O. Dolstra Foundation for Agricultural Plant Breeding (SVP), Wageningen Synthesis andfertilit y of xBrassicoraphanus andway so f transferring Raphanuscharacter s to Brassica Centre for Agricultural Publishing and Documentation Wageningen - 1982 'zcé^l^ CIP-GEGEVENS Dolstra, 0. Synthesis and fertility of xßrassicoraphanus and ways of transferring Raphanus characters to Brassica / 0. Dolstra. - Wageningen: Pudoc. - 111. - (Agricultural research reports; 917) Ook verschenen als proefschrift Wageningen, 1982. - Met lit. opg. ISBN 90-220-0805-3 SISO 632 UDC 633 Trefw.: plantenteelt. ISBN 90 220 0805 3 The author graduated on 29 October 1982 as Doctor in de Landbouwweten­ schappen at the Agricultural University, Wageningen, the Netherlands, on a thesis with the same title and contents. © Centre for Agricultural Publishing and Documentation, Wageningen, 1982. No part of this book may be reproduced or published in any form, by print, photoprint, microfilm or any other means without written permission from the publishers. Abstract Dolstra,0. ,1982 .Synthesi san dfertilit yo fxBrassicoraphanu san dway so ftransfer ­ ringRaphanu scharacter st oBrassica .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 econstitutio nAR ,AAR Ro rAR Rwer eob ­ tained fromove r1 500 0crosse sbetwee nBrassic a campestris (AAo rAAAA ,femal eparent ) andRaphanu ssativu s(R Ro rRRRR) .Th epoo rcrossabilit ywa sshow nt ob ea consequenc e ofvariou sbreedin gbarriers .Th ediploi dan dtetraploi dhybrid swer estudie d inform , fertility,chromosom eassociatio na tmeiosi san dcrossabilit ybot hamon gth ehybrid s andi ncrosse swit hth eparenta lspecie san dBrassic ananus .xBrassicoraphanu s (AARR) wasbackcrosse dtwic ewit hB .campestri s (AA),an dwa sals ocrosse dan dbackcrosse d withB .napu s (AACC).For man dchromosom eassociatio na tmeiosi si nAA Ran dAAC Rhy ­ bridswer estudied . Twopopulation so fxBrassicoraphanu s (AARR)wer epropagate d andstudie d inmor ede ­ tail,partl yfo rpossibl esuitabilit ya sa ne wcrop .Mos tplant swer ehighl ysteril e becauseo fbreedin gbarriers ,whic hwer esimila rt othos ei nth eorigina lintergeneri c crosses.Th eincreas ei nfertilit yan dchromosoma lstabilit ywer estudie d inth efirs t threegeneration so fth 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 . Contents 1 General introduction 1 1.1 Rationale 1 1.2 Species and allopolyploid hybrids 1 1.3 Scopeo fth eresearc hprogramm e 2 2 Crosses between Brassica campestris and Raphanus sativus 5 2.1 Introduction 5 2.2 Material andmethod s 6 2.3 Results 9 2.3.1 Settingo fsiliqua e and seeds 9 2.3.2 Seed size 9 2.3.3 Productiono fhybrid s andmatromorph s 10 2.3.4 Useo fgeneti cmale-sterilit y 13 2.3.5 Cultureo fembryo s 14 2.3.6 Genome constitution andnumbe ro fchromosome si n theprimar y hybrids 15 2.4 Discussion 17 3 Nature of breeding barriers 19 3.1 Introduction 19 3.2 Material andmethod s 19 3.3 Results 20 3.4 Discussion 22 4 Primary allodiploid ARhybrids and their progeny 24 4.1 Introduction 24 4.2 Material andmethod s 25 4.3 Results 26 4.3.1 Formo fA Rhybrid s 26 4.3.2 Chromosome association atmeiosi s 28 4.3.3 Male and seed fertility 30 4.3.4 Crossabilityo fA Rhybrid swit hvariou s species 31 4.3.5 Somatic andmeioti c doublingo fchromosom e number 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 associationi nAA R hybrids 67 8.3.2 Attemptst ointroduc e alienchromosome s 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 Hybrids from crossesbetwee n x-Brassicoraphanus and Brassica napus 73 9.3.2 Chromosome associationi nAAC Rhybrid s 75 9.3.3 Firstbackcros s generation 76 9.4 Discussion 76 10 Prospects 78 10.1 Resistancet obee teelwor m 78 10.2 xBrassicoraphanus 78 10.3 Introgression 79 Suvomary 80 Samenvatting 83 References 86 1 General introduction 1.1 RATIONALE Likemos tothe rcruciferou s crops, Brassica spp.ar ehost s toth ebee t eelworm (Heterodera schachtii Schm.)(de nOuden ,1954 ;Steele , 1965), which isdistribute d allove rth eworl d (Stelter,1973 )an d isth emos t seriousnematod e of sugar-beet (Beta vulgaris L.) inEurop e (Heijbroek, 1979). Inth eNetherlands , forexample ,a quarte r ofth e sugar-beetare a hasbee n infectedb y thisnematode ,especiall y inth e south-west and south-east (Heijbroek, 1979). Problems arisemainl y fromto onarro wcro p rotations.Th e growingo fsugar-bee t iseconomicall y attractive toth e farmers, and theywil l try tocontinu ewit h suchrotations ,despit e dis­ advantages from aphytosanitar y pointo fview . Underpresen tcircumstances ,n oplac e islef t for Brassica crops in rotationswit hbeets ,unles s resistantvarietie s arebre dwit hpreferabl y the ability toreduc e thepopulatio n density ofth eeelworm . Inth e genus Brassica, however,n o resistance hasbee n found sofar (Baukloh,1976 ; Lubberts& Toxopeus , 1982). Inth e 1960s,report s appeared inGerma n farming journals onresistanc e in Raphanus sativus L.var . oleiformis, theoil-see d radish.Th e early contradictory reportswer e reviewedb y Hirling (1976).Wit h oil-seed radish theeelwor m usuallymultiplie d slow­ ly,bu t ina fe wreport s rather quickly. Inrecen tyears ,th epresenc e ofresistanc e in Raphanus hasbee nconfirme db yBauklo h (1976)an dToxo ­ peus& Lubbert s (1979). Baukloh (1976)studie d the inheritance and assum­ ed asingl e dominant gene forresistance . Thegenu s Brassica includesmajo r crops for fodder,oil-see dproduc ­ tion, and greenmanure ,som eo fwhic hmigh t fitquit ewel l into cropro ­ tationswit h sugar-beet.S o aprogramm ewa s started onintergeneri c hy­ bridization between Brassica spp.an dnematode-resistan t formso f R. sa­ tivus with theultimat e goal oftransferrin g resistance to Brassica. A second argument forthi sprogramm e was thepresenc e ofresistanc e to club-root (Plasmodiophora brassicae Woron.)i noil-see d radish. 1.2 SPECIESAN DALLOPOLYPLOI D HYBRIDS Theprogramm e on intergeneric hybridization includes the species R. sativus (2n= 18 ,RR) , B. campestris (2n= 20,-AA )an d B. napus (2n =38 , AACC). Their genome constitutions and interrelationships areoutline d in Figure 1 (Mizushima, 1980). B. napus is anatura l allotetraploid andi s composed oftw o genomes of B. campestris andtw o of B. oleracea (2n =18 , CC). Table 1enumerate s the subspecies orbotanica lvarietie s distin­ guished inthes e species.Accordin g toHel m (1957), R. sativus comprises fivebotanica lvarieties ,whic h includevariou svegetable s andagricul ­ tural crops.Th ebes tknow n inEurop e are radish,gian tradish ,blac k radish, and oil-seed or fodder radish.Th e latter ishoweve r mainly used asgree nmanure .Mougri-radis h isgrow n in south-eastAsia , especially for its leaves andyoun gpod s (Banga, 1976). For B. campestris, the clas­ sification ofOlsso n (1954)ha sbee n followed.H e distinguished various subspecies,whic hwer e classified by other taxonomists as separate spe­ cies. B. campestris containsman y oriental leafyvegetables , and several root, oilseed and fodder crops. B. napus also includesmajo r root,oil ­ seed and fodder crops,althoug h itsdomesticatio n started only afe whun ­ dred years ago (Toxopeus,1974a ;McNaughton , 1976). Since the discovery ofth egenom e constitution of B. napus (U, 1935), this speciesha s often beenresynthesize d from itsdiploi d ancestors.Thi sha s increased genetic variation inth eexistin g crops andeve nresulte d ina headin g type cal­ led 'Hakuran' (Nishi, 1980). More detailed information onth e four spe­ cies inth epresen t study is found inBoswel l (1949), Herklots (1972), Simmonds (1976)an dNish i (1980). Intergeneric hybridsbetwee n R. sativus and Brassica species havea long andwel l knownhistory . Atpresen tvariou smeioticall y stableal ­ lopolyploids areknow n (Fig.1) .Plant s with thegenom e constitution RRCCwer e already obtainedb y Karpechenko (1927; 1928)an dname d Raphanobrassica. Someyear s later,Terasaw a (1932)wa s the firstt opro ­ duce allotetraploids withth e genome constitutionAARR ,whic hh e called Brassicoraphanus. Inthi s study, thename s xRaphanobrassica and xBrassi- coraphanus areuse d forplant swit hth egenom econstitutio n RRCC and AARR, respectively, irrespective ofth e femaleparen t inth e original crosses.Accordin g toth e international rules fornomenclature , those names should beprecede d bymultiplicatio n sign (International Bureau forPlan tTaxonom y andNomenclature , 1980).