INDUCTION ANDCHARACTERIZATIO N OFMICRONUCLE I IN PLANTCELL S
Perspectives for micronucleus-mediated chromosome transfer
INDUCTIE ENKARAKTERISERIN G VAN MICROKERNEN IN PLANTECELLEN
Perspectieven voorchromosoo m overdracht via microkernen
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0000 0330 8547 Promotoren:Dr .A .va n Kammen,hoogleraa r in demoleculair e biologie
Dr.B .d eGroot ,bijzonde r hoogleraar ind esomatisch e celgeneticava n dehoger eplan te n haar toepassingen aan deKatholiek eUniversitei t te Nijmegen NPöt2D\}l2äZ
HarrieA .Verhoeve n
INDUCTIONAN DCHARACTERIZATIO N OFMICRONUCLE I IN PLANTCELL S
Perspectivesfo rmicronucleus-mediate dchromosom etransfe r
Proefschrift ter verkrijging van de graad van doctor in de landbouwwetenschappen, opgeza gva n de rector magnificus, dr. H.C.va nde r Plas, in het openbaar te verdedigen op dinsdag 13jun i 1989 des namiddags te vier uur in de aula van de Landbouwuniversiteit te Wageningen „.RT LANDBOUWUNIVERSITEIT WAOENINGEN
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STELLINGEN
1 Het feit dat het karyotype van een cybride niet te onderscheiden isva n de kerndonor sluit de aanwezigheid van delen van het nucleaire genoom van de organeldonor niet uit. (Ichikawa et al., 1987)Theor . Appl.Genet . 74:746-752.
2 Voordetecti eva ndono rDN Ai nd erecipien t naasymmetrisch efusi e vanbestraald e donor protoplasten met onbestraalde acceptor-protoplasten verdient in-situ hybridisatie met soortspecifieke, repeterende probes devoorkeu r boven dot-blot hybridisaties. (Imamura et al., 1987)Theor . Appl. Genet. 74:445-450 .
3 Oryzaline isd e metafase remmer bij uitstek voor het isoleren van metafase chromosomen uit plantecellen. (Dit proefschrift).
4 Om het chromosoom aantal van planten te verdubbelen, kan beter van oryzaline gebruik gemaakt worden dan van colchicine. (Dit proefschrift). 5 De door Hertel waargenomen effecten van oryzaline en trifluralin worden verkeerd geïnterpreteerd. (Hertel et al., 1981) FEBS Letters 127: 37-39.
6 Intacte organellen kunnen rechtstreeks vanuit een donor-cel of -protoplast naar een recipiënte plantecel worden overgedragen via microinjectie. (Dit proefschrift).
7 De specifieke drie-dimensionale organisatie van chromosomen in de interfase kern van verschillende neurale celtypen is een aanwijzing voor een algemeen regulerende functie van de drie-dimensionale structuur bij de genexpressie. (Manuelidis and Borden, 1988) Chromosoma 96:397-410.
8 Het veelvuldigvoorkome n vansoortspecifieke , repeterende DNA sequenties,wijs t op een belangrijke functie van deze sequenties bij de soortbepaling.
9 Het feit dat de nog niet uitgestorven diepzee Coelacanth Latimeria chalumnae zijn kwastvinnen nietgebruik t omt elopen ,i sva nondergeschik t belangi nd ediskussi eove r een mogelijke verwantschap van kwastvinnigen met land-vertebraten. (Forey, 1988) Nature 336:727-732 . 10 De meest waardevolle DNA-sequenties in genetisch gemanipuleerde planten zullen voorlopigd e"copyright-sequenties "zijn , diegebruik t zullengaa nworde nom genenpiraterij tegen te gaan door genetisch gemanipuleerde planten te beschermen tegen illegaal kopiëren.
11 Introductie van met microprocessors uitgeruste meetapparatuur, waarvan de basis principese nd etoegepast egegevensverwerkin g niet bekendzij n bijd egebruikers ,verhoog t hetrisik oo p"Desk-to pResearch" ,waarbi j deresultate n oponverantwoord ewijz e verwerkt worden.
12 Het geringe succesva n "Artificial Intelligence" isvaa k tewijte n aan onderschatting van de eigen intelligentie door de (potentiële) gebruiker.
13 Het feit dat de meeste sportieve auto's zijn voorzien van opklapbare koplampen, wekt ten onrechte de indruk dat de wetten van de aërodynamica in het donker niet gelden.
14 De succesvolle introductie van de vele "light food" artikelen is een onverteerbare zaak in een wereld, waar minstens 75%va n de bevolking honger lijdt.
Stellingen, behorende bijhe t proefschrift " Induction and characterization ofmicronucle i in plant cells:perspective s for micronucleus-mediated chromosome transfer", door Harrie A. Verhoeven. Wageningen, 13jun i 1989 The cover shows a three dimensional reconstruction of a micronucleated suspension cell of N.plumbaginifolia, visualized by confocal laser scanning microscopy after vital staining with Acridin Orange.
The investigations were partly supported by funds from the CEC "Biotechnology Action Programme", contract BAP 0083-NL. Contents
Voorwoord V
Chapter 1:Genera l Introduction 1
Chapter 2: Efficient induction byamiprophos-methy l and flow-cytometric sorting of micronuclei inNicotiana plumbaginifolia. Planta (1987 ) 122:473-478 . 15 Chapter 3: Mitotic dynamics of micronuclei induced by amiprophos-methyl and prospects for chromosome-mediated gene transfer inplants . Theor.Appl . Genet. (1988) Z5_: 575-584. 27
Chapter 4:Compariso n of the effects of various spindle toxins on metaphase arrest and on formation of micronuclei in cell suspension cultures ofNicotiana plumbaginifolia 45
Chapter 5:Improve d accumulation of mitotic and micronucleated suspension cells ofNicotiana plumbaginifolia by amiprophos-methyl after pretreatment with DNA-synthesis inhibitors. 61 Chapter 6:Isolatio n and characterization of microprotoplasts from APM-treated suspension cells ofNicotiana plumbaginifolia. 79 Chapter 7:Direc t cell to cell transfer of organelles by microinjection: methodology and first results. 93
Chapter 8:Summar y and conlusions 105
Samenvatting 113
Curriculum vitae 117 VOORWOORD
Voor U ligt het tastbare resultaat van ruim vier jaar onderzoek, waaraan talloze mensen eenbijdrag e geleverd hebben.Di ti sda n ook degeschikt eplaat som iederee nt e bedanken, dieo penig ewijz ebetrokke n isgewees tbi jdi tonderzoek . Zonder iemand tekor t tewille n doen,wi li khie r een aantal personen met name noemen, dieva nbijzonder e betekenis zijn geweest.
Allereerstmij n ouders,voo rd emogelijkhede n diezi jgebode nhebbe nom t egaa n studeren.
Mijnvriendi nJenny ,e nhaa r ouders,di egee nmomen tgetwijfel d hebben aanhe twelslage n van deze onderneming.
Dr. Ir. Ad de Laat, die mij de schone kunsten van het chromosoom onderzoek heeft bijgebracht.
Dr. Lous van Vloten-Doting, die met haar niet aflatende belangstelling een essentiële bijdrage heeft geleverd bijhe t afronden van dit onderzoek.
Dr. Willem Stiekema, voor het stimuleren en in het rechte spoor houden van mijn experimenten.
Jan Blaas, die met zijn onmetelijke geduld de meest onwaarschijnlijke microinjectie experimenten tot een goed einde wist te brengen.
Dr. Kamisetti Sree Ramulu,voo r alle bijdragen op het gebied van de cytologie.
Paul Dijkhuis, voor zijn onverstoorbare hulp bij het eindeloze tellen van de talloze cytologische preparaten.
Dr. Ir. Henry van der Valk, voor de zeer plezierige samenwerking bij onze gezamelijke experimenten.
YvonneNollen ,di e altijd klaar stond omth e zorgendat er enorme hoeveelheden cellen en media beschikbaar waren.
Wil ten Broeke en Henk Eibers,voo r hun bijdrage op het gebied van de flowcytometrie.
Alle niet met name genoemde medewerkers van het Ital, en uiteraard speciaal de medewerkersva nprojec t 125,di ealle nwe lo péé n ofander ewijz e eensteentj e bijgedragen hebben.
Speciale dankbe n ikverde r nogverschuldig d aanDrs .Wilber t Boutse n Marjo Bouts,voo r alle steun en hulp bij het tot stand komen van dit boekje. Alslaatst ewi li kbesluite nme the tdanke nva nmij nbeid epromotoren ,Prof .Dr .Bra md e Groot,e nProf .Dr .A bva nKammen ,di emi jtelken swee r aanhe twer kwiste nt e krijgen methu nterecht ekritisch e opmerkingen. Maart1989 . CHAPTER 1
GENERAL INTRODUCTION CHAPTER 1
GENERAL INTRODUCTION
Outline
Theai mo fthi sstud yi st oinvestigat eth e induction, characterization, isolation,an d transfer of micronuclei in plant cells and to evaluate the possible application of micronuclei and microprotoplasts for transfer of chromosomes. Chapter one is an overview of the current knowledge on organelle transfer in both mammalian and plant cells.
Chapter two deals with the induction and characterization of micronuclei in plant cell suspension culturesb yth e herbicide amiprophos-methyl (APM).
Theeffect s ofAP Mo nmitoti cdivision so fsevera lsource so fplan tmaterial ,an dthei r effects on chromosome numbers and cellular DNA content are presented in chapter three.
To obtain more information on the mechanism which is involved in the formation of micronuclei, the effects of various spindle toxins on metaphase arrest and micronucleus formation are compared (chapter four).
The influence of cellcycl e manipulation byDNA-synthesi s inhibition on the mitotic index and the formation of micronuclei isth e subject of chapter five.
Chapter six describes the isolation and characterization of microprotoplasts from micronucleated suspension cells.
The possibilities for organelle transfer by microinjection are presented in chapter seven.
The results and conclusions are summarized in chapter eight. PARTIAL GENOME TRANSFER
Introduction Somatic cell hybridization.
In this chapter an overview is given of the Bysomati c hybridization, induced either by current state of both animal and plant cell chemical treatments, or by electrofusion, biology with respect to partial genome two complete genomes, including the transfer. The aim of this study is the cytoplasmic organelles, are joined in the development ofmethod s forpartia l genome fusion product.Th echromosom e numbero f transfer in plants by other means than the resulting somatichybri d will be the sum transfer of cloned genes.Th e application of of the chromosome numbers of the two molecular techniques for the isolation, parent cells, and the organelles from both cloning and transfer of identified single parents will be present. During the first genes is not considered within the scope of divisions of the fusion product, the thisthesis . composition of the cells with regard to chromosome composition, mitochondria Partial genome transfer can be and eventually plastids will be determined, accomplished bysomati c cell hybridization, and remain stable for the next cell by microcell-mediated chromosome generations. In some cases, when the two transfer, by chromosome-mediated gene genomes are closely related, the original transfer and by DNA-mediated gene fusion product can persist with minor transfer. The complexity of the genetic changes,a si sth ecas ei nSolanum tuberosum information is an important factor in (+ ) Solanum phureja fusions (Pijnacker et deciding which transfer systemwil lb e used. al., 1989), Solanum brevidens ( + ) In somatic hybridization and S.tuberosum fusions (Austin et al., 1985). microcell-mediated chromosome transfer, The plants, regenerated from these fusion where isolated protoplasts (plant cells from products, were fertile, notwithstanding the which the cell wall has been removed by fact that in several fusion products, one enzymatic digestion of the celluloses and chromosome (e.g. chromosome 2 from pectins) with intact plasmamembranes S.phureja, carrying the NOR region) was function as carrier, fusion will be applied. eliminated. When the two species are more For transfer of chromosomes or DNA distantly related, unilateral elimination will fragments, microinjection, chemically occur. In this case the chromosomes from induced uptake, or electroporation can be oneo fth e fusion partnerswil lb e eliminated used. In this overview, the differences preferentially (the "segregant" set),wherea s between the carrier-systems of genetic the chromosomes from the other partner information willb ediscussed ,alon gwit h the will all be retained (the "retained" set, techniques available for achieving transfer. Graves, 1984),a si sth e casewit h the human chromosomes in somatic rodent-man N.plumbaginifolia Fig. 1. Diagram, showing the 50 chromosome numbers of somatic hybrid calli,obtaine dafte r fusion of diploid N.plumbaginifolia (20 O o chromosomes) with diploid potato (S.tiiberosum, 24 chromosomes) 40 - o X X X 10 - Xx I I ^^ -xx 10 30 50 S.tuberosum fusions (Weissan dGreen , 1967;Ruddl e and chromosomes and few N.plumbaginifolia Creagan, 1975). In the case of fusions chromosomes (Fig.1) . between S.tuberosum ( + ) Nicotiana plumbaginifolia, no preferential loss of Recent cytological work on the sexual chromosomes from either species was hybrids between Hordeum vulgarean d Zea observed in regenerated plants (de Vries, mays has shown, that there is a preferential 1987). Although the mechanism of spatial arrangement of two incompatible chromosome elimination is still obscure, a genomes, which predicts which of the two correlation with the direction of genomes will be eliminated (Bennett, 1987; chromosome elimination and the ploidy 1988).Th egene so nth echromosome si nth e level of the partner genomeswa sfoun d in a periphery of the interphase nucleus are serieso fmouse-hamste r hybrids.Onl ywhe n expressed, but they appear to be more therati oo fth emouse:hamste r genomeswa s sensitive to chromosome elimination. below 1, the hamster chromosome set could Species specific chromosome arrangement be retained completely, and the mouse is also observed in somatic hybrids between chromosomes were eliminated (Graves, Atropa belladonna and Nicotiana chinensis 1984).A simila r observation hasbee n made (Glebae t al., 1983),an d couldb e eliminated recently with somatic hybrids between onlyb yrepeate d treatmentswit hcolchicine . diploid N.plumbaginifolia and dihaploid In this case, no correlation was noted S.tuberosum (Gilissen et al., 1989). Two between chromosome orientation and typeso fhybrid swer efound , onewit h excess unidirectional chromosome elimination. A N.plumbaginifoliachromosome san dwit ha n similar phenomenon is observed in incomplete set of potato chromosomes; and Hordeum X Secale hybrids the other type with excess S.tuberosum (Schwarzacher-Robinson et al., 1987). A genetically controlled spatial relationship an explanation of the occurrence of new appearst o existbetwee n the centromeres in RFLP bands in some recovered fusion normal metaphase plates (Heslop-Harrison products (Chetrit et al., 1985),recen t work and Bennett, 1983). has demonstrated that interspecific mitochondrial recombination isa rar e event Chromosome elimination of one of the (Kemble, 1988).A possible explanation for partners can be directed and stimulated by the new bands in the mitochondrial RFLP gamma-irradiation. In this way, the patterns is recombination within the chromosomes of the donor will be mitochondrial circular genome. This is fragmented, and according to cytological facilitated byth e large numbers of copieso f observations,onl yfragment s with functional the genome present in one mitochondrion centromeres are maintained stably during (Morgan and Maliga, 1987). Mitochondrial mitosis,whil e all other fragments appear to recombination is also observed after be lost. In most cases, only a few additional prolonged in-vitro culture (Shirzadegan et fragments and even intact-looking al., 1989). Transfer of spontaneously chromosomes were observed in the fusion occurring mitochondrial plasmids is product (Bates, 1987; Gleba et al., 1988). proposed as an alternative, explaining the Alternatively,fragment s from the irradiated newRFL P bands (Kemble, 1988). genome incorporate into the recipient genome as has been demonstrated by For chloroplasts, sorting out is observed in hybridization with species-specific highly most cases after somatic hybridization. In repetitive DNA-probes (Imamura et al., some rare cases,bot h parental chloroplasts 1987;Piastuch and Bates, 1988). coexist for a longer time, giving rise to The karyotypical analysis of the obtained mosaic chimaeric tissueswhe n they sort out hybrids is complicated, and often at a later time (Fluhr et al, 1983;reviewe d impossible, due to chromosome in Fluhr, 1983). The occurrence of such eliminations,rearrangements , deletions and mosaics in sexual crossings of Oenothera insertions. It has been shown by in-situ indicates that two different chloroplasts can hybridization with species-specific, highly persist during manydivisions . Alternatively, repetitive DNA-probes, combined with transfer of Chlamydomonas chloroplasts fluorescent labelling, that large intoDaucus protoplast sb yfusio n (Fowke et chromosome fragments of the heavily al., 1979)ha sshow ntha t incas eo f excessive irradiated N.plumbaginifolia donor have incompatibility between organelles and been integrated into the N.tabacum nuclear genes, the introduced chloroplasts acceptor genome (Piastuch and Bates, degenerate. 1988).I n this case, the metaphase plate did Specific transfer of organelles can be not show any significant aberrations in the obtained by heavy irradiation of the donor karyotype of N.tabacum. species, resulting in a complete fragmentation of the nuclear genome,whil e Beside the nuclear genes, also transfer of onlyintroducin ga relativel ysmal lnumbe ro f cytoplasmic organelles is accomplished by fragmented organellargenome s(Mencze le t somatic hybridization (reviewed in Galun al., 1982; Menczel, 1984). Although this and Aviv, 1986). Organelles from the two procedure seems to be astraighforwar d way parents are sorted out in most fusion for organelle transfer, and iswidel y used as products during the first divisions after such (Aviv et al., 1980; Sidorov et al.,1981 ; fusion, and chimaeric tissues will be Aviv et al., 1984; Aviv and Galun, 1986; obtained (reviewed in Fluhr, 1983). Glimeliusan dBonnett , 1986;Ichikaw ae tal. , Although recombination between the two 1987; Barsby et al, 1987; Menczel et al, parental mitochondria hasbee npropose d as 1987),i tha sth e disadvantage of introducing Fig. 2. Schematic diagram, showing the basic steps in microcell fusion experiments. Chromosomes APM become scattered through the cell byincubatio n with a spindle toxin (APM in this case), andfor m micronuclei. © Chromosome grouping Multinucleate protoplasts Donor cell are isolated from thetreate d suspension cells,an dthe y are subjected tocentrifugatio n in the presence of cytochalasin-B, and subprotoplasts are formed. Protoplasts Subprotoplasts containing APM micronuclei are isolated,an d fused to recipient Cytochalasin- B protoplasts. The centrifugation microprotoplast hybrids are Microcells Multinucleate cell selected and regenerated. Fusion Recipient cell Selection Microcell heterokaryon Microcell hybrid nuclear fragments as well (see discussion Microcell-mediated chromosome transfer. earlieri nthi schapter) .Th eus eo fcytoplasts , which are enucleated cells orprotoplasts ,a s organelle donor avoids this risk (Wallin et The number of transferred chromosomes al, 1979; Lörz et al., 1981; Maliga et al, can be limited by using microcells asdono r 1982).However , cytoplast preparations are in fusion experiments (Fig.2) . Microcells often contaminated with miniprotoplasts are prepared by prolonged exposure to (evacuolated protoplasts), introducing the metaphase inhibitors, such ascolchicin e or need for selection of the desired fusion Colchemid, orb ytreatin g mitotic cells with products afterwards. cytochalasin-B (Fournier, 1981). The metaphase chromosomes become scattered be mapped onto specific chromosomes (de throughout the cell, and decondensate Jonge et al., 1985;Sukoya ne t al., 1984).Th e individually or in small groups. A nuclear disadvantage of thisapproac h liesi nth e fact envelope is formed around the that linkage is not necessarily preserved decondensating chromosomes, resulting during chromosome fragmentation and into micronuclei, with varying numbers of incorporation. In the case, where two chromosomes per nucleus. By independent fragments are cointegrated, a centrifugation of micronucleated cells, misleading linkage will be the result, microcells can be isolated, with interphase whereas in case of intra-fragmental micronuclei containing a subset of the deletions,linkag ewil lno tb e detected atall . chromosomes.B yfusio nwit hrecipien tcells , Nevertheless, CMGT has advanced the karyologically simple hybrids can be mapping of the human genome constructed, in which the donor considerably,especiall yi nthos ecase swher e chomosomes are maintained mitotically important genes were localized in the stable (Lugo and Fournier, 1986). When a immediate vicinity of selectable genes suitable recipient is used, with a deficiency, (Klobutcher and Ruddle, 1981; Sukoyan et which can be complemented by a gene, al, 1984; de Jonge et al., 1985; Porteous, located on the desired donor chromosome, 1987). Even though this technique has genemappin gbecome spossible .I nthi sway , provedt ob eo fgrea tvalu efo rgen emappin g considerable progress has been made in the in animal cell lines, it has been almost localization of genes on human neglected in somatic plant genetics. Sofar, chromosomes by fusion with rodent few cases have been reported, in which recipient lines (Dhar et al., 1984; Saxon et isolated metaphase chromosomeshav ebee n al., 1985;Lug o and Fournier, 1986). used as donor material (Griesbach et al., 1982; Griesbach, 1987; de Laat and Blaas, Sofar, nosuc h systemwa savailabl efo r plant 1987; Dudits, 1988). In the first case, lily cells,du et oth elac ko f micronuclei-inducing chromosomes were introduced into agents,sinc ebot h colchicinean d Colchemid recipient protoplasts by PEG-induced did not induce a significant number of uptake, without any selectable marker, and micronuclei inplan t cells. divisions were never observed. Both Griesbach and De Laat employed Chromosome-mediated gene transfer microinjection as means to introduce the (CMGT). donor chromosomes intoth e recipient cells. Only Griesbach reported on the resulting putative hybrid lines obtained in this way, Whenisolate d metaphase chromosomes are and showed that although no cytological transferred to a recipient cell by chemically evidencefo r thesuccessful l transfer could be induced uptake, fragments of the obtained, the hybrids expressed genes of the chromosomesbecom e incorporated intoth e flavonoid biosynthesis pathway, not recipient genome (Klobutcher and Ruddle, previously present in the recipient cells of 1981). In this way, chromosome-specific Petunia hybrida. Dudits reported on the inserts can be obtained, which still show a transfer of isolated chromosomes from a more or less syntenic arrangement, i.e. metothrexate resistant carrot cell line into linked genes are found in a linked position protoplasts of Triticummonococcum, which in most of the inserts. This opens up the transferred the resistance, but no carrot opportunity to select for transformants, chromosomes could be retrieved in most expressing a complemented deficiency, in metothrexate resistant colonies, except for which also linked genes are present. Bythi s one colony, which contained one additional procedure, non-selectable properties could carrot chromosome. References Bennett, M.D. (1988). Genomic domains and hybrids between widely different species, in: Advanced technology and plant Austin, S., Baer, M., Ehlenfeldt, M., breeding strategy, 1988 Moet Hennesy Kazmierczak, P.J., Helgeson, J.P. (1985). Louis Vuitton conference, Communication Intra-specificfusion s inSolanum tuberosum. abstracts,pp .22-23 . Theor. Appl.Genet . 21:172-175. Chetrit, P.,Mathieu , C, Vedel,F , Pelletier, Aviv, D., Edelman, M., Galun, E. (1980). G, Primard, C.(1985) .Mitochondria l DNA Progenyanalysi so fth einterspecifi c hybrids: polymorphism induced byprotoplas t fusion Nicotiana tabacum (CMS) + Nicotiana in Cruciferae. Theor. Appl. Genet. 62: sylvestris with respect to nuclear and 361-366. chloroplast markers. Theor. Appl. Genet. 56:145-150. de Jonge, A.J.R, de Smit, S, Kroos, M.A, Reuser,A.J.J .(1985) .Cotransfe r ofsynteni c Aviv, D., Arzee-Gonen, P., Bleichman, S, human genes intomous ecell susin g isolated Galun, E. (1984). Novel alloplasmic metaphase chromosomes or cellular DNA. Nicotiana plants by "donor-recipient" Hum. Genet. 62:32-38 . protoplast fusion: cybridshavin g N.tabacum orN.sylvestris nuclea r genomesan d either or de Laat, A.M.M., Blaas, J. (1987). An bothplastome s and chondriomesfro m alien improved method for protoplast species.Mol. Gen . Genet. 126: 244-253. microinjection suitablefo r transfer of entire plant chromosomes. Plant Sei.5_Q : 161-169. Aviv, D., Galun, E. (1986). Restoration of male fertile Nicotiana by fusion of de Vries, S.E, Ferwerda, M.A, Loonen, protoplasts derived from two different A.E.H.M, Pijnacker, L.P, Feenstra, W.J. cytoplasmicmale-steril e cybrids.Plan t Mol. (1987). Chromosomes in somatic hybrids Biol.2 :411-417 . between Nicotiana plumbaginifolia and a monoploid potato. Theor. Appl. Genet._25_: Barsby, T.L., Chuong, P.V., Yarrow, S.A., 170-176. Sau-ChingWu ,Coumans ,M. ,Kemble , R.J., Powell,A.D. ,Beversdorf , W.D., Pauls,K.P . Dhar, V, Searle, B.M.,Athwal , R.S.(1984) . (1987).Th e combination of Polimacm s and Transfer of chinese hamster chromosome 1 cytoplasmic triazine resistance inBrassica to mouse cells and regional assignment of 7 napus.Theor .Appl .Genet . 23.:809-814 . genes: a combination of gene transfer and microcell fusion. Som.Cel lan d Mol. Genet. Bates, G.W., Hasenkampf, CA., Contolini, 1Q: 547-559. CL, Piastuch, W.C. (1987). Asymmetrie hybridization in Nicotiana by fusion of Dudits, D. (1988). Increase of genetic irradiated protoplasts. Theor. Appl. Genet. variability by asymmetric cell hybridization 21:718-726 . and isolated chromosome transfer, in: Progess in Plant Protoplast research, ed: Bennett, M.D. (1987). Ordered disposition Puite, K.J., Dons, J.J.M, Huizing, H.J, of parental genomes and individual Kool, A.J, Koornneef, M, Krens, F.A. chromosomes in reconstructed plant nuclei, Kluwer Academic publishers, Dordrecht, andthei r implications.Somat .Cel lan d Mol. Boston, London,pp .275-281 . Genet. 1$. 463-466. Fluhr, R. (1983). The segregation of organelles and cytoplasmic traits in higher 10 plant somatic fusion hybrids, p 85-92. in: Glimelius, K., Bonnett, H.T. (1986). Protoplasts 1983, lecture proceedings, 6th Nicotianacybrid swit h Petunia chloroplasts. int. Protoplast symp. Basel, Aug. 12-16, Theor.Appl .Genet . 22:794-798 . 1983, ed. I. Potrykus et al., Birkhäuser Verlag, Basel, Boston, Stuttgart. Graves, J.A.M. (1984). Chromosome segregation from cell hybrids. I. The effect Fluhr,R. , Aviv, D.,Edelman , M.,Galun , E. of parent cell ploidy on segregation from (1983). Cybrids containing mixed and mouse-Chinese hamster hybrids. Can. J. sorted-out chloroplasts following Genet. Cytol.26 :557-563 . interspecific somatic fusions in Nicotiana. Theor.Appl . Genet. 65_: 289-294. Griesbach, R.J., Malmberg, R.L., Carlson, P.S. (1982). Uptake of isolated lily Fournier, R.E.K. (1981). A general chromosomesb ytobacc oprotoplasts .Th eJ . high-efficiency procedure for production of ofHered . 22:151-152. microcell hybrids. Proc. Natl. Acad. Sei. USA.2S :6349-6353 . Griesbach, R.J. (1987). Chromosome- mediated transformation via microinjection. Fowke, L.C., Gresshoff, P.M., Marchant, Plant Sei.5_ß : 69-77. H.J. (1979). Transfer of organelles of the alga Chlamydomonas reinhardiiint o carrot Gupta, P.P.,Gupta , M.,Schieder , O.(1982) . cells by protoplast fusion. Planta 144: Correction of nitrate reductase defect in 341-347. auxotrophic plant cells through protoplast-mediated intergeneric gene Galun, E., Aviv, D. (1986). Organelle transfers. Mol.Gen . Genet. 18Ü: 378-383. transfer. Meth. in Enzym.US : 595-611. Heslop-Harrison, J.S., Bennett, M.D. Gilissen,L.J.W. ,Sre e Ramulu,K. , Dijkhuis, (1983).Th e positions of centromeres on the P.,Verhoeven , H.A.,Stiekema ,W.J .(1989) . somatic metaphase plate of grasses. J. Cell Application in somatic cell genetics of Sei.64:163-177 . potato marker lines produced through Agrobacterium transformation. XII. Ichikawa, H.,Tanno-Suenaga , L., Imamura, Eucarpia Congress, Göttingen, FRG, book J. (1987).Selectio n oiDauern cybrids based of poster abstracts,poste r N°30-10 . on metabolic complementation between X-irradiated D.capillifolius and Gleba, Y.Y., Momot, V.P., Okolot, A.N., iodoacetamide-treated D.carotab y somatic Cherep, N.N., Skarzhynskaya, M.V., Kotov, cellfusion . Theor.Appl .Genet .24:746-752 . V. (1983). Genetic processess in intergeneric cell hybrids Atropa + Imamura,J. ,Saul ,M.W. ,Potrykus ,I .(1987) . Nicotiana.Theor .Appl .Genet . 65.: 269-276. X-ray irradiation promoted asymmetric somatic hybridization and molecular Gleba, Y.Y., Hinnisdaels, S, Sidorov, V.A, analysiso fth eproducts .Theor .Appl .Genet . Kaleda, V.A., Parokonny, A.S., Boryshuk, 24:445-450 . N.V., Cherep, N.N.,Negrutiu , I.,Jacobs , M. (1988). Intergeneric asymmetric hybrids Kemble,R.J., Yarrow, S.A., Barsby, T.L. between Nicotiana plumbaginifolia and (1988). Cytoplasmic DNA stability and Atropa belladonna obtained by mitochondrial transformation in rapeseed "gamma-fusion". Theor. Appl. Genet. 26: somatic cybrids. in: The second 760-766. international congress of plant molecular 11 biology, Jerusalem, Nov. 13-18, 1988.Boo k species-specific repetitive DNA probes, in: of abstractsN °214 . The second international congress of plant molecular biology, Jerusalem, Nov. 13-18, Klobutcher, L.A., Ruddle, F.H. (1981). 1988.Boo k of abstracts,N ° 194. Chromosome mediated gene transfer. Ann. Rev.Biochem .5Q : 533-554. Pijnacker, L.P. ,Ferwerda ,M.A. ,Puite ,K.J. , Schaart, J.G. (1989). Chromosome Lörz, H., Paszkowski, J., Dierks-Ventling, elimination and mutation in tetraploid C, Potrykus, I. (1981). Isolation and somatic hybrids of Solanum tuberosum and characterization of cytoplasts and Solanum phureja.(submitted). miniprotoplasts derived from protoplasts of cultured cells.Physiol .Plant .52 :385-391 . Porteous, D.J. (1987). Chromosome mediated gene transfer: a functional assay Lugo, T.G., Fournier, R.E.K. (1986). for complex loci and an aid to human Microcell fusion and mammalian gene genome mapping.TIG .2:177-182 . transfer, in: Kucherlapati, R., ed. Gene transfer. Plenum Publishing Corp. , New Ruddle, F.H., Creagan, R.P. (1975). York, pp.79-93 . Parasexual approaches to the genetics of man.Ann . Rev. Genet. 9:407-486 . Maliga, P., Lorz, H., Lazar, G., Nagy, F. (1982). Cytoplast-protoplast fusion for Saxon, P.J., Srivatsan, E.S., Leipzig, V.G., interspecific chloroplast transfer in Sameshima, J.H., Stanbridge, E.J. (1985). Nicotiana. Mol.Gen . Genet. 185:211-215 . Selective transfer of individual human chromosomes to recipient cells. Mol. and Menczel,L , Galiba,G. , Nagy,F. ,Maliga ,P . Cell. Biol.5 : 140-146. (1982). Effect of radiation dosage on efficiency of chloroplast transfer by Schwarzacher-Robinson, T., Finch, R.A., protoplastfusio n inNicotiana. Genetic s Iflfl: Smith, J.B., Bennett, M.D. (1987). 487-495. Genotypic control of centromere positions of parental genomes in Hordeum XSecale Menczel, L. (1984). Inactivation of hybrid metaphases.J . Cell Sei.82 :291-304 . protoplasts before fusion to facilitate selective recovery of fusion-derived clones. Shirzadegan, M., Christey, M., Earle, E.D., Cell culture and somatic cell genetics of Palmer, J.D. (1989). Rearrangement, plants.1 :428-432 . amplification, and assortment of mitochondrial DNA molecules in cultured Menczel, L, Morgan, A.,Brown , S.,Maliga , cells of Brassica campestris. Theor. Appl. P. (1987). Fusion-mediated combination of Genet. 22:17-25 . Ogura-type cytoplasmic male sterility with Brassica napus plastids using X-irradiated Sidorov,V.A. ,Menczel ,L. ,Nagy ,F. , Maliga, CMSprotoplasts . Plant Cell Rep.6_ : 98-101. P. (1981). Chloroplast transfer inNicotiana based on metabolic complementation Morgan, A., Maliga, P. (1987). Rapid between irradiated and iodoacetate treated chloroplast segregation and recombination protoplasts. Planta 152:341-345 . of mitochondrial DNA in Brassicacybrids . Mol.Gen . Genet.2Q9_ : 240-246. Sukoyan, M.A., Matveeva, N.M., Belyaev, N.D.,Pack ,S.D. ,Gradov ,A.A. ,Shilov ,AG. , Piastuch,W.C. ,Bates ,G.W . (1988).Geneti c Zhdanova, N.S., Serov, O.L. (1984). analysis of asymmetric hybrids using Cotransfer and phenotypic stabilisation of 12 syntenic and asyntenic mink genes into mouse cellsb ychromosome-mediate d gene transfer. Mol.Gen . Genet. 126:97-104 . Szabados, L.G., Hadlaczky, G., Dudits, D. (1981). Uptake of isolated plant chromosomes by plant protoplasts. Planta 151:141-145 . Wallin, A., Glimelius, K., Eriksson, T. (1979).Formatio n ofhybri dcell sb y transfer of nuclei via fusion of miniprotoplasts from cell lines of nitrate reductase-deficient tobacco.Z . Pflanzenfysiol. 21: 89-94. Weiss, M.C., Green, H. (1967). Human-mouse hybrid cell lines containing partial complements of human chromosomesan dfunctionin g humangenes . Proc.Natl .Acad . Sei.USA .5£ :1104-1111 . 13 CHAPTER2 EFFICIENT INDUCTION BYAMIPROPHOS-METHY L AND FLOW CYTOMETRIC SORTING OF MICRONUCLEI IN NICOTIANA PLUMBAGINIFOLIA. A.M.M. deLaat ,HA .Verhoeven ,K .Sre eRamul u and P. Dijkhuis 15 EFFICIENT INDUCTION BYAMIPROPHOS-METHY L AND FLOW CYTOMETRIC SORTING OF MICRONUCLEI IN NICOTIANA PLUMBAGINIFOLIA. A.M.M. deLaat* ,H.A .Verhoeven ,K . SreeRamul uan dP .Dijkhui s ResearchInstitut eItal ,P.O .Bo x48,670 0A AWageningen ,Th eNetherland s Abstract genome transfer by transplantation of metaphase chromosomes (Malmberg and Griesbach, 1980; Szabados et al, 1981;D e Amiprophos-methyl (APM) is a potential Laat and Blaas, 1987) are largely hindered herbicide which acts at the level of by the tight association of chromosomes. microtubules. By exposure of suspension Micronuclei can be a promising alternative cells of Nicotiana plumbaginifolia to this for limited genome transfer by either agent,a hig h degree ofmetaphas e arrestwa s transplantation of isolated micronuclei observed and single as well as groups of containingon eo ra fe wchromosomes ,o rvi a chromosomes were scattered throughout fusion of microcells with recipient the cell, offering good prospects for protoplasts. The occurrence of application in cytology and chromosome micronucleate cells in mammalian cell isolation. After prolonged exposure to cultures after incubation with colchicine or APM, the chromosomes decondensed and Colcemidi sknow nfo rsevera lyear s(Phillip s micronuclei were formed. Based on their and Phillips, 1969; Ege and Ringertz 1974; DNA content, the micronuclei were sorted Fournier and Ruddle, 1977;Sekiguch i et al, byflo w cytometry. Prospects for application 1978). Several investigators have isolated a of isolated micronuclei for partial genome small subset ofth e totalgenom e byapplyin g transfer and gene mapping are discussed. enucleation procedures to micronucleate cellswhic h resulted inmicrocell s containing Key words: Amiprophos-methyl Flow varyingnumbe r of chromosomes (Ege et al, cytometry micronucleus (formation, 1977; McBride and Peterson, 1980; sorting) Microtubule poison Nicotiana Fournier, 1981). By subsequent microcell plumbaginifolia. fusions with recipient cells,th e transfer of a limited number of chromosomes was Introduction achieved. Amiprophos-methyl (APM) belongs to the class of phosphoric amide herbicides which are considered anti- Both for cytological research and for microtubule drugs in plant cells because of chromosomeisolatio ni nplants ,stickines so f the disappearance of microtubules and of chromosomes due to metaphase arrest by the disruption of microtubule-dependent spindle toxins is a major problem. In processes (Sumida and Ueda, 1976; particular, the isolation of individual Kiermayer and Fedtke, 1977;Robinso n and chromosomes for sorting by flow cytometry Herzog, 1977;Quade r etal , 1978;Koo pan d (De Laat and Blaas, 1984) and limited Kiermayer, 1980). Recently, it has been shown that APM is a specific drug which 'Present address: Koninklijk Kweekbedrijf en directly poisons microtubule dynamics in Zaadhandel D.J. van der Have B.V., P.O.Bo x 1,441 0 plant cells (Morejohn and Fosket 1984).W e AA Rilland, The Netherlands. have observed that APM treatment of cell 17 suspensions of Nicotiana plumbaginifolia treated cell suspensions were washed resulted in well scattered metaphase repeatedly (4 times) with culture medium chromosomes allowing the isolation of and the packed cellvolum ewa s determined single chromosomes (Verhoeven et al., at 1-7 daysafte r inoculation. 1986; 1987). In this paper we describe the massinductio n of micronucleib yprolonge d Protoplastisolation andculture. APM treatment, and a procedure for the isolation of micronuclei from multinucleate Suspension cells treated with APM were cells and sorting them by flow cytometry washed with culture medium and incubated based on DNA content. in 5% Driselase (Kyowa Hakko KogyoCo. , Tokyo, Japan) and 0.5% Pectolyase Y-23 Materials and methods (Seishin Pharmaceutical Co.,Tokyo , Japan) in 0.4 M mannitol for 2 h at 28°C on a gyratory shaker (30 rpm). Afterwards, Plantmaterial and culture conditions. protoplasts were isolated by centrifugation at6 0g fo r5 min .Purificatio n wascarrie d out Suspension cells of Nicotiana plumba as described by repeated washing in 0.4 M ginifolia("Doba "line ,kindl ysupplie d byDr . mannitolan dsubsequen t floatation on0. 4M R. Shields, Sittingbourne) were cultured in sucrose (De Laat and Blaas, 1987). thedar ka t28° Ci n"Doba "mediu m (Barfield Protoplasts were cultured at a density of et al., 1985)o n agyrator y shaker (120rpm) . 5x10 protoplasts/ml in "protoplast culture For sustained divisionactivity ,sub-culturin g medium 3" used by Kao et al. (1974) was carried out at 3 day-intervals. A fresh containing 0.4 Mglucos e and 0.8% agarose. solution of amiprophos-methyl (trade name After 6day so fcultur ea t28° Ci nth edar k the Tokunol M®; O-methyl-O-O- (4-methyl- plating efficiency (percentage of divided 6-nitrophenyl)-N-isopropyl-phosphoro protoplasts) was calculated. thioamidate) (Aya et al., 1975; Kiermayer and Fedtke, 1977) obtained from Bayer Isolation,flow cytometric analysis and Nederland B.V., Divisie Agrochemie, was sortingof nuclei and micronuclei. prepared in acetone (20 mg/ml) immediately before use. The chemical Nuclei and micronuclei for analysis and solutionwa sapplie d to cellsuspension s one sorting were isolated by the methods day after subculture. Mitotic index and the previously developed for the isolation of number of micronucleate cells were plant metaphase chromosomes (De Laat determined by staining the cell samples of and Blaas, 1984). For the isolation of suspension cultures directly with orcein multinucleate protoplasts, APM was added dissolved in lactic acid-propionic acid (1:1) to the enzyme and washing solutions as (Eriksson, 1966), or in Feulgen after fixing described in the preceding section. Purified in absolute alcohol: glacial acetic acid (3:1 protoplasts were incubated for 5mi no nice , v/v)fo r 24h (Sree Ramulu et al., 1985).Th e in chromosome isolation buffer (Malmberg mitotic index was expressed as the and Griesbach, 1980), ruptured by the percentage of nuclei undergoing mitosis in addition of 0.05% Triton-X-100 and the total number of nuclei scored in a repeated passage through a 25G epidermal sample. The number of cells analysed for needle. Cellular debris was removed by mitotic index and for scoring multinucleate passage through a 80 pm nylon filter. For cellsrange d from30 0t o60 0pe rsample .Th e flow cytometric analysis, the nuclei were relative growth rate of cell suspensions stained with the DNA-specific dye ethidium treated with APM was determined bromide (final concentration 10^g/ml).Th e according to Gilissen et al. (1983). The measurements of nuclear DNA content 18 Percentage of cells Treatment duration (h) Fig. 1.Mitoti c index (MI) and the percentage of micronucleate cells in cell suspensions ofN. plumbaginifolia treated with APM for various periods. -^—)j(—)|<—% :M I in control cell suspensions; g--[3- -Q-'E] : MI inAP M treated cell suspensions; -~| 1 ) (-•: Micronucleat e cells after APM treatment. Micronucleate cellswer e absent in the control. were made with a Fluorescence Activated Results Cell Sorter (FACS)-IV (Becton Dickinson, Sunnyvale,U.S.A. ) equipped with a Spectra Physics argon ion laser, model 164-05 Effect ofAPM onmitotic indexand operated at 0.3 W/488 nm with an LP-620 formation ofmicronuclei. filter in the emission beam. The DNA C-values corresponding to Gl and G2 Thefrequenc y ofmicronucleate dcell si ncel l nuclear phases of the cells were checked as suspensions ofN. plumbaginifolia depended reported previously (Sree Ramulu and on the concentration of APM, lower Dijkhuis, 1986).Th econtributio n of cellular concentrations (3-18 fiM) resulting in less and nuclear debris in the flow histograms than 10%micronucleat e cellsan dth e higher was removed by an interactive computer concentration (36 /iM) about 27%. We analysis (van der Linden, 1980).Th e nuclei analysedth etim e courseo f micronucleation were sorted on the basis of DNA content. in relation to the mitotic index after The sorted nuclei were concentrated on 0.2 treatment with the higher concentration of i*mnucleopor e poly-carbonate filters and 36fiM since the induction of large number stained with 4,6-diamidino-2-phenylindole of micronuclei is necessary for efficient (DAPI) or Hoechst 33342 (20 /*g/ml) for flow-cytometric analysis and sorting. The microphotography. results presented in Fig. 1sho w that in the control, the mitotic index, which was 3.0 at 19 B I 4 Äf t* «4 - *l •• • * > o • Fig.2 A-D .Microphotograph sillustratin gth eformatio n ofmicronucle i after APMtreatmen t ofcel l suspensions in N. plumbaginifolia. A: Metaphase cell showing scattered chromosomes. B: Metaphase chromosome groups.C: Décondensée!chromosomes . D: Micronucleated cell. Percentage of micronucleated cells IIB 2 3 4 5 6 7 8 9 10 11 12 13 >13 Number of micronuclei/cell Fig. 3. Frequency distribution of the number of micronuclei in micronucleate cells after treatment of N. plumbaginifolia cell suspensions with 18juM APM during 48 h. 20 (A) the start of the experiment, increased to 5.0 at 17h an dthereafte r decreased graduallyt o 1.2 at 50 h. In the presence of APM, the mitotic index increased significantly within two to four hours, reached a maximum of 13.2a t 17h and afterwards decreased to 2.0 at 50 h. Micronucleate cells were formed during APM treatment from 6 h onwards. Whereas the mitotic index dropped after approximately 20 h, the frequency of 4C micronucleate cells rised to 27%afte r 43h . 8C Examples of microphotographs are presented inFig .2 ,illustratin g the effects of APM on mitosis and the formation of ® micronuclei. Fig.2 A showsth e scattering of mitotic chromosomes throughout the cytoplasm. Single as well as groups of chromosomesdecondense d (Fig.2B ,C )an d subsequently, micronuclei were formed (Fig. 2D). The number of micronuclei per vA A-J><' \ cellvarie dfro m2 t oa sman ya s3 0dependin g on the chromosome number of a cell after -.D V.,, treatmentwit h 18^Mor36/ Effect ofAPM on cellgrowth and plating efficiencyof protoplasts. The effect of APM was analysed after treatments with various concentrations ranging from 1-18 ^M for 16-24 h. The results on the relative growthrat e of treated cellsuspension s showedtha t itdi d not differ significantly from that of the control (data not given). Also, the plating efficiency of 4C 8C protoplastswa sno taffecte d byAPM , except Fig.4 A-C .Flo w cytometric analysis of relative DNA at higher concentration where it decreased contents ofnucle i isolated from control (A) and APM slightly. treated cell suspensions (B) of N. plumbaginifolia. Subtraction of Fig. A from B gives C, which shows micronuclei and a highfrequenc y of8 Cnuclei .I nFig . Flowcytometric analysis andsorting of 4B, A-D refer to the sorting windows used for the micronuclei. separation ofentir e population ofsub-diploi d( < 2C) micronuclei (A), and the three different fractions of Nuclei were isolated from the purified sub-diploid micronuclei,i.e .th esmalles t micro-nuclei protoplasts prepared from cell suspensions (B), intermediate micronuclei (C) and the largest treatedwit hAP M(36^ 21 Fig.5 A-C .Photograph s ofsub-diploi dmicronucle ifraction s sorted byflowcytometry . A:Smalles t micronuclei. B:Intermediat e micronuclei. C:Larges t micronuclei. flowcytometry. The results on DNA such micronuclei with DNA contents histograms show that the untreated cell rangingfro m approximately 0.1Ct o2 Cwer e suspensions consisted of 4C and 8C nuclei sorted.Thes e differed insize ,a ssee ni n UV (Fig. 4A). Chromosome counts in light microscopy (see microphotographs in metaphases revealed the presence of Figs.5A-C) . predominantly tetraploid (2n = 4x = 40) cells,includin gsom ecell shypotetraploi d for Discussion 1-4 chromosomes. Thus, the nuclei with 4C and 8C correspond to Gl and G2 nuclear phases of the tetraploid cells, respectively. The results obtained show that APM is an Flow cytometric analysis of nuclei derived efficient chemical for high metaphase arrest from APM treated cells showed that the and for mass production of micronuclei in frequency of 8C nuclei had increased cell suspensions of N. plumbaginifolia. The considerably (Fig. 4B). This apparently metaphase cells showed grouping of resulted from the doubling of 4C cells. chromosomes, followed by decondensation Moreover,a sAP Mtreatmen t induced about leading to the formation of micronuclei. As 22% multinucleate cells, a large number of APM is a drug which specifically poisons nuclei with varying DNA contents were microtubule dynamics in plant cells by found (Fig. 4C). As the subdiploid (< 2C) inhibiting microtubule polymerization population of micronuclei is relevant for (Morejohn and Fosket, 1984), spindle partial genome transfer, three categories of disturbances can lead to irregular 22 chromosome movement, orientation and transferred genetic material. Both the grouping of chromosomes (Sree Ramulu et induction of micronuclei and the flow al.,1987) .Th efrequenc y of micronucleation sorting of sub-diploid micronuclei in plant seems to depend upon the type of cell systems, as demonstrated in N. suspensions treated with APM. The fast plumbaginifolia, open the way for the dividing cell suspensions of N. development of microcell hybrids which plumbaginifolia responded with a high constitutevaluabl e tools for partial genome frequency of multinucleate cells as transfer and gene mapping (Ege and comparedt oth erelativel yslow-growin gcel l Ringertz ,1974; Ege et al., 1977; Fournier, suspensionso fHaplopappus gracilis, Daucus 1982). carota and Solanum tuberosum (data not given). With regard to the other effects of Acknowledgements APM,th edat asnowe dtha tAP M treatments showed no appreciable reduction in cell growth or plating efficiency of protoplasts. Wewis h to thank Dr. L.va n Vloten-Doting Thus, because of low toxicity, APM for useful suggestions and Mr. W.R.R. ten treatments are compatible with genetic Broekefo r assistancei nflo wcytometry .Par t manipulation approaches. Moreover, the of this research was supported by funds of recent observations on themitoti c dynamics the"Biomolecula rEngineerin gProgramme " ofmicronucle i induced after treatment with of the Commission of the European APM (36^M , 48h )i n cellsuspension s ofTV . Communities, Contract No. GBI-4-109-NL. plumbaginifolia revealed that after removal We gratefully acknowledge the receipt of a ofAP Mb yrepeate dwashin gan d subculture, gift sample of APM from Bayer Nederland cellsrecovere d showingnorma l functioning B.V., Divisie Agrochemie. of spindle and continuity of mitotic cycles (Sree Ramulu et al., 1987). As demon References strated, flow cytometric sorting can be used to collect large numbers of micronuclei containing oneo r afe w chromosomes. Prior Aya, M.,Takise , I.,Kishino , S.,Kurihara , K. to using micronuclei for gene transfer, it is (1975). Amiprophos-methyl, a new necessary to remove the DNA intercalator herbicide in upland crops. Proc. 5th Asian ethidium bromide from the stained Pac. Weed Sei.Conf. ,pp . 138-141. micronuclei. To a large extent, this is achieved through dilution (20-fold) with the Barfield, D.G., Robinson, S.J., Shields, R. sheath fluid of the flow system added to the (1985). Plant regeneration from micronuclei suspension during flow sorting. protoplasts of long term haploid suspension Alternatively, a vital stain, such as Hoechst cultures of N. plumbaginifolia. Plant Cell 33258 can be used for flow sorting of Rep.4 : 104-107. micronuclei. Various methods, such as microinjection or fusion-like uptake De Laat. A.M.M., Blaas, J. (1984). Flow (Malmberg and Griesbach, 1980; Szabados cytometric characterization and sorting of et al., 1981; De Laat and Blaas, 1987; plantchromosomes .Theor .Appl .Genet .6JZ : Verhoeven and Blaas, 1987) and micro-cell 463-467. fusion (Fournier and Ruddle, 1977; Fourniere t al., 1979)ca nb e usedt o transfer De Laat, A.M.M., Blaas, J. (1987). An the desirable genes. In all these methods, improved method for protoplast availability of markers (phenotypic, microinjection suitable for transfer of entire genetic/cytological) is a prerequisite for plant chromosomes. Plant Sei.5_Q : 161-169. selection and identification of the 23 Ege, T., Ringertz, N.R. (1974). Preparation Kiermayer, O., Fedtke, C. (1977). Strong of microcells by enucleation of anti-microtubule action of micronucleate cells. Expt. Cell Res. S7_: amiprophos-methyl (APM) in Micrasterias. 378-382. Protoplasma 22: 163-166. Ege, T., Ringertz, N.R., Hamberg, H., Koop, H.U., Kiermayer, O. (1980). Sidebottom, E. (1977). Preparation of Protoplasmic streaming in the giant microcells, in: Methods in Cell Biology, 15 unicellular green alga Acetabularia (D.M. Prescott, ed.), Academic Press, New mediterranea II. Differential sensitivity of York,pp .339-358 . systems to substances acting on microfilaments and microtubuli. Eriksson, T. (1966).Partia l synchronization Protoplasma 1Q2: 295-306. of cell division in suspension cultures of Haplopappus gracilis. Physiol. Plant. 12: Malmberg, R.L., Griesbach, R.J. (1980). 900-910. The isolation of mitotic and meiotic chromosomes from plant protoplasts. Plant Fournier, R.E.K. (1981). A general high Sei.Lett .12 :141-147 . efficiency procedure for production of microcell hybrids. Proc. Natl. Acad. Sei. McBride, O.W., Peterson, J.L. (1980). USAIS:6349-6353 . Chromosome-mediated gene transfer in mammalian cells. Ann. Rev. Genet. 14_: Fournier, R.E.K. (1982). Microcell- 321-345. mediated chromosome transfer. In: Techniques in Somatic Cell Genetics (Ed. Morejohn, L.C., Fosket, D.E. (1984). J.W. Shay), Plenum Press, New York, Inhibition of plant microtubule London, pp.309-327 . polymerization in vitro by the phosphoric amide herbicide amiprophos-methyl. Fournier, R.E.K., Ruddle, F.H. (1977). Science 224:874-876 . Microcell-mediated transfer of murine chromosomesint omouse ,Chines e hamster, Phillips, S.G., Phillips, D.M. (1969). Siteso f and human somatic cells.Proc . Natl.Acad . nucleolus production in cultured Chinese Sei.USA ,24 : 319-323. hamster cells.J . Cell Biol.4 : 248-268. Fournier, R.E.K, Juricek, D.K., Ruddle, Quader, H.,Wagenbreth ,I. ,Robinson , D.G. F.H.(1979) .Somati ccel lgeneti c analysiso f (1978).Structure ,synthesi san d orientation transgenome integration. Somat. Cell of microfibrils. V. On the recovery of Genet. 5_: 1061-1077. Oocystis solitaria from microtubule inhibitor treatment. Cytobiologie IS: 39-51. Gilissen, L.J.W., Hänisch ten Cate, C.H., Keen, B. (1983). A rapid method of Robinson, D.G., Herzog, W. (1977). determining growth characteristics of plant Structure, synthesis and orientation of cellpopulation si nbatc hsuspensio nculture . microfibrils. III. A survey of the action of Plant Cell Rep.2 :232-235 . microtubule inhibitors on microtubules and microfibril orientation in Oocystis solitaria. Kao, K.N., Constabel, F., Michayluk, M.R., Cytobiologie 15.: 463-474. Gamborg, O.L. (1974). Plant protoplast fusion and growth of intergeneric hybrid Sekiguchi, T., Shelton, K., Ringertz, N.R. cells.Plant a 12Q: 215-227. (1978) .DN Aconten t ofmicrocell s prepared 24 from rat, kangaroo and mouse cells. Expt. isolation and flow cytometric sorting of Cell Res.112 :247-258 . metaphase chromosomes and micronuclei from cell suspension cultures of Nicotiana Sree Ramulu, K., Dijkhuis, P., Hänisch ten plumbaginifolia. In: Genetic and cellular Cate, Ch.H, de Groot, B. (1985). Patterns engineering of plants and micro-organisms of DNA and chromosome variation during important for agriculture, Commission of in vitro growth in various genotypes of the European Communities meeting, potato. Plant Sei.4J. : 69-78. Louvain-la-Neuve, March 23-26, pp. 119-120. Sree Ramulu, K, Dijkhuis, P. (1986). Flow cytometric analysis of polysomaty and in Verhoeven, H.A., Blaas, J. (1987). Direct vitro geneticinstabilit y inpotato .Plan t Cell cell to cell transfer of cytoplasm and Rep.2:234-237 . organelles by microinjection. Protoplasma (submitted). Sree Ramulu, K., Verhoeven, H.A., Dijkhuis, P. (1987). Mitotic dynamics of induced micronuclei and prospects for chromosome-mediated gene transfer in plants.Theor .Appl .Genet .25. : 575-584. Sumida, S., Ueda, M. (1976). Effect of 0-ethyl-0-(3-methyl-6-nitrophenyl)-N-sec -butyl phosphorothioamidate (S-2846), an experimental herbicide, on mitosis in Allium cepa. Plant and Cell Physiol. 17: 1351-1354. Szabados, L., Hadlaczky, G., Dudits, D. (1981). Uptake of isolated plant chromosomes by plant protoplasts. Planta 151:141-145. Van der Linden, P.M. (1980). Computer analysis of DNA-histograms, Ph.D. Thesis, State Univ. Utrecht. Verhoeven,H.A. ,d eLaat ,A.M.M. ,Blaas ,J . (1986). Methodological developments for chromosome-mediated gene transfer in higher plants. In: Bio-molecular Engineering in the European Community, achievements of the research programme (1982-1986), final rep. (Ed. E. Magnien), Martinus Nijhoff Publishers, Dordrecht, pp. 977-981. Verhoeven, H.A., de Laat, A.M.M., Sree Ramulu, K., Dijkhuis, P., van Vloten-Doting, L. (1987). Induction, 25 CHAPTER3 MITOTIC DYNAMICS OF MICRONUCLEI INDUCED BY AMIPROPHOS-METHYL AND PROSPECTS FOR CHROMOSOME-MEDIATED GENE TRANSFER IN PLANTS. K.Sre eRamulu ,H.A .Verhoeven ,P .Dijkhuis . 27 MITOTIC DYNAMICS OF MICRONUCLEI INDUCED BY AMIPROPHOS-METHYL AND PROSPECTS FOR CHROMOSOME-MEDIATED GENE TRANSFER IN PLANTS. K. SreeRamulu ,H.A .Verhoeve nan dP .Dijkhuis . ResearchInstitut eItal ,P.O .Bo x48,670 0A AWageningen ,Th eNetherlands . Summary specificchromosome san dgen emappin gar e indicated. Mitotic dynamics and the kinetics of mass Key words: Amiprophos-methyl; mass induction of micronuclei after treatment of induction; micronuclei; intact specific Nicotiana plumbaginifolia cell suspensions chromosomes; gene transfer. with the spindle toxin amiprophos-methyl (APM) are reported. The addition of APM Introduction to suspension cells resulted in the accumulation of a large number of metaphases. The course of mitosis was Cellular manipulation of specific strikingly different from normal. Metaphase chromosomes and chromosomal segments chromosomes showed neither centromere inmammal sha sprove n tob evaluabl e in the divisionno rseparatio no fchromatids .Singl e study of the regulation of gene expression, chromosomes and groups of two or more fine structural mapping of chromosomes, chromosomes were scattered over the molecularcharacterizatio n and construction cytoplasm. After five to six hours of APM of genomic libraries (Mc Bride and treatment, chromosomes decondensed and Peterson, 1980; Davies et al., 1981; Lebo, formed micronuclei. With increasing 1982; Fournier, 1982). Many interspecific treatment duration, the frequency of cells somaticcel lhybrid seliminat e chromosomes with micronuclei as well as those showing from one of the parents during culture.Thi s lobed micronuclei increased. Similarly,wit h preferential chromosome loss has allowed an increase in APM concentration the the assignment of genes to particular frequency of cells with micronuclei chromosomes (Kao, 1983). However, long increased. After removal of APM, periods of culture and recloning are often chromosome grouping disappeared, cells necessarybefore anaccurat e assignmentca n showing lobed micronuclei further be made. A more direct approach to the increased and mitoses with doubled production of cell lines possessing a limited chromosome numbers appeared in the next amount of genetic material of another cell division. species would be to introduce only a single Cytological observations and DNA donor chromosome into the recipient cells. measurements revealed that several Microcell hybridization is the only sub-diploid micronuclei containing one or a technique by which the transfer of single, few chromosomes can be obtained and that intact chromosomes has been achieved in flow cytometry candetec t and sort out these mammals (Fournier, 1982). Microcell micronuclei. The applications of hybridization is conceptionally and micronuclei for genetic manipulation of operationally distinct from traditional somatichybridization , intha t onlya fraction 29 of the donor genome is introduced into the spindle can cause segregation of recipient partner at the time of fusion. Ever chromosomesint oirregula rgroup sresultin g since the discovery of the phenomenon that in micronuclei of varying size in cell colchicine or Colcemid can induce a high suspensions of Medicago sativa (Lo Schiavo frequency ofmicronucle ii nmammalia n cell et al., 1980) and 4-epoxyethyl-l,2-epoxy- cultures, several investigators have isolated cyclohexane (VCH-diepoxide) produces micronuclei by applying enucleation micronuclei as a consequence of procedures to micronucleate cells. In this chromosome breakage in root-tip way, microcells are formed, containing meristems of Viciaf aba and Allium cepa micronuclei, and surrounded by a thin layer (NutiRonch ie tal. , 1986a ;b) .Moreover ,th e of cytoplasm and a plasma membrane mass isolation of individual metaphase (Levan, 1954; Phillips and Phillips, 1969; chromosomes for sorting by flow cytometry Ege and Ringertz, 1974; Fournier and or transfer into recipient protoplasts, is Ruddle, 1977;Eg e et al., 1977;Sekiguch i et greatly hindered by stickyness of al., 1978). By subsequent fusion of chromosomes that often occurs after microcells with recipient partners, treatment with spindle toxins (Malmberg karyotypically simple hybrid clones were and Griesbach, 1980; Szabados et al.,1981 ; constructed which contained only one or a De Laat and Blaas, 1984). Recently, we few introduced donor chromosomes observed that an organophosphorous (Fournier, 1982). Since the transferred herbicide amiprophos-methyl (APM) can genetic material can often be identified causescatterin go fmetaphas e chromosomes cytogenetically, microcells have been and induce micronuclei at high frequency in proven to be useful not only for gene plant cells (Verhoeven et al., 1986;1987 ;D e mapping, but also for studying the Laat et al., 1987). Amiprophos-methyl chromosomal sites of integration of foreign belongs to the class of phosphoric amide DNA (Fournier et al., 1979; Smiley et al., herbicides which are considered 1978).Microcel lfusio n canb euse dt ocreat e antimicrotubule drugs, because of the panels of monochromosomal hybrids in disappearance of microtubules after their which single, specific chromosomes are application, and of the disruption of maintained by direct selective pressure microtubule-dependent processes (Fournier and Ruddle, 1977; Fournier and (Morejohn and Fosket, 1984). It has been Frelinger, 1982). Microcell hybrid clones shown that APM directly disturbs made up of such a monochromosomal microtubule dynamics inplan t cells. hybrid panel have been shown to display This article presents data on the dynamic simple,stabl ean dhomogeneou s karyotypes. changes of chromosomes (condensation, The genes complementing recessive, decondensation) and the kinetics of mass conditional-lethal mutations or genes that induction of micronuclei after APM confer dominantdru gresistanc e phenotypes treatment of cell suspensions of Nicotiana have been mapped. plumbaginifolia. In plants such a microcell system has not Materials and methods been available so far, because the mitotic arresting-an d chromosome breaking agents induced micronuclei only rarely, the Genotype,cell culture andAPM treatment frequency of which is insufficient for fusion purposes (Levan, 1938; 1940; 1954; Nicotiana plumbaginifolia cell suspensions Hesemann and Fayed, 1982; Marshall and resistant to kanamycine were derived from Bianchi, 1983;D e Marcoe t al., 1986).Som e the "Doba" cell line, kindly supplied by Dr. chemicals,suc h asgriseofulvi n which affects R. Shields, Unilever Res. lab., Colworth 30 Home, Sharnbrook, Bedford, UK. These Ramulu and Dijkhuis, 1986). Suspension cellsuspension swer e cultured inth e dark at cellswer e submerged in thebuffe r for some 28°C in "Doba" medium (Barfield et al., time,drie d withblottin gpape r and chopped 1985) on a gyratory shaker (120 rpm). For with a sharp razor blade in a petridish. sustained division activity, subculturing was Afterwards, 1.0-1.5m lbuffe r solution(p H7) carried out at 3day-intervals . was added. All these operations have been carried out on ice. The cell materials were Amiprophos-methyl was obtained from pressed byglas sro d through an8 8fiia nylo n BayerNederlan d B.V.,Divisi e Agrochemie, filter and then through a 15 /tm nylon filter, Arnhem, The Netherlands (trade name centrifuged during 1mi n at 1000 rpm, and Tokunol M®; 0-methyl-0-0-(4-methyl-6- the pellet wasresuspende d in 0.5 ml buffer, nitrophenyl)-N-isopropyl-phosphoro stained with 5 n\ ethidium bromide (5 thioamidate) (Aya et al., 1975; Kiermayer mg/ml)an d added to 25/A Triton X-100(2 0 and Fedtke, 1977). Astoc k solution of APM %). The measurements of nuclear DNA in DMSO (20mg/ml ) wasprepare d and the content were made with a Fluorescence treatments at concentrations (in medium) Activated Cell Sorter (FACS)-IV (Becton rangingfro m 3.6/uM to 36nM were given to Dickinson, Sunnyvale, U.S.A.) equipped actively growing logphas e cell suspensions, witha Spectr aPhysic sargo nio nlaser ,mode l one dayafte r subculturing. 164-05 operated at 0.3 W/488 nm with a LP-620 filter in the emission beam. The Cytology DNAC-value scorrespondin g toG l and G2 nuclear phases of the cells were checked Thesample so fcontro lan dAP Mtreate d cell using control plants as reported previously suspensionswer efixe d inethanol/aceticaci d (Sree Ramulu and Dijkhuis, 1986). The (3:1 v/v) for 24h and examined for mitotic contributiono fcellula ran dnuclea rdebri si n division, chromosome behaviour and the flow histograms was removed by an micronucleus formation in Feulgen-stained interactive computer analysis (van der preparations (Sree Ramulu et al. 1985).Th e Linden, 1980). The micronuclei were sorted mitotic index was expressed as the on the basis of fluorescence intensity percentage of nuclei undergoing mitosis resulting from ethidium bromide staining. among the total number of nuclei scored in a sample.Th e number of cells analysed per DNAcytophotometry samplerange d from 600-1000.Mitoti c arrest or metaphase blocking observed after APM Feulgen microdensitometry was used to treatment were designated as C-mitosis or determine the relative DNA content of the C-metaphase inaccordanc ewit hth egenera l sorted fractions ofmicronuclei .Th e samples terminology proposed by Levan (1954). were fixed in absolute alcohol/glacial acetic Various terms, i.e. "scattered or exploded acid (3:1v/v ) for 24h.Th e staining and slide metaphases, star metaphases and preparations were carried out as reported reductional grouping of chromosomes", are earlier (Sree Ramulu et al., 1984). according to the definition byLeva n (1954). Hydrolysis of nuclei, spread and dried on a millipore filter (membrane filter SCWP Flow cytometry 02500),wa s carried out in 5 N HCl at room temperature for 55min .Nucle iwer e stained For flow cytometric analysis of nuclei and with Schiffs reagent. After 3 washes in SO2 micronuclei, cellsample swer e fixed for 24h water, 5 min each, the slides were in buffer solution described by Blumenthal dehydrated and mounted inCanad a balsam. et al. (1979) with some modification as Absorption values for the Feulgen-stained adopted previously for potato cells (Sree nuclei were measured with a Leitz MPV 31 * %> **• # m %• lîHrV ÄV& 1 #>>• ff* • #I «I* # f Fig. 1A-L. Microphotographs on the sequence of mitotic eventsleadin g to the formation of micronuclei after treatment of cell suspensions ofN. plumbaginifolia with APM, and recovery of cells after removal of APM. A. Scattering of metaphase chromosomes ("exploded metaphases"). B-C. Groups of two, three or more chromosomes (note single chromosomes). D. Decondensation of single and grouped chromosomes. E, F. Micronuclei in different phases. 32 * «• % Vf «•* .#**' ' -»" TV ^1 i Fig. 1 (contd.) G-I. Lobed micronuclei. J-L. Post C-mitotic cells after removal of APM by washing and subsequent culture (note multipolar metaphases). 33 Percentage of cells Fig. 2. A: Mitotic index (MI); B: the percentage of cells showing chromosome-groups and C: micronuclei after treatment with various concentrations of APM for different periods. The control suspension cells of N. plumbaginifolia showed no chromosome-groups or micronuclei(M Irange dfro m 1.5-5.0%) . 3.6/iU APM ••+•• 9.0^ M APM -B- 18^ M APM Fig. 2A. 10 15 20 Treatment duration (h) Percentage of cells -^- 3.6fiM APM •+•• 9.0^ M APM B 18^MAPM Fig. 2B. Treatment duration (h) Percentage of cells 3.6,uMAPM 9.0fiM APM 18 ^M APM Fig. 2C. Treatment duration (h) 34 Percentage ofcell s -^- Mitoticinde x -\- C-Metaphases -<^~ Groups Q- Micronuclei V^- Fused nuclei 20 30 40 50 Treatment duration (h) Fig. 3. Time course of inducton of micronuclei in relation to other mitotic events after treatment of cell suspensions ofN. plumbaginifolia with APM (36,«M) . In this,a s inFig .7 , C-Metaphases: Blocked metaphase cells with well spread or partially clumped chromosomes arranged in "a single group"; Chromosome-groups: Metaphase cells showing separate groups of chromosomes; Micronuclei: Cellsshowin g separate micronuclei; Fused nuclei:Cell swit h lobed micronuclei. compact microscope photometer at the theirregula rarrangemen t onth espindl ean d wave length of 548 nm and scanning stage in some cells they gathered into a single using a Hewlett Packard-85 computer. group in the centre of the cell. Ball These values were transformed to C-values metaphases with all chromosomes in the by taking as standard the DNA content of centreo fth ecel lwer eals oobserved .Severa l nuclei isolated from leaf cells of control cellsshowe d scattering of the chromosomes plants (Sree Ramulu et al., 1984). all over the cytoplasm (Fig. 1A) . In these scattered C-metaphases ("exploded Results metaphases") the chromosomes were arranged in characteristic groups of 2, 3 or more chromosomes ("reductional Mitoticarrest, chromosomegrouping and grouping"), including separated single micronucleation chromosomes (Fig. 1 B, C). In some cells several groups of almost radially arranged The cell suspensions of N. plumbaginifolia chromosomes were found, within each were treated with APM at concentrations group all chromosomes turned their rangingfro m 3.6t o3 6fiM i nth emediu m for centromere inwards towards one point; ina various periods. Characteristic few cases all chromosomes of one cell are modifications of mitosis were encountered arranged as star. In other cells from one or two hours after initiation of chromosome-groups were arranged in a APM treatment. The chromosomes lost wide ring or in a line. Mitosis after APM 35 Percentage of cells •2 :3-5 :6-9 : >9 Fig. 4. Relative percentage of cells showing various number of chromosome-groups (i.e. the number of chromosome-groups/cell) after treatment of cell suspensions of N. plumbaginifolia with APM (36fiM) for various periods.A s the data for the periods 3-9 h, 18-26 h and 33-48 h were similar, theywer e pooled. Percentageo fcell s ^ ;6-9 ^ ; >9 Fig.5 . Relative percentage of multinucleate cells showing various number of micronuclei (i.e. the number of micronuclei/multinucleate cell) after treatment of cell suspensions of N.plumbaginifolia with APM (36 ^M) for various periods. 36 Table 1. Percentage of groups that contained various number of chromosomes after treatment of cell suspensions ofM plumbaginifolia with APM for various periods Duration of Number of Relative percentage of groups showing various numbers of APM (36/ 3-9 h 139 (38) 50.6 2. 3.7 43.7 18 h 50 (10) 60.0 8.0 4.0 28.0 26 h 113 (16) 69.1 4.4 5.3 21.2 33-48 h 27(5) 37.2 7.4 7.2 48.2 (*): The number of cells that showed chromosome-groups are given in parantheses. treatment was devoid of anaphase, chromosome groups increased, whereas metaphase chromosomes entered directly those with 2 or 3-5 chromosome groups intorestitutio ntelophas ewithou t divisiono f decreased (Fig. 4).Wit h further increase in the centromeres and without separation of the incubation period (33-48 h), the chromatids. In the scattered C-mitoses the frequency of cells containing 2 metaphase to telophase transition could be chromosome-groups increased and that of followed with all its characteristic changes. cellswit h >9 group s decreased. In some cases, single metaphase Each of these groups contained varying chromosomes decondensed developing numbers of chromosomes. The data nuclear membrane and formed presented in Table 1sho w that the relative telophase-like micronuclei. In other cases, percentage of groups containing 1, 4 and twoo rmor echromosome sgroupe d together above4 chromosome swa shighe r than those in forming micronuclei (Fig. 1 D). Two with 2 or 3 chromosomes after treatment examples of cells containing several with APM for various periods. micronuclei in different phases are Micronuclei were formed from about 5-6 h presented inFigs .1 E ,F .Wit h anincreas ei n onwards, and the frequency of cells with theconcentratio n ofAP Mfrom3. 6 to 18^M, micronuclei increased gradually, with a therewa sa nincreas e inmitoti cinde xan d in corresponding decrease in mitotic index. the frequency of cells showing chromosome The maximum frequency of cells with groups and micronuclei (Fig. 2). As the micronuclei was about 28 % at 48 h after duration of treatment increased, mitotic APM treatment (36 fiM) (Fig. 3). With index dropped, but the percentage of cells prolongedincubatio no fcell si nth epresenc e with chromosome groups and micronuclei ofAP Munde r certaincondition s7 0% o fth e increased. cells showed micronucleation (data not Fig. 3 presents more detailed data on the given). time course of the induction of micronuclei The number of micronuclei per cell in relation to other mitotic events in cell generally varied from 2 to 13. In a few cells suspensions treated with36/< MAPM .U p to >2 0 micronuclei were also observed. The 9 h, mitotic index, C-metaphases and cells proportion of cells containing 2 or >9 showing chromosome-groups increased micronuclei decreased with an increase in with incubation time. Afterwards all these the duration of APM treatment (Fig.5) .O n decreased, except the frequency of the other hand, the percentage of cells C-metaphases, which did not decrease showing 3-5 micronuclei increased and before 18h. With increasing duration of those with 6-9 remained unchanged. APM treatment from 3 to 26 h, the relative Lobed nuclei often resulting from fusion of percentage of cells showing 6-9 two or more micronuclei, were observed 37 Percentage ofcell s idiploid tetraploid octoploid polyploid During treatment After washing Fig. 6. Relative percentage of diploid (2x= 20), tetraploid (4x= 40) , octoploid (8x= 80 ) and higher ploid (16x= 160)cell safte r treatment ofcel lsuspension so fN.plumbaginifolia withAP M (36ftM) for variousperiods , after washingou tAP M and subculturing. Chromosome numbersgenerall yvarie d by2 fo r diploid (i.e.inclusiv e of hypodiploid) and 4fo r tetraploid (inclusive of hypotetraploid) cells and 5 for octoploid or poly ploid cells. Percentage of cells Fig. 7. Mitotic index and frequency of cells showing C-metaphases, chromosome groups, micronuclei and fused (lobed) nuclei after washing out APM (36fiM) and subculturing of cells. X ^*- Mitotic index -j- C-metaphases 4^r Chromosome groups r^j Micronucleus index '->'- Fused nuclei Time after washing (h) from 9 h onwards (Figs. 1 G-I). With revealed that the cells underwent one or increasing duration of APM treatment, the more divisions with a consequent doubling proportion ofcell sshowin globe dnucle i also or quadrupling of the chromosomes increased (Fig.3) . complement. The proportion of octoploid cellsgraduall y increasedwit h corresponding Recoveryafter C-mitosis decrease of diploid and tetraploid cells. When APM was removed by washing the The control cell suspensions contained frequency ofoctoploi d andhighe rploi d cells predominantly tetraploid cells and some further increased. Thus, about 65 % of the diploid and octoploid cells (Fig. 6). The cells underwent doubling or quadrupling of analysis of chromosome numbers after the chromosome number (Fig. 6). It was various periods of APM treatment (36 ^M) further observed that after washing out 38 Percentage of nuclei Fraction : \.r. 2C 4C 8C 16C APM22h 1 0.2C 0.4C 0.6C 0.8C DNA content of nuclei (C-value) 1 Percentage of nuclei Fraction b lij I. ' Ui ,"'"\ 2C 4C 8C 16C y n V'-fJi.. 2C4C 8C Î6C Fig. 8. Flow cytometric analysis of relative DNA oc 0.2C 0.4C 0.6C 0.8C tc contents of nuclei isolated from control and APM (36 DNA content of nuclei (C-value) fiM: 22 h, 48 h) treated cell suspensions of N. Fig. 9. Feulgen microdensitometric measurement of plumbaginifolia. DNA contents of fraction-a and -b of subdiploid (<2C) micronuclei sorted by flow cytometry. The C-values were calibrated by the 2C level of control nuclei, isolated from leaves of diploid (2n= 2 x= 20) APM and subculturing for 50 h, plants of JV. plumbaginifolia chromosome grouping almost completely disappeared and C-metaphases and cells with micronuclei decreased gradually (Fig. DNA contentof micronuclei 7). By contrast, the frequency of cells showing lobed nuclei greatly increased. Flowcytometr ywa suse dno tonl yt omonito r Mitotic index was similar to that of the mass induction of micronuclei after APM control.Severa lcell sshowe d normal bipolar treatment, but also for sorting and mitoses,an d some multipolar mitoses.Figs . characterization of the micronuclei 1J- L show some examples of multinucleate containing one or a few chromosomes. Fig. cellsprogresse d into post C-mitotic stages. 8 shows the flow DNA histograms of the control and APM (36 ?iM) treated cell 39 suspensions. The control cell suspensions discovered as early as 1975 (cf. Aya et al., consisted of nuclei with 4C and 8C DNA 1975). Since then, this phosphoric amide contents. Chromosome counts in herbicide hasbee n investigated insevera l in metaphases of control squash preparations vitro and in vivo experiments dealing with revealed the presence of predominantly physiological or biochemical aspects and hypotetraploid to tetraploid cells microtubule polymerization (see Morejohn (2n= 4 x= 36-40) .Therefore , thenucle iwit h and Fosket, 1984), but so far not on the 4C and 8C DNA values correspond to Gl induction of micronuclei. andG 2nuclea rphase so fth e hypotetraploid As compared to colchicine blocked to tetraploid cells. metaphase cells, which mostly showed After APM treatment, sub-diploid (< 2C) chromosome clumping, APM treatment micronuclei were produced, which resulted in well scattered chromosomes increased in frequency with increasing allowing the isolation of single duration ofAP Mtreatment .I naddition , the chromosomes (Verhoeven et al., 1987).Th e proportion of nuclei showing 8C DNA isolation of a large number of individual content alsoincrease d andnucle i containing metaphase chromosomes is of considerable 16C DNA content appeared, due to importance for genetic manipulation of chromosome doubling of cells (Fig.8 ;AP M specific chromosomes by means of flow 48h). Fig. 9 gives data on Feulgen cytometric sorting,karyotypin g and genome microdensitometric measurements of DNA cloning (Mc Bride and Peterson, 1980; contents of the twofraction s (fraction-a and Davies et al., 1981;Lebo , 1982). -b)o fsub-diploi d micronucleisorte db yflo w Strikingly, the course of mitosis after APM cytometry. Eighty micronuclei from each of treatment was different from that of normal the fraction-a and -b were individually mitosiso rC-mitosi s inducedb ycolchicin ei n measured for DNA content and compared most plants. In normal mitosis the daughter with control nuclei (n= 80 ) isolated from chromosomesseparat e duringanaphas e and leaves of diploid (2n = 2x = 20) N. become surrounded bynuclea r membranes, plumbaginifolia plants. The results suggest directly as a group or at first as single that smaller micronuclei (fraction-b) chromosomes. This is followed by contain one or two chromosomes as can be decondensation of the chromosomes to seen from the position of the peak relative form the interphase nuclei. Colchicine to the 2Cvalue ,i.e .G l nuclei of the diploid inactivates the spindle leading to disturbed control plants. The fraction-a showed a chromatid distribution inanaphase , delayed broader distribution of micronuclei with a centromere division and the formation of peak around 2-4 chromosomes. restitution nuclei (Levan, 1954). Discussion By contrast, after APM treatment two additional features were observed.First , the chromosomes showed no centromere The results obtained in the present study division or chromatid separation. Second, show that APM is a highly efficient mitotis single chromosomes as well as grouped arresting agent which leads to the chromosomes remained scattered and accumulation of a large number of decondensed, developed nuclear metaphases and to the formation of high membranes and formed micronuclei. These frequencies of micronuclei. To our occurred irrespective of the APM knowledge, mass induction of micronuclei concentration, or the plant species (Sree by spindle toxins or chromosome-breaking Ramulu et al. unpublished results). The agents has not yet been reported in plant change of metaphase chromosomes into cells. Amiprophos-methyl has been micronuclei after APM treatment was 40 accompanied by structural and functional showed lobed micronuclei. The lobed changes in chromosomes resembling the condition might be due to incomplete progression of a telophase nucleus in the separation of chromosome-groups i.e. the normal cell cycle. This process has been groups which were not aggregated together previously reported in mammalian cells at the time of nuclear membrane formation treatedwit h colchicineo r Colcemid (Levan, or when passing into the resting stage. 1954;Obar a etal. , 1974),bu ti srar e inplant s Alternatively, it is also possible that two or (cf. Levan, 1954). more micronuclei fuse together forming In cell suspensions of Medicago sativa lobed nuclei. Light microscopic griseofulvin, which affects the spindle, observations also suggest that lobed nuclei induces metaphase arrest and often result from nuclear fusion. Previously, polyploidization. After separation of the some authors working on the induction of chromatids (during the recovery period) micronuclei by Colcemid in mammalian chromosomes segregate into irregular cells have reported fusion of micronuclei, groups resulting in micronuclei of varying vital functioning of micronuclei and size (Lo Schiavo et al., 1980). The continued proliferation of micronucleate spontaneous occurrence of spindle cells (Brues and Jackson, 1937; Klein et abnormalities in a cell suspension line of al.,1952 ; Levan, 1954; Stubblefield, 1964; Daucus carota also seems to result in Phillips and Phillips, 1969;Eg e et al.,1977) . metaphase arrest, chromosome scattering As the cells did not undergo centromere and multinucleate condition (Nuti Ronchi, division, chromatid separation or anaphase Personal Communication). movement in the present study, the chromosome number doubled in the next In Colcemid treated Chinese hamster cells, cell division and they continued growth lowering of Ca + promoted "telophasing", normally. i.e. the formation of the nuclear membrane around the chromosomes and increased the Cytological observations and DNA frequency of micronuclei (Matsui et al., measurements showed that several 1982). Several studies emphasized the sub-diploid micronuclei containing one or a importance of cellular Ca + in cell cycle few chromosomes can be obtained after progression, spindle functioning and in the APM treatment. These micronuclei can be reformation of nuclear membrane. From detected and sorted in extremely large previous investigations on the effects of numbers by flow cytometry. This opens the APM, it is known that the chemical can way to fusion and generation of microcell deregulate Ca + level and can inhibitC a hybrids suitable for genetic manipulation of uptake by mitochondria, the function of specific chromosomes and gene mapping in whichi simportan t for maintaining a normal plants. spatial relationship of the nuclear membrane to the chromatin (review in Acknowledgements Matsui et al., 1982). Therefore, it is likely that these effects of APM on cellular Ca play arol e in the formation of micronuclei. Wewis ht othan kProf .J . Sybenga and Dr.L . Recovery of cells from the van Vloten-Doting for useful suggestions "APM-metaphase block" can be seen as the andW.R.R .te nBroek efo rassistanc ei nflo w concentration decreases below the cytometry. Our thanks are also due to Prof. C-mitotic threshold with prolonged M.Terz i for valuable discussions on certain incubation. After washing out APM and phases of this work and to Prof. V. Nuti subculturing, recovery was more Ronchi for making available her papers and pronounced. Many multinucleate cells unpublished results. Part of this work was 41 supported by funds of the "Biotechnology De Laat, A.M.M., Verhoeven, H.A., Sree Action Programme" of the CEC contract Ramulu, K., Dijkhuis, P. (1987). Efficient BAP-0083-NL (GDF). We gratefully induction by amiprophos-methyl and flow acknowledge the receipt of a gift sample of cytometric sorting of micronuclei in APM from Bayer Nederland B.V., Divisie Nicotiana plumbaginifolia. Planta 122: Agrochemie. 473-478. References Ege, T., Ringertz, N.R. (1974). Preparation of microcells by enucleation of micronucleate cells. Expt. Cell Res. S2: Aya,M. ,Takise ,I. ,Kishino , S.,Kurihara , K. 378-382. (1975). Amiprophos-methyl, a new herbicide in upland crops. Proc. 5th Asian Ege, T., Ringertz, N.R., Hamberg, H., Pac. Weed Sei.Conf. ,pp . 138-141. Sidebottom, E. (1977). Preparation of microcells in: Methods in Cell Biology, 15 Barfield, D.G., Robinson, S.J., Shields, R. (D.M. Prescott ed.), Academic Press, New (1985).Plan t regeneration from protoplasts York, pp.339-358 . of long term haploid suspension cultures of N. plumbaginifolia. Plant Cell Rep. 4: Fournier, R.E.K. (1982). Microcell- 104-107. mediated chromosome transfer. In: Techniques in Somatic Cell Genetics (Ed. J Blumenthal, A.B.,Dieden ,J.D. ,Kapp ,L.N. , WShay) ,Plenu m Press,Ne wYork , London, Sedat, J.W. (1979). Rapid isolation of pp.309-327 . metaphase chromosomes containing high molecular weight DNA. J. Cell Biol. M: Fournier, R.E.K., Ruddle, F.H. (1977). 255-259. Microcell-mediated transfer of murine chromosomes intomouse ,Chines e hamster, Brues, A.M., Jackson, E.B. (1937). Nuclear and human somatic cells.Proc . Natl. Acad. abnormalities resulting from inhibition of Sei.US A74 : 319-323. mitosis by colchicine and other substances. Amer.J . Cancer3_Q : 504-511. Fournier, R.E.K., Frelinger, J.A. (1982). Construction of microcell hybrid clones Davies,K.E , Young,B.D. ,Elles ,R.G , Hill, containing specific mouse chromosomes. M.E, Williamson, R. (1981). Cloning of a Application to autosomes 8 and 17. Mol. representativegenomi clibrar yo fth ehuma n Cell Biol.2 :526-534 . X chromosome after sorting by flow cytometry. Nature 223.: 374-378. Fournier, R.E.K, Jurecek, D.K, Ruddle, F.H. (1979).Somati c cellgeneti c analysiso f De Marco, A., Romanelli, M., Stazi, M.A., transgenome integration. Somat. Cell Vitagliano, E. (1986). Induction of Genet.5 : 1061-1077. micronucleated cells in Vicia faba and Allium cepa root tips treated with Hesemann, C.U, Fayed, A.H. (1982). nitrilotriacetic acid. Mutat. Res. 171: Micronuclei in Viciafaba 1. The occurrence 145-148. and origin. Egypt. J. Genet. Cytol. U: 235-244. De Laat, A.M.M., Blaas, J. (1984). Flow cytometric characterization and sorting of Kao, F.T. (1983). Somatic cell genetics and plantchromosomes .Theor .Appl .Genet .67 : gene mapping. Int. Rev. of Cytology 8_5_: 463-467. 109-146. 42 Kiermayer, O., Fedtke, C. (1977). Strong Matsui, S., Weinfeld, H., Sandberg, A.A. anti-microtubule action of (1982). Factors involved in prophasing and amiprophos-methyl (APM) in Micrasterias. telophasing. In: "Premature chromosome Protoplasma 22:163-166. condensation", Application in basic, clinical and mutation research (P.N. Rao, R.T. Klein, G., Klein, E., Klein, E. (1952). The Johnson and K. Sperling, eds.), Academic viability and the average Press, New York,pp . 207-233. desoxy-pentosenucleic acid content of micronuclei-containing cells produced by Morejohn, L.C., Fosket, D.E. (1984). colchicine treatment in the Ehrlich ascites Inhibition of plant microtubule tumor. Cancer Research 12:484-489 . polymerization in vitro by the phosphoric amide herbicide amiprophos-methyl. Lebo, R.V. (1982). Chromosome sorting Science 224: 874-876. and DNA sequence localization. Cytometry 3_: 145-154. Nuti Ronchi, V., Bonatti, S., Turchi, G. (1986a). Preferential localization of Levan,A .(1938) .Th eeffec t ofcolchicin e on chemically induced breaks in root mitoses in Allium. Hereditas 24: heterochromatic regions of Vicia faba and 471-486. Allium cepa chromosomes. I. Exogenous thymidine enhances the cytologic effects of Levan, A. (1940). The effect of 4-epoxyethyl-l,2-epoxy-cyclohexane. acenaphthene and colchicine on mitosis of Environ. Expt. Bot.26 : 115-126. Allium and Colchicum. Hereditas 26: 262-276. Nuti Ronchi, V., Bonatti, S., Durante, M., Turchi,G . (1986b).Preferentia l localization Levan, A. (1954). Colchicine-induced of chemically induced breaks in C-mitosis in two mouse ascites tumors. heterochromatic regions of Vicia faba and Hereditas 4Q: 1-64. Allium cepa chromosomes II. 4-epoxyethyl-l,2-epoxy-cyclohexane Lo Schiavo, F., Nuti Ronchi, V., Terzi, M. interacts specifically with highly repetitive (1980). Genetic effects of griseofulvin on sequences of DNA inAllium cepa. Environ. plant cell cultures. Theor. Appl. Genet.5J£ : Expt. Bot.26:127-135 . 43-47. Obara, Y., Chai, L.S., Weinfeld, H., Mc Bride, O.W., Peterson, J.L. (1980). Sandberg, A.A. (1974). Synchronization of Chromosome-mediated gene transfer in events in fused interphase-metaphase mammalian cells. Ann. Rev. Genet. 14: binucleate cells: Progression of the 321-345. telophase-like nucleus.J . Natl.Cance r Inst. 53_: 247-259. Malmberg, R.L., Griesbach, R.J. (1980). The isolation of mitotic and meiotic Phillips, S.G., Phillips, D.M.(1969) .Site so f chromosomes from plant protoplasts. Plant nucleolus production in cultured Chinese Sei.Lett .12 : 141-147. hamster cells.J . Cell Biol.4 :248-268 . Marshall, I., Bianchi, M. (1983). Sekiguchi, T., Shelton, K., Ringertz, N.R. Micronucleus induction in Viciaf aba roots (1978) .DN Aconten t ofmicrocell s prepared 2. Biological effects of neutrons below 1 from rat, kangaroo and mouse cells. Expt. centigray. Int.J . Radiât. Biol.44. :163-174 . Cell Res.H3_ :247-258 . 43 Smiley, J.R., Steege, D.A., Juricek, K.D., Verhoeven, H.A., de Laat, A.M.M., Sree Summers, W., Ruddle, F.H. (1978). A Ramulu, K., Dijkhuis, P., van Herpes Simplex Virus 1 integration site in Vloten-Doting, L. (1987). Induction, the mouse genome defined by somatic cell isolation and flow cytometric sorting of genetic analysis.Cel l15_ : 455-468. metaphase chromosomes and micronuclei from cell suspension cultures of Nicotiana Sree Ramulu, K., Dijkhuis, P., Roest, S., plumbaginifolia. In: Genetic and cellular Bokelmann, G.S., De Groot, B. (1984). engineering of plants and micro-organisms Early occurrence of genetic instability in important for agriculture, Commission of protoplast cultures ofpotato .Plan tSei .Lett . the European Comunities meeting, 26:79-86 . Louvain-la-Neuve, March 23-26, pp. 119-120. Sree Ramulu, K., Dijkhuis, P., Hänisch ten Cate, Ch.H, De Groot, B. (1985). Patterns of DNA and chromosome variation during in vitro growth in various genotypes of potato.Plan t Sei.H : 69-78. Sree Ramulu, K., Dijkhuis, P. (1986). Flow cytometric analysis of polysomaty and in vitro genetic instability inpotato .Plan t Cell Rep. 3_: 234-237. Stubblefield, E. (1964). DNA synthesis and chromosomal morphology of Chinese hamster cells cultured in media containing colcemid. In "Cytogenetics of cells in culture" (R.T.C. Harris, ed.), Academic Press,Ne wYork ,pp .223-298 . Szabados, L., Hadlaczky, G., Dudits, D. (1981). Uptake of isolated plant chromosomes by plant protoplasts. Planta 151: 141-145. Van der Linden, P.M. (1980). Computer analysis of DNA- histograms, Ph. D Thesis, State Univ. Utrecht. Verhoeven,H.A. ,d eLaat ,A.M.M. ,Blaas ,J . (1986). Methodological developments for chromosome-mediated gene transfer in higherplants .In :Biomolecula r Engineering in the European Community, achievements of the research programme (1982-1986), final rep. (Ed. E. Magnien), Martinus Nijhoff Publishers, Dordrecht, pp.977-981 . 44 CHAPTER4 COMPARISON OF THE EFFECTS OF VARIOUS SPINDLE TOXINS ONMETAPHAS E ARRESTAN D ON FORMATION OF MICRONUCLEI IN CELL SUSPENSION CULTURES OF NICOTIANA PLUMBAGINIFOLIA. HA Verhoeven,K.Sre eRamulu ,P .Dijkhuis . 45 COMPARISON OF THE EFFECTS OF VARIOUS SPINDLE TOXINS ONMETAPHAS E ARRESTAN D ON FORMATION OF MICRONUCLEI IN CELL SUSPENSION CULTURES OF NICOTIANA PLUMBAGINIFOLIA. H.A.Verhoeven ,K.Sre eRamulu ,P .Dijkhuis . ResearchInstitut eItal ,P.O .Bo x48,670 0AA ,Wageningen ,Th eNetherlands . Summary transfer of isolated metaphase chromosomes results in the fragmentation of the chromosomes, and subsequent The effect of the anti-microtubular drugs incorporation of the fragments into the colchicine, amiprophos-methyl (APM) and recipient genome, microcell fusion was oryzalin on mitotic index, chromosome developed as a tool to transfer intact scattering and on the induction and yield of chromosomes into mammalian cells (Lugo micronuclei were compared. Results from and Fournier, 1986). With this technique, cytological analysisan d flowcytometry show micronuclei are induced by prolonged that oryzalin ismos t suitable for metaphase exposure to Colcemid, and the arrest, and consequently for isolation of micronucleated cells are centrifuged, to metaphase chromosomes, whereas APM is isolate subcells with a few micronuclei mosteffectiv e ininductio no fmicronuclei .A containinga smal lnumbe r ofchromosomes . hypothesis is presented, explaining the difference of action of APM and oryzalin Both chromosome- and microcell-mediated and the mechanism of micronucleus genetransfe r require efficient procedureso f induction byAPM . chromosomeisolatio nan dmas sinductio no f micronuclei in plants. A high mitotic index Keywords :metaphas e arrest, micronucleus, is necessary for the efficient isolation of spindle toxin, colchicine, herbicides, metaphase chromosomes from plant cells. Nicotiana. This can be achieved by utilizing the naturally occurring synchrony of Introduction gametophytic tissues, such as anthers or meiocytes(Malmber g andGriesbach ,1980) , or by metaphase arrest in other tissues Genetic manipulation of plants can be through spindle toxins, such as colchicine achieved by many techniques, ranging from (Malmberg and Griesbach, 1980; Szabados the introduction of single, cloned genes, to et al., 1981; Hadlackzy et al, 1981; 1983 the addition of the complete genomic Matthews, 1983; de Laat and Blaas, 1984 information of a donor species by somatic Guri et al, 1984; de Laat and Schel, 1986 hybridization. In between these extremes, Griesbach, 1987;Coniaetal. , 1987a; 1987b) transfer of chromosomes has already been availablefo r some time,bot h in mammalian Colchicine hasbee n shownt obin dwit hhig h cell systems and in plants (Klobutcher and affinity to tubulins in mammalian cells, and Ruddle, 1981;Griesbac he tal , 1982a).Sinc e induces metaphase arrest at concentrations 47 of 10"7 M and higher (Dustin, 1984). The Materials and methods concentration required for metaphase blocking in plant cells is a thousand-fold higher than that in animal cells. This has Cellculture been attributed to lower affinity of colchicine to plant tubulin heterodimers A kanamycin resistant suspension cell (Dustin, 1984). Podophyllotoxin has the culture ofN. plumbaginifolia, obtaine d after samemod eo factio na scolchicine ,an dbind s transformation of a haploid cell suspension toth esam esit eo nth etubuli n hetero-dimer, provided byDr .Shields ,Unileve r Res.Lab. , aswa sdemonstrate d bycompetitio n studies Colworth Home, Sharnbrook, Bedford, UK (Dustin, 1984).Th ecolchicin ebindin gsit ei s was cultured in the dark at 28°C in "Doba" poorlyconserve d duringevolution ,a sca nb e medium (Barfield et al., 1985)o n agyrator y concluded from the observation that shaker (120 rpm), and transferred to fresh colchicine binds with a lower affinity to the medium every week. All experiments were binding site on plant tubulins than to the performed with cells, grown for a week to binding site on mammalian tubulins. plateau phase. The cell suspensions from several flasks were mixed, and subcultured Some herbicides act on plant tubulin by a fourfold dilution with fresh "Doba" polymerization, and prevent cell cycle medium. progression through mitosis (reviewed in Dustin, 1984).Th e dinitroanilines trifluralin Chemicaltreatments and oryzalin, and amiprophos-methyl (S) (APM), a phosphoric amide, belong to this Amiprophos-methyl (Tokunol M ; classo fchemicals .Severa lo fthes einhibitor s 0-methyl-0-0-(4-methyl-6-nitrophenyl)- show very strong binding to plant tubulins, N-isopropyl-phosphoro thioami d ate) and arrest mitosis at concentrations of 10" (Kiermayer and Fedtke, 1977) was a gift M (Morejohn and Fosket, 1984; Morejohn from Bayer Nederland B.V., Divisie et al, 1987). Agrochemie, Arnhem, The Netherlands. Oryzalin was a gift from Eli-Lilly Co. On the basis of the finding that prolonged Colchicine was obtained from Jansen APM treatments induce micronucleation in Pharmaceuticals, Beerse, Belgium. Stock plant cells (Verhoeven et al., 1986; 1987;d e solutions of APM and oryzalin were Laat et al., 1987), it was suggested that the prepared at 20mg/m l inwater-fre e DMSO, disturbance of the mitochondrial calcium and could be stored for at least 6month s at uptake by APM (Hertel et al., 1980; 1981) -20°C,withou t losso f activity. Concentrated wasresponsibl e for micronucleus formation aqueous solutions of 100 mg/ml colchicine (Sree Ramulu et al., 1988a; this thesis, were prepared freshly for each experiment. chapter three). The deregulation of the All treatments were started 24 h after calciummetabolis mb ythes eherbicide sma y subcultureo fa on eweek -ol dcel lsuspensio n play an important role in the induction of culture. micronuclei. Cytology In this paper, the effects of various spindle-toxins on the accumulation of Cells were fixed in 3:1 ethanol : acetic acid mitotic cells and the induction of and prepared for cytology as described micronuclei are studied in the context of earlier (Sree Ramulu et al., 1985). Mitotic genetic manipulation using isolated index, micronucleus index (percentage of metaphase chromosomes, micronuclei and micronucleated cells) and number of microprotoplasts. micronuclei per cell were determined from 48 Mitotic index (%) Treatmentduratio n (h) Fig. 1. Mitotic index ofN.plumbaginifolia cell suspensions, treated with: 0.5 mM colchicine (—| 1-);5m M colchicine (^-^f); 3/ For flow cytometric determination of the Chromosomeisolation. nuclear DNA content, protoplasts were isolated from small samples of the cell Chromosomes were isolated from cell suspension. About 400 /A of an enzyme suspensions as described before (de Laat mixture, containing 25% Cellulase and Blaas, 1984). "Onozuka"RI O(Yakult ,Honsk a Co,Tokyo , Japan), 5 % Macerozyme RIOan d 1.2 Mo f Results sucrose, was added to 800 ^1 of cell suspension. After 30mi n incubation at28° C in a2. 5 cmpetridis h on agyrator y shaker at Mitotic index 30rpm ,th e enzymemixtur ewa s centrifuged for 5mi n at 60g . Floating protoplasts were Subculture of a cell suspension, grown to transferred to nuclear isolation buffer, plateau phase, results in a sudden increase containing 10 mM Tris-HCl, pH 7, with 10 ofth emitoti cinde xu pt o6% ,occurrin g after mM spermine-tetrahydrochloride, 2.5/ 49 4 * • * ;• «- * *4- V at, ..• > O'"* V *> d Fig. 2. Photomicrographs of a: cell with ball metaphase; b: cell with scattered chromosomes; c: cell with chromosomegrouping ;d :cel lwit hmicronuclei . 50 Ballmetaphase s (%o f mitotic cells) 100 • H 20 30 Treatment duration (h) Fig.3 . Frequency ofbal l metaphases (cellswit h clustered chromosomes), expressed aspercentag e of the total number of mitotic cells, after treatment with: 0.5 mM colchicine ( -|—-)-); 5mM colchicine (^—%); 3/ 51 t= 18 t= 44 Control 3,uM oryzalin 15fiM A. oryzalin 30^ M iL oryzalin 3fiM iL APM 32^ M APM 0.5 mM colchicine 5mM colchicine 4C 8C 4C 8C 16C Fig.4 .Flow-cytometri c histograms of nuclear DNA content of the control (upper row, t= 0,18 , 44h) , and the treated cellsuspension s (t= 18,44h) .O n thehorizonta l axisi sth e DNA content expressed inC-value ,i n which 1 C isth e DNA content of a haploid N.plwnbaginifolia; on the vertical axis isth e number of particles . 52 Scattered mitoses (%) Treatment duration (h) Fig. 5. Percentage of scattered mitoses (cells with scattered chromosomes and chromosome groups) after treatments with: 0.5 mM colchicine ( -(-—1~); 5mM colchicine (-^—»<); 3/ 53 Number of particles Fig. 6. Flowcytometric analysis of isolated chromosomes showing sister- chromatids (left peak) and metaphase chromosomes (right peak). On the horizontal axis is the DNA content expressed in C-value, inwhic h 1 Ci sth e DNA content of a haploid N.plumbaginifolia; on the vertical axis is the number of particles. 0.1C 0.2 C DNA-content (C-value) treatment. Duringchromosom e isolation an 500 cells, with an average number of 8 increased release of separate sister micronucleipe rmicronucleate d cell(Fig .8) . chromatids was obtained as a consequence of the infuence of the spindle toxins on the stabilityo fth e centromeres.Thi sresulte d in Discussion a large number of sister chromatids in the flow histogram (Fig. 6, left peak), together with the metaphase chromosomes (Fig. 6, The results presented in this paper, show right peak). that accumulation of metaphases can be increased significantly by using the Micronucleation anti-microtubular herbicides APM or oryzalin as compared to colchicine When the micronucleus index was treatments. Furthermore, APM treatment determined after the different treatments, results in a reduction of the frequency of striking differences were observed. cells showing chromosome clumping (ball Treatmentswit hcolchicin eresulte di nlevel s metaphases) to less than 30% of the of micronucleation up to 4%, whereas frequency of ball metaphases after 0.5 mM treatments with APM rendered colchicine treatment. The same effect was micronucleus indices up to 12% (Fig. 7). observed after incubation with low Although metaphase arrest was hardly concentrations of oryzalin, which is in affected by the APM or oryzalin agreement with reports of the effects of concentration, the micronucleus index oryzalin on mitotic endosperm cells of appeared to be influenced by the Haemantus katherinae (Morejohn et al., concentration of oryzalin. At a 1987). concentration of 3 fiM oryzalin, the induction of micronuclei is more efficient The accumulation of metaphases increases than at higher concentrations, which are from 30 to 35% with increasing more effective for ploidy doubling. The concentrations of oryzalin, however, optimal induction of micronuclei was relatively more ball metaphases are induced obtained by treatments with 32 / 54 Micronucleus index (%) Treatment duration (h) Fig. 7. Micronucleus index (percentage micronucleated cells) after treatments with: 0.5 mM colchicine ( -| 1-); 5mM colchicine ( X X )i 3 fiM oryzalin ( ---£>—); 15 fiM oryzalin (--X--); 30 ßM oryzalin(—Q--); 3pM APM OX- X)'> 32fiM APM (Q B) and untreated control ( + ). Chemicals were applied at t= 0 ,i.e .2 4h after subculture. Number of micronuclei 500 300 20 30 50 Treatment duration (h) Fig. 8. Total number of micronuclei per 500 cells after treatments with: 0.5 mM colchicine (-) 1-); 5mM colchicine (X X); 3^M oryzalin (~Q~- ); 15^M oryzalin (—X--); 30,uM oryzalin(—Q-); 3/iMAFM ()K X );32// M APM (j~ ] f3) and untreated control ( -|- ).Chemical swer eapplie d att = 0 ,i.e .2 4h after subculture. 55 formation is much stronger with APM or Ramulu et al, 1988a; b; this thesis, chapter oryzalin than with colchicine, resulting in a three). The micronucleus index is more efficient accumulation of metaphases. dependent on the chemical used for Disruption of the calcium storage in the metaphase arrest, and there seems to be a mitochondria has been proposed as the relation between the maximum level of causeo fth e destruction of tubulinsb yAP M mitotic index, and the level of the and oryzalin,a sha sbee n showni nth e green micronucleus index. APM is the most alga Chlamydomonas (Quader and Filner, effective inducer, followed by oryzalin, 1980). However, for deregulation of the whereas colchicine is the least effective calcium metabolism, 10-t o 100-fold higher (Figs. 7, Fig. 8). Another interesting concentrationso fth einhibito rwer e needed, observation is that micronuclei induced by than for disturbance of mitotic activities oryzalin persist for only a few hours before (Morejohn and Fosket, 1986). Recently, forming a restitution nucleus by fusing APMan doryzali nhav ebee nshow nt o affect together. Micronuclei, induced by APM, chromosome behaviour directly by binding persist longer, as can be deduced from Fig. toth etubuli nhetero-dimer s (Morejohn and 7,whic hshow stha t themicronucleu s indices Fosket, 1984;Morejoh n et al, 1987). for the 3 and 32^M APM treatment do not decrease for at least 10hours . The toxic effects of high concentrations of The difference in influence on calcium colchicine reduce the number of cycling uptake and accumulation might explain this cells, as can be concluded from the phenomenon, since the action of both flow-cytometry data. Since this does not chemicals on the spindle is comparable for occurwhe nAP Man doryzali nar eused ,hig h the tested concentrations. The effective mitotic indices can be induced, even with concentrations for inhibition of microtubule highconcentration so fthes eherbicides .Thi s assemblyar ei nth esubmicromola r range for is partly due to their very low solubility in both APM and oryzalin. Disruption of water (7 nM for oryzalin, and 225 fiM for calciumstorag e inth emitochondri a startsa t APM).Th e observation that concentrations about 5 fiM for oryzalin and APM, which higher than 7fiM oryzalin have additional makes it possible to manipulate the two effects mayb edu et oth elipophili cnatur eo f actionsmor e or lessindependently .Ther e is oryzalin, which causes an accumulation of a large difference in the effectiviness of the the chemical in the membranes (Morejohn herbicides compared to colchicine. et al, 1987).Thus , the additional effects of Colchicine has hardly anyinfluence , even at higher concentrations of oryzalin may be concentrations as high as 10 mM, whereas attributed to membrane effects, and most APMreduce smitochondria l calcium uptake likely the disruption of the accumulation of to 50% at 15 fiM (Hertel et al, 1980). calcium in the mitochondria. Oryzalin is slightly less effective than APM in inhibiting the calcium uptake by the Chromosome scattering is the first stage in mitochondria, and reduces the active the induction of micronuclei. As described excretion of calcium by the plasma earlier, chromosomes group together, and a membrane toles stha n60 %o fth e untreated nuclear membrane is formed around each control, whereas APM leaves this activity single chromosome and around grouped almost unchanged at 90% (Hertel et al, chromosomes in various species (Sree 1980). As a result, intracellular cytoplasmic Ramulu et al, 1988a; b). After prolonged calciumleve lwil lb ehighe r for oryzalin than treatments with APM, micronuclei were for APM treatments, resulting in a lower induceda thig hfrequenc y (Verhoeven et al, tendency to form nuclear membranes 1987;d eLaa t et al, 1987).Micronucle i fuse around single or grouped chromosomes together forming arestitutio n nucleus (Sree after oryzalin treatments. High cytoplasmic 56 calcium might also explain why micronuclei Acknowledgements disappearsoone ri noryzali ntreatment s than in APM treated cells, since high calcium concentrations promote the fusion of The skilfull assistance of Mrs. Yvonne membranes. Changes in the intracellular Nollen for in vitro culture is gratefully calcium concentrations are known to have acknowledged. Part of this work was effects on the formation of nuclear supported by the "Biotechnology Action membranes,a sha sbee nshow nb yth e effects Programme" of the CEC, contract on the transition from metaphase to BAP-0083-NL(GDF). The authors wish to anaphase in stamen hair cells of thankDr .L .va nVloten-Dotin gan dDr .W.J . Tradescantia (Wolniak et al., 1983;Wolnia k Stiekema for critical reading of the and Bart, 1985). Compartmentalization of manuscript. calcium ions by the smooth endoplasmatic reticulum plays a role in the regulation of References mitosis in barley cells (Hepler, 1980). Lowering of the cytoplasmic calcium concentration inducestelophasin gan dlead s Barfield, D.G., Robinson, S.J., Shield, R. to the formation of nuclear membranes (1985). Plant regeneration from long term around individual chromosomes in cultured haploid suspension cultures of TV. mammalian cells (Matsui et al., 1982). A plumbaginifolia.Plan t Cell Rep.1:104-107 . comparison on the effects of APM and oryzalin on the accumulation of calcium in Conia, J., Bergounioux, C, Perennes, C, the mitochondria and their influence on Brown, S., Muller, P., Gadal, P. (1987a). active excretion by the plasma membrane Chromosomes from protoplasts of Petunia might elucidate the mechanism involved in hybrida, flow analysis and sorting.Biol .Cel l the induction of micronuclei inplants . 5Ssuppl :5a . The dataobtaine d inthi sstud yindicat e that Conia, J., Bergounioux, C, Perennes, C, oryzalin at 3 to 15^ M is the most suitable Muller, P., Brown, S., Gadal, P. (1987b). chemical to be used for accumulation of Flowcytometri canalysi san dsortin go fplan t metaphase chromosomes, whereas 3 to 32 chromosomes from Petunia hybrida ^M APM treatments are more efficient for protoplasts. Cytometry ft 500-508. the induction of micronuclei. Large numbers of cells with scattered metaphase de Laat, A.M.M., Blaas, J. (1984). chromosomes, which can be obtained by Flow-cytometric characterization and oryzalin treatments, are useful for flow sorting ofplan t chromosomes.Theor .Appl . cytometricsortin g (de Laat and Blaas, 1984; Genet, 6J:463-467 . Conia et al., 1987a; 1987b) and chromosome-mediated gene transfer de Laat, A.M.M., Verhoeven, H.A., Sree (Griesbach, 1987). Micronucleated cellsca n Ramulu, K, Dijkhuis, P. (1987). Efficient be used to isolate microprotoplasts, induction by amiprophos-methyl and consisting of one or a few micronuclei, flow-cytometric sorting of micronuclei in surrounded by a small amount of cytoplasm Nicotiana plumbaginifolia. Planta 122: and an intact plasma membrane. Thus, a 473-478. systemanalogou st oth e microcell-mediated gene transfer in animal cells (Fournier, Dustin, P. (1984). Microtubules, 1982)woul d become feasible inplants . Springer-Verlag,Berlin ,pp .167-225 . 57 de Laat, A.M.M., Schel,J.H.N . (1986).Th e Hertel, C, Quader, H., Robinson, D.G., integrity of metaphase chromosomes of Marmé, D. (1980). Anti-microtubular Haplopappus gracilis(Nutt. ) Gray isolated herbicides and fungicides affect Ca byflo w cytometry.Plan t Sei.42:145-151 . transport inplan t mitochondria. Planta 149: 336-340. Falconer, M.M., Seagull, R.W. (1987). Amiprophos-methyl (APM): a rapid, Hertel, C, Quader, H., Robinson, D.G., reversible, anti-microtubule agent for plant Roos, I., Carafoli, E., Marmé, D. (198L|. cell cultures.Protoplasm a 136:118-124. Herbicides and fungicides stimulate Ca efflux from rat liver mitochondria. FEBS Fournier, R.E.K. (1982). Microcell- Letters 122:37-39 . mediated gene transfer. In: Techniques in somatic cell genetics. Ed.: Shay, J.W., Kiermayer, O, Fedtke, G (1977). Strong Plenum Press, New York, London, pp. anti-microtubule action of 309-327. amiprophos-methyl (APM) in Micrasterias. Protoplasma 22:163-166. Griesbach, R.J., Malmberg, R.L., Carlson, P.S. (1982a). Uptake of isolated lily Klobutcher, L.A., Ruddle, F.H. (1981). chromosomesb ytobacc oprotoplasts .Th eJ . Chromosome mediated gene transfer. Ann. ofHered .22 : 151-152. Rev. Biochem.5_Q : 533-554. Griesbach, R.J., Malmberg, R.L., Carlson, Lo Schiavo, F., Nuti Ronchi, V., Terzi, M. P.S. (1982b). An improved method for the (1980). Genetic effects of griseofulvin on isolation of higher plant chromosomes. plant cell cultures. Theor. Appl. Genet. 5_&: Plant Sei.Lett .24 :55-60 . 43-47. Griesbach, R.J. (1987). Chromosome- Lugo, T.G., Fournier, R.E.K. (1986). mediatedtransformatio n via microinjection. Microcell fusion and mammalian gene Plant Science 5Q: 69-77. transfer, in: Kucherlapati, R., ed. Gene transfer. Plenum Publishing Corp. , New Guri, A., Zelcher, A., Izhar, S. (1984). York, pp.79-93 . Induction of high mitotic index in Petunia suspension culturesb ysequentia l treatment Malmberg, R.L., Griesbach, R.J. (1980). with aphidicolin and colchicine. Plant Cell The isolation of mitotic and meiotic Reports 2:219-221 . chromosomes from plant protoplasts. Plant Sei.Lett . 12: 141-147. Hadlaczky, G., Sumner, A.T., Ross, A. (1981). Protein-depleted chromosomes. Matsui, S., Weinfeld, H., Sandberg, A.A. Chromosoma SI:537-555 . (1982). Factors involved in prophasing and telophasing. in: Premature chromosome Hadlaczky, G., Bistray, G., Praznovszky, T., condensation, application in basic, clinical Dudits, D. (1983). Mass isolation of plant and mutation research. Ed: Rao, P.N., chromosomes and nuclei. Planta 157: Johnson, R.T.,Sperling , K.Academi cPress , 278-285. New York. pp. 207-233. Hepler, P.K. (1980). Membranes in the Matthews, B.F. (1983). Isolation of mitotic mitotic apparatus of barley cells. The chromosomes from partially synchronized Journal of Cell Biol.Se :490-499 . carrot (D. carota) cell suspension cultures. Plant Sei.Lett . 21: 165-172. 58 Morejohn, L.C., Fosket, D.E. (1984). chromosomes by plant protoplasts. Planta Inhibition of plant microtubule 151: 141-145. polymerization in vitro by the phosphoric amide herbicide amiprophos-methyl. Verhoeven, H.A., de Laat, A.M.M,Blaas ,J . Science 224:874-876 . (1986). Methodological developments for chromosome-mediated gene transfer in Morejohn, L.C., Fosket, D.E. (1986). plants. In: Biomolecular engineering in the Tubulins from plants, fungi, and protists: a European Community., achievements of the review.In :Cel lan d molecularbiolog yo f the research programme (1982-1986), final cytoskeleton. ed: Shay, J.W. Plenum report. Ed.: Magnien, E. Martinus Nijhof Publishing Corp., NewYork . pp.257-329 . Publishers, Dordrecht,pp .977-981 . Morejohn, L.C., Bureau, T.E., Molé-Bajer, Verhoeven, H.A., de Laat, A.M.M., Sree J.,Bajer ,A.S ,Fosket ,D.E .(1987) .Oryzalin , Ramulu, K., Dijkhuis, P., van a dinitroaniline herbicide, binds to plant Vloten-Doting, L. (1987). Induction, tubulin and inhibits microtubule isolation and flowcytometric sorting of polymerization in vitro. Planta 172: metaphase chromosomes and micronuclei 252-264. from cell suspension cultures of Nicotiana plumbaginifolia. In: Genetic and cellular Quader, H., Filner, P. (1980).Th e action of engineering of plants and micro-organisms antimitotic herbicides on flagellar important for agriculture, Comission of the regeneration inChlamydomonas reinhardtii: European Communities meeting, a comparison with the action of colchicine. Louvain-la-Neuve,pp . 119-120. Eur. J. of Cell Biol.21 : 301-304. Wolniak, S.M., Hepler, P.K., Jackson, W.T. Sree Ramulu, K., Dijkhuis, P., Hänisch ten (1983). Ionic changes in the mitotic Cate, C.H., de Groot, B.(1985) .Pattern s of apparatus at the metaphase/anaphase DNA and chromosome variation during in transition. The Journal of Cell Biol. 26.: vitro growth invariou s genotypes of potato. 598-605. Plant Sei.41 :69-78 . Wolniak, S.M., Bart, K.M. (1985). The Sree Ramulu, K., Verhoeven, H.A., buffering of calcium with quin2 reversibly Dijkhuis, P. (1988a). Mitotic dynamics of forestalls anaphaseonse ti nstame nhai rcell s micronuclei induced by amiprophos-methyl of Tradescantia. Eur. J. of Cell Biol. 22: and prospects for chromosome-mediated 33-40. gene transfer inplants .Theor .Appl . Genet. 25_: 575-584. Sree Ramulu, K., Verhoeven, H.A., Dijkhuis, P., Gilissen, L.J.W. (1988b). Chromosome behaviour and formation of micronuclei after treatment of cell suspension cultures with amiprophos-methyl invariou splan t species. Plant Sei.5_6_ : 227-237. Szabados, L., Hadlaczky, G., Dudits, D. (1981). Uptake of isolated plant 59 CHAPTER 5 IMPROVED ACCUMULATION OF MITOTIC AND MICRONUCLEATED SUSPENSION CELLS OF NICOTIANA PLUMBAGINIFOLIA BYAMIPROPHOS-METHY L AFTER PRETREATMENT WITH DNA-SYNTHESIS INHIBITORS HA Verhoevenan dH.C.P.M .va nde rVal k 61 IMPROVED ACCUMULATION OF MITOTIC AND MICRONUCLEATED SUSPENSION CELLS OF NICOTIANA PLUMBAGINIFOLIA BYAMIPROPHOS-METHY L AFTER PRETREATMENT WITH DNA-SYNTHESIS INHIBITORS HA Verhoevenan dH.C.P.M .va nde rVal k ResearchInstitut eItal ,P.O.Bo x48,670 0AA ,Wageningen ,Th eNetherlands . Summary Passage through the cell cycle can be prevented eitherb ystarvatio nprocedure s or chemical inhibitors, acting on different The accumulation of suspension cells of stages of the DNA synthesis. N.plumbaginifolia at metaphase and the induction of micronuclei was optimized by Forinhibitio nb ystarvation ,suspensio ncell s inhibition of DNA synthesis, preceding the are grown to plateau phase, and partial amiprophos-methyl (APM) treatment. The synchrony is induced by adding fresh effects of a starvation pretreatment and medium (reviewed in King, 1980; Gould, varyingconcentration s ofhydroxyure a (HU) 1984). Starvation results in a drastic or aphidicolin (APH) on cellular DNA reduction of the mitotic index. After content, mitotic index (MI) and replacement of the medium, the mitotic micronucleus index (MNI) were index rises sharply. During the starvation investigated, resulting in a procedure for period, growth can be retarded by accumulation of up to 50% for both carbohydrate, phosphate or nitrogen metaphases and micronuclei. limitation. Starvation isa simple procedure, however, it seldom yields a satisfactorily Key words: DNA synthesis inhibition, high accumulation of cells in a particular hydroxyurea, aphidicolin,Nicotiana, mitoti c phase of the cell cycle.Cell s are arrested at index, micronucleus. the Gl/S boundary or in G2 phase, and not at one specific point, resulting in poor Introduction synchrony. A similar approach is based on the Accumulation of plant cells in a specific observation that isolated protoplasts show phaseo fth ecel lcycl ei sa nimportan t tooli n synchronous division after removal of the many biological investigations. cell wall-degrading enzymes (Weber and Simultaneous division is important Schweiger, 1985). especially in the case of mass isolation of metaphase chromosomes (Griesbach et al., Most chemical treatments for cell cycle 1982; Hadlaczky et al., 1983, Matthews, arrest interfere with DNA synthesis during 1983; de Laat and Blaas, 1984), and for the S-phase. Two different mechanisms are induction of micronuclei (Verhoeven et al., known for inhibition of DNA synthesis. 1986;1987 ;d eLaa te tal. ,1987 ;Sre eRamul u et al., 1988a). Firstly, the enzymes involved in the production of the nucleotides can be inhibited by substrate analogues, such as 63 hydroxyurea, 5-aminouracil and Aphidicolin (APH) belongs to the second fluorodeoxyuridine. In this way, the group of DNA synthesis inhibitors (Galli endogenous pool of intermediates hast o be and Sala, 1982). APH inhibits DNA exhausted, before the inhibition becomes a-polymerase directlyan dreversibl y (Salae t effective. As a consequence, also DNA al.,1980) .I ncontras tt oothe rDN Asynthesi s repair is inhibited. inhibitors, aphidicolin does not block DNA repair mechanisms (Sala et al., 1982). By Secondly, the DNA a-polymerase enzyme using aphidicolin, followed by a colchicine itself can be inhibited. Once all enzyme treatment, high mitotic indices, up to 65 %, molecules have bound the inhibitor, DNA were obtained in Petunia cell suspensions synthesis is blocked completely, but the (Guri et al., 1984). DNA repair mechanisms remain operational. The nucleotides, necessary for In thispaper , a studyi spresente d about the DNA repair, are still available because effects ofstarvatio n combinedwit h different nucleotide synthesis is unaffected by the concentrations of HU and APH on mitotic inhibitor.Sinc eDN Arepai ri sachieve dwit h index (MI) and micronucleus index (MNI). a different DNA polymerase, for which the For this purpose, the division activity was substrates are still available, no DNA evaluated cytologically and by flow damage will be accumulated during cytometric DNA analysis. Besides, the G2-phase. synchronizing treatments were combined with amiprophos-methyl blockage Hydroxyureai sa ninhibitor ,belongin gt oth e (Verhoeven et al., 1986)t oaccumulat e cells first class, acting on the ribonucleotide in metaphase and study the process of reductaseenzym e(Kihlma ne tal. ,1966 ;Kei l induction of micronuclei (de Laat et al., and Chaleff, 1983).Durin g HU treatments, 1987). abberations are induced in the nuclear DNA. The generation of abberations by Materials and methods hydroxyurea seems to occur especially during G2 phase, by inhibition of DNA repair mechanisms owing to lack of Cellculture precursors (Anderson, 1983). It has also been shown that the action of HU can be A suspension cell culture of N. reversed when the duration of the plumbaginifolia, kindly provided by Dr. treatmentsi s2 4h o rles s(Coyl ean dStrauss , Shields, Unilever Res. Lab., Colworth 1970). Besides, work in our laboratory has Home, Sharnbrook, Bedford, UK was shown, that damage to the chromosomes of cultured in the dark at 28°C in "Doba" Happlopappus gracilis due to 24 h medium (Barfield et al., 1985)o n agyrator y hydroxyurea treatment, could not be shaker (120rpm) .Fro m thiscel l suspension, estimated by electron microcoscopic a kanamycin resistant line was derived by evaluation, or by alkaline gradient transformation with Agrobacterium centrifugation (de Laat and Schel, 1986). tumefaciens as described before (de Laat Prolonged exposure (> 2 4h )o fbot h animal and Blaas, 1987). For optimal growth, the and plant cells to hydroxyurea induces suspension was subcultured twice a week, chromosome breaks, and leads to the with a fourfold dilution with fresh medium. formation of sister chromatid exchanges Starvation experiments were performed (Kihlman et al., 1966; Coyle and Strauss, withcells ,grow nfo ra wee kt oplatea uphase . 1970; Andersson, 1983), which are The materialfro m severalflask swa spooled , irreversible. mixed, and subcultured byfourfol d dilution with fresh "Doba" medium. 64 O 12 19 24 30 36 46 52 56 61 67 72 t= 0 I I t=6 ! I t=1 2 I \ t=1 9 1 I I l t=2 4 ^•mmm t=3 0 t=3 6 I \ —ka mmmm* I t=4 6 \ *—mmé t=5 2 t=5 6 \ I t=6 1 I ! t= 6 7 \ Fig.1 .Tim eschedul efo rth edeterminatio n ofth epercentag e ofcells ,whic hg oint odivisio ndurin gth e indicated intervals.AP M isadde d to identically treated cultures, at the times indicated byth e big arrows.Sample s were taken at the times indicated byth e small arrows, and mitotic indices were determined. The increase in mitotic indexfollowin g the addition ofAP M was considered to be the number of divided cells inth e test period. Chemicaltreatments. Cytology DNA synthesis was blocked by various Squash slides were prepared by staining a concentrations of hydroxyurea (HU) or small amount of dried suspension cellswit h aphidicoline (APH)(both supplied by a solution of 1% orceine in 1:1 lactic Sigma). HU was added as a concentrated, acidpropioni c acid, followed by heating of freshly prepared solution in medium (40 theslide st oabou t60° C(Eriksson , 1966)an d mg/ml). APH was added from a stock gentle squashing. Mitotic index was solution (20 mg/ml) in dimethylsulphoxide determined by counting between 300 and (DMSO). Amiprophos-methyl (Tokunol 600cells .Al lstage sfro m prophaseu pt o and M ; 0-methyl-0-0-(4-methyl-6-nitro including telophasewer e counted asmitoti c phenyl)-N-isopropyl-phosphoro cells. thioamidate, Kiermayer and Fedtke, 1977) was a gift from Bayer Nederland B.V., Flow cytometry Divisie Agrochemie, Arnhem, The Netherlands. Stock solutions of APM were For flow cytometric determinations of the prepared at 20 mg/ml in waterfree DMSO, nuclear DNA content, protoplasts were and could be stored for at least 6month s at isolated from small samples of the cell -20°C,withou t loss of activity. suspension. To 800 /A of cell suspension, 40Qalo fa nenzym emixture ,containin g25 % 65 subculture addition ofinhibito r washing 1 • 1 j L o 12 19 24 30 36 46 52 56 61 67 71 Fig.2 .Tim e schedule for experiments on the influence of DNA-synthesis inhibitors.Sample s for FCM and MI were taken at the times,indicate d by arrows.A t 24h after subculture, chemicals were added, and removed at 46h after subculture bywashin gwit h excess medium (5volumes) . Cellulase "Onozuka" RIO (Yakult, Honska and half of the cell masswa s subcultured in Co, Tokyo, Japan), 5 % Macerozyme RIO fresh medium with 32/« MAPM . and 1.2 M of sucrose, was added. After 30 minincubatio n ina 2. 5c mpetridish ,a t28°C , Results on a gyratory shaker at 30 rpm, the enzyme mixture was centrifuged for 5 min at 60 g. Floating protoplasts were transferred to Starvation nucleus isolation buffer, containing 10 mM Tris-HCl, pH 7, with 10 mM spermine- The effect of the starvation period is tetrahydrochloride, 2.5wg/ml4,6-diamidino - represented in Fig. 3. Control cell 2-phenylindole (DAPI), 10 mM NaCl, 200 suspension cultures were subcultured as mM hexyleneglycol and 0.025% usual on the third day of culture.Th e 3da y Triton-XlOO. Samples were analyzed on a old culture showed amitoti cinde xo f4 % at PartecPAS-I Iflowcytometer , with the UG5 the time of subculture (t= 0 )an d recovered excitation filter, TK420 and TK520 dichroic rapidly as revealed by the rapid increase of mirrors,an dGG43 5longpas sfilter . Channel MI to 7%. Mitotic index was increased analysis was performed with the standard already 4 h after subculture, and reached a software package of the PAS-II. maximum during log-phase close to 7% (8-24 h after subculture). In the starved, 7 Timeschedule day old culture, mitotic index remained below 2% until 20 h after subculture. From The number of cells, going into division this moment on the mitotic index gradually during the various stages of the treatment, increased, reaching a value of 8% at 52h was determined by adding APM to a final after subculture. concentration of 16^M to different flasks with cells. Mitotic index and DNA content Division activity were determined at regular intervals (Fig. 1).Th eincreas ei nmitoti cinde xdurin geac h Fig. 4 represents the percentage of mitotic incubation period was determined, and the cells (the sum of cells in mitosis or with average was calculated from thisvalu e with micronuclei) after subculture, determined the value for the corresponding interval by accumulating all mitotic cells during from the previous treatment. APM treatment. The treatments were For the evaluation of the effects of the startedaccordin gt oth eschem ei nFig .1 ,an d inhibitors on mitotic index, samples were the average of the increase during two taken for cytological analysis according to subsequent intervals was graphically the scheme of Fig. 2. After the chemical represented in Fig.4 .Thi s graph shows that inhibition of DNA synthesis, cells were recovery from starvation treatment lasts at washed with 5volume s of culture medium, least2 0h .The n asudde n risei nth e division 66 Mitotic index (%) Timeafte r subculture (h) Fig.3 . Effect of starvation of a cell suspension culture on the mitotic index after subculture. ( 0> 0>): 3-day old culture; (-)|<—Sfe): starved, 7-day old culture. Percentage of cells Timeafte r subculture (h) Fig. 4. Division activity (^—^) of a starved (7-day old) cell suspension culture at different times after subculture,an d thetota lnumbe r ofcell swhic h haveundergon e celldivisio n attha t time (0—ED)-^ ar indicates the duration of DNA-synthesis inhibition, and the arrow the percentage of divided cells at the end of the treatment. activityi sobserved ,whic hi sles spronounce d 50% of the cells would have undergone in the mitotic index of the control without division during the HU and APH APM treatment (Fig. 3). The burst of 25% treatments which took place from 24t o4 6h of divided cells isfollowe d by a divison rate (indicated by the bar in Fig.4) . of 5-17%. The graph shows that more than 67 Mitotic index (%) Timeafte rsubcultur e(h ) Fig.5 .Mitoti cinde xo fa starve dcel lsuspensio na tdifferen t timesafte rsubculture ,an dth einfluenc eo f different concentrationso fHU ,combine dwit hAP Mtreatmen t after washing(—(-"• •) : untreated control;(- | 1-): 1 mMHU ;(•*—*•) :2 m MHU ;(Q-Q) : 5m MHU ;(-X— X) :1 0m MHU ;(•<>-£>•) :2 0m MHU .Ba rindicate s periodo fH Utreatments . Mitotic index (%) Ji Timeafte r subculture (h) Fig.6 .A sFig .5 ,wit hAP Hinstea do fHU .( H 1-):3^M APH ; (^ ):6^ MAPH ;(Q-B) : 15fiM APH ; (X X) :30,u MAPH ;( O O):6 0pM APH.Ba rindicate sperio do fAP Htreatments . Chemicalinhibition respectively. For this experiment, a suspension culture was used, which was The effects of the addition of different grown to plateau phase at the time of concentrations of HU and APH on mitotic subculture (t = 0). A concentration index are represented in Figs. 5 and 6, dependent inhibition is observed, which 68 Inhibition (%) Inhibition (%) 5 10 15 20 25 10 20 30 40 50 HUconcentratio n (mM) APHconcentratio n («M) Fig.7 .Inhibitio n ofdivisio na sa consequence of DNA Fig.8 .A s Fig.7 ,wit hAP H instead of HU. synthesis inhibition by various concentrations of HU, determined at various times after start of the treatment.100 %inhibitio n causesa M Io f0% ,an d 0% X X t= 6 h inhibition results in a MI identical to the untreated t= 12h control. --X-"*- t= 22 h shows some characteristic differences subculture), and in case of 1 mM HU an between HU and APH. The inhibition was increase of the MIt o 136%o fth e untreated calculated by dividing the mitotic index of controlwa sobserve d (seeFig .5) .Th e effects the treated cells by the mitotic index of the of APH treatment are comparable to the control cell suspension. The result was HU treatment, with two differences. expressed as percentage and subtracted Inhibition is already 100% at sixhou r after from 100%. The resulting values are thestar t of the treatment for concentrations presented inFigs .7 an d 8,a t different times of 15JUM and higher.Th e sampleso f 12 h of of treatment (6,12 and 22h of treatment). treatmentsho wa patter nsimila rt oth elowe r Treatment with HU results in a HU treatments,bu tth e 22h samplessho wa concentration dependent reduction of the strongdecreas eo fth einhibition ,resultin ga t mitotic index, which was apparent after six 3 fiM in doubling of the mitotic index as hours of treatment (t = 30 h after compared to the control (see Fig.6) . subculture). Higher concentrations showed astronge rinhibition .Afte r 12h o f treatment After washing, the mitotic index remains at (t= 3 6h afte r subculture),al l concentrations a low level for all concentrations of the tested, except ImM, resulted in 100% DNA-synthesis inhibitors, which were still inhibition of mitosis.Th e effect of the lower activea tth etim eo fwashin g(10,2 0m M HU concentrations (1 mM, 2 mM and 5mM ) is and 15,30an d 60^MAPH) .Thi s effect lasts reduced after 22h oftreatmen t (t= 4 6 after at least until t= 5 7 h after subculture (llh 69 Micronucleated cells (%) Fig. 9. Micronucleus index (MNI) caused by 32/< M APM treatment, after pretreatment with varying concentrations of HU. ( -|- ): untreated control; (-| (-): 1m M HU; (-)K X-): 2 mM HU; (Q-B): 5 mM HU; (-^^): 10 mM HU; «> O): 20 mM HU. 10 15 20 Timeafte rwashin g (h) Micronucleated cells (%) Fig.10 .A sFig .9 ,wit hAP H instead of HU. (H h): 3 fiM APH; (^-)K): 6fiM APH; (B"B): 15 IJM APH; (—-*--): 30fiM APH; (O 0):60/*MAPH- 10 15 20 Timeafte rwashin g (h) Table 1. Relation between Time after HU Concentration (mM) MI and MNI at various washing (h) 1 2 5 10 20 times after washing the MI MNI MI MNI MI MNI MI MNI MI MNI suspension cell cultures 0 9.1 0 4.7 0 0.4 0 0 0 0 0 free from HU orAPH . The 6 10.0 0 4.2 0 0.6 0 0 0 0 0 concentrations of HU and 11 14.2 3.4 14.7 3.9 2.7 0.3 0 0 0 0 APH are indicated. 15 20.6 3.4 19.6 1.9 17.1 0 0.7 0 0.4 0 21 25.1 18.5 34.9 19.3 48.9 6.1 25.7 0 3.7 0 26 24.4 31.8 18.9 39.0 31.9 26.9 51.6 6.4 36.8 2.2 APH Concentration (p M) 3 6 15 30 60 Ml MNI MI MNI MI MNI MI MNI MI MNI 0 13.3 0 5.8 0 2.8 0 0 0 0 0 6 11.0 0 6.9 0 2.6 0 0 0 0 0 11 27.9 2.2 24.5 1.0 3.3 0.3 0 0 0 0 15 43.6 2.9 52.6 1.8 48.5 0.2 50.2 0 12.0 0 21 27.7 25.0 26.0 30.5 41.0 16.3 53.7 7.6 57.4 0.3 26 25.9 29.0 12.8 43.8 9.4 45.2 20.1 38.1 31.1 24.2 70 Divided cells (%) Fig.11 .Th epercentag eo fdivide d cells following HU treatment, determined byth e sum of theM I andth eMNI .(••••-) - }:untreate d control; (-| h): 1 i»M HU; (•#-*•): 2m MHU ; (B~B): 5 mM HU; (X *••): 10 mM HU; (O O):20mMHU. 10 15 20 Timeafte rwashin g (h) Dividedcell s (%) Fig. 12. As Fig. 11, after APH treatment. (-] 1-): 3fiM APH; (•*-*•): 6 AIM APH; (-B-B): 15 pM APH; (y V): 30 fiM APH; (O 0-):6 0f*M APH . Timeafte rwashin g (h) after washing), and the mitotic index rises treatments result in comparable levels of sharply at t= 6 1h after subculture for the 5 mitotic index, but with HU the increase is mM HU and the 15, 30 and 6f>M APH slower as compared to APH treatments, treatments. A significantly longer period is resulting in a less efficient accumulation of necessary to recover from the inhibition metaphases and an insufficiently separated after 10 and 20 mM HU treatments. occurrence of micronuclei and metaphase Maximum mitotic index (about 50%) is chromosomes.Th erelatio nbetwee n MIan d reached at 67 h after subculture (21 h after MNI is represented in Table 1. The table washing)fo rth e HU treatments,wherea s all shows,tha t a high MIi s a prerequisite for a APH treatments exhibit the maximum high MNI, and that there is a delay of 12h mitoticinde x(5 3% )fro m t= 6 1t ot = 67 ,an d between the rise in MI and the rise in MNI show a sharp decrease at t = 72 after (see alsoFigs .9 and 10).Whe n the MIrises subculture (26h after washing). slowly, as after 10 and 20 mM HU treatments, the appearance of metaphases Remarkable is the rapid decrease of the andmicronucle ii sinsufficientl y separated in mitotic index due to formation of time. In both treatments, a high MI (40%) micronuclei (Figs. 9 and 10).H U and APH occurs simultaneously with a high MNI 71 Percentageo fcell s Fig. 13. Influence of the delayed addition (6 h after washing, instead of immediately after washing) of 32 fiU APM on the MI and MNI. (.)K-..)](.) :MI , APM added at t= 0 ; (•Q--B-): MNI, APM added at t= 0 ; ()K )K-):MI , APM added at t= 6 afte r washing; (3-0): MNI, APM added at t= 6 after washing. Timeafte rwashin g (h) (6%).Treatmen twit h 15fiMAPH ,however , treatment, is due to incomplete recovery shows a very sharp transition from from inhibition. A similar effect can be metaphase arrest to accumulation of observed with 60fiM APH, which reaches micronuclei between t= 15 and t =2 6 h the 55% level later than all other APH after washing (MNI rises from 0% to45% , treatments. while mitotic index decreases from 48% to 9%).I n allcase s agoo d yield of micronuclei Fig. 13show s the effect of addition of APM is achieved when mitotic index shows a at6 h afte r washing,compare dt oadditio no f synchronous increase, whereas a poor APM directly after washing, on the mitotic micronucleation is obtained after a slow index and the occurrence of micronuclei. increase of mitotic index. From Figs. 9 and The graphshows ,tha t iti spossibl et o obtain 10 can be concluded, that micronucleus a cell suspension with a high mitotic index induction requires an exposure time of and a low micronucleus index by adding mitoticcell st oAP Mfo r atleas t 11 h, before APM at 6 hours after washing, without a significant transition from mitotic cells to affecting the level of the mitotic index. micronucleated cells takes place. Flowcytometri canalysi sreveale dth e strong The total number of mitotic cells and inhibition of cellular DNA synthesis by 10 micronucleated cells(M Ian dMNI added )i s and2 0m Mconcentration s ofH U and3 0an d represented in Figs. 11an d 12,fo r HU and 60,«M AP H at the time ofwashin g (Figs.1 4 APH respectively.Th e graphs show that for and 15, respectively). In all treatments the all of the treatments except for 20m M HU, G2 phase is completely absent, which is in thetota lnumbe r ofmitoti ccell sha sreache d agreement with the data on mitotic index. a value of about 55%, which is in good Lower concentrations did not inhibit DNA agreement withth etota l number of dividing synthesis completely, as is demonstrated by cells,determine d earlier.Th elowe rvalu eo f the presence of a G2 peak in the DNA mitotic cells obtained with 20mM HU histograms. At the time of flow cytometric 72 t=0 t=6 t=11 t=15 t=21 1 mM 2 mM 5 mM 10 mM 20 mM Fig. 14. DNA histograms of the cellular DNA content at the time of washing (t= 0 ) and later, for all concentrations ofH U tested.Th e DNAconten t ispu t onth e horizontal axis,an d thenumbe r ofcell so nth e verticalaxis . Thepositio no fth eG1/G 0leve li sindicated ,a swel la sth epositio n ofth eG 2level . analysis, DNA synthesis had already 4 at t= 7 2 h after subculture (26 h after restarted during the very short duration of washing). protoplast isolationprocedur e inth e2 an d5 mMH U treatments,an di nth e 15,30an d 60 The duration of S and G2-phase can be fiMAP Htreatments ,a sca nb esee nfro m the deduced from the histograms of the 30^ M number of cells passing through S-phase at APH treatment. Since inhibition was still t= 0 (arrows in t= 0 row). The number of 100% with this treatment at the time of cyclingcell s isdetermine d byth e difference washing, and most of the cells were already in the number of G1/G0 cells in the 60 f*M passing through S-phase at the time of APH treatment sample at t= 21 h after analysis, this represents the least affected washing with the number of G1/G0 cells in situation in this experiment. Six hours after the same treatment, at t= 0 after washing, washing, all cycling cells (> 80%) had which is 80%. This number is in good already reached the G2-phase (Arrows in agreementwit hth enumbe rindicate d inFig . t= 6 row) .Thi s indicatestha t S-phase lasts6 73 <îl/0 G2 t=0 t=6 ik IA LiÂ. t=11 Li Li Lx L A t=15 t=21 3 MM 6 MM 15 pM 30 MM 60 UM Fig. 15.A s Fig. 14, but with APH instead of HU. ho rless .Th esam etim ewa srequire dfo r the Discussion passage through S-phase with the 60fiM APH treatment. It took less than 11 hours before the MIstarte d to increase, indicating Most protocols for accumulation and the occurrence of the first divisions. The isolationo fmetaphas echromosomes ,d ono t G2-phase must therefore be somewhat less provide information onth echaracteristic so f than 11hours .Th e total time necessary for thecel l materialused ,suc ha sth enumbe r of the cells to pass through S- and G2-phase cyclingcell san d the duration ofth e relevant was about 17 h. Only the higher HU stages of the cell cycle (Griesbach et al., concentrations (10 and 20 mM) required a 1982;Hadlaczk y et al, 1983;Szabado se tal. , longer time for recovery,an dfro m the DNA 1981;Matthews , 1983).T oobtai na noptima l histogramsca nb e concluded,tha t especially induction of synchronous division without S-phase was prolonged: in case of 20 mM knowledge of these parameters is almost HU evenwit h more than 6hours . impossible without performing many 74 laboriousempirica l experiments.Th eai mo f of the inhibited cells. With cell cultures our experiments was to establish efficient which were not grown to plateau phase, a procedures for the induction of less synchronous division was observed, simultaneousmitosis ,necessar yfo r themas s resulting in a lower number of cells which isolation of metaphase chromosomes, and canb earreste d duringa 2 4h treatmen t with induction andisolatio n ofmicronuclei .Bot h DNA-sythesis inhibitors.A s a consequence, approaches require levels of mitotic cells the history of the culture together with its andmicronucleate d cells,a shig ha spossible . growth characteristics are important Cell cycle synchronization implicates that parameters,determinin gth emaximu mleve l the cell material used is of a homogeneous of the mitotic index. compositionwit h regard to the length of the cellcycle .Onl y cellswit h the same duration Secondly, the inhibition of DNA-synthesis of thecel lcycl e canb e synchronized (Gould was examined by cytological and flow 1984). cytometric DNA analysis. Flow cytometry However, a truly synchronous passage has the advantage, that large numbers of through the cell cycle is not required for cells can be analysed with a high degree of isolation of metaphase chromosomes and accuracy in aver y short time (Crissma n and micronuclei, since for thispurpos e a double Tobey, 1974;Galbraith et al., 1983; inhibition can be applied, using both DNA Bergounioux et al., 1988). Especially the synthesis blockers and metaphase arresting effects of DNA-synthesis inhibitors can be agents. analysed readily (Foxe t al., 1987).Fro m the results,presente d in this paper, conclusions Firstly, the number of actually cycling cells can be drawn on the number of cycling cells during the period of accumulation was (> 80%), together with their cell cycle determined, since this number limits the parameters (S-phase 6 h; G2-phase 11 h), maximum accumulation of cells at a given without the need of radioactive treatments stage of the cell cycle. For thispurpose , cell in labelling studies (Wang et al., 1986).Th e division activity was determined by APM information obtained is also relevant for a addition at different times after subculture, more efficient way to establish procedures to otherwise identically treated cultures.B y for accumulation of mitotic cells. Since the counting accumulated metaphases during number of cycling cells can be estimated, treatment, and later also micronucleated either cytologically or by flow cytometry cells,a n accurate picture wasobtaine d from during any period, the best period for division activity at various times during the DNA-synthesis inhibition can be defined. experiment. It turned out that the cells Furthermore, the effect and the duration of needed 30h befor e theyha drecovere d from the treatment can be analysed easily, the starvation treatment, resulting in a allowing the assay of many chemicals and sudden increase in mitotic activity between their concentrations. t= 3 0an d t= 46 .Thi s period coincided with DNA synthesis inhibition, and resulted in The results from the present analysis show accumulation of more than 50%o f the cells that both HU and APH are efficient DNA in or at the beginning of S-phase, when synthesis blockers, but at completely inhibition was sufficiently strong. The cells different concentrations. going into division after t= 46 , were only The effects of both HU and APH treatment partially accumulated: only the cells which arefull y comparable.A tlo wconcentrations , would have been in S-phase during the the inhibition lasts only a short time, due to inhibition would have accumulated. Since degradation of the inhibitor in the case of S-phase lasts for about 6 hours, this will APH (Sala et al., 1983). A degradation result in afurthe r increase with 5% to55 % mechanism for HU is not known, but our 75 datasugges t that such amechanis m existsi n isolation of metaphase chromosomes, thecel lsupensio no fN.plumbaginifolia. A sa micronuclei and microprotoplasts consequenceo fth edecrease dinhibition ,th e (Verhoeven et al., 1988). simultaneous division of the treated cell suspension is impaired. Acknowledgements The difference in time, necessary for maximum inhibition may reflect the difference in polarity of both compounds. H.C.P.M. van der Valk is supported by a HU ishighl ypolar , dissolves easilyi nwater , grant from the "Biotechnology Action and penetrates membranes only slowly, Programme" of the CEC, contract whereasAP H ishighl yapolar ,dissolve sonl y BAP-0083-NL. The authors thank Dr. W.J. as a trace in water, and crosses membranes Stiekema and Dr. L.va n Vloten-Doting for easily.Wit hAPH ,a highe r mitoticinde xca n critical reading of the manuscript. The be obtained than with HU, and skilful assistance of Mrs. Y. Nöllen is synchronization is also better, resulting in a gratefully acknowledged. better control of the appearance of micronuclei.Thi si s due to the fact that HU References does not arrest cells at the beginning of S-phase, but somewhere in S-phase, resulting in poor synchrony after release of Anderson, H.C. (1983). Hydroxyurea the HU block (Walters et al., 1976).AP H is induces sister chromatid exchanges in G2: theinhibito ro fchoic efo rth ecel llin etested , implications for the formation of because of its low cytotoxicity and the fact chromosomal aberrations. Hereditas 9_&: that DNA repair is not affected (Sala et al.,' 61-64. 1982). Barfield, D.G., Robinson, S.J., Shields, R. With the procedures described, suspension (1985). Plant regeneration of long term cultures of Nicotiana plumbaginifolia and haploid suspension cultures of N. other plant species (Sree Ramulu et al., plumbaginifolia. Plant Cell Rep.4:104-107 . 1988b) can be analyzed rapidly, resulting in aprotoco l for theinductio n of simultaneous Bergounioux, G, Perennes, C, Brown, S.C., division. The pretreatment with DNA Sarda, C, Gadal, P. (1988). Relation synthesis inhibitors (lOmM HU or 15nM between protoplast division, cell-cycle stage APH during 24h) ,followe d bywashin g and and nuclear chromatin structure. the addition of APM, results in a Protoplasma 142: 127-136. synchronous increase of MI up to 50%. Twelve hours after its maximum, MI Coyle, M.B., Strauss, B. (1970). Cell killing decreases and the MNI rises to 40%. By and the accumulation ofbreak s in the DNA varyingth emomen t ofadditio n ofAPM ,th e of HEp-2 cells incubated in the presence of time at which metaphase chromosomes or hydroxyurea. Cancer Res.2Û : 2314-2319. micronuclei appear can be controlled accurately. In this way, metaphase Crissman, H.A., Tobey, R.A. (1974). chromosomes can be isolated with low Cell-cycle analysis in 20 minutes. Science contamination by micronuclei, and vice 184: 1297-1298. versa. Furthermore, both the number of micronucleated cells and the number of de Laat, A.M.M., Blaas, J. (1984). Flow micronuclei per cell are significantly cytometric characterization and sorting of improved after cell cycle arrest. This may plantchromosomes .Theor .Appl .Genet .6_Z : open up the way for a more efficient 463-467. 76 de Laat, A.M.M., Schel,J.H.N . (1986).Th e Guri, A., Zelcher, A., Izhar, S. (1984). integrity of metaphase chromosomes of Induction of high mitotic index in Petunia Haplopappus gracilis(Nutt. ) Gray isolated suspension culturesb ysequentia l treatment byflo w cytometry. Plant Sei.4J:145-151 . with aphidicolin and colchicine. Plant Cell Rep.2 :219-221 . de Laat, A.M.M., Blaas, J. (1987). An improved method for microinjection of Hadlaczky, G., Bisztray, G., Praznovsky, T., plantprotoplasts .Plan tScienc e 50:161-169. Dudits, D. (1983). Mass isolation of plant chromosomes and nuclei. Planta 15JZ: de Laat, A.M.M., Verhoeven, H.A., Sree 278-285. Ramulu, K., Dijkhuis, P. (1987). Efficient induction by amiprophos-methyl and flow Keil, R.L., Chaleff, R.S. (1983). Genetic cytometric sorting of micronuclei in characterization of hydroxyurea-resistant Nicotiana plumbaginifolia. Planta 172: mutants obtained from cell cultures of 473-478. Nicotiana tabacum. Mol. Gen. Genet. 192: 218-224. Eriksson, T. (1966).Partia l synchronization of cell division in suspension cultures of Kiermayer, O., Fedtke, C. (1977). Strong Haplopappus gracilis. Physiol.Plant. 19.: anti-microtubule action of 900-910. amiprophos-methyl (APM) in Micrasterias. Protoplasma 22: 163-166. Fox, M.H., Read, R.A., Bedford J.S. (1987). Comparison of synchronized Chinese Kihlman, B.A. Eriksson, T., Odmark, G. hamster ovary cells obtained by mitotic (1966). Effects of hydroxyurea on shake-off, hydroxyurea, aphidicolin, or chromosomes, cell division and nucleic acid methotrexate. Cytometry &315-320 . synthesis in Vicia faba. Hereditas 5_5_: 386-397. Galbraith, D.W., Harkins, K.R., Maddox, J.M., Ayres, N.M., Sharma, D.P., King,P.J .(1980) .Plan ttissu ecultur ean dth e Firoozabady, E. (1983). Rapid flow cell cycle,in : Fiechter A. (ed.) Advances in cytometric analysis of the cell cycle in intact biochemical engineering IS: 1-38. Springer plant tissue.Scienc e 22Q: 1049-1051. Verlag, Berlin. Galli, M.G., Sala, F. (1982). Aphidicolin as Matthews, B.F. (1983). Isolation of mitotic synchronizing agent inroo t tipmeristem s of chromosomes from partially synchronized Haplopappus gracilis. Plant Cell Rep. 2: carrot (D.carota) cell suspension cultures. 156-159. Plant Science Letters 21: 165-172. Gould,A.R . (1984).Contro l ofth e cellcycl e Puite, K.J., Ten Broeke, W.R.R. (1983). in cultured plant cells. In: CRC critical DNA staining of fixed and non-fixed plant Reviewsi nPlan t Sciences.Ed :Conger , B.V. protoplasts for flow cytometrywit h Hoechst CRC Press, Boca Raton, FL.pp .315-344 . 33342.Plan t Sei.Lett . 22: 79-88. Griesbach, R.J., Malmberg, R.L., Carlson, Sala, F., Parisi, B., Burroni, D., Amileni, P.S. (1982).A n improved technique for the A.R., Pedralli-Noy, G., Spadari, S. (1980). isolation of higher plant chromosomes. Specific and reversible inhibition by Plant Sei.Lett . 21: 55-60. aphidicolin ofthea-lik eDN Apolymeras eo f plant cells.FEB S letters HZ:93-98 . 77 Sala, F., Magnien, E., Galli, M.G., Louvain-la-neuve, March 23-26, pp. Dalschaert, X.,Pedrali-Noy ,G. , Spadari, S. 119-120. (1982). DNA repair synthesis in plant protoplasts is aphidicolin-resistant. FEBS Verhoeven, H.A., Sree Ramulu, K.,va n der lettersI2S :213-217 . Valk, H.C.P.M., Dijkhuis, P., Blaas, J. (1988). Isolation of microprotoplasts. In: Sala, F., Galli, M.G., Nielsen, E., Progess in Plant Protoplast research. Ed: Pedrali-Noy, G., Spadari, S. (1983). Puite, K.J., Dons, J.J.M., Huizing, HJ., Inactivation of aphidicolin by plant cells. Kool, A.J., Koornneef, M., Krens, F.A. Plant Cell Rep.2 :265-268 . Kluwer Academic publishers, Dordrecht, Boston, London,pp .297-298 . Sree Ramulu, K., Verhoeven, H.A., Dijkhuis, P. (1988a). Mitotic dynamics of Walters, R.A., Tobey, R.A, Hildebrand, micronuclei induced by amiprophos-methyl CE. (1976). Hydroxyurea does not prevent and prospects for chromosome-mediated synchronizedG l Chinesehamste rcell sfro m gene transfer inplants .Theor .Appl . Genet. entering the DNA synthetic period. 25_: 575-584. Biochem. and Biophys. Res. Comm. 6_9_: 212-217. Sree Ramulu, K., Verhoeven, H.A., Dijkhuis, P., Gilissen, L.J.W. (1988b). Wang, A.S., Phillips, R.L., Mi, C.C. (1986). Chromosome behaviour and formation of Cell cycle parameters and accumulation of micronuclei after treatment of cell metaphase cells in maize suspension suspension cultures with cultures. Plant Science,4 & 53-61. amiprophos-methyl invariou splan t species. Plant Science 5_6_: 227-237. Weber, G., Schweiger, H.G. (1985). Synchronization of cells from higher plants. Szabados, L., Hadlaczky, G., Dudits, D. Naturwissenschaften 22:438-439 . (1981). Uptake of isolated plant chromosomes by plant protoplasts. Planta 151:141-145. Verhoeven,H.A. ,d eLaat ,A.M.M. ,Blaas ,J . (1986). Methodological development for chromosome-mediated gene transfer in higher plants, in: Magnien, E. (ed.) Biomolecular engineering in the European Community. Martinus Nijhoff Publishers, Dordrecht, Boston, Lancaster. Verhoeven, H.A., de Laat, A.M.M., Sree Ramulu, K., Dijkhuis, P., van Vloten-Doting, L. (1987). Induction, isolation, and flow cytometric sorting of metaphase chromosomes and micronuclei from cell suspension cultures of Nicotiana plumbaginifolia. In: Genetic and cellular engineering of plants and micro-organisms important for agriculture, Commission of the European Communities meeting, 78 CHAPTER6 ISOLATION AND CHARACTERIZATION OF MICROPROTOPLASTS FROM APM-TREATED SUSPENSION CELLS OF NICOTIANA PLUMBAGINIFOLIA. HA,Verhoevenan dK . SreeRamul u 79 ISOLATION AND CHARACTERIZATION OF MICROPROTOPLASTS FROM APM-TREATED SUSPENSION CELLS OF NICOTIANA PLUMBAGINIFOLIA. HAVerhoevenan dK . SreeRamul u ResearchInstitut eItal ,P.O.Bo x48,670 0A AWageningen ,Th eNetherlands . Summary Introduction Aprocedur e isdescribe d for the isolationo f Recent progress in the field ofplan t genetic subprotoplasts with a DNA content of less manipulation hasresulte d ina wid erang eo f than the Gl level, so called micro- techniques for the introduction of foreign protoplasts.Microprotoplast s consist of one genes. Protoplast fusion can be used when or more micronuclei surrounded by a thin polygenically determined complex rim of cytoplasm and an intact plasma characters are of interest, or when the membrane. Micronuclei are induced after molecularbasi so fa particula r trait isno tye t treatment of a suspension culture of known. However, since the entire donor N.plumbaginifolia with amiprophos-methyl genome is added to the recipient, the (APM). Protoplasts are fractionated on a resulting hybrid constitutes a complex continuous iso-osmotic gradient of Percoll, genetic system, which can not be analyzed resulting insevera lvisibl e bands.Th e bands properly. Therefore, X-ray irradiation is were analyzed, and data on cytology, DNA often applied to induce chromosome loss content of individual (sub)protoplasts, from the donor (Bates et al., 1987). Recent vitality and membrane integrity are work has shown however, that a large presented.Th eresult srevea lth epresenc eo f proportion of the fragmented genome up to 40% microprotoplasts in the fraction, incorporates as small fragments into the just below the major visible band. These recipient genome (Imamura et al., 1987). microprotoplasts have DNA content less than Gl level, some showing an amount Mammalian somatic cell genetics employed equivalent to that of one or a few twoadditiona ltechnique sfo rgen emapping : chromosomes. The application of chromosome mediated gene transfer microprotoplasts for chromosome mediated (Klobutcher and Ruddle, 1981) and gene transfer inplant s is discussed. microcell-mediated chromosome transfer (Fournier, 1982). So far, only Key words: Cellular manipulation, microcell-mediated chromosome transfer Micronucleus, Microprotoplast, has resulted into stable maintenance of Evacuolation, Chromosome-mediated gene donor chromosomes,whil e still transferring transfer. only a small fraction of the donor genome, even single chromosomes (Lugo and 81 Fournier, 1986).Th eprocedur e involves the Materials and methods induction of micronucleation in cell lines carrying selectable markers, through modified C-mitosis after colchicine or Cellculture Colcemid treatment. Single or groups of chromosomes undergo decondensation and A suspension cell culture of N. form micronuclei. Subsequently, microcells plumbaginifolia, kindly provided by Dr. are prepared by either treatment with Shields, Unilever Res. Lab., Colworth cytochalasin-B, or by centrifugation in the Home, Sharnbrook, Bedford, UK was presence of cytochalasin-B, and fused with cultured in the dark at 28°C in "Doba" the recipient cells (Ege et al., 1977; medium (Barfield et al., 1985)o n agyrator y Fournier, 1981). shaker (120rpm) .Fro m thiscel l suspension, a kanamycine resistant line was developed Until recently, no treatment or agent after transformation withA. tumefaciens as (physical or chemical) was known for the described before (de Laat and Blaas, 1987). efficient induction of micronuclei in plants, This cell line (designated as 1-125-1) is a which prevented the development of a tetraploid and responds well to treatments microprotoplast system, analogous to the with cell synchronizing agents, such as microcell system in mammals. Since the hydroxyurea (HU) and aphidicolin (APH). finding, that the herbicide APM induces micronuclei at high frequency in cell APM treatments suspension cultures of N.plumbaginifolia (Verhoeven et al., 1986,1987;d eLaa t et al., Amiprophos-methyl (Tokunol M ; 1987), the application of microprotoplasts 0-methyl-0-0-(4-methyl-6-nitrophenyl)- for limited genome transfer became N- isopropyl-phosphorothioamidate) feasible. APM is efficient in mass induction (Kiermayer and Fedtke, 1977)wa sobtaine d of micronucleation and induction of high from Bayer Nederland B.V., Divisie number of micronuclei per micronucleated Agrochemie, Arnhem, The Netherlands. cell.I t interferes with the polymerization of Stocksolution s ofAP Mwer eprepare d at2 0 plant cortical and spindle microtubules mg/ml in water-free DMSO, and could be (Morejohn and Fosket, 1984;Falcone r and stored for at least 6month s at -20CCwithou t Seagull, 1987). Normal mitosis and growth any appreciable loss of activity. were resumed after removal of APM by washing out and subculturing of cells, Micronucleation demonstrating that its inhibitory effect on the mitotic spindle is reversible (Sree Micronucleiwer einduce d byth e additiono f Ramulu et al., 1988a). The induction of APM at 32 fiM to cell suspensions on the micronucleib yAP Mwa sobserve d ina wid e second day after subculture. For optimal range of genotypes and species (Sree synchronization, this treatment was Ramulu et al., 1988b). preceded by a 24 h incubation with 10 mM HU. After that, cellswer ewashe d free from Inthi spaper , we report on the isolation and HU andAP Mwa sadde d toth efres h culture characterization of microprotoplasts from medium. APM treated suspension cultures of N. plumbaginifolia. Some preliminary results Protoplast isolation have been communicated before (Verhoeven et al., 1988). Protoplasts were isolated by treating 1g o f suspension cells with an enzyme solution, containing 5% Cellulase RIO and 1% 82 Macerozyme RIO, and 0.4 M mannitol as Flowcytometry osmoticum. To prevent the fusion of micronuclei during protoplast isolation, All measurements were made with a Partec APM (32 nM) was added to the enzyme PAS II flowcytometer, equipped with a mixture.Cytochalasin- B(10mg/l )wa sadde d HBO-100 mercury lamp. Protoplast to disrupt microfilaments during protoplast membrane integrity was assessed by isolation. Protoplasts were purified as incubation of protoplasts with 10 ,«g/ml described before (de Laat et al., 1987). fluorescein-diacetate (FDA) prior to centrifugation. Sampleswer ecollecte d from Centrifugation thegradien t and analysedfo r the fluorescein content of the fractionated protoplasts. For Continuous iso osmotic gradients of Percoll this analysis, the FTTC filter-set (BP 450, were prepared by adding 7.2% (w/v) of dichroic mirrors TK500 and TK590 with mannitol to Percoll solution (Pharmacia), BP520a sbarrie r filter) was used. and centrifuged during 30 min in a Christ Omega ultra centrifuge (170,000 g), using Nuclear DNAconten t ofsubprotoplast swa s the 6X5m l swing-out rotor. For calibration determined bydilutin gth esample sfro m the purposes,densit ymarke rbead s (Pharmacia, gradient with the Partec stain solution for code 17-0459-01) were used. The top layer milkcell s(1 0volume so fbuffe r on 1volum e of2- 5m mwa sremove d from the preformed of cells). The filter combination of UG5, gradient, and the protoplast suspension TK420,TK59 0 and GG435wa s used. containing 10 mg/1 cytochalasin-B was For calibration of the histograms, nuclei layered on top. Cell fractionation was isolated from leaves of control achieved by centrifugation during 1 h at N.plumbaginifolia (2n = 2x = 20) plants, 170,000 g at a temperature of 25 °C. The stained with the same buffer were used. gradient was collected in small samples using a peristaltic pump. For cytological Subprotoplastculture analysis of subprotoplasts, 1 ftg/nû 4',6-diamidino-2-phenylindole (DAPI, To determine viability, about 500 Sigma chemical co.) was added to the subprotoplasts, collected from several gradient. fractions of the gradient were cultured at very low densities (1000protoplast s per ml) Microdensitometry in Millicell-CM Culture plate Inserts (Millipore, Cat. No. PICM 01250). The Feulgen microdensitometry in combination Inserts were put in a 5 cm petridish, withcytologica lanalysi so fth e celltype swa s containing3 m l of K3cultur e medium (Kao carried outt odetermin eth erelativ e nuclear et al., 1974) with 10 protoplasts/ml of DNAconten t ofth eindividua lprotoplast si n N.plumbaginifoliaa s feeder. each fraction. Samples were fixed by the addition of formaldehyde to a final Results concentration of4% .Fixe dprotoplast swer e collected onto amembran e filter (Millipore SCWP 02500) or on object slides, coated Isolationof micronucleated protoplasts with eggprotein-glycerine. Feulgen staining and microdensitometry were carried out as Prolonged exposure of suspension cells of described earlier (Sree Ramulu et al. 1984). N.plumbaginifoliat oAP M resulted ina hig h frequency of micronucleation. With APM only, up to 15 % of the cells became micronucleated. After HU treatment, 83 Micronucleatedcell s (%) Density (g/ml) Hourso fincubatio n Distancefro mmeniscu s (mm) Fig. 1. Percentage of cells with micronuclei after Fig. 2. Density profiles of Percoll solution before treatment with APM (32/nM) only( X X) or after centrifugation ( ), after 30 min. preformation treatment with HU (10m M during 24h) , followed by at 170,000 g (XX) and after protoplast washingan dAP M (32pM) treatment (Q •) incel l fractionation (90 min. at 170,000 g) (~f—|-). The suspension cultures of kanamycin resistant arrow indicates the position of the main band of N.plumbaginifolia. The 0 h point refers to the starting subprotoplasts after protoplast fractionation. of incubation time after 24 h HU treatment and washing. followed byth e addition ofAPM , this value micronucleated protoplasts (data not was more than doubled (Fig. 1), and shown). micronucleationwa smor e efficient in terms of number of nuclei per cell (data not Fractionationof protoplasts shown). During protoplast isolation, the addition of APM (32 pM) to the enzyme After a short incubation in 0.4 M mannitol mixture resulted in the maintenance of the with 10 (Mg/ml cytochalasine-B, the number of micronucleated cells at the same protoplasts were layered on top of the levela si nth etreate d suspensioncells .I nth e preformed Percoll gradient.Th e preformed absence of APM, micronuclei in the gradient was analyzed, using the Density protoplasts tend to fuse together, forming Marker Beads. After centrifugation, the multilobed nuclei. The addition of patterno fth ebead swa sagai nrecorde d (Fig. cytochalasin-B induced a slight but 2). In thisway ,eac h band was related to the significant increase in the number of specific density of the gradient at the positiono fth eband .Fig .3 give sth e relation 84 Fig.3 .Diagra mo fth epositio n of the band in a Percoll ' a gradient after protoplast 1 ' b fractionation. The bars • c illustrate the position of the 2 ' d e fractionsan dth ecode suse di n 3 f thetext . 4 , g h ' 5 i Band 6 j ; 7 k 8 1 ; 9 m 10 n : 11 0 12 P 13 . 14 d . 15 r 16 s Solid Percoll DAPI/FDArati o betweenth efractio n number andpositio ni n 1000 the gradient. Fractionation of protoplasts : was carried out in four different :.".-.•:. ::•./:•. experiments. For microdensitometric and /•••• cytological analyses, the lower fractions of / 100 the gradient (I-IV) were used. For more detailed analysis, the whole gradient was ':•••' separated into 16 fractions (1-16). Flow cytometric analysis was performed with 10 ir 0 another gradient separated into 19 fractions :-. . - (a-s). • •.. / Flowcytometric analysis •••:•>.$. A Protoplasts, stained with FDA were ;Q^— ~^ analyzed by flowcytometer directly after isolation. More than 95%o f the protoplasts wereintensel y stained,an d thusintact . Each 0.1 ! I I I I I I of the 19 fractions (a-s) was split into two abcdefghijklmoprs equal parts, one used for analysis of the Fraction number fluorescein content, and the other for determination of the nuclear DNA content. Fig. 4b. Ratio of the number of DAPI-positive The visible bands were collected into subprotoplasts and the number of FDA-positive different samples.Th eban di nfractio n cwa s subprotoplasts. DAPI only stains nuclei, and FDA onlyvisibl eafte r FDAstainin go na U V-box , only stains subprotoplasts with an intact plasmamembrane. The numbers were derived from andcontaine d onlyvacuole san dvacuoplast s thediagram s infig 4a . withver yfe wcontaminatin g nuclei(Fig .4a) . The next band (fraction k) consisted of evacuolated protoplasts with varying DNA 85 FDAIntensit y DAPIIntensit y contents (Fig. 4a).Thi s band contained the highest number of subprotoplasts. The nuclear DNA content of the subprotoplasts was determined by DAPI staining, in combination with Triton-XlOO,t o make the membranes permeable to DAPI, which cannot cross an intact plasma membrane. The number of particles still containing fluorescein after centrifugation, is higher than the DAPIpositiv eparticle s in fractions a to k (fraction k contains the major band), while these numbers are equal in fraction 1 (Fig. 4a). Down from fraction 1, less FDA positive structureswer eobserve d compared to the number of DAPI positives.Th e ratio ofth enumbe ro fDAP Ipositiv eparticle san d BÉitouttiÉ—HÈÉkiÉiiite-. . the number of FDA-positive particles is represented in Fig. 4b. This graph shows, Gl G2 that allsubprotoplast s inth e mainband ,an d in the fractions just below the main band (from fraction a down to and including fraction 1), still possess an intact plasma membrane (Ratio < 1). Fractions m-r contain increasing numbers of damaged subprotoplasts or isolated nuclei. ik à, ülL Microdensitometry Microdensitometric analysis revealed the presence of relatively high numbers of microprotoplasts in orjus t below the dense L band, depending on the size of the fraction. Fig. 5 shows the cumulative distribution of the DNA content of intact looking subprotoplasts of the band (fraction I) and MihfcMtl» tm, thethre elowe rregion s(fraction s II-IV),an d Fig.6 show sthi sare adivide d over 9 samples (fractions 6-16). These data show that just below the main band (fraction 6) a high proportion (upto 40%) of intact microprotoplasts with less than Gl DNA Membrane integrity DNA content content was observed in fraction 7 (Fig. 6). In Fig. 5 a lower frequency (20%) of Fig. 4a. Intact subprotoplasts, determined by microprotoplasts with 86 %o fcell s 100 10 15 20 25 30 35 40 45 DNAconten t (arbitrary units) Fraction 1 I Fraction 2 ~~7[\ Fraction 3 I [ Fraction4 Fig. 5. Cumulativedistributio no fnuclea rDN Aconten t ofsubprotoplas t isolatedfro m gradientfraction s I-IV. Thefrequenc y (%)o feac hvalu eo fDN Aconten t was added to the precedingvalu e to obtaincumulativ eDN A distribution.Th ebar sindicat eth eG lan d G2 DNA levels, as calculatedfro m the DNAhistogram sfro mdiploi d leafnucle ifro m N.plumbaginifolia, measured by microdensitometryunde r thesam econditions . % of subprotoplasts 00 G0/G1 G2/M rtdgK^H*^ 80 - 60 - ,fy«£pH^ ..../ ï-L_-—-~"''"~^ 40 - u— ^7\ 20 ,/ , , r—i I 1 ,^f 0^ Ep"l i I i I I I I I I I I I I I ! ' i M 1.-1.11 i 111 i i i | 11 i..| i 11 10 20 25 30 35 40 45 55 DNAconten t (arbitrary units) Fraction 7 I FractionS Fig.6 . Cumulativedistributio no fnuclea rDN Aconten to fsubprotoplast sisolate dfro m gradientfraction s6-16 . Fractions 13-16 gave similar patters of DNA distribution and therefore only the data on fraction 13 are presented,a stypica lexample .Bar sindicat eth eDN Aconten t of the Gl andG 2level . 87 Table 1.Frequenc y ofvariou s typeso f subprotoplasts isolated from gradient fractions I-IV and the number ofmicrocall i developedfro m cultured subprotoplasts. percentage distribution of various types of subprotoplasts ingradien t fraction types of sub Fractions protoplasts I II III IV Cytoplasts 7 9 6 9 Evacuolated 70 84 86 78 protoplasts Micro pps < G2 2 3 4 4 Micro pps < Gl 21 4 4 9 Number of microcalli Microcalli XX 90 49 35 XX:Los t duet o contamination. Cytology All cell types were recorded, together with the nuclear DNA content, allowing the analysis of the different subprotoplasts (Table 1). The percentage of cytoplasts is almostconstan t inal lfou rfractions , whereas the frequency ofsubprotoplast s with a DNA content lesstha nth e Gl leveli shig honl yi n fraction I,whic h contains the dense band of \ the gradient. The analysis of the DAPI stained subprotoplasts showed the presence of microprotoplasts with single micronuclei, butals osom ewit hmultipl emicronucle i (Fig 7a,b). Fig 7c shows an incompletely fractionated protoplast, which already lost its vacuole. Micronuclei can be seen on a thin thread of cytoplasm, pulled out of the Fig. 7. Photomicrograph of a microprotoplast after evacuolated protoplast. centrifugation. a: phase contrast image; b: fluorescence image, showing the DAPI-stained micronuclei; c: incompletely fractionated Subprotoplastculture microprotoplast, with the nuclei visible on the strands of cytoplasm. From the fractions I, II, III and IV, respectively 1000, 1000, 200 and 35 FDA positive subprotoplasts were isolated, and cultured inMillicell-C MPlat eInserts ,usin g N.plumbaginifolia cell suspension derived higher yield of evacuolated, intact protoplastsa sfeede r inth epetri-dish .I nthi s subprotoplasts and better fractionation way, microcalli were obtained from each under the present experimental conditions. fraction tested, except fraction I, which was Themai nban d appeared tocontai n 100%o f lost due to contamination (Table 1). The evacuolated, intact (sub)protoplasts, as can microcalli were transferred to solid bededuce d from thedat ao nflo wcytometry . MS-medium with 1 % sucrose when they The upper band, with aver ylo w percentage were 0.5-1m m in diameter. of nuclei contains only vacuoplasts. This is evident from the fact that only vacuoles Discussion surrounded by an intact protoplast membrane, retain FDA efficiently (Lesney et al., 1986). From the main band Previous work with the evacuolation of downwards, the amount of FDA-positive protoplastsha sdemonstrate d theviabilit yo f structures decreases strikingly.I n fraction 1, the products of the evacuolation procedure the ratio FDA-positive: DAPI-positive (Wallin et al., 1979; Lörz et al., 1981; particles is about one, and decreases in Griesbach and Sink, 1983; Burgess and fraction m and lower. Also, particles with a Lawrence, 1985; Lesney et al., 1986). The lower DNA content than the Gl level, are different stages of the evacuolation still present in fraction 1, whereas they procedure, as reported by Griesbach and completely disappear from the lower Sink (1983), also apply to the fractionation fractions. This pattern is confirmed by the of subprotoplasts presented in this paper. cytological data, which show a high First, the protoplasts band at their proportion of subprotoplasts containing corresponding density,become s oblong due micronucleii nfractio n 1.Thi sfindin g isals o toth e accumulationo forganelle s and nuclei consistent with the results of two different at the lower end of the protoplast. The microdensitometric analyses of the vacuolepinche sof f andrises t oth eto po fth e gradients: the highest frequency of gradient. The evacuolated protoplasts are subprotoplasts with DNA content less than completely devoid of a cytoskeleton due to Gl level is found in or just below the main the action of APM and cytochalasin-B, and band, depending on the sizeo f the sample. more susceptible to deformation by the The formation of microcalli from gravity field (Fig 7.c) .Th e micronuclei can subprotoplasts demonstrates their viability. be seen as small beads on the strand of Microprotoplast culture might have a cytoplasm. By continued centrifugation, valuableapplicatio ni nth eproductio no fcel l thesecytoplas m strandsdivid et ogiv eris e to lineswit h partial genomes and in studies of individual microprotoplasts. Due to a genome function in cell differentiation. decrease in the cytoplasm to nucleus ratio, With the procedure for the isolation of the microprotoplasts will band at a higher microprotoplasts, described in this paper, a densityi nth egradien tbelo wth emai n band. system for transfer of chromosomes by microprotoplast fusion, becomes feasible. The main difference between the Up to 40% of the subprotoplasts, isolated procedures employed by the previous from the fraction just below the main band, authors,an dth eon epresente d inthi spaper , has a DNA content less than the Gl level, is the use of cytoskeleton disrupting agents, some showingamount s equivalent to thato f namely APM for the microtubuli and one or afe w chromosomes.Thi smean s that cytochalasin-B for the microfilaments. The 60% o f the protoplasts still containsa t least negative effects of cytochalasin-B on the the normal DNA content. Consequently, it regeneration of subprotoplasts, reported by is desirable to fuse and culture the Lörz et al.(1981), are compensated by the microprotoplasts in a controllable way.Th e 89 fusion of individual protoplasts, such as De Laat, A.M.M., Verhoeven, H.A., Sree developedb yKoo pe tal .(1983) ,provide sa n Ramulu, K., Dijkhuis, P. (1987). Efficient elegant system for fusion of induction by amiprophos-methyl and microprotoplasts with recipient protoplasts. flow-cytometric sorting of micronuclei in Thiswoul dope nu pth epossibilit yt otransfe r Nicotiana plumbaginifolia. Planta 172: single or a few chromosomes for gene 473-478. mapping and expression studies in plants, and for the direct construction of addition Ege, T., Ringertz, N.R., Hamburg, H., lines important for use inplan t breeding. Sidebottom, E. (1977). Preparation of microcells.in :Prescott , D.M.(Ed ) Methods Acknowledgements inCel lBiology ,vo l 15.Academi cPress ,Ne w York, London, pp.339-358 . We thank Mrs. Yvonne Nollen for her Falconer, M.M., Seagull, R.W. (1987). skilfull assistance with regard to cell Amiprophos-methyl (APM): a rapid, cultures, and Mr Paul Dijkhuis for help in reversible, anti-microtubule agent for plant cytology.Par t ofthi sresearc hwa sfunde d by cell cultures. Protoplasma 126: 118-124. the CEC "Biotechnology Action Programme", contract BAP-0083-NL Fournier, R.E.K. (1981). A general (GDF). The authors thank Dr.Lous van high-efficiency procedure for production of Vloten-Dotingan dDr .Wille mStiekem afo r microcell hybrids. Proc. Natl. Acad. Sei. critical reading of the manuscript. The USA. IS:6349-6353 . receipt of a sample of APM from Bayer Nederland B.V., Divisie Agrochemie, is Fournier, R.E.K. (1982). Microcell- gratefully acknowledged. mediated gene transfer. In: Shay,J.W . (Ed) Techniquesi nsomati ccel lgenetics .Plenu m References Press, New York, London, pp.309-327 . Griesbach, R.J., Sink, K.C. (1983). Barfield, D.G., Robinson, S.J., Shields, R. Evacuolation of mesophyll protoplasts. (1985). Plant regeneration from long term Plant Sei.Lett . 3_Q: 297-301. haploid suspension cultures of N.plumbaginifolia. Plant Cell Rep.4 : Imamura,J. ,Saul ,M.W. ,Potrykus ,I .(1987) . 104-107. X-ray irradiation promoted asymmetric somatic hybridization and molecular Bates, G.W., Hasenkampf, CA., Contolini, analysiso fth eproducts .Theor .Appl .Genet . CL., Piastuch, W.C (1987). Asymmetrie 24:445-450 . hybridization in Nicotiana by fusion of irradiated protoplasts. Theor. Appl. Genet. Kao, K.N., Constabel, F., Michayluk, M.R., 74:718-726 . Gamborg, O.L. (1974). Plant protoplast fusion and growth of intergeneric hybrid Burgess,J. ,Lawrence ,W .(1985) .Studie s on cells.Plant a 12Q: 215-227. the recovery of tobacco mesophyll protoplats from an evacuolation treatment. Kiermayer, O., Fedtke, C. (1977). Strong Protoplasma 126.:140-146 . anti-microtubule action of amiprophos-methyl (APM) in Micrasterias. de Laat, A.M.M., Blaas, J. (1987). An Protoplasma 22: 163-166. improved method for microinjection of plantprotoplasts .Plan tScienc e_5J} : 161-169. 90 Klobutcher, L.A., Ruddle, F.H. (1981). micronuclei after treatment of cell Chromosome mediated gene transfer. Ann. suspension cultures with Rev.Biochem . 5Q: 533-554. amiprophos-methyl invariou splan t species. Plant Sei.56:227-237 . Koop, H.U., Dirk, J., Wolff, D., Schweiger, H.G. (1983). Somatic hybridization of two Verhoeven, H.A., deLaat , A.M.M,Blaas ,J . selected single cells. Cell Biol. Int. Rep. 2: (1986). Methodological developments for 1123-1128. chromosome-mediated gene transfer in plants. In: Biomolecular engineering in the Lesney, M.S., Callow, P.W., Sink, K.C. European Community., achievementso f the (1986). A technique for bulkproductio n of research programme (1982-1986), final cytoplasts and miniprotoplasts from report. Ed.: Magnien, E. Martinus Nijhof suspension culture-derived protoplasts. Publishers, Dordrecht, pp.977-981 . Plant CellRe p 5_: 115-118. Verhoeven, H.A., de Laat, A.M.M., Sree Lorz, H., Paszkowski, J., Dierks-Ventling, Ramulu, K., Dijkhuis, P., van C, Potrykus, I. (1981). Isolation and Vloten-Doting, L. (1987). Induction, characterization of cytoplasts and isolation and flowcytometric sorting of miniprotoplasts derived from protoplasts of metaphase chromosomes and micronuclei cultured cells.Physiol .Plant .52 :385-391 . from cell suspension cultures of Nicotiana plumbaginifolia. In: Genetic and cellular Lugo, T.G., Fournier, R.E.K. (1986). engineering of plants and micro-organisms Microcell fusion and mammalian gene important for agriculture, Comission of the transfer, in: Kucherlapati, R., ed. Gene European Communities meeting, transfer. Plenum Publishing Corp. , New Louvain-la-Neuve, March 23-26, pp. York, pp.79-93 . 119-120. Morejohn, L.C., Fosket, D.E. (1984). Verhoeven, H.A., Sree Ramulu, K.,va n der Inhibition of plant microtubule Valk, H.C.P.M., Dijkhuis, P., Blaas, J. polymerization in vitro by the phosphoric (1988). Isolation of microprotoplasts. In: amide herbicide amiprophos-methyl. Progress in Plant Protoplast Research. Ed: Science 224:874-876 . Puite, K.J., Dons, J.J.M., Huizing, HJ., Kool, A.J., Koornneef, M., Krens, F.A. Sree Ramulu, K., Dijkhuis, P., Roest, S., Kluwer Academic Publishers, Dordrecht, Bokelmann G.S.,d e Groot, B.(1984) . Early Boston, London,pp .297-298 . occurrence of genetic instability in protoplast cultures of potato. Plant Sei Lett Wallin, A., Glimelius, K., Eriksson, T. 26:79-86 . (1979).Formatio n ofhybri d cellsb y transfer of nuclei via fusion of miniprotoplasts from Sree Ramulu, K., Verhoeven, H.A., cell lines of nitrate reductase-deficient Dijkhuis, P. (1988a). Mitotic dynamics of tobacco.Z . Pflanzenfysiol. 9J.:89-94 . micronuclei induced by amiprophos-methyl and prospects for chromosome-mediated gene transfer inplants .Theor .Appl . Genet. 25: 575-584. Sree Ramulu, K., Verhoeven, H.A., Dijkhuis, P., Gilissen, L.J.W. (1988b). Chromosome behaviour and formation of 91 CHAPTER7 DIRECT CELL TO CELL TRANSFER OF ORGANELLES BY MICROINJECTION: METHODOLOGY ANDFIRS T RESULTS. H.A .Verhoeve nan dJ .Blaas . 93 DIRECT CELL TO CELL TRANSFER OF ORGANELLES BY MICROINJECTION: METHODOLOGY ANDFIRS T RESULTS. H.A .Verhoeve nan dJ .Blaas . ResearchInstitut eItal ,P.O .Bo x48,670 0A A Wageningen,Th eNetherlands . Summary the application of microinjection of DNA fragments into plant cells and protoplasts has received considerable attention A novel technique for transfer of organelles (Crossway et al., 1986; Reich et al., 1986; between plant cellsi spresented . Organelles Spangenberg et al., 1986; Morikawa and are removed from evacuolated donor Yamada, 1985), the transfer of organelles protoplasts by micromanipulation, and has been limited to the injection of mass microinjected directly into acceptor cells. isolated chromosomes (Griesbach, 1987;d e Firstresult sobtaine dwit h thistechniqu e for Laat et al., 1987).Th e major problems using transfer of chloroplasts and fluorescently microinjection for plant organelle transfer labelled mitochondria are presented. are the rigid cell wall of plant cells, the fragile nature of protoplasts, and the Key words: Microinjection, Micro presence of large vacuoles in most plant manipulation, Organelle transfer, cells, which interfere with the demand for Complementation. large bore capillaryneedles .Althoug h some microinjection systems are suitable for Introduction transferring mass isolated organelles (Griesbach, 1987;D e Laat and Blaas, 1987), no system has been described by which The exchange of organelles between two organelles and chromosomes can be taken plant species or varieties is a complicated out of a donor protoplast and transferred procedure in conventional plant breeding. into a recipient cell. Using this approach, Transfer by means of protoplast fusion is damage to the transferred organelles might now available for some years (reviewed in be minimized. Galun and Aviv, 1986), but the undesirable nuclear genes from one of the partners have In thispape r wepresen t an injection system tob eeliminated ,e.g .b yirradiation .On ewa y whichallow sth edirec t cellt ocel ltransfe r of tolimi tth enumbe r ofpossibl e combinations organelles. Data are presented which show is to use cytoplasts as the organelle donor that protoplasts and cells survive injections (Maliga et al., 1982;Lörz et al., 1981).Als o with cytoplasm, and go into division. First uptake of isolated organelles by means of resultsdemonstratin gth esuccessfu l transfer fusion-like processes hasbee n reported, but of chloroplasts, leading to the no evidence for the functionality of the complementation of a plastome deficient introduced organelleswa spresente d (Giles, albino tobacco cell line are presented, and 1978). In animal cells, microinjection has the prospects for this microinjection been applied successfully to the technique are discussed. Part of this work transplantation of organelles, and even hasbee n communicated earlier (Verhoeven entire nuclei (Willadsen, 1986). Although and Blaas, 1988;Verhoeve n et al, 1988). 95 Materialsan dmethod s protoplastsan dth elin e9 2dono r protoplasts were isolated after overnight incubation of leaves in anenzym e mixture,containin g 1% Plantmaterial Cellulase RIO, 0.2% Macerozyme RIO and 0.4M mannitol .Protoplast swer epurifie d as For the cell to cell transfer of cytoplasm, a described before (de Laat and Blaas, 1987), cell suspension of transformed Nicotiana and resuspended in a small amount of K3 plumbaginifolia, was used as donor and (Kao et al., 1974) medium. recipient.Th e original supensionwa skindl y provided by Dr. R.Shields, Sittingbourne, Protoplast evacuolation UK. The microfilaments ofth edono r protoplasts In chloroplast complementation were disrupted by a 15 min. 10 ^g/ml experiments, the VBW albino line of cytochalasin-B (Sigma) pretreatment. N.tabacum was used as acceptor. This cell Before staining,pretreate d protoplastswer e line contains deficient chloroplasts, and can evacuolated by centrifugation on a be complemented byth e green chloroplasts preformed, isoosmotic Percoll gradient (0.4 from the donor tobacco line 92, as was M mannitol in Percoll (Pharmacia)) during demonstrated by fusion experiments (Aviv 60 min at 25 °C at 170,000 g, in a 6X5 et al., 1984). The donor cell line 92 contains swing-out rotor. chloroplasts, conferring streptomycin and spectinomycin resistance, and Protoplaststaining mitochondria, both originating from N. undulata, in a N.tabacum nuclear To visualize the transfer of mitochondria, background. Both cell lines were kindly donorprotoplast swer etreate dwit h20/ 96 Fig.1 Illustratio n ofth e injection system, operating at different pressures during an injection cyclus, a: no over pressure; b: mercury forced into the tipb y 4 bar overpressure; c: uptake of cytoplasm by reduction of the overpressure to 3 bar; d: injection of the cellular material by increasing the overpressure to 4ba r again. D Microinjectionsystem Results Thebasic so fth emicroinjectio n systemhav e been described elsewhere (De Laat and Methodology Blaas, 1987). For precise control of suction and injection with large-diameter bevelled The use of mercury in combination with an capillaries, the system was modified air pressure injection system allows the according to the same principles described precise regulation of suction and injection by Hiramoto (1962) for the introduction of over aver y large range, from femto-liter up sperm cells into sea urchin eggs. A small to micro-liter volumes. Also liquids with droplet ofmercur ywa spu t inth ecapillaries , high viscosities, or liquids containing near the tip of the needle (Fig. 1). This particles,ca nb eaccuratel yhandled .Th e use droplet could be moved by controlling the of sharp, bevelled glass needles minimizes pressure from about 0.5ba r to 4.0ba r in the damage to the regenerating protoplasts, pneumatic injection system, which was resulting in up to 80% survival in a typical adapted to these pressures. The experiment, after injection with a needle of compensation pressure forced the mercury 5uva diameter . intoth e tipo fth ecapillary ,an dwa s adjusted until the mercury was close to the opening; Several days after the isolation of the suction was achieved by reducing the acceptor protoplasts, the freshly isolated pressure. For injections, the pressure was donor protoplasts were immobilized in the again increased (Fig. 1). same petridish. This short period of preculture of the acceptor wasnecessar y for 97 To reduce the risk ofvacuol epunctures , the donor protoplasts were evacuolated (Griesbach andSink , 1983).Pretreatmen to f the donor protoplasts with cytochalasin-B improved the yield of evacuolated significantly. An additional effect of this microfilament disrupting treatment appeared to be the reduction of viscosity of the cytoplasm, facilitating handling of the organelles during extraction and injection (data not shown). However, all of the cytoplasm could not always be removed from the needle after each injection. Therefore, the needles could be used for an average of five injections. With these modifications, up to 50% of the cytoplasm from an evacuolated protoplast could be Fig. 2 Fluorescence photomicrograph of a removed from the donor, and injected into DIODC-stainedprotoplas t ofN.plumbaginifolia. the recipient. Organelle transfer partial regeneration of the cell wall. It appeared almost impossible to inject The transfer of mitochondria was cytoplasm into freshly isolated protoplasts, •demonstrate d by using stained donor and remove the needle without destruction mitochondria with the mitochondrial of the membrane. A partially regenerated membrane specific stain DIODC (Matzke cell wall protected the membranes and Matzke, 1986) (Fig.2) . sufficiently. Visual preselection of well Rapid redistribution of the fluorescent dye regenerating protoplasts appeared to be a from the injected mitochondria to the successful approach, and madei tpossibl e to unstained mitochondria of the acceptor was apply microinjection to protoplasts with a used as an indicator of a true cytoplasmic lowplatin g efficiency. injection: when the fluorescent cytoplasm was deposited against the outer surface of To visualize the transfer of mitochondria, the recipient, nostainin go fth eaccepto rwa s the first experiments were performed with observed. Both regenerating protoplasts DIODC-stained donor mitochondria from a (Fig. 3 a-d) and already divided cells could cell suspension of N.plumbaginifolia, and be used as acceptor cell (Fig. 3 e-h), and unstained protoplasts of the same species as subsequent divisionswer eobserve d (Fig.3d , acceptor.Albin oVB Wwa suse d as acceptor 3h). Occasionally, green chloroplasts were andtobacc olin e 92a sdono r to demonstrate seen to leave the injected clump by chloroplast transfer. cytoplasmic streaming (data not shown). After some time, divisions could be Initially,normal ,freshl y isolated protoplasts observed in injected cells, finally leading to were used as organelle donor. However, it the formation of microcalli, which were was difficult to extract a sufficiently large collected by micromanipulation and amount of cytoplasm without damaging the transferred onto callus growth medium. vacuole. Since the cell sytem used for the demonstration of the transfer of 98 h Fig.3 :Injectio n of mitochondria into a regenerating protoplast (a-d),an d into an already divided regenerating protoplast (e-h). a: protoplast prior to injection, after 1 day of culture; b: mitochondria injected into the protoplast (arrow); c:afte r 3day s of culture (twoday s after injection); d: after seven days of culture; e: divided cell prior to injection; f: fluorescence micrograph of the injected cell, showing the fluorescently labelled mitochondria (arrow);g :on e day after injection; h: after 10day s of culture. 99 Fig. 4 Uptake of cytoplasm from an evacuolated mesophyll protoplast, containing mature green chloroplasts. J» o' • u Fig. 5 Uptake of proplastids and mitochondria from an evacuolated suspension donor protoplast. Fig. 6 Green mesophyll chloroplasts injected into an albino recipient protoplast. oo mitochondria did not contain any useful transported in the cell by cytoplasmic genetic marker, the albino VBW and the streaming. It was possible to inject up to 10 tobacco line 92wer e used to investigate the mature chloroplasts,wherea s the number of fate of the injected organelles. Evacuolated mitochondria and proplastids could not be mesophyll leaf protoplasts and suspension estimated visually. cell protoplasts of line 92 were used as organelle donor, to transfer mature From an albino VBW acceptor protoplast, chloroplasts and proplastids together (Fig. injected with cytoplasm from the 4),an dproplastid sonl y(Fig .5) , respectively. evacuolated mesophyll donor protoplasts, In both cases, also transfer of mitochondria one microcalluswa sobtaine d which showed islikel y to occur. sorting out of the red fluorescence of chlorophyll (data not shown). The number The injected green chloroplasts from the ofinjecte d green chloroplastswa sfa r below mesophyll donor could be discriminated the number of red fluorescing chloroplasts from the albino chloroplasts during several inth e microcallus.Henc e atleas tpar t of the daysafte r the injection (Fig.6) .Som e of the red fluorescence originated from new chloroplastslef t theinjecte d clumpan dwer e chloroplasts, derived from the injected 100 Fig. 7a Photo micrograph of a microcallus, Fig. 7b Fluorescence photo micrograph of the regenerated from an albino protoplast, four weeks microcallus offig. 7a , showing the sectors with red after theinjectio no fsuspensio ncel ldono rcytoplasm . fluorescence due to the newly synthesized chlorophyll. plasties.Sortin gou twa sals oobserve d inth e Knowles 1974).However ,Paramecium cells case where donor cytoplasm from the cell are much larger than plant protoplasts, suspensiono flin e92 ,whic hdoe sno t contain impeding the application of this technique anymature ,gree n chloroplasts,wa s injected with plant cells. The only reports on into the albino, indicating the complete de organelle transfer to plant cells by novo synthesis of chlorophyll (Fig. 7 a,b). microinjection employ mass isolated Out of fifty injections, five microcalli were chromosomes (Griesbach, 1987; De Laat obtained snowing the red fluorescence: two and Blaas, 1987).Thi s approach carries the from injections with mesophyll protoplast risko forganell edamag edurin gth e isolation cytoplasm, and three from injections with procedure. The risk is largely absent in the suspension cell protoplast cytoplasm. With direct cell to cell transfer. Furthermore, the mock injected albino protoplasts, this red precise control over the injection flow and fluorescence was never observed. the possibility to use very large orifices, reduce the damage due to shearing forces Discussion during the injection of fragile organelles (Griesbach, 1987). Although polyethylene- glycol induced uptake of isolated Transfer of cytoplasm and organelles chloroplasts has been reported (Giles, (nuclei, microchondria) by microinjection 1978), no evidence for the functionality of has already been established in animal the introduced organelles has been researchan dprotozoa nbiology .Nucle i from presented sofar. sheep egg cells have been removed by micromanipulation techniques, followed by This is the first report on complementation microinjection to introduce new genetic of acytoplasmi c albino trait byothe r means material (Willadsen, 1986). Transfer of than fusion. The albino was successfully cytoplasm has been developed for research complemented by injection with mesophyll in Paramecium using similar techniques as protoplast organelles, and by injection of described in this paper (Koizumi,1974 ; suspension protoplast cytoplasma swell . No 101 evidence could be obtained for a manipulation. Preliminary results, obtained preferential donor, since both mesophyll in our laboratory, demonstrate the andsuspensio n cellplastid swer eefficien t in possibility to extract metaphase complementation. The green chloroplasts chromosomes and micronuclei (Verhoeven from the mesophyll donor gradually et al., 1986; 1987;d e Laat et al., 1987) from bleached and could no longer be stained, immobilized protoplasts (data not distinguished after about one week shown). This may be a valuable new following the injection. Nevertheless, approach for the aimed introduction of complementation was observed in the case donor chromosomes, useful for of injected mature chloroplasts. This chromosome mapping and genome indicates that complementation is interaction studies. accomplished by proliferating proplastids which were present in the injected Acknowledgements cytoplasm, or by dedifferentiated mature chloroplasts, since mature chloroplasts are presumed to havelos t their division activity. Part of this research was funded by the A similar phenomenon is often observed "Biotechnology Action Programme" of the after fusion ofmesophyl lan dsuspensio ncel l CEC, contract BAP-0083-NL. The skilfull protoplasts. assistance of Mrs. Y. Nöllen with the cell cultures isgratefull y acknowledged. We like Out of a total of fifty injections, five to thank Dr. D.Avi van d Prof.Dr . E. Galun microcalli were obtained which showed red for the plant material of the VBW and line fluorescence. Even with the low number of 92, and for their useful suggestions.W e also injections,whic hca nb edon eo non eda y(u p thankDr .L .va nVloten-Dotin gan dDr .W.J . to five survivingprotoplasts) , this isa useful Stiekema for critical reading of the frequency for achieving transfer of manuscript. cytoplasmic traits. References. Protoplast isolation is no longer a prerequisite for genetic manipulation, since thecel lwal lo fplan ttissue se.g .embryo s can Aviv, D., Arzee-Gonen, P., Bleichman, S., be penetrated by microinjection (Neuhaus Galun, E. (1984). Novel alloplasmic et al., 1987). Thisreduce s theproblem swit h Nicotiana plants by "donor-recipient" the regeneration of protoplasts from protoplast fusion: cybridshavin gN.tabacum difficult species, like monocots, and orN.sylvestris nuclea rgenome s andeithe r or circumvents the concomitant somaclonal bothplastome s and chondriomes from alien variation (Sree Ramulu et al., 1985).I n this species. Mol.Gen . Genet. 126: 244-253. way,ne w approaches to the manipulation of the organelle composition of a given plant Barfield, D.G. Robinson, S.J. Shields, R. species orvariet y maybecom e feasible. The (1985). Plant regeneration from protoplasts directly visible segregation patterns, along of long term haploid suspension cultures of with the possibility to control the amount of N. plumbaginifolia. Plant Cell Rep. 4_: donorcytoplasm ,create sne wpossibilitie st o 104-107. study the interaction between the nucleus and the cytoplasmic organelles. Crossway, A., Oakes, J.V., Irvine, J.M., Ward, B.,Knauf , V.Can d Shewmaker, CK. Additionally, the application of this (1986). Integration of foreign DNA microinjection technique for plant cell following microinjection of tobacco researchopen su pne wway sfo rplan tgeneti c 102 mesophyll protoplasts. Mol. Gen. Genet. Lörz, H., Paszkowski, J., Dierks-Ventling, 202:179-185. C, Potrykus, I. (1981). Isolation and characterization of cytoplasts and de Laat, A.M.M., Blaas, J. (1987). An miniprotoplasts derived from protoplasts of improved method for protoplast cultured cells.Physiol .Plant .52 : 385-391. microinjection suitable for transfer of entire plant chromosomes.Plan t Sei.5J} :161-169 . Maliga, P., Lörz, H., Lazar, G., Nagy, F. (1982). Cytoplast-protoplast fusion for de Laat, A.M.M., Verhoeven, H.A., Sree interspecific chloroplast transfer in Ramulu, K, Dijkhuis, P. (1987). Efficient Nicotiana. Mol.Gen . Genet.i£5_:211-215 . induction by amiprophos-methyl and flowcytometric sorting of micronuclei in Matzke, M.A., Matzke, J.M. (1986). N.plumbaginifolia. Planta 122:473-478 . Visualization of mitochondria and nuclei in living plant cells by the use of a Galun, E., Aviv, D. (1986). Organelle potential-sensitive fluorescent dye. Plant, transfer. Methods in Enzymology 118: Cell and Environm. 9_: 73-77. 595-611. Morikawa,H. ,Yamada ,Y .(1985) .Capillar y Giles,K.L .(1978) .Th e uptake of organelles microinjection into protoplasts and and microorganisms by plant protoplasts: intranuclear localization of injected old ideas but new horizons. In: Proc. Int. material.Plan t CellPhysiol .26 :229-236 . Congr. Plant Tissue Cell Cult. 4th, Calgary, Alberta,pp .67-74 . Neuhaus, G., Spangenberg, G., Mittelsten Scheid, O., Schweiger, H.-G. (1987). Griesbach, R.J., Sink, K.C. (1983). Transgenic rapeseed plants obtained by the Evacuolation of mesophyll protoplasts. microinjection of DNA into Plant Sei.Lett . 2Q: 297-301. microspore-derived embryoids. Theor. Appl.Genet .25. : 30-36. Griesbach, R.J. (1987). Chromosome- mediatedtransformatio n via microinjection. Reich, T.J., Iyer, V.N., Miki, B.L. (1986). Plant Sei.5Ü : 69-77. Efficient transformation of alfalfa protoplasts by the intranuclear injection of Hiramoto, Y. (1962). Microinjection of the Ti plasmids.Bio/Techn .4:1001-1004 . live spermatozoa into sea urchin eggs.Exp . CellRes .22 :416-426 . Spangenberg, G., Neuhaus, G., Schweiger, H. (1986). Expression of foreign genes in a Kao, K.N., Constabel, F., Michayluk, M.R., higher plant cell after electrofusion- Gamborg, O.L. (1974). Plant protoplast mediated cell reconstitution of a fusion and growth of intergeneric hybrid microinjected karyoplast and a cytoplast. cells.Plant a 120:215-227. Eur. J. Cell Biol.42 : 236-238. Koizumi, S. (1974). Microinjection and Sree Ramulu, K., Dijkhuis, P., Hänisch ten transfer of cytoplasm in Paramecium. Exp. Cate, Ch. H., de Groot, B. (1985). Patterns CellRes .S£ : 74-78. of DNA and chromosome variation during in vitro growth in various genotypes of Knowles, J.K.C. (1974). An improved potato. Plant Sei.41 : 69-78. microinjection technique in Paramecium aurelia. Exp.Cel l Res.8j£ : 79-87. Verhoeven,H.A. ,d eLaat ,A.M.M. ,Blaas ,J . (1986). Methodological developments for 103 chromosome-mediated gene transfer in higher plants. In: Magnien, E. (Ed) Biomolecular engineering in the European Community, achievements of the research programme (1982-1986). Nijhoff, Dordrecht, pp.977-981 . Verhoeven, H.A., de Laat, A.M.M., Sree Ramulu, K., Dijkhuis, P., van Vloten-Doting, L. (1987). Induction, isolation and flowcytometric sorting of metaphase chromosomes and micronuclei from suspension cell cultures of N.plumbaginifolia.In : Genetic and cellular engineering of plants and micro-organisms important for agriculture, Commission of the European Communities meeting, Louvain-la-Neuve,pp . 119-120. Verhoeven,H.A .an dBlaas ,J .(1988) .Direc t cell to cell transfer of mitochondria by microinjection. In: Progress in plant protoplast research, éd. K.J.Puite et al., Kluwer Academic publishers, Dordrecht, Boston, London, pp.299-300 . Verhoeven, H.A., Blaas,J. ,Aviv ,D. ,Galun , E.(1988). Direct cell to cell transfer of chloroplasts by microinjection. In: The second international congress on plant molecular biology, Jerusalem, novenber 14-19,Boo k ofAbstracts , Abstract N° 136. Willadsen, S.M. (1986). Nuclear transplantation in sheep embryos. Nature 320:63-65 . 104 CHAPTER8 SUMMARYAN D CONCLUSIONS 105 SUMMARYAN D CONCLUSIONS In this thesis, micronucleation in plant cells research on mammalian cells, it is known has been investigated and systems for that these phenomena occur with a lower isolation and transfer of organelles have frequency after microcell-mediated been established. chromosome transfer (Fournier, 1981). If microprotoplasts would become available Thediscovery ,describe d inchapte rtwo ,tha t for plant genetic manipulation, transfer of a the phosphoric amide herbicide limited number of chromosomes by amiprophos-methyl induced micronuclei at microprotoplast fusion would offer an a high frequency in cell suspensions of alternative to the use of gamma-irradiation. N.plumbaginifolia, has opened the With the finding that APM induces possibility to develop a microcell-mediated micronuclei at high frequency in plants, chromosome transfer system analogous to transfer of low numbers of chromosomes that inmammalia n cell lines.I n mammalian after micronucleation canno wb e tested for cells,micronucle i are induced byprolonge d usei nplan tgeneti cmanipulation .Th e APM exposure of cells to spindle toxins treatment wasfoun d tob ereversible , aswa s (colchicine, Colcemid), resulting in up to demonstrated by washing the cell 60% micronucleated cells (Matsui et al, suspension cultures free from APM. After 1982). Micronucleated cells are isolated by washing, normal growth and cell division the "shake-off method, and subjected to were soon resumed, with some abnormal, high speed centrifugation, which results in multipolar spindles in the first division after fractionation of the cells into microcells, washing. This observation is in good containing micronulei with one or a few agreement withth eth e reversible inhibition chromosomes. Subsequently, microcells are of microtubule polymerization by APM fused to the recipient cells.Th e transferred (Falconer and Seagull, 1987). This low chromosomes were found to remain intact cytotoxicity makes APM ausefu l tool in the andmitoticall y stable (Fournier, 1982).Thi s induction of micronuclei inplants . techniqueha shithert ono tbee navailabl e for plant cells or protoplasts, due to the lack of The flow cytometric analysis of the nuclear efficient procedures to induce micronuclei. DNA content of APM-treated eel Gamma-irradiation isno wofte n used in the suspension cultures of N.plumbaginifolia construction ofmonochromosoma l addition revealed the presence of many micronuclei lines by somatic hybridization (Bates et al., with a DNA content equivalent to one 1987),t oinduc echromosom edamag ewhic h metaphase chromosome (which consists of promotes chromosome elimination from both sister chromatids). Similar one of the fusion partners. As has been observations have been made in pointed out in the introduction (chapter micronucleated rat kangaroo cells after one), ionizing radiation induces treatment with Colcemid (Sekiguchi et al., chromosome rearrage ments, deletions and 1978). Sorting of the micronuclei on the insertions (Menczel et al., 1982). From basis of the fluorescence of 107 ethidium-bromide, followed by analysis of is responsible for the accumulation of cells the DNA content by Feulgen staining at metaphase. The concentrations of the (chapter three), shows that it is possible to inhibitors required for complete metaphase separate micronuclei on the basis of their arrest,var yfro m 3// Mfo r APMan d oryzalin DNA content by flowcytometry, like it has to 500fiM for colchicine, as a consequence been shown for isolated plant metaphase of differences in binding specificity (Hertel chromosomes. Chromosome identification et al, 1980; Dustin 1984).Th e differences in is sometimes possible with isolated the percentage of ball metaphases indicate metaphase chromosomes (de Laat and specific effects of the above mentioned Blaas, 1984; Conia et al., 1987a; 1987b). inhibitors on chromosome scattering. Apart Identification of chromosomes present in a from the disruption of the microtubules, particular micronucleus isno tpossible .Thi s APM and oryzalin have been shown to is due to different degrees of chromosome influence theaccumulatio no fcalciu mi nth e decondensation in the micronuclei (which mitochondria (Hertel et al., 1981). influences the fluorescence signal of the Moreover, oryzalin disturbs the active fluorochrome-DNA complex by excretion of calcium by the plasma quenching), and due to the various membrane.Thes ecombine d effects resulti n combinations of chromosomes in an increased cytoplasmic calcium micronuclei containing more than one concentration (Hertel et al, 1980), which metaphase chromosome. This is illustrated will be higher after oryzalin treatment than by the DNA histograms of isolated after APM treatment, due to the reduction micronuclei in chapter two, which lack the of active calcium excretion byoryzalin . Our specific chromosome peaks, present in data suggest that the APM or oryzalin metaphase chromosome preparations induced increase of the cytoplasmic calcium (chapter four). When micronuclei are concentration is involved inbot h formation present in large numbers, the overall DNA andfusio n ofmicronuclei .Colchicine ,whic h histogram will show no appreciable does not influence the cytoplasmic calcium contribution of a particular type of concentration, is not effective in the chromosome combination in micronuclei, induction of micronuclei. The higher since chromosome grouping appears tob e a cytoplasmic calcium levels after oryzalin randomprocess ,a swa sshow nb yth eanalysi s treatment, would increase the fusogenic of the number of micronuclei per cell in properties ofth enuclea r membranes,whic h chapter two, and by cytological data in would explain why micronuclei exist for a chapter two and three. Furthermore, the shorter time after oryzalin treatment as reduction of the number of micronuclei per compared to APM treatment. This micronucleated cell,whic happear st ob e the hypothesis will be tested in future consequence of fusion of micronuclei into a experiments bytreatment s with the calcium lobed restitution nucleus, gives rise to even ionophore A23187 in combination with the more combinations of chromosomes. calcium-specific chelator ethyleneglycol- The processes, involved in the formation of bis-(2-aminoethylether)-N,N'-tetra acetic micronuclei, are studied in chapter three acid (EGTA), with simultaneous and four. The effects of the measurements of the cytoplasmic calcium anti-microtubular herbicides APM, oryzalin concentrationswit hth ene wcalciu m specific and the alkaloid colchicine, used for fluorochromes Fluo-3 and Rhod-2 metaphase arrest and induction of (Haugland, 1989). micronuclei in mammalian cells, on the mitotic index and micronucleus formation In order to obtain both large numbers of arecompared .Th e disruption of the spindle micronucleated cells, and large numbers of bydirec tinhibitio n ofmicrotubul e assemble micronuclei per micronucleated cell, the 108 effect of DNA synthesis inhibitors was could overcome this problem (Koop et al., investigated. The results in chapter five 1983). This control is essential for the show, that a considerable increase in the efficient application of microprotoplasts, number of micronucleated cells can be since the DNA content per microprotoplast achieved by HU or APH treatments, and will depend upon the DNA content per that the time at which micronuclei appear micronucleus in the cell suspension. can be controlled. The results further Microprotoplast fusion willresul ti n transfer indicatetha tmetaphase s havet ob e exposed of a part of the total number of to APM for at least 12h, before chromosomes, directly followed by micronucleation occurs, and that their spontaneouschromosom e eliminationwhe n lifetime is in the same order. These data twodistantl y related species arefused , since demonstrate that iti spossibl et o manipulate chromosome elimination seems to be the conditions of the treatments in order to directed by genome dose effects (Graves, obtain either a high yield of metaphase 1984; Gilissen et al., 1989). Sofar no chromosomes,o ra hig hyiel do fmicronuclei , successful fusion experiments have been with little contamination by micronuclei or performed, whichmake si timpossibl e at the chromosomes, respectively. In this way, it moment to comment on the usefulness of becomespossibl e todetermin e the moment microprotoplasts in chromosome transfer. atwhic h the number of micronuclei per cell However, fusion experiments with isa t itsmaxima l value. karyoplasts indicate that it is possible to perform fusions in a controlled way The isolation and characterization of (Spangenberg et al., 1987). microprotoplasts from micronucleated suspension cells is described in chapter six. In addition to the microprotoplast fusion, Data obtained from DNA content microinjection wasdevelope dfo r transfer of measurements and flow cytometry organelles and micronuclei. Glass needles demonstrate the presence of up to 40% of with a large orifice (5^M) were prepared, subprotoplastswit ha DN Aconten t lesstha n along with a pressure system, based on the the Gl-level ofth eAP Mtreate d suspension application of mercury. With the injection cells. This indicates that genome system, described in chapter seven, it is fractionation has occurred, and the data on possiblet osuc kdono rmateria lfro m adono r the FDA-staining show that most of the protoplast, and inject this directly into the subprotoplasts stillposses s an intact plasma recipient.Th e data onth e complementation membrane, since FDA can not be retained ofth e albino tobacco byinjectio n of mature byvacuola rmembrane s only(Lesney , 1986). green chloroplasts or etiolated plastids, The viability of the microprotoplasts and indicate that protoplasts can survive the other types of subprotoplasts isindicate d by injection treatment, and that the injected the successful culture after gradient plastidsca nb ereplicate d byth erecipient .I n fractionation. Asi ti simpossibl e to measure thisway ,th e organellest ob e transferred are the DNA-content of microprotoplasts in a not subjected to damaging isolation non-destructive way, no preselection could procedures and they can be preselected be performed to use only microprotoplasts visually. Selective transfer of organelles for fusion. In a mass fusion system, the offers a number of advantages when smallest microcellswil l be the least likely to compared to fusion techniques, or transfer fuse when electrofusion is used, because of isolated genes. One of the advantages is their small diameter will prevent alignment the protective nature of the membranes and membrane breakdown, which are both associated with chloroplasts, mitochondria related to particle diameter (Zimmermann andnuclei .Althoug h structural integrity and et al., 1982).Individua l selection and fusion functionality has been demonstrated for 109 isolated chloroplasts and mitochondria, iti s hybridization in Nicotiana by fusion of not known whether isolated organelles are irradiated protoplasts. Theor. Appl. Genet. still physiologically intact. The isolation of 24:718-726 . intact nuclei from plant cells has also been described, with data indicating their Conia, J., Bergounioux, C, Perennes, C, structural integrity, aswel l asthei r ability to Brown, S., Muller, P., Gadal, P. (1987a). transfer genes into recipient protoplasts Chromosomes from protoplasts of Petunia (Saxena et al., 1986). Transfer of marker hybrida, flow analysis and sorting.Biol . Cell genes does not necessarily implicate the 5Ssuppl :5a . functional integrity of isolated nuclei, since transfer ofmarke r genesca n be achievedb y Conia, J., Bergounioux, C, Perennes, C, uptake of isolated genomic DNA. Muller, P., Brown, S., Gadal, P. (1987b). Preliminary results obtained from Flowcytometri canalysi san dsortin go fplan t experiments with microinjection of chromosomes from Petunia hybrida micronuclei, indicate that it is possible to protoplasts.Cytometr y &500-508 . remove micronuclei from the donor by suction. Sofar, transfer into a recipient has Dustin, P. (1984). Microtubules, not been achieved. The Springer-Verlag, Berlin,pp . 167-225. kanamycine-resistance, which was introduced into N.plumbaginifolia by De Laat. A.M.M., Blaas, J. (1984). Flow transformation with Agrobacterium cytometric characterization and sorting of tumefaciens, will be used as selectable plantchromosomes .Theor .Appl .Genet .62 : marker after transfer of micronuclei. The .463-467. transfer of chromosomes willb e tested with species specific repetitive DNA probes, Falconer, M.M., Seagull, R.W. (1987). which are able to discriminate between the Amiprophos-methyl (APM): a rapid, donor genome (N.plumbaginifolia and the reversible, anti-microtubule agent for plant recipient (eitherLycopersicon esculentum o r cell cultures.Protoplasm a IM: 118-124. Solanum tuberosum). Several probes with the required specificity have already been Fournier, R.E.K. (1981). A general characterized from a series of highly high-efficiency procedure for production of repetitive sequences, isolated from microcell hybrids. Proc. Natl. Acad. Sei. N.plumbaginifolia(dat a not shown). USA. 28:6349-6353 . With the methods, described in this thesis, Fournier, R.E.K. (1982). Microcell- thetransfe r ofchromosome svi amicronucle i mediated gene transfer. In: Techniques in has come within reach. Future work will somatic cell genetics. Ed.: Shay, J.W., focus on achieving transfer, and study the Plenum Press, New York, London, pp. fate of the introduced micronuclei. This 309-327. should provide an answer whether micronuclei can be used as chromosome Gilissen, L.J.W., Sree Ramulu, K., Dijkhuis, carriersi nplants ,a sha salread y been shown P, Verhoeven, H.A., Stiekema,W.J .(1989) . inmammalia n somatic cell genetics. Application in somatic cell genetics of potato marker lines produced through References Agrobacterium transformation. XII. Eucarpia Congress, Göttingen, FRG, book of poster abstracts,poste r No.30-10 . Bates, G.W., Hasenkampf, CA., Contolini, CL., Piastuch, W.C. (1987). Asymmetrie 110 Graves, J.A.M. (1984). Chromosome isolated nucleiint oplan tprotoplasts . Planta segregation from cell hybrids. I. The effect 168: 29-35. of parent cell ploidy on segregation from mouse-Chinese hamster hybrids. Can. J. Sekiguchi, T., Shelton, K., Ringertz, N.R. Genet. Cytol.26 : 557-563. (1978).DN Aconten t ofmicrocell sprepare d from rat, kangaroo and mouse cells. Expt. Haugland, R.P. (1989). Handbook of Cell Res.112 :247-258 . fluorescent probes and research chemicals, Molecular Probes,Inc . Zimmermann, U. (1982). Electric field-mediated fusion and related electrical Hertel, G, Quader, H., Robinson, D.G., phenomena.Biochim .e tBiophys .Acta ,69_4_ : Marmé, D. (1980). Anti-microtubular 227-277. herbicides and fungicides affect Ca + transport inplan t mitochondria. Planta 149: 336-340. Hertel, G, Quader, H., Robinson, D.G., Roos, L, Carafoli, E., Marmé, D. (1981). Herbicides and fungicides stimulate Ca efflux from rat liver mitochondria. FEBS Letters 122:37-39 . Koop, H.U., Dirk, J., Wolff, D., Schweiger, H.G. (1983). Somatic hybridization of two selected single cells. Cell Biol. Int. Rep. 2: 1123-1128. Lesney, M.S., Callow, P.W., Sink, K.G (1986). A technique for bulk production of cytoplasts and miniprotoplasts from suspension culture-derived protoplasts. Plant Cell Rep 5_: 115-118. Matsui, S., Weinfeld, H., Sandberg, A.A. (1982). Factors involved in prophasing and telophasing. In: "Premature chromosome condensation", Application inbasic , clinical and mutation research (P.N. Rao, R.T. Johnson and K. Sperling, eds.), Academic Press,Ne w York,pp . 207-233. Menczel,L. ,Galiba ,G. , Nagy,F. ,Maliga ,P . (1982). Effect of radiation dosage on efficiency of chloroplast transfer by protoplastfusio n inNicotiana. Genetic s 100: 487-495. Saxena,P.K. , Mii,M. ,Crosby ,W.L. ,Fowke , L.C., King, J. (1986). Transplantation of 111 SAMENVATTING 113 SAMENVATTING In dit proefschrift zijn de resultaten hoofdstuk wordt het aantal cellen met gepresenteerd van onderzoek naar de microkernen, enhe taanta lmicrokerne n per inductie, isolatie en overdracht van gemicronucleëerde cel, alsmede het microkernen. Het onderzoek is uitgevoerd DNA-gehalte per microkern onderzocht. met het doel een systeem te ontwikkelen, Uit deze resultaten blijkt, dat er vele waarmee slechts één of enkele microkernen voorkomen, die slechts één chromosomen van een donor plant via metafase chromosoom bevatten. Dit wordt microkernen naar een recipient kunnen bevestigd door het sorteren van de worden overgedragen. Met een dergelijke microkernen. De toxische effecten van de benadering ishe tmogelij k gebleken, slechts behandeling blijken bij de gebruikte APM enkele menselijke chromosomen over te concentraties nietva n belang. brengen naar een muizecellijn. Met de verkregen cellijnen konden de genen op de In hoofdstuk drie wordt het gedrag van menselijke chromosomen in kaart worden mitotische cellen tijdens en na de gebrachtvi ahe t opheffen vandeficiëntie s in APM-behandelingnade ronderzocht .Ui t de de muizecellijn door de genen op de resultaten blijkt, dat de chromosomen chromosomen van de mens. Behalve voor tijdens de metafase geen metafaseplaat het opstellen van een chromosomenkaart, vormen, maar in het hele cytoplasma zijn plantecellijnen met één extra, vreemd verspreid komen te liggen. Tijdens de donor chromosoom van belang voor de metafase vindt er geen deling van de planteveredeling. centromeren plaats, zodat zuster Chromatiden aan elkaar blijven zitten. Door In hoofdstuk een wordt de doelstelling van het ontbreken van de anafase, begint in de het onderzoek uiteengezet, en worden telofase devormin gva nee n kernmembraan literatuurgegevens over chromosoom om groepjes en vrijliggende chromosomen, overdracht bij dierlijke en plantaardige watuiteindelij k resulteert ind evormin gva n cellen besproken. microkernen. ZodraAP Mverwijder d wordt door uitwassen, vinden er al snel, De inductie van microkernen door hoofdzakelijk normale delingen plaats, met behandelingen van cel-suspensie cultures een afnemend aantal multipolaire van N.plumbaginifolia met het herbicide spoelfiguren. amiprophos-methyl (APM) is het onderwerp van hoofdstuk twee. Het blijkt In hoofdstuk vier wordt de werking van de dat dit herbicide de vorming van de herbicides APM en oryzaline vergeleken spoelfiguur verstoort, waardoor de met colchicine, dat gebruikt wordt om bij chromosomendoo rd ece lversprei d worden. dierlijke cellen microkernen te induceren. Na ongeveer twaalf uur wordt dit gevolgd Onderzocht zijn de effecten van colchicine, door de vorming van microkernen. In dit oryzaline en APM op de mitotische index, 115 chromosoom spreiding en de vorming van het uitwassen van de DNA-synthese microkernen. De resultaten tonen aan, dat remmers toe te voegen. colchicine niet geschikt is om cellen in metafase op te hopen voor de isolatie van Hoofdstuk zes beschrijft de isolatie van chromosomen.D eresultate ntone noo kaan , microprotoplasten. Microprotoplasten zijn dat er door colchicine weinig microkernen subprotoplasten met een DNA-gehalte dat gevormd worden in plantecellen. Onder overeenkomt met dat van slechts enkele invloed van oryzaline wordt een hoge chromosomen. Hiertoe worden van een mitotischeinde xbereikt , die ditmidde l zeer gemicronucleëerde celsuspensiecultuur geschikt maakt voor chromosoomisolatie. protoplasten geïsoleerd, die vervolgens na Tijdens de oryzaline behandeling worden een korte cytochalasine-B behandeling op wel microkernen gevormd, maar deze eenPercol lgradien tgefractioneer d worden. fuseren bij hogere oryzaline concentraties Na centrifugatie zijn er verschillende snel tot een restitutiekern. Hierdoor bandeni nd egradien t aanwezig,waarbi j een verkrijgen de cellen een verdubbelde bepaalde fractie meer dan 40% hoeveelheid DNA. Dit is niet het geval bij microprotoplasten bevat. Via behandelingen met APM, waarbij flowcytometrie en microdensitometrie is microkernen ongeveer twaalf uur kunnen aangetoond dat de microprotoplasten een blijven bestaan alvorens te fuseren tot laag DNA-gehalte hebben, en dat ze tevens restitutiekern .He tgelijktijdi g optredenva n een intacte plasmamembraan hebben. De verspreide chromosomen en microkernen levensvatbaarheid vand esubprotoplaste n is maakt APM minder geschikt voor het bewezen door ze in kweek te brengen, isoleren van metafasechromosomen dan waarbij van elke fraktie van een gradient oryzaline. microcalli verkregen konden worden. Dit toont aan, dat microprotoplasten gebruikt Om de inductie van microkernen te kunnen worden als fusiepartn'er in fusie optimaliseren, is het effect van een experimenten. behandelingme tDNA-syntheseremmer s op de mitotische index en de frequentie van Hoofdstuk zevenbehandel t de ontwikkeling microkernen onderzocht (hoofdstuk vijf). en de eerste resultaten van een nieuw Door de periode van DNA-synthese- microinjectie systeem. Een grote remmingz ot ekiezen ,da the thoogst eaanta l naaldopening (groter dan 5 ,«m) in delende cellen door de remming in S-fase combinatie met een op kwik gebaseerd opgehoopt wordt, is het mogelijk de druksysteem, maakt het mogelijk donor maximale mitotische index te bereiken na organellen op te zuigen uit de donor cellen, hetuitwasse nva nd e remmer. Deze periode en deze direct weer te injecteren in de kan eenvoudig bepaald worden via de in dit receptor cel. Met deze techniek is hoofdstuk beschreven methode. Alle cytoplasma met groene chloroplasten delende cellen kunnen in metafase geïnjecteerd in albino protoplasten, waarna opgehoopt wordendoo r direkt na uitwassen verscheidene microcalli verkregen zijn, die APM toe te voegen. De opgehoopte de roodfluorescentie van nieuw mitotische cellen vormen dan twaalf uur na gesynthetiseerd chlorofyl bezitten. het beginva nd e toename vand e mitotische index microkernen. Dit resulteert in een Hoofdstuk acht geeft de resultaten en opbrengstva n45 %celle nme t microkernen. conclusies van het onderzoek weer, en Microkern inductie kan ten behoeve van de plaatst deze tegen de doelstellingen, zoals isolatie van metafase chromosomen die in hoofdstuk een geformuleerd zijn. uitgesteld worden door APMpa s zesuu r na 116 Curriculum Vitae Harrie Verhoeven werd geboren op 14 november 1956 te Valkenswaard. Na het behalen vanhe t Gymnasium diploma, metbiologi e alsachtst evak ,gin ghi ji n 1976 Electrotechniek studeren aan de Technische Hogeschool Eindhoven. Na een periode van twee jaar veranderde hij van studierichting, en begon in 1977 met zijn studie Biologie aan de Landbouwhogeschool te Wageningen. Hij behaalde het kandidaats diploma in de richting Celbiologie in mei 1981. In 1984 studeerde hij af met als doctoraal vakken dierfysiologie en plantemorfologie en -anatomie. Sinds april 1984i s hij werkzaam als Celbioloog bij de Stichting Ital te Wageningen, eerst op een door de EEG gefinancierd project, en sinds oktober 1987al sprojectleide r van het onderzoeksproject Organel Transfer. 117