Isolation andcharacterisation ofsomatic hybrids between LycopersiconesculentumandLycopersiconperuvianum Isolatieenkarakterisering van somatische hybriden van LycopersiconesculentumenLycopersiconperuvianum

ONTVANGEN

2 0 NOV. t-59 CB-KARDEX

CENTRALE LANDBOUWCATALOGUS

000003590946

Promotor:

dr. C. Heyting hoogleraar in de generatieve en somatische celgenetica

Co-promotor: dr. ir. M. Koornneef universitair hoofddocent

Jelle Wijbrandi

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,Friesche rymlerye)

Met dank aande "Stichting Hendrik Nannes- enCatrijn Epes-leen" teBolsward, die het uitgeven van dit proefschrift middels een financiële bijdrage mogelijkheeft gemaakt.

^ J o ? z o i .1*320

S T E L L I N G E N

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 éénvan beide fusieouders.

3.

Aneuploldie in somatische hybridenkan leiden tot incongruentie.

4.

Interspecifieke somatische hybridisatie verhoogt de genetische variatie van een gewas.

Als modelgewas

voor

celgenetisch

geschikter dan Nicotiana

onderzoek

is de cultuurtomaat

soorten, vanwege de goed ontwikkelde genetica

bijde tomaat. Dit proefschrift

6.

In geval van niet-groeiende of niet-regenererende fusieproducten van plantecellen,

verdient

het

aanbeveling

de

term

somatische

incompatibiliteit tevervangen door somatische incongruentie. Harms CT (1983)TheQuarterly Review ofBiology 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 inde 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 bijeengroot publiek. In: Vandersteen W (1987) Suske en Wiske. De woeste wespen, Standaard UitgeverijAntwerpen, p3.

10. Mede gezien de zorgvuldige procedures die het onderzoeksinstituut Ital gevolgd heeft alvorens een proefveld met transgene aardappels in te richten,kan gesteld worden dat 'HetZiedende Bintje'halfgaar 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: characterisation of somatic hybrids between Lycopersicon Lycopersicon peruvianum." Wageningen, 29november 1989.

"Isolation and esculentum and

VOORWOORD

Dit proefschrift beschrijft de resultaten van een promotie-onderzoek aangaande protoplastenfusie bij de tomaat. Dit boekje was niet verschenen zonderde hulpvanvele mensen,die ikhierbijwil bedanken. Inde 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 copromotor,die me met veel inzet en enthousiasme heeft begeleid tijdens alle fasen van het onderzoek; Corrie Hanhart en Patty van LoenenMartinetSchuringa, 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 mijmet 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 indekas inleven of intoomhebbengehouden. 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 alsvrijwilligsterveelwerk verzetheeft. 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 ende goedewerksfeer. De collega's van andere vakgroepen van de LUW, andere universiteiten en institutenwil ikbedankenvoor overleg en samenwerking. Ik ben BION en het Löhnisfonds zeer erkentelijk voor het financieren van een studiereis naarAmerika. Verder bedank ik mijn familie-, vrienden- en kennissenkring; met name Monique, Erik,Piet enMarieke voor degezellige 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. Chapter2.

Chapter3.

Chapter4.

Chapter5.

Chapter6.

Generalintroduction Selectionandcharacterisationofsomatichybrids between Lycopersicon esculentum and Lycopersicon peruvianum '

11

Analysisofprogeniesderivedfromsomatichybrids between Lycopersicon esculentum and Lycopersicon peruvianum

29

Asymmetricsomatichybridsbetween Lycopersicon esculentum andirradiated Lycopersicon peruvianum I. Cytogeneticsandmorphology

41

Asymmetricsomatichybridsbetweenlycopersicon *•esculentumandirradiated Lycopersicon peruvianum II. Analysiswithmarkergenes

57

Asymmetricsomatichybridsbetween Lycopersicon esculentum andirradiated Lycopersicon peruvianum III.Analysiswithrestrictionfragmentlength polymorphisms

71

Chapter7. Summary

Generaldiscussion

91 97

Samenvatting

100

Curriculumvitae

104

CHAPTER1 G E N E R A L

Partial

I N T R O D U C T I O N

genome transfer

by somatic

hybridisation

Somatic hybridisation is a technique by which nuclear genomes of different species can be combined. Interspecific somatic hybrids have been obtained of various animal cell lines (Ringertzand Savage 1976), fungi (Peberdy 1979)and plants (Glimelius 1988). Plant somatic hybrids originate from fusion of protoplasts, 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 importantthatundesirabletraitsarenottransferredtothecultivatedspecies, or thatthey canbe removed from thehybrids.After sexualhybridisation it is often possible to achieve this by repeated backcrossing of the hybrid to the cultivatedparent.Inthisrespectsomatichybridisationhassomedisadvantages, since most of the fertile somatic hybrids are stable polyploids. Preferential elimination of the chromosomes of one of the parental species, like has been reported for several mammalian somatic hybrids (Ringertz and Savage 1976), rarelyoccursinsomatichybridsofplants(GlebaandSytnik1984).Thepolyploid state of the hybrids may cause sexual incongruity with the cultivated parent. Crossing ofan interspecific somatic hybrid to the cultivated parental species isthereforenotalwayspossible.Thisproblemmaybecircumventedbyasymmetric somatic hybridisation; inthis case,untreated protoplasts ofone species,the recipientoracceptor,arefusedwithprotoplastsofanotherspecies,thedonor, whosenucleargenome isreduced.Theconstructionofasymmetric somatic hybrids canbe done invarious ways: (i)bytheuseofhaploid cellsfromadonorspecies.Thesecellscanbederived 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, inwhich introgression of donor genes 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 hybridistheisolationofmicroprotoplastsfrommicronucleated cells (Verhoeven 1989). Micronuclei are inducedbyexposure ofmitotic cells toaspindletoxin; protoplastsofthesecellsarefractionatedintomicroprotoplasts,whichcontain 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 ofthecontributionofthedonorgenome tothatofthe hybrids canalsobeachieved bytreatment ofthedonor cellswithhighdosesof ionisingradiation(Röntgen-andgamma-rays),whichfragmentisesthechromosomes. Thismethodwas originally developed tocompletely eliminate thenuclear donor genome in order to obtain cybrids (fusion products that contain only the cytoplasm of thedonor; 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 replicatedandmaintained iftheycontainacentromere regionand telomeres.If chromosome fragments lack such structures they can still be rescued by recombinationwith otherdonor 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 eliminationofmostofthechromosomesofoneparenthad occurred, theremainingdonorchromosomes couldbeidentified bycytogenetic 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 intomammalian cells bymicrocell fusion; this procedure has also been used successfully for gene mapping (Lugo and Fournier 1986). Irradiationofonefusionparenthasnotonlybeenused toenhance preferential eliminationofchromosomesfromoneparent (Pontecorvo 1971),buthasalsobeen appliedforregionalmappingofchromosomes;thus,thelinearorderofdifferent markergenesonasamechromosome (region)couldbedetermined bymeasuring the frequency ofthepresence ofthese genes inalarge populationofhybrids (Goss and Harris 1975).

Asymmetric

somatic hybrids

of

plants

Partial genome transfer byasymmetric somatic hybridisation has been described for several plant species. Asymmetric nuclear hybrids were isolated from cybridisation experiments,asunintended by-products (Zelcer etal. 1978;Aviv

and Galun 1980;Sidorov et al. 1981;Menczel et al.1982,1983;Hamill etal. 1984). Besides,asymmetric hybrids were purposely obtained from experiments in which selection was based on a nuclear encoded character of the irradiated donor.Efficient selectablemarkersarerequired toselectforfusionproducts, because, in general, the frequencies of asymmetric hybrids obtained were much lower than those of symmetric hybrids obtained after fusion of unirradiated protoplasts. Inthefirstreported,successful asymmetric somatic hybridisation experiment, albino carrot protoplasts were fused with irradiated parsley protoplasts; the regenerated greenplanthad one additional chromosome (Dudits etal. 1980). Inmanyoftheasymmetrichybridisationexperiments,theselection wasbasedoncomplementationofthenitratereductasedeficiencyoftherecipient species (e.g. Gupta et al. 1982;Gleba 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.Interspecificasymmetricsomatichybridisationexperiments.Unirradiated protoplasts from a (recipient) species were fused with irradiated protoplasts from another (donor)species.

Asymmetric somatic nuclear hybrids recipient (+)donor

References

Intrageneric hybrids:

Brassica

campestris

(+) B.

oleracea*

Nicotiana glauca (+)JV. langsdorffii N. plumbaginifolia (+)JV. sylvestris* JV. plumbaginifolia (+)JV. tabacum* JV. tabacum (+)JV. paniculata JV. tabacum (+)JV. plumbaginifolia* Solanum tuberosum (+)S. pinnatisectum

Yamashita et al. 1988 Itohand Futsuhara 1983 Famelaer et al. 1989 Koornneef et al. 1988 Müller-Gensert and Schieder 1987 Bates etal.1987 Sidorov et al. 1987

Intergeneric, intrafamiliarhybrids:

Daucus carota (+) Petroselinum hortense* Datura innoxia (+) Physalis minima* N. tabacum (+) Datura innoxia JV. tabacum (+) Physal is minima

Dudits et al. 1980 Gupta et al. 1984 Imamura et al. 1987 Gleba et al. 1988 Gupta etal.1982 Gupta et al.1982

Interfamiliarhybrids: JV. tabacum (+) Daucus carota* JV. tabacum (+) Hordeum vulgare*

Dudits et al. 1987 Somers et al. 1986

Hyoscyamus muticus JV. plumbaginifolia

(+)JV. tabacum (+) Atropa belladonna*

asymmetric hybrid plants obtained

combination of parents. Inmost of the intrageneric combinations,a limited eliminationwasobserved,whereasintheunrelatedcombinationstheelimination wasmoreincreased.Inmostoftheintergenerichybrids,oneorafewchromosomes wereretained,whileintheinterfamiliarhybridsonlyafewtraits(Duditset al.1987)orevenonetrait(Somersetal.1986)ofthedonorspeciescouldbe detected.Whendifferentdoseswereusedinthesameexperiment,nocleareffect oftheapplieddosewasobservedontheeliminationofdonorgenome (Glebaet al. 1988; Yamashita et al. 1988; Famelaer et al. 1989). Five different experiments have been described, where asymmetric hybrids were fertile when selfedofbackcrossedtotherecipientspecies(Somersetal.1986;Batesetal. 1987; Duditsetal.1987;Sidorovetal.1987;Yamashitaetal.1988). Intwo otherexperimentsbackcrosseshadtoberescuedbyembryoculture(Glebaetal. 1988;Famelaeretal.1989).

Scope of the

study

Asymmetricsomatichybridshaveanimportantpotentialuseforplantbreeders whowanttotransfermono-andmultigenictraitsfromamoreorlessunrelated (donor)speciestoarecipientcropspecies.MonogenictraitsofwhichtheDNA hasbeen cloned canbemore easily transferred by other techniques,suchas Agrobacterium transformationanddirectgenetransfer (HohnandSchell 1987). To evaluate the possibilities and difficulties of somatic hybridisation, experimentswereperformedwithaplantspeciesthatcanbeanalysedgenetically indetail,namelythecultivated tomato (Lycopersicon esculentum). Protoplast fusionswere carried outwith the tomato and itswild relative Lycopersicon peruvianum. Thesetwospeciesaredifficulttohybridisesexually.Regeneration capacityfrom L. peruvianum waschosenasselectabletraitandalsoasanexample of a multigenic trait which might be desirable to transfer by somatic hybridisation.Theaimsoftheexperimentswere: (i)theisolationofsomatichybridsbetweenbothspeciesinanefficientway andthesubsequentcharacterisationoftheseplants; (ii) the isolation of asymmetric hybrids between both species, using L. peruvianum asdonorspecies; (iii)thecharacterisationoftheseasymmetrichybridsinordertodeterminethe degreeofeliminationofdonorgenomeand,inaddition,toevaluatetheiruse forbreedingpurposesandmappingstudies.

Lycopersicon Lycopersicon

L. esculentum

esculentum Hill., the cultivated tomato, and peruvianwn (L.) Mill., "the most variable and least exploited tomato species" (Rick and L. peruvianum

are both members of the genus Lycopersicon

1979a) of

thefamily Solanaceae. Thisgenusiscloselyrelatedtothegenus Solanum and consistsofeightspecies,whicharedividedinthe'esculentum-complex'andthe 'peruvianum-complex' (Taylor 1986). The species oftheformer complexcanbe easilycrossedwith L. esculentum, whereasthespeciesofthelattercomplex, namely L. peruvianum and L. chilense, canonlybecrosseswithgreatdifficulty. All Lycopersicon speciesshowahighdegreeofhomologyintheirchromosomes, whichallowsmeioticrecombinationinspecieshybrids (Rick1979b). Thecultivatedtomatoisanimportantfoodcrop.Moreover,itisafavorite modelsystemforgeneticstudiesinplants,becausethecropiseasytoculture, hasarelativelyshortlifecycle,ishighlyself-fertileandisasimplediploid (2n=2x=24)whosechromosomesaredistinguishableatthepachytenestageof meiosis (Rickand Butler 1956;Gill 1983;Rick andYoder 1988;Hilleetal. 1989).Thelinkagemapoftomatocontainsmorethan300morphological,isozymeanddiseaseresistancemarkers(Mutschleretal. 1987),towhichatleast300 restrictionfragmentlengthpolymorphism(RFLP)markershavebeenaddedinrecent years(YoungandTanksley1989). L. peruvianum isanoutbreedingwildrelativeofthecultivatedtomatowith ahighlevelofvariability (Rick1979).Therearemanyaccessions,whichhave agreatnumberofcharactersdesirablefortomatoimprovement,suchasdisease resistances (Rick1982).Becauseofthecrossingbarriersbetweenbothspecies (Hogenboom1972a),onlyafewresistances,namelyagainsttheroot-knotnematode Heloidogyne

incognita

(Gilbert and McGuire 1956), Pyrenochaeta

lycopersici

(corkyroot;Szteyn1962),tobaccomosaicvirus(Alexander 1963), Cladospoxium (KerrandBaily1964)andcurlytopvirus (Martin 1969), couldbeintroduced intothetomato.WhenL.peruvianumisusedaspistillateparent,growthofthe L.esculentumpollentubesisinhibited.When L. peruvianum isthestaminate parent,fertilisationtakesplace;however,theembryosoonabortsduetothe degenerationoftheendosperm(BarbanoandTopoleski1984).Thecrossingbarriers wereovercomebytherescueoftheabortiveembryobytissueculturetechniques (Smith1944;deNettancourtetal.1974;ThomasandPratt1981)andtheselection forrareI.peruvianumgenotypeswithareducedpollentubeinhibition(Hogenboom 1972b;Rick1983). L. peruvianum alsohas desirablecellandtissueculture properties.Its

callus growth and shoot regeneration capacity are superior to those of L. esculentum (Zapataetal.1977;Mühlbach1980;MorganandCocking1982).The latterspeciesisverydifficulttoregeneratefromprotoplastandestablished callus cultures. An L. esculentum genotype with the good callus growth and superior regeneration capacity from L.peruvianum was obtained by classical breeding (Koornneefetal. 1986).Thegeneticanalysisofthisplantrevealed thatthe favourable traits ofL.peruvianumwere dominant,that regeneration capacitywascontrolledbytwogenesandthatthecallusgrowthcharacteristics wereshowntobecontrolledbydifferentloci(Koornneefetal. 1987).

Somatic hybrids

of

Lycopersicon

Alreadyin1978hybridsbetweentomatoandpotatowereobtained(Melchersetal. 1978); however,thesehybridswere notfertile andformed fruitsnortubers. Morerecently,othersomatichybridplantsbetweendifferent Lycopersicon species andbetween Lycopersicon and Solanum specieswereobtained (Table2).Theaim ofthesesomatichybridisationexperimentswastoby-passcrossingbarriersand possiblytointroducevaluableagronomictraitsintothetomato.Althoughthese hybridswerevigorousplantsandoftendidflowerandsetfruits,noprogenies were described. Somatic hybrids between L. esculentum and L. peruvianum were alsoobtained;however,from21differentfusionexperimentsonlytwohybrids were isolated,because thesehybridscould onlybe identified assuchatthe plantlevelandnotincellculture (Kinsaraetal. 1986).Asymmetricsomatic hybridsoftomatohavenotbeenreportedsofar.

Table2.Somatichybridisationexperimentswith Lycopersicon species. Fusioncombination

References

I. esculentum esculentum esculentum

(+) L. (+) L. (+) S.

L.

(+) S. nigrum

L. L.

esculentum

pennellii peruvianum lycopersicoides

L. L.

esculentum esculentum

(+) (+)

S. S.

rickii tuberosum

L.

pennellii

(+)

L.

peruvianum

L.

pimpinellifol

ium (+)

S.

tuberosum

O'ConnellandHanson1987 Kinsaraetal.1986 Handleyetal.1986 Tan1987 Gurietal.1988 Jainetal.1988 O'ConnellandHanson1986 Melchersetal.1978 Shepardetal.1983 AdamsandQuiros1985 Tan1987 Okamura1988

Outline

of the

thesis

Two types of fusion experiments with protoplasts of L. esculentum and L. peruvianumwerecarriedout.Aselectionstrategywasdeveloped,toobtain efficiently somatic hybrids.Selectionagainst L. peruvianum wasachievedby the use of kanamycin resistant L. esculentum genotypes and the subsequent selectionforkanamycinresistanthybrids.These"symmetric"somatichybridswere characterised cytogenetically,biochemically andmorphologically (Chapter2). Since these hybridswere fertile,their progeny could be characterised also (Chapter3). In the second series of protoplast fusion experiments, L. peruvianum was irradiatedbeforefusioninordertoachievethetransferofonlypartofthe genome ofI.peruvianum. Inthese experiments,the effect ofdifferentdoses ofgamma-raysontheeliminationofdonorgenomewasanalysed.Allasymmetric hybridswerecharacterisedcytogeneticallyandmorphologically (Chapter 4).To determinetheamountoftransferredL.peruvianumgenome,theasymmetrichybrids wereanalysedforthepresenceof L. peruvianum specificgenes(Chapter 5). In addition,fifteenasymmetricsomatichybridswerecharacterised indetailwith 30chromosomespecificRFLPmarkers(Chapter6). Finally, the significance of the somatic hybrids obtained and of the asymmetrichybridisationtechniquearediscussed (Chapter7).

References Adams TL,QuirosCF (1985)Somatic hybridizationbetweenLvcopersicon peruvianum and Lvcopersiconpennellii: regenerating ability and antibiotic resistance asselection systems.PlantScience 40:209-219 Alexander LJ (1963)Transfer of a dominant type of resistance to the fourknown Ohio pathogenic strainsof tobaccomosaic virus (TMV), fromLvcopersicon peruvianum toL.esculentum. Phytopathology 53:869 Aviv D, Galun E (1980)Restoration of fertility in cytoplasmic male sterile (CMS)Nicotiana sylvestris by fusionwithX-irradiated N. tabacumprotoplasts.TheorApplGenet 58:121-127 BarbanoPP.TopoleskiLD (1984)PostfertilizationhybridseedfailureinLvcopersiconesculentumxLvcopersicon peruvianum ovules.JAmerSocHortSei 109:95-100 BatesGW,HasenkampfCA,ContoliniCL,PiastuchWC (1987)Asymmetriehybridization inNicotianaby fusionof irradiated protoplasts.TheorApplGenet 74:718-726 de Nettancourt D, DevreuxM, Laneri U,Cresti M, Pacini E,Sarfatti G (1974)Genetical and ultrastructural aspects of self and cross incompatibility in interspecific hybrids between self-compatible Lvcopersicon esculentum and self-incompatible L.peruvianum. TheorApplGenet 44:278-288 DuditsD, Fejer 0,Hadlaczky GY,KonczCS,Lazar G, Horvath G (1980)Intergeneric gene transfermediated by plantprotoplast fusion.MolGenGenet 179:283-288 DuditsD,MaroyE,Praznovszky T,OlahZ,Gyorgyey J,CellaR (1987)Transferofresistancetraitsfromcarrot into tobaccoby asymmetric somatic hybridization:Regeneration of fertileplants.Proc Natl Acad SeiUSA 84:8434-8438 FamelaerI,GlebaÏY,SidorovVA,KaledaVA,ParakonnyAS,BoryshukNV,CherupNN,NegrutiuI,JacobsM (1989) Intrageneric asymmetric hybrids between Nicotiana plumbaginifolia and Nicotiana sylvestris obtained by 'gamma-fusion'.PlantScience 61:105-117

Gilbert JC,McGuire DC (1956)Inheritance of resistance to severe root knot fromMeloidoRvne incognita in commercialtypetomatoes.FrocAm SocHorticSei68:437-442 GillBS (1983)Tomato cytogenetics -A search fornew frontiers.In:SwaminathanMS,GuptaPK,SinhaU (eds) Cytogenetics ofcropplants.MacMillanIndiaLtd,NewDelhi,pp455-480 GlebaYY, Hinnisdaels S, Sidorov VA,Kaleda VA, Parokonny AS,Boryshuk NV, Cherup NN, Negrutiu I, JacobsM (1988)IntergenericasymmetrichybridsbetweenNicotianaplumbaginjfoliaandAtropabelladonnaobtainedby "gamma-fusion". TheorApplGenet 76:760-766 GlebaYY,SytnikKM (1984)Protoplast fusion.Genetic engineering inhigherplants.SpringerVerlag,Berlin GlimeliusK (1988)Potentialsofprotoplastfusioninplantbreedingprograms.In:PuiteKJ,DonsJJM,Huizing HJ,KoolAJ,KoornneefM,KrensFA (eds)Progressinplantprotoplast research,Kluwer,Dordrecht,pp159168 Goss SJ,HarrisH (1975)Newmethod formapping genesinhuman chromosomes.Nature 255:680-684 GuptaPP,GuptaM,Schieder0(1982)Correctionofnitratereductasedefectinauxotrophicplantcellsthrough protoplast-mediated intergeneric genetransfers.MolGenGenet 188:378-383 Gupta PP, Schieder 0, Gupta M (1984) Intergeneric nuclear gene transfer between somatically and sexually incompatibleplantsthroughasymmetric protoplast fusion.MolGenGenet 197:30-35 GuriA,LeviA,SinkKC (1988)Morphological andmolecular characterizationofsomatic hybridplantsbetween Lvcopersicon esculentum andSolanumnigrum.MolGenGenet 212:191-198 HamillJD,Pental D,Cocking EC (1984)The combination of anitrate reductase deficient nuclear genomewith a streptomycin resistant chloroplast genome,inNicotiana tabacum. byprotoplast fusion. J Plant Physiol 115:253-261 Handley LW, Nickels RL, Cameron MW, Moore PP, Sink KC (1986) Somatic hybrid plants between Lycopersicon esculentum andSolanum lvcopersicoides. TheorApplGenet 71:691-697 HilleJ,KoornneefM,RamannaMS,ZabelP (1989)Tomato:a crop species amenable to improvement by cellular andmolecularmethods.Euphytica 42:1-23 Hogenboom NG (1972a) Breaking breeding barriers in Lycopersicon. 1. The genus Lvcopersicon. its breeding barriers andtheimportance ofbreaking thesebarriers.Euphytica 21:221-227 Hogenboom NG (1972b)Breakingbreeding barriers inLycopersicon. 4. Breakdown ofunilateral incompatibility betweenL.peruvianum (L.)Mill,andL. esculentumMill..Euphytica21:397-404 HohnT,SchellJ (1987)PlantDNA infectious agents.Springer-Verlag,WienNewYork ImamuraJ,SaulMW,PotrykusI(1987)X-rayirradiationpromotedasymmetricsomatichybridisationandmolecular analysisoftheproducts.TheorApplGenet 74:445-450 ItohK, Futsuhara Y (1983) Interspecific transfer of only part of genome by fusion between non-irradiated protoplasts ofNicotiana glauca andX-ray irradiated protoplasts of N. langsdorffii. Jpn JGenet58:545553 JainSM,ShahinEA,SunS (1988)Interspecificprotoplast fusion forthetransferofatrazineresistancefrom Solanumnigrumtotomato(LycopersiconesculentumL.). In:PuiteKJ,DonsJJM,HuizingHJ,KoolAJ,Koornneef M,Krens FA (eds)Progress inplantprotoplast research,Kluwer,Dordrecht,pp221-224 Kerr EA,Bailey DL (1964)Resistance toCladosporiumfulvumCke.obtained fromwild species oftomato.CanJ Bot 42:1541-1554 KinsaraA,PatnaikSN,CockingEC,PowerJB (1986)Somatichybrid plantsofLycopersicon esculentumMill,and LvcopersiconperuvianumMill..JPlantPhysiol 125:225-234 KoornneefM, Hanhart CJ, Jongsma M, Toma I,WeideR, Zabel P, Hille J (1986)Breeding of atomato genotype readily accessible togeneticmanipulation. PlantScience 45:201-208 KoornneefM, HanhartCJ,Martinelli L (1987)Agenetic analysisofcell culturetraits intomato.TheorAppl Genet 74:633-641 KoornneefM,denBoerB,vanLoenen-MartinetP,vanRoggenP,WijbrandiJ(1988)Protoplastfusionofkanamycin resistant, cnx"Nicotiana plumbaginifoliawith streptomycin resistant N. tabacum (SRI)and the effect of irradiationofthetabacumparent.In:PuiteKJ,DonsJJM,HuizingHJ,KoolAJ,KoornneefM,KrensFA (eds) Progress inplantprotoplast research,Kluwer,Dordrecht,pp287-288 LeeCH,Power JB (1988)Intra-and interspecific gametosomatic hybridisationwithin thegenusPetunia.Plant Cell TissOrgCult 12:197-200 Lugo TG, Fournier REK (1986) Microcell fusion and mammalian gene transfer. In: Kucherlapati R (ed) Gene transfer.Plenum Publishing Corp,NewYork,pp79-93

MartinNW(1969)InheritanceofresistancetocurlytopinthetomatobreedinglineC/F,.Phytopathology 59:1040 MelchersG, SacristanMD,Holder AA (1978)Somatic hybrid plantsofpotato and tomatoregenerated fromfused protoplasts.CarlsbergResCommun 43:203-218 Menczel L,GalibaG,Nagy F,MaligaP(1982)Effectofradiationdosageonefficiency ofchloroplasttransfer byprotoplast fusion inNicotiana.Genetics 100:487-495 MenczelL,NagyF,LâzârG,MaligaP(1983)Transferofcytoplasmicmalesterilitybyselectionforstreptomycin resistance afterprotoplast fusion inNicotiana.MolGenGenet 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 speciesoftomato.Planta 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,Rick CM (1987)Linkagemaps of the tomato (Lvcopersicon esculentum).Rep Tomato GenetCoop 37:5-34 O'Connell MA, Hanson M (1986)Regeneration of somatic hybrid plants formed between Lvcopersicon esculentum andSolanumrickii.TheorApplGenet 72:59-65 O'Connell MA, Hanson M (1987)Regeneration of somatic hybrid plants formed between Lvcopersicon esculentum andL.pennellii.TheorApplGenet 75:83-89 OkamuraM(1988)RegenerationandevaluationofsomatichybridplantsbetweenSolanumtuberosumandLvcopersicon pimpinellifolium.In:PuiteKJ,DonsJJM,HuizingHJ,KoolAJ,KoornneefM,KrensFA(eds)Progressinplant protoplast research,Kluwer,Dordrecht,pp213-214 Peberdy JF (1979)Protoplast fusion -A new approach to interspecific genetic manipulation and breeding in fungi. In:Ferenczy L,FarkasGL (eds)Advance inprotoplastresearch.PergamonPress,Oxford,pp63-72 PentalD,MukhopadhyayA,GroverA,PradhanAK (1988)Aselectionmethod forthesynthesisoftriploidhybrids by fusionofmicrosporeprotoplasts (n)with somatic cellprotoplasts (2n).TheorApplGenet 76:237-243 PentalD,Cocking EC (1985)Some theoretical andpracticalpossibilities ofplantgenetic manipulationusing protoplasts.Hereditas (Suppl)3:83-92 PirrieA, Power JB (1986)Theproductionof fertile,triploid somatichybrid plants (Nicotiana«lutinosa (n) +N,tabacutn (2n))viagametic:somaticprotoplast fusion.TheorApplGenet 72:48-52 PontecorvoG (1971)Inductionofdirectional chromosome elimination insomatic cellhybrids.Nature230:367369 RickCM (1979a)Potentialimprovementoftomatoesbycontrolled introgressionofgenesfromwildspecies.Proc ConfBroadeningGenetBaseCrops,Wageningen, 1978.Pudoc,Wageningen,pp 167-173 Rick CM (1979b)Biosystematic studies inLvcopersicon and closelyrelated species ofSolanum. In:HawkesJG, LesterRN,SkeldingAD (eds)Thebiology and taxonomyoftheSolanaceae.Academic Press,NewYork,pp667677 RickCM (1982)Thepotentialofexoticgermplasm fortomatoimprovement.In:VasilIK,ScowcroftWR,FreyKJ (eds)Plantimprovement and somatic cellgenetics.Academic Press,NewYork,pp 1-28 RickCM (1983)CrossabilitybetweenL.esculentumandanewraceofL.peruvianum.RepTomatoGenetCoop33:13 RickCM,Butler L (1956)Cytogenetics ofthetomato.AdvGenet8:267-382 Rick CM, Yoder JI (1988)Classical and molecular genetics of tomato: highlights and perspectives. Ann Rev Genet 22:281-300 RingertzNR,SavageRE (1976)Chromosomepatternsinhybridcells.In:Cellhybrids.AcademicPress,NewYork, pp 162-179 Shepard JF,Bidney D, Barsby T,KembleR (1983)Genetic transfer inplants through interspecific protoplast fusion.Science 219:683-688 Sidorov VA, Menczel L, Nagy F, Maliga P (1981) Chloroplast transfer in Nicotiana based on metabolic complementationbetweenirradiated and iodoacetate treated protoplasts.Planta 152:341-345* SidorovVA,ZubkoMK,KuchkoAA,KomarnitskyIK,GlebaYY (1987)Somatichybridizationinpotato:useofgammairradiatedprotoplastsofSolanumpinnat.i^ftrt.nmingenetic reconstruction.TheorApplGenet 74:364-368 SmithPG (1944)Embryocultureofatomatospecieshybrid.ProcAmerSocHortSei 44:413-416 Somers DA,NarayananKR, Kleinhofs A,Cooper-Bland S,Cocking EC (1986)Immunological evidence for transfer of thebarley nitrate reductase structural gene toNicotiana tabacumby protoplast fusion.MolGenGenet 204:296-301

10 SzteynK (1962)Interspecific crosses inthegenusLycopersicon. I.Backcrosses toLycopersiconglandulosum. Euphytica 11:149-156 Tan M C (1987) Somatic hybridization and cybridization in some Solanaceae. Ph D Thesis, Free University Amsterdam,TheNetherlands TaylorIB (1986)Biosystematicsofthetomato.In:AthertonJG,RudichJ (eds),Thetomatocrop.A scientific basis for improvement.ChapmanandHall,London/NewYork,pp 1-34 ThomasBR,PrattD (1981)EfficienthybridizationbetweenLycopersiconesculentumandL.peruvianumviaembryo callus.TheorApplGenet 59:215-219 VerhoevenHA(1989)Inductionandcharacterizationofmicronucleiinplantcells.Perspectivesformicronucleusmediated chromosome transfer.PhDThesis,AgriculturalUniversityWageningen,TheNetherlands. Yamashita Y, Terada R, Nishibayashi S, Shimamoto K (1989)Asymmetric somatic hybrids of Brassica: partial transfer offf.campestrisgenomeintoB.oleraceaby cellfusion.TheorApplGenet 77:189-194 Young ND, Tanksley SD (1989)Restriction fragment length polymorphism maps and the concept of graphical genotypes.TheorApplGenet 77:95-101 ZapataFJ,EvansPK,PowerJB,CockingEC (1977)Theeffectoftemperatureonthedivisionofleafprotoplasts ofLycopersiconesculentum andL.peruvianum. PlantScience Lett8:119-124 Zelcer A, Aviv D, Galun E (1978) Interspecific transfer of cytoplasmic male sterility by fusion between protoplastsofnormalNicofrianasylvestris andX-ray irradiated protoplastsofmale-sterile N,tabacum. Z Pflanzenphysiol 90:397-407

11 CHAPTER2 S E L E C T I O N A N D

C H A R A C T E R I S A T I O N

SOMATIC HYBRIDS BETWEEN ESCUI^ENTZJM

A N D

Z^YCOPERSICON

L.Y COPE RS

O F

ICON PERZJVIANUM

J.Wijbrandi,W.vanCapelle,C.J.Hanhart,E.P.vanLoenenMartinet-Schuringa, M.Koornneef

Summary.Somatichybridsofthecultivatedtomato, Lycopersicon esculentum, and awildspecies, L. peruvianum, wereobtainedbyfusionofleafprotoplastsfrom bothspeciesinthepresenceofpolyethyleneglycolorinanelectricfield. The somatic hybrids were selected on the basis of kanamycin resistance of L.esculentumandtheplantregenerationcapacityof L. peruvianum. Chromosome countsinroottipsandthedeterminationofthenumberofchloroplastsinguard cellpairsrevealedthatthemajorityofthesehybridswastetraploid (2n-4x =48).Theremaininghybridswereatthehexaploidlevelwithchromosomenumbers between64and72.Thehybridnatureoftheregeneratedplantswasconfirmedby analysisofisozymemarkersandbytheirmorphology.Mosthybridsdidflowerand setfruitsandseedsafterselfing.AccordingtoRFLPanalysis6outofthe10 hexaploid hybrids contained twogenomes of L. esculentum andfourgenomesof L.peruvianum.Oneofthesehexaploidshadgenomesoftwodifferent L. peruvianum genotypesandwasthereforeconsidered tobederivedfromatripleprotoplast fusion.Thehexaploid plantswerelessfertile thanthetetraploids andmore resembledL.peruvianum.

Introduction Somatichybridisationisanalternative tosexualhybridisationwhencrossing barriers between parental species exist. By means of various cell culture techniques,protoplastsfromdifferentspeciescanbeinducedtofusewitheach other. Several procedures have been applied to select somatic hybrids after fusion.Somatichybridsof Solanum and Lycopersicon specieshavebeenselected eithermechanicallybymicromanipulation(Puiteetal.1986),byselectionbased onspecificgrowthcharacteristicsincellculture(Gleddieetal.1986;Handley etal.1986),orbytheidentificationofhybridsattheplantlevel(Austinet al.1985;Shihachakr1989;Tan1987).Regenerationcapacitythatderivedfrom one of the parentshas beenusedas selectable markerby several authorsin protoplastfusionexperimentsof Nicotiana (Maligaetal.1977), Petunia (Itoh and Futsuhara 1983), Brassica (Terada et al. 1987)and Lycopersicon species (AdamsandQuiros1985;Kinsaraetal. 1986). In the present experiments, protoplasts of Lycopersicon esculentum, the cultivated tomato, and L. peruvianum, a wild tomato species with several

12 desirableagriculturaltraits(Rick1982a),werefused.Thesespeciescannotbe crossedeasilywitheachother.When L. esculentum isusedasstaminateparent, pollen tube elongation is inhibited in the L. peruvianum style (unilateral stylarincompatibility orincongruity (Hogenboom1973).When L. peruvianum is thepollinator,fertilisationtakesplace.However,theembryosoonabortsdue tothedegenerationoftheendosperm(BarbanoandTopoleski1984).Inthelatter combination sexual hybrid plantswere obtained by the application of tissue culturetechniquessuchasembryoculture(Smith1944)andembryocallusculture (ThomasandPratt1981).BackcrossesofsuchhybridswithI.esculentumwerenot possiblewithouttheapplicationofadditionaltissueculturetechniques(Smith 1944;ThomasandPratt1981). Theaimofthepresentexperimentswastheproductionofsymmetricsomatic hybridsbetweenthetwospeciesinanefficientandreproduciblemannerbythe developmentofadoubleselectionstrategybasedonthedominantregeneration capacityof L. peruvianum (AdamsandQuiros1985;Kinsaraetal.1986;Koornneef etal.1987a)andthedominantkanamycinresistanceof L. esculentum. Thelatter selectionmarkerhadbeenintroducedbyleafdisctransformationof L. esculentum with Agrobacterium tumefaciens. Thehybridsobtainedwerecharacterisedonthe basisofchromosomenumbers,isozymepatterns,morphology,fertilityand,insome cases,bytheanalysisofrestrictionfragmentlengthpolymorphisms (RFLPs).

Materialandmethods Plant material SeedsofL.escuientumcv.Bellina, L. esculentum genotypeLA1182 (homozygous forrecessivemarkergenes sy, sf (chr.3)and alb (chr.12)(Rick1982b)and L. peruvianum PI128650werekindlyprovidedbyRijkZwaanSeedCompany(deLier, The Netherlands), Prof. CM. Rick (Tomato Stock Center,Davis,USA)and the Institute of Horticultural Plant Breeding (Wageningen, The Netherlands), respectively. Kanamycin resistant transformants of these genotypes,obtained afterleafdisctransformationwith A. tumefaciens containingtheplasmidAGS112 (Koornneefetal. 1987b),wereavailableandweredesignatedATW3001,ATW3003 (Bellina),ATW3052(LA1182)andATW2002(PI128650).Fourfusioncombinationswere made:ATW3001/-3003/-3052(+)PI128650andBellina (+)ATW2002. Isolation, fusion and culture of protoplasts Shootculturesweregrownasepticallyinglasscontainersatalightintensity of30W/m2 (16h), at25°ConmediumcontainingMSsalts(MurashigeandSkoog 1962),Tvitamins(Tewesetal.1984)and10g/1sucrose,solidifiedwith9g/1 agar (shootculture medium). Plantswerekeptinthedarkonedaybeforethe harvestofleaflets,whichthenwerefloatedinthedarkat4°Cfor24hours on a pre-incubation medium: hx MSsalts, lxTvitamins, 1mg/1 2,4dichlorophenoxyacetic acid (2,4-D) and 0.5mg/1 benzylaminopurine (BA). Subsequently,theleaveswerecutinsmallpiecesandincubatedfor16hoursin the dark at 25°C in an enzyme solution: 10g/1 CellulaseRIO and 1.5g/1 MacerozymRIO(bothenzymesfromYakultLtd.,Japan),CPWsalts(Frearsonetal. 1973), 100mg/1 2[N-morpholino]-ethane-sulfonate (MES)and 137g/1 sucrose, pH5.6.Theprotoplastswereseparated fromcelldebrisbyfiltrationthrough

13 anylonfilterwithaporesizeof50 pmandpurifiedbyfloatationonasolution of 137g/1 sucrose + CPW salts + 100mg/1 MES by centrifugation for 5min. at 70x g. The floating protoplastswere pelleted twice inW5 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 describedbyMenczeletal.,exceptthat30%PEGMW4000insteadof40%PEG 6000 was used. In the experiments where electrofusion was used, the protoplast floatation stepwas 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 10 6protoplastsperml). 60//1oftheprotoplast suspensionwastransferred toafusionchamberwith a1mmwidegapbetweentwoparallel brass electrodes, glued inaglassPetridish.Thefusionapparatuswasacombinationofafunction generator, a custom-built generator of direct current (D.C.) pulses and the fusionchamber.Theelectrofusionwasperformedasfollows:theprotoplastswere aligned in an alternating current (A.C.) field (1Mhz) of 200V/cm, one D.C. 'pulseof2000V/cm (25 ßsec.) wasapplied,whereaftertheA.C.fieldwasslowly reduced to zero.Finally theprotoplasts were transferred toa Petridishwith culture medium. The protoplasts were cultured at a density of 1-2 x lO5/1"! in TMpmedium, which is a modified TM-2medium (Shahin 1985) containing 103g/1 sucrose and 0.5 mg/1 BA (instead of zeatin riboside), in thedarkat 25 °C.Whenthe first celldivisionswere observed,mostlyafter 3days,thecultureswere exposed to dim light and diluted (1:1 or less)once or twice aweek with TMd, which is a modifiedTMpmediumcontaining68.5 g/1sucroseand0.1 mg/1a-naphtylaceticacid (NAA). Four weeks after protoplast isolation microcalli were transferred onto TMc,which isamodified TM-3medium (Shahin1985)with2.5 g/1 sucrose,36 g/1 mannitol,0.1 mg/1NAA,0.5 mg/1BAand 8g/1 purifiedagar.After twoweeksof culturing,weaddedkanamycintothemediaforhybridselection;inliquidmedia the final concentration of kanamycin was 50mg/1, in TMc 100mg/1. When the microcallionTMc mediumhaddeveloped togreencalliofseveralmillimeters in diameter, they were transferred to a shoot induction medium, which is shoot culturemediumwith20g/1 sucrose (instead of10g/1)supplementedwith1mg/1 zeatin and 0.1 mg/1 indole-acetic acid (IAA). After 4 weeks the calli were transferred to the same medium without IAAand subcultured every 4weeks until well developed shoots were present. The shoots were rooted on shoot culture medium. After rooting the régénérants were maintained in vitro, tested on kanamycinresistance (100mg/1)andtransferred tosoil inaheated greenhouse.

Cytogenetic

analysis

Root tipswere collected fromgreenhouse-grownplants,treated for 3-4hoursat 15-20 °Cwith 290mg/18-hydroxyquinolinetoarrestmetaphase plates,and fixed inamixture ofethanolandaceticacid (3:1)at4 °C.Theroottipswere either maceratedinINHClfor4-5minutesat60 °Candthensquashedinlacto-propionic orcein (Dyer 1963) or they were treated with an enzyme solution (1g/1 Cellulase RS (fromYakult,Japan)+ 1g/1 Pectolyase Y23 (from Seishin, Japan) +1g/1Cytohelicase (fromIBF,France)in2.5 g/1Na-citratepH 4.8)for3hours 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 stripsfromleavesofgreenhouse-grownplantsasdescribedbyKoornneef et al. (1989).

Isozyme and RFLP

analysis

Young leaves from greenhouse-grown plants were used for isozyme and RFLP analyses. Acidphosphatase (APS;E.C.3.1.3.2)activitywasanalysed by electrophoresis of 50/ilcrude extract,supplementedwith 5 pi electrode bufferand 5 pi marker dye (0.5%bromophenol blue in 20%glycerol)ona 10%Polyacrylamide (PAA)slab gel.Theextractswereobtainedbysqueezingthawingleafmaterial,thathadbeen

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 fewhours,gentle shaking), prepared according toVallejos (1983). DNAwas isolated from several plants according toDellaporta et al. (1983), digestedwith Dra I,separatedbyagarosegelelectrophoresis,blottedontoGene 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, CornellUniversity, Ithaca,USA),asdescribed inChapter 6.

Morphology and

fertility

Thefollowingfeaturesoftheplants,growninanunheatedgreenhouseunderDutch summer conditions, were determined: growth habit, leaf morphology, sympodial index (=meannumber ofnodes between two subsequent inflorescences),presence of stipules and bracts, size of the flower parts,pollen viability, fruit and seed set.Pollengrains (atleast 100perflower)were stained with a solution oflactophenolacidfuchsin;viablepollenstainedpurple,nonviablepollendid notstain.Observationsweremadeonsomatichybridplants,several 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,were used. The somatic hybridplantswereselfedandcrossedwith L. esculentum (diploidandtetraploid) andwith 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 purplegreencolouredcalli.Themajorityofthesecalliproduced shootswithin4weeks onshoot inductionmedium; someofthem evenshowed shootprimordiawhilebeing cultured onTMc.The shootscould easilyberooted.Whenkanamycinwasadded to the liquid culture, all cell colonies stopped growing and in cases where microcalli were put onTMc withkanamycin theybecame 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 brownwithin a few weeks onshoot inductionmedium. No regenerated plants could be obtained bymeans of thedescribed 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 culturemostcoloniesstoppedgrowing,whereasothersdidnot.Amajorityofthe microcalli,thatweretransferredtoTMc +kanamycin,developedintogreencalli. Most of these formed shoot primordiawithin 4weeks 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 to0H40)were rooted. Eight of the calli were derivedfromelectrofusiontreatedprotoplasts,whiletheremainderwasobtained by PEG treatment. The kanamycin resistance of the putative hybrid shoots was confirmedlateronbytheirabilitytorootonshootculturemedium supplemented withkanamycin. One other putative hybrid, named 0H1, was obtained after PEG-fusion of protoplastsfromL. esculentum

cv. BellinaandL. peruvianumATW2002.Thishybrid

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 inthe greenhouse andwere analysed forone ormore characters, together with the parental genotypes.

Cytogenetic

analysis

Both parental species have chromosome numbers of 2n= 2x- 24.The chromosome numbers of 47 shootsderived from 23different hybrid calliweredetermined in metaphaseplatesofroottipcells.Mostshootsweretetraploidwiththeeuploid numberof2n= 4x= 48(Fig. 1A+ 2 ) ;fewwereaneuploidatthetetraploidlevel. Aminorityoftheshootswasatthehexaploidlevel (Fig. IB+ 2 ) ;halfofthese 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 tobeatetraploid. Thefewhyper-tetra/hexaploidnumbersmighthave been obtained by the unintended counting of themacrosatellites (short arms of the chromosomes 2)as independent chromosomes. The number of chloroplasts in the guard cell pairs can be used as an additionalmeasurefortheploidylevelin Lycopersicon

plants(Koornneefetal.

1989).However,therelationbetweenchloroplastnumberandploidyleveldiffers between L. esculentum

and L. peruvianum

(Fig. 3; Koornneef et al. 1989). The

chloroplastnumbersofthesomatichybridsalsoareshowninFig. 3.Theaverage chloroplast number of tetraploid hybrids (18.1), isintermediate to that of

16

.apt.' 1jfc

WW

i fj "

%«

if

i •

-

•••

B

-

.

/

;

*

*

*

•

Fig.1.Metaphaseplatesofroottipcellsfromsomatichybridsof L. esculentum andL.peruvianum.A.Thetetraploidhybrid0H4,2n=4x=48.B.Thehexaploid hybrid0H14,2n=6x=72.

(I)

C

a 0)

E 3

24

36

48

60

72

84

96

Chromosome number Fig.2.Frequencydistributionofaveragechromosomenumbersofdifferentshoots fromsomatichybridcalli,derivedfromprotoplastfusionsbetween 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 ploidy classes overlap,which indicates that this character does not always allow an unambiguous distinction betweenboth ploidygroups. We classified the ploidy level of the somatic hybrids on the basis of chromosome numbers, or, in some cases, chloroplast counts (taking 10)wereobservedonone plant. Twenty-three progeny plants set seeds, in general after spontaneous

Setting seeds 23

38 seifing.Backcrosseswithdiploid L. esculentum as staminateparentwerenot successful.

Discussion Oneofthefactorsthatareimportantforthesuccessfulapplicationofsomatic hybridisationtotheimprovementofcropsisthefertilityofthehybridswhen theyareselfedorbackcrossedtothecultivatedparent.Theresultspresented inthispapershowthatsomatichybridsbetweenL.esculentumand L. peruvianum arefertilewhenselfed.Backcrossesoftetraploidhybridsaspistillateparent todiploid L. peruvianum didsucceed,whereasthosetodiploid L. esculentum did not. The backcrosses of tetraploid hybrids as staminate parents to diploid L. peruvianum didnotsucceed,whilethosetodiploidL.esculentumonlyyielded abortive seeds.These results are inagreement with those obtained by Soost (1958)andSzteyn(1962),whoisolatedallotriploidsexualhybridsfromcrosses oftetraploid L. esculentum withdiploid L. peruvianum. Theyfoundthatcrosses oftheseallotriploidsaspistillate parentstodiploidL.peruvianumyielded viableoffspring,butthosetodiploid L. esculentum didnot.However,Szteyn (1962)foundthatiftheallotriploidswereusedasstaminateparents,viable offspringcouldbeobtainedfromcrosseswithdiploid L. esculentum. Allodiploids of L. esculentum xL.peruvianum canalsobebackcrossed to L. esculentum as pistillate parent (Ancora etal. 1981).Wefound thatthebackcrosses ofthe tetraploidhybridstoL.esculentumaspistillateparentyieldedseveralfruits withmany abortive seeds.It isquite possible that if suchbackcrossesare repeated on a larger scale, viable triploid offspring will be obtained. Subsequent backcrosses of these triploids to diploid L. esculentum are then probablyrelativelyeasilyobtained (RickandButler1956). Another important factor is the occurrence of homoeologous pairing and recombinationinthesomatichybrids.Ourresultsprovidesomeindicationsfor tetrasomicinheritanceofseveraltraitsinthehybrids.Thevariancesofthe s.p.i.andoftheratioofsepalandpetallengthswassignificantlylargeramong theprogenyoftheselfedtetraploidhybridsthanamongthehybridsthemselves. Withrespecttoothertraits,suchas Aps-1 andfruitcolour,phenotypeswere presentamongtheprogenyoftheselfingsthatwereabsentamongtheparental hybrids.Withrespecttothemorphological characteristics,theseresultscan beattributedatleastinparttoheterozygosityoftheparentsofthehybrids: L. esculentum cv.BellinaisanFxhybridand L. peruvianum isaveryvariable, outbreedingspecies.Butthisexplanationdoesnotholdforthesegregationof Aps-1 isozymepatternsandfortheappearanceofpurplepigmentedfruitsinthe

39 progeny of the selfedhybrids.We therefore tentatively conclude thatat least Aps-1,

butprobably othermarkers aswell,haveatetrasomic inheritance inthe

hybrids.Thisimpliesthattherelevanthomoeologouschromosomesprobablypaired in the tetraploid somatic hybrids.A more detailed analysis of the segregation of monogenic inherited markers, and of the behaviour of chromosomes at metaphase Iinthehybrids isrequired to support thisconclusion,however. Thekanamycinresistance marker (orNPT IIlocus)was present asone 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 (withonehemizygouskanamycinresistance locus)were selfed,the ratio ofresistanttosensitiveprogenydiffered significantlyfrom3:1andwascloser to2:1.Thisdeviating ratiocanbe explainedbythemore frequent transmission of the corresponding L. peruvianum

chromosome than of the L.

esculentum

chromosome containing theNPT IIlocus.Deviating segregationratiosof several traits were also observed in F 2 generations of sexual hybrids of tomato and I. pennellii

(Bernatzky andTanksley 1986;GadishandZamir 1987)and of tomato

and L. chilense

(Rick 1963), all in favour of characters of the wild species.

Inbackcross progenies of tomato specieshybridsdeviating segregation ratios ineither directionhave been observed (Rick 1963,1969;Vallejos and Tanksley 1983; Rick et al. 1988). The phenomenon was ascribed to differential survival ofthemalegametesortodifferentialzygoticlethality(Rick1963,1969;Gadish and Zamir 1987). Both explanations are conceivable for ourresults. 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 canbe achieved more efficiently by the pre-selection of fertile progeny plants with some L. esculentum

with L.

specifictraitsandthesubsequentcrossingoftheseprogenyplants

esculentum.

Acknowledgements. Thisresearchwas supported bytheFoundationfor Fundamental BiologicalResearch (BION),which issubsidisedbytheNetherlands Organisation for Scientific Research (NWO). We thank Corrie Hanhart, Patty van Loenen Martinet-SchuringaandRenéRijken for doingpart of the experiments,Dr. J.H. de Jong and Prof. C.Heyting for critically reading of the manuscript.

References AncoraG,SaccardoF,CappadociaM,SreeRamuluK (1981)BackcrossprogeniesfromLvcopersiconesculentumL.x hybrid (L.esculentumxL.peruvianum Mill.). ZPflanzenzüchtg 87:153-157

40 BernatzkyR,TanksleySD (1986)MajorityofrandomcDNAclonescorrespondtosinglelociinthetomatogenome. MolGenGenet203:8-14 Ehlenfeldt MK, HelgesonJP (1987)Fertility of somatic hybrids fromprotoplast fusions of Solanum brevidens andS. tuberosum. TheorApplGenet 73:395-402 EvansDA, Bravo JE,Kut SA,Flick CE (1983)Genetic behaviour of somatic hybrids inthe genusNicotiana:N. otophora+N.tabacum andN. sylvestris+N.tabacum. TheorApplGenet 65:93-101 FahlesonJ,RâhlénL,GlimeliusK(1988)AnalysisofplantsregeneratedfromprotoplastfusionsbetweenBrassica nanus andEruca sativa.TheorApplGenet 76:507-512 GadishI,ZatnirD (1987)Differential zygotic abortioninaninterspecific Lvcopersiconcross.Genome29:156159 Gleddie S,KellerWA,SetterfieldG (1986)Production andcharacterizationofsomatic hybridsbetweenSolanum melongenaL.and S.sisymbriifolium Lam.. TheorApplGenet 71:613-621 Grimbly P (1986) Disorders. In: Atherton JG, Rudich J (eds), The tomato crop. A scientific basis for improvement.Chapman andHall,London/NewYork,pp 369-389 HamillJD,Pental D,Cocking EC (1985)Analysis of fertility insomatic hybrids ofNicotiana rustica andN,. tahartimandprogeny overtwosexualgenerations. TheorApplGenet 71:486-490 KinsaraA,Patnaik SN,CockingEC,PowerJB (1986)SomatichybridplantsofLycopersicon esculentumMill,and LvcopersiconperuvianumMill.. JPlantPhysiol 125:225-234 Koornneef M, JongsmaM,Weide R, Zabel P, Hille J (1987)Transformation of tomato. In:Nevins DJ,Jones RA (eds)Tomatobiotechnology.AlanR Liss, Inc,NewYork,pp 169-178 MelchersG, SacristanMD,Holder AA (1978)Somatic hybrid plantsofpotato andtomato regenerated fromfused protoplasts.CarlsbergResCommun 43:203-218 Menzel MY (1964) Preferential chromosome pairing in allotetraploid Lvcopersicon eseulentum-Solanum lycopersicoides.Genetics 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,FerwerdaMA (1984)GiemsaC-banding ofpotato chromosomes.CanJGenetCytol 26:415-419 Primard C, Vedel F, Mathieu C, Pelletier G, Chèvre AM (1988) Interspecific somatic hybridization between Brassicananus andBrassicahirta (Sinapsis alba L.). TheorApplGenet 75:546-552 RickCM (1963)Differential zygotic lethality inatomato specieshybrid.Genetics 48:1497-1507 Rick CM (1969) Controlled introgression of chromosomes of Solanum pennellii into Lycopersicon esculentum: segregation andrecombination.Genetics 62:753-768 Rick CM (1982)Stock list.RepTomatoGenetCoop 32:3-10 RickCM,Butler L (1956)Cytogenetics ofthetomato.AdvGenet8:267-382 Rick CM,ChetelatRT,DeVerna JW (1988)Recombination in sesquidiploid hybrids ofLvcopersicon esculentum x Solanumlycopersicoides andderatives.TheorApplGenet 76:647-655 Rick CM,KhushGS (1962)Preferentialpairing intetraploid tomato specieshybrids.Genetics 47:979-980 Schieder0 (1980)SomatichybridsofDaturainnoxiaMill.+DaturadiscolorBernh.andofDaturainnoxiaMill. +DaturastramoniumL.var.tatulaL.II.Analysisofprogeniesofthree sexualgenerations.MolGenGenet 179:387-390 SmithHH,KaoKN,CombattiNC(1976)InterspecifichybridizationbyprotoplastfusioninNicotiana.Confirmation and extension.JHeredity 67:123-128 SoostRK(1958)ProgeniesfromsesquidiploidFjhybridsofLvcopersiconesculentumandL.peruvianum.JHeredity 49:208-213 SundbergE,LandgrenM,GlimeliusK (1987)Fertility and chromosome stability inBrassicanapusresynthesised byprotoplast fusion.TheorApplGenet75:96-104 SzteynK (1962)Interspecific crosses inthegenusLvcopersicon. I.Backcrosses toLvcopersiconglandulosum. Euphytica 11:149-156 TewesA,GlundK,Walther R,Reinbothe H (1984)Highyield isolation and rapid recovery ofprotoplasts from suspensionculturesoftomato (Lycopersicon esculentum). ZPflanzenphysiol 113:141-150 Vallejos CE (1983)Enzyme activity staining. In:Tanksley SD,Orton TJ (eds)Isozymes inplantgenetics and breeding,partA. Elsevier,Amsterdam,pp 469-515 VallejosCE,TanksleySD(1983)Segregationofisozymemarkersandcoldtoleranceinaninterspecificbackcross oftomato.TheorApplGenet 66:241-247

41 CHAPTER4 A S Y M M E T R I C S O M A T I C H Y B R I D S B E T W E E N I.Y COPE RS ICON ESCUIENTUM A N D I R R A D I A T E D IYCOPERSICON PERUVIANZJM

I.

CYTOGENETICS AND

MORPHOLOGY

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

Summary.Asymmetricsomatichybridsof Lycopersicon

esculentum and Lycopers icon

peruvianum were obtained by fusion ofleaf protoplasts from both species after irradiation ofprotoplasts orleaf tissue of L. peruvianum with 50,300or1000 Gyofgamma-rays.These irradiationdoseswere sufficient toabolishthe growth oftheL.peruvianumprotoplasts.Thehybridswere selected ontheirabilityto regenerate plants; this regeneration capacity derived from L. peruvianum. All asymmetrichybridplantswereaneuploid.Theploidylevel,themorphologyaswell as the regeneration rate were analysed in relation to the irradiation dose, appliedtoL.peruvianum.Afteralowdose (50Gy)mosthybridshadnear-triploid chromosome numbers,whereasafterahighdose (300or1000Gy)mosthybridshad near-pentaploid numbers. The morphology of the asymmetric hybrids was intermediatebetweenthatof L. esculentum andsymmetric somatichybridsofboth species (obtainedwithout irradiation treatment),andapproached themorphology of L. esculentum more afterahighdose of irradiation. Theasymmetric hybrids regenerated more slowly than the symmetric hybrids,and regeneration proceeded moreslowlyafterahighdosethanafteralowdoseofirradiation.Thehighdose hybrids also grew more slowly, flowered less and set fruits less than the low dose hybrids.No seeds could be obtained from any asymmetric hybrid.

Introduction

The transfer ofdesirable traits fromwild into cultivated species isamethod toimprove crops.Forthispurpose,interspecific sexualorsomatic hybridsmay be constructed. The formation of sexual hybrids is limited to closely related species. Symmetric somatichybrids,obtainedbyfusionofuntreated 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 thewild species.These problems may be circumvented by asymmetric somatichybridisation.Bythisprocedure protoplasts ofonespecies, the recipient, are fused with inactivated (mostly by Röntgen- or gammairradiation) protoplasts of another species, the donor. The donor genome will befragmentedandtheasymmetrichybridswillcontainthecompletegenomeofthe recipient species and a small part of thedonor genome.The advantages of this

42 procedurecouldbethathybridsarisewithfewunwanteddonortraits,andthat fewerbackcrossesoftheasymmetrichybridsarerequiredtogetridofthese. Several asymmetric hybrids were obtained in other studies (Chapter1).The fractionofdonorgenomethatwastransferredvariedfromoneorafewtraits (e.g. Duditsetal.1987),oneorafewchromosomes(e.g.Guptaetal.1984)to many chromosomes (e.g. Gleba et al. 1988). Fertile asymmetric hybrids were reportedinseveralcasesandirrespective oftheamountoftransferreddonor 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 hasmanydesirablecharactersfortomatoimprovement(Rick1982a), butonlyafewofthese(e.g. Mi- andTMV-resistance)havebeenintroducedinto thecultivatedtomato,becausesexualhybridsofbothspeciesareverydifficult to obtain (Taylor 1986). In the present experiments we obtained asymmetric somatichybridsbyfusingL.escuientumprotoplastswithirradiated L. peruvianum protoplasts.Thehybridswereselectedforthedominantregenerationcapacity characterofL.peruvianum(Koornneefetal.1987a;Chapter 2).Ourpurposewas toanalysethecytogenetics,morphologyandfertilityoftheregeneratedhybrids inrelationtotheirradiationdose,appliedto L. peruvianum.

Materialsandmethods Plant materials Asrecipient (Lycopersicon esculentum) theDutchhybridcultivarBellina(kindly providedbyRijkZwaanSeedCompany,deLier,TheNetherlands)andthegenotypes LA291,LA1164,LA1166,LA1182,LA1189,LA1444andLA1665fromtheTomatoGenetics StockCenterinDavis,USA(kindlyprovidedbyProf.C M . Rick)wereused.The latter genotypes allow observations on the complementation of monogenic morphological mutant phenotypes by the donor (Chapter5). Plants from the Lycopersicon peruvianum accession PI128650 (received from the Institute of HorticulturalPlantBreeding,Wageningen,TheNetherlands)wereusedasdonor. KanamycinresistantplantsofL.peruvianumand L. esculentum cv.Bellinawere also available; these had been obtained by leaf disc transformation with Agrobacterium tumefaciens containingtheplasmidAGS112(Koornneefetal.1987b). Kanamycinresistancein L. esculentum wasusedtoselectforsymmetricsomatic hybrids(Chapter2);kanamycinresistantL.peruvianumwasusedtomonitorthe lossoftheresistanceintheasymmetricsomatichybrids(Chapter5). Fusion combinations The protoplast fusion experiments are listed in Table1. L. peruvianum was irradiatedwithgammaraysfroma60Cosourceatadoserateofapproximately 2000 Gray/hour at the Pilot-Plant for Food Irradiation, Wageningen (The Netherlands).Threedifferentdoseswereused:50,300or1000Gy (=5,30or 100kRad).Putativehybridsthatresultedfromthesefusions,areindicatedas 5H, 30Hand100H,respectively.Symmetrichybrids,whichresultedfromfusions ofunirradiatedprotoplasts,areindicatedas0H-hybrids.The irradiationwas carried out either to protoplasts, suspended at 1-3 x 105/ml inW5solution (Menczeletal.1981)onetothreehoursbeforefusion,ortoleafletsoneday beforefusion.

43 Table 1.ThegenotypeoftherecipientspeciesI. esculentum andthe irradiation treatment ofthedonor species L. peruvianum, involved intheprotoplast fusion experiments. The LA-genotypes are multiple marker lines,homozygous recessive formorphological marker genes (Rick 1982b). The protoplast fusionmethod used is given for each combination; M = method according toMenczel et al. (1981), CMS=methodaccordingtoNegrutiuetal. (1986).Thenumberofcalli,whichdid regenerate shoots,isgiven inthe rightmost column. Recipient

Irradiation ofdonor material

cv. Bellina cv. Bellina cv. Bellina cv. Bellina LA291 LA1164 LA1166 LA1182 LA1182 LA1189 LA1189 LA1444 LA1665 LA1665

protoplasts protoplasts protoplasts protoplasts protoplasts protoplasts leaflets leaflets protoplasts leaflets protoplasts protoplasts leaflets protoplasts

dose (Gy)

0 50 300 1000

300 300 300 300 300 300 300 300 300 300

Number of experiments

1* 3 3 3 4 1 2 1 3 1 2 2 4 1

Fusion method

Regenerating calli

M M M M M/CMS

M M M M M M CMS M M

11 53 25 13 31 16 14 8 12 3 7 9 13 1

this experiment was used to determine the regeneration frequency, as shown inFig. 1;therecipient (ATW3003)waskanamycinresistent to selectagainst theunirradiateddonor species;onlyasampleof14calliwastested onshoot 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 inseveral experimentswithout negative effects. Protoplast fusionwas carried out either according to Negrutiu et al. (1986; the CMSmethod) or according to Menczel et al. (1981), except that 30%PEG 4000was used instead of 40%PEG 6000 (Table 1 ) . The ratio of L. esculentum to t.peruvianum protoplasts during fusionwas 1:1 to 2:1. Ineach fusion experiment 1x 10 6to 3 x 10 6protoplastswere involved. Ineachexperiment,cultures ofthe parental protoplasts,both separate and mixed,were started inparallel with the fusion cultures (i.e.culturesofmixedprotoplastsofrecipientanddonor,afterfusion treatment);insomeexperimentsalsodonorprotoplastsweresubjectedtoafusion treatment. Rooting of regenerated shoots was induced on shoot culture medium (MS salts (Murashige and Skoog 1962), Tvitamins (Tewes et al. 1984), 10 g/l sucrose), without hormones or supplemented with 0.1 mg/1 indole-butyric-acid. The regeneration capacity of a number of asymmetric hybrids was tested as described byKoornneef et al. (1987a).

Plant

characterisation

Chromosome numbers ofroot tip cellswere determined in squashpreparations or bymeans ofa slightly modified procedure ofPijnackerand Ferwerda (1984; see also Chapter 2 ) .Several 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. TheLA genotypes differed somewhat intheir protoplast culture responses.Most ofthemshowedlimitedcelldivisionandveryfewmicrocallideveloped.However, growth of these L. esculentum

calli seemed to be improved in cultures where

they were mixed with irradiated L. peruvianum

protoplasts, which apparently

behavedasfeeder-cells.InthecaseofLA1182atetraploidplantwiththeLA1182 phenotype could be regenerated from suchamixed culture. The L. peruvianum

protoplasts that were irradiated with 50, 300 or 1000 Gy

ofgammarays,formedcellwalls.Someofthesecellsstronglyincreased 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,we obtained three calli;these didnot regenerate plants. Thefusionculturesyieldedmanycalliofwhichseveral formedshoots.Within each experiment there was a large variation in callus as well as shoot morphology.Fig. 1showsthetimecourseofshootformationbyasymmetrichybrid calli,resulting fromfusionswheredifferent irradiationdoseswereapplied 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.Characteristicsofdifferenthybridcalli,thatregeneratedshoots:root formationofshoots (in vitro), establishmentinsoilinthegreenhouse (atleast onemonth),flowering,fruitandseedset.Allhybridswereobtainedafterfusion ofprotoplastsfrom L. esculentum withprotoplastsfrom L. peruvianum, whichhad not been irradiated (OH)or irradiated with 50,300or 1000Gray of gamma-rays (5H, 30H or 100H,respectively)before fusion. Hybri ds

OH 5H 30H 100H

Regenerati ng calli

ND 53 139 13

Root formation

40 38 63 9

Greenhouse plants

32* 27 32 3

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

Flowers

31 21 15 1

Fruits

Seeds

28 13 5 1

21 0" 0***

0

45

uu

>

Ü C 0) zs ö" 0)

80-

./OH

60-

5H

c

o

40^

100H

(Ü

c 3cm).Also the colour of the fruits differed between hybrids (Table 4 ) . Most fruits were orange, which is intermediate between the fruitcolour of the symmetric hybrids (yellow)and of the cultivated tomato (red). No seed set was obtained in the asymmetric hybrids after selfing or backcrossingwiththeparental species (Table 2 ) ;the symmetrichybrids,onthe contrary, did set seeds. In some fruits of five 5H-hybrids and one 30H-hybrid very small,abortive seedswere observed.

52 Table4.Thecolour ofthefruits obtained fromdifferent asymmetricsomatic hybrids.Thehybridswereobtainedafterprotoplastfusionof L. esculentum with L. peruvianum, whichwasirradiatedwith50,300or1000Grayofgamma-rays(5H, 30Hor100H,respectively)beforefusion.Eachobservationwasdoneononeto severalshootsfromonehybridcallus. Plants

Fruitcolour yellow

5H-hybrids 30H-hybrids 100H-hybrids

5 0 0

orange 6 4 1

red 1 1 0

Discussion Thepresentexperimentsshowthatasymmetricsomatichybridplantscanbederived from protoplast fusions between L. esculentum and irradiated L. peruvianum. Selectionofthesehybridswaspossibleonthebasisoftheregenerationcapacity derived from the irradiated species. The asymmetric hybrid nature could be confirmed by several morphological characteristics, which were intermediate betweenthesymmetricsomatichybrids(Chapter2)and L. esculentum, andbythe analysis of specific marker genes (Chapter 5). However, the irradiation of L.peruvianumbeforefusiondidnothavethedesiredeffect,namelyelimination ofthedonorgenometosuchanextent,thatonlyasmallfractionwasconserved intheasymmetrichybrids. Allasymmetrichybridswereaneuploid.Fromthecytogeneticandmorphological analysis,wetentatively conclude thatmostasymmetrichybridscontainedone, twoorthreediploid L. esculentum genomes,supplementedwithonepartialdiploid genomeof L. peruvianum. For15asymmetrichybridsthiswasconfirmedbyRFLP analysis(Chapter 6).The5H-hybrids,ofwhichmosthadonediploidI. esculentum genome,hadontheaverage16presumed L. peruvianum chromosomes (range:5to 22);the30H-hybrids,whichinmostcasesprobablycontainedatetraploidgenome of L. esculentum, hadameanof13(range:2to22) L. peruvianum chromosomes; andboth100H-hybridsprobablyhadatetraploid L. esculentum genomeand,onthe average,7(range:4to12)L. peruvianum chromosomes.Althoughthereisnoclear correlationbetweentheirradiationdoseandeliminationofthe L. peruvianum genome,the30H-hybridsresembledL.esculentummorethanthe5H-hybrids.This isnotnecessarilyduetotheeliminationof L. peruvianum chromosomes,butcan alsobeascribedtothepresenceofalargernumberof L. esculentum genomesin most30H-hybrids.Anyway,arelativelylargefractionofthe L. peruvianum genome

53 isretained,eveninthelOOH-hybrids.Limitedchromosomeelimination,together withaweakdoseeffect,wasalsoobservedinasymmetricsomatichybridsofother species.Retentionofthedonorchromosomesrangedfrom11%to90%(ofadiploid donor genome)in Nicotiana plumbaginifolia (recipient) (+) Atropa belladonna (donor)hybrids(100-1000Gy;Glebaetal.1988),8%-75%in N. plumbaginifolia (+) N. sylvestris hybrids (100-1000Gy;Famelaeretal.1989)and 25%-100%in Brassica oleracea (+)B. campestris hybrids(100-800Gy;Yamashitaetal.1989). Inallthosecaseschromosome rearrangements and/ordeleteddonorchromosomes werereported.InsomeoftheL.esculentum(+)L.peruvianumasymmetrichybrids reported here, we observed fragments. It was often difficult to identify fragmentswithcertainty,becauseofthesmallsizeofthetomatochromosomes. However,RFLPanalysis,appliedto15hybrids,clearlyindicated thepresence ofincompletechromosomesinalltestedplants(Chapter6). Theasymmetric 5H-hybridsweremoreviablethanthe30H-and100H-hybrids, irrespective whether shoot regeneration, root formation or morphological characteristicswereusedascriteria,whereasallasymmetrichybridswereless 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 onlyprimarytrisomiesandsomemonosomiesareviable (Rickand Butler 1956),whereasaneuploidyistoleratedbetterbyprimitivetomatoesand specieshybridsofthetomatothaninthecultivated tomato (Soost1958;Rick and Notani 1961;Györffy and Mako 1963). In general the asymmetric somatic hybridswerenearthetriploidorpentaploidlevel;the5H-hybridsusuallyhad adiploidL. esculentum genome,whereasmost30H-and100H-hybridspresumably hadatetraploidrecipientgenome.Inotherasymmetrichybridisationexperiments thatresultedinlimitedchromosomeelimination(Glebaetal.1988;Famelaeret al.1989;Yamashitaetal.1989),therecipientgenomeintheasymmetrichybrids usuallywasaroundtetraploidorhexaploid.Anexplanationofthiscouldbethat cellswithpolyploidised recipient genomes,whichmayhave arisenduringthe tissue culture phase, better tolerate additional and abnormal chromosomes (rearrangementsandfragments). The variation in chromosome number within each of the asymmetric hybrids mightindicateinstabilityattheplantlevel.Probablyrearrangedandincomplete chromosomestendtogetlostmorefrequently.Theobservedlossofregeneration capacityinestablishedcallusculturesofsome30H-hybridsmightbeexplained thisway.Anotherexplanationiseliminationofthistraitintheestablished calluscultures. Theasymmetricsomatichybridswerenotfertile.Somefruitscontainedsmall abortiveseed-likestructures.Embryorescuetechniqueswereappliedtwotofour weeksafterpollination(selfingorbackcrosswith L. esculentum pollen).Intwo

54 casescalluswasinducedontiny,prematurelyisolatedseeds;oneoftheseformed shoot primordia. However,these callimight also descend from maternal tissue. The limited elimination of donor chromosomes is a serious drawback for the applicationofasymmetrichybridsinplantbreeding,becauseseveral backcrosses to the recurrent parent are still required to get rid of the unwanted donor traits.Crosseswillbeimpossibleoratleasthamperedstronglybythesterility and the polyploidy of these hybrids. The sterility is probably due to the cytogeneticaberrations,suchasaneuploidy andrearrangements.Thelatterwere morefrequent afterhigher irradiationdoses (Chapter 6 ) .Despite thefact that several tens of asymmetric hybrids were obtained and analysed, none of the plantshad justone or two chromosomes above thediploid level,which probably wouldhaveallowedbackcrossingtothediploidrecipient.Ifthepolygenicnature 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 complementingauxotrophicmutations,wouldhelptoovercometheproblem. Ifnot, other ways toenhance elimination ofdonor chromosomes should be looked for or very large populations ofasymmetric hybridshave tobe evaluated.

Acknowledgements. Thisresearchwassupported by theFoundationfor Fundamental BiologicalResearch (BION),which issubsidised bytheNetherlands Organisation for Scientific Research (NWO). We are very grateful to Corrie Hanhart, Henny Verhaar andAnne-marie Wolters fordoing part of the experiments,and Prof.C. Heyting for critically reading of the manuscript.

References DuditsD,MaroyE,PraznovszkyT,OlahZ,GyorgyeyJ.CellaR C1987)Transferofresistancetraitsfromcarrot into tobaccoby asymmetric somatichybridization:Regeneration of fertile plants.Proc Natl Acad Sei USA 84:8434-8438 FamelaerI,GlebaYY,SidorovVA,KaledaVA,FarakonnyAS,BoryshukNV,CherupNN,NegrutiuI,JacobsM (1989) Intrageneric asymmetric hybrids between Nicotiana olumbaginifolia and Nicotiana sylvestris obtained by 'gamma-fusion'.PlantScience 61:105-117 Gleba YY,Hinnisdaels S, Sidorov VA, Kaleda VA, Parokonny AS,Boryshuk NV, Cherup NN,Negrutiu I, JacobsM (1988)Intergeneric asymmetrichybridsbetweenNicotianaplumbaeinifoliaandAtropabelladonnaobtainedby "gamma-fusion".TheorApplGenet 76:760-766 Grimbly P (1986) Disorders. In: Atherton JG, Rudich J (eds), The tomato crop. A scientific basis for improvement.ChapmanandHall, London/NewYork,pp 369-389 Gupta PP, Schieder 0, Gupta M (1984) Intergeneric nuclear gene transfer between somatically and sexually incompatibleplants through asymmetric protoplast fusion.MolGenGenet 197:30-35 Györffy B, Mako J (1963) Two aneuploid progenies from a sesquidiploid tomato hybrid after uncontrolled pollinations.TGCRep 13:36-37 KoornneefM,HanhartCJ,Martinelli L (1987a)Agenetic analysisofcellculturetraitsintomato.TheorAppl Genet 74:633-641

55 KoornneefM, JongsmaM,WeideR, ZabelP,Hille J (1987b)Transformation of tomato. In:NevinsDJ,JonesRA (eds)Tomatobiotechnology.AlanR Liss, Ine,NewYork,pp 169-178 MenczelL,NagyF,KissZR,MaligaP (1981)Streptomycinresistantandsensitive somatichybridsofNj-cotiana tabacum+Nicotianaknightiana:correlationofresistancetoN.tabacumplastids.TheorApplGenet59:191195. Murashige T, Skoog F (1962)A revised medium for rapid growth and bioassays with tobacco tissue cultures. PhysiolPlant15:473-497. Negrutiu I,DeBrouwer D,WattsJW,SidorovVI,DirksR,JacobsM (1986)Fusionofplantprotoplasts:astudy using auxotrophicmutantsofNicotianaplumbaginifoliaViviani.TheorApplGenet 72:279-286 PijnackerLP,FerwerdaMA (1984)GiemsaC-bandingofpotato chromosomes.CanJGenetCytol26:415-419 RickCM (1982a)Thepotentialofexoticgermplasm fortomatoimprovement. In:VasilIK,ScowcroftWR,FreyKJ (eds),Plant improvement andsomatic cellgenetics.Academic Press,NewYork,pp 1-28 Rick CM (1982b)Stock list.RepTomatoGenetCoop32:3-10 Rick CM,Butler L (1956)Cytogenetics ofthetomato.AdvGenet8:267-382 RickCM,NotaniNK (1961)Thetolerance ofextrachromosomesbyprimitive tomatoes.Genetics46:1231-1235 SoostRK(1958)ProgeniesfromsesquidiploidF!hybridsofLvconersiconesculentumandL.peruvianum.JHeredity 49:208-213 Taylor IB (1986)Biosystematicsofthetomato.In:AthertonJG,RudichJ (eds),Thetomatocrop.A scientific basis for improvement.Chapman andHall,London/NewYork,pp 1-34 TewesA,GlundK, WaltherR,Reinbothe H (1984)High yield isolation and rapid recovery ofprotoplasts from suspensionculturesoftomato (Lycopersicon esculentum). ZPflanzenphysiol 113:141-150 Yamashita Y, Terada R, Nishibayashi S, Shimamoto K (1989)Asymmetric somatic hybrids of Brassica: partial transfer ofB.campestrisgenomeintoB.oleraceaby cellfusion.TheorApplGenet 77:189-194

57 CHAPTER5

ASYMMETRIC LYCOJPEJEZSICON L.YCOPERSICON

II.

SOMATIC HYBRIDS

BETWEEN

ESCUI^ENTUM A N D I>EKLTV IANUM

I R R A D I A T E D

A N A J L . Y SI S W I T H M A R K I E R . G E N E S

J.Wijbrandi,A.M.A.Wolters,M.Koornneef

Summary.Asymmetricsomatichybridsof Lycoper sicon esculentum and Lycopersicon peruvianum wereanalysedfortheretentionofgenesandalleles,specificfor L. peruvianum. The hybrids were obtained by fusion of protoplasts from L. esculentum withthoseof L. peruvianum (thedonor),whichhadbeenirradiated before fusionwith 50,300or1000Gyofgamma-rays.The retentionofthree different types of genes or alleles was analysed: (1)The gene coding for kanamycinresistance,whichisdominantandhadbeenintroducedinmostofthe L. peruvianum donorplantsbytransformation.Itwaspresentatonelocusin16 L. peruvianum donorplantsandattwoloci inonedonorplant. (2)Thegenes codingforacidphosphatase,locus Aps-1, andglutamateoxaloacetatetransaminase (GOT);differentallelesofthesegenesareco-dominant,andweredetectedby isozymeanalysis.(3)Eighteensinglegenemorphologicalmarkers,forwhichmost ofthe L. esculentum genotypesusedwerehomozygousrecessive. Kanamycinresistancefromthedonorplantswithonelocuswasretainedinabout 50% of the asymmetric 30H-hybrids (the donor was irradiated with 300Gy). L. peruvianum specificallelesof Aps-1 andGOTwerepresentinatleast70%of thehybrids;theretentionofdonoralleleswaslowerin30H-thanin5H-hybrids (donorirradiatedwith50Gy).Ontheaverage,74%ofthe L. peruvianum specific alleles (oneorboth)ofthemorphological markersweredetected inthe30Hhybrids.Severalofthe L. esculentum genotypesusedwerehomozygousrecessive fortwomorphologicalmarkersonthesamechromosome.In36%ofthe30H-hybrids 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. Onthewhole,thisanalysisconfirms the conclusionfromthe cytogeneticand morphologicalanalysisoftheseasymmetrichybrids,namelythatirradiationprior tofusioneliminatedtheL.peruvianumgenomeonlytoalimitedextent.

Introduction Partial genome transfer by asymmetric somatic hybridisation, which involves fusionofprotoplastsofarecipientspecieswithirradiatedprotoplastsofa donor species,has beendescribed for several plants.The transferred donor genomevariedfromafewtraits (e.g.Duditsetal.1987)tomanychromosomes (e.g.Gleba et al. 1988). The amount of transferred donor genome was often assessed on the basis of chromosome counts. This isnot always anaccurate estimation,becausediscriminationbetweenthespeciesspecificchromosomesand identificationofincompletechromosomesarenotalwayspossible.

58 InChapter4wedescribed the cytogenetic andmorphological analysis ofa series of asymmetric hybrids of L. esculentum (the cultivated tomato)and L. peruvianum (awild species;the donor). Thesehybrids appeared tocontain stillarelatively largenumber ofL.peruvianum chromosomes.Inmost ofthe asymmetrichybrids,thechromosomenumberwasaroundthetriploidorpentaploid level;probably,thegenomeofthesehybridsconsistedofadiploidortetraploid genomefrom L. esculentum andseveralchromosomesfrom L. peruvianum. Theamount ofdonorchromosomesvariedanddidnotcorrelatestronglywiththeirradiation dose applied to the L.peruvianum protoplasts (50, 300 and 1000 Gray, respectively).Tostudytheeliminationofgeneticmaterialofthedonorspecies moreindetail,thesameasymmetrichybridswereanalysedfortransferofsingle genemarkers.Thiskindofanalysiscanbeperformedverywell inthetomato, because of its detailed linkage map. The map consists of more than 300 morphological,isozyme-anddiseaseresistancegenes(Mutschleretal.1987)and atleast300RFLPmarkers (YoungandTanksley 1989).Wetriedtoestimatethe fractionofthe L. peruvianum genomethatwasconservedintheasymmetricsomatic hybridsbyanalysingtheretentionofthreetypesofgenesoralleles: (i)theneomycinphosphotransferase II(NPTII)gene,whichcausesresistance totheantibiotickanamycin.Thisisadominantgene,whichhadbeenintroduced into the nuclear genome of most of the L.peruvianum donor plants by transformation; (ii)the isozymemarkers acid phosphatase (Aps-1) and glutamate oxaloacetate transaminase (GOT); (iii)singlegenedeterminedmorphologicalmarkers,presentinmostofthetomato genotypesused.

Materialandmethods Thegenotypes of L. esculentum, therecipient species,were theDutchhybrid cultivar Bellina (kindly provided by Rijk Zwaan Seed Company, de Lier,The Netherlands)andsevenmultiplemarkerlines(Table1)fromtheTomatoGenetics StockCenterinDavis,USA(kindlyprovidedbyProf.C M .Rick).Plantsfromthe L. peruvianum accessionPI128650(receivedfromtheInstituteofHorticultural Plant Breeding, Wageningen, The Netherlands)were used as donor. Seventeen kanamycinresistant L. peruvianum plants,obtainedbyleafdisctransformation with A. tumefaciens containingtheplasmidpAGS112(Koornneefetal.1987b)were available;theseplantsweredesignatedATW2001toATW2027.Theisolationofthe asymmetric somatichybridswasdescribed inChapter4.Theasymmetrichybrids weredesignatedaccordingtotheirradiationdoseappliedtoL.peruvianumbefore protoplastfusion:5H-,30H-andlOOH-hybrids,whichwereirradiatedwithadose of50,300and1000Gyofgamma-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. Theresultingseedsweredecontaminatedbytreatmentfor10"in

59 Table1.Themultiplemarkerlinesof L. esculentum usedinthepresentstudy. Themorphologicalmarkergenesaregivenwiththeirchromosomallocation(Rick 1982).*indicatesacharacterwhichwasusedintheanalysisoftheasymmetric somatichybridsof L. esculentum (recipient)and L. peruvianum (donor). Tomato genotype

Markers [chromosomearm]

LA291

ms-2 (=malesterile)[2L] hi (-hairless,nolargetrichomes)[IIS] a(-anthocyaninless)[11L] var (-variabilis,leavesemergeyellow)[7S] not (=notabilis,leaveswilting)[7L] ah (=anthocyaninless)[9L] maim(-marmorata,leavesmarbledwhite-green)[9L] clau (=clausa,leavessubdivided)[4S] di (-divergens,stemsslenderandwhitish)[4L] icn (-incana,leaveswithwhitishmargins)[10S] ag (=anthocyaningainer,laminaeabaxiallypigmented)[10L] sy (=sunny,leavesemergeyellow)[3S] sf (-solanifolia,leafletsentireandconcave)[3L] alb (-albescent,strongwhite-greenvariegation)[12S] mua(=multifurcata,dullgreeninterveinalchlorosis)[12L] yv (-yellowvirescent,leavesemergeyellow)[6L] c(-potatoleaf,fewerleafsegments)[6L] af (-anthocyaninless)[5S] tf (=trifoliate,leaves3-segmented)[5S] wv(-whitevirescent,leavesemergewhite)[2L] d(-dwarf)[2L] dgt (-diageotropica, stemsandrootsdiageotrophic)[IL] 1 (-lutescent,leavesyellowing)[8S] al (-anthocyaninloser,pigmentedonlyatnodeslater)[8L]

LA1164

LA1166

LA1182

LA1189 LA1444

LA1665

70/Sethanol and 20'in5xdiluted commercial bleach (10%NaCIO),andwashed severaltimesinsterilewater.Theseedsweretransferredtoplasticcontainers withshootculturemedium(Chapter2)containing100mg/1kanamycinandincubated inthedark.Afterafewdaystheseedsgerminatedandweretransferredtolight. Twoweekslatertheseedlingswerescoredforgrowth(-resistance). To determine the kanamycin resistance in calli, derived from protoplast cultures ofa L. esculentum genotype and one ofthe L. peruvianum ATW-plants (withandwithoutfusiontreatment),weslicedthecalliandsubculturedonepart onthecallusmediumTMc(Chapter4)andtransferredtheotherparttoTMcwith 100mg/1kanamycin.Kanamycinsensitivecalliturnedbrownonthelattermedium. Totestkanamycinresistanceofsomatichybridplants,cuttingsofhybridshoots weretransferredtoshootculturemediumsupplementedwith100mg/1kanamycin. Sensitiveshootsformednorootsandbleachedafterafewweeks. Southern blot analysis DNA was isolated from several plants according to Dellaporta et al. (1983), digestedwith DraI,separatedbyagarosegelelectrophoresis,blottedontoGene ScreenPLUS (NewEnglandNuclear)andhybridisedwithaprobe fortheNPTII gene; as probe was used the Cla l-Sal Ifragment,which contained theNOSpromoterand the structural gene,ofthe T-region ofplasmid pAGS112 (kindly provided by Dr. P.J.M. van den Elzen, MOGEN Leiden, The Netherlands). The hybridisationprocedureisdescribedinChapter6.

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 slabgels (Chapter 2 ) .Enzyme activitywas stained according to Vallejos et al. (1983).

Morphological

markers

Fig. 1 shows the position of the recessive morphological markers,used in the present study,onthelinkagemapofL. escuientum.The L. esculentum genotypes thatwereusedforthefusions,werehomozygousfortwotofourofthesemarkers located onone to twodifferent chromosomes (Table 1 ) .The asymmetric hybrids, preferably greenhouse-grown plants, were assayed for the phenotype of the relevant markers todetermine the retention of thedominant L. peruvianum wild type alleles.The presence of some markers could not be assayed, because they weretypicalhypocotyledonmarkers (e.g. ag) orbecausethehybridandaneuploid nature of thehybrids interferedwith the expression of themutant phenotype.

4

7

5

°-rc/au

8

10 11 12 0-

Qot-3

\*--af 27'

Qot-1

J4j£yi'

2*- -ah 'aot-4

40- -not

4«- -sy

48--»/

mi--a/

marm

a9-- di 104-- c 111.-5/

\ot--g

\si--dgt

Fig. 1.Linkagemapofthemorphologicalandisozymemarkersusedintheanalysis of the asymmetric somatic hybrids. The shaded areas indicate the centromeres. All positions are according toMutschler et al. (1987).

61 Results Number of kanamycin resistance

loci in L. peruvianum

plants

Thesegregationratiosofkanamycinresistanceinthetestcrossof L. peruvianum ATW-plantswithwildtype L. peruvianum aregiveninTable2.SixteenATW-plants showedaratioof1:1,whichindicatesthepresenceofoneNPTIIlocus,whereas oneplant,ATW2002,hadaratioof3:1,whichindicatesthatNPTIIgeneswere insertedattwodifferent unlinked loci.Thiswasconfirmedby Southernblot analysiswiththeNPT IIgeneasprobe (Fig.2):ATW2002 showed twodistinct fragments;thetestedkanamycinresistanthybridsofBellina(+)ATW2002,namely onesymmetricandtenasymmetrichybrids,hadeitheroneorbothofthesebands; thebandssegregatedindependently.Theonlytestedkanamycinsensitivehybrid ofthesameparentalgenotypes,5H10,hadnosuchband(s).

Table2. Segregation of kanamycin resistance in the testcross of kanamycin resistant L. peruvianum PI128650plants(ATW2001toATW2011,ATW2014toATW2016, ATW2020,ATW2021 andATW2027)as female parent withwild type L. peruvianum PI128650,andamongregenerating30H-hybridcalli,whichderivedfromprotoplast fusionsbetween L. esculentum and300Gygamma-irradiatedkanamycinresistant L. peruvianum. Theassayswere performed onmedia supplemented with 100mg/1 kanamycin.Thecalliwereassayedbeforeregenerationoccurred. Km11,kanamycinresistant;Kms,kanamycinsensitive. L.peruvianum genotype

Segregationratiointestcross KmK:Kma

[x*{l:l}]w

ATW2001 ATW2003 ATW2004 ATW2005 ATW2006 ATW2007 ATW2008 ATW2009 ATW2010 ATW2011 ATW2014 ATW2015 ATW2016 ATW2020 ATW2021 ATW2027

29:35 35:29 32:32 73:53 41:53 31:33 36:27 33:32 33:31 31:33 59:66 31:33 29:34 29:33 31:41 32:32

[0.56] [0.56] [0.00] [3.17] [1.53] [0.06] [1.29] [0.02] [0.06] [0.06] [0.39] [0.06] [0.40] [0.26] [1.39] [0.00]

ATW2002

47:17

[14.06]**

Ratioin30H-calli KmK:Kms 1:3 1:0 9:3 6:2 2:1 1:0 3:0 7:5 3:0 4:1 5:3 1:3 1:1 13:1 5:3 3:2 total65:28

segregationratiodoesnotdeviatesignificantlyfrom1:1, if x2 ,whichisco-dominantandpresentinall L. esculentum genotypes used,isincludedinTable4.ThecorrespondinggeneofL.peruvianum is B(= Beta,increaseofß-caroteneandreductionoflycopene).Theredcolour(caused byahighleveloflycopene)of L. esculentum fruitsindicatesabsenceof B.The symmetric hybrids,containing complete genomes ofbothparental species,had yellow fruits (Chapter2).The asymmetric hybrids set yellow, orange orred fruits(seeTable4inChapter4).So,atleastone Balleleispresentinthe plantsthatsetyellowandorangefruits;thiswasthecasein92%(11outof 12)ofthe5H-hybridsand80%(4outof5)ofthe30H-hybrids.

66 Table4. Complementation of marker genes in asymmetric somatic hybrids of L. esculentum and 300Gygamma-irradiatedL. peruvianum. The table showsthe numberofhybridshavingretained L. peruvianum allele(s)ofeachofaseries ofmarkergenes,"absent"meansthatnocomplementationwasobservedinanyof theanalysed shoots;"present"meansthatcomplementationwasobservedinall analysedshoots;"mixed"meansthatcomplementationwasobservedinpartofthe analysedshootsofahybrid.Observationsweremadeongreenhouse-grownplants, derived from 71hybrid calli,andonshoots invitro.Thelatter shootswere assayedonlyforclearlyscorablephenotypes. Marker gene

Corresponding L. peruvianum allele(s) inasymmetrichybrids

Chromosome position

absent dgt sy sf

clau di af yv c

b* var not

1 al ah marm hi a alb

1-152 3-46 3-111 4- 0 4-89 5-14 6-34 6-104 6-106 7- 0 7-40 8- 0 8-67 9-24 9-62 11-48 11-68 12- 0 total

present

1 4 4 1 0 0 0 3 1 0 0 0 1 4 0 1 6

-L

1 4 6 1 2 1 2 0 4 4 2 1 5 6 3 12 9 _5

27

68

mixed

b, low ß-caroteneandhighlycopene afewsectorsofoneplantwere marm oneshootinvitroofeachhybridwas alb

Eachofthe L. esculentum multiplemarkerlinescontained twomarkersona singlechromosome (Table 1). Therefore,itwaspossibletodeterminewhethera given L. peruvianum chromosome was transferred completely by analysing both markers.Thefrequencyofcomplementationofno,oneorbothofthesegenesis shown in Table5 (Aps-1 and B data included). In 36% of the plants complementation ofonly onegenewas observed.This suggeststhepresenceof fragmentsorincomplete,deletedchromosomesof L. peruvianum intheseasymmetric hybrids.

1** 2 ~Aft*

67 Table 5. Complementation of L. esculentum marker genes located on a same chromosomebycorresponding L. peruvianum alleles.Observationswerecarriedout onasymmetric somatichybridsof L. esculentum and L. peruvianum irradiatedwith 300Gy. Only those plants were included where both markers could be scored unambiguously.Thegene symbolssupplementedwith+ indicatethepresenceofthe wild typegenederivedfromL.peruvianum intheasymmetrichybrids. 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 tobe present, while B means the presence of the L. peruvianum specific allele(s), because of the observation of orange fruits.

Chromosome

3

Phenotypic observations sy

sf

sy

3

4

clau

di

yv

7

c

8 9 11

ApsE b 1 var 1 ah hi

not 0 al 0 marm 0 a 0

4

di

clau* di 0

1 c+

yv

0

6

sy* sf 0 +

clau

0

6

sf* 1

0 ApsE B 0

var 1

not* 0

ah hi

12

clau* di* 1

50%

ApsF b 0

ApsP B 4

0%

var*

not* 2

33%

0

1* al* 1

ah* marm 0

ah* marm* 2

50%

hi* a 3##

3

62«

1

a* 2*

12*5%

100%

+

marm+

sf* 4

yv* c* 0

-A *

0

sy*

yv+ c 2*

var*not

at

Single comp lementation

al

17

0%

12/33 - 36%

one of bothalsowas ApsP c (hybrid 30H37) concerning one subclone, other subclone was var*not* (hybrid 30H36) only sectors of one shoot,other shootswere ah marm (hybrid 30H36) * one subclone of onehybrid, other subclone was hl*a* (hybrid 30H33) ** one subclone of one hybrid, other subclonewas hi a (hybrid 30H22)

Discussion The results presented in this paper show that a large amount of L.

peruvianum

genomeisretainedinasymmetric somatichybridsofL. esculentumand irradiated L.peruvianum. This is in agreement with the relatively large number of donor chromosomes, observed in these hybrids (Chapter 4 ) .From the isozyme analysis

68 ofthehybrids,itappearsthatthehigherdosehybrids(30Hand100H)retained less L. peruvianum specificallelesthanthelowerdosehybrids(5H).Thisagrees withtheobservationthatthehigherdosehybridsresemble L. esculentum more thanthelowdosehybridsingeneralmorphologicalappearance (Chapter4). Thelimitedeliminationofdonorgenomeinourasymmetricsomatichybridsis incontrastwithasymmetrichybridsofotherspecies,whichhadretainedonly oneorafewdonorchromosomes(Batesetal.1987;Duditsetal.1980;Guptaet al.1984),orevenoneorafewtraits(Duditsetal.1987;Somersetal.1986). Thelimitedeliminationinthetomatoasymmetrichybridscanbeaconsequence oftheunintendedselectionforgoodcallusgrowth,becausethistraitisbetter in L.peruvianum than in L. esculentum, is multi-genic and not linked to regeneration capacity (Koornneef et al. 1987a). Another explanation forthe limitedeliminationcouldbethelackofsomaticincongruity.Inthecaseswith much elimination, relatively unrelated species (from different genera or families)werefused.Nosymmetrichybridscouldbeobtainedfromthesespecies. Therefore,ifsomaticincongruityoccurs,onlyhybridswithaminimalamountof donorgenomecansurvive.Whenrelated specieswerefused,theeliminationof donorgenomewas often limited (e.g.Famelaer et al. 1989;Yamashitaetal. 1988).Thosespeciesweresomaticcongruent,becausealsosymmetrichybridscould be obtained.Anexceptionare some of the asymmetric hybrids oftherelated species Nicotiana tabacum (+) N. plumbaginifolia (donor),thatcontainedonly onedonorchromosome (Batesetal.1987). Kanamycin resistance was retained in46 percent of the 30H-hybrids,that derivedfromadonorparentwithoneNPTIIlocus.Thispercentageisanaverage, obtainedfromtheanalysisof16independentkanamycinresistant L. peruvianum genotypes.IftheNPTIIgeneintegratesrandomlyinthe L. peruvianum genome, thiswouldimplythatanyalleleofthedonorgenomeisretained inabout50% ofthehybrids.Incontrastwiththesinglekanamycin resistance allele,the isozyme and morphological markers were represented by two alleles in the L. peruvianum donorgenome.Whenahybridisozymepatternorcomplementationof amutantphenotypewasobserved,eitheroneortwodonoralleleswerepresent. Theaveragechancethatacertainalleleisretained,canbededucedfromthe numberofhybridswhichhadlostbothhomologousalleles.Theisozymemarkers (Table3)andmorphologicalmarkers(Table4)weredistributedmoreorlessat randomoverthegenome.Thefrequencesoflossofbothdonorallelesofagiven locusinthe30H-hybridswas,ontheaverage,21%to30%("mixed"isconsidered aslostinthelatter).Anindividual L. peruvianum alleleisthuslostwitha frequency of 0.21 to 0.30 = 46% to 55%,and is therefore retained with a frequency of45%to 54%.These frequencies agreewellwith the frequencyof retentionofthekanamycinresistancealleles.

69 Wefrequentlyobservedthatshoots,whichderivedfromasamehybridcallus, differedwithrespecttooneormoreortheanalysedmarkers,namelyintwoof the38tested5H-hybrids (5%),10ofthe6330H-hybrids (16%)andoneofthe9 lOOH-hybrids (11%).Apparently,thehigherdosehybridsshowedthisphenomenon more often. The segregation occurred most probably in the hybrid callus. Segregationattheplantlevelwasshowninonehybridshootcontainingsectors intheleafthatwere marmorata, thephenotypeoftherecipientLA1164(Table4). Somaticsegregationhasalsobeenobservedinasymmetrichybridcalliandplants of Nicotiana plumbaginifolia (+)(irradiated)N. sylvestris forseveralisozymes (Famelaeretal.1989). Becausethecomplementationofonlyoneoftwogenesonthesamechromosome occurredratherfrequently(Table5),alargefractionoftheasymmetricsomatic hybridsmusthave contained incomplete chromosomes of L. peruvianum. Ineach fusioncombinationthiscouldbeanalysedforonlyoneortwochromosomes.Even when both markerswere complemented by L. peruvianum alleles,thismayhave resultedfromtheretentionoftwoincompletechromosomes.Therefore,weconclude that thehigh doseofgamma irradiation (300Gy)inducedmany breaks inthe L. peruvianum chromosomes.Thiswasconfirmed bytheRFLPanalysis of730Hhybrids, in which, on the average, at least 12 of the 18 retained donor chromosomeswereincomplete (Chapter6). Theasymmetricsomatichybridswereselectedonregenerationcapacity.This traitofL.peruvianumissupposedtobegovernedbytwounlinked,dominantgenes (Koornneefetal.1987a).Theoretically,itshouldhavebeenpossibletolocate these regeneration genes inour experiments,because ofthe availability of markergenesforeachofthechromosomes.Genesof L. peruvianum linkedtothe regenerationgenes,shouldbepresentintheasymmetrichybrids.However,wewere notabletolocatethesegenes.Thiscouldhavebeencausedby:(1)thepresence ofmany L. peruvianum chromosomes in the asymmetric hybrids; (ii)the small number of asymmetric hybrids that had lost any given gene; (iii)the high frequencyofbreakageofthedonorchromosomes,sothatonlycloselylinkedgenes couldbeusedforthisanalysis.Nevertheless,wecanstatethattheregeneration genesarenotcloselylinkedto sy, sf (bothmappedonchromosome 3), hi anda (bothchromosome 11),becauseboth L. peruvianum allelesofthesegeneswere absentinalargefractionoftheasymmetrichybrids.

Acknowledgements. ThisresearchwassupportedbytheFoundationforFundamental BiologicalResearch(BION),whichissubsidisedbytheNetherlandsOrganisation for Scientific Research (NWO). We are very grateful to Prof. C M . Rick for supplyingthetomatotesterlines,toDr.P.J.M.vandenElzenforprovidingthe plasmidpAGS112,toJoséKok,PattyvanLoenenMartinet-Schuringa,AnjaPosthuma, RenéRijkenandJannyVosfordoingpartoftheexperiments,andProf.C.Heyting forcriticallyreadingofthemanuscript.

70 References BatesGW,HasenkampfCA,ContoliniCL,PiastuchWC (1987)Asymmetriehybridization inNicotianabyfusionof irradiatedprotoplasts.TheorApplGenet74:718-726 DellaportaSL,WoodJ,HicksJB(1983)AplantDNAminipreparation:versionII.PlantMolecularBiologyReporter 1:19-21 DuditsD,Fejer0,HadlaczkyGY,KonczCS,LazarG, HorvathG (1980)Intergeneric gene transfermediatedby plantprotoplast fusion.MolGenGenet 179:283-288 DuditsD,MaroyE,PraznovszkyT,OlahZ,Gyorgyey J,CellaR (1987)Transferofresistancetraitsfromcarrot into tobaccoby asymmetric somatic hybridization:Regeneration of fertile plants.Proc Natl Acad SeiUSA 84:8434-8438 FamelaerI,GlebaYY,SidorovVA,KaledaVA,ParakonnyAS,BoryshukNV,CherupNN,NegrutiuI,JacobsM (1989) Intrageneric asymmetric hybrids between Nicotjana plurobaRinifolia and Nicotiana sylvestris obtained by 'gamma-fusion'.PlantScience 61:105-117 GlebaYY,Hinnisdaels S, Sidorov VA,KaledaVA, Parokonny AS,Boryshuk NV, CherupNN,Negrutiu I, JacobsM (1988)Intergeneric asymmetrichybridsbetweenNicotianaplumbaginifoliaandAtropabelladonnaobtainedby "gamma-fusion".TheorAppiGenet 76:760-766 Gupta PP, Schieder 0, Gupta M (1984) Intergeneric nuclear gene transfer between somatically and sexually incompatibleplantsthroughasymmetricprotoplastfusion.MolGenGenet 197:30-35 KoornneefM,HanhartCJ,MartinelliL (1987a)Agenetic analysisofcellculturetraitsintomato.TheorAppl Genet 74:633-641 KoornneefM, JongsmaM, WeideR, Zabel P, HilleJ (1987b)Transformation of tomato. In:NevinsDJ,JonesRA (eds)Tomatobiotechnology.AlanR Liss, Inc.NewYork,pp 169-178 Mutschler MA, Tanksley SD,Rick CM (1987)Linkage maps of the tomato (Lvcopersicon esculentum).Rep Tomato GenetCoop 37:5-34 RickCM (1982)Stock list.RepTomatoGenetCoop 32:3-10 RickCM (1983)Tomato (Lvcopersicon).In:TanksleySD,OrtonTJ (eds)Isozymesinplantgeneticsandbreeding, partB. Elsevier,Amsterdam,pp 147-165 SomersDA,NarayananKR,KleinhofsA,Cooper-Bland S,CockingEC (1986)Immunologicalevidencefortransferof the barley nitrate reductase structural gene to Nicotiana tabacum by protoplast fusion. Mol Gen Genet 204:296-301 SuursLCJM,JongedijkE,TanMMC (1989)Polyacrylamidegradient-gelelectrophoresis:aroutinemethodforhigh resolution isozymeelectrophoresis ofSolanum andLvcopersiconspecies.Euphytica 40:181-186 Vallejos CE (1983)Enzyme activity staining. In:Tanksley SD,Orton TJ (eds)Isozymes inplant genetics and breeding,partA.Elsevier,Amsterdam,pp 469-515 Yamashita Y, Terada R, Nishibayashi S, Shimamoto K (1989)Asymmetric somatic hybrids of Brassica: partial transfer ofB. canroestrisgenome intoB.oleraceaby cell fusion.TheorApplGenet 77:189-194 Young ND, Tanksley SD (1989) Restriction fragment length polymorphism maps and the concept of graphical genotypes. TheorApplGenet 77:95-101

71 CHAPTER6 A S Y M M E T R I C Z^YCOPEJRJSICON UYCOFERSICON

S O M A T I C H Y B R I D S B E T W E E N ESCUUENTUM A N D I R R A D I A T E D PEISLTVIANUM

III. ANALYSIS WITH RESTRICTION LENGTH POLYMORPHISMS

FRAGMENT

J.Wijbrandi,P.Zabel,M.Koornneef

Summary.Thegenomecompositionofasymmetricsomatichybrids,obtainedbyfusion of leaf protoplasts from Lycopersicon esculentum and gamma-irradiated leaf protoplastsfrom L. peruvianum,wascharacterisedbySouthernblotanalysiswith 29RFLPmarkersandarDNAprobe.Eight"lowdosehybrids"andseven"highdose hybrids" (irradiationdose 50Gyand 300Gy,respectively)wereanalysed.By densitometric scanningoftheautoradiographs,thenumberofallelesforeach locus of the component species was established. Ingeneral, elimination of allelesfromtheirradiatedL.peruvianumdonorgenomewaslimitedandranged from 17%to69%.Three L. peruvianum loci,located onchromosome 2,4and7, respectively,werepresentineachasymmetrichybrid,whichmaysuggestlinkage totheregenerationcapacitytraitwhichwasusedinselectingtheasymmetric hybrids.Thelossofdonorgenomewasdosedependent.Lowdosehybridscontained morealleles,lociandcompletechromosomesfrom L. peruvianum thanhighdose hybrids,whereas thehighdosehybridscontainedmore incompletechromosomes. Inmosthybridssome L. esculentum alleleswerelost.Theamountofribosomal DNAfrom L. peruvianum retainedintheasymmetrichybridsvariedstrongly:in onehybridamplificationhadoccurred,whereasinothers L. peruvianum rDNAwas eitherabsentor(in)completelypresent.TheamountofrDNAfrom L. esculentum wasdecreased'inmostasymmetric hybrids.Thepossible implicationsofthese resultsfortheuseofasymmetrichybridsinplantbreedingarediscussed.

Introduction Variousstrategieshavebeendevelopedforcombiningthegeneticinformationof twoplantspecieswhichcannotbehybridisedsexually.WhileDNAtransformation techniquesareparticularlydesignedtoallowtheintroductionofdefinedbut small-sizedDNAfragmentsintoarecipientspecies,protoplastfusionandrelated cellular techniques are the obviousmeans for transferring large chromosomal segmentsorevencompletegenomes(forareviewconcerningtomato,seeHilleet al.1989).However,inmostprotoplastfusionexperiments,onlyalimitednumber oftraitsfromthedonorspeciesisofinterestandeliminationofthebulkof thedonorgenomehastobeachieved.Inmammaliansomaticcellhybrids,preferential loss of chromosomes from one parent species often occurs spontaneously (RingertzandSavage1976). Inplantsomaticcellhybrids,however,chromosome elimination is limited and, in general, does not concern specifically the

72 chromosomes of one of the component species (Gleba and Sytnik 1984). Inan attempt to decrease the amount of donor genome, several investigators have irradiatedthedonorprotoplastsbeforefusionwithRöntgen-orgamma-rays,so astoinducelossofentirechromosomesorchromosomesegments.Insomecases, oneorafewdonorchromosomeswerefoundtoberetainedwithinthehybrid(Bates et al. 1987; Dudits et al. 1980; Gupta et al. 1984), whereas in others, eliminationofthe irradiated genome appeared tobelimited (Famelaer etal. 1988;Glebaetal.1988;Imamuraetal.1987;Müller-GensertandSchieder1987; Yamashitaetal.1989). In the present paper we have analysed the effects of irradiation of L.peruvianumontheeliminationofthe L. peruvianum genomeinL. esculentum (+) L. peruvianum somatichybrids.Forthispurpose L. peruvianum protoplasts wereirradiatedbeforefusionwitheitheraloworahighdoseofgamma-rays, andtheasymmetric somatichybridswere selectedonthebasisofthesuperior regenerationcapacityofL.peruvianum.Previously,evidencehasbeenpresented showingthattheregenerationcapacitytraitof L. peruvianum isdominant(Adams andQuiros1985;Kinsharaetal.1986;Koornneefetal.1987a;Wijbrandietal. 1988)andlikelytobecontrolledbyalimitednumberofgenes(Koornneefetal. 1987a).Bymeansof30RFLPmarkersofknownchromosomallocation,thegenome constitutionofanumberofasymmetrichybridswasestablished.

Materialsandmethods Plant material Somatichybridplantswereobtainedfromindependentprotoplastfusionevents (Wijbrandi et al. 1988). The parental genotypes of thehybrids are shownin Table1. Seeds of the tomato cultivar Belllna, the LA-genotypes and the L.peruvianumaccessionPI128650werekindlyprovidedbyRijkZwaanSeedCompany (deLier,TheNetherlands),Prof.C.M.Rick(TomatoGeneticsStockCenter,Davis, USA) and the Institute of Horticultural Plant Breeding (Wageningen, The Netherlands),respectively.TheL. peruvianum parentwasirradiatedbeforefusion withadoseofeither50Gray("low dosehybrids",designatedas5H)or300Gray ("highdosehybrids",30H),using a60Cosource atadose rate ofabout2000 Gray/hour(atthePilot-PlantforFoodIrradiation,Wageningen,TheNetherlands). The symmetric somatic hybrid 0H1 (2n=4x),L. esculentum cv.Bellina, and L. peruvianum PI128650-ATW2002wereusedascontrolsinDNAanalysis. Hybridisation probes Thefollowingtomatosinglecopycloneswereused:(i)thecDNAclonesCD1,CD14, CD15, CD27, CD41,CD50, CD56, CD59 (Bernatskyand Tanksley 1986)and "Adh2" (pCB25E6,containingapartoftheAdh-2codingregion;ChaseandWilliams1986, seealsovanDaelenetal.1989). (ii)thegenomicclonesTG8,TG9,TG16,TG20, TG27,TG30,TG31,TG34,TG37,TG42,TG43,TG44,TG45,TG50,TG54,TG61,TG62, TG63,TG68,TG69 (Tanksleyetal.1988)and "Adhl" (pTAdh-1,agenomic1.8kb fragmentclonedinthe Eco RI-Sal IsiteofpTZ18R).TheCD-andTG-cloneswere kindlysuppliedbyDr.S.D.Tanksley,CornellUniversity,Ithaca(USA),pCB25E6 wasagiftfromDr.Th.ChaseJr.,RutgersUniversity,NewJersey (USA),and pTAdh-1fromDr.E.Lifschytz,Technion-Israel Institute ofTechnology,Haifa

73 Table 1. The parental genotypes of the somatic hybrids and the dose of gammairradiationapplied to thedonor. Hybrid

Recipient

(L.

esculentum)

Donor

(L.

peruvianum)

Dose (Gray)

0H1

cv. Bellina

PI128650-ATW2002*

0

5H2, 5H3,5H5, 5H7, 5H13, 5H16, 5H28

cv. Bellina

PI128650-ATW2002*

50

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 akanamycin resistance gene (seeKoornneef et al. 1987b). tomatogenotype,homozygous recessive for sy, sf (chr. 3 ) ,and alb (chr. 12) (Rick 1982). tomato genotype,homozygous recessive for hi and a (chr.11) (Rick 1982).

(Israel).Apeagenomic clonefor45SrRNA, "R45S" (pBsARl),wasagiftfromDr. J.P. Nap, Research Institute Ital, Wageningen (The Netherlands). The map positionsoftheclonesused inthehybridanalysis,areshowninFig. 1.Except for the ribosomal clone, the insert of each clone was excised from the vector withtheappropriate restrictionendonucleases andseparated fromthevector on a 1%agarosegel.DNAfragmentswereradiolabeledwith [32P]dATPbymeansofthe random-priming method (Feinberg and Vogelstein, 1983).

Plant DNA analysis Total DNAwas extracted from leaves (fresh or freeze dried)of plantsgrown in the greenhouse,asdescribed byDellaporta et al. (1983). DNA samples (2.5/ig) were digested to completion with Eco RI, Eco RV, Hin dill, Dra I or Pst I (5hours 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)bymeansofanalkalineblottingprocedure (ReedandMann1985). Hybridisation in10%dextransulphate,1%SDSandIMNaCl,at65 °C,wascarried out for 18hours under conditions as recommended by the manufacturer of the membrane. After hybridisation, blots were washed under stringent conditions: three 5min.-washes in 2xSSC at 20 °C, two 20min.-washes in 2xSSC+ 1% SDS at 65 °C, two 30min.-washes inO.lxSSC + 1% SDSat 65 °C,and two 15 min.-washes inO.lxSSC at 20 °C. Subsequently, the blotswere exposed toX-ray film (Kodak X-OmatAR 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 thusbeused at least four times. To obtain quantitative data on the relative intensity ofbands derived from both parents, autoradiographs were scanned with a LKBUltrascanXL Laser Densitometer connected toanOlivetti M24personal computer,whichwas equipped with the LKB 2400GelScan XL software. The following calculations weremade: (i)therelativenumber of L. peruvianum andL. esculentum specificalleles for a given probe in each asymmetric hybrid was determined as the ratio of the absorptionvalues(peakareas)oftherelevantbandsontheautoradiographs.This ratio was divided by the corresponding ratio in the symmetric hybrid 0H1 that wasused asa reference(=1);

74

1

2

3

4

5

6

7

8

9

°TCD4f°-TCD14

1&.R45S 4 - -TG31 10- -CD1

10

1 1 1 2

•TG9

•CD59 M-\-TG44

™--TG20

TG43 .CD50 3 g_ -Adh2 43- -TGS4

tgU-COTS

*1--CD27

B\--Adh1 59. S6-X-TG42

TG16

52--TGS0

TG62

7 3+TG69 'TGS

75. 1'

7 3-i-rag TG45 *S->-TG3Cn-TQ6S

103-L TG37

io6J_r0 Fig.3.Densitographsof EcoRI-digestedDNAfromthelowdosehybrid5H22,the highdosehybrids 30H1,30H3,30H6and the symmetric hybrid 0H1,probedwith TG31.TheautoradiographshowninFig.2A,upperpanel,wasscanned (leftside 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) werecalculated.Theratioofthepeakareaswasdetermined (E/P)andnormalisedbyacorrectionfactordeducedfromtheratioofthepeak areasof0H1,whichwasusedasareference (E/P=•1), Theseratiosareshown inthetablebelowthedensitographs;theratiosofalleles intheasymmetric hybridsweredeterminedfromthesecorrectedvalues.

79 Table3. The amount of L. peruvianum genome (number of loci, alleles and chromosomes)inthe"lowdose"-and"highdose"asymmetrichybrids,asdetermined by autoradiographic analysis and densitometric scanning of Southern blots hybridised with 29 single copy clones (29RFLPs = 29loci - 58alleles).A chromosomewasconsideredcompletewhenforeachlocusofthechromosomethesame numberofalleleswasfound. Lowdosehybrids (n-8)

Highdosehybrids (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%)

Completechromosomes

13.4+2.8

Incompletechromosomes*

4.9±1.6

Lostchromosomes

5.8+3.6

7.1±4.1 11.4±3.2 6.9±3.9

significant difference between the two groups ofhybrids,as testedwith Student'sttest(P