee nAA Rhybrid san dBrassic a campestris (2x)i n197 7an d1978 .

Year Numbero fplant swit h2 n= Total number 25+f* 26 27 28+f 29 44 48 ? ofplant s

1977 1 1 114 1978 13 1 2 7 Total 1 13 1 1 1 1 2 11

']f = fragmen t

70 8.4 DISCUSSION

The introduction of single Raphanus chromosomes into B. campestris pro­ ved asye t impossible.M y difficulties inobtainin g aBC ,generatio nwer e similar tothos e ofBannero t et al. (1974)an dMcCollu m (1979)i n attempts tocros s CCR hybrids with B. oleracea (CC).M yproblem s mightb e overcome byusin gmor e AAR hybrids and awide r range ofpollinators . InBC, , thevariatio n inchromosom e numberwa s large,a sother s found forCC R hybrids (Bannerot et al., 1974;McCollum , 1979). Even repeated backcrossingb y those authors with B. oleracea asrecurren tparen tdi dno t result inmonosomi c additions.Bannero te t al. (1974)obtaine d such an additionb y culture invitr o of immature embryos.Th e same approachma y benecessar y to obtain additions in B. campestris. Analysis of chromosome association duringmeiosi s inAA R hybrids sho­ wed thatonl yhomologou s A chromosomes paired as arule .Allosyndeti c and non-homologous autosyndetic pairingwa s rare.Th epresen tdat a confirmed thehypothesi s ofMizushim a (1980)tha tth eR genomema y giveris e toon e autosyndetic bivalent.Th e frequencywa s about0.08 6bivalent s per cell. Thedegre e of allosyndetic pairingwa spresumabl y evenlowe rbu t atleas t oneR chromosom e seemed sometimes tob eboun d to anA chromosome.Thi s agreeswit h the findings on chromosome association ina nAR R hybridb y Mizushima (1980). Comparison ofchromosom e association inA R andAAR R hybrids indicates thatth e degree of allosyndesis inth eAA R hybridswa s reduced,becaus e ofpreferentia l pairingbetwee nhomologou sA chromosomes inth ehybrid s (Tables 14 and 33). Preferential pairingprobabl y affected especially thedegre e of allosyndesis,becaus e thedegre e of autosyndesis within theA genomewa s already small (Mizushima,1980 )an dther ewa sn o reason for adecreas e in autosyndesiswithi n theR genome . Inconclusion , transfer of Raphanus characteristics to B. campestris byth emetho d chosen isver y difficult and time-consuming,becaus e of difficulties of introducing singleR chromosomes into B. campestris and theexpecte d low degree ofrecombinatio nbetwee nR an dA chromosomes .

7] 9 Ways of transferring Raphanus characteristics to Brassica napus

9.1 INTRODUCTION

Directtransfe ro f Raphanus characteristicst o B. napus bycrossin g thetw ospecie san dbackcrossin gth eF ,hybrid swit hth elatte ri sdif ­ ficultbecaus eo fth epoo rcrossabilit ybetwee nthes especie s(Chopinet , 1942;1944 ;Fukushima ,1945 ;Becker ,1951 ;Heyn ,1973 ;Valdivi a& Badilla , 1977;McNaughto n& Ross ,1978 ;Rousselle ,1979 ;Takeshit âe tal. ,1980 ; myresults) .Som eauthors ,however ,obtaine da fe whybrids .Takeshit ae t al.(1980 )culture dsevera lovule sfro mreciproca lcrosse si nvitr oan d raisedon eplant .Crossabilit ycoul db esomewha tbetter ,i ftetraploi d formso f R. sativus wereuse d(Turesso n& Nordenskiöld ,1943 ;Chopinet , 1944).Hybrid sfro msuc hcrosses ,however ,appea rles ssuitabl ea s startingmateria lfo rtransfe ro f Raphanus genes,becaus eo fpreferen ­ tialpairin gbetwee nR chromosome san dprobabl ya poo rcrossabilit ywit h B. napus. Alternativeway sfo rintrogressio nar epossibl ewit hth evariou sin - tergenerichybrid sfro mcrosse sbetwee n Brassica speciesan d R. sativus, i.e.allopolyploid swit hth egenom econstitutio nCCRR ,AARR ,o rAACCRR . Anattractiv ewa yi sa backcros sprogramm eo f xRaphanobrassica (CCRR) with B. napus, because xRaphanobrassica hybridizesreadil ywit htha t species(Karpechenko ,1937 ;Rousselle ,1979) .I nthi sway ,Stefansso n (Winnepeg,Canada ,cite db yHeyn ,1978 )ha ssucceede di ntransferrin g thewhite-flowe rtrai to fradis ht orape .Simila rwor ki si nprogres si n Germany(Heyn ,1978 )an dFranc e (Rousselle,1979 )t otransfe rfertility - restorergene sfro m Raphanus torap ewit hcytoplasmi cmal esterility . Heyn(1978 )als ocrosse dsuc hmale-steril erap ewit ha nallohexaploi d withth egenom econstitutio nAACCRR ,tha tha dbee nproduce db yChopinet . As xBrassicoraphanus seemeda goo dbridg efo rtransfe ro f Raphanus characteristicst o B. napus, abackcros sprogramm ewit h B. napus as recurrentparen twa sse tu pwit hspecia lattentio nt oform ,fertilit y andchromosom eassociatio na tmeiosi so fhybrid sbetwee n xBrassicoraph- anus and B. napus.

9.2 MATERIALAN DMETHOD S

Initialmateria lfo rth ebackcros sprogramm ewa s2 4male-steril e hybridsfro mcrosse so f xBrassicoràphanus (perviridis and chinensis

72 hybrids)wit h 'Zephyr', aCanadia n rapevariet y (Table20) .Befor e flowering, theseplant swer e transplanted into two isolation cages in aheate d greenhouse, inwhic h continuously floweringplant s of 'Zephyr', the recurrentparen t inth ebackcros s programme,wer epresent .Honey ­ beeswer ekep t inth ecage s forpollinatio n and soman y flowers of the hybridplant s werepollinate d with 'Zephyr'pollen . Some embryos were raised invitr o by themetho d ofHarber d (1969,1971 )i norde r to shorten thelife-cycle . The firstbackcros s generation,partl y raisedb y embryo culture,wa s grownunde r isolation in aheate d greenhouse togetherwit h 'Zephyr'.Abou t 100hand-pollination s were done oneac hplan tt oobtain d asecon d backcross generation.

9.3 RESULTS

9.3.1 Hybrids from crosses between xBrassicoraphanus and Brassica napus

Table 36 summarizes the data on somatic chromosome numbers inhybrid s from reciprocal crossesbetwee n xBrassicoraphanus and rape.Plant s with 38chromosome spresumabl y had the expected genome constitutionAAC R and arecalle dAAC R hybrids.A small group ofplant s from crosseswit h B. napus as female,however ,ha d about 57chromosome s andprobabl y had the genome constitutionAAACCR . Also aplan twit h 31chromosome swa s found, whose originwa s notclear . MostAAC R hybrids grewvigorousl y andha d abalance d habito fgrowth . Theplant s exhibitedman y characteristics ofrape ,suc ha s anobviou s

rape 'Zephyr'an dset t ingo fsiliqua ea ; ndseed s ofth eF i after pollinationwit hrap e 'Zephyr' (BCj).

Originó f Chromosome Numbero f BC1 F hybrids numberi n F plants F, Numbero f Number of Numbero f seed-pro­ siliquae seeds ducing harvested plants perviridis hybridsx 31 1 0 0 0 'Zephyr' 38 14 5 129 44 9 1 1 142 52 chinensishybrid sx 38 3 2 61 24 'Zephyr' ? 1 1 63 37

'Zephyr'x chinensi s 38 1 1 378 57 hybrids 57 1 1 339 102 58 2 0 0 0

73 't"&JJ "i

•I-<' , •-*"• '"«• WK * U>

Figure 15.Hybrid sfro mcrosse sbetwee nBrassic anapu san dAAR Rhybrids . A.Offsprin go fcrosse sbetwee nrap ecv .Zephy ran dchinensi shybrid s inth evegetativ e stage.To pro wfro mlef tt oright :rape ;hybri dwit h2 n= 38 ;hybri dwit h2 n= 57 . Bottomro wfro mlef tt oright ;tw ohybrid swit h2 n= 58 ;rape . B.AAC Rhybri di nth egenerativ estage .

stem inth evegetativ e stage and leaves thatwer e dull greenbecaus e of amarkedl ywax y layer (Fig.15A) .Mos tAAC R hybrids grew rather tall (upto2 m )an d flowered abundantly (Fig.15B) ,bu t a fewdi d not flower atall .Th e flowers ofth ehybrid swer e somewhat larger than in xBrassi- coraphanus andwer ebrigh t andwhite .Marke d veins,a s in xBrassicorapha- nus, were absent.A few flowers had ayello w sector onth epetals .Th e siliquae ofAAC Rhybrid s werebivalved , as in Brassica, andha d a length ofabou t3 cm , including abea k of about 1.5 cm.Th e aberrantplan t with 31chromosome s remained small,gre w irregularly and did not flower at all.

The small group ofhexaploid s consisted ofon ewel lbalance d and two irregularly shapedplant s (Fig. ISA). The formerha d 57chromosomes ,wa s similart o arelate dAAC Rhybri d and flowered abundantly. The two other Plantsha d 58chromosomes , floweredpoorl y and hadplai nwhit e flowers, althoughon eplan tals oproduce d someyello w flowers.Onl y the hexaploid with 57chromosome s yielded siliquae,whos e size and shapewer e similar tothos e ofth eAAC Rhybrids .

All Fl hybridswer e completelymale-steril e and, after pollination

74 withpolle n from rape, set siliguae and seedpoorly : abouthal f theAAC R hybrids and one hexaploid yielded seed (Table36) .Howeve r a considerable number of seedswa s obtained,becaus e ofth e large-scalepollinations . Mostproductiv e were the twoF .hybrid s with 'Zephyr'a s femaleparent . The totalnumbe r of siliguae harvested and of seedspe rhybri d were somewhatreduced ,becaus e 53 siliguae ofAAC R hybrids and4 0 siliguae of thehexaploi d with 2n = 57wer e used forembry o culture.Th e respective numbers ofembryo s obtained were 49 and 17.Mos tembryo s had reached the walking-stick stage (Wilmar& Hellendoorn , 1968)o rwer ematur e already. Intotal ,2 1 and 5plants ,respectively , were obtained by embryoculture .

9.3.2 Chromosome association in AACR hybrids

Meiosis inAAC R hybrids (2n = 38)wa s analysed ina fewpolle nmothe r cells (Table 37).I fonl y homologous pairingwer e tooccur ,te nbivalent s (twoA genomes)an d 18univalent s (oneC and oneR genome )woul db eex ­ pected.Th e degree of association,however ,wa s higher,th emea n numbers per cell ofbivalents ,trivalent s and guadrivalentsbein g 10.59, 0.75 and 0.02, respectively. Consequently thenumbe r ofunivalent spe r cell, 14.48,wa s less than expected. The number ofchromosom e configurations exceeded 10 in8 7% of allpolle nmothe r cellswit h amaximu m of 13biva -

Table37 .Chromosom eassociatio n inmetaphas e Io ftw oAAC R hybrids (2n= 38 )fro mcrosse sbetwee nxBrassicoraphanu san d rape.

Chromosome Numbero fpolle nmothe rcell s configurations" Plant1 Plant2 Total 18I + 7I I+ 2 II I

18I + 10 II 0 1 17I + 9 II + 1 III 1 0 16I + 8 II + 2 III 1 0 16I + 9 II + 1 IV 1 0 15I + 7 II + 3 III 0 1 16I + 11 II 8 15I + 10 II + 1 III 4 1 14I + 9 II + 2 III 0 2 13I + 8 II + 3 III 0 2 1 14I + 12 II 5 4 13I + 11 II + 1 III 2 3 12I + 10 II + 2 III 0 3

12I + 13 II 2 0 11I + 12 II + 1 III 0 1

Total 25 19 44 '•]I ,II ,II Ian d IVar esymbol s forunivalents ,bivalents , trivalentsan dquadrivalents ,respectively .

75 lents andmultivalents .Thi snumbe r indicates thedegre e of autosyndesis and allosyndesiswithi n andbetwee n the C andR genome, respectively. Thenumbe r oftrivalent s ina cel l ranged up to3 .A n increasing number ofmultivalent s ina cel lwa s associatedwit h areductio n innumbe r of bivalents intha tcell .S omos ttrivalent sprobabl y resulted from pairing betweenon eC o rR chromosom e and onepai r ofA chromosomes .

9.3.3 First backcross generation

A fewBC. ,plant s derived fromAAC R andAAACC R hybridswer e raised, partlyb yembry ocultur e (Table 38). Both typeso fhybri d gave riset o highlyvariabl e offspring. Someplant s showed slight abnormalities, such asdeforme d leaves,bu t all flowered.Th e flowerswer e usuallywhite , although inth e firstbackcros s generation derived fromAAC R andAAACC R hybrids somewer eyello w (Table 38).Als owhit e flowerswit h ayello w sector occurred occasionally inth e firstbackcros s generation, even more frequently thani nth eparen thybrids . Apromisin gphenomeno nwa s the occurrence of somepartl y male-fertile plants inth e firstbackcros s generation (Table 38). Oneplant , derived fromth eonl y fertileAAACC R hybrid, evenha d apolle n stainability of 59% .Th esee d seto fth e firstbackcros s generationwa s low in crosses withrape .

9.4 DISCUSSION

Introgressiono f Raphanus characteristics into rapeb y repeated back- crossing of xBrassicoraphanus with rape asth erecurren tparen t isprob ­ ably feasible.Majo r drawbacks are low fertility in successive backcross generations, and rather infrequent associationbetwee n Raphanus and Brassica chromosomes.Th e adaptedmetho d for large-scale crossing ofAAC R

Table38 .Flowe rcolour ,polle nfertilit yan dsee dfertilit yi nB C plantsf r about10 0hand-crosse so feithe rAAC Ro rAAACC Rhybrid swit hBrassic anapu s 'Zephyr'an draise dfro msee do rb yembry oculture .

Cross Numbero f Modeo f Flower ccilou r Numbero f flowering culture

plants white yeillo w plants plants BC2 with produ­ seed; some cing stainable seed pollen

AACR xAAC C 15 embryo 12 3 2 5 20 14 seed 10 4 1 9 41

AAACCRx AAC C 4 embryo 2 2 1 2 75

76 andAAACC R hybrids with rapeb y growing hybrids andrap e together ina n isolation cage proved tob e useful forproductio n of a firstbackcros s generation of areasonabl e size,despit epoo r fertility ofF .hybrids . The infertility in successivebackcros s generations,whic hprobabl y will be less severe,ma yb e overcome in asimila rway . Successful transfer of Raphanus genes dependsmainl y onth epairin g abilitybetwee n Raphanus and Brassica chromosomes.Th echance s forpair ­ ingan d exchange between R andA chromosomes inAAC R hybrids areprobabl y assmal l as inAA R hybrids because ofpreferentia l pairingbetwee nhomo ­ logousA chromosomes.Th e ratherhig h frequencyo fmultivalent s wasprob ­ ablymainl y due topairin g betweenA andC chromosomes ,becaus e inAA C hybrids similar numbers ofmultivalent s occurred (Namai, 1976;1978 )an d inAA R hybrids only a few.O n the other hand,AAC R hybrids aremor e favourable for exchange betweenR and Cchromosomes .Th e frequency of pairingbetwee n such chromosomes isdifficul t toestimat ebecaus e the degree of autosyndesis inth eR and Cgenom e isunknown .O n thebasi s of detailed genomic analyses,Mizushim a (1980)estimate d thatth emaximu m number of autosyndetic pairswa s two inth e Cgenom e and one inth eR genome.Th ebivalen t frequency inth eAAC R hybrids due to non-homologous autosyndesis wasprobabl y low,becaus e suchpairin g only occurs sporadic­ ally inAA R andAA Chybrid s (myresults ;Namai ,1976 ; 1978). Consequently mostpairin g involving Co rR chromosomes,o n average atleas t 1.34biva ­ lentspe rpolle nmothe r cell,wa sprobabl y causedb y allosyndesis.Th e number ofcell s examined was too small to justify conclusions aboutth e maximum number of Raphanus chromosomes thatcoul dpai rwit h aC chromo ­ some.Howeve r theobservatio n ofu p to 9bivalent s inC Rhybrid s indi­ cates that several R chromosomes are capable ofpairin g allosyndetically (Harberd &McArthur , 1980;Namai ,1976 ;1980 ;McNaughton , 1973).

77 10 Prospects

10.1RESISTANC ET OBEE TEELWOR M

Theabsenc eo fresistanc et obee teelwor mi n Brassica andit spres ­ encei nsom efodde rradis hvarietie swa sth emai nmotiv efo rth epresen t researchprogramme .Littl ewa sknow nabou tth einheritanc ean dmechanis m ofth eresistanc ewhe nth eprogramm estarted .Bauklo h (1976)presume d simplemonofactoria ldominan tinheritance . Toxopeus& Lubbert s(1979 )showe dtha tth emultiplicatio nrat eo fbee t eelwormwa slo wo nmos tform so f R. sativus. Raphanus populationsrespond ­ edwel lt oselectio nfo rresistanc ean dToxopeu san dhi scolleague sa t theFoundatio nfo rAgricultura lPlan tBreedin g (SVP),Wageningen ,hav e producedpopulation so nwhic hth enematod emultiplie dmuc hslowe rtha n onth eorigina lstoc k(Lubbert s& Toxopeus ,1982) .Recen tstudie shav e showntha tresistanc eresult sfro ma shif ti nth ese xrati oo fth eeel ­ wormi nfavou ro fth emale s (Veerman,1981 )Th eeffec to fgrowin gpar ­ tiallyresistan tfodde rradis hpopulation so nbee teelwor mpopulation s inth esoi li sbein gstudie di nth eNetherlands . Thepresen tpopulation so f xBrassicoraphanus alsoshowe dmor eresist ­ ancetha n Brassica species.Th ecys tproductio npe rplant ,however ,wa s highlyvariabl ean dth emultiplicatio nrat eo fth eeelwor mwa so naverag e considerablyhighe rtha nfo rth e Raphanus parents.T oimprov eresistance , selectionfo rresistanc ei sproceedin gi nthos epopulations .Th eexpe ­ riencewit h Raphanus suggeststha tconsiderabl eimprovemen ti spossible .

10.2XBRASSICORAPHANU S

Insynthesizin ga ne wforag ecrop , xBrassicoraphanus, severalproblem s arosesuc ha sth epresenc eo fbreedin gbarrier sbetwee nth eparenta lspe ­ cies,poo rfertilit yi nth efirs tthre egeneration san doccurrenc eo f aneuploidan dhexaploi dplants .Extensiv estudie shav eshow ntha tsuc h difficultieshampe rth erapi destablishmen to ffertil ean dbroadl ybase d populations.Howeve rproductio no fsuc hpopulation si slikel yt ob epos ­ sibleb yselectio nfo rimprove dfertilit yi nth eavailabl ebreedin gpopu ­ lationsan dresynthesi so f xBrassicoraphanus inorde rt obroade nth egene ­ ticbasis .Resynthesi smigh tb efacilitate db yexploitatio no fth egeneti c variation incrossibilit y in Brassica campestris and Raphanus sativus, useo fmale-steril e B. campestris plantsi ncrosse san dimprovemen to f embryo-culturetechnique sfo rsuc hcrosses . Theagricultura lvalu eo f y-Brassicoraphanus couldno tye tb eassessed , becauseth enumbe ro fseed savailabl edi dno tpermi tfiel dtrials .Howeve r growthhabi tan dlife-cycl ear ereason sfo roptimis mo nsuitabilit ya sa foragecrop .Howeve ryiel dan dqualitativ easpect slik epalatability ,di ­ gestibility,crud eprotei nconten tan dpossibl eoccurrenc eo ftoxi ccon ­ stituentsmus tb eteste da sfo r xRaphanobrassica (McNaughton,1979) . Insummary , xBrassicoraphanus mightbecom ea productiv eforag ecrop , highlyresistan tt obee teelworm ,powder ymilde w (Erusiphe cruciferarum) andt oclub-root .I nth eagricultura lsystem so fNort hWester nEurope , sucha cro pmigh tfi tint oth esam enich ea sstubbl eturnips ,fodde rrap e andfodde rradish ,catc hcrop sgrow nfo rforag eproductio no rgree nma ­ nuring.

10.3INTROGRESSIO N

Firststep sfo rtransfe ro f Raphanus traitst o B. campestris and B. napus weremad ebu tintrogressio no fresistanc et obee teelwor man dclub - roothav eno tye tbee naccomplishe dbecaus eo fth elong-ter mnatur eo f suchaim san dth eproblem sencountered . Transfero f Raphanus genest o B. campestris provedt ob edifficult , becauseproductio no falie nmonosomi caddition sfailed .Onl ywit hconsi ­ derableeffort ssuc haddition smigh tb eobtained .Alie nchromosome si n suchplants ,however ,woul dpai ronl ysporadicall ywit h Brassica chromo­ somes,i fa tall .S oth ewa ychose nha sprove dunattractive . Theprospect sfo rintrogressio no f Raphanus genesint o B. napus were morepromising . xBrassicoraphanus proveda usefu lbridg ebetwee n R. sati- vus and B. napus, becauseth ecrossabilit ywit h B. napus wasrathe rgoo d andth econdition sfo rexchang ebetwee n Raphanus and Brassica chromosomes weremor efavourabl ei nth eAAC Rhybrids .Howeve rdifficultie si nachie ­ vingbackcrosse srestricte dsucces st osom eextent . Finallyincrease dknowledg eo fth emechanis mo fresistanc et oth ebee t eelwormmad etransfe rfa rles sfeasibl etha ni tseeme dt ob ea tth estar t ofth eprogramme .Th epartia lan dpossibl ypolygeni ccharacte ro fresist ­ ancewil lhampe rtransfe rt o B. campestris and B. napus.

79 Summary

InDutc hagriculture ,cruciferou sarabl ecrop sar eo flittl esignifi ­ cance,althoug hth egenu s Brassica doescompris eattractiv ecrop slik e rape.Th esmal linteres ti sno tsurprisin gi narea swit ha nemphasi so n cultivationo fsugar-beets ,becaus ebee teelwor m (Heterodera schachtii) isabl et omultipl yo n Brassica crops.Breedin go fcruciferou scrop swit h resistancet othi snematod ewoul dremov ea majo robjectio nt osuc hcrops . Thisconsideratio nwa sth emai nmotiv efo rth epresen tprogramm eo nin - tergenericcruciferou shybrids . Theinvestigation swer edirecte dt otransfe ro fgene sfro mfodde rra ­ dish (Raphanus sativus) toforag erap eo roil-see drap e (Brassica napus) andstubbl eturni p (Brassica campestris), especiallygene sfo rresistanc e tobee teelwor man dclub-root ,an dt oproductio no fa fertil eartificia l allotetraploid, xBrassicoraphanus. Suchhybrid sfro mth ecros s B. cam­ pestris x R. sativus couldhav eagricultura lvalu ea sa forag ecrop ,b y combiningth erapi dgrowth ,resistanc et obee teelwor man dclub-roo to f fodderradis han dpalatabilit yo ffoliag eo f B. campestris. Ona larg escale , 2x x2x ,4 xx 4x ,4 xx 2 xan d2 xx 4 xcrosse sbe ­ tween B. campestris (AAo rAAAA )an d R. sativus (RRo rRRRR )wer emade , alwayswit hth eforme rspecie sa sfemal eparent .Thi sresulte di n0.020 , 0.024,0.00 1an d0.00 2hybri dpe rpollination ,respectively :i ntotal , 196hybrids .Moreover ,anothe r5 8hybrid swer eobtaine db yembry ocul ­ ture. Thepoo rcrossabilit ybetwee nth etw ospecie sresulte dfro mpoo rfer ­ tilizationan darreste dembry ogrowt han ddevelopment .O nth estigma , growthan dpenetratio no fmos tpolle ntube sint ostigm apapilla ewer e disturbed.Suc hevent slimite dconsiderabl yth enumbe ro fpolle ntubes , penetratingth estyle .Eve ns ote no rmor epolle ntube spe rstyl ewer e countedi nmos tpistils .Bu dpollinatio nha dn opositiv eeffec to nthi s number.Poo rfertilizatio nwa spredominantl ydu et oerrati cgrowt ho f pollentube swithi nth eovary .I naggregate ,1 t o2 ovule spe rpisti l werefertilized .Th enumbe ro fdevelope dovule si n2 xx 2 xan d4 xx 4 x crosseswit hrathe reasil ycrossabl eaccession so f B. campestris wasal ­ mosta shigh .Th eovule swer esmal lan dofte ncontaine da nembry owit h arrestedgrowt h(Tabl e 9).Howeve rsom eovule swer ebigge ran dcontaine d anormall ydevelope dembryo .Th eembryo sfro mthos eovule sgav eris eonl y tomatromorphs ,an dth eembryo sfro msmal lovule sonl yt ohybrids . Forlarge-scal eproductio no fhybrids ,cultur eo fembryo si nvitr o

80 seemed attractive, since otherwise agrea tman y embryos diedbefor ematu ­ rity.Result s ofembry o culture,however ,wer e onth ewhol e ratherpoor . Alarge r effectwa s obtained by selection ofmor e easily crossable geno typeso f B. campestris. The laborious work of crossing couldb e cut down byusin g genetic male-sterile plants of B. campestris. The genome constitution ofth e hybrids was AR,AR R orAARR . The cros­ ses2 xx 4 x and4 xx 2x gave rise to a fewAAR R hybrids.Th e intergene- richybrid swer e ingenera l vigorous, formed a leafrosett e atth evege ­ tative stage and hadwhite ,purpl e oryello w flowers,whic hwer e mostly distinctly veined. Despite deranged meiosis with only 0.47 bivalents per cell, 38% of allA Rhybrid s stillproduce d some stainable pollenb y formation of 2n gametes,probabl y as aconsequenc e of formation of restitution nuclei during interkinesis. Intercrossing ofA R hybrids resulted in fewoff ­ springwit h abouthal fhexaploid s (AAARRR). Intercrossing ofsuc hoff ­ spring gave rise to a few seeds. Insom e of theA R hybrids,th enumbe r of chromosomes was doubledb y colchicine treatments of rooted cuttings orb y crossing ofA R hybrids withAAR R hybrids (meiotic doubling). TheAAR R hybrids were highly sterile;crosse sbetwee n suchhybrid s resulted in0.00 8see dpe r pollination.Th e low fertilitywa s notcause d bymeioti c irregularities,bu tb y breedingbarriers ,whic hwer ever y sim­ ilart o thebarrier s inth e original intergeneric crosses.S oth e incong­ ruity systems of B. campestris and R. sativus were apparently stillac ­ tive in xBrassicoraphanus. Someplant s proved self-compatible, i.e.th e number ofpolle n tubes inth e style after self-pollination was no less thanafte r cross-pollination. Twopopulation s of xBrassicoraphanus were established and exposed forthre e generations to natural selection.Bot hpopulation s improved forpolle n stainability, production of siliquae and seeds,an d seed-set per siliqua.Howeve r fertility was still low afterthre egenerations . Thebes tpopulatio n gave 72 seedspe rplant .Variatio n in fertility traitswa s still large, so that aconsiderabl e improvementwa s stillex ­ pected in later generations. . Beside good fertility, astabl e number ofchromosome s isprerequisit e forcommercia l use of xBrassicoraphanus. The occurrence ofhexaploid s (AAARRR)an d the formation ofunivalent s endanger chromosomal stability. The dangerwil l probably decrease as the fertility of «Brassicoraphanus improves. . ,7 TheA R andAAR R hybrids were crossedwit hvariou s species »anni hvbridizedreadily ,in ­ completely sterile,x Brassicoraphanus and B. napus nyu 81 the intergeneric hybridswer euse d as femaleparent .Th e reciprocal cros­ sesresulte dmainl y inmatromorph san donl y somehybrids ,mostl yhexa - ploid,wit hth e genome constitutionAAACCR . TheF .hybrid s (AACR and AAACCR)wer e subsequently twicebackcrosse d with B. napus. Studies of chromosome association inth emeiosi s ofAA R andAAC R hybrids showed, thatth e formertype swer erathe rpromisin g forth e introgression of Raphanus genes into a Brassica species.

82 Samenvatting

Ind eNederlands e landbouw is de teeltva n kruisbloemigelandbouwge ­ wassenva n geringe betekenis,hoewe l het geslacht Brassica aantrekkelijke landbouwgewassen omvat,zoal s koolzaad. Instreke nme t een intensieve teeltva n suikerbieten is de geringe belangstelling voor landbouwcrucife- renechte r niet zoverwonderlijk , omdathe tbietecystenaaltj e (Heterodera schachtii) zich opBrassica-gewasse nvermeerdert .He t kwekenva n rassen vanlandbouwcrucifere nme t resistentie tegen dit aaltje zalnaa rverwach ­ tingee nbelangrijk e beperkende factorvoo r de teeltva ndergelijk ege ­ wassenwegnemen .Dez e overweging vormde hethoofdmotie fvoo rhe tverwe ­ zenlijkenva n een soortkruisingsprogramma. Hetonderzoe k was gericht opprobleme n die samenhangenme td eover ­ drachtva ngene nui tbladramena s (Raphanus sativus) naarbladkoo l of koolzaad (Brassica napus) en stoppelknol (Brassica campestris), inhe t bijzonder genenvoo r resistentie tegenhe tbietecystenaaltj e enknolvoet , enhe tproducere n van een fertiele kunstmatige allotetraploid, xBrassi- coraphanus. Zulke hybriden uitd e kruising B. campestris x R. sativus kunnennaa rverwachtin g landbouwkundige waarde hebben als voedergewas doorcombinati e van de snelle groei enresistenti e tegenhe tbietecysten ­ aaltje enknolvoe tva n R. sativus met de smakelijkheid van B. campestris. Tussen B. campestris (AAo fAAAA )e n R. sativus (RRo fRRRR )zij no p grote schaal 2x x 2x,4 x x 4X, 4xx 2xe n2 xx 4 xkruisinge ngemaakt ; steedsme t de eerstgenoemde soort alsmoeder .Di t leverderespectieve ­ lijk 0,020, 0,024, 0,001 en 0,002hybride npe rbestuivin g op;i ntotaa l 196hybriden .Toepassin g van embryocultuur leverde nog eens 58hybriden . De slechte kruisbaarheid tussen de twee soortenwa s hetgevol gva n (1)ee n slechte bevruchting en (2)ee ngeremd e embryogroei en-ontwikke ­ ling,o p de stempel waren de groei end epenetrati e ind estempelpapil - lenva n demeest e pollenbuizen in sterkemat e gestoord.Dergelijk ereac ­ tiesveroorzaakte n een aanzienlijke beperking vanhe taanta lpollenbui ­ zen, dat de stijl wistbinne n te dringen.Toc hwerde n inhe tgrootst e deelva n de stampers tien ofmee rpollenbuize npe r stijl geteld.Knop - bestuiving had geenpositiev e invloed op dit aantal.D e slechtebevruch ­ ting werdvoora l veroorzaakt dooree ngestoord e oriëntatieva nd epollen - buisgroei inhe tvruchtbeginsel .Gemiddel d werden 1à 2 zaadknoppe npe r stamperbevrucht .He t aantaluitgegroeid e zaadknoppenwa sbijn ane tz o grooti n2 xx zxe n4 x x4 x kruisingenme tgoe d kruisbare B. campestns- herkomsten.D e zaadknoppenware nklei n enbevatte nvaa kee nembryo ,waar - vand e groeimeesta l voortijdig stagneerde (Tabel 9).Sommig e zaadknoppen waren echter aanzienlijk groter enbevatte n eennormaa l ontwikkeld embryo. Uitdez egrot e zaadknoppenwerde n door embryocultuuruitsluiten dmatro - morfenverkrege n enui td e kleine zaadknoppen alleenhybriden . Voor grootschaligeprodukti e vanhybride n leek invitr o opkweek van hybride-embryo's aantrekkelijk, omdater gvee l embryo's inee nonvol ­ groeid stadium afsterven.D e resultatenva n embryocultuur waren echter overhe tgehee l nogal teleurstellend. Betere resultaten werden verkregen door de selectieva n goed kruisbare B. campestris-genotypen.He ttijdro ­ vende kruisingswerk konvoort sworde nvermede n doorgenetisc h mannelijk steriele B. campes tris-planten tegebruiken . De genoomsamenstelling van deverkrege n hybridenwa sAR ,AR R ofAARR . Hetwa s opmerkelijk dat2 xx 4 xe n4 xx 2 xkruisinge n enkeleAARR-hybri - denopleverden .D e geslachtshybriden waren overhe t algemeen groeikrach- tig,vormde n inhe tvegetatiev e stadium eenbladroze t enhadde nwitte , paarse ofgel ebloemen ,di emeesta l duidelijk geaderdwaren . Ondanksee nvolledi g gestoordeméios eme tgemiddel d slechts 0,47bi ­ valentenpe r celproduceerd e tochno g 38% va n alleAR-hybride n enig kleurbaar stuifmeel.Di thiel dverban d methe tontstaa nva n 2n-gameten waarschijnlijk alsgevol gva nd evormin gva n restitutiekernen tijdens de interkinese.Onderling e kruisingva nAR-hybride n leverde eenklein enako ­ melingschap op,di evoo r ietsmee r dand ehelf tui thexaploide n (AAARRR) bestond. Onderlingebestuivin g van deplante nui tdez e nakomelingschap gafee nklei n aantalzaden . Van eendee lva n deAR-hybride nwer d het aantal chromosomen verdubbeld door (1)okselknoppe nva nbeworteld e stekkenme tcolchicin e tebehande ­ lene n (2)doo r kruisingva nAR-hybride nme tAARR-hybride n (meiotische verdubbeling). DeAARR-hybride n waren inhog emat e steriel;onderling e kruisingen leverdengemiddel d 0,008zade npe rbestuivin g op.D e geringe fertiliteit werd nietveroorzaak t door onregelmatigheden ind eméiose ,maa r door kruisingsbarrières dievee l overeenkomstvertoonde nme t debarrière s in deoorspronkelijk e geslachtskruising. Ditwijs t erop dat in xBrassicora- phanus de incongruentiesystemen van B campestris en R. sativus nog actief zijn. Eendee lva n deplante nblee kbi j zelfbestuivingzelf-compatibel , d.w.z. hetaanta lpollenbuize n ind e stijlwa s na zelfbestuiving niet lager dann a kruisbestuiving. Tweepopulatie sva n xBrassicoraphanus zijn samengesteld enzij ngedu ­ rende driegeneratie smi no fmee rovergelate n aannatuurlijk e selectie. Beidepopulatie svertoonde nverbeterin g vanpollenfertiliteit , hauw- en zaadproduktie enzaadzettin gpe rhauw .N a drie generaties was hetferti - liteitsniveau echterno g laag.D ebest epopulati e leverde gemiddeld on­ geveer 72zade npe rplan top .D evariati e voor deverschillend e fertili- teitskenmerkenwa sno ggroot ,zoda tnaa rverwachtin g de fertiliteit in devolgend e generaties nog aanzienlijk zalverbeteren . 84 Naastee ngoed efertilitei ti see nstabie lchromosoomaanta lee nnood ­ zakelijkevoorwaard evoo rcommerciee lgebrui kva n xBrassicoraphanus. Het ontstaanva nhexaploide n (AAARRR)e nd evormin gva nUnivalente nzij nee n potentieelgevaa rvoo rd echromosomal estabiliteit .Naa rverwachtin gwor ­ dendez egevare nminder ,indie nd efertilitei tva n xBrassicoraphanus ver­ betert. DeAR -e nAARR-hybride nzij nme tdivers esoorte ngekruis t (Tabel1 7 en20) . xBrassicoraphanus istweemaa lme tsucce steruggekruis tme t B. cam- pestris, teneindei n B. campestris eenreek smonosom eadditie st eprodu ­ ceren.D eT„-plante nhadde nevenwe lnie the tgewenst eaanta lchromosome n enware nvolledi gsteriel .Kruisin gva n xBrassicoraphanus met B. napus verliepopvallen dgoe dme td egeslachtshybrid eal smoeder .D ereciprok e kruisingenleverde ndaarentege nhoofdzakelij kmatromorfe no pe nslecht s enkele,mees thexaploid ehybride nme td egenoomsamenstellin gAAACCR .D e

F1-hybriden(AAC Re nAAACCR )werde nvervolgen stweemaa lme tsucce sterug - gekruistme t B. napus. Studiesva nd echromosoomassociati ei nd eméios e vanAAR -e nAACR-hybride ntone naan ,da td elaatstgenoemd etype nvri j kansrijkzij nme tbetrekkin gto td eintrogressi eva n Raphanus-genen in eenSrassica-soort .

85 References

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