Isolation andcharacterisatio n ofsomati c hybrids between Lycopersicon esculentum andLycopersicon peruvianum

Isolatie enkarakteriserin g van somatische hybriden van Lycopersiconesculentum enLycopersicon peruvianum

ONTVANGEN 2 0 NOV. t-59

CB-KARDEX

CENTRALE LANDBOUWCATALOGU S

0000035 9094 6 Promotor: dr. C. Heyting hoogleraar in de generatieve en somatische celgenetica

Co-promotor: dr. ir. M. Koornneef universitair hoofddocent Jelle Wijbrand i

Isolation and characterisation of somatic hybrids between Lycopersicon esculentum and Lycopersicon peruvianum

Proefschrift ter verkrijging van de graad van doctor in de landbouwwetenschappen, op gezag van de rector magnificus, dr. H. C. van der Plas, in het openbaar te verdedigen op woensdag 29 november 1989 des namiddags te twee uur in de aula van de Landbouwuniversiteit te Wageningen BlßUOTKECK tANDBOUWUNIVERSI'l'liM .WA(;ENL\(;KN \sJ foyitS Slioechc in rioecht

Joe deys leyC de wrâd omkere Yn klear duwbbeld-hertigheyt. Jou uwz Lan wer ljxve HEERE d'Ade roune yenfadigheyt.

(G. Japicx, 1681,Friesch e rymlerye)

Met dank aand e "Stichting Hendrik Nannes- enCatrij n Epes-leen" teBolsward , die het uitgeven van dit proefschrift middels een financiële bijdrage mogelijk heeft gemaakt. ^Jo?zoi.1*32 0

STELLINGEN

Genlocalisatie via asymmetrische protoplastenfusie, waarbij de protoplasten van de donor met een hoge dosis ioniserende straling behandeld worden, is niet efficient, omdat er relatief veel chromosoomherrangschikkingen plaats vinden.

Dit proefschrift.

Het verdient aanbeveling om meer onderzoek te verrichten naar het proces van chromosoomeliminatie in asymmetrische fusieproducten, verkregen na bestraling van éénva n beide fusieouders.

3. Aneuploldie in somatische hybriden kan leiden tot incongruentie.

4. Interspecifieke somatische hybridisatie verhoogt de genetische variatie van een gewas.

Als modelgewas voor celgenetisch onderzoek is de cultuurtomaat geschikter dan Nicotiana soorten, vanwege de goed ontwikkelde genetica bijd e tomaat.

Dit proefschrift

6. In geval van niet-groeiende of niet-regenererende fusieproducten van plantecellen, verdient het aanbeveling de term somatische incompatibiliteit te vervangen door somatische incongruentie.

Harms CT (1983)Th e Quarterly Review of Biology 58:325-353. Hogenboom NG (1984). In: Linskens HF, Heslop-Harrison J (eds) Cellular Interactions, Springer-Verlag Berlin, pp640-654 . 7. Het feit dat de klassieke genetica bij de aardappel bijna niet ontwikkeld is, maakt dat dit gewas niet goed bruikbaar is voor fundamenteel moleculair-genetisch onderzoek.

Gezien de grote aantrekkingskracht van afwijkende kleuren en vormen van traditionele groenten bij de consument, verdient het aanbeveling de grote variatie binnen de tomaat wat betreft vruchtkleur en -vorm te benutten ind e veredeling.

De definitie van genetische manipulatie door professor Barabas is onvolledig en ten dele onjuist. Gezien de grote verspreiding van de publicatie waarin zijn definitie vermeld wordt, leidt dit tot een vertekend beeld van genetische manipulatie bij een groot publiek.

In: Vandersteen W (1987) Suske en Wiske. De woeste wespen, Standaard UitgeverijAntwerpen , p 3.

10. Mede gezien de zorgvuldige procedures die het onderzoeksinstituut Ital gevolgd heeft alvorens een proefveld met transgene aardappels in te richten,ka n gesteld worden dat 'Het Ziedende Bintje'halfgaa r is.

11. Indien de huidige trend om alleen te bezuinigen door natuurlijk verloop zich doorzet en indien de migratie van Groningse biologen naar Wageningen in hetzelfde tempo doorgaat als in de afgelopen jaren, is de sluiting van het Biologisch Centrum van de Rijksuniversiteit Groningen onafwendbaar.

Stellingen bij het proefschrift van J. Wijbrandi: "Isolation and characterisation of somatic hybrids between Lycopersicon esculentum and Lycopersicon peruvianum." Wageningen, 29novembe r 1989. VOORWOORD

Dit proefschrift beschrijft de resultaten van een promotie-onderzoek aangaande protoplastenfusie bij de tomaat. Dit boekje was niet verschenen zonder de hulp vanvel e mensen,di e ikhierbi jwi l bedanken. In de eerste plaats de medewerkers van de vakgroep Erfelijkheidsleer, die aan dit proefschrift hebben bijgedragen in de vorm van adviezen, goede voorzieningen en een gezellige sfeer. Enkele mensen wil ik speciaal vermelden: mijn promotor Christa Heyting, die in korte tijd al mijn manuscripten vakkundig heeft gecorrigeerd; Maarten Koornneef, mijn co- promotor, di e me met veel inzet en enthousiasme heeft begeleid tijdens alle fasen van het onderzoek; Corrie Hanhart en Patty van LoenenMartinet - Schuringa, die zorgden voor gezelligheid, de goede organisatie van het lab en het accuraat uitvoeren van vele experimenten; Henny Verhaar, die hielp bij het opsporen van chromosomen; Johan van Ooijen, die mij met veel geduld heeft leren omgaan met de p.c.; Arend Arends, Willem van Blijderveen, Henk Kuiper, Jan Laurens, Evert van de Wardt en Gerrit van IJmeren, die met toewijding mijn planten ind eka s in leven of in toom hebben gehouden. Naast de vaste vakgroepmedewerkers hebben ook tijdelijke medewerkers bijgedragen: the guest-scientists Lucia Martinelli and Aäron Zelcer, whom I thank for their co-operation and the interesting discussions; Bart den Boer, Witte van Capelle, José Kok, Frans Mulckhuijse, Maaike Posthuma, René Rijken, Annette Vergunst, Janny Vos, Michel Wissink en Anne-marie Wolters, die als Studenten mijn begeleiding doorstaan hebben en voor dit proefschrift een groot aantal belangrijke experimenten hebben uitgevoerd; Anja Posthuma, die als vrijwilligster veel werk verzet heeft. De vakgroep Moleculaire Biologie bedank ik voor het mogen uitvoeren van experimenten binnen de tomatengroep: Els Hulsebos voor het zeer precies aanleren van moleculaire technieken; Pim Zabel voor de moeite om leesbaar Engels van mijn 'proza' te maken; Jac Aarts, Raymond van Daelen, Ruud Verkerk, Rob Weide, Ellen Wisman en de overige medewerkers voor adviezen, hulp end e goede werksfeer. De collega's van andere vakgroepen van de LUW, andere universiteiten en instituten wil ikbedanke n voor overleg en samenwerking. Ik ben BION en het Löhnisfonds zeer erkentelijk voor het financieren van een studiereis naar Amerika. Verder bedank ik mijn familie-, vrienden- en kennissenkring; met name Monique, Erik, Piet enMariek e voor de gezellige weekendjes. Tot slot wil ik mijn ouders en Elly bedanken voor hun belangstelling en steun. The investigations were supported by the Foundation for Fundamental Biological Research (BION), financed by the Netherlands Organisation for the Advancement of Scientific Research (NWO) and performed at the Departments of Genetics and Molecular Biology, Agricultural University Wageningen, The Netherlands. CONTENTS

Chapter1 . Generalintroductio n

Chapter2 . Selectionan dcharacterisatio no fsomati chybrid s between Lycopersicon esculentum and Lycopersicon peruvianum ' 11

Chapter3 . Analysiso fprogenie sderive dfro msomati chybrid s between Lycopersicon esculentum and Lycopersicon peruvianum 29

Chapter4 . Asymmetric somatichybrid sbetwee n Lycopersicon esculentum andirradiate d Lycopersicon peruvianum I. Cytogeneticsan dmorpholog y 41

Chapter5 . Asymmetric somatichybrid sbetwee nlycopersico n *•esculentu man dirradiate d Lycopersicon peruvianum II. Analysiswit hmarke rgene s 57

Chapter6 . Asymmetric somatichybrid sbetwee n Lycopersicon esculentum andirradiate d Lycopersicon peruvianum III.Analysi swit hrestrictio nfragmen tlengt h polymorphisms 71

Chapter7 . Generaldiscussio n 91

Summary 97

Samenvatting 100

Curriculumvita e 104 CHAPTER1

GENERAL INTRODUCTION

Partial transfer by somatic hybridisation

Somatic hybridisation is a technique by which nuclear of different species can be combined. Interspecific somatic hybrids have been obtained of various animal cell lines (Ringertz and Savage 1976), fungi (Peberdy 1979)an d plants (Glimelius 1988). Plant somatic hybrids originate from fusion of , which are plant cells whose walls have been removed by enzymatic digestion. Somatic hybridisation is of potential interest for the improvement of crops, because it provides the possibility to circumvent sexual crossing barriers, to mix cytoplasmons of different species, and to generate novel nucleus-cytoplasm combinations (Glimelius 1988). Thus, desirable traits of a wild species can be introduced into a crop species. It is, however, equally importanttha tundesirabl e traitsar eno ttransferre d toth ecultivate dspecies , or that they canb e removed from the hybrids.Afte r sexual hybridisation it is often possible to achieve this by repeated backcrossing of the to the cultivatedparent .I nthi srespec tsomati chybridisatio nha ssom edisadvantages , since most of the fertile somatic hybrids are stable polyploids. Preferential elimination of the of one of the parental species, like has been reported for several mammalian somatic hybrids (Ringertz and Savage 1976), rarelyoccur si nsomati chybrid so fplant s(Gleb aan dSytni k1984) .Th epolyploi d state of the hybrids may cause sexual incongruity with the cultivated parent. Crossing of an interspecific somatic hybrid to the cultivated parental species istherefor eno talway spossible .Thi sproble mma yb ecircumvente db yasymmetri c somatic hybridisation; in this case,untreate d protoplasts of one species,th e recipient oracceptor ,ar efuse dwit hprotoplast so fanothe r species,th edonor , whosenuclea r genome isreduced .Th e construction ofasymmetri c somatic hybrids can be done invariou s ways: (i)b yth eus e ofhaploi d cellsfro ma dono r species.Thes e cellsca nb ederive d either from haploid plants or from microspores in pollen tétrades (Pental and Cocking 1985). The latter technique is called gameto-somatic fusion, and has been successfully applied to Nicotiana and Petunia species (Pirrie and Power 1986; Lee and Power 1988; Pental et al. 1988). Such fusions result in allotriploid plants, inwhic h introgression of donor into the recipient genome may occur by meiotic recombination and by further backcrosses to the recipient species; (ii) another way to reduce the contribution of donor genome to that of the hybridi sth eisolatio no fmicroprotoplast sfro mmicronucleate d cells (Verhoeven 1989). Micronuclei are induced by exposure ofmitoti c cells toa spindle toxin; protoplastso fthes ecell sar efractionate d intomicroprotoplasts ,whic hcontai n micronuclei with one or a few chromosomes. Procedures for the isolation of microprotoplasts of Nicotiana plumbaginifolia have recently been developed by Verhoeven (1989); (iii)finally , reduction of the contribution of thedono r genome totha t of the hybrids canals ob eachieve d by treatment of thedono r cellswit hhig hdose s of ionisingradiatio n(Röntgen -an dgamma-rays) ,whic hfragmentise sth echromosomes . This method was originally developed to completely eliminate the nuclear donor genome in order to obtain cybrids (fusion products that contain only the cytoplasm of the donor; Zelcer et al. 1978). However, in several cases it has been shown that partial elimination of the donor genome by irradiation of the donor cells is also possible. Fragmented chromosomes can only be stably replicatedan dmaintaine d ifthe y containa centromer e regionan d telomeres.I f fragments lack such structures they can still be rescued by recombination with other donor or recipient chromosomes. Asymmetric somatic hybrids can also be used for mapping of genes. This has been well established for mammalian cell hybrids. In such hybrids where preferential elimination ofmos to f the chromosomes of oneparen tha d occurred, theremainin gdono rchromosome s could be identified bycytogeneti c analysis. In this way, asymmetric hybrids containing a complete rodent genome and a small number of human chromosomes in different combinations were obtained (Ringertz and Savage 1976). Another approach was to transfer directly small numbers of donor chromosomes into mammalian cells by microcell fusion; this procedure has also been used successfully for mapping (Lugo and Fournier 1986). Irradiationo fon e fusionparen tha sno t onlybee nuse d toenhanc e preferential eliminationo f chromosomes from oneparen t (Pontecorvo 1971), butha sals o been appliedfo rregiona lmappin go fchromosomes ;thus ,th elinea rorde ro fdifferen t marker geneso na same chromosome (region)coul d bedetermine d bymeasurin g the frequency of the presence of these genes ina larg e population ofhybrid s (Goss and Harris 1975).

Asymmetric somatic hybrids of plants

Partial genome transfer by asymmetric somatic hybridisation has been described for several plant species. Asymmetric nuclear hybrids were isolated from cybridisation experiments,a sunintende d by-products (Zelcer et al. 1978;Avi v and Galun 1980; Sidorov et al. 1981;Mencze l et al. 1982,1983 ; Hamill etal . 1984). Besides,asymmetri c hybrids were purposely obtained from experiments in which selection was based on a nuclear encoded character of the irradiated donor. Efficient selectablemarker sar e required toselec tfo rfusio nproducts , because, in general, the frequencies of asymmetric hybrids obtained were much lower than those of symmetric hybrids obtained after fusion of unirradiated protoplasts. Inth efirs t reported, successful asymmetric somatic hybridisation experiment, albino carrot protoplasts were fused with irradiated parsley protoplasts; the regenerated green plant had one additional chromosome (Dudits etal . 1980). Inman yo fth easymmetri chybridisatio nexperiments ,th e selection wasbase do ncomplementatio no fth enitrat ereductas edeficienc yo fth erecipien t species (e.g. Gupta et al. 1982;Gleb a et al. 1988). Thus, asymmetric nuclear hybrids were selected from combinations of species differing with respect to their phylogenetic relatedness (Table 1): intrageneric, intergeneric + intrafamiliar, and interfamiliar. The applied irradiation doses ranged from 50 to 1000 Gray. The elimination of donor genome depended strongly on the

Table 1.Interspecifi casymmetri csomati chybridisatio nexperiment s. Unirradiate d protoplasts from a (recipient) species were fused with irradiated protoplasts from another (donor)species .

Asymmetric somatic nuclear hybrids References recipient (+)dono r

Intrageneric hybrids: Brassica campestris (+) B. oleracea* Yamashita et al. 1988 Nicotiana glauca (+) JV . langsdorffii Itoh and Futsuhara 1983 N. plumbaginifolia (+) JV . sylvestris* Famelaer et al. 1989 JV. plumbaginifolia (+) JV . tabacum* Koornneef et al. 1988 JV. tabacum (+) JV . paniculata Müller-Gensert and Schieder 1987 JV. tabacum (+)JV . plumbaginifolia* Bates et al. 1987 Solanum tuberosum (+)S . pinnatisectum Sidorov et al. 1987

Intergeneric, intrafamiliarhybrids : Daucus carota (+) Petroselinum hortense* Dudits et al. 1980 Datura innoxia (+) Physalis minima* Gupta et al. 1984 Hyoscyamus muticus (+) JV . tabacum Imamura et al. 1987 JV. plumbaginifolia (+) Atropa belladonna* Gleba et al. 1988 N. tabacum (+) Datura innoxia Gupta et al. 1982 JV. tabacum (+) Physal is minima Gupta et al. 1982

Interfamiliar hybrids: JV. tabacum (+) Daucus carota* Dudits et al. 1987 JV. tabacum (+) Hordeum vulgare* Somers et al. 1986

asymmetric hybrid plants obtained combination of parents. Inmos t of the intrageneric combinations,a limited eliminationwa sobserved ,wherea si nth eunrelate dcombination sth eeliminatio n wasmor eincreased .I nmos to fth eintergeneri chybrids ,on eo ra fe wchromosome s wereretained ,whil ei nth einterfamilia rhybrid sonl ya fe wtrait s(Dudit se t al.1987 )o reve non etrai t (Somerse tal .1986 )o fth edono rspecie scoul db e detected.Whe ndifferen tdose swer euse di nth esam eexperiment ,n oclea reffec t ofth eapplie ddos ewa sobserve d onth eeliminatio no fdono rgenom e (Glebae t al. 1988; Yamashita et al. 1988; Famelaer et al. 1989). Five different experiments have been described, where asymmetric hybrids were fertile when selfedo fbackcrosse dt oth erecipien tspecie s(Somer se tal .1986 ;Bate se tal . 1987; Duditse tal .1987 ;Sidoro ve tal .1987 ;Yamashit ae tal .1988) . Intw o otherexperiment sbackcrosse sha dt ob erescue db yembry ocultur e(Gleb ae tal . 1988; Famelaere tal .1989) .

Scope of the study

Asymmetric somatichybrid shav ea nimportan t potential usefo rplan tbreeder s whowan tt otransfe rmono -an dmultigeni ctrait sfro ma mor eo rles sunrelate d (donor)specie st oa recipien tcro pspecies .Monogeni ctrait so fwhic hth eDN A has been cloned can be more easily transferred by other techniques,suc ha s Agrobacterium transformation anddirec tgen e transfer (Hohnan d Schell 1987). To evaluate the possibilities and difficulties of somatic hybridisation, experimentswer eperforme dwit ha plan tspecie stha tca nb eanalyse dgeneticall y indetail ,namel y thecultivate d (Lycopersicon esculentum). fusions were carried out with the tomato and its wild relative Lycopersicon peruvianum. Thesetw ospecie sar edifficul tt ohybridis esexually .Regeneratio n capacityfro m L. peruvianum waschose na sselectabl etrai tan dals oa sa nexampl e of a multigenic trait which might be desirable to transfer by somatic hybridisation.Th eaim so fth eexperiment swere : (i)th eisolatio no fsomati chybrid sbetwee nbot hspecie s ina nefficien twa y andth esubsequen tcharacterisatio no fthes eplants ; (ii) the isolation of asymmetric hybrids between both species, using L. peruvianum asdono rspecies ; (iii)th echaracterisatio no fthes easymmetri chybrid si norde rt odetermin eth e degreeo feliminatio no fdono rgenom eand ,i naddition ,t oevaluat ethei rus e forbreedin gpurpose san dmappin gstudies . Lycopersicon esculentum Hill., the cultivated tomato, and Lycopersicon peruvianwn (L.) Mill., "the most variable and least exploited tomato species" (Rick 1979a)

L. esculentum and L. peruvianum are both members of the genus Lycopersicon of thefamil y Solanaceae. Thisgenu si sclosel yrelate d toth egenu s Solanum and consistso feigh tspecies ,whic har edivide di nth e'esculentum-complex 'an dth e 'peruvianum-complex' (Taylor 1986). The species of the former complex canb e easilycrosse dwit h L. esculentum, whereasth especie so fth elatte rcomplex , namely L. peruvianum and L. chilense, canonl yb ecrosse swit hgrea tdifficulty . All Lycopersicon species showa hig hdegre e ofhomolog y inthei rchromosomes , whichallow smeioti crecombinatio ni nspecie shybrid s (Rick 1979b). Thecultivate d tomatoi sa nimportan tfoo dcrop .Moreover ,i ti sa favorit e modelsyste mfo rgeneti cstudie si nplants ,becaus eth ecro pi seas yt oculture , hasa relativel yshor tlif ecycle ,i shighl yself-fertil ean di sa simpl ediploi d (2n= 2 x= 24 )whos echromosome sar edistinguishabl ea tth epachyten estag eo f meiosis (Rick and Butler 1956;Gil l 1983;Ric k and Yoder 1988;Hill e et al. 1989).Th elinkag ema po ftomat ocontain smor etha n30 0morphological ,isozyme - anddiseas e resistancemarker s (Mutschlere tal . 1987),t owhic ha tleas t30 0 restrictionfragmen tlengt hpolymorphis m(RFLP )marker shav ebee nadde di nrecen t years(Youn gan dTanksle y1989) . L. peruvianum isa noutbreedin gwil drelativ eo fth ecultivate dtomat owit h ahig hleve lo fvariabilit y (Rick1979) .Ther ear eman yaccessions ,whic hhav e agrea tnumbe ro fcharacter sdesirabl efo rtomat oimprovement ,suc ha sdiseas e resistances (Rick1982) .Becaus eo fth ecrossin gbarrier sbetwee nbot hspecie s (Hogenboom1972a) ,onl ya fe wresistances ,namel yagains tth eroot-kno tnematod e Heloidogyne incognita (Gilbert and McGuire 1956), Pyrenochaeta lycopersici (corkyroot ;Sztey n1962) ,tobacc omosai cviru s(Alexande r 1963), Cladospoxium (Kerr and Baily 1964)an d curly topviru s (Martin 1969), couldb eintroduce d intoth etomato .Whe nL .peruvianu mi suse da spistillat eparent ,growt ho fth e L.esculentu m pollen tubes is inhibited.Whe n L. peruvianum isth estaminat e parent,fertilisatio ntake splace ;however ,th eembry osoo nabort sdu et oth e degenerationo fth eendosper m(Barban oan dTopolesk i1984) .Th ecrossin gbarrier s wereovercom eb yth erescu eo fth eabortiv eembry ob ytissu ecultur etechnique s (Smith1944 ;d eNettancour te tal .1974 ;Thoma san dPrat t1981 )an dth eselectio n forrar eI .peruvianu mgenotype swit ha reduce dpolle ntub einhibitio n(Hogenboo m 1972b;Ric k1983) . L. peruvianum alsoha s desirablecel lan dtissu ecultur e properties.It s callus growth and shoot capacity are superior to those of L. esculentum (Zapatae tal .197 7;Mühlbac h1980 ;Morga nan dCockin g1982) .Th e latterspecie si sver ydifficul tt oregenerat efro mprotoplas tan destablishe d callus cultures. An L. esculentum genotype with the good callus growth and superior regeneration capacity from L.peruvianu m was obtained by classical breeding (Koornneefe tal . 1986). Thegeneti canalysi so fthi splan treveale d that the favourable traits ofL .peruvianu m were dominant,tha t regeneration capacitywa scontrolle db ytw ogene san dtha tth ecallu sgrowt hcharacteristic s wereshow nt ob econtrolle db ydifferen tloc i (Koornneefe tal . 1987).

Somatic hybrids of Lycopersicon

Alreadyi n197 8hybrid sbetwee ntomat oan dpotat ower eobtaine d(Melcher se tal . 1978); however,thes e hybridswer e not fertile and formed fruitsno rtubers . Morerecently ,othe rsomati chybri dplant sbetwee ndifferen t Lycopersicon species andbetwee n Lycopersicon and Solanum specieswer e obtained (Table2) .Th eai m ofthes esomati chybridisatio nexperiment swa st oby-pas scrossin gbarrier san d possiblyt ointroduc evaluabl eagronomi ctrait sint oth etomato .Althoug hthes e hybridswer evigorou splant san dofte ndi dflowe ran dse tfruits ,n oprogenie s were described. Somatic hybrids between L. esculentum and L. peruvianum were alsoobtained ;however ,fro m 21differen tfusio nexperiment sonl ytw ohybrid s were isolated,becaus e thesehybrid s could onlyb e identified assuc ha tth e plantleve lan dno t incel lcultur e (Kinsarae tal . 1986).Asymmetri c somatic hybridso ftomat ohav eno tbee nreporte ds ofar .

Table2 .Somati chybridisatio nexperiment swit h Lycopersicon species.

Fusioncombinatio n References

I. esculentum (+) L. pennellii O'Connell andHanso n198 7 L. esculentum (+) L. peruvianum Kinsarae tal .198 6 L. esculentum (+) S. lycopersicoides Handleye tal .198 6 Tan198 7 L. esculentum (+) S. nigrum Gurie tal .198 8 Jaine tal .198 8 L. esculentum (+) S. rickii O'Connellan dHanso n198 6 L. esculentum (+) S. tuberosum Melcherse tal .197 8 Sheparde tal .198 3 L. pennellii (+) L. peruvianum Adamsan dQuiro s198 5 Tan198 7 L. pimpinellifol ium (+) S. tuberosum Okamura198 8 Outline of the thesis

Two types of fusion experiments with protoplasts of L. esculentum and L. peruvianumwer e carriedout .A selectionstrateg ywa sdeveloped ,t oobtai n efficiently somatic hybrids. Selection against L. peruvianum was achieved by the use of kanamycin resistant L. esculentum genotypes and the subsequent selectionfo rkanamyci nresistan thybrids .Thes e"symmetric "somati chybrid swer e characterised cytogenetically,biochemicall y andmorphologicall y (Chapter2) . Since these hybrids were fertile, their progeny could be characterised also (Chapter3) . In the second series of protoplast fusion experiments, L. peruvianum was irradiated beforefusio ni norde rt oachiev eth etransfe ro fonl ypar to fth e genome ofI .peruvianum . Inthes e experiments,th e effect ofdifferen tdose s ofgamma-ray s onth eeliminatio no fdono rgenom ewa sanalysed .Al lasymmetri c hybridswer echaracterise d cytogeneticallyan dmorphologicall y (Chapter 4).T o determineth eamoun to ftransferre dL .peruvianu mgenome ,th easymmetri chybrid s wereanalyse dfo rth epresenc eo f L. peruvianum specificgene s (Chapter 5). In addition,fiftee nasymmetri csomati chybrid swer echaracterise d indetai lwit h 30chromosom e specificRFL Pmarker s (Chapter6) . Finally, the significance of the somatic hybrids obtained and of the asymmetrichybridisatio ntechniqu ear ediscusse d (Chapter7) .

References

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TheorApp lGene t 58:121-127 BarbanoPP .Topolesk iL D (1984)Postfertilizatio nhybri dsee dfailur ei nLvcopersico nesculentu mx Lvcopersico n peruvianum ovules.J Ame r SocHor t Sei 109:95-100 BatesGW ,Hasenkamp f CA,Contolin i CL,Piastuc hW C (1987)Asymmetri ehybridizatio n inNicotian ab y fusiono f irradiated protoplasts.Theo r ApplGene t 74:718-726 de Nettancourt D, Devreux M, Laneri U, Cresti M, Pacini E,Sarfatt i G (1974)Genetica l and ultrastructural aspects of self and cross incompatibility in interspecific hybrids between self-compatible Lvcopersicon esculentum and self-incompatible L.peruvianum . TheorApp lGene t 44:278-288 Dudits D, Fejer 0, Hadlaczky GY,Konc zCS ,Laza r G, Horvath G (1980)Intergeneri c gene transfer mediated by plant protoplast fusion.Mo lGe nGene t 179:283-288 DuditsD ,Maro yE ,Praznovszk y T,Ola hZ ,Gyorgye y J,Cell aR (1987)Transfe ro fresistanc e traitsfro mcarro t into tobacco by asymmetric somatic hybridization: Regeneration of fertile plants. Proc Natl Acad SeiUS A 84:8434-8438 Famelaer I,Gleb aÏY ,Sidoro vVA ,Kaled aVA ,Parakonn yAS ,Boryshu kNV ,Cheru pNN ,Negruti u I,Jacob sM (1989) Intrageneric asymmetric hybrids between Nicotiana plumbaginifolia and Nicotiana sylvestris obtained by 'gamma-fusion'. Plant Science 61:105-117 Gilbert JC,McGuir e DC (1956) Inheritance of resistance to severe root knot from MeloidoRvne incognita in commercialtyp e tomatoes. FrocA m Soc Hortic Sei 68:437-442 GillB S (1983)Tomat o -A search forne w frontiers.In :Swaminatha nMS ,Gupt a PK,Sinh a U (eds) Cytogenetics of cropplants .MacMilla n IndiaLtd ,Ne wDelhi ,p p455-48 0 Gleba YY, Hinnisdaels S, Sidorov VA,Kaled a VA, Parokonny AS,Boryshu k NV, Cherup NN, Negrutiu I, Jacobs M (1988)Intergeneri casymmetri chybrid sbetwee nNicotian aplumbaginjfoli aan dAtrop abelladonn aobtaine db y "gamma-fusion". TheorApp lGene t 76:760-766 Gleba YY, SytnikK M (1984)Protoplas t fusion.Geneti c engineering inhighe r plants.Springe r Verlag,Berli n GlimeliusK (1988)Potential so fprotoplas t fusioni nplan tbreedin gprograms .In :Puit eKJ ,Don sJJM ,Huizin g HJ,Koo lAJ ,Koornnee fM ,Kren s FA (eds)Progres s inplan tprotoplas t research,Kluwer ,Dordrecht ,p p159 - 168 Goss SJ,Harri sH (1975)Ne wmetho d formappin g genes inhuma n chromosomes.Natur e 255:680-684 GuptaPP ,Gupt aM ,Schiede r0 (1982)Correctio no fnitrat ereductas edefec ti nauxotrophi c plantcell sthroug h protoplast-mediated intergeneric gene transfers.Mo lGe nGene t 188:378-383 Gupta PP, Schieder 0, Gupta M (1984) Intergeneric nuclear gene transfer between somatically and sexually incompatibleplant s through asymmetric protoplast fusion.Mo lGe nGene t 197:30-35 Guri A,Lev iA ,Sin kK C (1988)Morphologica l andmolecula r characterization of somatic hybrid plants between Lvcopersicon esculentum and Solanumnigrum .Mo lGe nGene t 212:191-198 Hamill JD,Penta l D, Cocking EC (1984)Th e combination of anitrat e reductase deficient nuclear genomewit h a streptomycin resistant chloroplast genome, inNicotian a tabacum. by protoplast fusion. J Plant Physiol 115:253-261 Handley LW, Nickels RL, Cameron MW, Moore PP, Sink KC (1986) Somatic hybrid plants between Lycopersicon esculentum and Solanum lvcopersicoides. TheorApp lGene t 71:691-697 Hille J, Koornneef M,Ramann a MS, ZabelP (1989)Tomato : a crop species amenable to improvement by cellular andmolecula rmethods .Euphytic a 42:1-23 Hogenboom NG (1972a) Breaking breeding barriers in Lycopersicon. 1. The genus Lvcopersicon. its breeding barriers andth e importance ofbreakin g thesebarriers .Euphytic a 21:221-227 Hogenboom NG (1972b)Breakin g breeding barriers in Lycopersicon. 4. Breakdown of unilateral incompatibility betweenL .peruvianu m (L.)Mill , andL . esculentumMill. . Euphytica 21:397-404 Hohn T,Schel lJ (1987)Plan tDN A infectious agents.Springer-Verlag ,Wie nNe wYor k ImamuraJ ,Sau lMW ,Potryku sI (1987 )X-ra y irradiationpromote d asymmetric somatichybridisatio nan dmolecula r analysis of theproducts .Theo rApp lGene t 74:445-450 Itoh K, Futsuhara Y (1983) Interspecific transfer of only part of genome by fusion between non-irradiated protoplasts ofNicotian a glauca andX-ra y irradiated protoplasts of N. langsdorffii. Jpn JGene t58:545 - 553 JainSM ,Shahi nEA , SunS (1988)Interspecifi c protoplast fusion forth etransfe r ofatrazin e resistance from Solanumnigru mt otomat o(Lycopersico nesculentu mL .) . In:Puit eKJ ,Don sJJM ,Huizin gHJ ,Koo lAJ ,Koornnee f M,Kren s FA (eds)Progres s inplan tprotoplas t research,Kluwer ,Dordrecht ,p p221-22 4 Kerr EA,Baile y DL (1964)Resistanc e toCladosporiu m fulvumCke .obtaine d fromwil d species of tomato.Ca nJ Bot 42:1541-1554 KinsaraA ,Patnai k SN,Cockin g EC,Powe rJ B (1986)Somati c hybrid plantso fLycopersico n esculentumMill ,an d Lvcopersiconperuvianu m Mill.. JPlan t Physiol 125:225-234 Koornneef M, Hanhart CJ, Jongsma M, Toma I,Weid e R, Zabel P, Hille J (1986)Breedin g of a tomato genotype readily accessible togeneti cmanipulation . Plant Science 45:201-208 KoornneefM , HanhartCJ ,Martinell i L (1987)A genetic analysis of cell culture traits intomato . TheorApp l Genet 74:633-641 KoornneefM ,de nBoe rB ,va nLoenen-Martine tP ,va nRogge nP ,Wijbrand iJ (1988)Protoplas tfusio no fkanamyci n resistant, cnx"Nicotian a plumbaginifolia with streptomycin resistant N. tabacum (SRI) and the effect of irradiationo fth etabacu mparent .In :Puit eKJ ,Don sJJM ,Huizin gHJ ,Koo lAJ ,Koornnee fM ,Kren sF A (eds) Progress inplan tprotoplas t research,Kluwer ,Dordrecht ,p p 287-288 LeeCH ,Powe r JB (1988)Intra - and interspecific gametosomatic hybridisation within thegenu sPetunia . Plant Cell TissOr gCul t 12:197-200 Lugo TG, Fournier REK (1986) Microcell fusion and mammalian gene transfer. In: Kucherlapati R (ed) Gene transfer.Plenu m Publishing Corp,Ne wYork ,p p 79-93 MartinN W (1969)Inheritanc eo fresistanc et ocurl yto pi nth etomat obreedin glin eC/F, .Phytopatholog y 59:1040 MelchersG , SacristanMD ,Holde r AA (1978)Somati c hybrid plantso fpotat o and tomato regenerated fromfuse d protoplasts.Carlsber gRe sCommu n 43:203-218 Menczel L,Galib aG ,Nag y F,Malig a P (1982)Effec to fradiatio ndosag eo n efficiency of chloroplast transfer byprotoplas t fusion inNicotiana .Genetic s 100:487-495 MenczelL ,Nag yF ,Lâzâ rG ,Malig aP (1983 )Transfe ro fcytoplasmi cmal esterilit yb yselectio nfo rstreptomyci n resistance afterprotoplas t fusion inNicotiana .Mo lGe nGene t 189:365-369 Morgan A, Cocking EC (1982) Plant regeneration from protoplasts of Lvcopersicon esculentum Mill.. Z Pflanzenphysiol 106:97-104 Mühlbach HP (1980) Different regeneration potentials of mesophyll protoplasts from cultivated and a wild specieso f tomato.Plant a 148:89-96 ' Müller-Gensert E, Schieder 0 (1987) Interspecific T-DNA transfer through plant protoplast fusion. Mol Gen Genet 208:235-241 Mutschler MA, Tanksley SD,Ric k CM (1987)Linkag e maps of the tomato (Lvcopersicon esculentum). Rep Tomato GenetCoo p 37:5-34 O'Connell MA, Hanson M (1986)Regeneratio n of somatic hybrid plants formed between Lvcopersicon esculentum andSolanu mrickii .Theo rApp lGene t 72:59-65 O'Connell MA, Hanson M (1987)Regeneratio n of somatic hybrid plants formed between Lvcopersicon esculentum andL .pennellii .Theo rApp lGene t 75:83-89 OkamuraM (1988)Regeneratio nan devaluatio no fsomati chybri dplant sbetwee nSolanu mtuberosu man dLvcopersico n pimpinellifolium.In :Puit eKJ ,Don sJJM ,Huizin gHJ ,Koo lAJ ,Koornnee fM ,Kren sF A (eds)Progres si nplan t protoplast research,Kluwer ,Dordrecht ,p p 213-214 Peberdy JF (1979) Protoplast fusion - A new approach to interspecific genetic manipulation and breeding in fungi. In:Ferencz y L,Farka sG L (eds)Advanc e inprotoplas tresearch .Pergamo nPress ,Oxford ,p p 63-72 PentalD ,Mukhopadhya yA ,Grove rA ,Pradha nA K (1988)A selectionmetho d forth esynthesi so ftriploi dhybrid s by fusiono fmicrospor e protoplasts (n)wit h somatic cellprotoplast s (2n).Theo rApp lGene t 76:237-243 Pental D, Cocking EC (1985)Som e theoretical andpractica lpossibilitie s ofplan t genetic manipulationusin g protoplasts. Hereditas (Suppl)3:83-9 2 PirrieA , Power JB (1986)Th eproductio no f fertile,triploi d somatic hybrid plants (Nicotiana «lutinosa (n) + N, tabacutn (2n))vi agameti c:somati c protoplast fusion.Theo rApp lGene t 72:48-52 Pontecorvo G (1971)Inductio no fdirectiona l chromosome elimination insomati c cellhybrids .Natur e230:367 - 369 RickC M (1979a)Potentia limprovemen to ftomatoe sb ycontrolle d introgressiono fgene sfro mwil dspecies .Pro c ConfBroadenin gGene tBas e Crops,Wageningen , 1978.Pudoc ,Wageningen ,p p 167-173 Rick CM (1979b)Biosystemati c studies inLvcopersico n and closely related species of Solanum. In:Hawke sJG , LesterRN , SkeldingA D (eds)Th ebiolog y and taxonomyo fth eSolanaceae .Academi c Press,Ne wYork ,p p667 - 677 Rick CM (1982)Th epotentia lo f exotic germplasm for tomato improvement.In :Vasi l IK,Scowcrof tWR , FreyK J (eds)Plan t improvement and somatic cellgenetics .Academi c Press,Ne wYork ,p p 1-28 RickC M (1983)Crossabilit ybetwee nL . esculentum anda ne wrac eo fL .peruvianum .Re pTomat oGene tCoo p33:1 3 Rick CM,Butle r L (1956)Cytogenetic s of thetomato .Ad vGene t 8:267-382 Rick CM, Yoder JI (1988)Classica l and molecular genetics of tomato: highlights and perspectives. Ann Rev Genet 22:281-300 RingertzNR ,Savag eR E (1976)Chromosom epattern s inhybri d cells.In :Cel lhybrids .Academi c Press,Ne wYork , pp 162-179 Shepard JF,Bidne y D, Barsby T, KembleR (1983)Geneti c transfer inplant s through interspecific protoplast fusion. Science 219:683-688 Sidorov VA, Menczel L, Nagy F, Maliga P (1981) Chloroplast transfer in Nicotiana based on metabolic complementation between irradiated and iodoacetate treated protoplasts.Plant a 152:341-345* SidorovVA ,Zubk oMK ,Kuchk oAA ,Komarnitsk y IK,Gleb aY Y (1987)Somati chybridizatio n inpotato :us eo fgamma - irradiated protoplasts of Solanum pinnat.i^ftrt.n mi ngeneti c reconstruction. TheorApp lGene t 74:364-368 SmithP G (1944)Embry o cultureo f atomat o specieshybrid .Pro cAme rSo cHor tSe i 44:413-416 Somers DA,Narayana n KR, Kleinhofs A,Cooper-Blan d S, Cocking EC (1986)Immunologica l evidence for transfer of thebarle y nitrate reductase structural gene toNicotian a tabacum by protoplast fusion.Mo l Gen Genet 204:296-301 10

SzteynK (1962)Interspecifi c crosses inth egenu s Lycopersicon. I. Backcrosses toLycopersico nglandulosum . Euphytica 11:149-156 Tan MC (1987) Somatic hybridization and cybridization in some Solanaceae. Ph D Thesis, Free University Amsterdam, TheNetherland s Taylor IB (1986)Biosystematic s ofth etomato . In:Atherto nJG ,Rudic hJ (eds),Th etomat o crop.A scientific basis for improvement.Chapma n and Hall, London/New York,p p 1-34 ThomasBR ,Prat tD (1981)Efficien thybridizatio nbetwee nLycopersico nesculentu man dL .peruvianu mvi aembry o callus. TheorApp lGene t 59:215-219 VerhoevenH A(1989 )Inductio nan dcharacterizatio no fmicronucle ii nplan tcells .Perspective sfo rmicronucleus - mediated chromosome transfer.P hD Thesis ,Agricultura l University Wageningen,Th eNetherlands . Yamashita Y, Terada R, Nishibayashi S, Shimamoto K (1989) Asymmetric somatic hybrids of Brassica: partial transfer offf. campe str is genom e intoB . oleraceab y . TheorApp lGene t 77:189-194 Young ND, Tanksley SD (1989) Restriction fragment length polymorphism maps and the concept of graphical genotypes. TheorApp lGene t 77:95-101 ZapataFJ ,Evan sPK ,Powe r JB,Cockin gE C (1977)Th eeffec to ftemperatur eo nth edivisio no flea fprotoplast s ofLycopersico n esculentum andL .peruvianum . Plant Science Lett8:119-12 4 Zelcer A, Aviv D, Galun E (1978) Interspecific transfer of cytoplasmic male sterility by fusion between protoplasts ofnorma lNicofrian asylvestri s andX-ra y irradiated protoplasts ofmale-steril e N,tabacum . Z Pflanzenphysiol 90:397-407 11

CHAPTER2

SELECTION AND CHARACTERISATION OF SOMATIC HYBRIDS BETWEEN L.Y COPE RS ICON ESCUI^ENTZJM AND Z^YCOPERSICON PERZJVIANUM

J.Wijbrandi ,W .va nCapelle ,C.J .Hanhart ,E.P .va nLoene nMartinet-Schuringa , M.Koornnee f

Summary.Somati chybrid so fth ecultivate dtomato , Lycopersicon esculentum, and awil dspecies , L. peruvianum, wereobtaine db yfusio no flea fprotoplast sfro m bothspecie s inth epresenc eo fpol yethylen eglyco lo ri na nelectri cfield . The somatic hybrids were selected on the basis of kanamycin resistance of L.esculentu man dth eplan tregeneratio ncapacit yo f L. peruvianum. Chromosome countsi nroo ttip san dth edeterminatio no fth enumbe ro fchloroplast si nguar d cellpair sreveale dtha tth emajorit yo fthes ehybrid swa stetraploi d (2n- 4 x =48).Th eremainin ghybrid swer ea tth ehexaploi dleve lwit hchromosom enumber s between6 4an d72 .Th ehybri dnatur eo fth eregenerate dplant swa sconfirme db y analysiso fisozym emarker san db ythei rmorphology .Mos thybrid sdi dflowe ran d setfruit san dseed safte rselfing .Accordin gt oRFL Panalysi s6 ou to fth e1 0 hexaploid hybrids contained twogenome s of L. esculentum and fourgenome so f L.peruvianum .On eo fthes ehexaploid sha dgenome so ftw odifferen t L. peruvianum genotypesan dwa stherefor econsidere d tob ederive dfro ma tripl eprotoplas t fusion.Th ehexaploi d plantswer e less fertile thanth e tetraploids andmor e resembledL .peruvianum .

Introduction

Somatichybridisatio n isa nalternativ e tosexua lhybridisatio nwhe ncrossin g barriers between parental species exist. By means of various cell culture techniques,protoplast sfro mdifferen tspecie sca nb einduce dt ofus ewit heac h other. Several procedures have been applied to select somatic hybrids after fusion.Somati chybrid so f Solanum and Lycopersicon specieshav ebee nselecte d eithermechanicall yb ymicromanipulatio n(Puit ee tal .1986) ,b yselectio nbase d onspecifi cgrowt hcharacteristic si ncel lcultur e(Gleddi ee tal .1986 ;Handle y etal .1986) ,o rb yth eidentificatio no fhybrid sa tth eplan tleve l (Austine t al. 1985;Shihachak r 1989;Ta n 1987).Regeneratio ncapacit ytha tderive dfro m one of the parentsha s beenuse d as selectable marker by several authorsi n protoplastfusio nexperiment so f Nicotiana (Maligae tal . 1977), Petunia (Itoh and Futsuhara 1983), Brassica (Terada et al. 1987)an d Lycopersicon species (Adamsan dQuiro s1985 ;Kinsar ae tal . 1986). In the present experiments, protoplasts of Lycopersicon esculentum, the cultivated tomato, and L. peruvianum, a wild tomato species with several 12 desirableagricultura ltrait s(Ric k1982a) ,wer efused .Thes especie scanno tb e crossedeasil ywit heac hother .Whe n L. esculentum isuse da sstaminat eparent , pollen tube elongation is inhibited in the L. peruvianum style (unilateral stylar incompatibility orincongruit y (Hogenboom 1973). When L. peruvianum is thepollinator ,fertilisatio ntake splace .However ,th eembry osoo nabort sdu e toth edegeneratio no fth eendosper m(Barban oan dTopolesk i1984) .I nth elatte r combination sexual hybrid plants were obtained by the application of tissue culturetechnique ssuc ha sembry ocultur e(Smit h1944 )an dembry ocallu scultur e (Thomasan dPrat t1981) .Backcrosse so fsuc hhybrid swit hI .esculentu mwer eno t possiblewithou tth eapplicatio no fadditiona ltissu ecultur etechnique s(Smit h 1944;Thoma san dPrat t1981) . Theai mo fth epresen texperiment swa sth eproductio no fsymmetri csomati c hybridsbetwee nth etw ospecie si na nefficien tan dreproducibl emanne rb yth e development ofa doubl e selection strategybase d onth edominan t regeneration capacityo f L. peruvianum (Adamsan dQuiro s1985 ;Kinsar ae tal .1986 ;Koornnee f etal .1987a )an dth edominan tkanamyci nresistanc eo f L. esculentum. Thelatte r selectionmarke rha dbee nintroduce db ylea fdis ctransformatio no f L. esculentum with Agrobacterium tumefaciens. Thehybrid sobtaine dwer echaracterise d onth e basiso fchromosom enumbers ,isozym epatterns ,morphology ,fertilit yand ,i nsom e cases,b yth eanalysi so frestrictio nfragmen tlengt hpolymorphism s (RFLPs).

Materialan dmethod s Plant material Seeds ofL .escuientu mcv .Bellina , L. esculentum genotype LA1182 (homozygous forrecessiv emarke rgene s sy, sf (chr.3 )an d alb (chr.12 )(Ric k1982b )an d L. peruvianum PI128650wer ekindl yprovide db yRij kZwaa nSee dCompan y(d eLier , The Netherlands), Prof. CM. Rick (Tomato Stock Center, Davis,USA )an d the Institute of Horticultural (Wageningen, The Netherlands), respectively. Kanamycin resistant transformants of these genotypes,obtaine d afterlea fdis ctransformatio nwit h A. tumefaciens containingth eplasmi dAGS11 2 (Koornneefe tal . 1987b),wer eavailabl e andwer edesignate dATW3001 ,ATW300 3 (Bellina),ATW305 2(LA1182 )an dATW200 2(PI128650) .Fou rfusio ncombination swer e made:ATW3001/-3003/-305 2(+ )PI12865 0an dBellin a (+)ATW2002 . Isolation, fusion and culture of protoplasts Shootculture swer egrow nasepticall yi nglas scontainer sa ta ligh tintensit y of3 0W/m 2 (16h) , at2 5° Co nmediu mcontainin gM Ssalt s (Murashigean dSkoo g 1962),T vitamin s (Tewese tal .1984 )an d1 0g/ 1sucrose ,solidifie dwit h9 g/ 1 agar (shootcultur e medium). Plantswer ekep t inth edar kon eda ybefor eth e harvesto fleaflets ,whic hthe nwer efloate d inth edar ka t4 ° Cfo r2 4hour s on a pre-incubation medium: hx MSsalts , lxT vitamins , 1mg/ 1 2,4- dichlorophenoxyacetic acid (2,4-D) and 0.5mg/ 1 benzylaminopurine (BA). Subsequently,th eleave swer ecu ti nsmal lpiece san dincubate dfo r1 6hour si n the dark at 25° C in an solution: 10g/ 1 CellulaseRI O and 1.5g/ 1 MacerozymRI O(bot henzyme sfro mYakul tLtd. ,Japan) ,CP Wsalt s(Frearso ne tal . 1973), 100mg/ 1 2[N-morpholino]-ethane-sulfonate (MES) and 137g/ 1 sucrose, pH5.6 .Th eprotoplast swer e separated fromcel ldebri sb yfiltratio nthroug h 13 anylo nfilte rwit ha por esiz eo f5 0 pman dpurifie db yfloatatio no na solutio n of 137g/ 1 sucrose + CPW salts + 100mg/ 1 MES by centrifugation for 5 min. at 70x g. The floating protoplasts were pelleted twice inW 5 solution (Menczel et al. 1981) and a sample was counted in a haemocytometer to determine the concentrâtion . Fusion of protoplasts in poly-ethylene-glycol (PEG) was carried out as describedb yMencze l etal .,excep ttha t30 %PE G MW 4000instea do f40 %PE G 6000 was used. In the experiments where electrofusion was used, the protoplast floatation step was carried out four times with a solution of 137g/ 1 sucrose without salts and MES. The layer of protoplasts was transferred to a solution of 64g/ 1 mannitol supplemented with 73.5mg/ 1 CaCl2'2H20 (final concentration h.-\ x 106protoplast spe r ml). 60//1o fth eprotoplas t suspensionwa s transferred toa fusio nchambe rwit h a 1m mwid e gapbetwee ntw o parallel brass electrodes, glued ina glas sPetr idish .Th efusio napparatu swa sa combinatio no fa functio n generator, a custom-built generator of direct current (D.C.) pulses and the fusionchamber .Th eelectrofusio nwa sperforme da sfollows :th eprotoplast swer e aligned in an alternating current (A.C.) field (1Mhz ) of 200V/cm , one D.C. 'pulseo f200 0V/c m (25 ßsec.) wasapplied ,wher eafte r theA.C . fieldwa s slowly reduced to zero.Finall y the protoplasts were transferred to a Petri dish with culture medium. The protoplasts were cultured at a density of 1-2 x lO5/1"! in TMp medium, which is a modified TM-2 medium (Shahin 1985) containing 103 g/1 sucrose and 0.5 mg/1 BA (instead of zeatin riboside), in the dark at 25 °C.Whe n the first celldivision swer e observed,mostl y after 3days , theculture s were exposed to dim light and diluted (1:1 or less) once or twice awee k with TMd, which is a modifiedTM p mediumcontainin g68. 5 g/1sucros ean d0. 1 mg/1a-naphtylaceti caci d (NAA). Four weeks after protoplast isolation microcalli were transferred onto TMc,whic h isa modifie d TM-3 medium (Shahin1985 )wit h 2.5 g/1 sucrose,3 6 g/1 mannitol, 0.1 mg/1NAA ,0. 5 mg/1 BAan d 8g/ 1 purified agar.Afte r twoweek s of culturing,w eadde dkanamyci nt oth emedi afo rhybri d selection;i nliqui dmedi a the final concentration of kanamycin was 50mg/1 , in TMc 100 mg/1. When the microcalli onTM c mediumha ddevelope d togree ncall io fsevera lmillimeter s in diameter, they were transferred to a shoot induction medium, which is shoot culturemediu m with 20 g/1 sucrose (instead of 10g/1 ) supplemented with 1mg/ 1 zeatin and 0.1 mg/1 indole-acetic acid (IAA). After 4 weeks the calli were transferred to the same medium without IAA and subcultured every 4week s until well developed shoots were present. The shoots were rooted on shoot culture medium. After rooting the régénérants were maintained in vitro, tested on kanamycin resistance (100mg/1 )an d transferred tosoi l ina heate d greenhouse.

Cytogenetic analysis Root tipswer e collected from greenhouse-grownplants ,treate d for 3-4hour sa t 15-20 °Cwit h 290mg/ 1 8-hydroxyquinoline toarres tmetaphas e plates,an d fixed ina mixtur e ofethano l andaceti caci d (3:1)a t4 °C.Th eroo t tipswer e either maceratedi nI N HClfo r4- 5minute sa t6 0 °Can dthe nsquashe di nlacto-propioni c orcein (Dyer 1963) or they were treated with an enzyme solution (1 g/1 Cellulase RS (from Yakult, Japan)+ 1g/ 1 Pectolyase Y23 (from Seishin, Japan) +1 g/ 1Cytohelicas e (fromIBF ,France )i n2. 5 g/1Na-citrat ep H 4.8)fo r3 hour s at 20 °C. In the latter case chromosome preparations were made by the cell spreading technique and chromosomes were stained with Giemsa, according to Pijnacker and Ferwerda (1984). The number of chloroplasts per guard cell pair was determined in lower epiderm strips fromleave so fgreenhouse-grow nplant sa sdescribe db yKoornnee f et al. (1989).

Isozyme and RFLP analysis Young leaves from greenhouse-grown plants were used for isozyme and RFLP analyses. Acid phosphatase (APS;E.C.3.1.3.2 )activit ywa sanalyse d by electrophoresis of 50/i lcrud e extract, supplemented with 5 pi electrode buffer and 5 pi marker dye (0.5%bromopheno l blue in 20%glycerol ) ona 10%Polyacrylamid e (PAA) slab gel.Th eextract swer eobtaine db ysqueezin gthawin glea fmaterial ,tha tha dbee n 14

stored at -80 °C. For analysis of glutamate oxaloacetate transaminase (GOT; E.C.2.6.1.1)activity , samples were prepared according to Suurs et al. (1989) and electrophoresed on a 7.5% PAA slab gels. Electrophoresis was performed in vertical gels (Desaphor VA equipment, Desaga). The stacking gel contained 5% Polyacrylamide. The electrode buffer consisted of 0.04 M tris-HCl and 0.1 M glycin, pH 8.9. After running, the gels were incubated for 15 minutes in the appropriate staining buffer with gentle shaking. Subsequently, they were incubated in the corresponding staining solutions for one of both isozymes (a few hours,gentl e shaking), prepared according toVallejo s (1983). DNA was isolated from several plants according to Dellaporta et al. (1983), digestedwit h Dra I,separate db yagaros ege lelectrophoresis ,blotte dont oGen e Screen PLUS (New England Nuclear) and hybridised with the tomato single copy clones TG16 and TG63 (Tanksley et al. 1988) (kindly supplied by Dr. S.D. Tanksley, Cornell University, Ithaca,USA) ,a s described inChapte r 6. Morphology and fertility Thefollowin gfeature so fth eplants ,grow ni na nunheate dgreenhous eunde rDutc h summer conditions, were determined: growth habit, leaf morphology, sympodial index (=mea nnumbe r ofnode s between two subsequent inflorescences),presenc e of stipules and bracts, size of the flower parts, pollen viability, fruit and seed set.Polle n grains (at least 100 per flower)wer e stained with a solution oflactopheno l acidfuchsin ;viabl epolle nstaine dpurple ,no nviabl epolle ndi d notstain .Observation swer emad eo nsomati chybri dplants ,severa l L. peruvianum régénérants and the parental genotypes mentioned before. Also seedlings of a tetraploid plant of the tomato cultivar Moneymaker and of a tetraploid L. peruvianum plant, both derived from tissue culture,wer e used. The somatic hybridplant swer eselfe dan dcrosse dwit h L. esculentum (diploidan d tetraploid) andwit h L. peruvianum (diploid and tetraploid).

Results

Cell culture and plant regeneration

Upon isolation and culture with the procedures described, L. peruvianum protoplasts formed cell walls and grew prosperously. The cultures had to be diluted at least every 3-4 days during the first 4 weeks of culture. The resulting microcalli, that were put on the TMc-medium, developed into purple- greencoloure d calli.Th emajorit y ofthes ecall iproduce d shootswithi n4 week s onshoo t inductionmedium ; some ofthe m even showed shoot primordiawhil e being cultured onTMc . The shoots could easily be rooted.Whe nkanamyci nwa sadde d to the liquid culture, all cell colonies stopped growing and in cases where microcalli were put on TMc withkanamyci n they became brown. L. esculentum cells divided at a lower frequency and developed more slowly than those of L. peruvianum. Only half of the microcalli transferred to TMc developed into small green calli. These calli turned brown within a few weeks on shoot inductionmedium . No regenerated plants could be obtained by means of the described procedures. The mixed cultures (protoplasts of both species without fusion treatment) resulted in many microcalli that appeared to be of L. peruvianum origin, as 15

judged by the growth characteristics. Self-fused L. peruvianum cultures did produce many microcalli also. From both culture types, shoots could be regenerated. The fusion cultures (protoplasts of both species with fusion treatment) yielded many well growing colonies. When kanamycin was added to the liquid culturemos tcolonie s stoppedgrowing ,wherea s othersdi dnot .A majorit yo fth e microcalli,tha twer etransferre dt oTM c + kanamycin,develope dint ogree ncalli . Most of these formed shoot primordia within 4week s on shoot induction medium. The shoots were characterized by broader leaflets than the L. peruvianum régénérants and formed roots easily. One to several shoots from 39 different putative hybrid calli (named 0H2 to 0H40)wer e rooted. Eight of the calli were derivedfro melectrofusio ntreate dprotoplasts ,whil eth eremainde rwa sobtaine d by PEG treatment. The kanamycin resistance of the putative hybrid shoots was confirmedlate ro nb ythei rabilit yt oroo to nshoo tcultur emediu m supplemented withkanamycin . One other putative hybrid, named 0H1, was obtained after PEG-fusion of protoplastsfro mL . esculentum cv. BellinaandL. peruvianumATW2002 .Thi shybri d was selected on the basis of callus morphology (green and well-growing) and regeneration capacity. Shoots from 33 hybrid calli and from 12 L. peruvianum derived calli were grown in the greenhouse and were analysed for one or more characters, together with the parental genotypes.

Cytogenetic analysis

Both parental species have chromosome numbers of 2n = 2x - 24.Th e chromosome numbers of 47 shoots derived from 23differen t hybrid calli were determined in metaphase plateso froo tti pcells .Mos tshoot swer etetraploi dwit hth eeuploi d numbero f2 n= 4x= 48(Fig . 1A+ 2);fe wwer eaneuploi da tth etetraploi dlevel . Aminorit y ofth eshoot swa sa tth ehexaploi d level (Fig. IB+ 2);hal fo fthes e had a chromosome number slightly below 72. We found an octaploid chromosome number in one shoot derived from a cutting of a plant that we had previously determined tob ea tetraploid . Thefe whyper-tetra/hexaploi dnumber smigh thav e been obtained by the unintended counting of the macrosatellites (short arms of the chromosomes 2)a s independent chromosomes. The number of chloroplasts in the guard cell pairs can be used as an additionalmeasur efo rth eploid yleve li n Lycopersicon plants(Koornnee fe tal . 1989). However,th erelatio nbetwee nchloroplas tnumbe ran dploid yleve ldiffer s between L. esculentum and L. peruvianum (Fig. 3; Koornneef et al. 1989). The chloroplastnumber s ofth e somatichybrid sals oar eshow ni nFig . 3.Th eaverag e chloroplast number of tetraploid hybrids (18.1), is intermediate to that of 16

.apt.'

WW 1jfc

ifj %«

" if i

• ••• -/ * * *

B -.;*• Fig.1 .Metaphas eplate so froo tti pcell sfro msomati chybrid so f L. esculentum andL .peruvianum .A .Th etetraploi dhybri d0H4 ,2 n= 4 x= 48 .B .Th ehexaploi d hybrid0H14 ,2 n= 6 x= 72 .

(I) C a

0) E 3

24 36 48 60 72 84 96

Chromosome number

Fig.2 .Frequenc ydistributio no faverag echromosom enumber so fdifferen tshoot s fromsomati chybri dcalli ,derive dfro mprotoplas tfusion sbetwee n L. esculentum and L. peruvianum. 17 tetraploid L. esculentum and L. peruvianum plants and lower than that of hexaploid hybrids (24.9). However, the frequency distributions of both classes overlap, which indicates that this character does not always allow an unambiguous distinction between both ploidy groups. We classified the ploidy level of the somatic hybrids on the basis of chromosome numbers, or, in some cases, chloroplast counts (taking <20 as tetraploid and 25-30 as hexaploid). 54 shoots regenerated from 21hybri d calli were tetraploid, 18shoot sfro m 9call iwer ehexaploi d and 3shoot sfro m 2call i were tetra- or hexaploid; the latter plants had an intermediate chloroplast number. From one callus one tetraploid and one hexaploid shoot derived. These were considered as two independent fusion products in further analyses. The fusionmetho d used had no clear effect on the ploidy level distribution of the obtained somatic hybrids (Table1) .

I L. esculentum Ü diploid If) k -t-1 C L. peruvianum JÇ | tetraploid CL M— m Ji | hexaploid O Somatic hybrids | octaploid E 3 0 ?-ploid

1'0 Ï5 2Ö 2'5 30 35 40 Mean number of chloroplasts in guard cell pairs

Fig. 3. Mean chloroplast number in the guard cell pairs of the leaf epiderm: upper panel,L . esculentum plants,tha twer ediploid , tetraploid ando funknow n ploidy level (= no chromosome counts available); middle panel, I. peruvianum plants, thatwer ediploid , tetraploid ando funknow nploid ylevel ;lowe rpanel , Somatic hybrid (of both species mentioned above) shoots,tha t were tetraploid, hexaploid and of unknown ploidy level. 18

Table1 .Th eploid yleve lo fhybri d cloneso f L. esculentum and L. peruvLanum foreac ho fbot hfusio nmethod sused ,eithe rPEG-fusio n (Menczele tal .1981 ) orelectrofusion .Th eploid yleve lwa sdetermine di nshoot sregenerate dfro mth e hybridcall iusin gchromosom ecount so rth emea nnumbe ro fchloroplast si nth e guard cell pairs.Mixe d means that bothtetraploi d andhexaploi d shootswer e derivedfro mon ecallus .

Method Ploidy1 eve 1 ofhybri d clone s tetraploid hexaploid mixed PEG-fusion 17 7 1 Electrofusion _4 _2 _0 Total 21 9 1

Isozyme analysis The Aps-1 (acidphosphatase ,locu s Aps-1; adimeri cenzym e(Ric k1983 )pattern s of L. esculentum, L. peruvianum andsom esomati chybrid sar eshow ni nFig .4A . L.esculentu mha don efas tmigratin gband . L. peruvianum plantsha don eo ftw o differentpatterns :eithe rthre eband smigratin gmor eslowl ytha nth etomat oban d (patternA ) or one heavy band which migrates as fast as the tomato band (patternB ). O f the hybrid plants tested 24ha d one heavy band similar to L.peruvianu m patternB (for instance,0H 8 inFig .4A ). Eigh t hybridsha da n Aps-1 patterno f6 band so fwhic hthre ewer ea tth esam epositio na sth eband s ofpatter nA o fL . peruvianum, onewa s similar toth eL . esculentum band,an d twower e"new" ;0H 1i nFig .4 Ai sa nexampl eo fthi sclas so fhybrids .W esuppos e thatthes e"new "band scontai ndimer so fI .peruvianu man dL .esculentu menzym e subunits. One of the hybrids (0H14)ha d this same pattern of sixband swit h differentintensitie s (Fig. 4A). Isozyme patternso fglutamat e oxalate transaminase (GOT;4 loc iar eknown , ofwhic ha tleas t 3contai ngene s coding fordimeri c (Rick 1983)ar e showni nFig .4B . L. esculentum hadfou rGO Tband so fwhic hon ewa softe nver y fainto rabsent .Again ,ther ear etw o L. peruvianum patterns:som eL .peruvianu m plantsha d5 bands ,o fwhic h2 wer esimila rt o L. esculentum bands(patter nA) ; others had 7GO T bands (pattern B), of which 5 coincided with the bandso f patternA an d3 wit hth etomat obands .W eanalyse dth eGO Tisozym epattern so f hybridplant sderive dfro m2 2differen tcalli .Thes ehybrid sha dcomple xisozym e patternso f9 band s(fo rinstance ,0H 2an d0H1 4i nFig .4B) , ofwhic hon eo rtw o didno tcoincid ewit han yo fth eparenta l GOTbands ;thes eprobabl yrepresen t interspecificheterodimers . 19

The simultaneous occurrence of isozyme bands from both parents in the putativehybrid san dth eoccurrenc eo fpresume dinterspecifi cheterodime rband s for Aps-1 aswel la sGO Tindicat eth ehybri dnatur eo fth eregenerate dplants .

Le LpA 0H1 LpB 0H8 0H14 Le 0H14 0H2 Lph Lp •I

Fig. 4.A .Isozym epattern so faci dphosphatase ,locu s Aps-1, of(fro mlef tt o right)L. esculentum cv. Bellina(Le) ,on e L. peruvianum PI128650(Lp A),somati c hybrid 0H1,anothe r L. peruvianum PI128650 (Lpg),:somati chybri d 0H8,somati c hybrid0H14 .B .Isozym epattern so fglutamat eoxaloacetat etransaminas eo f(fro m left to right)L .esculentu m cv.Bellin a (Le),somati c hybrid 0H14, somatic hybrid 0H2,on e L. peruvianum PI128650 (Lpa), another L.peruvianu m PI128650 (LPA)•

RFLP analysis Toestablis hth econtributio no feac hparenta lgenom et otha to fth ehybrids , sixhexaploi dsomati chybrid san dfou rtetraploi dsomati chybrid swer esubjecte d toRFL Panalysis .Fo r thispurpos ew euse d TG16an dTG63 , singlecop yprobe s located on chromosome 8 and 10,respectively , as markers. In addition,tw o L.esculentu mgenotypes ,cv .Bellin aan dLA1182 ,an dtw o L. peruvianum plants, differing in Aps-1 and GOTpatterns ,wer eanalyse dwit hth e sameprobes .Th e resultsar eshow ni nFig .5 .Th eRFL Ppattern so fth etw otomat ogenotype swer e identical,wherea sthos eo fbot h L. peruvianum plantswer edifferent .Al lhybrid s showeda composit epatter nfo rbot hRFL Pprobes .Fiv ehexaploi dhybrid sdisplaye d thesam epatter na sth etetraploi dhybrids ,bu twit hth eL . peruvianum specific band(s)bein grelativel y more intense.W etherefor e tentatively concludetha t theseshoot scontaine d4 genome so f L. peruvianum and2 genome so fL .esculentum . Hexaploidhybri d0H1 4ha dth eRFL Ppattern so fbot hL .peruvianu mplant sa swel l as the tomato specific band (Fig.5B) .Apparently , this hybrid arose froma fusionproduc to fthre egenotypicall ydifferen tprotoplasts .Th eresult sobtaine d 20 withhybri d0H 3wer edeviating :0H 3wa sclassifie da stetraploi d onth ebasi s ofchromosom enumbe ran dchloroplas tcounts .RFL Panalysi so fthi shybri dwit h TG16 yielded a typical tetraploid pattern,wherea s analysis with probe TG63 produceda hexaploi dpatter n(Fig . 5).

1 2 3 4 5 6 7 8 9 10 11 12 13 14

PA -2.0kb E 5 PA+PB '•I - •- • •

-0.6kb A: TG16

-6,7kb

PB- -4.4 kb

i PA+PU V -2.3 kb B: TG63

Fig.5 .Souther nblo tanalysi so fsom e somatichybrid san dthei rparent swit h A. probe TG16an d B.prob e TG63. DraI digest s of the DNAs of the following genotypes were analysed: lane 1, L. esculentum cv.Bellina ; lane 2, one L. peruvianum PI128650 (A);lan e 3,tetraploi d hybrid 0H4;lan e 4,hexaploi d hybrid0H14 ;lan e5 ,hexaploi dhybri d0H19a ;lan e6 ,hexaploi dhybri d0H27 ;lan e 7,anothe r L. peruvianum PI128650 (B);lan e 8,hexaploi d hybrid 0H7;lan e 9, tetraploidhybri d0H8 ;lan e10 , L. esculentum LA1182;lan e11 ,hexaploi dhybri d 0H2;lan e12 ,tetraploi dhybri d0H3 ;lan e13 ,hexaploi dhybri d 0H32;lan e14 , tetraploidhybri d0H1 .Th eparenta lgenotype so fth ehybrid swere :Bellin a(+ ) L.peruvianu m plantA , 0H4, 0H19a, OH27 and 0H1; Bellin a (+) L .peruvianu m plantB ,0H 7an d0H8 ;LA118 2(+ )L. peruvianum plantB ,0H2 ,0H 3an d0H32 ;LA118 2 (+) L. peruvianum plantA (+ ) L. peruvianum plantB ,0H14 .Th especie sspecifi c bands are indicated at the left side (E, L.escuientum ; PA, t.peruvianu m plantA ;P B, L. peruvianum plant B),an dth epositio no fth e Hin dillfragment s ofphag elambd aDN Aa tth erigh tside . 21

Plant morphology Thesomati chybrids ,represente db y6 0shoot sderive dfro m3 2differen tfusio n products,wer ever yvigorousl ygrowin gplants .Th elea fmorpholog y(Fig .6 )wa s intermediatebetwee nbot hparents ,althoug hi nth eLA118 2hybrid sth eleave swer e more serrated thani nLA1182 ,indicatin g thecomplementatio no fth erecessiv e solanifolia (sf) traito fthi sgenotyp e(th elatte rhybrid swer eals owil dtyp e

Fig.6 .A .Th eleave so f (fromlef tt oright )L .esculentu mcv .Bellina , the tetraploid somatic hybrid 0H36, the hexaploid somatic hybrid 0H22 and L. peruvianum PI128650.B . The leaves of (from left to right)L .esculentu m LA1182,th etetraploi dsomati chybri d0H3 ,th ehexaploi dsomati chybri d0H3 2an d L.peruvianu mPI128650 . 22 for sy, sunny- yellow colour of young leaflets,an d alb, albescent =whit e sectors).Th eleave so fth ehexaploi dhybrid swer ei ngenera lsomewha tsmaller , less incised anddarke r greentha nth e leaveso f the tetraploid hybrids.Th e sympodial index seemed to be a dominantly expressed trait derived from L.escuientu mi nth etetraploi dhybrid s(Fig . 7A);however ,thi strai twa smor e intermediate in the hexaploid hybrids (Fig.7A) .Th e presence of stipules appearedt obehav ea sa dominan t L. peruvianum character(Fig . 7B),wherea sth e presenceo fbract swa sintermediat ebetwee nbot hparent si nth esomati chybrid s (Fig. 7C). In half of the shoots variegation of grey-green spots and sectors was observed. From several hybrid calli both variegated and normal shoots were regenerated.Eleve nhybri dcall iforme donl ynorma lshoots .Severel yvariegate d shootsha dleave stha twer esmalle ran dirregularl yshaped .

Flower morphology Shootsfro mal lhybri d calli,excep t for onehexaploi d hybrid,hav e flowered (Table 2). The flowerso f the cultivated tomatoha d long sepals,pal eyello w petalsan dn oprotrudin gstyle .Th e L. peruvianum flowersha dshor tsepals ,dar k

A B c Sympodial index Stipules Bracts L. esculentum

c " lEÇi ,_ _szp_ H diploid L. peruvianum -Q_*> • | tetraploid ° 0. f Somatic co II hexaploid hybrids "§ w

12 3 4 1 2 3 Mean number Class of nodes

Fig.7 .Morphologica l characterso f L. esculentum plants, L. peruvianum plants andsomati chybrid so fdifferen tploid ylevel .A .Sympodia l index:mea nnumbe r ofnode sbetwee ntw osubsequen tinflorescences .B .Presenc eo fstipules :leaflet s onth este ma tth enodes .C .Presenc eo fbracts :leaflet si nth einflorescenc e atth enodes .Classificatio n ofB an d C: 1= absence,2 = presence ata fe w nodes,3 = presenc ea teac hnode . 23

Table2 .Th epresenc eo fflowers ,fruit san dseed si nth esomati chybri dclone s (on at least one shoot of each fusion callus), divided in ploidy groups. Observationswer emad eo ngreenhouse-grow nplants .See dse tafte rcontrolle do r spontaneousselfing ,unles sotherwis ementioned .

Ploidyleve l Numbero fsomati chybri dclone s total flowering settingfruit s settingseed s Tetraploid 20 20 20 16 Hexaploid 10 9 6 4* Tetra-o rhexaploi d _2 JL _2 JL Total 32 31 28 21 2ou to f4 onl yafte rbackcrossin gwit htetraploi dL .peruvianu ma s staminateparen t

yellow petals and aprotrudin g style.Th e flowers of the somatic hybrids in general had medium-sized sepals, dark yellow large petals and a slightly protrudingstyle .Th elength so fth edifferen tflowe rpart sar egive ni nTabl e3 . Thesepa llengt ho fth ehybrid swa s intermediate.Th epetals ,th estamen san d thepisti l ofth ehybrid swer e larger thanthos e ofbot hdiploi d parentsan d comparable with those of thetetraploi d L.peruvianu mplants ,excep tfo rth e pistilwhic hwa slonge ri nth elatter .Th ehexaploi dhybrid sha da mor eexerte d style thanth e tetraploid hybrids,becaus e of slightly shorter stamens anda slightlylonge rpistil .

Table3 . Characteristics of flower parts of L. esculentum, L. peruvianum and somatichybrids .Polle nviabilit y wasdetermine d bylactopheno l acidfuchsi n staining.Eac hmeasuremen twa sth eaverag eo fa tleas ttw oflower sfro mth esam e plant.2x ,diploid ;4x ,tetraploid ; 6x,hexaploid ; SD,standar ddeviation .

Genotypes Meanlength s(i nmillimeter s±SD ) Pollenviabilit y (percentage±SD ) sepals petals stamens pistil L. esculentum 2x (n-11) 13.9 ±2.1 17.3 ±2.4 11.1 ±0.8 10.7 ±0.9 77.4 ±18.2 4x (n=2 ) 14.4 ±0.1 17.7 ±1.0 11.8 ±0.3 9.1 ±1.3 35.7 ±32.3 L.peruvianu m 2x (n-20) 5.1 ±0.4 16.7 ±2.7 10.8 ±0.9 12.9 ±1.1 75.0 ±19.3 4x (n=4 ) 5.8+1.0 20.3 ±3.0 13.8 ±3.2 15.1 ±3.7 44.6 ±23.8 Somatichybrid s 4x (n=30) 9.0 ±1.3 20.7 ±2.6 13.6 ±1.2 14.0 ±1.2 50.6 ±21.9 6x (n=8 ) 7.8 ±0.9 19.8 ±2.4 13.2 ±1.1 14.2 ±1.8 26.9 ±20.6 24

Fertility All hybrid flowers produced pollen,althoug h less abundant thanth eparenta l flowers. Staining with lactophenol fuchsine was used as a measure ofpolle n viability.A sshow ni nTabl e3 ,th epolle nviabilit y ofth ehybrid swa slowe r thantha to fbot hdiploi dparent san dhighe rtha ntha to ftetraploi dplant so f bothparenta lspecies .Th etetraploi dhybrid sha da highe rpercentag eo fviabl e pollentha nth ehexaploi dones . Plantsfro m2 8o fth e3 1hybri dcall iwhic hproduce dflowerin gshoot sdi dse t fruits; the 3fruitles shybrid swer ehexaploid s (Table 2). The fruits ofth e somatichybrid swer e smallan dyellow ,wherea stomat oproduce d bigre dfruit s and L. peruvianum small green ones. A very variegated hybrid plant produced irregular shaped, yellow-green variegated fruits. When the hybrid fruits containedseed sthe yofte nwer eslightl ylarge rtha nnorma l L. peruvianum fruits. Plants from 21 of the 28 fruit setting hybrid clones set several intermediate-sized seeds (Table 2). Inmos t cases,th e seed setresulte dfro m controlledan dspontaneou sselfing .Backcrosse so fth ehybrid swit hdiploi dan d tetraploid tomato did not result in seed set. Backcrosses with diploid L. peruvianum asmal eparen tyielde da fe wseed si ntw otetraploi dhybrids ,whil e backcrosseswit hpolle no fa tetraploi d L. peruvianum yieldedsom eseed si nthre e hexaploids (all 2/3 L. peruvianum genome). The analysis of the progenies is reported inChapte r3 .

Discussion

The experiments described in this paper show that somatic hybrids of L.esculentu m and L. peruvianum can be effectively selected on the basis of kanamycinresistance ,introduce d inth e L. esculentum genotypesused ,an dth e regeneration capacity of L. peruvianum. All régénérants that survived the selectionan d thatwer eanalyse d biochemicallyand/o rmorphologically ,prove d tob ehybrids .A nanalogou sselectio nmetho dwa suse db yAdam san dQuiro s(1985) , who obtained somatic hybrids between L. peruvianum and L. pennellii after selectionfo rregeneratin gcapacit yo fth eforme rspecie san dfo rresistanc et o theantibioti cG41 8presen ti na spontaneou svarian tcel lclon eo f L. pennellii. Kinsara et al. (1986) also isolated somatic hybrids of L. esculentum and L. peruvianum. They obtained hybrid shoots from two calli. This low yield probablywa sdu et oth eabsenc eo fa selectio nschem eagains tth e L. peruvianum parenta tth ecellula rlevel .Thei rhybri dshoot swer eal lhexaploid ,probabl y due to the tetraploid origin of the suspension culture from which the L.peruvianu m protoplasts derived. In our experiments most shoots that were 25 derived from hybrid calliwer e tetraploid (22ou t of 34hybri d clones)an d the remaining hybrids were hexaploid. No obvious effect of the fusion method used on the ploidy level of the hybrids was observed. We found two cases of aneuploidy at the tetraploid level. The only shoot of 0H26ha d4 1chromosome san don eshoo to f0H1 3ha d4 5chromosomes ;wherea sanothe r shoot from the 0H13 callus had 48 chromosomes. It ispossibl e that spontaneous chromosome loss occurred during the latter stages of the cell culture ora t the plant level. Inhexaploi d hybrids we observed more cases of aneuploidy. Six of the 12 hexaploid shoots (from 7 calli) counted were hypohexaploid. Also some differences between shoots of the same callus (2 out of 4 lineswit h more than one shoot counted)wer e observed.Apparentl yhexaploi d hybridswer eles s stable for chromosome number than the tetraploid hybrids. It was not possible to classify all hybrid plants as either tetraploid or hexaploid on the basis of the number of chloroplasts inguar d cell pairs; this confirms the observation of Frandsen (1968)o n tetraploid and hexaploid plants. A number of extreme deviations between chromosome number and expected chloroplastnumbe rma y indicate chimerismo fdifferen t tissuelayer s (Koornneef et al. 1989; Sree Ramulu et al. 1976). According to the RFLP analysis, all 6 hexaploid hybrids contained one L. esculentum genome and two L. peruvianum genomes. In hexaploid 0H14 the two L. peruvianum genomes were different; this suggests that this hybrid has originatedfro ma tripl efusio nbetwee nthree ,geneticall ydifferen tprotoplasts . This conclusionwa s further supported byth e Aps-1 isozyme pattern of 0H14 (see Fig. 4A), which resembled the typical pattern of the hybrid between both L. peruvianumplant s involved (datano t shown). Iti sconceivabl e thatth eothe r 5hexaploi d hybrids alsooriginate d from triple fusion,althoug ha n alternative explanation is that the genome of these hybrids arose after doubling of the L. peruvianum genome either before fusion or before karyogamy. One euploid tetraploid shoot had the "tetraploid" restriction fragment pattern for one of theprobes ,whil efo rth eothe rprobe ,TG63 ,th eL . esculentum specificban dwa s fainter than expected. Possibly this hybrid lacked part of a L. esculentum chromosome 10,containin g the TG63 marker (Tanksley et al. 1988). Themorphologica l characteristicso fth esomati chybrid sar esimila rt othos e described for diploid sexual hybrids of L. esculentum x L. peruvianum (de Nettancourte tal .1974 ;Maheswara ne tal .1986 ;Smit h1944 ;Thoma san dPrat t 1981). Ingeneral ,th ehexaploi dsomati chybrid swer esomewha tles svigorou stha n the tetraploid hybrids. The analysed hexaploid hybrids (6 out of 10) possessed more L. peruvianum than L. esculentum genome; this explains why they resembled L. peruvianum moretha nth etetraploi dhybrid s insevera lmorphologica laspects . 26

Many hybrid shoots showed a grey-green variegation. This may be similar to what isdescribe d as silvering,whitehea d orchimera ,whic h sometimes occurs in sometomat ogenotype so fnorth-wes tEurop ean dca nb einduce db ylo wtemperature s (Grimbly 1986). Thisdisorde rwa sno tobserve d inth e L. peruvianum régénérants, whereas it was in some explant régénérants of tomato (data not shown). I n the latterplant san d inth esomati chybrids ,thi sphenomeno nwa sapparentl y induced by thetissu e culture phasean dno tdu e toth ehybri dnatur e ofth eplants .Whe n thesymptom swer ever ysevere ,th eplan tdi dno tflowe ro rha ddeformed ,seedles s fruits, and irregularly shaped leaves; this so-called leaf distortion is also known tob e related to silvering (Grimbly 1986). The percentage of viable pollen of the somatic hybrids (27-51%) was comparable with that of the somatic hybrids ofKinsar a et al. (45%) (1986)an d lower than that of some sexual hybrids (77 to 90%) (Cappadocia and Sree Ramulu 1980; Maheswaran et al. 1986). Most of the selected somatic hybrids were self- compatible: 17 out of 22 tetraploid lines and 2ou t of 10hexaploi d lines.Th e hexaploids of Kinsara et al. (1986) were also self-compatible, whereas the diploid sexual hybrids of both species were self-incompatible (de Nettancourt et al. 1974;Maheswara n et al. 1986; Smith 1944;Thoma s and Pratt 1981). This can be due to the polyploid state of the somatic hybrids. Tetraploid L. peruvianum plants are self-compatible too,wherea s they are strictly self- incompatible asdiploids .Mos t somatic hybrid plants set small,mostl y seedless fruits. Parthenocarpy might result from an interspecific genomic combination and/or high temperatures (Stevens and Rick 1986), which were both the case in the present experiments (at summer on sunny days the temperature in the greenhouse could be above 40°C) . Themoderat e fertilityo fth e somatichybrid scoul db epromisin g fortransfe r of traits from L. peruvianum toL . esculentum.Althoug h the backcrosses of the hybrids to tomato were unsuccessful, many seeds derived from selfings. If segregation for species specific traits can occur in the latter progeny, it is conceivable that some of these plants are crossable with tomato.

Acknowledgements. This researchwa s supported by theFoundatio n for Fundamental Biological Research (BION),whic h issubsidise d by theNetherland s Organisation for Scientific Research (NWO). We thank Rijk Zwaan Seed Company for supplying Bellina seeds,Ja nMaasse nan dFran sMulckhuijs e forconstructin gan d optimising the electrofusion-equipment, Anja Posthuma, Janny Vos and René Rijken for assistance, Dr. S.D. Tanksley for providing the single copy clones,Dr .F.H.M . Derks for supplying DNA samplesan d Dr.J.H . de Jong,Dr .P . Zabel andProf . C. Heyting for critically reading of the manuscript. 27

References

AdamsTL ,Quiro sC F(1985 )Somati chybridizatio nbetwee nLvcopersico nperuvianu man dL .pennellii :regeneratin g ability and antibiotic resistance as selection systems.Plan t Science 40:209-219 Austin S,Bae rMA ,Helgeso n JP (1985)Transfe r ofresistanc e topotat o leafrol lviru s fromSolanu mbreviden s intoSolanu m tuberosumb y somatic fusion. Plant Science 39:75-82 AustinA ,Ehlenfeld tMK ,Bae rMA ,Helgeso nJ P (1986)Somati chybrid sproduce db yprotoplas t fusionbetwee nS . tuberosum andS .brevidens :phenotypi c variationunde r field conditions.Theo rApp lGene t 71:682-690 BarbanoPP ,Topolesk iL D(1984 )Postfertilizatio nhybri d seedfailur ei nLvcopersico nesculentu mx Lvcopersico n peruvianum ovules.J Ame r Soc HortSe i 109:95-100 Cappadocia M, SreeRamul uK (1980)Plan t regeneration from invitr o cultures of anthers and stem internodes in an interspecific hybrid, Lvcopersicon esculentum L.x L.peruvianu m Mill, and cytogenetic analysis of the regenerated plants.Plan t SeiLet t20:157-16 6 DellaportaSL ,Woo dJ ,Hick sJ B(1983 )A plan tDN Aminipreparation :versio nII .Plan tMolecula rBiolog yReporte r 1:19-21 De Nettancourt D, Devreux M, Laneri U, Cresti M, Pacini E, Sarfatti G (1974)Genetica l and ultrastructural aspects of self and cross incompatibility in interspecific hybrids between self-compatible Lvcopersicon esculentum and self-incompatible L.peruvianum . TheorApp lGene t 44:278-288 Dyer AF (1963)Th e use of lacto-propionic orcein in rapid squash methods for chromosome preparations. Stain technol 38:85-90 FishN ,Kar pA , JonesMG K (1988)Productio n of somatic hybridsb y electrofusion inSolanum . Theor ApplGene t 76:260-266 FrandsenN O (1968)Di e Piastidenzahl alsMerkma lbe ide rKartoffel .Theo rApp lGene t 38:153-167 FrearsonEM ,Powe rJB ,Cockin g EC (1973)Th eisolation ,cultur ean dregeneratio no fPetuni a leafprotoplasts . Dev Biol 33:130-137 GleddieS ,Kelle rWA ,Setterfiel d G (1986)Productio n and characterization of somatichybrid sbetwee nSolanu m melon«ena L.an d S. sisymbriifolium Lam. TheorApp lGene t 71:613-621 GritnblyP (1986 )Disorders .In :Atherto nJG ,Rudic hJ (eds )Th etomat ocrop .A scientifi cbasi sfo rimprovement . Chapman and Hall, London/New York,p p 369-389 Guri A, Levi A, SinkK C (1988)Morphologica l andmolecula r characterization of somatic hybrid plants between Lvcopersicon esculentum and Solanum nigrum.Mo lGe nGene t 212:191-198 Guri A, Sink KC (1988) Interspecific somatic hybrid plants between eggplant (Solanummelongena )an dSolanum , torvum. TheorApp lGene t 76:490-496 Handley LW, Nickels RL, Cameron MW, Moore PP, Sink KC (1986) Somatic hybrid plants between Lvcopersicon esculentum and Solanum lycopersicoides. TheorApp lGene t 71:691-697 Hogenboom NG (1973) Incongruity and incompatibility in intimate partner relationships. Ph.D. Thesis, Agricultural University Wageningen, theNetherland s ItohK , Futsuhara Y (1983)Restoratio n of the ability to regenerate shootsb y somatic hybridization between Petuniahybrid a andP . parodii. JapanJ Bree d 33:130-137 KinsaraA ,Patnai k SN,Cockin g EC,Powe rJ B (1986)Somati chybri d plantso fLvcopersico n esculentumMill ,an d Lvcopersicon peruvianum Mill. JPlan t Physiol 125:225-234 KoornneefM ,Hanhar tCJ ,Martinell i L (1987a)A geneti c analysis ofcel lcultur e traits intomato .Theo rApp l Genet 74:633-641 Koornneef M, JongsmaM , WeideR , Zabel P, Hille J (1987b)Transformatio n of tomato. In:Nevin s DJ, JonesR A (eds)Tomat obiotechnology . AlanR Liss, NewYork ,p p 169-178 KoornneefM ,va nDiepe nJAM ,Hanhar tCJ ,Kieboom-d eWaar tAC ,Martinell iL ,Schoenmaker sHCH ,Wijbrand iJ (1989 ) Chromosomal instability incell - andtissu e cultures oftomat ohaploid s anddiploids .Euphytic a 43:179-186 Maheswaran G, Perryman T, Williams EG (1986) Use of an interspecific hybrid in identifying a new allelic specificity generated atth eself-incompatibilit y locusafte r inbreeding inLycopersico nperuvianum . Theor ApplGene t 73:236-245 MaligaP ,Lâzâ rG ,Jo óF ,Nag yAH ,Mencze lL (1977 )Restoratio no fmorphogeni cpotentia li nNicotian ab ysomati c hybridisation.Mo lGe nGene t 57:291-296 Menczel L,Nag y F,Kis s ZR,Malig a P (1981)Streptomyci nresistan t and sensitive somatichybrid s ofNicotian a tabacum+ Nicotian aknishtiana : correlationo fresistanc e toN .tabacu mplastids .Theo rApp lGene t 59:191- 195 28

Murashige T, Skoog F (1962)A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473-497 O'CormellMA ,Hanso nM (1986)Regeneratio no fsomati chybri dplant sforme dbetwee nLycopersico nesculentu man d Solanum rickii.Theo rApp lGene t 72:59-65 Pijnacker LP,Ferwerd aM A (1984)Giems aC-bandin g ofpotat o chromosomes.Ca nJ Gene tCyto l 26:415-419 Puite KJ, Roest S, Pijnacker LP (1986)Somati c hybrid potato plants after electrofusion of diploid Solanum tuberosum andSolanu mphureia . PlantCel lRe p 5:262-265 RickC M (1982a)Th epotentia l ofexoti c germplasm fortomat o improvement. In:Vasi l IK,Scowcrof tWR ,Fre yK J (eds)Plan t improvement and genetics.Academi c Press,Ne w York,p p 1-28 Rick CM (1982b)Stoc k listRe pTomat oGene tCoo p 32:3-10 RickC M (1983)Tomat o (Lycopersicon).In :Tanksle y SD,Orto nT J (eds)Isozyme si nplan tgenetic san dbreeding , partB . Elsevier,Amsterdam , pp 147-165 ShahinE A (1985)Totipotenc y oftomat oprotoplasts .Theo rApp lGene t 69:235-240 SihachakrD ,Haicou rR ,Chapu tMH ,Barriento s E,Ducreu xG ,Rossigno l L (1989)Somati c hybrid plantsproduce d by electrofusionbetwee n Solanummelongen a L. and Solanum torvum Sw. Theor ApplGene t 77:1-6 SmithP G (1944)Embry ocultur eo fa tomat o specieshybrid . ProcAme r SocHor tSe i 44:413-416 SreeRamul uK ,Devreu xM ,Ancor aG ,Laner iU (1976)Chimeris mi nLycopersico nperuvianu mplant sregenerate dfro m invitr o cultureso f anthers and stem internodes.Z Pflanzenzücht g 76:299-319 StevensMA ,Ric kC M (1986)Genetic san dbreeding .In :Atherto nJG ,Rudic hJ (eds)Th etomat ocrop .A scientifi c basis for improvement.Chapma n and Hall, London/New York,p p 35-109 SuursLCJM ,Jongedij k E,Ta nMM C (1989)Polyacrylamid e gradient-gelelectrophoresis :a routin emetho d forhig h resolution isozyme electrophoresis ofSolanu m andLycopersico n species.Euphytic a 40:181-186 Tan MMC (1987) Somatic hybridization and cybridization in some Solanaceae. Ph D Thesis, Free University Amsterdam, theNetherlands ,p p49-6 6 Tanksley SD,Mille r JC,Paterso nA ,Bernatzk y R (1988)Molecula r mapping ofplan t chromosomes. In:Gustafso n JP,Appel sR (eds)Chromosom e structure and function.Plenu m Press,Ne wYork ,p p 157-173 Terada R, Yamashita Y, Nishibayashi S, Shimamoto K (1987) Somatic hybrids between Brassica oleracea and B. ramp^t-.ris - selectionb y theus e of iodoacetamide inactivation andregeneratio n ability. TheorApp lGene t 73:379-384 TewesA , GlundK , Walther R, Reinbothe H (1984)Hig h yield isolation and rapid recovery of protoplasts from suspension cultures of tomato (Lycopersicon esculentum). ZPflanzenphysio l 113:141-150 Thomas BR and Pratt D (1981) Efficient hybridization between Lycopersicon esculentum and L.peruvianu m via embryo callus.Theo rApp lGene t 59:215-219 Vallejos CE (1983)Enzym e activity staining. In:Tanksle y SD,Orto n TJ (eds)Isozyme s inplan t genetics and breeding,par tA . Elsevier,Amsterdam , pp 469-515 29

CHAPTER3

ANALYSI S OF PROGENIES DERIVED FROM SOMATIC HYBRIDS BETWEEN IYCOPERSICON ESCUIENTUM AND I^YCORERS ICON PERUVIANUM

J.Wijbrandi ,M .Posthuma ,M .Koornnee f

Summary. The fertility of tetraploid and hexaploid somatic hybrids between Lycopersicon esculentum, thecultivate d tomato,an dI . peruvianum wasanalyse d afterselfin gan dafte rbackcrossin gt obot hfusio nparents .Mos to fth ehybrid s werefertil eupo nselfin gan dyielde dman yseeds ,o fwhic h79 %germinated .Thes e progenyplant swer evigorou san dofte nfertil eafte r selfing.Backcrossin go f thesomati chybrid swit hth eI .esculentu mparen tdi dno tyiel dan yviabl eseeds ; backcrossing with L. peruvianum yielded afe w germinating seeds,bu t onlyi f L. peruvianum wasuse da sstaminat eparent .Th eplant sderive dfro mth ebackcros s hexaploidhybri dx tetraploi d L. peruvianum hada pentaploi dchromosom enumbe r (2n= 5 x= 60 )an dwer evigorous ,wherea sth eplant sderive dfro mth ebackcros s tetraploidhybri dx diploi dL .peruvianu mgre wretarded .T oobtai ninformatio n aboutth ebehaviou ro fchromosome si nth ehybrids ,th eprogenie so fth eselfing s and backcrosses were analysed for the segregation of several traits,namel y kanamycinresistance ,isozym e patterns foraci dphosphatase ,locu s Aps-1, and some morphological characteristics. Most of the progenies segregated for kanamycinresistanc ea sexpecte do nth ebasi so fth enumbe ro floc ipresen ti n thekanamyci n resistant fusionparent ,wherea si nsom eprogenie sth efractio n ofkanamyci nresistan tplant swa ssmalle rtha nexpected .Th esegregatio no fth e Aps-1 isozyme patterns and the variation for some of the morphological characteristicsindicate da tetrasomi cinheritanc eo fa tleas tpar to fth egene s inth ehybrids .Th eimplication so fthes efinding sfo rth eus eo fthes esomati c hybrids for the introgression of desirable I. peruvianum traits into the cultivatedtomat oar ediscussed .

Introduction

Somatic hybridisation is a technique for bringing together the genomes of sexually incongruent species. Most of the somatic hybrids that have been described arebetwee nspecie so fth esam egenu so rbetwee n specieso frelate d genera;som eo fthe mwer ecompletel ysteril e(e.g .Melcher se tal .1978 ;Gleddi e etal .1986) ,wherea sothe r (mostlyeuploid )hybrid swer efertil ean dcoul db e selfed or backcrossed to one or both of the parental species. Examples are hybridsi nth e Brassicaceae family(Sundber ge tal .1987 ;Fahleso ne tal .1988 ; Primarde tal .1988) , of Datura species (Schieder 1980), of Nicotians species (e.g. Smith et al. 1976;Evan s et al.1983 ;Hamil l et al. 1985), of Solanum tuberosum and S. brevidens (Ehlenfeldtan dHelgeso n1987) ,an do f L. esculentum and L. peruvianum (Kinsarae t al. 1986;Chapte r 2).Somati c hybridisation is potentially importantfo rcro pimprovement ,becaus ei tallow sth eintroductio n 30 of desired traits from related wild species into cultivated species in cases where sexual Hybridisation isno t possible. However,no t only the introduction ofdesire d traits,bu tals oth eremova l ofundesire d ones isimportant .Thi sca n be achievedb y repeatedbackcrossing , but onlyi fth e polyploidised hybrids are fertile when backcrossed with the cultivated parent, and if pairing and recombinationbetwee nth e relevanthomoeologou s chromosomes occur. Homoeologous pairing has been observed in low frequencies in tetraploid hybrids of Lycopersicon esculentum x L. pennellii and L. esculentum x Solanum lycopersicoides, obtained after sexual crossing and subsequent doubling of the chromosome number by colchicine (Rick and Khush 1962;Menze l 1964). Recently, we obtained somatic hybrids of L. esculentum, the cultivated tomato, and the wild species L. peruvianum (See Chapter 2).Thes e hybrids grew vigorouslyan dprove dt ob etetraploi do rhexaploid . Inth eexperiment sdescribe d inthi s Chapter,w e tested the fertility of thehybrid s after selfing or after backcrossing toon eo fth e parents.I naddition ,a provisiona l characterisation ofth eobtaine d progeny plantswa s carried out.Th e variation of several traits in the progeny of selfed hybrids was analysed in order to find out whether homoeologous pairing occurred during meiosis in the somatic hybrid plants.

Material and methods

Theisolatio nan dcharacterisatio no fth esomati chybrid swer edescribe d earlier (Chapter 2).Th ehybrid s that produced seeds,wer e classified infou rgroup s on the basis of the parental genotypes,a s shown inTabl e 1. Assay for kanamycin resistance Seedso f the somatichybrid s and theparenta l genotypeswer e surface sterilized (10 sec.70 %ethanol ,2 0min .5 xdilute d commercial bleach (10%NaCIO) , several washes in sterile water) and transferred to shoot culture medium (MS salts (Murashige and Skoog 1962), Tvitamin s (Tewes et al. 1984), 10g/ 1 sucrose) in plastic containers, in the dark. After a few days the seeds germinated and the containers were transferred to the light. Shoot tips of the seedlings were transferred to shoot culture medium supplemented with 100mg/ 1 kanamycin. Resistant shoots formed roots and grew, whereas sensitive shoots did not form roots and died. The remainder of the seedlings was transferred to soil in a greenhouse.I nsevera l cases,seed so fth eparenta lgenotype swer e sowndirectl y onshoo tcultur emediu msupplemente dwit h10 0mg/ 1kanamycin ;resistan tseedling s developed normally, whereas sensitive seedlings were retarded in growth and showed poor root development.

Isozyme analysis Leaf material from greenhouse-grown plants was used for the analysis of acid phosphatase, locus Aps-1. Crude extracts,whic hwer e obtained by the squeezing of thawing leaf material that had been stored at -80 °C, were electrophoresed on vertical 10% Polyacrylamide slab gels (Chapter 2). Enzyme activity was determined by means of the staining technique of Vallejos (1983). Plant characterization Chromosomenumber so froo tti pcell swer edetermine d incel lsprea d preparations 31

Table 1. The parental genotypes of the somatic hybrids,fro m whichprogen y was analysed; the hybrids were subdivided on the basis of their ploidy level. The numbers of different shoots,derive d from individual somatic hybrid calli, are givenbetwee nparentheses .Al lsomati chybrid swer eobtaine dafte r selectionfo r kanamycin resistance and regeneration capacity, except for the only hybrid of Bellina (+)ATW2002 ; the latter hybrid was selected for callus colour and regeneration capacity. The hexaploid somatic hybrids all contained a diploid genome of L. esculentum and a tetraploid genome of L. peruvianum.

L. esculentum L. peruvianum Somatic hybrids tetraploid hexaploid

cv. Bellina-ATW3001** PI128650 5 (9) 1 (1)

cv. Bellina-ATW3003** PI128650 7 (13) 2 (2)

LA1182*-ATW3052** PI128650 2 (2) 1 (2)

cv. Bellina PI128650-ATW2002** 1 (1) 0

tomatogenotype ,homozygou srecessiv e for sy, sf (chr. 3),an d alb (chr. 12) (Rick 1982). transgenic plants containing theneomyci n phosphotransferase IIgene ,whic h causes resistance to the antibiotic kanamycin (Koornneef et al. 1987).

(Chapter 2).I n addition, growth habit and leaf morphology were analysed, and sympodial index (=mea nnumbe r ofnode s betweentw osubsequen t inflorescences), size of the different flower parts, pollen viability, fruit and seed set were determined. Pollen was stained in a solution of lactophenol acid fuchsin to establish their viability; at least 100 pollen grains per flower were scored.

Results

Progeny of the somatic hybrids

The somatic hybrids were selfed and crossed with diploid and tetraploid plants of L. esculentum and L. peruvianum, both as pistillate and staminate parents (Table 2).Th e selfings,eithe r spontaneous orcontrolled ,yielde d manynormal - looking seeds in most of the somatic hybrids (See Table 2 in Chapter 2).Th e crosses with L. esculentum as pistillate parent yielded fruits that contained several toman yabortiv e seeds.Th ebackcrosse s to L. peruvianum only succeeded whenth e somatichybrid swer euse da spistillat e parent.75 %t o90 %o fth e seeds germinated (Table2) .

Segregation of kanamycin resistance

Table 3show sth esegregatio nratio so fkanamyci nresistance/sensitivit y forth e offspring of the kanamycin resistant fusion parents (X. esculentum and 32

Table2 . The results of the crosses with the somatic hybrids (SH) of L. esculentum (Le)an d L. peruvianum (Lp), andth efrequenc yo fgerminatio no f theproduce d seeds.Th eploid yleve lo fth eplant si sgive nbetwee nbrackets . Thenumbe ro fdifferen tshoots ,derive dfro mth eindividua lsomati chybri dcalli , aregive nbetwee nparentheses .On ecros srepresent son epollinate dflower .Th e 828seed so f"SH[4x ]selfed "wer ederive dfro m1 7hybri dclone s (27differen t shoots);th eseed sfro m1 5(23 )wer etested .Th eseed so f"SH[4x ]x Lp[2x] "wer e derivedfro m3 clone s( 3shoots) , "SH[4x]x Lp[4x] "fro m2(2) ,"SH[6x ]selfed " from2 (3 )an d "SH[6x]x Lp[4x] "fro m3 (3) .

Crosses No.o fhybrid s No.o f No.o f No.o f Germinating 9x 6 (shoots) crosses fruits seeds seeds Le[2x xLp[2x ] - 9 4 2* 0% Le[2x xLp[4x ] - 1 1 0 Lp[2x xLe[2x ] - 52 0 Lp[4x xLe[2x ] - 9 0 SH[4x selfed 21 (45) ND 333 828 79%* SH[4x xLe[2x ] 21 (40) 264 7 0 SH[4x xLe[4x ] 12 (15) 53 0 SH[4x xLp[2x ] 18 (24) 71 32 4* * 75% SH[4x xLp[4x ] 14 (19) 37 4 rx ** 0% Le[2x xSH[4x ] 16 (21) 50 18 0 Le[4x xSH[4x ] 9 (10) 30 7 1* 0% Lp[2x xSH[4x ] 10 (13) 26 1 0 Lp[4x xSH[4x ] 2 (2) 3 0 SH[6x selfed 9 (14) ND 32 37 84%* * SH[6x xIe[2x ] 7 (11) 71 3 0 SH[6x xLe[4x ] 6 (7) 25 0 SH[6x xLp[2x ] 6 (6) 17 0 SH[6x xLp[4x ] 5 (6) 10 3 20 90% Le[2x xSH[6x ] 5 (6) 14 2 0 Le[4x xSH[6x ] 3 (3) 10 1 0 Lp[2x xSH[6x ] 1 (1) 2 0 ND notdetermined ,becaus emajorit yo ffruit swa sse tspontaneousl y verysmal lfla tseed s flatseed s * 376seed swer eteste d ** 32seed swer eteste d

L. peruvianum) ando fth esomati chybrid shavin gon ekanamyci nresistan tparen t (either L. esculentum or L. peruvianum; Table 1).Th e resistant parentswer e obtainedb ylea fdis c transformation (Koornneefe tal . 1987)o f L. esculentum cv.Bellina ,o fL . esculentum LA1182an do fth e L. peruvianum accessionPI128650 . The segregation ratio for the progeny of the selfed kanamycin resistant L. esculentum genotypeATW300 1i s3 resistan t :1 sensitive ,whic hi sconsisten t withon ehemizygou skanamyci n resistance locus inthi sgenotype .Accordingly , thesegregatio nrati ofo rth eprogen yo fth eselfe dtetraploi dhybrid sdescendin g fromthi sgenotyp ei sals o3:1 .Th eprogen yobtaine db yselfin go fATW300 3als o showed a ratio of 3:1. However, the ratio for the progeny of the selfed tetraploidhybrid sdescendin gfro mATW300 3deviate dsignificantl yfro m3:1 ;th e 33

Table 3.Segregatio nratio so fkanamyci nresistanc e inseedling so fth eorigina l ATW-plants and of the somatic hybrids thatha d aATW-plan t as parent. TheATW - plantso f L. esculentum (Le),ATW3001 ,ATW300 3an dATW3052 ,wer e selfed,wherea s the L. peruvïanum (Lp) ATW2002wa scrosse dwit ha kanamyci nsensitiv e plant.Th e somatic hybrids (SH)wer e selfed or backcrossed withL . peruvianum. The ploidy level of the plants isgive n betweenbrackets . KmR, kanamycin resistant; Kms, kanamycin sensitive.

Crosses Kanamycin resistant parental genotype ATW3001 ATW3003 ATW3052 ATW2002 KmR:Kms KmR:Kms KmR:Kms KmR:Kms

Le[2x]R selfed 32:8* 36:13* Lp[2x]R x Lp[2x]s 63:18*

SH[4x]R selfed 92:37* 73:41* 4:0 33:1* * SH[4x]R x Lp[2x]s 1:0 SH[6x]R selfed 2:0 12:8 SH[6x]R x Lp[4x]s 1:2 2:4 4:4

kanamycin resistant plant kanamycin sensitive plant segregation ratio does not deviate significantly from 3:1 (x2 < ^(l, 0.95) = 3.84); consistent with the presence of one locus of the kanamycin resistance (NPT II)gen e segregation ratio deviates significantly from 3:1 (x2 = 7.31) segregation ratio does not deviate significantly from 3:1; consistent with twounlinke d NPT II loci segregation ratio doesno tdeviat e significantly from 15:1;consisten t with twounlinke d NPT II loci fractiono fkanamyci nresistan t plantswa s smaller thanexpecte d (Table 3).Th e testcross of the kanamycin resistant L. peruvianum parent ATW2002 yielded a progeny with a segregation ratio of 3resistan t :1 sensitive,whic h indicates the presence of two unlinked hemizygous kanamycin resistance loci in this genotype. The observed ratio of 33:1 in the progeny obtained by selfing of the single somatic hybrid descending fromATW200 2 is inagreemen t with this (Table 3). We could only harvest a few seedless fruits from ATW3052; the segregation ratioso fth eprogen yo fth ehybrid sdescendin gfro mthi sgenotyp ei sconsisten t withth epresenc e ofon ehemizygou skanamyci nresistanc elocu si nthes ehybrids .

Analysis of acid phosphatase-1

The different Aps-1 isozyme patterns of the analysed genotypes are represented in Fig. 1 (See also Chapter 2): L. esculentum had one fast migrating band (pattern E); L. peruvianum had two different patterns, either three slow migrating bands (patternA )o r one band similar to the L. esculentum band, but much darker (pattern B); the somatic hybrids had, depending on their I. peruvianum parent, either one band like pattern B or a pattern of six bands which can be interpreted as a combination of the L. esculentum band, pattern A 34

Aps- 1 Lp Lp hybrids A B E+A (E+)A+B

Fig.1 .Representatio no fth edifferen tpattern so f acid phosphatase-1 foundi n L. esculentum (Le), L. peruvianum (Lp) , their somatic hybrids and the sexual progenyo fth elatter . Le hason eband ,patter nE ,whil e Lpha stw odifferen t patterns,eithe r three bands (patternA )o r one heavy band (patternB ). Fou r different hybrid patterns were distinguished: (i) combination of E and A, resultingi n6 bands ;(ii )combinatio no fE an dpar to fA ,resultin gi n3 bands ; (iii)combinatio nof E+ A + Bor A +B ,resultin gi n3 stron gan d3 wea kbands ; (iv)combinatio no fE + par to fA + B o rpar to fA + B ,resultin g in3 stron g bands.Not etha tpattern sE an dB diffe ronl yi nintensit yo fth eban dan dtha t thecombinatio no fE an dB i na hybri di sno tdifferen tfro mpatter nB .

Table4 .Isozym epattern so faci dphosphatase ,locu s Aps-1, insexua lprogenie s of L. esculentum (Le), L. peruvianum (Lp) and their somatic hybrids (SH);th e ploidy level of the selfed or (back)crossed plants is given between square brackets.Th eisozym epattern so fth eparent sar eshow nbetwee nparentheses .Se e Fig.1 fo rexplanatio no fth epatterns .Th esecon dcolum ngive sth edesignation s of the individual somatic hybrid OH-plants; thenumber s indicate independent hybridclone san dth elowe rcas eletter sindependen tshoot sfro mth esam eclone .

Crosses (Aps-1) Hybridplant s Aps-1 patterno fprogen y Le(E) hybrid Lp(A) Lp(B) Le[2x](E)selfe d 12 Lp[2x](A)x Lp[2x](B ) 31 SH[4x](EA)selfe d 0H4a,b,c,d,e; 101 0H16a;0H17a ; 0H18a;0H19 c SH[4x](EB)x Lp[2x](? ) 0H6a;OHIO c SH[6x](EAB)selfe d 0H14a,b 15 SH[6x](EAB)x Lp[4x](B ) 0H14b 5 Segregationrati odoe sno tdeviat e significantlyfro m1:34:1 ; X2- 3.3 5< x 2(2,0.95 )= 7.8 2 35 of L. peruvianum and two intermediate bands. In several cases two bands of patternA an d one intermediate bandwer e lacking inth ehybri d pattern;hybrid s of bothI . peruvianum types also showed patterns of six or threebands . The segregation of Aps-1 patterns among the progenies is given for each ploidyleve lan d parental patterntyp e (Table 4).Th e offspring of the parental genotypes had patterns as expected. In 4 out of 105 tested progeny plants of selfed tetraploid somatic hybrids having ahybri d pattern of 6bands ,w e found theL . peruvianum specific patternA . The 101othe r progeny plantsha d ahybri d pattern.Althoug hw edi dno t find theL . esculentum specific pattern,th e ratio of Aps-1 patterns in this progeny did not deviate significantly from a segregationrati oo f1:34: 1(Tabl e 4),whic hwa sexpecte di ncas eo fa tetrasomi c inheritance of^ips- l in thesehybrids . The isozyme patterns of twoplants ,eac h ofwhic h resulted from a backcross ofa tetraploi d hybrid (pattern B)wit ha diploi d L. peruvianum plantwer e also analysed:on eha dpatter n B,wherea sth eothe rshowe da hybri dpatter n(Tabl e4) . The latter pattern indicates that at least one of both plants derived from a backcross and not from an unintended selfing ; the progeny of the selfed tetraploidhybrid swit hpatter n Bal lals oha dpatter n B (43plant stested ;dat a not shown). We also analysed the segregation of the Aps-1 pattern in the progeny of hexaploid hybrid 0H14, which probably contains one diploid genome of L. esculentum, one of L. peruvianum (patternA ) and one of L. peruvianum (pattern B) (Chapter 2).Mos t of the progeny plants resulting from selfing of this hybrid had a hybrid pattern, while a few had pattern B (Table 4).A s expected,th eprogen yfro mth ebackcros so f0H1 4wit ha tetraploi dL . peruvianum (pattern B), showed a larger proportion of pattern B than the progeny derived from selfing (Table4) .

Horphology

Theprogen yplant so fth eselfe dhybrid san do fth ebackcrosse s "SH[6x]x Lp[4x]" werever yvigorou swit hlarg eleave san dflowers ,simila rt oth esomati chybrids . Theexpecte dpentaploi dchromosom enumbe ro fthes ebackcros splant swa sconfirme d by chromosome counts of 5plant s (atleas t oneplan t of each three crosses)ou t of 15grow n in the greenhouse; all had 60 chromosomes. Two plants derived from the cross "SH[4x]x Lp[2x]" grew slowly and had small leaves and small flowers with very exerted styles. Inman y of the somatic hybrids variegation ofgrey-gree n spotswa s observed, whichresemble dth edisorde rcalle dchimer ao rsilvering ,occurrin gi nglasshous e tomatoes (Grimbly 1984). The progenies of several of these hybrids showed the same symptoms (Table 5);thi s indicates agenetica l basis of the disorder. 36

Table 5. The presence of grey-green variegation, "chimera", in progeny plants ofsomati chybrid s (SH)o f L. esculentum and L. peruvianum (Lp) ;th eploid yleve l of the selfed or (back)crossed plants isgive nbetwee nbrackets .Th e individual somatic hybrid plants (OH)ar e added; the numbers indicate independent hybrid clones and the lower case letters independent shoots from the same clone. The symptoms of "chimera" in the hybrids are indicated; severe symptoms are the presence of large grey sectors and irregularly shaped leaves. +, "chimera" symptoms; -,n o symptoms.

Crosses of somatic hybrids "Chimera" "Chimera" seedlings symptoms

SH[4x] selfed: OHlb; 0H4c,e; 0H6c; 0H8a,d; 0H13a; 160 0H15b; 0H16a; 0H18a,b; 0H19c; 0H24c; 0H31a; 0H36a 0H4a; OHIOb; 0H17a; 0H24a moderate 4 47 0H4b; 0H25a,c severe 39 0

SH[4x]x Lp[2x]: OHIOc no 0H6a severe SH[6x] selfed: 0H14a,b moderate 18 0H19a severe 0

SH[6x]x Ip[4x]: 0H7c; 0H14b; 0H27a moderate

For a few characters, e.g. the sympodial index (s.p.i.), we observed more variation among the progeny of the tetraploid somatic hybrids than among these hybrids themselves (Fig. 2A).Th e means of the s.p.i. of the progeny plants (2.83)di d not differ significantly from that of the hybrids (2.86), but the variances (<72;0.36 4an d0.118 ,respectively )di d (variance ratiotest : F= 3.0 8 > F(65, 38,0.95 ) = 1.65). A similar result was obtained for the sizes of the flower parts; e.g. the average ratios of the lengths of sepals and petals (Fig. 2B) was 0.43 (CT= 0.06) for the tetraploid somatic hybrids and 0.44 (a= 0.11) for the progeny obtained by selfing of these hybrids. The variances ofthi srati ower esignificantl ydifferen tfo rbot hexperimenta lgroup s (F= 2.65 > F(56, 30,0.95 ) = 1.75)

Fertility

The average pollen viability of the progeny plants obtained by selfing of the somatichybrid swa ssimila rt otha to fthes ehybrids ,namel y30 %t o50 %(Se eals o Chapter 2).Onl y thetetraploi d progeny plants setmatur e fruits (Table 6).Th e small, often parthenocarpic fruits were usually yellow; one plant had pink- yellow fruits. Three progeny plants had fruits with much purple pigmentation, 37 A B Sympodial index Sepals/petals 30-

SH[4x] 20- to -M § 10- Q_

O 0_.

0 SH[4x] |20. selfed -5

10-

0, —i r 12 3 4 0.2 0.4 0.6 0.8 Mean number Ratio of lengths of nodes Fig.2 . Distribution of some morphological characteristics among tetraploid somatichybrid s (SH[4x])o f L. esculentum and L. peruvianum andthei rprogen y afterselfin g(SH[4x ]selfed) .A .Sympodia linde x( =mea nnumbe ro fnode sbetwee n two subsequent inflorescences).B . Ratio between the lengths of sepals and petals.

Table6 . The presence of flowers,fruit san d seeds inth e progeny ofsomati c hybridso f L. esculentum and L. peruvianum. Thesomati chybrid s (SH)ha dbee n selfedo rbackcrosse dwit h L. peruvianum (Lp); theploid yleve lo fth eparenta l plants isgive nbetwee n brackets.Observation swer e made ongreenhouse-grow n plants.Th efrui tan dsee dyiel d(afte rselfing )o fth e"SH[4x ]selfed "progen y was19 4an d348 ,respectively .

Progenyplant s Total Flowering Settingfruit s Setting (+buds) (+immature) seeds SH[4x]selfe d 70 68 24(+13 ) 23 SH[4x]x tp[2x ] 2 2 0 SH[6x]selfe d 8 4 (+3) 0 (+1) SH[6x]x Lp[4x ] 15 12(+2 ) 0

like L. peruvianum fruits.Trusse swit hman yfruit s( >10 )wer eobserve do non e plant. Twenty-three progeny plants set seeds, in general after spontaneous 38 seifing.Backcrosse swit hdiploi d L. esculentum as staminate parent wereno t successful.

Discussion

Oneo fth efactor stha tar eimportan tfo rth esuccessfu lapplicatio no fsomati c hybridisationt oth eimprovemen to fcrop si sth efertilit yo fth ehybrid swhe n theyar eselfe do rbackcrosse d toth ecultivate dparent .Th eresult spresente d inthi spape rsho wtha tsomati chybrid sbetwee nL .esculentu man d L. peruvianum arefertil ewhe nselfed .Backcrosse so ftetraploi dhybrid sa spistillat eparen t todiploi d L. peruvianum didsucceed ,wherea sthos et odiploi d L. esculentum did not. The backcrosses of tetraploid hybrids as staminate parents to diploid L. peruvianum didno tsucceed ,whil ethos et odiploi dL .esculentu monl yyielde d abortive seeds.Thes e results are in agreement with those obtained by Soost (1958)an dSztey n(1962) ,wh oisolate dallotriploi dsexua lhybrid sfro mcrosse s oftetraploi d L. esculentum withdiploi d L. peruvianum. Theyfoun dtha tcrosse s ofthes eallotriploid sa spistillat e parentst odiploi dL .peruvianu myielde d viable offspring,bu tthos et odiploi d L. esculentum didnot .However ,Sztey n (1962)foun d that ifth eallotriploid swer euse da sstaminat eparents ,viabl e offspringcoul db eobtaine dfro mcrosse swit hdiploi d L. esculentum. Allodiploids of L. esculentum xL .peruvianu m canals o bebackcrosse d to L. esculentum as pistillate parent (Ancora etal . 1981). We found that thebackcrosse s ofth e tetraploidhybrid st oL .esculentu ma spistillat eparen tyielde dsevera lfruit s withman y abortive seeds. It isquit e possible that if such backcrosses are repeated on a larger scale, viable triploid offspring will be obtained. Subsequent backcrosses of these triploids to diploid L. esculentum are then probablyrelativel yeasil yobtaine d (Rickan dButle r 1956). Another important factor is the occurrence of homoeologous pairing and recombinationi nth esomati chybrids .Ou rresult sprovid esom eindication sfo r tetrasomic inheritance of several traits inth ehybrids .Th evariance s ofth e s.p.i.an do fth erati oo fsepa lan dpeta llength swa ssignificantl ylarge ramon g theprogen yo fth eselfe dtetraploi dhybrid stha namon gth ehybrid sthemselves . Withrespec tt oothe rtraits ,suc ha s Aps-1 andfrui tcolour ,phenotype swer e presentamon gth eprogen yo fth e selfingstha twer eabsen tamon gth eparenta l hybrids.Wit hrespec tt oth emorphologica l characteristics,thes eresult sca n beattribute da tleas ti npar tt oheterozygosit yo fth eparent so fth ehybrids :

L. esculentum cv.Bellin ai sa nF xhybri dan d L. peruvianum isa ver yvariable , outbreedingspecies .Bu tthi sexplanatio ndoe sno thol dfo rth esegregatio no f Aps-1 isozymepattern san dfo rth eappearanc eo fpurpl epigmente dfruit si nth e 39

progeny of the selfed hybrids.W e therefore tentatively conclude that at least Aps-1, butprobabl y other markers aswell ,hav e a tetrasomic inheritance inth e hybrids.Thi simplie stha tth erelevan thomoeologou schromosome sprobabl ypaire d in the tetraploid somatic hybrids.A more detailed analysis of the segregation of monogenic inherited markers, and of the behaviour of chromosomes at metaphase Ii n thehybrid s isrequire d to support this conclusion,however . The kanamycin resistance marker (or NPT II locus)wa s present as one or two unlinked hemizygous loci in one of the parents of the hybrids. This marker segregated as expected among the progeny of most of the selfed or backcrossed kanamycin resistant hybrids. However, if tetraploid hybrids descending from ATW3003 (withon ehemizygou skanamyci n resistance locus)wer e selfed,th e ratio ofresistan tt osensitiv eprogen ydiffere d significantlyfro m3: 1an dwa s closer to 2:1.Thi sdeviatin g ratio canb e explained by themor e frequent transmission of the corresponding L. peruvianum chromosome than of the L. esculentum chromosome containing theNP T IIlocus .Deviatin g segregationratio so f several

traits were also observed in F2 generations of sexual hybrids of tomato and I. pennellii (Bernatzky andTanksle y 1986;Gadis han d Zamir 1987)an d of tomato and L. chilense (Rick 1963), all in favour of characters of the wild species. Inbackcros s progenies of tomato species hybrids deviating segregation ratios in either direction have been observed (Rick 1963,1969 ;Vallejo s and Tanksley 1983; Rick et al. 1988). The phenomenon was ascribed to differential survival ofth emal egamete so rt odifferentia l zygoticlethalit y (Rick1963 ,1969 ;Gadis h and Zamir 1987). Both explanations are conceivable for our results. The good fertility of some progeny plants derived from selfed tetraploid somatic hybrids and the assumed tetrasomic inheritance of some traits, is an indication that such plants might be used for introgression of L. peruvianum specific characters into the cultivated tomato. This probably can be achieved more efficiently by the pre-selection of fertile progeny plants with some L. esculentum specifictrait san dth esubsequen tcrossin go fthes eprogen yplant s with L. esculentum.

Acknowledgements. This researchwa s supported by theFoundatio n for Fundamental Biological Research (BION),whic h issubsidise d by theNetherland s Organisation for Scientific Research (NWO). We thank Corrie Hanhart, Patty van Loenen Martinet-Schuringa and René Rijken for doing part of the experiments,Dr . J.H. de Jong and Prof. C. Heyting for critically reading of the manuscript.

References

AncoraG ,Saccard oF ,Cappadoci aM ,Sre eRamul uK (1981)Backcros sprogenie s fromLvcopersico nesculentu mL .x hybrid (L.esculentu m xL .peruvianu m Mill.). ZPflanzenzücht g 87:153-157 40

Bernatzky R,Tanksle yS D (1986)Majorit yo frando m cDNAclone scorrespon d tosingl eloc i inth etomat ogenome . MolGe nGene t 203:8-14 Ehlenfeldt MK, HelgesonJ P (1987)Fertilit y of somatic hybrids from protoplast fusions of Solanum brevidens andS . tuberosum. TheorApp lGene t 73:395-402 EvansDA , Bravo JE,Ku t SA, Flick CE (1983)Geneti c behaviour of somatic hybrids inth e genus Nicotiana: N. otophora+ N . tabacum andN . sylvestris +N .tabacum . Theor ApplGene t 65:93-101 FahlesonJ ,Râhlé nL ,Glimeliu sK (1988)Analysi so fplant sregenerate dfro mprotoplas tfusion sbetwee nBrassic a nanus andEruc a sativa. TheorApp lGene t 76:507-512 Gadish I,Zatni rD (1987)Differentia l zygotic abortion ina ninterspecifi c Lvcopersicon cross.Genom e29:156 - 159 Gleddie S,Kelle rWA ,Setterfiel d G (1986)Productio n andcharacterizatio n of somatic hybridsbetwee n Solanum melongena L.an d S. sisymbriifolium Lam.. TheorApp lGene t 71:613-621 Grimbly P (1986) Disorders. In: Atherton JG, Rudich J (eds), The tomato crop. A scientific basis for improvement.Chapma n andHall , London/New York,p p 369-389 Hamill JD,Penta l D, Cocking EC (1985)Analysi s of fertility in somatic hybrids of Nicotiana rustica andN, . tahartiman dprogen y over two sexual generations. TheorApp lGene t 71:486-490 KinsaraA ,Patnai k SN,Cockin g EC,Powe rJ B (1986)Somati c hybridplant so fLycopersico n esculentumMill ,an d Lvcopersiconperuvianu m Mill.. JPlan t Physiol 125:225-234 Koornneef M, Jongsma M, Weide R, Zabel P, Hille J (1987) Transformation of tomato. In:Nevin s DJ, Jones RA (eds)Tomat obiotechnology . AlanR Liss, Inc,Ne wYork ,p p 169-178 MelchersG , SacristanMD ,Holde r AA (1978)Somati c hybrid plantso f potato andtomat o regenerated from fused protoplasts.Carlsber gRe sCommu n 43:203-218 Menzel MY (1964) Preferential chromosome pairing in allotetraploid Lvcopersicon eseulentum-Solanum lycopersicoides.Genetic s 50:855-862 Murashige T, Skoog F (1962)A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant15:473-497 . Pijnacker LP,Ferwerd aM A (1984)Giems aC-bandin g ofpotat o chromosomes.Ca nJ Gene tCyto l 26:415-419 Primard C, Vedel F, Mathieu C, Pelletier G, Chèvre AM (1988) Interspecific somatic hybridization between Brassicananu s andBrassic ahirt a (Sinapsis alba L.). TheorApp lGene t 75:546-552 Rick CM (1963)Differentia l zygotic lethality ina tomat o specieshybrid .Genetic s 48:1497-1507 Rick CM (1969) Controlled introgression of chromosomes of Solanum pennellii into Lycopersicon esculentum: segregation and recombination.Genetic s 62:753-768 Rick CM (1982)Stoc k list.Re pTomat oGene tCoo p 32:3-10 Rick CM,Butle r L (1956)Cytogenetic s of the tomato.Ad vGene t8:267-38 2 Rick CM, ChetelatRT , DeVerna JW (1988)Recombinatio n in sesquidiploid hybrids of Lvcopersicon esculentum x Solanum lycopersicoides andderatives .Theo rApp lGene t 76:647-655 Rick CM,Khus hG S (1962)Preferentia lpairin g intetraploi d tomato specieshybrids .Genetic s 47:979-980 Schieder0 (1980)Somati chybrid so fDatur ainnoxi aMill .+ Datur adiscolo rBernh .an do fDatur ainnoxi aMill . + Datura stramonium L.var .tatul aL . II.Analysi so fprogenie s ofthre e sexualgenerations .Mo lGe nGene t 179:387-390 SmithHH ,Ka oKN ,Combatt iN C(1976 )Interspecifi chybridizatio nb yprotoplas tfusio ni nNicotiana .Confirmatio n and extension.J Heredit y 67:123-128 SoostR K (1958)Progenie sfro msesquidiploi dF jhybrid so fLvcopersico nesculentu man dL .peruvianum .J Heredit y 49:208-213 Sundberg E,Landgre nM ,Glimeliu sK (1987)Fertilit y and chromosome stability inBrassic anapu sresynthesise d by protoplast fusion. TheorApp lGene t75:96-10 4 SzteynK (1962)Interspecifi c crosses inth e genusLvcopersicon . I.Backcrosse s to Lvcopersicon glandulosum. Euphytica 11:149-156 TewesA ,Glun d K, Walther R, Reinbothe H (1984)Hig hyiel d isolation and rapid recovery of protoplasts from suspension cultures of tomato (Lycopersicon esculentum). ZPflanzenphysio l 113:141-150 Vallejos CE (1983)Enzym e activity staining. In:Tanksle y SD,Orto n TJ (eds)Isozyme s inplan t genetics and breeding,par tA . Elsevier,Amsterdam ,p p 469-515 VallejosCE ,Tanksle yS D(1983 )Segregatio no fisozym emarker san dcol dtoleranc ei na ninterspecifi cbackcros s of tomato. TheorApp lGene t 66:241-247 41

CHAPTER4

ASYMMETRIC SOMATIC HYBRIDS BETWEEN I.Y COPE RS ICON ESCUIENTUM AND IRRADIATED IYCOPERSICON PERUVIANZJM I. CYTOGENETICS AND MORPHOLOGY

J. Wijbrandi,A . Posthuma, J.M. Kok,R . Rijken, J.G.M.Vos ,M . Koornneef

Summary.Asymmetri csomati chybrid so f Lycopersicon esculentum and Lycopers icon peruvianum were obtained by fusion of leaf protoplasts from both species after irradiation ofprotoplast s orlea f tissue of L. peruvianum with 50,30 0o r 1000 Gy ofgamma-rays .Thes e irradiationdose swer e sufficient toabolis hth e growth of theL . peruvianum protoplasts.Th ehybrid swer e selected onthei rabilit y to regenerate plants; this regeneration capacity derived from L. peruvianum. All asymmetrichybri dplant swer eaneuploid .Th eploid ylevel ,th emorpholog ya swel l as the regeneration rate were analysed in relation to the irradiation dose, appliedt oL . peruvianum.Afte ra lo wdos e (50Gy )mos thybrid sha dnear-triploi d chromosome numbers,wherea s after ahig hdos e (300o r 1000Gy )mos thybrid s had near-pentaploid numbers. The morphology of the asymmetric hybrids was intermediatebetwee ntha to f L. esculentum andsymmetri c somatichybrid so fbot h species (obtainedwithou t irradiation treatment),an dapproache d the morphology of L. esculentum more after a highdos e of irradiation. The asymmetric hybrids regenerated more slowly than the symmetric hybrids,an d regeneration proceeded more slowlyafte ra hig hdos etha nafte ra lo wdos eo firradiation .Th ehig hdos e hybrids also grew more slowly, flowered less and set fruits less than the low dose hybrids.N o seeds could be obtained from any asymmetric hybrid.

Introduction

The transfer of desirable traits from wild into cultivated species isa method to improve crops.Fo r thispurpose , interspecific sexual or somatic hybridsma y be constructed. The formation of sexual hybrids is limited to closely related species. Symmetric somatic hybrids,obtaine d byfusio no funtreate d protoplasts of different species, can be more easily made in some combinations. However, species hybrids, both sexual and somatic, contain many unwanted traits of the wild species besides the desired ones, and are often sterile. Fertility is required to perform several backcrosses with the cultivated species to remove unwanted characters of the wild species.Thes e problems may be circumvented by asymmetric somatichybridisation .B y thisprocedur e protoplasts ofon especies , the recipient, are fused with inactivated (mostly by Röntgen- or gamma- irradiation) protoplasts of another species, the donor. The donor genome will befragmente d andth easymmetri chybrid swil lcontai nth ecomplet egenom eo fth e recipient species and a small part of the donor genome. The advantages of this 42 procedurecoul db etha thybrid saris ewit hfe wunwante ddono rtraits ,an dtha t fewerbackcrosse so fth easymmetri chybrid sar erequire dt oge tri do fthese . Several asymmetric hybrids were obtained in other studies (Chapter 1).Th e fractiono fdono rgenom e thatwa stransferre dvarie d fromon eo ra fe wtrait s (e.g. Duditse tal .1987) ,on eo ra fe wchromosome s(e.g .Gupt ae tal .1984 )t o many chromosomes (e.g. Gleba et al. 1988). Fertile asymmetric hybrids were reported insevera l casesan d irrespective ofth eamoun to ftransferre ddono r genome (Chapter1) . We are interested in the transfer of traits of the wild tomato species Lycopersicon peruvianum to the cultivated tomato, Lycopersicon esculentum. L. peruvianum hasman ydesirabl echaracter sfo rtomat oimprovemen t(Ric k1982a) , butonl ya fe wo fthes e(e.g . Mi- andTMV-resistance )hav ebee nintroduce dint o thecultivate dtomato ,becaus esexua lhybrid so fbot hspecie sar ever ydifficul t to obtain (Taylor 1986). In the present experiments we obtained asymmetric somatichybrid sb yfusin gL .escuientu mprotoplast swit hirradiate d L. peruvianum protoplasts.Th ehybrid swer e selected for thedominan t regenerationcapacit y charactero fL .peruvianu m(Koornnee fe tal .1987a ;Chapte r 2).Ou rpurpos ewa s toanalys eth ecytogenetics ,morpholog yan dfertilit yo fth eregenerate dhybrid s inrelatio nt oth eirradiatio ndose ,applie dt o L. peruvianum.

Materialsan dmethod s Plant materials Asrecipien t (Lycopersicon esculentum) theDutc hhybri dcultiva rBellin a(kindl y providedb yRij kZwaa nSee dCompany ,d eLier ,Th eNetherlands )an dth egenotype s LA291,LA1164 ,LA1166 ,LA1182 ,LA1189 ,LA144 4an dLA166 5fro mth eTomat oGenetic s StockCente ri nDavis ,US A (kindlyprovide db yProf .CM . Rick)wer eused .Th e latter genotypes allow observations on the complementation of monogenic morphological mutant phenotypes by the donor (Chapter 5). Plants from the Lycopersicon peruvianum accession PI128650 (received from the Institute of Horticultural PlantBreeding ,Wageningen ,Th eNetherlands )wer euse da sdonor . Kanamycinresistan tplant so fL .peruvianu man d L. esculentum cv.Bellin awer e also available; these had been obtained by leaf disc transformation with Agrobacterium tumefaciens containingth eplasmi dAGS11 2(Koornnee fe tal .1987b) . Kanamycinresistanc e in L. esculentum wasuse dt oselec tfo rsymmetri csomati c hybrids (Chapter 2);kanamyci nresistan tL .peruvianu mwa suse dt omonito rth e losso fth eresistanc e inth easymmetri csomati chybrid s (Chapter5) .

Fusion combinations The protoplast fusion experiments are listed in Table1 . L. peruvianum was irradiatedwit hgamm aray sfro ma 60 Co source ata dos e rateo fapproximatel y 2000 Gray/hour at the Pilot-Plant for Food Irradiation, Wageningen (The Netherlands).Thre edifferen tdose swer eused :50 ,30 0o r100 0G y (=5 ,3 0o r 100kRad) . Putativehybrid stha tresulte dfro mthes efusions ,ar eindicate da s 5H, 30Han d100H ,respectively .Symmetri chybrids ,whic hresulte dfro mfusion s ofunirradiate d protoplasts,ar e indicated as0H-hybrids .Th e irradiationwa s carried out either to protoplasts, suspended at 1-3 x 105/ml inW 5solutio n (Menczele tal .1981 )on et othre ehour sbefor efusion ,o rt oleaflet son eda y beforefusion . 43

Table 1.Th egenotyp e ofth erecipien t speciesI . esculentum andth e irradiation treatment of thedono r species L. peruvianum, involved inth eprotoplas t fusion experiments. The LA-genotypes are multiple marker lines,homozygou s recessive for morphological marker genes (Rick 1982b). The protoplast fusion method used is given for each combination; M = method according to Menczel et al. (1981), CMS= metho d according toNegruti ue tal . (1986). Thenumbe r ofcalli ,whic hdi d regenerate shoots, isgive n in the right most column.

Recipient Irradiation of donor Number of Fusion Regenerating experiments method calli material dose (Gy) cv. Bellina protoplasts 0 1* M 11 cv. Bellina protoplasts 50 3 M 53 cv. Bellina protoplasts 300 3 M 25 cv. Bellina protoplasts 1000 3 M 13 LA291 protoplasts 300 4 M/CMS 31 LA1164 protoplasts 300 1 M 16 LA1166 leaflets 300 2 M 14 LA1182 leaflets 300 1 M 8 LA1182 protoplasts 300 3 M 12 LA1189 leaflets 300 1 M 3 LA1189 protoplasts 300 2 M 7 LA1444 protoplasts 300 2 CMS 9 LA1665 leaflets 300 4 M 13 LA1665 protoplasts 300 1 M 1

this experiment was used to determine the regeneration frequency, as shown inFig . 1;th e recipient (ATW3003)wa skanamyci n resistent to select against theunirradiate d donor species;onl ya sampleo f1 4call iwa s tested onshoo t induction medium

Cell culture The isolation and culture of protoplasts are described in Chapter 2. The pretreatment of leaf material, in the dark and on pre-incubation medium, was omitted in several experiments without negative effects. Protoplast fusionwa s carried out either according to Negrutiu et al. (1986; the CMS method) or according to Menczel et al. (1981), except that 30%PE G 4000 was used instead of 40%PE G 6000 (Table 1). The ratio of L. esculentum to t. peruvianum protoplasts during fusionwa s 1:1 to 2:1. In each fusion experiment 1x 106t o 3 x 106protoplast swer e involved. Ineac h experiment,culture s of the parental protoplasts, both separate and mixed, were started inparalle l with the fusion cultures (i.e.culture so fmixe dprotoplast so frecipien tan ddonor ,afte rfusio n treatment);i nsom eexperiment sals odono rprotoplast swer esubjecte dt oa fusio n treatment. Rooting of regenerated shoots was induced on shoot culture medium (MS salts (Murashige and Skoog 1962), T vitamins (Tewes et al. 1984), 10 g/l sucrose), without or supplemented with 0.1 mg/1 indole-butyric-acid. The regeneration capacity of a number of asymmetric hybrids was tested as described byKoornnee f et al. (1987a). Plant characterisation Chromosome numbers of root tip cells were determined in squash preparations or by means of a slightly modified procedure of Pijnacker and Ferwerda (1984; see also Chapter 2).Severa l morphological characters distinguishing both species as well as fertility were monitored as described in Chapter 2. As controls served diploid and tetraploid plants of the parental species and symmetric somatic hybrids of both species, derived from fusion experiments without irradiation treatment. 44

Results

Cell culture and plant regeneration

With the culture procedures employed, calli could be obtained from Bellina protoplasts. These calli did not regenerate shoots on shoot induction medium. The LA genotypes differed somewhat in their protoplast culture responses.Mos t ofthe mshowe dlimite dcel ldivisio nan dver yfe wmicrocalli developed .However , growth of these L. esculentum calli seemed to be improved in cultures where they were mixed with irradiated L. peruvianum protoplasts, which apparently behaveda sfeeder-cells .I nth ecas eo fLA118 2a tetraploi d plantwit hth eLA118 2 phenotype could be regenerated from such a mixed culture. The L. peruvianum protoplasts that were irradiated with 50, 300 or 1000 Gy ofgamm arays ,forme dcel lwalls .Som eo fthes ecell sstrongl yincrease d insize , while some others divided once or twice. In one out of 12 experiments where L. peruvianum protoplasts, irradiated with 300 Gy, were subjected to a fusion treatment,w e obtained three calli; these did not regenerate plants. Thefusio nculture syielde dman ycall io fwhic hsevera l formed shoots.Withi n each experiment there was a large variation in callus as well as shoot morphology.Fig . 1show sth etim ecours eo fshoo tformatio nb yasymmetri c hybrid calli,resultin g from fusionswher edifferen t irradiation doseswer e applied to the donor protoplasts. The rate of shoot regeneration as well as the fraction of calli that could form shoots decreased with the irradiation dose. The

Table 2.Characteristic so fdifferen thybri dcalli ,tha tregenerate dshoots :roo t formationo fshoot s (in vitro), establishment insoi li nth egreenhous e (atleas t onemonth) ,flowering ,frui tan dsee dset .Al lhybrid swer eobtaine dafte rfusio n ofprotoplast sfro m L. esculentum withprotoplast sfro m L. peruvianum, whichha d not been irradiated (OH)o r irradiated with 50,30 0 or 1000 Gray of gamma-rays (5H, 30H or 100H, respectively)befor e fusion.

Hybri ds Regenerati ng Root Greenhouse Flowers Fruits Seeds calli formation plants OH ND 40 32* 31 28 21 5H 53 38 27 21 13 0" 30H 139 63 32 15 5 0*** 100H 13 9 3 1 1 0

ND not determined not all rooted hybrids were transferred to soil 5 with abortive seeds 1 with abortive seeds 45

uu

> Ü 80- C ./OH 0) zs ö" 60- 0)

c 5H o 40^

(Ü c 100H

Fig. 1.Regeneratio n frequency, i.e. the percentage ofcall i tested that formed shootso nshoo t inductionmedium .Th ecall iwer ederive dfro m cultureso fmixed , fusion-treated protoplasts of L. esculentum cv. Bellina and L. peruvianum PI128650-ATW2002, which latter were irradiated with 50, 300 or 1000 Gray of gamma-rays (5H, 30H, 100H; 48, 66, 37 calli tested, respectively) and of L. esculentum cv. Bellina-ATW3003 and unirradiated L. peruvianum PI128650 (OH; 14 calli tested). The OH-hybrid calli derived from a fusion experiment of a kanamycin resistant Bellina and a L. peruvianum sensitive to this antibiotic, so that selection against the L. peruvianum parent was possible.

regeneration efficiency of the symmetric somatic hybrids (OH in Fig. 1) is similar to that ofunirradiate d L. peruvianum (data not shown). The characteristics of the putative hybrid calli that regenerated shoots, with respect to root formation, establishment in soil, flowering and seed set, are shown in Table 2 for each irradiation dose. The 5H-hybrids could be transferred to soil more efficiently than the 30H- and lOOH-hybrids. Only one out of 14 5H-hybrid plants tested could not regenerate shoots from established callus (Table 3),wherea s 3 out of 6 30H-hybrids did not. The controls reacted as expected (Koornneef et al. 1987a); L. esculentum was non- responsive,wherea s all tested L. peruvianum plants and OH-hybrids showed plant regeneration from established callus cultures. 46

Table3 .Regeneratio n capacity, measureda sshoo t formationo nshoo t induction mediumo festablishe d callus cultures,whic h were inducedo nlea f expiantso f L. esculentum, L. peruvianum andsomati c hybrids. Thenumbe r of independent hybridcalli ,fro mwhic hth eteste dplant sderived ,i sgive nbetwee nparentheses . The hybrids were obtained after fusion of L. esculentum protoplasts with L. peruvianum protoplasts,whic hha dbee nunirradiate d (OH)o rirradiate d with 50,30 0o r100 0Gra yo fgamma-ray s (5H,30 Ho r100H ,respectively )befor efusion . +, callus regenerating; -,callu sno tregenerating .

Plants Regeneration capacity

L. esculentum 0 4 L. peruvianum 7 0 0H-hybrids 2(2 ) 0 5H-hybrids 14(9 ) 1 (1) 30H-hybrids 6(5 ) 3 (3) lOOH-hybrids 1(1 ) 0

Cytogenetics

The parental species bothhav e2 n- 2 x- 24,relativel y small chromosomes.Th e L. peruvianum chromosomesar ehomoeologou st oth eL . esculentumchromosome s(Ric k and Butler 1956). Mosto fth esymmetri c somatic hybrids between these species hada chromosom e numbero f4 8o r7 2(2 n= 4 xan d6x , respectively; Chapter2) . The chromosome numberso fth easymmetri c somatic hybrids varied from2 9t o 85, as presented inFig .2 an d3 .Th emajorit y ofth e5H-hybrid s hadchromosom e numbers below thetetraploi d level (shoots from 13ou to f2 1hybri d calli), whereas the30H -an dth e100H-hybrid s usually were above thetetraploi d level (shoots from 17ou to f2 530H-call i andbot h lOOH-calli).A fe whybrid s even exceeded thehexaploi d level (shoots from one 5H-an dthre e 30H-hybrid calli). The countingo fth e chromosomeswa shampere db ythei r small size.I nsom e cases very small particles,mos tprobabl y centric chromosome fragments,wer e observed (e.g.Fig . 2A).I ngenera l the chromosome numbero feac h shootvarie d slightly, withinan dbetwee n the analysed roots.I nsevera l cases largerdifference s were observed, mostly occurring between different roots.I nthes e cases chromosome numbers differedb yte nt otwenty . Normally theshoot s fromon efusio n callus had similar chromosome numbers.Als o when shootso fdifferen t subcloneso fth e same hybrid callus were present, they mostly (4ou to f5 calli )ha dsimila r chromosome numbers. However, onehybrid , 30H28, deviated: four well-looking shootsfro mon esubclon eha d4 1an da fift hha d7 0chromosomes ,wherea s theonl y shoot from theothe r subclone probably had2 9 chromosomes (only twocell s counted);th eshoot s with2 9an d7 0chromosome s grewwors e than the four other shoots derived from the same callus. 47

f

.'.<"- ** . V * -

B

• • », "•jr.: v\ • • • •

te. 'S » • ,*

Fig.2 .Metaphas eplate so froo tti pcell sfro masymmetri e somatichybrid so f L. esculentum andirradiate d L. peruvianum. A.5H2 4(irradiatio ndos eo f5 0G y was applied), 2n= abou t 66; notice the small particles which probably are centric chromosome fragments (arrows). B.5H15 , 2n= 43 . C.5H19 , 2n= 5 7 D.30H4 6 (dose of 300Gy) , 2n- 56 . E.30H28 , 2n= 70 . F.100H 3 (dose of 1000Gy) , 2n- 57 . 48

25 5H—hybrids

15 -M c

a 5 0. Q) 30H—hybrids E 5 0. 5 100H-hybrids

24 36 48 60 72 84 96

Chromosome number

Fig. 3.Frequenc ydistributio no faverag echromosom enumber so fdifferen t shoots from asymmetric somatic hybrids of L. esculentum and L. peruvianum (the donor species). Upper panel: 5H-hybrids (donor irradiated with 50Gy) ;middl e panel: 30H-hybrids (300Gy) ;lowe r panel: lOOH-hybrids (1000Gy) .

Plant morphology

The asymmetric hybrid plants were less vigorous than bothparenta l species and the symmetric hybrids. Especially the 30H- and 100H-hybrids were very retarded in growth and often died after transfer to soil. The leaves of the asymmetric hybrids differed much in colour (from light- to grey-green), shape and size (Fig. 4).Generall y the leaveswer e smaller than those of the parental species (Fig. 5A).Leave s of the 30H- and lOOH-hybrids mostly were smaller than those ofth e5H-hybrids .Usuall yth easymmetri chybrid sha dleave swit hdee pincisions , similar to L. esculentum, and secondary leaflets, as I. esculentum and thé symmetric hybrids.N o stipuleswer e present onth e stems of the 30H-hybrids and only a few small ones on the stems of the 5H-hybrids (Fig. 5B); the same holds for the presence of bracts (leaflets in the inflorescences; Fig. 5C). The sympodial index (i.e. the mean number of nodes between two subsequent inflorescences)o f theasymmetri c hybrids ingenera lwa s three,simila r totha t of the cultivated tomato and the symmetric hybrids. A grey-green variegation was observed in 29 out of 51 5H-shoots and 13 out of4 5 30H-shoots.Som eshoot swer ever yaffecte d andha d small irregularleaves . 49

Fig.4 .Th eleave so ftwelv edifferen tasymmetri csomati chybrids , L. esculentum cv. Bellina(left )an d L. peruvianum PI128650(right) .Al lhybrid swer eobtaine d afterprotoplas tfusio no fL . esculentum cv.Bellin awit hL .peruvianum ,whic h wasirradiate dwit h5 0Gra yo fgamma-ray sbefor efusion .

Nine5H-hybri dcall i(33 %o ftotal )an d1 130H-call i (55%)produce dplant stha t wereno tvariegated .Th esam elea fvariegatio nha sals obee nobserve d inman y symmetric hybrids (Chapter 2) and could be similar to the disorder, named silvering,whit ehea do rchimer a (Grimbly 1984),whic hoccur si nsom eEuropea n tomatocultivars .

Flower morphology Amajorit yo fth egreenhouse-grow n5H-hybrid san da minorit yo fth egreenhouse - grown 30H- and lOOH-hybrids formed flowers (Table2) .Th e asymmetric hybrids floweredles sabundantl ytha nth eparent san dth esymmetri chybrids .Th ecoroll a ofth e5H-hybri dflower swer ei ngenera ldar kyellow ,lik etha to f L. peruvianum andth esymmetri chybrids ,wherea smos t30H-hybrid san dth eonl yflowerin g100H - 50 A B C % Leaf size Stipules Bracts IOOJ Le so . 1. 1. I. c Lp a 4— .1 1 1 0 OH ö) -.1. 5H c ü 30H i. i. 12 3 4 5 1 2 3 1 2 3 Classification

Fig.5 .Frequenc ydistributio no fsom emorphologica lcharacter so f L. esculentum (Le;1 6plant stested) ,L .peruvianu m(Lp ;2 2tested) ,symmetri csomati chybrid s ofbot hspecie s(OH ;6 0tested )an dasymmetri csomati chybrid so fbot hspecies , inwhic hoccasio n L. peruvianum wasirradiate dbefor efusio nwit h5 0an d30 0Gra y ofgamma-ray s (5Han d30H ;4 1an d3 4shoot stested ,respectively) . A.Lea fsize :A narbitrar yclassification .1 = ver ysmal lleave s(< 2cm )t o5 = verylarg eleave s(>5 0cm) .B .Presenc eo fstipules :Leaflet so nth este ma tth e nodes.C .Presenc eo fbracts :Leaflet si nth einflorescence .Classe so fB + C : 1= absence ;2 = presenc ea ta fe wnodes ;3 = presenc ea teac hnode .

hybrid had paleyello w flowers,suc h as L. esculentum ones. The size ofth e flowerpart sa swel la sth eratio so fth esize so fdifferen tflowe rpart svarie d stronglybetwee nhybrid s(Fig .6 A+ 6B) .Th eaverag esiz eo fth eflower so fth e 5H-hybridswa ssimila rt otha to fth eparenta lspecies ,wherea sth e30H-hybrid s hadsmalle rflowers .Th erelativ esepa llengt ho fth easymmetri chybrid susuall y wasintermediat ebetwee n L. esculentum andth esymmetri chybrids .I nmos tplant s ofbot hasymmetri chybri dclasse sth episti lwa ssmalle rtha nth estamens ;thi s wasals oobserve di ntetraploi d L. esculentum plants (Table3 i nChapte r2) . 51

Size of petals Sepals/petals % 100 Le WT Lp L

OH CD Ol co T*T i 5H c - CD u 30H CD a W # WW T 6 14 22 30 0.2 0.6 1.0 1.4 6 14 22 30 A Length (mm) Ratio Length (mm)

Fig. 6. Frequency distribution of some sizes of flower parts of L. esculentum (Le;1 2plant s tested), L. peruvianum (Lp; 20tested) ,symmetri c somatichybrid s ofbot h species (OH;3 9 tested)an d asymmetric somatic hybrids ofbot hspecies , inwhic hoccasio n L. peruvianum wasirradiate dbefor efusio nwit h5 0an d30 0Gra y of gamma-rays (5Han d 30H; 28an d 7 shoots tested, respectively). A. Size of the petals and relative length of the sepals (with respect to the petal length). B. Size of the stamens and relative length of the pistil (with respect to the stamen length). Eachmeasuremen t was theaverag e ofa tleas t two flowers from the same plant.

Fertility

The flowers of the asymmetric somatic hybrids produced no or small amounts of pollenwhic hwer eno tviable ,a s testedwit hlactopheno l acid fuchsinstaining . Several asymmetric hybrids set fruits (Table 2)wit h adiamete r from ahal f to a few centimeters.Usuall y theywer e smaller thanL . peruvianum fruits (1-2 cm)an d much smaller than tomato fruits (>3 cm).Als o the colour of the fruits differed between hybrids (Table 4). Most fruits were orange, which is intermediate between the fruit colour of the symmetric hybrids (yellow)an d of the cultivated tomato (red). No seed set was obtained in the asymmetric hybrids after selfing or backcrossingwit h theparenta l species (Table 2);th e symmetric hybrids,o nth e contrary, did set seeds. In some fruits of five 5H-hybrids and one 30H-hybrid very small,abortiv e seedswer e observed. 52

Table4 .Th e colour ofth e fruits obtained fromdifferen t asymmetric somatic hybrids.Th ehybrid swer eobtaine dafte rprotoplas tfusio no f L. esculentum with L. peruvianum, whichwa sirradiate dwit h50 ,30 0o r100 0Gra yo fgamma-ray s(5H , 30Ho r100H ,respectively )befor efusion .Eac hobservatio nwa sdon e onon et o severalshoot sfro mon ehybri dcallus .

Plants Fruitcolou r yellow orange red 5H-hybrids 5 6 1 30H-hybrids 0 4 1 100H-hybrids 0 1 0

Discussion

Thepresen texperiment ssho wtha tasymmetri csomati chybri dplant sca nb ederive d from protoplast fusions between L. esculentum and irradiated L. peruvianum. Selectiono fthes ehybrid swa spossibl eo nth ebasi so fth eregeneratio ncapacit y derived from the irradiated species. The asymmetric hybrid nature could be confirmed by several morphological characteristics, which were intermediate betweenth esymmetri csomati chybrid s(Chapte r2 )an d L. esculentum, andb yth e analysis of specific marker genes (Chapter 5). However, the irradiation of L.peruvianu mbefor efusio ndi dno thav eth edesire deffect ,namel yeliminatio n ofth edono rgenom et osuc ha nextent ,tha tonl ya smal lfractio nwa sconserve d inth easymmetri chybrids . Allasymmetri chybrid swer eaneuploid .Fro mth ecytogeneti can dmorphologica l analysis,w etentativel y conclude thatmos t asymmetric hybridscontaine done , twoo rthre ediploi d L. esculentum genomes,supplemente dwit hon epartia ldiploi d genomeo f L. peruvianum. For 15asymmetri chybrid sthi swa sconfirme db yRFL P analysis(Chapte r 6).Th e5H-hybrids ,o fwhic hmos tha don ediploi dI . esculentum genome,ha do nth eaverag e 16presume d L. peruvianum chromosomes (range:5 t o 22);th e30H-hybrids ,whic hi nmos tcase sprobabl ycontaine da tetraploi dgenom e of L. esculentum, hada mea no f1 3(range :2 t o22 ) L. peruvianum chromosomes; andbot h100H-hybrid sprobabl yha da tetraploi d L. esculentum genomeand ,o nth e average,7 (range :4 t o12 ) L. peruvianum chromosomes.Althoug hther ei sn oclea r correlationbetwee nth e irradiationdos ean d elimination ofth e L. peruvianum genome,th e30H-hybrid sresemble dL .esculentu mmor etha nth e5H-hybrids .Thi s isno tnecessaril ydu et oth eeliminatio no f L. peruvianum chromosomes,bu tca n alsob eascribe dt oth epresenc eo fa large rnumbe ro f L. esculentum genomesi n most30H-hybrids .Anyway ,a relativel ylarg efractio no fth e L. peruvianum genome 53 isretained ,eve ni nth elOOH-hybrids .Limite dchromosom eelimination ,togethe r witha wea kdos eeffect ,wa sals oobserve di nasymmetri csomati chybrid so fothe r species.Retentio no fth edono rchromosome srange dfro m11 %t o90 %(o fa diploi d donor genome) in Nicotiana plumbaginifolia (recipient) (+) Atropa belladonna (donor)hybrid s (100-1000Gy ;Gleb ae tal .1988) ,8%-75 %i n N. plumbaginifolia (+) N. sylvestris hybrids (100-1000Gy ;Famelae r etal . 1989)an d 25%-100%i n Brassica oleracea (+) B. campestris hybrids(100-80 0Gy ;Yamashit ae tal .1989) . Inal lthos ecase s chromosome rearrangements and/ordelete ddono rchromosome s werereported .I nsom eo fth eL .esculentu m(+ )L .peruvianu masymmetri chybrid s reported here, we observed fragments. It was often difficult to identify fragmentswit hcertainty ,becaus eo fth esmal lsiz eo fth etomat ochromosomes . However,RFL Panalysis ,applie d to 15hybrids ,clearl y indicated thepresenc e ofincomplet echromosome si nal lteste dplant s (Chapter6) . Theasymmetri c 5H-hybridswer emor eviabl etha nth e 30H-an d100H-hybrids , irrespective whether shoot regeneration, root formation or morphological characteristicswer euse da scriteria ,wherea sal lasymmetri chybrid swer eles s viable than the symmetric hybrids. A possible explanation for this is the unbalanced genome of the asymmetric hybrids. It has been reported that in L. esculentum onlyprimar ytrisomie san d somemonosomie sar eviabl e (Rickan d Butler 1956),wherea saneuploid yi stolerate dbette rb yprimitiv etomatoe san d specieshybrid so fth etomat otha ni nth ecultivate d tomato (Soost1958 ;Ric k and Notani 1961;Györff y and Mako 1963). In general the asymmetric somatic hybridswer enea rth etriploi do rpentaploi dlevel ;th e5H-hybrid susuall yha d a diploid L. esculentum genome,wherea smos t 30H-an d 100H-hybridspresumabl y hada tetraploi drecipien tgenome .I nothe rasymmetri chybridisatio nexperiment s thatresulte di nlimite dchromosom eeliminatio n(Gleb ae tal .1988 ;Famelae re t al.1989 ;Yamashit ae tal .1989) ,th erecipien tgenom ei nth easymmetri chybrid s usuallywa saroun dtetraploi do rhexaploid .A nexplanatio no fthi scoul db etha t cellswit h polyploidised recipient genomes,whic hma yhav e arisendurin gth e tissue culture phase, better tolerate additional and abnormal chromosomes (rearrangementsan dfragments) . The variation in chromosome number within each of the asymmetric hybrids mightindicat einstabilit ya tth eplan tlevel .Probabl yrearrange dan dincomplet e chromosomesten dt oge tlos tmor efrequently .Th eobserve dlos so fregeneratio n capacityi nestablishe dcallu sculture so fsom e30H-hybrid smigh tb eexplaine d thisway .A nothe rexplanatio ni seliminatio no fthi strai ti nth eestablishe d calluscultures . Theasymmetri csomati chybrid swer eno tfertile .Som efruit scontaine dsmal l abortiveseed-lik estructures .Embry orescu etechnique swer eapplie dtw ot ofou r weeksafte rpollinatio n(selfin go rbackcros swit h L. esculentum pollen).I ntw o 54 casescallu swa sinduce do ntiny ,prematurel yisolate dseeds ;on eo fthes eforme d shoot primordia. However, these calli might also descend from maternal tissue. The limited elimination of donor chromosomes is a serious drawback for the applicationo fasymmetri chybrid si nplan tbreeding ,becaus esevera l backcrosses to the recurrent parent are still required to get rid of the unwanted donor traits.Crosse swil lb eimpossibl eo ra tleas thampere dstrongl yb yth esterilit y and the of these hybrids. The sterility is probably due to the cytogenetic aberrations, sucha saneuploid y andrearrangements .Th elatte rwer e more frequent afterhighe r irradiation doses (Chapter 6).Despit e the fact that several tens of asymmetric hybrids were obtained and analysed, none of the plants had just one or two chromosomes above the diploid level,whic h probably wouldhav eallowe dbackcrossin gt oth ediploi drecipient .I fth epolygeni cnatur e of the selectable donor traits (both callus growth and regeneration characteristics) is the main cause of this limited elimination, the use of simpler selectable markers, such as antibiotic resistance or alleles complementingauxotrophi c mutations,woul dhel pt oovercom e theproblem . Ifnot , other ways toenhanc e elimination ofdono r chromosomes should be looked for or very large populations of asymmetric hybrids have tob e evaluated.

Acknowledgements. Thisresearc hwa s supported by theFoundatio n for Fundamental Biological Research (BION),whic h issubsidise d byth eNetherland s Organisation for Scientific Research (NWO). We are very grateful to Corrie Hanhart, Henny Verhaar andAnne-mari e Wolters fordoin g part of the experiments,an d Prof. C. Heyting for critically reading of the manuscript.

References

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KoornneefM , JongsmaM ,Weid eR , Zabel P,Hill e J (1987b)Transformatio n of tomato. In:Nevin sDJ ,Jone sR A (eds)Tomat obiotechnology . AlanR Liss, Ine,Ne wYork ,p p 169-178 MenczelL ,Nag y F,Kis sZR ,Malig aP (1981)Streptomyci nresistan tan dsensitiv e somatichybrid so fNj-cotian a tabacum+ Nicotian aknightiana : correlationo fresistanc e toN .tabacu mplastids .Theo rApp lGene t59 :191 - 195. Murashige T, Skoog F (1962)A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant15:473-497 . Negrutiu I,D eBrouwe r D,Watt sJW , SidorovVI ,Dirk sR , JacobsM (1986)Fusio no fplan tprotoplasts :a stud y using auxotrophic mutants ofNicotian a plumbaginifoliaViviani .Theo rApp lGene t 72:279-286 Pijnacker LP,Ferwerd aM A (1984)Giems aC-bandin g ofpotat o chromosomes.Ca nJ Gene tCyto l26:415-41 9 Rick CM (1982a)Th epotentia lo fexoti c germplasm fortomat o improvement. In:Vasi lIK ,Scowcrof tWR ,Fre yK J (eds), Plant improvement and somatic cellgenetics .Academi c Press,Ne wYork ,p p 1-28 Rick CM (1982b)Stoc k list.Re pTomat oGene tCoo p 32:3-10 Rick CM,Butle r L (1956)Cytogenetic s of the tomato.Ad vGene t8:267-38 2 Rick CM,Notan i NK (1961)Th etoleranc e of extra chromosomesb y primitive tomatoes.Genetic s 46:1231-1235 SoostR K (1958)Progenie sfro msesquidiploi dF !hybrid so fLvconersico nesculentu man dL .peruvianum .J Heredit y 49:208-213 Taylor IB (1986)Biosystematic so fth etomato . In:Atherto nJG ,Rudic hJ (eds), Thetomat o crop.A scientific basis for improvement.Chapma n and Hall,London/Ne w York,p p 1-34 Tewes A,Glun d K, Walther R, Reinbothe H (1984)Hig h yield isolation and rapid recovery ofprotoplast s from suspension cultures of tomato (Lycopersicon esculentum). ZPflanzenphysio l 113:141-150 Yamashita Y, Terada R, Nishibayashi S, Shimamoto K (1989)Asymmetri c somatic hybrids of Brassica: partial transfer ofB .campestri sgenom e intoB .olerace ab y cell fusion.Theo rApp lGene t 77:189-194 57

CHAPTER5

ASYMMETRIC SOMATIC HYBRIDS BETWEEN LYCOJPEJEZSICON ESCUI^ENTUM AND IRRADIATED L.YCOPERSICON I>EKLTV IANUM II. ANAJL.YSI S WITH MARKIER. GENES

J.Wijbrandi ,A.M.A .Wolters ,M .Koornnee f

Summary.Asymmetri csomati chybrid so f Lycoper sicon esculentum and Lycopersicon peruvianum wereanalyse d forth eretentio no fgene san dalleles ,specifi cfo r L. peruvianum. The hybrids were obtained by fusion of protoplasts from L. esculentum withthos eo f L. peruvianum (thedonor) ,whic hha dbee nirradiate d before fusionwit h 50,30 0 or 1000G y ofgamma-rays .Th e retention of three different types of genes or alleles was analysed: (1)Th e gene coding for kanamycinresistance ,whic hi sdominan tan dha dbee nintroduce d inmos to fth e L. peruvianum donorplant sb ytransformation .I twa spresen ta ton elocu si n1 6 L. peruvianum donor plants anda ttw oloc i inon edono r plant. (2)Th egene s codingfo raci dphosphatase ,locu s Aps-1, andglutamat eoxaloacetat etransaminas e (GOT);differen tallele so fthes egene sar eco-dominant ,an dwer edetecte db y isozymeanalysis .(3 )Eightee nsingl egen emorphologica lmarkers ,fo rwhic hmos t ofth e L. esculentum genotypesuse dwer ehomozygou srecessive . Kanamycinresistanc efro mth edono rplant swit hon elocu swa sretaine di nabou t 50% of the asymmetric 30H-hybrids (the donor was irradiated with 300Gy) . L. peruvianum specificallele so f Aps-1 andGO Twer epresen ti na tleas t70 %o f thehybrids ;th eretentio no fdono rallele swa slowe ri n30H -tha ni n5H-hybrid s (donorirradiate dwit h5 0Gy) .O nth eaverage ,74 %o fth e L. peruvianum specific alleles (one orboth )o f themorphologica l markerswer e detected inth e30H - hybrids.Severa lo fth e L. esculentum genotypesuse dwer ehomozygou srecessiv e fortw omorphologica lmarker so nth esam echromosome .I n36 %o fth e30H-hybrid s derived from them, only one of these markers was complemented by the L. peruvianum allele. This is an indication for frequent breakage of the L. peruvianum chromosomes. Several hybrid calli regenerated genotypically differentshoots . On thewhole ,thi sanalysi s confirms the conclusion from the cytogenetican d morphologicalanalysi so fthes easymmetri chybrids ,namel ytha tirradiatio nprio r tofusio neliminate d theL .peruvianu mgenom eonl yt oa limite dextent .

Introduction

Partial genome transfer by asymmetric somatic hybridisation, which involves fusiono fprotoplast s ofa recipien t specieswit hirradiate dprotoplast so fa donor species,ha s been described for several plants.Th e transferred donor genomevarie d froma fe wtrait s (e.g.Dudit se tal . 1987)t oman ychromosome s (e.g. Gleba et al. 1988). The amount of transferred donor genome was often assessed on the basis of chromosome counts. This isno t always an accurate estimation,becaus ediscriminatio nbetwee nth especie sspecifi cchromosome san d identificationo fincomplet echromosome sar eno talway spossible . 58

InChapte r4 w e described the cytogenetic andmorphologica l analysis ofa series of asymmetric hybrids of L. esculentum (the cultivated tomato) and L. peruvianum (awil d species;th e donor). Thesehybrid s appeared tocontai n stilla relativel y large number ofL .peruvianu m chromosomes. Inmos t ofth e asymmetrichybrids ,th echromosom enumbe rwa saroun dth etriploi do rpentaploi d level;probably ,th egenom eo fthes ehybrid sconsiste do fa diploi do rtetraploi d genomefro m L. esculentum andsevera lchromosome sfro m L. peruvianum. Theamoun t ofdono rchromosome svarie dan ddi dno tcorrelat estrongl ywit hth eirradiatio n dose applied to the L.peruvianu m protoplasts (50, 300 and 1000 Gray, respectively).T ostud yth eeliminatio no fgeneti cmateria lo fth edono rspecie s morei ndetail ,th esam easymmetri chybrid swer eanalyse dfo rtransfe ro fsingl e genemarkers .Thi skin do fanalysi s canb eperforme d verywel l inth etomato , because of its detailed linkage map. The map consists of more than 300 morphological,isozyme -an ddiseas eresistanc egene s(Mutschle re tal .1987 )an d atleas t 300RFL Pmarker s (Youngan dTanksle y 1989).W etrie dt oestimat eth e fractiono fth e L. peruvianum genometha twa sconserve di nth easymmetri csomati c hybridsb yanalysin gth eretentio no fthre etype so fgene so ralleles : (i)th eneomyci nphosphotransferas e II (NPTII )gene ,whic hcause sresistanc e toth eantibioti ckanamycin .Thi si sa dominan tgene ,whic hha dbee nintroduce d into the nuclear genome of most of the L.peruvianu m donor plants by transformation; (ii)th e isozyme markers acid phosphatase (Aps-1) and glutamate oxaloacetate transaminase (GOT); (iii)singl egen edetermine dmorphologica lmarkers ,presen ti nmos to fth etomat o genotypesused .

Materialan dmethod s The genotypes of L. esculentum, the recipient species,wer e theDutc hhybri d cultivar Bellina (kindly provided by Rijk Zwaan Seed Company, de Lier,Th e Netherlands)an dseve nmultipl emarke rline s(Tabl e1 )fro mth eTomat oGenetic s StockCente ri nDavis ,US A(kindl yprovide db yProf .CM .Rick) .Plant sfro mth e L. peruvianum accessionPI12865 0(receive dfro mth eInstitut eo fHorticultura l Plant Breeding, Wageningen, The Netherlands) were used as donor. Seventeen kanamycinresistan t L. peruvianum plants,obtaine db ylea fdis ctransformatio n with A. tumefaciens containingth eplasmi dpAGS11 2(Koornnee fe tal .1987b )wer e available;thes eplant swer edesignate dATW200 1t oATW2027 .Th eisolatio no fth e asymmetric somatichybrid swa sdescribe d inChapte r4 .Th easymmetri chybrid s weredesignate daccordin gt oth eirradiatio ndos eapplie dt oL . peruvianumbefor e protoplastfusion :5H- ,30H -an dlOOH-hybrids ,whic hwer eirradiate dwit ha dos e of50 ,30 0an d100 0G yo fgamma-rays ,respectively .

Kanamycin resistance assays To determine the number of NPT II loci in the independent L. peruvianum transformants, they were backcrossed as pistillate parent to wild type L. peruvianum. Theresultin gseed swer edecontaminate db ytreatmen tfo r10 "i n 59

Table1 .Th emultipl emarke rline so f L. esculentum usedi nth epresen tstudy . Themorphologica lmarke rgene sar egive nwit hthei rchromosoma llocatio n(Ric k 1982).* indicate sa characte rwhic hwa suse di nth eanalysi so fth easymmetri c somatichybrid so f L. esculentum (recipient)an d L. peruvianum (donor).

Tomato Markers [chromosomearm ] genotype LA291 ms-2 (=malesterile )[2L ] hi (-hairless,n olarg etrichomes )[IIS ] a (-anthocyaninless)[11L ] LA1164 var (-variabilis,leave semerg eyellow )[7S ] not (=notabilis,leave swilting )[7L ] ah (=anthocyaninless)[9L ] maim(-marmorata ,leave smarble dwhite-green )[9L ] LA1166 clau (=clausa,leave ssubdivided )[4S ] di (-divergens,stem sslende ran dwhitish )[4L ] icn (-incana,leave swit hwhitis hmargins )[10S ] ag (=anthocyaningainer ,lamina eabaxiall ypigmented )[10L ] LA1182 sy (=sunny,leave semerg eyellow )[3S ] sf (-solanifolia,leaflet sentir ean dconcave )[3L ] alb (-albescent,stron gwhite-gree nvariegation )[12S ] mua(=multifurcata ,dul lgree ninterveina lchlorosis )[12L ] LA1189 yv (-yellowvirescent ,leave semerg eyellow )[6L ] c(-potat oleaf ,fewe rlea fsegments )[6L ] LA1444 af (-anthocyaninless)[5S ] tf (=trifoliate,leave s3-segmented )[5S ] wv(-whit evirescent ,leave semerg ewhite )[2L ] d(-dwarf )[2L ] LA1665 dgt (-diageotropica, stemsan droot sdiageotrophic )[IL ] 1 (-lutescent,leave syellowing )[8S ] al (-anthocyaninloser ,pigmente donl ya tnode slater )[8L ]

70/Sethano l and 20'i n 5x diluted commercial bleach (10%NaCIO ), an d washed severaltime si nsteril ewater .Th eseed swer etransferre dt oplasti ccontainer s withshoo tcultur emediu m(Chapte r2 )containin g10 0mg/ 1kanamyci nan dincubate d inth edark .Afte ra fe wday sth eseed sgerminate dan dwer etransferre dt olight . Twoweek slate rth eseedling swer escore dfo rgrowt h (-resistance). To determine the kanamycin resistance in calli, derived from protoplast cultures ofa L. esculentum genotype and one of the L. peruvianum ATW-plants (withan dwithou tfusio ntreatment) ,w eslice dth ecall ian dsubculture don epar t onth ecallu smediu mTM c(Chapte r4 )an dtransferre dth eothe rpar tt oTM cwit h 100mg/ 1kanamycin .Kanamyci nsensitiv ecall iturne dbrow no nth elatte rmedium . Totes tkanamyci nresistanc eo fsomati chybri dplants ,cutting so fhybri dshoot s weretransferre dt oshoo tcultur emediu msupplemente dwit h10 0mg/ 1kanamycin . Sensitiveshoot sforme dn oroot san dbleache dafte ra fe wweeks . Southern blot analysis DNA was isolated from several plants according to Dellaporta et al. (1983), digestedwit h Dra I,separate db yagaros ege lelectrophoresis ,blotte dont oGen e Screen PLUS (NewEnglan dNuclear )an dhybridise dwit ha prob e for theNP TI I gene; as probe was used the Cla l-Sal I fragment, which contained theNOS - promoter and the structural gene,o f the T-region of plasmid pAGS112 (kindly provided by Dr. P.J.M. van den Elzen, MOGEN Leiden, The Netherlands). The hybridisationprocedur e isdescribe d inChapte r6 . 60

Isozyme analysis Leaf material from greenhouse-grown plants was used for the analysis of acid phosphatase, locus Aps-1, and glutamate oxaloacetate transaminase (GOT), loci Got-1, Got-2, Got-3 and Got-A; the positions of these loci on the linkage map of tomato are shown in Fig. 1. Crude extracts and extracts prepared according to Suurs et al. (1989), respectively, were electrophoresed on vertical Polyacrylamide slab gels (Chapter 2).Enzym e activity was stained according to Vallejos et al. (1983).

Morphological markers Fig. 1 shows the position of the recessive morphological markers,use d in the present study, onth elinkag ema po fL . escuientum.Th e L. esculentum genotypes thatwer euse d forth efusions ,wer ehomozygou sfo rtw ot ofou ro fthes emarker s located on one to twodifferen t chromosomes (Table 1).Th e asymmetric hybrids, preferably greenhouse-grown plants, were assayed for the phenotype of the relevant markers todetermin e the retention of thedominan t L. peruvianum wild type alleles. The presence of some markers could not be assayed, because they weretypica lhypocotyledo nmarker s (e.g. ag) orbecaus eth ehybri dan daneuploi d nature of the hybrids interfered with the expression of the mutant phenotype.

4 5 7 8 10 11 12 0- °-rc/au Qot-3 \*--af 2*- -ah 27' 'aot-4 Qot-1 J4j£yi' 40- -not 4«- -sy 48--»/

mi--a/ marm

a9-- di

104-- c 111.-5/ \ot--g

\si--dgt

Fig. 1.Linkag ema po fth emorphologica l andisozym emarker suse d inth eanalysi s of the asymmetric somatic hybrids. The shaded areas indicate the centromeres. All positions are according toMutschle r et al. (1987). 61

Results

Number of kanamycin resistance loci in L. peruvianum plants Thesegregatio nratio so fkanamyci nresistanc ei nth etestcros so f L. peruvianum ATW-plantswit hwil dtyp e L. peruvianum aregive ni nTabl e2 .Sixtee nATW-plant s showeda rati oo f1:1 ,whic hindicate sth epresenc eo fon eNP TI Ilocus ,wherea s oneplant ,ATW2002 ,ha da rati oo f3:1 ,whic hindicate stha tNP TI Igene swer e inserted at twodifferen t unlinked loci.Thi swa s confirmed by Southernblo t analysiswit h theNP T IIgen ea sprob e (Fig.2) :ATW200 2 showed twodistinc t fragments;th eteste dkanamyci nresistan thybrid so fBellin a(+ )ATW2002 ,namel y onesymmetri can dte nasymmetri chybrids ,ha deithe ron eo rbot ho fthes ebands ; theband ssegregate d independently.Th eonl yteste dkanamyci nsensitiv ehybri d ofth esam eparenta lgenotypes ,5H10 ,ha dn osuc hband(s) .

Table2 . Segregation of kanamycin resistance in the testcross of kanamycin resistant L. peruvianum PI128650plant s(ATW200 1t oATW2011 ,ATW201 4t oATW2016 , ATW2020,ATW202 1 and ATW2027)a s female parent withwil d type L. peruvianum PI128650,an damon gregeneratin g30H-hybri dcalli ,whic hderive dfro mprotoplas t fusionsbetwee n L. esculentum and 300G ygamma-irradiate d kanamycinresistan t L. peruvianum. The assayswer e performed onmedi a supplemented with 100mg/ 1 kanamycin.Th ecall iwer eassaye dbefor eregeneratio noccurred . Km11,kanamyci nresistant ;Km s,kanamyci nsensitive .

L.peruvianu m Segregationrati oi ntestcros s Ratioi n30H-call i genotype KmK:Kma [x*{l:l}]w KmK:Kms ATW2001 29:35 [0.56] 1:3 ATW2003 35:29 [0.56] 1:0 ATW2004 32:32 [0.00] 9:3 ATW2005 73:53 [3.17] 6:2 ATW2006 41:53 [1.53] 2:1 ATW2007 31:33 [0.06] 1:0 ATW2008 36:27 [1.29] 3:0 ATW2009 33:32 [0.02] 7:5 ATW2010 33:31 [0.06] 3:0 ATW2011 31:33 [0.06] 4:1 ATW2014 59:66 [0.39] 5:3 ATW2015 31:33 [0.06] 1:3 ATW2016 29:34 [0.40] 1:1 ATW2020 29:33 [0.26] 13:1 ATW2021 31:41 [1.39] 5:3 ATW2027 32:32 [0.00] 3:2 total65:2 8 ATW2002 47:17 [14.06]** segregationrati odoe sno tdeviat esignificantl yfro m1:1 , if x2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 • .f • * • -67 kb

-4.4 kb

- • ï . •••• •':•. -2.3k b

Fig. 2. Southern blot analysis of some somatic hybrids and their parents with a probe of the NPT II gene. Dra I digests of the following genotypes were analysed: lane 1, 5H2; lane 2, 5H3; lane 3, 5H5; lane 4, 5H7; lane 5, 5H13; lane 6, 5H16;lan e 7,5H28 ; lane 8,5H10 ;lan e 9, 30H1;lan e 10,30H3 ; lane 11, 30H7; lane 12, L. esculentum cv. Bellina; lane 13,0H1 ; lane 14, L. peruvianum PI128650-ATW2002. All hybrids derived from protoplast fusions between L. esculentum cv. Bellina and ATW2002. The latter was either unirradiated (symmetric hybrid 0H1), irradiated with5 0G y ofgamma-ray s (asymmetric hybrids 5H2 to 5H28) or irradiated with 300 Gy of gamma-rays (asymmetric hybrids 30H1 to 30H7)befor e fusion. The position of the Hin dill fragments of phage lambda DNA is indicated at the right.

Retention of kanamycin resistance loci in asymmetric hybrids

The ratio of kanamycin resistant to kanamycin sensitive regenerating calli derived from fusions of L. esculentum and irradiated L. peruvianum containing one NPT II locus, is shown in Table 2 (right column). Seventy percent of the tested calli (65ou to f93 )wer ekanamyci nresistant .Th eassa ywa srepeate d for shoots thatha d regenerated from 12resistan t 30H-calli;onl y aminorit y of the 30H-hybridswer e tested at theplan t level,becaus e of thever y lowgrowt h rate ofman y of these hybrids and becausew e preferred totransfe r the shoots to the greenhouse. Shoots from 7 of these 12 resistant calli were still resistant, whereasth eshoot sfro m4 resistan t calliwer ekanamyci n sensitive.Fro m another resistant hybrid callus one resistant and one sensitive shoot was regenerated. So, inon e third of the 30H-hybridstha twer e kanamycin resistant at the callus level, the resistance was lost inth e shoots.Assumin g thatth e chance toloos e thistrai twa sth esam ei nal lresistan t30H-hybrids ,w eestimat etha t4 6percen t (~43ou t of 93) of the 30H-hybrids had retained the kanamycin resistance from L. peruvi anum. The retention of kanamycin resistance in nine 30H-hybrids (all nine tested atth eplan tlevel )derive d fromATW200 2 (withtw oNP T IIloci) ,wa shigher :al l shoots from eight hybrids were resistant and those from one hybrid were sensitive. 63

Isozyme analysis Bothparenta l species, L. esculentum andI . peruvianum, haddifferen t isozyme patterns(Fig .3 )fo r Aps-1, alocu swhic hencode saci dphosphatase-1 ,adlraeri c enzyme(Ric k1983) .I nth easymmetri csomati chybrids ,thre edifferen tpattern s were found (Fig.3) :on e pattern resembled the tomato patternwit h one fast migratingband ;anothe rpatter nha dsi xbands ,al lband sfro mbot hspecie san d twonovel ,presume dheterodimeri cones ;th ethir dpatter nconsiste do fth esingl e band from tomato, one of the L. peruvianum specific bands and one presumed interspecificheterodimeri cband .Th elatte rpatter nindicate sth epresenc eo f a single allele of the L. peruvianum Aps-1 in the hybrid. The number of asymmetric hybrids which have lost or retained Aps-1 alleles from thedono r speciesar e shown inTabl e3 .Mos t of thehybrid scontaine d allelesfro mth e donorspecies .A large rfractio no fth ehighe rdos ehybrid s(30 Han d100H )tha n ofth elo wdos e(5H )hybrid slacke d L. peruvianum alleles.Fo rtw ohybri dcalli , differencesbetwee nth eseparat e shootswer eobserved .W ecoul dno tdetermin e thenumbe ro fretaine ddono ralleles ,becaus eth ehybri dpatter no fsi xband s canresul tfro mon eo rtw o L. peruvianum alleles (datano tshown) .

5H16 5H2 30H3 Le Lp S

+ Fig.3 . Isozyme patterns of acid phosphatase, locus Aps-1, of (from left to right)asymmetri csomati chybrid s5H16 ,5H 2an d30H3 , L. esculentum cv.Bellin a (Le) and L. peruvianum PI128650 (Lp).Th e asymmetric hybrids derived from protoplastfusion sbetwee nL . esculentum and L. peruvianum, irradiatedwit h5 0G y (5H2an d5H16 )o r30 0G y (30H3)o fgamma-rays . 64

Table3 . The presence of L. peruvianum specific and hybrid bands in isozyme patterns of asymmetric somatic hybrids of L.esculentu m and L. peruvianum, irradiatedwit h50 ,30 0an d100 0G yo fgamma-ray s (5H-,30H -an dlOOH-hybrids , respectively). The tested isozymes were acid phosphatase, locus Aps-1, and glutamate oxaloacetate transaminase (GOT). The GOTband swer e assayed intw o parts: the two slow migrating bands and the three fast migrating bands (see Fig.4) ."mixed "mean stha tth eassaye dband swer eabsen t inon eshoo tfro ma hybridcallus ,wherea sthes eband swer epresen ti nanothe rshoo tfro mth esam e callus.Th enumbe ro fhybrid s inwhic hno tal l L. peruvianum andhybri dband s werepresent ,i sindicate dbetwee nparentheses .

Genotypes Aps-1 absent present mixed 5H-hybrids 1 23(5 ) 0 30H-hybrids 2 7 1 lOOH-hybrids 1 0 1(1 )

Genotypes t Slow •GO Tband s absent present mixed 5H-hybrids 1 7 0 30H-hybrids 7 14(8 ) 3(2 ) lOOH-hybrids 0 2(2 ) 0

Genotypes i Fast 'GO Tband s absent present mixed

5H-hybrids 1 6 (4) 1(1 ) 30H-hybrids 2 20(8 ) 2(1 ) lOOH-hybrids 2 0 0

GOT

slow

«« fast

Fig.4 . Representation of the isozyme pattern of glutamate oxaloacetate transaminase, found in symmetric somatic hybrids between L. esculentum and L.peruvianum .A tth erigh tsid ear eindicate dth e L. esculentum specific(E) , the L. peruvianum specific (P)an dhybri d specific (H)bands .Th e latter two typeso fband swer edivide di nslo wmigratin g (*) andfas tmigratin g(**) . 65

Forglutamat eoxaloacetat etransaminas e (GOT),fou rloc io nthre edifferen t chromosomes (Fig.1 )ar e known; at least 3ar e dimeric enzymes (Rick 1983). Isozymeanalysi s ofth ecultivate d tomatoshow sfou rGO Tbands ,whil e thato f L. peruvianum shows5 o r7 bands ,o fwhic h2 o r3 wer ea tth esam epositio na s tomatoband s (seeFi g4 Bi nChapte r 2).A typica lhybri dpattern ,a sexpresse d insymmetri c somatichybrid s(Chapte r 2),containe d 9band san di srepresente d inFig .4 .W e couldno t assesswhic hband s correlated witheac ho fth eloci . Therefore,th e L. peruvianum specifican dhybri dband swer edivide di nslo wan d fastmigrating ,becaus e thesesegregate d independently.Th epresenc e ofthos e bandsi nth easymmetri chybrid swa sdetermine d(Tabl e3) .A si nth ecas eo f Aps- 1, mosthybrid sha dretaine ddono ralleles .Som ehybri d calliha dregenerate d shootstha tdiffere dwit hrespec tt oth eretaine ddono rbands .Th elos so fslo w migratingGO Tband swa sdos edependent .Th efas tband swer eretaine dmor eofte n and the frequency of their loss was not clearly dose dependent. Probably, productsfro mmor etha non elocu sar epresen ti nth efas tmigratin gbands .Fou r hybridsha da complet ehybri dpatter no f9 band san dth epattern so fonl ytw o hybrids resembled the L. esculentum pattern. The latter hybrids do not necessarily lack all L. peruvianum specific alleles, because several L. peruvianum bands comigrate with L.esculentu m bands on the Polyacrylamide gels.

Morphological analysis For the analysis of the retention of L. peruvianum alleles of morphological markergenes ,w eha duse d L. esculentum genotypestha twer ehomozygou srecessiv e for suchgene sa srecipien t (Table 1); L. peruvianum carriesdominan tallele s foral lmorphologica lmarke rgene sinvolved .Th ecomplementatio no fth erecessiv e L. esculentum allelesi nth easymmetri c30H-hybrid si sgive ni nTabl e4 .I nth e majority of these hybrids, the recessive markers were complemented by L. peruvianum specificalleles .Severa lhybri dcall i( 7ou to f102 )regenerate d shootswhic hdiffere dwit hrespec tt oth eretentio no f L. peruvianum alleles. Themarke r£> , whichi sco-dominan tan dpresen ti nal l L. esculentum genotypes used,i sinclude d inTabl e4 .Th ecorrespondin g geneo fL .peruvianu m is B( = Beta,increas eo fß-caroten ean dreductio no flycopene) .Th ere dcolou r(cause d bya hig hleve lo flycopene )o f L. esculentum fruitsindicate sabsenc eo f B. The symmetric hybrids,containin g complete genomes ofbot h parental species,ha d yellow fruits (Chapter 2).Th e asymmetric hybrids set yellow, orange or red fruits(se eTabl e4 i nChapte r4) .So ,a tleas ton e Ballel ei spresen ti nth e plantstha tse tyello wan dorang efruits ;thi swa sth ecas ei n92 %(1 1ou to f 12)o fth e5H-hybrid san d80 %( 4ou to f5 )o fth e30H-hybrids . 66

Table4 . Complementation of marker genes in asymmetric somatic hybrids of L. esculentum and 300G ygamma-irradiate d L. peruvianum. The table showsth e numbero fhybrid shavin gretaine d L. peruvianum allele(s)o feac ho fa serie s ofmarke rgenes ,"absent "mean stha tn ocomplementatio nwa sobserve d inan yo f theanalyse d shoots;"present "mean stha tcomplementatio nwa sobserve d inal l analysedshoots ;"mixed "mean stha tcomplementatio nwa sobserve di npar to fth e analysedshoot so fa hybrid .Observation swer emad eo ngreenhouse-grow nplants , derived from 71hybri d calli,an d onshoot s invitro .Th elatte r shootswer e assayedonl yfo rclearl yscorabl ephenotypes .

Marker Chromosome Corresponding L. peruvianum allele(s) gene position inasymmetri chybrid s absent present mixed dgt 1-152 1 1 sy 3-4 6 4 4 sf 3-111 4 6 clau 4- 0 1 1 di 4-8 9 0 2 af 5-1 4 0 1 yv 6-3 4 0 2 c 6-104 3 0 b * 6-106 1 4 var 7- 0 0 4 not 7-4 0 0 2 1 8- 0 0 1 al 8-6 7 1 5 ah 9-2 4 4 6 marm 9-6 2 0 3 1** hi 11-4 8 1 12 2 a 11-6 8 6 9 alb 12- 0 -L _5 ~Aft*

total 27 68 b, low ß-carotenean dhig hlycopen e afe wsector so fon eplan twer e marm oneshoo ti nvitr oo feac hhybri dwa s alb

Eacho fth e L. esculentum multiplemarke rline scontaine d twomarker so na singlechromosom e (Table 1). Therefore,i twa spossibl et odetermin ewhethe ra given L. peruvianum chromosome was transferred completely by analysing both markers.Th efrequenc yo fcomplementatio no fno ,on eo rbot ho fthes egene si s shown in Table5 (Aps-1 and B data included). In 36% of the plants complementation of only one genewa s observed. This suggests the presenceo f fragmentso rincomplete ,delete dchromosome so f L. peruvianum inthes easymmetri c hybrids. 67

Table 5. Complementation of L. esculentum marker genes located on a same chromosomeb ycorrespondin g L. peruvianum alleles.Observation swer ecarrie dou t onasymmetri c somatichybrid so f L. esculentum and L. peruvianum irradiatedwit h 300Gy . Only those plants were included where both markers could be scored unambiguously. Thegen e symbols supplementedwit h+ indicateth epresenc eo fth e wild typegen ederive dfro mL . peruvianum inth easymmetri c hybrids. ApsE means an Aps-1 isozyme pattern with only the L. esculentum specific band, and ApsP means that L. peruvianum specific and hybrid bands were also present; b means observation of red fruits indicating only L. esculentum specific alleles to be present, while B means the presence of the L. peruvianum specific allele(s), because of the observation of orange fruits.

Chromosome Phenotypic observations Single comp lementation

3 sy sf sy sf* sy* sf sy* sf* 3 1 0 4 12*5%

4 clau di clau di+ clau* di clau* di* 0 1 0 1 50%

6 yv c yv c+ yv+ c yv* c* 0 0 2* 0 100 %

6 ApsE b ApsE B ApsF b ApsP B 1 0 0 4 0 %

7 var not var not* var*no t var* not* 0 0 -A * 2 33 %

8 1 al 1 at 1+ al 1* al* 0 0 0 1 0 %

9 ah marm ah marm+ ah* marm ah* marm* 0 0 2 50%

11 hi a hi a* hi* a 0 2* 3## 3 62«

4 12 17 12/33 - 36%

one of both also was ApsP c (hybrid 30H37) concerning one subclone, other subclone was var*not* (hybrid 30H36) only sectors of one shoot,othe r shoots were ah marm (hybrid 30H36) * one subclone of one hybrid, other subclone was hl*a* (hybrid 30H33) ** one subclone of one hybrid, other subclone was hi a (hybrid 30H22)

Discussion

The results presented in this paper show that a large amount of L. peruvianum genome isretaine d inasymmetri c somatichybrid so fL . esculentuman d irradiated L. peruvianum. This is in agreement with the relatively large number of donor chromosomes, observed in these hybrids (Chapter 4).Fro m the isozyme analysis 68 ofth ehybrids ,i tappear stha tth ehighe rdos ehybrid s(30 Han d100H )retaine d less L. peruvianum specificallele stha nth elowe rdos ehybrid s(5H) .Thi sagree s withth eobservatio ntha tth ehighe rdos ehybrid sresembl e L. esculentum more thanth elo wdos ehybrid si ngenera lmorphologica lappearanc e (Chapter4) . Thelimite deliminatio no fdono rgenom ei nou rasymmetri csomati chybrid si s incontras twit hasymmetri chybrid so fothe r species,whic hha d retainedonl y oneo ra fe wdono rchromosome s(Bate se tal .1987 ;Dudit se tal .1980 ;Gupt ae t al.1984) ,o reve non eo ra fe wtrait s(Dudit se tal .1987 ;Somer se tal .1986) . Thelimite deliminatio n inth etomat oasymmetri chybrid sca nb ea consequenc e ofth eunintende dselectio nfo rgoo dcallu sgrowth ,becaus ethi strai ti sbette r in L.peruvianu m than in L. esculentum, is multi-genic and not linked to regeneration capacity (Koornneef et al. 1987a). Another explanation for the limitedeliminatio ncoul db eth elac ko fsomati cincongruity .I nth ecase swit h much elimination, relatively unrelated species (from different genera or families)wer efused .N osymmetri chybrid scoul db eobtaine dfro mthes especies . Therefore,i fsomati cincongruit yoccurs ,onl yhybrid swit ha minima lamoun to f donorgenom eca nsurvive .Whe nrelate d specieswer efused ,th eeliminatio no f donor genome was often limited (e.g.Famelae r et al. 1989;Yamashit a etal . 1988).Thos especie swer esomati ccongruent ,becaus eals osymmetri chybrid scoul d be obtained. An exception are some of the asymmetric hybrids of the related species Nicotiana tabacum (+) N. plumbaginifolia (donor),tha tcontaine donl y onedono rchromosom e (Batese tal .1987) . Kanamycin resistance was retained in 46 percent of the 30H-hybrids, that derivedfro ma dono rparen twit hon eNP TI Ilocus .Thi spercentag ei sa naverage , obtainedfro mth eanalysi so f1 6independen tkanamyci nresistan t L. peruvianum genotypes.I fth eNP TI Igen eintegrate srandoml yi nth e L. peruvianum genome, thiswoul d implytha tan yallel eo fth edono rgenom ei sretaine d inabou t50 % ofth ehybrids .I ncontras twit h the singlekanamyci n resistance allele,th e isozyme and morphological markers were represented by two alleles in the L. peruvianum donorgenome .Whe na hybri disozym epatter no rcomplementatio no f amutan tphenotyp ewa sobserved ,eithe ron eo rtw odono rallele swer epresent . Theaverag echanc etha ta certai nallel e isretained ,ca nb ededuce dfro mth e numbero fhybrid swhic hha dlos tbot hhomologou salleles .Th eisozym emarker s (Table3 )an dmorphologica lmarker s(Tabl e4 )wer edistribute dmor eo rles sa t randomove rth egenome .Th efrequence so flos so fbot hdono rallele so fa give n locusi nth e30H-hybrid swas ,o nth eaverage ,21 %t o30 %("mixed "i sconsidere d aslos ti nth elatter) .A nindividua l L. peruvianum allelei sthu slos twit ha frequency of 0.21 to 0.30 = 46% to 55%,an d is therefore retained with a frequency of 45%t o 54%.Thes e frequencies agree well with the frequency of retentiono fth ekanamyci nresistanc ealleles . 69

Wefrequentl yobserve dtha tshoots ,whic hderive dfro ma sam ehybri dcallus , differedwit hrespec tt oon eo rmor eo rth eanalyse dmarkers ,namel yi ntw oo f the3 8teste d5H-hybrid s (5%),1 0o fth e6 330H-hybrid s (16%)an don eo fth e9 lOOH-hybrids (11%).Apparently ,th ehighe rdos ehybrid sshowe dthi sphenomeno n more often. The segregation occurred most probably in the hybrid callus. Segregationa tth eplan tleve lwa sshow ni non ehybri dshoo tcontainin gsector s inth elea ftha twer e marmorata, thephenotyp eo fth erecipien tLA116 4(Tabl e4) . Somaticsegregatio nha sals obee nobserve di nasymmetri chybri dcall ian dplant s of Nicotiana plumbaginifolia (+)(irradiated )N. sylvestris forsevera lisozyme s (Famelaere tal . 1989). Becauseth ecomplementatio no fonl yon eo ftw ogene so nth esam echromosom e occurredrathe rfrequentl y(Tabl e 5),a larg efractio no fth easymmetri csomati c hybrids must have contained incomplete chromosomes of L. peruvianum. Ineac h fusioncombinatio nthi scoul db eanalyse dfo ronl yon eo rtw ochromosomes .Eve n when both markers were complemented by L. peruvianum alleles,thi s may have resultedfro mth eretentio no ftw oincomplet echromosomes .Therefore ,w econclud e that the high dose of gamma irradiation (300Gy )induce d many breaks inth e L. peruvianum chromosomes.Thi swa sconfirme d by theRFL Panalysi s of 730H - hybrids, in which, on the average, at least 12 of the 18 retained donor chromosomeswer eincomplet e (Chapter6) . Theasymmetri c somatichybrid swer eselecte do nregeneratio ncapacity .Thi s traito fL .peruvianu mi ssuppose dt ob egoverne db ytw ounlinked ,dominan tgene s (Koornneefe tal .1987a) .Theoretically ,i tshoul dhav ebee npossibl et olocat e these regeneration genes inou r experiments,becaus e of the availability of markergene sfo reac ho fth echromosomes .Gene so f L. peruvianum linkedt oth e regenerationgenes ,shoul db epresen ti nth easymmetri chybrids .However ,w ewer e notabl et olocat ethes egenes .Thi scoul dhav ebee ncause dby :(1 )th epresenc e of many L. peruvianum chromosomes in the asymmetric hybrids; (ii)th e small number of asymmetric hybrids that had lost any given gene; (iii)th e high frequencyo fbreakag eo fth edono rchromosomes ,s otha tonl yclosel ylinke dgene s couldb euse dfo rthi sanalysis .Nevertheless ,w eca nstat etha tth eregeneratio n genesar eno tclosel ylinke dt o sy, sf (bothmappe do nchromosom e 3), hi anda (bothchromosom e 11),becaus e both L. peruvianum alleles of thesegene swer e absenti na larg efractio no fth easymmetri chybrids .

Acknowledgements. Thisresearc hwa ssupporte db yth eFoundatio nfo rFundamenta l BiologicalResearc h (BION),whic hi ssubsidise db yth eNetherland sOrganisatio n for Scientific Research (NWO). We are very grateful to Prof. CM. Rick for supplyingth etomat otesterlines ,t oDr .P.J.M .va nde nElze nfo rprovidin gth e plasmidpAGS112 ,t oJos éKok ,Patt yva nLoene nMartinet-Schuringa ,Anj aPosthuma , RenéRijke nan dJann yVo sfo rdoin gpar to fth eexperiments ,an dProf .C .Heytin g forcriticall yreadin go fth emanuscript . 70

References

BatesGW ,Hasenkamp f CA,Contolin iCL ,Piastuc hW C (1987)Asymmetri ehybridizatio n inNicotian ab y fusion of irradiated protoplasts. TheorApp lGene t 74:718-726 DellaportaSL ,Woo dJ ,Hick sJ B(1983 )A plan tDN Aminipreparation :versio nII .Plan tMolecula rBiolog yReporte r 1:19-21 DuditsD ,Feje r0 , Hadlaczky GY,Konc zCS ,Laza rG , HorvathG (1980)Intergeneri c gene transfer mediated by plantprotoplas t fusion.Mo lGe nGene t 179:283-288 DuditsD ,Maro yE ,Praznovszk y T,Ola hZ ,Gyorgye y J,Cell aR (1987)Transfe ro fresistanc etrait sfro m carrot into tobaccob y asymmetric somatic hybridization: Regeneration of fertile plants. Proc Natl Acad Sei USA 84:8434-8438 FamelaerI ,Gleb aYY ,Sidoro vVA ,Kaled aVA ,Parakonn yAS ,Boryshu kNV ,Cheru pNN ,Negruti u I,Jacob sM (1989) Intrageneric asymmetric hybrids between Nicotjana plurobaRinifolia and Nicotiana sylvestris obtained by 'gamma-fusion'.Plan t Science 61:105-117 Gleba YY, Hinnisdaels S, Sidorov VA,Kaled a VA, Parokonny AS,Boryshu k NV, Cherup NN, Negrutiu I, Jacobs M (1988)Intergeneri c asymmetric hybridsbetwee nNicotian a plumbaginifolia andAtrop abelladonn aobtaine db y "gamma-fusion". TheorApp iGene t 76:760-766 Gupta PP, Schieder 0, Gupta M (1984) Intergeneric nuclear gene transfer between somatically and sexually incompatibleplant s through asymmetric protoplast fusion.Mo lGe nGene t 197:30-35 KoornneefM ,Hanhar tCJ ,Martinell iL (1987a)A geneti c analysis ofcel lcultur e traits intomato .Theo rApp l Genet 74:633-641 KoornneefM , JongsmaM , Weide R, Zabel P, Hille J (1987b)Transformatio n of tomato. In:Nevin s DJ, JonesR A (eds)Tomat obiotechnology . AlanR Liss, Inc.Ne wYork ,p p 169-178 Mutschler MA, Tanksley SD,Ric k CM (1987)Linkag e maps of the tomato (Lvcopersicon esculentum). Rep Tomato GenetCoo p 37:5-34 Rick CM (1982)Stoc k list.Re pTomat oGene tCoo p 32:3-10 RickC M (1983)Tomat o (Lvcopersicon).In :Tanksle y SD,Orto nT J (eds)Isozyme s inplan tgenetic san dbreeding , partB . Elsevier,Amsterdam , pp 147-165 SomersDA ,Narayana nKR ,Kleinhof sA ,Cooper-Blan d S,Cockin gE C (1986)Immunologica l evidencefo rtransfe ro f the barley nitrate reductase structural gene to Nicotiana tabacum by protoplast fusion. Mol Gen Genet 204:296-301 SuursLCJM ,Jongedij kE ,Ta nMM C (1989)Polyacrylamid e gradient-gelelectrophoresis :a routin emetho d forhig h resolution isozyme electrophoresis of Solanum and Lvcopersicon species.Euphytic a 40:181-186 Vallejos CE (1983) Enzyme activity staining. In:Tanksle y SD,Orto n TJ (eds) Isozymes inplan t genetics and breeding,par tA . Elsevier,Amsterdam ,p p 469-515 Yamashita Y, Terada R, Nishibayashi S, Shimamoto K (1989) Asymmetric somatic hybrids of Brassica: partial transfer ofB . canroestrisgenom e into B.olerace a by cell fusion.Theo rApp lGene t 77:189-194 Young ND, Tanksley SD (1989) Restriction fragment length polymorphism maps and the concept of graphical genotypes. TheorApp lGene t 77:95-101 71

CHAPTER6

ASYMMETRIC SOMATIC HYBRIDS BETWEEN Z^YCOPEJRJSICON ESCUUENTUM AND IRRADIATED UYCOFERSICON PEISLTVIANUM III. ANALYSIS WITH RESTRICTION FRAGMENT LENGTH POLYMORPHISMS

J.Wijbrandi ,P .Zabel ,M .Koornnee f

Summary.Th egenom ecompositio no fasymmetri csomati chybrids ,obtaine db yfusio n of leaf protoplasts from Lycopersicon esculentum and gamma-irradiated leaf protoplastsfro m L. peruvianum,wa scharacterise db ySouther nblo tanalysi swit h 29RFL Pmarker san da rDN Aprobe .Eigh t"lo wdos ehybrids "an dseve n"hig hdos e hybrids" (irradiation dose 50G yan d 300Gy ,respectively )wer e analysed.B y densitometric scanning ofth eautoradiographs ,th enumbe r ofallele sfo reac h locus of the component species was established. In general, elimination of allelesfro m theirradiate dL .peruvianu mdono rgenom ewa slimite dan drange d from 17%t o69% .Thre e L. peruvianum loci,locate d onchromosom e 2,4 an d7 , respectively,wer epresen ti neac hasymmetri chybrid ,whic hma ysugges tlinkag e toth eregeneratio ncapacit y traitwhic hwa suse d inselectin g theasymmetri c hybrids.Th elos so fdono rgenom ewa sdos edependent .Lo wdos ehybrid scontaine d morealleles ,loc ian dcomplet e chromosomesfro m L. peruvianum thanhig hdos e hybrids,wherea s thehig hdos ehybrid scontaine d more incompletechromosomes . Inmos thybrid s some L. esculentum alleleswer elost .Th eamoun to fribosoma l DNAfro m L. peruvianum retained inth easymmetri chybrid svarie dstrongly :i n onehybri damplificatio nha doccurred ,wherea si nother s L. peruvianum rDNAwa s eitherabsen to r(in)completel ypresent .Th eamoun to frDN Afro m L. esculentum wasdecrease d'i nmos t asymmetric hybrids.Th e possible implications ofthes e resultsfo rth eus eo fasymmetri chybrid si nplan tbreedin gar ediscussed .

Introduction

Variousstrategie shav ebee ndevelope dfo rcombinin gth egeneti cinformatio no f twoplan tspecie swhic hca nno tb ehybridise dsexually .Whil eDN Atransformatio n techniquesar eparticularl ydesigne d toallo wth e introductiono fdefine dbu t small-sizedDN Afragment sint oa recipien tspecies ,protoplas tfusio nan drelate d cellular techniques are the obvious means for transferring large chromosomal segmentso reve ncomplet egenome s(fo ra revie wconcernin gtomato ,se eHill ee t al.1989) .However ,i nmos tprotoplas tfusio nexperiments ,onl ya limite dnumbe r oftrait sfro mth edono rspecie si so finteres tan deliminatio no fth ebul ko f thedono rgenom eha st ob eachieved .I nmammalia nsomati ccel lhybrids ,preferen ­ tial loss of chromosomes from one parent species often occurs spontaneously (Ringertzan dSavag e 1976). Inplan tsomati ccel lhybrids ,however ,chromosom e elimination is limited and, in general, does not concern specifically the 72 chromosomes of one of the component species (Gleba and Sytnik 1984). Ina n attempt to decrease the amount of donor genome, several investigators have irradiated thedono rprotoplast sbefor efusio nwit hRöntgen -o rgamma-rays ,s o ast oinduc elos so fentir echromosome so rchromosom esegments .I nsom ecases , oneo ra fe wdono rchromosome swer efoun dt ob eretaine dwithi nth ehybri d(Bate s et al. 1987; Dudits et al. 1980; Gupta et al. 1984), whereas in others, elimination of the irradiated genome appeared to belimite d (Famelaer et al. 1988;Gleb ae tal .1988 ;Imamur ae tal .1987 ;Müller-Genser tan dSchiede r1987 ; Yamashitae tal .1989) . In the present paper we have analysed the effects of irradiation of L.peruvianu mo nth eeliminatio no fth e L. peruvianum genomei nL . esculentum (+) L. peruvianum somatichybrids .Fo rthi spurpos e L. peruvianum protoplasts were irradiated beforefusio nwit heithe ra lo wo ra hig hdos eo fgamma-rays , andth easymmetri c somatichybrid swer e selected onth ebasi so fth esuperio r regenerationcapacit yo fL .peruvianum .Previously ,evidenc eha sbee npresente d showingtha tth eregeneratio ncapacit ytrai to f L. peruvianum isdominan t(Adam s andQuiro s1985 ;Kinshar ae tal .1986 ;Koornnee fe tal .1987a ;Wijbrand ie tal . 1988)an dlikel yt ob econtrolle db ya limite dnumbe ro fgene s(Koornnee fe tal . 1987a).B ymean so f3 0RFL Pmarker so fknow nchromosoma l location,th egenom e constitutiono fa numbe ro fasymmetri chybrid swa sestablished .

Materialsan dmethod s Plant material Somatichybri dplant swer eobtaine dfro m independentprotoplas tfusio nevent s (Wijbrandi et al. 1988). The parental genotypes of thehybrid s are shown in Table1 . Seeds of the tomato cultivar Belllna, the LA-genotypes and the L.peruvianu maccessio nPI12865 0wer ekindl yprovide db yRij kZwaa nSee dCompan y (deLier ,Th eNetherlands) ,Prof .C.M .Ric k(Tomat oGenetic sStoc kCenter ,Davis , USA) and the Institute of Horticultural Plant Breeding (Wageningen, The Netherlands),respectively .Th e L. peruvianum parentwa sirradiate dbefor efusio n witha dos eo feithe r5 0Gra y("lo w dosehybrids" ,designate da s5H )o r30 0Gra y ("highdos ehybrids" ,30H) ,usin g a60 Cosourc e at ados e rate ofabou t 2000 Gray/hour(a tth ePilot-Plan tfo rFoo dIrradiation ,Wageningen ,Th eNetherlands) . The symmetric somatic hybrid 0H1 (2n= 4x ), L . esculentum cv.Bellina , and L. peruvianum PI128650-ATW2002wer euse da scontrol si nDN Aanalysis . Hybridisation probes Thefollowin gtomat osingl ecop yclone swer eused :(i )th ecDN Aclone sCD1 ,CD14 , CD15, CD27, CD41,CD50 , CD56, CD59 (Bernatsky and Tanksley 1986)an d "Adh2" (pCB25E6,containin ga par to fth eAdh- 2codin gregion ;Chas ean dWilliam s1986 , seeals ova nDaele ne tal .1989) . (ii)th egenomi cclone sTG8 ,TG9 ,TG16 ,TG20 , TG27,TG30 ,TG31 ,TG34 ,TG37 ,TG42 ,TG43 ,TG44 ,TG45 ,TG50 ,TG54 ,TG61 ,TG62 , TG63,TG68 ,TG6 9 (Tanksley etal . 1988)an d "Adhl" (pTAdh-1,a genomi c1. 8k b fragmentclone di nth e Eco RI-Sal Isit eo fpTZ18R) .Th eCD -an dTG-clone swer e kindlysupplie db yDr .S.D .Tanksley ,Cornel lUniversity ,Ithac a(USA) ,pCB25E 6 wasa gif tfro mDr .Th .Chas eJr. ,Rutger sUniversity ,Ne wJerse y (USA),an d pTAdh-1fro mDr .E .Lifschytz ,Technion-Israe l Institute ofTechnology ,Haif a 73

Table 1. The parental genotypes of the somatic hybrids and the dose of gamma- irradiation applied to thedonor .

Hybrid Recipient Donor Dose (L. esculentum) (L. peruvianum) (Gray)

0H1 cv. Bellina PI128650-ATW2002* 0

5H2, 5H3,5H5 , 5H7, cv. Bellina PI128650-ATW2002* 50 5H13, 5H16, 5H28

5H22 cv. Bellina PI128650 50

30H1, 30H3, 30H7 cv. Bellina PI128650-ATW2002* 300

30H6, 30H12, 30H27 LA 1182** PI128650-ATW2003* 300

30H26 LA 291*** PI128650-ATW2001* 300

transgenic plants containing akanamyci n resistance gene (seeKoornnee f et al. 1987b). tomato genotype,homozygou s recessive for sy, sf (chr. 3),an d alb (chr. 12) (Rick 1982). tomato genotype,homozygou s recessive for hi and a (chr. 11) (Rick 1982).

(Israel).A peagenomi c clone for45 SrRNA , "R45S" (pBsARl),wa sa gif t fromDr . J.P. Nap, Research Institute Ital, Wageningen (The Netherlands). The map positionso fth eclone suse d inth ehybri danalysis ,ar e showni nFig . 1.Excep t for the ribosomal clone, the insert of each clone was excised from the vector with the appropriate restrictionendonuclease s and separated from thevecto r on a 1%agaros e gel.DN Afragment swer e radiolabeled with [32P]dATPb ymean s of the random-priming method (Feinberg and Vogelstein, 1983). Plant DNA analysis Total DNAwa s extracted from leaves (fresh or freeze dried)o f plants grown in the greenhouse, as described by Dellaporta et al. (1983). DNA samples (2.5/ig ) were digested to completion with Eco RI, Eco RV, Hin dill, Dra I or Pst I (5hour s at 37 °C in buffers according to Maniatis et al. 1982), separated by electrophoresis on 0.7% agarose gels and transferred to Gene Screen PLUS (New EnglandNuclear )b ymean so fa nalkalin eblottin gprocedur e (Reedan dMan n 1985). Hybridisation in10 %dextra nsulphate ,1 %SD San dI MNaCl ,a t6 5 °C,wa s carried out for 18hour s under conditions as recommended by the manufacturer of the membrane. After hybridisation, blots were washed under stringent conditions: three 5min.-washe s in 2xSSC at 20 °C, two 20min.-washe s in 2xSSC+ 1% SDS at 65 °C, two 30min.-washe s in O.lxSSC + 1% SDS at 65 °C,an d two 15 min.-washes in O.lxSSC at 20 °C. Subsequently, the blots were exposed toX-ra y film (Kodak X-OmatA R or Konica H7A) at -80 °C with intensifying screens. After autoradiography, blots were deprobed in 0.4N NaOH at 42 °C according to the manufacturer. The blots could thus be used at least four times. To obtain quantitative data on the relative intensity of bands derived from both parents, autoradiographs were scanned with a LKBUltrasca n XL Laser Densitometer connected toa nOlivetti M24persona l computer,whic hwa s equipped with the LKB 2400 GelScan XL software. The following calculations weremade : (i)th e relativenumbe r of L. peruvianum andL . esculentum specific alleles for a given probe in each asymmetric hybrid was determined as the ratio of the absorptionvalue s (peakareas )o fth erelevan tband so nth eautoradiographs .Thi s ratio was divided by the corresponding ratio in the symmetric hybrid 0H1 that was used as a reference(=1) ; 74

1 2 3 4 5 6 7 8 9 10 1112 1&.R45S °TCD4f°-TCD14 •TG9 4- -TG31 10- -CD1 •CD59 ™--TG20 M-\-TG44 TG43 .CD50 tgU-COTS 3g_ -Adh2 *1--CD27 43- -TGS4 B\--Adh1 TG16 52--TGS0 59. TG62 S6-X-TG42

7 3+TG69 75. 7 3-i-rag 'TGS 1 ' TG45 *S->-TG3Cn-TQ6S

103-L TG37 io6J_r

M6--TG34

W±-TG27 Fig.1 .Linkag ema po fth eRFL Pmarker suse di nth eanalysi so fth easymmetri c somatichybrids .Th eshade darea sindicat eth ecentromer eregions .Al lposition s areaccordin gt oYoun gan dTanksle y (1989).

(ii)th erati oo fth eabsorptio nvalue so fth eL . esculentum specificban di n eachasymmetri chybri dan dtha ti nth esymmetri chybri d0H 1wa sdetermine dfo r a given probe. If all L. esculentum genomes in the asymmetric hybrid were complete,thi srati oshoul db eth esam efo reac hprob e thatwa shybridise dt o thesam eblot ;th eus eo fth eratio sfro mon eblo texclude dpossibl evariation s inth eamoun to fDN Aloaded/transferre dfro meac hhybrid .Deviatin gratio sfo r individual probes in an asymmetric hybrid were therefore interpreted as deviationsi nth eamoun to f L. esculentum specificDNA/alleles .

Results

Restriction fragment length polymorphisms (RFLPs) between L. esculentum and L. peruvianum Toestablis hprobe-restrictio n enzyme combinationswhic hrevea l polymorphisms betweenth etw oparents ,Souther nblot so fDN Afro mL .escuientum ,L . peruvianum and0H1 ,a symmetri csomati chybri dbetwee nbot hspecies ,wer ehybridise dt oeac h ofth eRFL Pmarker smentione d inth eMaterial san dmethod ssection .Eac hprob e was tested on DNA digested with the restriction enzymes Eco RI, Hin dill or 75

Dra I;i naddition ,1 3an d2 2probe swer eteste do n Eco RV-an d Pst I-digests, respectively (Table 2). Foreac ho fth eenzyme s tested,approximatel ya thir d of theprobe s showeda "useful"polymorphis m intha t eacho f thetw oparent s displayedon eo rmor euniqu ebands .Approximatel ya nothe rthir do fth eprobe s revealed a polymorphism, referred to as "not useful",wit h only one species

Table2 .Restrictio nfragmen tlengt hpolymorphism s(RFLPs )betwee n L. esculentum and L. peruvianum. PlantDN Awa sdigeste dwit h Eco RI, EcoRV , Hin dill, DraI or Pst I,fractionate db yagaros ege lelectrophoresis ,transferre dt oGen eScree n PLUSan dhybridise dwit hDN Afro mth eclone sindicated .+ - "useful "RFLP ,bot h parentshav e(a )uniqu eband(s) ;+/ -= "no tuseful "RFLP ,onl yon eo fth eparent s hasa uniqu eband ; -= n oRFLP ;n d= no tdetermined .Th erigh tcolum n(total ) indicateswhethe ra "useful "RFL Pwa sfoun dfo reac hprob ewit hth erestrictio n enzymestested .Th elowe rpane lshow sth etotal so f"useful" ,"no tuseful "an d noRFLP sfo reac hrestrictio nenzyme .

Probe Eco RI Eco RV Hin dlII Dra I PstI Total

TG8 +/- nd + + + + TG9 nd + - nd + TG16 +/- +/- +/- + - + TG20 +/- nd + +/- - + TG27 + nd + +/- + + TG30 +/- nd + +/- nd + TG31 + nd - - - + TG34 +/- nd + +/- - + TG37 +/- nd +/- + - + TG42 + +/- +/- +/- +/- + TG43 +/- + +/- +/- - + TG44 + - +/- - - + TG45 - - - + - + TG50 +/- nd +/- + + + TG54 +/- nd +/- + +/- + TG61 +/- + - +/- +/- + TG62 - + - - - + TG63 + nd - + - + TG68 + - + - nd + TG69 + + +/- +/- nd + CD1* - + +/- + nd + CD14 - nd + + nd + CD15 + nd + + + + CD27 + nd - + - + CD41 +/- + +/- +/- +/- + CD50 - + + +/- nd + CD56 +/- nd +/- - nd +/ CD59 + nd +/- - nd + "Adhl" +/- nd +/- +/- + + "Adh2" + + + + nd + "R45S" - nd - + - + + 11 8 11 13 5 30 +/- 13 2 13 11 4 1 7 3 7 7 12 0 excisiono fth einser to fCD 1wit hPs tI yielde dthre efragment so f0. 5kb , 1.0k ban d1. 8kb ,respectively ;th e1. 0k bfragmen twa suse di nth e hybridisationexperiments . 76 containing one ormor e specific bands.B y testing the five enzymesmentione d above,a polymorphis mwa sfoun dfo reac ho fth e3 1probes ,3 0o fwhic hshowe d atleas ton e "useful"RFL P (Table2 ;th ema pposition so fth e "useful"clone s areshow ni nFig .1) .Seve n(TG8 ,TG34 ,TG63 ,TG68 ,TG69 ,CD5 0an d"Adhl" )ou t ofth e3 1probe sproduce da hybridisatio nsigna lwit hDN Afro m L. peruvianum that was markedly weaker thanwit h L.esculentu m DNA. As expected, the symmetric somatichybri d0H 1showe dth ecomposit epatter nfo ral lclone stested .

Analysis of asymmetric somatic hybrids Southernblot so fappropriat erestrictio nenzym edigest so fDN Afro meigh t"lo w dose hybrids" (5H-hybrids), seven "high dose hybrids" (30H-hybrids)an d the controls L. esculentum, 0H1an d L. peruvianum werehybridise dwit h2 9singl ecop y clonesan da rDN Aclone .Wit hrespec tt oth enumbe ro fL .peruvianu m specific fragmentshybridising ,tw oclasse so frestrictio npattern swer edistinguished , thesingl ean dmulti-fragmen tpatterns ,respectively .A sa nexampl eo fth efirs t class,hybridisatio npattern s oftw oclone slocate d onchromosom e 2,TG3 1an d TG34,ar eshow ni nFig .2 Aan d 2B.Bot hclone sproduce d asimpl eRFL Ppatter n with each species exhibiting a single, unique fragment. The L. esculentum specificTG3 1an dTG3 4band swer epresen ti nal lhybrids ,a swa sth ecas ewit h the L. peruvianum specificTG3 4band . Incontrast ,th e L. peruvianum specific TG31ban dwa sabsen ti nfou r"hig hdos ehybrids" .Th esecon dclass ,th emulti - fragment patterns,wa s represented by eleven probes (TG27, TG62, TG37, CD41, CD14, 'Adh2', TG20, TG16, TG30,TG4 4an d TG50)an d showed a pattern sucha s exemplifiedb yclon eTG5 0i nFig .2C :on eban dpresen ti nbot hparenta lspecies , oneL .esculentu mspecifi cban dan dtw omajo r L. peruvianum specificbands .Th e asymmetrichybrid ssurveye dfo rth epresenc eo fth eL .peruvianu mTG5 0alleles , were found to contain either none, one, or two bands, which indicates heterozygosityfo rth eTG5 0locu si n L. peruvianum andlos so fspecifi callele s inth ehybrids . For all probes,a marked variation in the intensity of the hybridisation signalbetwee nL .peruvianu man d L. esculentum specificband swa sobserve damon g theasymmetri chybrids .T oquantif ythes edifferences ,th eautoradiograph swer e scannedwit ha densitometer .Th erati oo fth eban dintensitie si nth esymmetri c hybrid was used as a reference. As an example of such an analysis, the densitographs of the TG31 patterns of four asymmetric hybrids are shown in Fig.3 .Th erati oo fth eban dintensitie sfro mbot hparents ,a scalculate dfro m thepea karea swithi non elane ,wa s(L . esculentum : L. peruvianum) 1:1(hybri d 5H22),2: 1(30H3) ,4: 1(30H1 )an dx: 0(30H6) .Similarly ,thes eratios ,whic hca n beinterprete da srelativ enumber so fL . peruvianum allelescontaine dwithi nth e hybrids,wer ecalculate dfo ral lclone shybridisin gt oth efiftee nasymmetri c 77

Asymmetric hybrids f J3* L.esc. (+) L.per. J&

Probe: 50 Gy 300 Gy «r «p CxT V V v x Eco Rl TG31 -3.5k b (2- 2) "3.0k b

xHindlll TG34 •3.0 kb B (2-124) I '2.Bk b

x Dral TG50 9.4 kb (12-52) P, 6.7 l 4.4

3 2.2-0

0.6

Fig.2 .Surve yfo rth epresenc eo fth e L. peruvianumspecifi callele so fTG31 , TG34,an dTG5 0i nasymmetri chybrid sb ySouther nblo tanalysis .DN Afro meigh t lowdos ehybrid s (5H2,5H3 ,5H5 , 5H7, 5H13,5H16 ,5H2 2an d 5H28;lane s 1-8) sevenhig hdos ehybrid s (30H1,30H3 ,30H6 ,30H7 ,30H12 ,30H2 6an d30H27 ;lane s 9-15), L. esculenCum cv.Bellin a (lane 16),symmetri chybri d0H 1(lan e17 )an d L. peruvianum PI128650-ATW2002 (lane 18) was digested with the restriction enzymes indicated, electrophoresed, blotted and hybridised with the clone indicated. A. Hybridisation with clone TG31. A single fragment was detected in the respectiveparents .Th esiz eo fth efragment si sindicate da tth eright . B. Hybridisation with clone TG34. A single fragment was detected in the respectiveparents .Th esiz eo fth efragment si sindicate da tth eright . C.Hybridisatio nwit hclon eTG50 .P xan dP 2a tth elef tindicat eth etw omajo r L. peruvianum specificband san dE th eI . esculentum specificband .Th epositio n ofth e Hin dill fragmentso fphag elambd aDN Ai sindicate da tth eright . 78 hybrids.Mos thybrid sshowe dratio so feithe rx:0 ,4:1 ,2: 1o r1:1 .On ehybri d (30H12) showed ratios of x:0, 6:1 and 3:1, suggesting a higher number of L. esculentum allelesfo reac hlocu stha ni nth eothe rhybrids .I na fe wcase s (TG61i nhybri d 5H7,TG1 6an dTG4 5 inhybri d 5H28)a rati oo f1: 2wa sfound , indicating the presence of two L. peruvianum alleles for each L. esculentum allele.I nsom eothe rcase s(TG4 3i nhybrid s30H3 ,30H 7an d30H27 ;TG9 ,TG8 ,CD2 7 andTG5 0i nhybri d30H12 )ratio shighe rtha n4: 1(o r6:1 )wer efound ,suggestin g totaleliminatio no fthes e L. peruvianum allelesi npar to fth eplant . Thus, by determining the relative number of a series of L.peruvianu m specificalleles ,w ecoul dasses sth egeneti cmake-u po feac hasymmetri chybrid . Becauseth echromosom enumber ,a scounte di nmetaphas eplate so froo tti pcells , was knownfo r eachhybrid ,w e could also deduce the absolute number ofeac h allele(Tabl e3 ,4 an d 5).A sexpected ,th elo wdos ehybrid scontaine dmor eloc i and alleles from L. peruvianum than the high dose hybrids (Table 3). This

Hybrid Peakarea s Factor E/Px facto r Allelerati o E P E/P (E:P) 0H1 2.70 2.59 1.04 0.96 1 1:1 5H22 2.43 2.55 0.95 0.91 1:1 30H1 2.82 0.63 4.48 4.29 4:1 30H3 1.67 0.91 1.84 1.76 2:1 30H6 5.27 0 00 x:0" xi sa nintege r> 0 Fig.3 .Densitograph so f Eco RI-digestedDN Afro mth elo wdos ehybri d5H22 ,th e highdos ehybrid s 30H1, 30H3, 30H6an d the symmetric hybrid 0H1,probe d with TG31.Th eautoradiograp hshow ni nFig .2A ,uppe rpanel ,wa sscanne d (leftsid e of each scan corresponds to the low molecular weight region of the autoradiograph) and the species specific peak areas (P, L. peruvianum; E, L. esculentum) were calculated.Th e ratioo fth epea karea swa sdetermine d (E/P)an dnormalise db ya correctio nfacto rdeduce dfro mth erati oo fth epea k areaso f0H1 ,whic hwa suse da sa referenc e (E/P= •1) , Theseratio sar eshow n inth etabl ebelo w thedensitographs ;th eratio so fallele s inth easymmetri c hybridswer edetermine dfro mthes ecorrecte dvalues . 79

Table3 . The amount of L. peruvianum genome (number of loci, alleles and chromosomes)i nth e"lo wdose" -an d"hig hdose "asymmetri chybrids ,a sdetermine d by autoradiographic analysis and densitometric scanning of Southern blots hybridised with 29 single copy clones (29RFLP s = 29loc i - 58alleles) . A chromosomewa sconsidere dcomplet ewhe nfo reac hlocu so fth echromosom eth esam e numbero fallele swa sfound .

Lowdos ehybrid s Highdos ehybrid s (n-8) (n-7)

Loci(n-29 )* 27.3± 3. 4(94% ) 20.4± 4. 3 (70%)

Alleles(n-58 ) 38.6± 7. 8 (67%) 30.6±11. 2(53% )

Completechromosome s 13.4+2.8 7.1± 4. 1 Incompletechromosome s* 4.9± 1. 6 11.4± 3. 2 Lostchromosome s 5.8+3.6 6.9±3.9 significant difference between the two groups of hybrids, as tested with Student'st tes t( P< 0.01) .

differencewa sals oobviou sa tth echromosom elevel ;a chromosom ewa sconsidere d completewhe nfo reac hlocu so fth echromosom e thesam enumbe ro fallele swa s present.Lo wdos ehybrid scontaine dmor ecomplet eL .peruvianu mchromosome stha n highdos ehybrids ,wherea s thehig hdos ehybrid scontaine d ahighe rnumbe ro f incomplete chromosomes. Surprisingly, the number of lost L. peruvianum chromosomeswa slo wan dvirtuall yth esam efo rbot hhybri dtype s(Tabl e 3). In general,th echromosom enumber sderive dfro mth eRFL Pmappin gdat aapproximate d thenumbe ro fchromosome sa sdetermine db ycountin gmetaphas eplate so froo ttip s (Table 4). Inte nou to ffiftee nasymmetri chybrids ,th etota lnumbe ro fcomplet e chromosomesfro mL .esculentu man d L. peruvianum asdetermine db yRFL Panalysi s was somewhat lower than thenumbe r of counted chromosomes,wherea s thetota l numbero fcomplet eplu sincomplet echromosome swa ssomewha thigher . In most hybrids, one to several L. esculentum specific alleles were lost (determineda sdescribe di nMaterial san dmethod ssection) ,a sshow ni nTabl e4 . Except for twolo wdos ehybrid s (5H3an d 5H5), no euploidnumbe r ofcomplet e L. esculentum chromosomeswa sfoun damon g theasymmetri chybrids .Fou rhybri d plants had lost one to three complete L. esculentum chromosomes, either by missingon eou to ftw o(5H28 ,chromosom e 8), oneou to ffou r(5H16 ,chromosome s 3,4 an d9 ;30H7 ,chromosom e 9),on eou to fsi x(30H12 ,chromosom e3 )o rtw oou t ofsi x(30H12 ,chromosom e 9)homologou schromosomes . 80

Table4 .Th enumbe ro f L. esculentum (E)an d L. peruvianum (P)chromosome si n 8lo wdos easymmetri chybrid s (5H2t o5H28 )an d7 hig hdos easymmetri chybrid s (30H1t o30H27) ,a sdetermine db yRFL Panalysi swit h2 9singl ecop yclone san d by chromosome counting inmetaphas e plates ofroo t tip cells.Th echromosom e numberso fth eparenta lspecie san da symmetri chybri d (0H1)ar eliste dbelow . The total number ofpresume d complete chromosomes,indicate d as "c",an d the total number of incomplete chromosomes, "+inc" between parentheses, of each genotypear egive na swell .

Genotype Chromosome number RFLP analysis metaphases complete incompl etelos t total 2n E P E P E P c(+inc)

5H2 23 13 1 8 0 3 36(+9) 37-40 5H3 24 14 0 4 0 6 38(+4) 37-41 5H5 24 16 0 4 0 4 40(+4) 39-43 5H7 20 13 4 4 0 7 33(+8) 41-43 5H13 45 13 3 6 0 5 58(+9) 59-63 5H16 45 7 0 4 3 13 52(+4) 56-58 5H22 23 16 1 6 0 2 39(+7) 39-45 5H28 23 14 0 6 1 4 37(+6) 35-42

30H1 45 6 3 15 0 6 51(+18) 56-62 30H3 44 7 4 8 0 9 51(+12) 54-59 30H6 44 4 4 7 0 13 48(+ll) 50-58

30H7 45 6 2 11 1 7 51(+13) 55-61 30H12 62 6 7 12 3 7 68(+19) 76-85 30H26 43 16 5 14 0 0 59(+19) 64-70 30H27 43 7 5 11 0 6 50(+16) 68-70

L.esculentu m 24 0 0 0 0 0 24(+0) 24 0H1 24 24 0 0 0 0 48(+0) 48 L.peruvianu m 0 24 0 0 0 0 24(+0) 24 81

Torevea la possibl elinkag eo fthes eloc iwit hregeneratio ncapacity ,whic h traitwa suse dt oselec tth easymmetri chybrids ,th epresenc eo f3 0individua l L. peruvianum lociamon gth efiftee nasymmetri chybrid swa sdetermine d(Tabl e5 ). Onlythre eloci ,o nchromosom e 2 (TG34),4 (CD59)an d 7 (TG61),respectively , werepresen ti nal lhybrids .Non eo fth eloc iwa sabsen tfro mal lhybrids . Weals oanalyse dth erepresentatio no findividua l L. peruvianum chromosomes inth easymmetri chybrids .Th eresult sfo rchromosom e6 ar eshow ni nFig .4 .Wit h the exception of hybrid 5H16, all hybrids contained L. peruvianum specific alleleso fchromosom e6 (Fig .4) .I nte nhybrid sal lL .peruvianu mloc iteste d were present,wherea s in the remaining hybrids one ormor e of the lociwer e missing. Hybrids 30H3, 30H6 and 30H7 provided evidence for rearrangements of L. peruvianum loci. In 30H3 and 30H7, the L. peruvianum TG54 alleles were present,whil e on the other hand Adh2 alleles were lacking. Because Adh2i s located more closely to the centromere and chromosome fragments without centromere are not stably maintained, the chromosomal fragment bearing the L.peruvianu mTG5 4allele smus thav ebee ntranslocate dt oanothe rchromosom eo r toa centromere-containin gfragment .Similarly ,i nhybri d30H6 ,th eAdh 2an dTG5 4 allelesfro mL .peruvianu mwer eretained ,bu tno tth e Aps-1 andCD1 4loc iwhic h are more proximal to and on either side of the centromere of chromosome 6

Table5 .Distributio no f3 0 L. peruvianum lociamon g1 5asymmetri chybrids ,a s deduced from the L. peruvianum specific bands on Southern blots following hybridisation with 29 single copy probes and one repeat probe. Between parenthesesi sgive nth echromosom eo nwhic heac hprob e( =molecula rmarker )i s located.

Presenti nno .o f Molecularmarke r(chromosome ) asymmetrichybrid s 15hybrid s TG34 (2); CD59 (4); TG61 (7)

14hybrid s TG54 (6); TG16 (8); TG8 (9); TG43(10) ; TG63 (10); TG44(11) ; TG30(11) ; TG50(12 ) 13hybrid s R45S (2); Adhl (4); TG20 (7)

12hybrid s CD15 (1); TG27 (1); CD50 (3); TG37 (4); Adh2 (6); CD27(12 ) 11hybrid s TG31 (2); CD1 (2); TG42 (3); TG62 (4); CD41 (5); CD14 (6); TG45 (8) 10hybrid s TG69 (5); TG68(12 )

9hybrid s TG9 (9) 82

Numbero f L. peruvianum alleles

lowdos ehybrid s high dose hybrids (5H-) (30H-) 2 3 5 7 13 16 22 28 1 3 6 7 12 26 27 CHR.6

0- -CDU 22212012 0 0 0 2 2 2 2

5- •Apsl + + + + + + (isozyme)

39- -Adh2 11212021 2 0 10 2 4 1 43- -TG5A 12212011 2 1112 2 1

•B + +n f + + +n f +n f nf +n f + (fruit colour) Fig.4 .Surve yfo rth epresenc eo f L. peruvianum specificallele so ffiv eloc i locatedo nchromosom e6 i neigh tlo wdos ehybrid san dseve nhig hdos ehybrids . Thenumbe ro f L. peruvianum allelescorrespondin gt oth eloc i CD14, Adh2an dTG54 wasdetermine d byRFL Panalysis .Th epresenc e (+)o rabsenc e (-) of theaci d phosphatase-1locu s (Apsl) and the^-caroten e locus (B)o f L. peruvianum was detected by isozyme analysis and observation of the colour of the fruits (if present),respectively ;nf ,n ofruit sobserved .Th eligh tshade dare aindicate s thecentromer eregion .Ma pposition sar eaccordin gt oYoun gan dTanksle y(1989) ; thepositio no f Bi snea rth een do fth elon garm ,a t10 6c Mo nth eclassica l map(Mutschle re tal . 1987).Not eth epresenc eo fth e Adh2 allelean dth eTG5 4 allelei nhybri d 30H6,whil e CDU, Apsl and Bar eabsent .

(Fig.4) .Thes e phenomena were not typical for loci on chromosome 6 of L.peruvianum .Simila rresult swer eobtaine dfo rothe rL .peruvianu mchromosomes , wheremor etha ntw oprobe swer euse d (seeFig . 1).

Analysis of ribosomal DNA in the asymmetric hybrids

Theribosoma lRN Agene s(rDNA )o ftomat oar earrange di ntande mrepeatin gunit s ata singlelocu s onth e shortar m ofchromosom e 2 (Dobrowolski etal .1989 ; Ganal et al. 1988;Vallejo s et al. 1986; Zabel et al. 1985) and represent approximately 3%o fth egenome .Ribosoma l DNAwa s included inou ranalysi st o determinewhethe r the rDNAs ofbot hspecie swer e presenta scomplet e setsi n the asymmetric hybrids. We found a useful polymorphism between the DraI - digestionpattern so fth erDN Ao f L. esculentum and L. peruvianum (Table 2).I n 83

addition to common bands at 1.8 and 4.0 kb, L. esculentum and L. peruvianum showed unique bands at 3.8 kb and 4.6 kb, respectively (lanes 16 and 18i n Fig.5) .Th e total fragment lengths of 9.6 kb (L.esculentum ) and 10.4 kb (L. peruvianum) compareswel lwit hth erepeatin guni tlengt ho f8. 6 to11. 0k b described for Lycopersicon species (Dobrowolski et al. 1989;Vallejo se tal . 1986). Except for two low dose hybrids (5H3 and 5H28), all asymmetric hybrids contained the L.peruvianu m specific 4.6 kb fragment. The ratio between L. peruvianum andL .esculentu malleles ,a scalculate d from thepea karea so f thecharacteristi cbands ,range dfro m0 t o2. 1(se eFig . 5).Value slarge rtha n 1.0 suggest that either amplification of L. peruvianum specific or loss of L. esculentum specific rDNA sequencesha d occurred. Inorde r toquantif y the

Asymmetric hybrids ^ Lese. (+) Lper f

er e? o 50 Gy 300G y V V v -6.7 kb —«• - 4.4 E- I -2.3 -2.0

0.6 P/E l0.4 0 2.02. 10. 7 0.2 0.8 0 10.50.50. 40. 3 0.5 0.7 0.51 0 1 »

Fig.5 . L. peruvianum specific ribosomal DNA sequences inasymmetri chybrids . DNA from eight lowdos ehybrid s (5H2 , 5H3, 5H5, 5H7, 5H13,5H16 ,5H22 , 5H28; lanes1-8) ,seve nhig hdos ehybrid s(30H1 ,30H3 ,30H6 ,30H7 ,30H12 ,30H26 ,30H27 ; lanes9-15) ,L .esculentu mcv .Bellin a(lan e16) ,symmetri chybri d0H 1(lan e17 ) and L. peruvianum PI128650-ATW2002 (lane 18) was digested with DraI , electrophoresed,blotte dan dhybridise dwit hpBsARl ,a ribosoma lDN Aclon efro m pea.P an dE a tth elef tindicat eth e L. peruvianum and L. esculentum specific band,respectively .Belo weac hlan eth erati obetwee nth e L. peruvianum alleles andth eL .esculentu mallele si sgive n (P/E)a scalculate dfro mth epea karea s ofdensitometri c scans (seeFig .3) .Th erati oi nth esymmetri chybri d0H 1wa s used asreferenc e (P/E= 1). The position ofth e Hin dill fragments ofphag e lambdaDN Ai sindicate da tth eright . 84

amount of L. esculentum rDNA ineac hhybrid ,w e determined the densitometric absorptionvalu eo fth e L. esculentum specificban d relativet oth esam eban d inth esymmetri chybri d0H 1(se eMaterial san dmethod ssection) ,assumin gtha t thesymmetri chybri d0H 1containe dth ecomplet eset so frDN Arepeat sfro mbot h parents. The calculations revealed a large variation in the amount of L.esculentu mspecifi crDN Ai nth easymmetri chybrid s (Table 6).Amon gth esi x lowdos ehybrid scontainin ga diploi d L. esculentum genome(5H2 ,5H3 ,5H5 ,5H7 , 5H22an d 5H28), three (5H3,5H 5 and 5H22)ha d the expected two sets ofrDN A repeatsfro m L. esculentum, whereas theothe rthre eha da lowe rnumber .Eigh t hybrids (5H13, 5H16, 30H1, 30H3, 30H6, 30H7, 30H26 and 30H27) contained a tetraploidgenom eo f L. esculentum, butthei r L. esculentum specificrDN Aconten t waslowe rtha nth eexpecte dfou rset san drange dfro m0. 6t o3.2 .Th eplan twit h ahexaploi d L. esculentum genome (30H12)als oha da lowe rnumbe ro frDN Aset s

Table6 .Th ecompositio no fth eribosoma lDN Ai nth easymmetri chybrids ,8 lo w dosehybrid s (5H2t o5H28 )an d7 hig hdos ehybrid s (30H1t o30H27) .Th eamoun t ofeac hparenta lrDN Ai sgive ni nsets ;on ese tcorrespond st oon ecomplet erRN A codingregio nlocate do non echromosom e 2.Th enumbe ro fset so f L. esculentum was determined by the ratio of the densitometric absorption values of the L. esculentum bandi neac hasymmetri chybri dwit htha ti nth esymmetri chybri d 0H1.Thi srati owa sdivide db yth eratio sdetermine dafte rhybridisin gth esam e blotwit hth esingl ecop yclone sTG5 0an dTG54 ;th edifferenc ebetwee nth eratio s obtainedwit hTG5 0an dTG5 4wa sles stha n10 %fo reac hasymmetri chybrid .I twa s assumedtha t0H 1containe dtw ocomplet erDN Aset sfro meac hspecies .Th eamoun t of L. peruvianum setswa sdeduce dfro mth eP/ Erati oi nFig .5 .Th enumber so f complete (c)plu s incomplete (inc)chromosome s 2,base d onRFL Panalysi swit h threechromosom e2 specifi csingl ecop yprobes ,ar egive nbetwee nbracket sfo r eachgenotype .

Genotype Numbero frDN Aset s [c+ in c chr.2 ] L. esculentum L. peruvianum 5H2 1.2 [2+0] 0.5 [1+1] 5H3 1.8 [2+0] 0.0 [1+0] 5H5 2.1 [2+0] 4.2 [1+0] 5H7 1.2 [1+1] 2.5 [1+0] 5H13 0.6 [4+0] 0.4 [1+1] 5H16 3.2 [4+0] 0.8 [1+0] 5H22 2.2 [2+0] 1.9 [2+0] 5H28 1.2 [2+0] 0.0 [1+1] 30H1 3.1 [3+1] 1.6 [0+1] 30H3 2.6 [4+0] 1.3 [0+2] 30H6 2.3 [4+0] 1.0 [0+1] 30H7 2.8 [4+0] 1.0 [1+0] 30H12 4.2 [5+1] 2.0 [0+1] 30H26 2.9 [4+0] 2.1 [0+2] 30H27 2.4 [3+1] 1.2 [0+1] 0H1 2 [2+01 2 [2+01 85 thanexpected . Theamoun to f L. peruvianum specificrDN A(expresse di nset so frepeats )wa s calculatedfro mth e L. peruvianum/L. esculentum ratioshow ni nFig .5 an dfoun d torang efro m0 t o4.2 .I nvariou shybrid s(5H5 ,5H7 ,30H 1an d30H12 )th enumbe r of rDNA sets was higher than the number of chromosomes 2 (complete and incomplete);thi ssuggest seithe rpreferentia lretentio no ramplificatio no fth e L. peruvianum rDNArepeatin gunits .I nhybri d 5H5,th e L. peruvianum rDNAwa s clearlyamplified .

Discussion

RFLPs between L. esculentum and L. peruvianum In this paper we have shown that RFLP analysis is an effective means of characterizing,qualitativel ya swel la squantitatively ,th egenom ecompositio n of asymmetric somatic hybrids obtained after fusion of protoplasts of the divergentspecie s L. esculentum andt .peruvianua i(Se eals oYoun ge tal .1988) . Usingonl ya limite dnumbe ro frestrictio nenzymes ,w ecoul dreadil yestablis h diagnosticRFLP sfo r3 0o fth e3 1molecula rmarker stested .Thes eRFLP sallo w eachchromosom eo fth erespectiv e Lycopersicon speciest ob eidentifie di nth e hybrid by at least twomarkers . Inconjunctio n with studies by Young et al. (1988),whic hsho wtha ta monogeni ctrai tresidin go na smal l L. peruvianum DNA segmentintrogresse d into L. esculentum canb erecognise di nth e L. esculentum DNAbackgroun db yvirtu eo fRFLP sassociate dwit hth especifi ctarge tgene ,w e conclude that, in principle, any L. peruvianum sequence, introduced into L. esculentum shouldb eidentifiabl eb yRFL Panalysis . While the level of variability, as detected by RFLPs, is high among Lycopersicon species,L. esculentumgenotype ssho wfe wpolymorphism s(Helentjari s etal .1985) .Thi sha sbee nattribute dt oth einbre dcharacte ro f L. esculentum. Because L. peruvianum isa n outbreeding species,w e expected ahig hleve lo f RFLPsi nthi sspecies ,an dthi swa sindee dwha tw efound .Th e L. peruvianum plant used inth epresen texperiments ,whic hwa sderive d froma naccessio ntha twa s maintained by sibmating alimite dnumbe r ofplant s forman y generations,wa s heterozygous forth e isozymeaci dphosphatase- 1 (Aps-1; Chapter 2)an d for1 1 ofth e3 0DN Aprobe stested .Besides ,wit h2 0ou to f2 2probe spolymorphism swer e detected inanothe r individual from the same population (datano t shown); in addition,tw o isozymes (Aps-1 andglutamat e oxaloacetate transaminase)showe d different patterns forbot hplant s (Chapter 2).Thes eobservation sagre ewit h thoseo fYoun ge tal . (1988),wh odescribe dRFLP sfo rtw oloc ibetwee nsevera l individualso fth esam eaccessio no f L. peruvianum. 86

Genomic constitution of the asymmetric somatic hybrids

One of the major conclusions of the present experiments is that asymmetric somatic hybrids obtained by fusion of L. esculentum protoplasts with gamma- irradiated L. peruvianum protoplasts and selected for the regenerationtrai t of L. peruvianum, contain asubstantia l amount ofth edono rgenome .Eliminatio n of the L. peruvianum genome was limited in the 15 asymmetric hybrids tested: 17% to 69%o f the donor alleles,a s determined by RFLP analysis,an d 13%t o 88%o f the donor chromosomes, as determined by counts of metaphase plates, was eliminated. Similar results have been reported for asymmetric somatic hybrids of other plant species. Thus, on the basis of chromosome numbers, elimination ofth edono rgenom ewa sfoun dt orang efro m10 %t o89 %i n Nicotiana plumbaginifo- lia (+) Atropa belladonna hybrids (recipient (+)donor ; 100-1000 Gy; Gleba et al. 1988), 40%-92% in N. plumbaginifolia (+) N. sylvestris hybrids (500an d 1000 Gy; Famelaer et al. 1989) and 0%-75% in Brassica oleracea (+) B. campestris hybrids (100-800 Gy; Yamashita et al. 1989). In all those cases, chromosome rearrangements and/or deleted donor chromosomes were observed. To account for the limited elimination of the L. peruvianum genome in the L. esculentum (+) L. peruvianum asymmetric hybrids, several explanations are conceivable: (i)chromosom ebreak swer erepaire dbefor eand/o rafte rfusion .Repai ro fsingle - strandedbreak swithi non ehou ro firradiatio nha sbee nshow nt ooccu r incarro t protoplastsfollowin g treatmentwit h 200G yo fgamm airradiatio n (Howland eta l. 1975); (ii)hybri d cells with a high proportion of donor genome were better balanced (around the tri-,penta - or hepta-ploid level) and therefore grew better than cells with a low proportion of donor genome; (iii) in selecting the fusion products, we were biased to good callus growth, a multi-genic trait from L. peruvianum which is not linked to regeneration capacity (Koornneef et al. 1987a). The amount ofdono r DNAdetecte d inth e fusion productswa sdependen t onth e dose of irradiation applied, though the differences between the high- and low dose hybrids were not dramatic. Low dose hybrids contained more loci, alleles and complete chromosomes from L. peruvianum than high dose hybrids (Table 3), while the latter class contained more incomplete L. peruvianum chromosomes (probably due toa highe r frequency of chromosome breaks). For plant asymmetric somatic hybrids, it is assumed that the donor genome is retained as fragments bearing a centromere and that exchange events are rare, since the chromosomes ofbot h parentsar eno t mixed inth e first divisions (Hinnisdaels et al. 1988). Our results, however, provide substantial evidence showing that chromosome fragmentsgenerate db yirradiatio nwer einvolve di ninducin grearrangements ,lik e 87 translocations: (i)a schromosom e fragmentswithou ta centromer ear eno t stablymaintaine di n the absence of rearrangements, L. peruvianum loci located at the ends of chromosomalarm sar eexpecte dt ob efoun dles sfrequentl yi nasymmetri chybrid s followingfragmentatio nb yirradiatio ntha nloc imor eproxima lt oth ecentromere . No relationwa s found,however ,betwee n the elimination ofloci/allele sfro m L.peruvianu man dthei rchromosoma lma pposition .Hybri d30H6 ,fo rexample ,wa s missingth e L. peruvianum Aps-1an dCD1 4allele swhic har elocate da teithe rsid e ofth ecentromer e ofchromosom e 6,bu tdi dcontai nth eAdh- 2an dTG5 4allele s residing more distal from the centromere (Fig.4) .Besides , telomeres are necessaryfo rstabl emaintenanc eo fchromosom efragment sa swel l(Blackbur nan d Szostak 1984); (ii)th etota lnumbe ro fcomplet eplu s incomplete chromosomesa sestimate db y RFLPanalysi s usuallywa slarge r thanth enumbe r counted inmetaphas eplate s (Table 4); (iii)i na fe wcases ,amplificatio no fallele sha doccurre da sshow nb yth eAdh 2 alleleso nchromosom e6 i nhybri d30H2 6(Fig .4 )an dth elocu sTG5 0o nchromosom e 12i nhybri d 30H1(uppe rban dlan e9 ,Fig .2C) . Inmos to fth ehybrids ,als oa fe wallele so fth erecipien tL .esculentu mgenom e were absent and in some cases even complete chromosomes (Table4) .Maybe , exchangeevent swit h L. peruvianum chromosomeso rchromosom efragment sha dtake n place shortly after fusion, followed by elimination of translocated parts. Anotherexplanatio nfo rth elos so fL . esculentum chromosomesan dfragment smigh t be that rearrangements, such as structural chromosome mutations and non­ disjunction,occurre d atth etissu ecultur e phase (Pijnackere tal . 1986;Le e andPhillip s1988 ;Evan se tal . 1989).

Ribosomal DNAin the asymmetric hybrids

In the present study a large variation in the amount of ribosomal DNA was detected in the asymmetric somatic hybrids. Calculations were based on the assumptiontha tth eparenta lspecie sdi dno tvar yi nthei rrDN Aconten tan dtha t thesymmetri c somatichybri d0H1 ,whic hwa suse da sa reference ,containe dth e completeset so frDN Afro mbot hspecies .Variatio ni nth eamoun to frDN Aha ss o far not been reported for Lycopersicon genotypes, although variation was demonstrated ina few individuals ofpopulation s following regenerationfro m tissue culture (potato,Landsman n and Uhrig 1985; ,Brettel le t al. 1986);howeve ri nanothe rstudy ,plant sregenerate dfro mtissu ecultur eshowe d novariatio n (carrot andartichoke ,Has e etal .1982) . Onth e otherhand ,i n symmetric somatic hybrids of Solanum tuberosum and S. phureja, thenucleola r 88

chromosomes of the latter species were either eliminated or had rearranged satellites (Pijnackere tal . 1987).Variatio ni nrDN Awa sals ofoun di nsymmetri c hybrids of L. esculentum and Solanum lycopersicoides (Moorean d Sink 1988): the amount of rDNA (with respect to total DNA)range d from 28%t o 106%relativ e to the sexual hybrid between these species; in 6 out of 12 somatic hybrids this amountwa sno t significantly different from that inth e sexualhybrid . Inthes e experiments, the S. lycopersicoides protoplasts derived from a suspension culture; suspension cultures are known to accumulate various chromosomal aberrations (e.g.Pijnacke re tal . 1986).W eassume dtha ti nou rreferenc eplant , the symmetric hybrid 0H1, no significant changes had occurred in the rDNA contributiono feithe rparent ,becaus eth eparenta lprotoplast sderive dfro mlea f tissue,whic h is expected to be stable at the chromosome level,an d because of the short tissue culture phase involved in generating this hybrid. In our asymmetric hybrids, the amount of rDNA ranged from 25% to 158% as compared to that in the symmetric hybrid 0H1. Even if the symmetric hybrid 0H1 had deviating amounts of ribosomal DNA, still the large variation in the asymmetrichybrid si sstriking .Th erDN Acontributio no f L. esculentum in1 2ou t of1 5asymmetri chybrid sanalysed ,wa sdecrease drelativ et oth enumbe ro fcopie s of chromosome 2 from L. esculentum. The L. peruvianum rDNA was absent in 2 hybrids, whereas itwa s present invariou s amounts, irrespectively of the copy number of L. peruvianum chromosomes 2, in the other hybrids. The observed variationmigh tb ecause db yth eirradiatio n treatment (incas eo f L. peruvianum rDNA), the tissue culture conditions (Leean d Phillips 1988;Evan s et al. 1989) and/or the interspecific genome combination (Pijnacker et al. 1987).

Linkage of molecular markers to regeneration capacity

Originally, it was anticipated that gamma-irradiation of the L. peruvianum protoplasts would lead to elimination of the bulk of the donor genome and identification of chromosomal markers associated with the regeneration trait. However,eliminatio no fth e L. peruvianum genomewa srathe rlimited .Al lmarker s representing the 12 chromosomes of L. peruvianum, were recovered ina t least 9 out of 15asymmetri c hybrids surveyed. Since only a limited number of genes is known to be involved in regeneration as shown by classical genetic analysis (Koornneef et al. 1987a), this result suggests that extensive chromosomal elimination by irradiation did not occur and/or that L. peruvianum loci, which are not associated with the regeneration trait, were retained on their own (incomplete) chromosomes or by translocation to other chromosomes. Three L. peruvianum loci, TG34 (chromosome 2), CD59 (chromosome 4) and TG61 (chromosome 7),wer e present in all asymmetric hybrids; this suggests linkage of these loci to those which are essential for regeneration. 89

In general, even with the rather limited number of asymmetric hybrids surveyed,ou rstudie scas tseriou sdoubt so nasymmetri chybridisatio na sa usefu l means of introducing desirable traits into a recipient species. The aneuploid character of the fusion products and the numerous chromosomal rearrangements involved -phenomen awhic hbot har eknow nt ocaus e sterility -rende rth ehybri d plants inaccessible to backcrossing (Chapter4) .

Acknowledgements. This researchwa s supported by theFoundatio n for Fundamental Biological Research (BION),whic h issubsidise d byth eNetherland s Organisation forScientifi cResearc h (NWO).W ethan kDr .Tanksley ,Dr .Chas ean dDr .Lifschyt z for providing the clones,Ms .E .Hulsebo s for technical advices,Mr .R . Busink for instructing thedensitomete r and Prof.C .Heytin g forcriticall y reading of the manuscript.

References

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Hille J, KoornneefM ,Ramann a MS, Zabel P (1989)Tomato : a crop species amenable to improvement by cellular andmolecula rmethods .Euphytic a 42:1-23 Hinnisdaels S, Negrutiu I, Jacobs M, Sidorov V (1988) Plant somatic cell hybridizations: evaluations and prospectives.Newslette r IAPTCJul y 1988:2-10 HowlandGP ,Har tRW ,Yett eM L (1975)Repai ro fDN Astran dbreak safte rgamma-irradiatio no fprotoplast sisolate d from culturedwil d carrot cells.Mutatio nRes .27:81-8 9 ImamuraJ ,Sau lMW ,Potryku sI (1987 )X-ra y irradiationpromote d asymmetric somatichybridisatio nan dmolecula r analysis of theproducts .Theo r ApplGene t 74:445-450 KinsaraA ,Patnai k SN,Cockin g EC,Powe rJ B (1986)Somati chybri d plantso fLycopersico nesculentu m Mill, and Lycopersiconperuvianu mMill. . JPlan t Physiol 125:225-234 KoornneefM ,Hanhar tCJ ,Martinell i L (1987a)A geneti c analysis of cellcultur etrait s intomato .Theo rApp l Genet 74:633-641 KoornneefM , JongsmaM ,Weid eR , Zabel P, Hille J (1987b)Transformatio n of tomato. In:Nevin s DJ, JonesR A (eds)Tomat obiotechnology . AlanR Liss, Inc,Ne wYork ,p p 169-178 Landsmann J, Uhrig H (1985)Somaclona l variation inSolanu m tuberosum detected at themolecula r level. Theor ApplGene t 71:500-505 LeeM ,Phillip sR L (1988)Th echromosoma lbasi s ofsomaclona lvariation .An nRe v Plant PhysiolPlan tMo lBio l 39:413-437 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Laboratories. Cold Spring Harbor,Ne wYor k MoorePP ,Sin kK C (1988)Molecula ranalysi so fsingl ecop yan drepetitiv egene so nchromosom e2 i nintergeneri c tomato somatic hybrid plants.Plan tMolecula r Biology 11:139-145 Müller-GensertE ,Schiede r0 (1987)Interspecifi c T-DNAtransfe rthroug hplan tprotoplas tfusion .Mo lGe nGene t 208:235-241 MutschlerMA ,Tanksle ySD ,Ric kC M(1987 )Linkag emap so fth etomat o (Lycopersiconesculentum) .Re pTomat oGene t Coop 37:5-34 Pijnacker LP,Ferwerd aMA ,Puit eKJ ,Roes tS (1987)Eliminatio no fSolanu mphurej anucleola r chromosomes inS . tuberosum + S.phure.j asomati c hybrids.Theo rApp lGene t 73:878-882 Pijnacker LP,Hermelin k JHM,Ferwerd aM A (1986)Variabilit y ofDN A content andkaryotyp e in cell cultures of aninterdihaploi d Solanum tuberosum. PlantCel lReport s 5:43-46 ReedKC ,Man nD A (1985)Rapi d transfero fDN A from agarosegel st onylo nmembranes .Nuclei cAcid sRe s13:7207 - 7221 Rick CM (1982)Stoc k list.Re p TomatoGene tCoo p 32:3-10 RingertzNR ,Savag eR E(1976 )Gen emappin gan dgen ecomplementatio n analysis.In :Cel lhybrids .Academi cPress , NewYork ,p p224-24 4 Tanksley SD,Mille r JC,Paterso nA , Bernatzky R (1988)Molecula r mapping of plant chromosomes. In:Gustafso n JP,Appel sR (eds)Chromosom e structure and function.Plenu m Press,Ne wYork ,p p 157-173 Vallejos CE,Tanksle y SD,Bernatzk y R (1986)Localizatio n in the tomato genome of DNA restriction fragments containing sequences homologous to the rRNA (45s), themajo r chlorophyll a/b binding polypeptide and the ribulosebisphosphat e carboxylase genes.Genetic s 112:93-105 VanDaele nRAJ , Jonkers JJ, Zabel P (1989)Preparatio n of megabase-sized tomato DNA and separation of large restriction fragments by field inversion gel electrophoresis (FIGE). PlantMo lBio l 12:341-352 Wijbrandi J, Vos JGM, Koornneef M (1988) Transfer of regeneration capacity from Lycopersicon esculentum to L.peruvianu mb yprotoplas t fusion.Plan tCel l TissueOrga nCultur e 12:193-196 Yamashita Y, Terada R, Nishibayashi S, Shimamoto K (1989) Asymmetric somatic hybrids of Brassica: partial transfer ofB ,campestri sgenom e intoB .olerace a by cell fusion. TheorApp lGene t 77:189-194 Young ND, Tanksley SD (1989) Restriction fragment length polymorphism maps and the concept of graphical genotypes. TheorApp lGene t 77:95-101 YoungND ,Zami rD ,Gana lMW ,Tanksle y SD (1988)Us eo fisogeni c lines and simultaneousprobin g to identifyDN A markers tightly linked to theTm~2 agen e intomato .Genetic s 120:579-585 ZabelP ,Meye rD ,va nd eStolp e0 ,va nde rZaa lB ,Ramann aRS ,Koornnee fM ,Kren sF ,Hill eJ (1985)Toward sth e construction of artificial chromosomes for tomato. In: van Vloten-Doting L, Groot GSP, Hall TC (eds) Molecular form and function of theplan t genome.Plenu m Press,Ne wYork ,p p609-62 4 91

CHAPTER7

GENERAL DISCUSSION

The experiments presented in this thesis show that the favourable plant regeneration capacity of L. peruvianum is an efficient selectable marker in protoplast fusion experiments; this confirms the results of Adams and Quiros (1985)an dKinsar ae tal . (1986).Symmetri csomati chybrid swer eeasil yobtained . These plants were vigorous and produced fertile progenies upon selfing. The asymmetric somatichybrid swer emor edifficul tt oobtain ,les svigorou san dver y variable. They were characterised in detail by chromosome specific markers. Especiallyth eRFL Panalysi swa sver yuseful ,becaus eeac hdono rchromosom ecoul d bedetecte d bya tleas t twomarkers .Suc ha detaile d analysis ofsomati c hybrids has not been reported before. The analysis revealed that a large number of L. peruvianum chromosomes, which were often incomplete, was retained in the asymmetric hybrids.

Application of somatic hybrids of tomato in breeding programs ?

The tetraploid symmetric somatichybrid s ofbot h Lycopersicon speciesuse d were self compatible; their progeny showed some segregation towards both fusion parents (Chapter 3).N o backcrosses of these hybrids to tomato did succeed. However, it seems feasible that a plant sexually congruent with L. esculentum canb efoun damon gth elarg epopulatio no fprogen yplant sderive dfro mth eselfe d tetraploid hybrids or among a next generation of these plants, because of the observed variation. The asymmetric somatic hybrids obtained in the present study were sterile (Chapter 4).Th e reasons are most probably the aneuploidy of these plants and the assumed frequent occurrence of rearrangements. Besides, these hybrids retained a lot of chromosomes from the donor. When very large numbers of asymmetric hybrids are assayed, maybe some highly asymmetric hybrids and/or fertile plants canb e found.

Somatic mapping by asymmetric hybridisation ?

It was anticipated that highly asymmetric hybrids, which contained a limited number of more or less undamaged donor chromosomes, could arise from the 92 described experiments. In that case, the genes coding for the regeneration capacity of L. peruvianum could be mapped to particular chromosomes and to particularchromosoma lregions .However ,th eamoun to fretaine ddono rchromosome s waslarg e(Chapte r4 ,5 an d6) .I naddition ,th eirradiatio ntreatmen tgenerate d many fragmented chromosomes, which probably induced rearrangements of chromosomes, also involving L. esculentum chromosomes (Chapter 5 and6) . Incorporationo fdono rfragment s inth erecipien tgenom emigh tb eestablishe d by in situ hybridisationwit hspecie sspecifi crepetitiv eDN Aprobes ,i fthes e mightbecom eavailabl efo r Lycopersicon species.I nsuc ha manner ,integratio n of Nicotiana plumbaginifolia fragments in N. tabacum chromosomes has been demonstratedfo rasymmetri chybrid so fthes especie s(Piastuc han dBate s1988) . The asymmetric hybrids between L. esculentum and L.peruvianu m showed some instabilitywit h respect to the chromosome numbers counted inroo t tipcell s (Chapter4 )an dwit hrespec tt oth eretentio no f L. peruvianum specifictrait s indifferen tshoot sfro ma sam ehybri dclon e (Chapter 5).Thi shamper ssomati c mappingb yasymmetri chybridisatio nafte rfragmentatio no fth edono rgenom eb y high doses of ionising radiation. On the other hand, regional mapping (i.e. mapping of traits incertai n small chromosome regions)shoul d befeasibl e in plantasymmetri chybrid sa sha salread ybee nshow nfo rmammalia ncel lhybrid s (Gossan dHarri s1975) .Fo rthi spurpose ,on eneed sa sufficien tnumbe ro fmarke r genest odefin especifi cregion san don eha st otes tman yhybrids .Th emappe d RFLP markers of the tomato (Tanksley et al. 1988)ar e appropriate for such studies. The only statement that can be made about the localisation of the "regeneration genes" of L. peruvianum is that these genes are probably not mapped to chromosomes 1, 3, 5 and 11, because markers specific for these chromosomeswer elos ti na larg efractio no fth easymmetri chybrid s (Chapter5 and 6). An alternative method for mapping these "regeneration genes" to particularchromosomes ,i sth eanalysi so fsexua lpopulation stha tsegregat efo r this trait (Koornneef et al. 1987). This analysis canb e performed withth e abundantly availablemorphologica l andbiochemica lmarker s (isozyme andRFLP ) oftomato .

Elimination of chromosomes in the somatic hybrids

Thesymmetri csomati chybrid sbetwee n L. esculentum and L. peruvianum rarelylos t chromosomes spontaneously (Chapter 2).I n contrast, all asymmetric hybrids between these species had lost chromosomes, as indicated by the aneuploid chromosomenumber san dth elos so f L. peruvianum traits (Chapter4 ,5 an d6) . 93

This clearly shows that high doses of ionising radiation cause elimination of chromosomes.However ,n ohighl y asymmetric hybridswit honl ya fe wdono r traits or chromosomeswer e obtained.Mos t of thehybrid sha d retained atleas thal f of a diploid I. peruvianum genome. Several explanations are conceivable for this limited elimination: (i)th e selectable marker used is not appropriate for the selection of highly asymmetric hybrids. Regeneration capacity is important only in the last stage ofth ecel lcultur ephase .Marker stha tac ta searl ya spossibl eafte rprotoplas t fusion, such as recessive auxotrophic mutations in the recipient (e.g.nitrat e reductasedeficiency )o rresistance st oantibiotic sintroduce db ytransformatio n in the donor species, might be more effective for the selection of highly asymmetric hybrids; (ii)th e choice ofparenta l speciesma yno thav e been suitable toobtai nhighl y asymmetric hybrids. The donor species L. peruvianum was for all cell culture traits superior to the recipient L. esculentum. The selection on the basis of several favourable L. peruvianum genes, which code for callus growth characteristics and regeneration capacity, may have lead to the retention of large numbers of donor chromosomes; (iii)th echromosom enumber sten dt ob emor eo rles sbalance daroun dth etriploi d or pentaploid level. There might be selection against aneuploid chromosome numbers that deviate too much from these numbers. In addition, the somatic congruity between the two species can imply a lack of selection against high numbers of donor chromosomes. To verify or exclude these possible explanations, fusion experiments might be performed inwhic hunirradiate d protoplasts ofL . peruvianum are fused with irradiated protoplasts ofa kanamyci nresistan tL . escuientum.Th ehybrid shav e to be selected for kanamycin resistance in an early stage of the cell culture phase.I fth eobtaine dasymmetri chybrid swoul dcontai nfe wtrait so rchromosome s of L. esculentum, this would support explanations (i) and (ii). If the elimination of L. esculentum chromosomes islimited , explanation (iii)i smor e likely.

Other methods to achieve chromosome elimination

Highdose s of gamma-rays were not sufficient,despit e the frequent breakage of L. peruvianum chromosomes, to eliminate the bulk of the donor genome in the presented experiments. Moreover, the chromosome lesions probably induced chromosome rearrangements. Other methods, which have hardly been tested in plants, to get highly asymmetric hybrids could be tested: 94

(i)fragmentatio n by premature chromosome condensation (PPC). This is induced inprotoplast s ofinterphas e cellswhe nthe yar efuse dwit hprotoplast so fcell s inmetaphas e (Szabados and Dudits 1980); (ii)fragmentatio no f the donor genome by chemical agents,fo r example Acridin Orange (Pedersen et al. 1988)an d BUdR (5-bromodeoxyuridine;use d in mammalian cell hybrids, Pontecorvo 1971). Both chemicals are incorporated into DNA, photosensitise the DNAan d therefore cause chromosome breakswhe nth e cellsar e exposed tolight ; (iii) gameto-somatic fusion and subsequent backcrossing (Pental et al. 1988). Haploid microspore protoplasts, which are isolated from young pollen tetrads, are fused with protoplasts of diploid somatic cells. The resulting triploid hybrids have to be backcrossed to achieve further elimination of the donor chromosomes; this prerequisite limits the method to more or less congruent combinations.Gameto-somati cfusio nha sbee ntrie d insevera lexperiment so fth e present study (data not shown). Microspore protoplasts could be isolated from small flowerbud s ofL . peruvianum andwer e fused withmesophyl l protoplasts of L. esculentum. However,th efusio nproduct sa swel la sunfuse d cellsi nth esam e culture did not grow. Apparently, the presence of microspore protoplasts inhibited cell growth; (iv)microprotoplast-protoplas t fusion (Verhoeven 1989). The microprotoplasts, containing one or a few chromosomes or fragments, are isolated from micronucleated cells,whic har e induced bytreatmen t witha nanti-mitoti c drug. This method isattractive , because elimination is achieved before fusion. The methods (iii) and (iv) probably also suffer less from chromosome rearrangements. Thus,althoug hhighl yasymmetri c somatichybrid so f Lycopersicon specieshav e notye tbee nconstructed ,ther ear estil lsevera lprocedure savailabl etha tcoul d be tried to achieve this.

References

Adams TL,Quiro sC F (1985)Somati c hybridization between Lycopersicon peruvianum and Lycopersicon pennellii: regenerating ability and antibiotic resistance as selection systems.Plan t Science 40:209-219 Goss SJ,Harri sH (1975)Ne wmetho d formappin g genes inhuma n chromosomes.Natur e 255:680-684 KinsaraA ,Patnai k SN,Cockin g EC,Powe r JB (1986)Somati chybri d plants ofLycopersico n esculentumMill , and Lycopersicon peruvianum Mill. JPlan t Physiol 125:225-234 KoornneefM , HanhartCJ ,Martinell i L (1987)A genetic analysis of cell culture traits intomato .Theo rApp l Genet 74:633-641 PedersenHC ,Larse nAB ,Vamlin gK ,Keime rB (1988)Inactivatio no fsugarbee tprotoplast susin gAcridi nOrange , anagen t for late selection of fusionproducts . In:Puit eKJ ,Don s JJM,Huizin g HJ,Koo l AJ,Koornnee fM , Krens FA (eds)Progres s inplan t protoplast research,Kluwer ,Dordrecht ,p p 265-266 95

PentalD ,Mukhopadhya yA ,Grove rA ,Pradha nA K (1988)A selectio nmetho d forth esynthesi so ftriploi dhybrid s by fusion ofmicrospor e protoplasts (n)wit h somatic cellprotoplast s (2n).Theo rApp lGene t 76:237-243 Piastuch WC, Bates GW (1988) Genetic analysis of asymmetric hybrids using species-specific repetitive DNA probes. In: The second international congress of plant molecular biology, Jerusalem, Nov 13-18. Book of abstracts,no . 194 PontecorvoG (1971)Inductio n ofdirectiona l chromosome elimination insomati c cellhybrids .Natur e230:367 - 369 Szabados L, Dudits D (1980)Fusio nbetwee n interphase andmitoti c plant protoplasts. Induction of premature chromosome condensation. ExpCel lRe s 127:442-446 Tanksley SD,Mille r JC,Paterso n A,Bernatzk y R (1988)Molecula r mapping ofplan t chromosomes. In:Gustafso n JP,Appel sR (eds)Chromosom e structure and function. Plenum Press,Ne wYork ,p p 157-173 VerhoevenH A(1989 )Inductio nan dcharacterizatio no fmicronucle ii nplan tcells .Perspective sfo rmicronucleus - mediated chromosome transfer. PhD Thesis ,Agricultura l University Wageningen, theNetherlands . 97 SUMMARY

Several desirable traits, such as disease resistances, have been introduced frommor eo rles srelate dwil d Lycopersicon species,int oth ecultivate dtomato , L. esculentum, by classical breeding techniques. Somatic hybridisation by protoplast fusion canenhanc e the germplasm pool available for tomato breeding, because this procedure allows to by-pass sexual crossing barriers. Especially the technique of asymmetric hybridisation, by which untreated recipient protoplasts are fused with donor protoplasts of which the major part of the genome has been eliminated, for instance by irradiation, might be very useful. Theresultin gasymmetri chybrid swil l containonl ya smal l fractiono fth edono r genome,and ,therefore ,th enumbe ro fbackcrosse srequire dt oeliminat eundesire d donor traits might be relatively small; it might also be possible to breed at the(near )diploi dleve lwit hsuc hhybrids .Asymmetri csomati chybrid smigh tals o be useful for chromosome mapping. In this thesis several aspects of somatic hybridisation that are important for the application of this technique to the improvement of the tomato,wer e analysed indetail .

The efficient isolation of symmetric somatic hybrids between L. esculentum and the wild species L. peruvianum by means of a double selection strategy is described in Chapter 2. The symmetric hybrids were selected on the basis of kanamycin resistance of L. esculentum andth e superior regeneration capacity of L. peruvianum. These hybrids were very vigorous plants; most of them had a tetraploid chromosome number of 48 (2n= 4x). A minority of the hybrids was at thehexaploi d level with chromosome numbers from 64t o 72.Th ehybri d nature of all regenerated plants was confirmed by analysis of isozyme markers, by their intermediate morphology and, in some cases, by the analysis of restriction fragment length polymorphisms (RFLPs). According to RFLP analysis 6 hexaploid hybrids thatwer e tested all contained one diploid genome of L. esculentum and twodiploi dgenome so fL . peruvianum.On eo fthes ehexaploid sha dgenome so ftw o differentL . peruvianumgenotype san dwa stherefor econsidere dt ob ederive dfro m a triple protoplast fusion. The hexaploid plants resembled L. peruvianum more than the tetraploids did. The hybrids did flower and set fruits. The fertility of the tetraploid and hexaploid somatic hybrids was analysed afterselfin gan dafte rbackcrossin g toeac ho fbot hfusio nparent s (Chapter3) . Most of the somatic hybrids, especially the tetraploids, were fertile upon selfing and yielded many seeds, of which 79%germinated . These progeny plants were vigorous and often fertile after selfing. Backcrossing of the somatic hybrids with the L. esculentum parent did not yield any viable seeds; 98 backcrossing with L. peruvlanurn yielded a few germinating seeds, but only if L. peruvianum wasuse da sstaminat eparent .Th eplant sderive dfro mth ebackcros s hexaploid hybrid x tetraploid L. peruvianum had a pentaploid chromosome number (2n— 5x— 60)an dwer evigorous ,wherea s theplant s derived from the backcross tetraploid hybrid x diploid L. peruvianum grew retarded. To obtain information about thebehaviou r ofchromosome s inth ehybrids ,th eprogenie so fth eselfing s and backcrosses were analysed for the segregation of several traits, namely kanamycin resistance, isozyme patterns for acid phosphatase, locus Aps-1, and some morphological characteristics. Most of the progenies segregated for kanamycin resistance as expected onth e basis of the number of loci present in the kanamycin resistant fusion parent, whereas in some progenies the fraction ofkanamyci n resistant plantswa s smaller thanexpected . The segregation ofth e Aps-1 isozyme patterns and the variation for some of the morphological characteristics among the progeny plants indicated a tetrasomic inheritance of at least part of the genes in thehybrids .

Asymmetric somatichybrid sbetwee n L. esculentum and L. peruvianum wereselecte d onth ebasi so fth eregeneratio ncapacit yo f L. peruvianum (Chapter 4).Selectio n against growth of L. peruvianum was effected by lethal doses (50, 300 or 1000 Gray)o fgamma-irradiatio napplie dt othi sspecie sbefor eprotoplas tfusion .Th e asymmetrichybrid sneede da longe rregeneratio ntim etha nth esymmetri chybrids , and showed a large variation in callus morphology, plant morphology and chromosomenumbers .Al lasymmetri chybri dplant swer eaneuploid .Afte ra lo wdos e (50Gy )mos thybrid sha da near-triploi dchromosom enumber ,wherea safte ra hig h dose (300o r 1000Gy )mos t of thehybrid s were near pentaploid. Inspit e of the aneuploid chromosomenumbers ,man yplant sgre wvigorously ,flowere dand ,i nsom e cases, set fruits. In general the morphology of the asymmetric hybrids was intermediate between that of L. esculentum and symmetric somatic hybrids of both species,an d approached the morphology of L. esculentum more after a high dose of irradiation. The high dose hybrids also grew more slowly, flowered and setfruit sles stha nth elo wdos ehybrids .N oviabl eseed scoul db eobtaine d from any asymmetric hybrid. Theasymmetri c hybridswer eals oanalyse dfo rth eretentio no fspecifi c genes and alleles ofth edono r L. peruvianum (Chapter 5).Abou t 50%o f the asymmetric 30H-hybrids (L. peruvianum parent irradiated with 300 Gy) had retained the kanamycin resistance trait from donor plants with one hemizygous resistance locus. L. peruvianum specificallele so fth e isozymemarker s Aps-1 andglutamat e oxaloacetate transaminase were present in at least 70% of the hybrids; the retentiono fdono rallele swa slowe r in30H-tha ni n5H-hybrid s (donor irradiated with 50Gy) .O n the average, 74%o f the L. peruvianum specific alleles (one or 99 both) of 18 morphological markers, which were located on 10 of the 12 tomato chromosomes,wer edetecte d inth e 30H-hybrids.I twa sestimate dtha teac hallel e of a given marker was, on the average, present inhal f of the 30H-hybrids. In 36% of the 30H-hybrids, only one of both recessive morphological markers that were located on a same L. esculentum chromosome was complemented by the corresponding L. peruvianum allele.Thi s isa nindicatio n for frequent breakage ofth e L. peruvianum chromosomes.Severa lhybri dcalli regenerate dgenotypicall y different plants,whic h suggested that some segregation occurred inthes e calli before shoot regeneration. Fifteen asymmetric hybrids, eight 5H- and seven 30H-hybrids, were analysed with 29RFL P markers (Chapter 6);eac htomat o chromosome was represented by at least two such markers.Retentio n of alleles from the irradiated L. peruvianum donor genome in the asymmetric hybrids ranged from 31%t o 83%.O n the average, 67% of the 58allele s of the 29RFL P lociwa s present inth e 5H-hybrids and53 % in the 30H-hybrids. The 5H-hybrids contained more complete L. peruvianum chromosomes, as determined by the retention ofRFL P alleles of the loci on one chromosome ofth edonor ,tha nth e30H-hybrids ,wherea sth e30H-hybrid s contained more incomplete chromosomes; this indicated a more frequent breakage of L. peruvianum chromosomes inth e30H-hybrids .I nmos thybrid s some L. esculentum alleles were lost.Thre e L. peruvianum loci, located on chromosome 2,4 and 7, respectively,wer e present ineac hasymmetri c hybrid,whic hma y suggest linkage to the regeneration capacity trait which was used in selecting the asymmetric hybrids. The asymmetric hybrids were also analysed with a probe for ribosomal DNA (rDNA). Theamoun to frDN Afro m L. peruvianum retained inth ehybrid svarie d strongly: in one hybrid amplification had occurred, whereas in others L. peruvianum rDNA was either absent or (in)corapletely present. The amount of L. esculentum specific rDNA was decreased inmos t asymmetric hybrids.

The analyses of the asymmetric somatic hybrids showed that irradiation before fusioneliminate dth eL . peruvianum genomeonl yt oa limite dextent .I naddition , thehybrid swer e sterile despite their ability toflowe r and set fruits. Itca n be concluded therefore that application of these hybrids in breeding programs isver yrestricted .However ,fertil eprogen yplant sderive dfro mselfe dsymmetri c hybrids might be used for this purpose, because segregation for L. esculentum specific traits was observed. The use of the asymmetric hybrids for somatic mapping is not favourable, because of the limited elimination of the donor genome, the large number of incomplete donor chromosomes and the probable presenceo frearrange dchromosomes .I ti spossibl etha thighl yasymmetri chybrid s can be obtained by the use of other selectable markers or other procedures to induce elimination of the donor genome. 100

SAMENVATTING

DE ISOLATIE EN KARAKTERISERING VAN SOMATISCHE HYBRIDEN VAN IY'COPERSICON ESCZJJLENTUM EN LYCOPERSICON PERUVIANZJM

Bijd everedelin gva nd etomaat , Lycopersicon esculentum, wordtgebrui kgemaak t van verwante, wilde Lycopersicon soorten om gunstige eigenschappen, zoals ziekteresistenties,ui t deze soorten via kruisingen over te brengen naar de cultuurtomaat. Eenalternatiev e methode om gewenste eigenschappen uitander e soortenove rt edrage ni ssomatisch ehybridisati eo fprotoplastenfusie .Hierbi j worden protoplasten, ongeslachtelijke cellen (bijvoorbeeld uit bladweefsel) waarvan de celwand verwijderd is,to t fusie aangezet. Fusieproducten worden vervolgensopgekweek tto tcalluswee fsel ,va nwaarui tplante ngeregenereer dkunne n worden.Eé nva nd evoordele nva ndez etechnie ki sda thybride nverkrege nkunne n worden van niet-kruisbare soorten. Een nadeel is dat de ontstane somatische hybridend ecomplet eaantalle nchromosome nva nbeid esoorte nbevatten ,waardoo r er,z odi ta lmogelij kis ,vel eterugkruisinge nnodi gzij nme td egecultiveerd e soort om alle ongewenste eigenschappen van de wilde soort te verwijderen. Asymmetrische protoplastenfusie,waarbi jd ecelle nva nd een esoor tbestraal d zijn met een hoge dosis gamma- of Röntgen-straling, zou dit nadeel kunnen omzeilen.D echromosome nva nd ebestraald ecelle nworde ngefragmenteerd ,waardoo r dezesne lgeëlimineer dworde ni nee nfusieproduct .Dez easymmetrisch esomatisch e hybridenzoude nminde rterugkruisinge nnodi ghebben .I ndi tproefschrif tzij n experimenten beschreven waarin wordt nagegaan wat de mogelijkheden en moeilijkhedenzij nva nd eoverdrach tva neigenschappe nui tee nwild esoor tnaa r de cultuurtomaat met behulpva n protoplastenfusie. De volgende aspecten,di e vanbelan gzij nvoo rd etoepassin gva nd eprotoplastenfusi e bijd everedelin g vand etomaa tzij ni ndetai londerzocht : (i)he tontwikkele nva nee ndoeltreffend e selectiemethodevoo rd eisolati eva n somatischehybriden ; (ii)he tisolere nva nasymmetrisch esomatisch ehybride nwaarbi jd ewild esoor t bestraaldword tvoo rd efusie ; (iii)he tkarakterisere nva nd ehybride no mt ebepale nwa td ebijdrag e isva n de(bestraalde )wild esoor taa nhe tgenoo mva nd ehybriden . Alswild e soortwer d Lycopersicon peruvianum gebruikt. Deze iszee rmoeilij k kruisbaar met de tomaat en heeft vele voor de veredeling waardevolle 101 eigenschappen.Bovendie nheef t L. peruvianum, integenstellin g totd etomaat , eenzee rgoe dregeneratievermogen ;di t ishe tvermoge nplante nt eregenerere n uit protoplasten- en calluscultures. Deze eigenschap werd gebruikt in de beschrevenexperimente no mfusieproducte ntusse nbeid esoorte nt eselecteren .

Hoofdstuk 2 beschrijft de efficiënte isolatie van symmetrische somatische hybriden van L. esculentum en L. peruvianum via eendubbe l selectiesysteem. Hybridenwerde ngeselecteer d opgron d vanresistenti e tegenhe t antibioticum kanamycine (eeneigenscha pva n L. esculentum) enhe tvermoge nva n L. peruvianum om uit celkweek planten te regenereren.D e geregenereerde plantenware nzee r groeikrachtige nd emeerderhei dha dee ntetraploi dchromosoomaanta l (2n- 4 x- 48);ee naanta lplante nware n(hypo)hexaploi dme t2 n- (64-)72 .D ehybrid eaar d vand e plantenblee kui thu n intermediaire morfologie enwer d aangetoond met behulpva nisozymanalys ee nvoo rsommig eplante nm.b.v .analys eva nrestrictie - fragment-lengte-polymorfismes (RFLP's).Volgen sdez eRFLP-analys ebevatte nd e 6 geteste hexaploide hybriden één diploid genoom van L. esculentum en twee diploïde genomen van L. peruvianum. Eén van deze hexaploiden had genomisch materiaal van twee verschillende L. peruvianum genotypen; deze hybride moet ontstaanzij nui tee nfusi eva ndri everschillend eprotoplasten .D ehexaploid e hybriden leken morfologisch iets meer op L. peruvianum dan de tetraplolde hybriden.D esomatisch ehybride nbloeide nvolo pe nvormde nvruchten . Devruchtbaarhei dva nd etetraplold ee nhexaploid esomatisch ehybride nwer d onderzocht via zelfbevruchtingen en terugkruisingen met beide fusie-ouders (Hoofdstuk 3). Demeest ehybriden ,voora ld etetraplolden ,vormde nvee lzade n nazelfbevruchtingen ;d ezade nware nlevensvatbaa r(79 %kieming) .D enakomelinge n hieruitware nzee rgroeikrachti g envormde nvaa k zadenn azelfbevruchting .D e terugkruisingen van de somatische hybriden met L. esculentum leverden geen levensvatbare zaden op.D e terugkruisingen met L. peruvianum leverdenenkel e levensvatbarezade no pmit s L. peruvianum alsstuifmeelplan twer dgebruikt .D e planten afkomstig van de terugkruising hexaploide hybride x tetraplolde L. peruvianum hadden een pentaplold chromosoomaantal (2n= 5 x- 60 ) en waren groeikrachtig, terwijl de plantenui t de terugkruising tetraplolde hybride x diploïde L. peruvianum vertraagd groeiden. De uitsplitsing van enkele eigenschappenwerde nbi jd enakomelinge ngeanalyseer do minformati et ekrijge n over het chromosoomgedrag in de somatische hybriden. De nakomelingen vand e zelfbevruchte tetraplolde somatische hybriden vertoonden uitsplitsing voor isozympatronen,vanzur efosfatas e (locus Aps-1) engave nee ngroter evariati e voorenkel emorfologisch ekenmerke nt ezie nda nd esomatisch ehybriden ;di twijs t optetrasom everervin gva ntenminst e Aps-1. 102

De isolatie van asymmetrische somatische hybriden van L. esculentum en L. peruvianum was gebaseerd op selectie voor regeneratievermogen (Hoofdstuk 4). Selectie tegen I. peruvianum werd uitgevoerd door deze te bestralen voor protoplastenfusie met lethale doses (50, 300 of 1000 Gray) gamma-straling. De asymmetrische hybriden hadden meer tijd nodig voor plantregeneratie dan de symmetrische hybriden, en vertoonden een grote variatie in callusmorfologie, plantmorfologie en chromosoomaantallen. Alle asymmetrische hybriden waren aneuploïd; na een lage dosis (50 Gy) hadden de meeste hybriden chromosoomaantallen rondomhe t triploïde niveau, terwijl na eenhog edosi s (300 of 1000 Gy) de chromosoomaantallen van de meeste planten zich rondom het pentaploïde niveau bevonden. Ondanks de aneuploidie waren veel plantenvitaal , bloeidene nsommig evormde nvruchten .D ealgemen emorfologi eva nd easymmetrisch e hybride planten was intermediair tussen die van de tomaat en die van de symmetrischehybriden .N aee nhog edosi sleke nd ehybride nmee ro pL . esculentum, groeiden slechter, bloeiden minder en vormden minder vruchten dan na een lage dosis bestraling. De kruisingen met en zelfbevruchtingen van de asymmetrische hybriden leverden geen levensvatbare zaden op. Deasymmetrisch e somatischehybride nwerde noo kgeanalyseer d op aanwezigheid vangene n enallele nva nd edono r L. peruvianum (Hoofdstuk 5). Ongeveer 50%va n de asymmetrische 30H-hybriden, welke ontstaanware n na fusie van L. esculentum en L. peruvianum, bestraald met 300 Gy en hemizygoot voor één copie van het kanamycine-resistentie-gen, bezat kanamycine-resistentie. In minstens 70% van deasymmetrisch ehybride nWare n L. peruvianum specifieke allelenva nd e isozymen Aps-1 en glutamaat oxaalacetaat transaminase aanwezig; de 30H-hybriden (donor bestraald met 300Gy )bezate nminde r vandez eallele nda nd e 5H-hybriden (donor bestraald met 50 Gy). Gemiddeld 74% van de 18 recessieve morfologische eigenschappen, die van L. esculentum afkomstig waren endi e gelocaliseerd zijn op 10 van de 12 tomatechromosomen, waren gecomplementeerd door de corresponderende L. peruvianum allelen(éé no fbeide )i nd e30H-hybriden .Volgen s berekening is elk willekeurig donor allel van een bepaalde isozym- of morfologische eigenschap gemiddeld ind ehelf tva nd e 30H-hybriden aanwezig. In 36% vand e 30H-hybriden was maar één van beide ophetzelfd e chromosoom gelegen morfologische eigenschappen gecomplementeerd door een corresponderend L. peruvianum allel; dit wijst op het vaak voorkomen van breuken in de donor chromosomen. Uitsplitsing voor bepaalde eigenschappen moet opgetreden zijn in de asymmetrische hybride klonen voor regeneratie optrad, omdat verscheidene klonen genotypisch verschillende scheuten vormden. InHoofdstu k 6word t van vijftien asymmetrische hybriden (acht 5H- en zeven 30H-hybriden) een zeer gedetailleerde analyse beschreven met 29 RFLP-merkers. Elk tomatechromosoom is vertegenwoordigd met twee tot vier van dergelijke 103 merkers. 31%to t 83%va n de L. peruvianum specifieke allelen van deze merkers was aanwezig ind e asymmetrische hybriden. Gemiddeld was 67%va n de 58 allelen vand e 29RFLP-loci aanwezig ind e 5H-hybriden en 53%i nd e 30H-hybriden. Ind e 30H-hybriden werden meer incomplete L. peruvianum chromosomen gevonden dan in de 5H-hybriden; kennelijk bevatten de van L. peruvianum afkomstige chromosomen in de 30H-hybriden meer breuken dan die in de 5H-hybriden. De meeste hybriden haddenoo k enkeleL . escuientum specifieke allelenverloren .Dri eRFLP-loci van L. peruvianum, gelocaliseerd op de chromosomen 2, 4 en 7, waren in elke asymmetrische hybride aanwezig; mogelijk zijn deze loei gekoppeld aan de L. peruvianum specifieke genen voor hét regeneratievermogen, waarop de hybriden geselecteerd waren. De asymmetrische hybriden werden ook geanalyseerd met een RFLP-probevoo rhe tribosomal eDN A (rDNA).D easymmetrisch ehybride nverschilde n onderling sterkwa tbetref t dehoeveelhei d L.peruvianu m specifieke rDNA inhu n genoom. Inhe t genoom van één hybride was de relatieve hoeveelheid groter dan inhe t L. peruvianum genoom;i nda tva nander ehybride nontbra khe t L. peruvianum specifieke rDNA geheel, of de hoeveelheid was kleiner dan of even groot als in hetL .peruvianu m genoom. Dehoeveelhei d L. esculentum specifiek rDNAwa s ind e meeste hybriden iets afgenomen.

Uit de resultaten blijkt dat de eliminatie van L. peruvianum chromosomen vrij beperkt was ind e asymmetrische somatische hybriden. Ondanks het feit dat vele asymmetrische hybriden in staat waren te bloeien, is er geen fertiele plant verkregen. Daarom zijn deze hybriden van een zeer beperkte waarde voor veredelingsdoeleinden. Echter de vruchtbare nakomelingen van de zelfbevruchte tetraploïde symmetrische hybriden zouden bruikbaar kunnen zijn vanwege de waargenomenuitsplitsin gvoo renkel e tomate-eigenschappen.Ee nander e mogelijke toepassingva nasymmetrisch ehybride ni she tlocalisere nva ngene ndoo rkoppelin g van de selectie-eigenschap met andere reeds gelocaliseerde genenaa n te tonen. Het isnie t aan te raden om dehie r beschreven asymmetrische hybriden voor dit doel tegebruike n omdatd e eliminatie vandono r chromosomen beperktwas ,e r een groot aantal incomplete chromosomen van de donor aanwezig was en er waarschijnlijk ook herrangschikkingen van de chromosomen opgetreden waren. Met andere selectiemethoden of met andere technieken om eliminatie te bevorderen, zouden misschien sterker asymmetrische hybriden verkregen kunnenworden . 104

CURRI CXJLXJM VI TAE

Jelle Wijbrandi werd geboren op 26 maart 1959 in Bolsward. Na het behalen van het VWO-diploma aan het Bogerman College te Sneek, begon hij in 1977 met de studie Biologie aan de Rijksuniversiteit Groningen. In december 1981 werd het kandidaatsdiploma B5b (2e hoofdvak bodemkundige geologie) behaald. De doctoraalfase omvatte het hoofdvak Celgenetica en de bijvakken Plantenfysiologie en Plantensystematiek. In het kader van het hoofdvak werden gedurende twee maanden experimenten uitgevoerd op het 'Rothamsted Experimental Station' in Harpenden (Groot-Brittannië). Eind augustus 1985 haalde hij het doctoraalexamen. Per 1 september van hetzelfde jaar werd hij aangesteld als assistent-onderzoeker bij de Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO; voorheen ZWO), op een 3-jarig onderzoeksproject gesteund door de Stichting voor Biologisch Onderzoek in Nederland (BION, werkgemeenschap GEFYBO). Het onderzoek werd uitgevoerd onder leiding van dr. ir. M. Koornneef op de vakgroepen Erfelijkheidsleer en Moleculaire Biologie van de Landbouwuniversiteit Wageningen. Eind oktober 1989 treedt hij in dienst als celbioloog bij het plantenbiotechnologiebedrijf Keygene te Wageningen.