Interaction of E. Coli DNA with Tobacco Mesophyll Protoplasts 9 3
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PROTOPLASTS AND DNA STUDLES TOWARDS THE GENETIC MODIFICATION OF PLANT CELLS PROTOPLASTS AND DNA * STUDIES- TOWARDS THB GËNÉTIG MODIFICATION OF PLANT CELLS PROEFSCHRIFT TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE WISKUNDE EN NATUURWETENSCHAPPEN AAN DE RIJKS- UNIVERSITEIT TE LEIDEN,OP GEZAG VAN DE RECTOR MAGNIFICUS DR. A. E. COHEN , HOOGLERAAR IN DE FACULTEIT DER LETTEREN,VOLGENS BESLUITVAN HET COLLEGE VAN DEKANEN TE VERDEDIGEN OP WOENSDAG 1 OCTOBER 1975 TE KLOKKE 14.15 UUR DOOR RUDOLF FRANS HEYN GEBOREN TE EINDHOVEN IN 1944 1975 DRUK : KRIPS REPRO MEPPEL PROMOTOR : PROF. DR. H. VfLDSTRA Het ir> dit proefschrift beschreven onderzoek werd verricht binnen de werkgroep "Molekuiaire Basis van Differentiatie en Oncogenese bij Planten" geleid door Dr. R.A.Schilperoort (eerste coreferent) STELLINGEN I De aanname van Schiess en Goebel, dat de in vitro tcanscriptio. van Col El DNA geen streng-specificiteit vertoont, is allerminst "redelijk" en maakt hun numerieke conclusies twijfelachtig. W. Schiess en W. Goebel, FEBB Lett. 47(1974)356-359. 17. Het onvermeld laten van de aangelegde criteria doet ernstige afbreuk aan de waarde van uitspraken aangaande de vitaliteit van planteprotoplasten. T. Hibi ei al. Virology 64(1975)308-318. H.T. Bonnett en T. Eriksson, Planta 120(1974)71-79. III. De waarneming van Babula en Galsky, dat cyclisch-AMP de crown gall vorming door Agvobaeteriwn tïmefaeiens kan remmen, mag geen duidelijke aanwijzing heten voor een rol van deze stof in da tumor inductie. M.J. Babula en A.G. Galsky, Plant Cell Physiol. 16(1975)357-360. IV Het verschijnen van een radioaktieve MA, fraktiemét afwijkende zweefdïcht- heid (1,724 g.cm ) na behandeling van Matphiota.inaand'kLem.plap.tjès (1,698) met P-gemerkt T.DNA is geert bewijs voor opname en integratie van T.DNA. W.;;Rebel et al. ZyNdtürförsch. 28c(l973)473-^474. : ."• •' ' :: '" '• • ' ;: ••••:v:!'- •••••:' .'• • ^••'••" .' •.• '• • ' •• Het onderzoek van tciderbeck naar de invloed van zogenaamde protoplasten- media op de tumor inductie in Kalanohoë bladeren is voor het bepalen van cytimale condities yöbr de in vitro triansformatie van planteprotoplasten irrelevant en verklaart geenszins het falen vaii de experimenten van Schilde-Rentschler. R. Beiderbeck, Z.Naturforsch, 30c(1975)73-76. L. Schilde-Rentschler, Coll.Intevn.C.N.R.S. 212(1973)t79-483. Het vermeende specifieke verlies van complete SV40 ONA-cRNA hybriden van . ni.trocei lul'pse filters had, vooral "i.v.tn.. de belangrijke impl'it-atips ërvarij bevestigd moeten worden door het aantonen van die verdwenen hybriden in de hybridisatie oplossing. >i. Haas ei al. Pro-, ted-l. Aaad.S^i, U.S.A. 69(1972)2160-2164. VII De geneesmiddelenreclame, waarbij niet de consument maar zijn wettelijk verplichte adviseur beïnvloed worde, kan gevaren inhouden voor de patiënt- consument. Naast het verhogen van de weerhaarheid van geneeskundigen togen deze reclame, is het wenselijk dat in dit speciale geval zowel vorm als inhoud %Tan reclamemateriaal onderworpen worden aan wettelijke voorschriften. F. Kalsbeek, Hed.T.Gsn&esk. 117(1973)141-145. VIII De proefresultaten van Sacristan rechtvaardigen niet haar gebruik van de term kloon. M.D. Sacristan, tiatwnoiss. 62(1975)139-140. IX Het is niet •'.eker of een goede oplossing voor het twijfelachtige "Publish ar perish" gevonden kan worden in Nutman's verwensing "Perish the publishers". P.S. Nutman, J.E-xp-.Bot. 26(1975)477. ; Leiden, 1 oktober 1975. R.F. Heyn. Scanning electron micrograph of a freshly isolated tobacco Protoplast;; real diameter ;ca.50 mi fixation i ^ 113(1973)21-2/. VI CONTENTS VIII CHAPTER I Genera 1, introduction CHAPTER IT Prospects in genetic enginecjring of plants Qycrt.Rrr.'Si-iphyn. 7(1974)35-73 CHAPTER III Rapid and efficient isolation of highly polymerized plant DNA Plan: S^i.Lati. 2(1974)73-78 44 CHAPTER IV The use of protoplasts to follow the fate of Agrobat-toviian tv.mcfaeiens DNA on incubati-on with tobacco eel3s Coll.Intern.C.U.B •?. 212(1973)385-395 51 CHAPTER V Tobacco mesophyll protoplasts: isolation DNA synthesis and plant regeneration CHAPTER'V".' Attempts to detect expression of ColEi DNA in tobacco mesophyll protoplasts 82 CHAPTER VII Interaction of E. coli DNA with tobacco mesophyll protoplasts 9 3 CHAPTER V1I1 General discussion i 1 1 VII ABBREVIATIONS A. turn. Agt'obzeteriiSn tn:--tx. fujiena • (Smith and' Townsend) Conn cpm counts per Minute cRSA complementary RNA, synthesized in &liiK> D dalton; unit of weight equal to 1/16 of the weight o£ a single oxygen a com (ca. 16 x 10•"2 8 kg) DEAE-dextran div'thylaminocthyl-dextran DNA deoxyribiiinucleic; acid -.. •'•• '-....;'.' v-'- .: -^ ::t . ' ' dpni disintegratibr.s per minute E. coli Esoheriakia aoli '.;.. ..'....•.,. v,,;- • .. - '-..-,,.:.-. ..- - EDTA e,thylene--aiamine--tetra-acet:ate : EtBr ethidium bromide x g number times gravity (at centrifugecube midpoint) M.W. molecular weight pps protcplast(s) RNA ribonucleic acid rpm rotations per minute SDS sodium dodecyl sulphate •,-.:...•.--.•..-- J SSC standard saline citrate (0.15 MNaCl, 0.015 M tri-sadium • _; •_; : : .,: citrate, pH7.3).; • :.;•,•..,.'>-•:"•.-.. ;••.- "•-'•• •-.- : TCA trichloroacetic acid .- : - ; : .. : ': Tris tris(hydroxjnnethyl;-aniinomethane -'• •: -';•;••;•;. UV. ultraviolet ; • /.-.•_• • : : ' - •:•;:.:"'•."•' ' : X()T/T % relative pyrimidine-dimer content VIII GHAPTER I GENERAL INTRODUCTION Since the formulation of the working hypothesis, that one way of inducing the pLant tumor crown gall may be the transfer of nucleic acid from the indu- cing bacterium (A.turn.) to the plant cell, followed by its stable and in- heritable incorporation in the plant's genome (9), much effort has been de- voted to proving or disproving this "transformation" hypothesis (2, 3, 16). Nevertheless the central question (as formulated by Schilperoort in his the- sis (IS): "Are we dealing in fact with a real transformation of a normal cell into a tumor cell in the" sense or a generic transformation ':" still remains unanswered. Ideas about the kind of DNA, which could possibly carry the TIP (tumor inducing principle)(1) or be identical with it, evolved from total bacterial DNA (19), via that of phage PS8 (13, 20) to the very large A.turn, plasmid harbored,by all virulent strains yet examined (11) and very much implicated, though always indirectly, in the tumor induction process. If (plasmid) DNA is the carrier of the genetic information for the TIP, then the best demon- stration of that fact would be the induction of tumor cells with purified DNA. The long-range aim of the research efforts reported in this thesis has there- fore been che definition of conditions inducive of DNA uptake by tobacco cells, possibly followed by tumor cell induction if "virulent" DNA could be prepared in sufficient amounts. The administration of purified DNA to plant cells resulting in "transfor- mation" (12) has attracted considerable interest during the last ten years. The very bad reproducibility of this work has generally provoked much criticism (6, 8, 14, 21), But the introduction into living plant cells of foreign DNA in a biologically active form and under controlled conditions, could be effected by other means than DNA application. For example both somatic cell fusion and the use of transfecting phages could be useful as well. A review |of the pos- sibilities and the new tools presently available is given in chapter II, while the most tfv-ent approaches (up to August (975) are included in the discussion (chapter V 111). "liit UM' vi nucleic acid preparations from A.tuin. for the induct inn of plant I'ID.TS lus ri'v'tnt ly boon critically try-examined (17), resulting in a complete l.ii-V i>i" evidence for the induction of tumors with total A. turn.UNA. During tin? inception phase of our work .the experiments performed by Kovoor (10.) with '• •: •-••.•' V; .'.'•.; have been repeated with tobacco tissue. Very large a- "vuntr- v>t" A. i nni. PNA weft- more or less smeared on pieces of norni'il tobacco ..•JIIHS tirt^vu1, previously "wounded" by making deep incisions in the middle. Our "success" was even greater than that of Kovoor: about 90% of the pieces treated either with DNA oi* LKT.U C.I S-i'C became phytohorsnone independent and remained so after 8 subcultures, while about all the pieces survived and grew well when the application of UNA or buffer was followed by one subculture, on medium containing 1/10 of the regular hormones (7) before transfer to hormone- less medium. The -.-tobacco ..tissue used had been in culture for more than 8 years and our conclusion was that it had become very prone to "habituation" (see also A). Phillips and Butcher(17) arrived at the same conclusion commenting Kovoor's work. The criterium of phytohormone-independence in establishing the tunorous nature of plant cells and tissues should be used with great care. It is probably only valid (in conjunction with other criteria) when applied to cells which have been brought into culture recently. In view of setting up a model system for crown gall induction using puri- fied DMA, we developed a reliable method for the isolation of plant DNA on a micro scale (chapter III). Since its introduction this method has been suc- cessfully used with a great variety of plant materials other than those used for its development. The usefulness of the method has been illustrated by the subsequent publication of two similar versions (5, 15). -When exposing intact plant cells to DNA solutions in order to effect up- take, a rigourous distinction has to be made between DNA merely sticking to the plant cell wall and DNA penetrating into the cytoplasm of the cells. De- gradation of the DNA and reutilization of the breakdown products should not be confounded with integration." .These problems are studied in chapter IV. Similar studies have recently been performed with the alga Chlamydomonas reinkardi, resulting in evidence for irreversible association of bacterial DNA with the ceils, but no evidence for integration of detectable amounts of donor DNA into the host cell genome (14).