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). After the first International Symposium on Protoplasts and Fusion of Soma- f,c Pljnt Colls (Versailles, 1972) we decided, on the advice of many workers in the pps field, to switch to the use of mesophyll pps instead of cultured rolls in the study of DNA uptake. One of the .mportant considerations was the need for so-called conditioned cells for successful crown gall induction. With some experience in the aseptic isolation and culture of these pps and after determination of the onset of DNA synthesis in treshly isolated pps (chapter V), we attempted to obtain expression of a small bacterial plasmid in these pps (chapter VI). These studies showed the necessity of optimalizing uptake of those UNA molecules which enter the cells in a biologically »,i5e- fi.il" way (chapter VII).
In chapter VIII we discuss our findings against the background of the most recent approaches in pps research.' ' ".,..•' V j. ? " ."': r'z:^ :., : ' ^\.-'''.''':'\\. REFERENCES
1. Braun, A.C.- A^?'.,'. r,i. 34 (1947)234-240. . • • 2. Brauu, A.C. (vol.ed.) • i;>\.••j-F-xp-.'Swo)' Ft\s. 15(1972) Karger, *. Butc-iwr, D.N. in: riant Tissue and Cell Culture (H.E, Street, ed.) .• -;>.'•; V. ». v*-";?'"' 11(1973)356-391. Blackwell Sc-i .Publ. .Oxford. ^ i. Oons, J.J.M., \alentijn, M., Sclii. lpon-orc, R.A. and Dui jn, P.van, -j-7 • • V "' .^?. 89(1974)^83-294. 5. Hell, F.B., Gamborg, O.L., Ohyama, K. ,md Pelchur, L.(lQ74) in : -*•.'.•.' i. ."•'..•'. :>. : •' :• ' - -•'• * v .'.r?- (H.E. Street,edv) Academic Press, London, p.301-327. 6. Hotta, Y. and Stern, H.(1971) in : Infor'maiiiK Mol-sczilee in Biological ?rj3:.er:S (L. Ledoux, ed.) North Holland, Amsterdam, p.176-184, 7. Jaspars, E.M.J. and Veldstra, H., Pfiysiol.Plant. 18(1965)626-634. 8. Kado, C.I. and Lurquin, P.F., bioonem.SiophjB.Beo.Commitn. 64(1975) 175-183. 9. Klein, R.M. and Link, G.K.K., Quart.ZeV-.Biol. 30(1955)207-277. 10. Kovoor, A., C.B.Aaad.Sai. jSer.D 265C !96", ; 1623-1626. 11. Larebeke, N.van, Engler, G., Holsters, M., Elsacker, S.van do, Zaenen, I., Schilperoort, R.A. and Schell, J., Mature 252(1974) 169-170. 12. Ledoux, L., PvogJluel. Acrid Res.Mel.Biol. 4(1965)231-267. 13. Leff, J. and Beardsley, R.E., C.R.Aead.Sei;,Ser.J) 270(1970)2505-2507. 14. Lurquin, P.P. and Behki," R.M.» Mutation Res. 29(1975)35-51. 15. Lurquin, P.F., Tshitenge, G., Delaunoit, G. and L?doux, L., Anal.
16. Meins jr., F.,(I974) in : Tissue Culture and Plant Science 1974 (H.E. Street, ed.) Academic Press, London, p.233-264. 17. Phillips, R. and Butcher, V.K., J.Gen.MieroMol. 86(1975)311-318.- 18. Schilperoort, R.A. (1969) ?fest's, State University of Leid-n. 19. Schilperoort, R.A., Veldstra, H., Warnaar, S.O., Mulder, G. and Cohen, J.A., Bioaktm.Biophys.Asia 145(1967)523-525. 20. Schilperoort, R.A., Sittert, N.J.van, and Schell, J., Ew.J'.Bioshem. 33(1973;l-7. 21. Stern, H., (1975) in': 2nd John Innes Symp. "Modification of the information content of plant
Prospects in genetic engineering of plants
R. FKANS HEYN, ARTHUR RORSCH AND ROBBERT A. SCHILPEKOORT Department of Biochemistry and Laboratory of Molecular Genetics, Leiden State University, Leiden, The Netherlands
I. INTRODUCTION , II. SURVEY OF CONVENTIONAL AND UNCONVENTIONAL APPROACHES 8 2.1. The limitations of classical genetics 2.2. Handling eggs 2.3. Cloning of higher organisms 2.4. Haploid and diploid organisms 2.5. Somatic cell hybridization 2.6. Direct transformation by DNA 2.7. Definition of genetic engineering
III. BACTERIAL GENETICS 1 2 3.1. The Escherichia coii system 3.2. Translocation: mixing genes in vivo 3.3. Mutator phage Mu
IV. THE PROCESSING OF DNA IN VITRO 17 4.1. Enzymic fusion of genomes 4.2. Synthesis and isolation of genes 4.3. Genetic engineering of bacteria
V. INFORMATION TRANSFER BY PHYTOPATHOGENIC 21 BACTERIA 5.1. The Agrobacterium system 5.2; The/Rhizobiutn system VI. THE QUALITIES or- tin-: PLANT SYSTEM 24 6.1. Growth and tori potency of plant cells 6.3. Cult>'"aiiar of haphid plants 6.3. Phmt protoplasts 6.4. DXA transformation 6.5. Somatic cell hybridization 6.(>. Gene transfer and expression of foreign D.\'A in plant cells
VII. FURTHER CONSIDERATIONS AND CONCLUSIONS 36
VIII. REFERENCES 3 8
I. INTRODUCTION Genetic engineering has quite rightly an image of science fiction. The time when new species with any wanted combination of genetic properties can be ordered from an animal or plant breeding factory seems far away. The layman's view that the science fiction of today is the reality of tomorrow is certainly an insufficient argument to justify optimism. If this were so, we should by now be able to produce hybrids between members of the animal and plant kingdom as was foreseen by a nineteenth-century equivalent of Fred Hoyle. (see Fig. i). Despite the scepsis expressed by the prominent scientist Sir Macfarlane Burnet in his book Genes, Dreams and Realities (1971),/recent advances in molecular genetics have raised new enthusiasm (and uneasiness) which make people speak of genetic engineering as something to aim at as an approach to correct inborn errors of metabpUsm; iThis will^hdwever, not be our principal dish if we restrict ourselves to a vegetarian menu. We view genetic engineering of plantsnot pnjy_ as a future methodto improve speciesrbut also as a fundamental approach to the study of gene expression, especially with respect to cell differentiation. If we consider the term literally, the definition of genetic engineering might be any intentional genetic manipulation to alter species or to rnake new ones. In this sense genetic engineering was practised long before Mendel presented his laws (1865). In the seventeenth century the bulb growers of the Low Countries produced new varieties of tulips for which prices of a thousand florins each were paid (at the 1636 price index!). We notice here already a strong impact of genetic engineering on society; the so-called crazy tulip trade caused a financial disaster on the Amster- dam stock-exchange comparable to the Wall Street crash in the 1930s. -••-'•• ••-.-' : ..- Tiaerliliui Teriibilis Fig. i. TigerlMia Terribills.ioim&m the valley of Verrikwje, near Lake Odd- Phoenix Publ. Co., Bern, 1863, reprirted 1948.)
If .genetic engineering is d^fined[ as above, jj^^sMQ applied fenetic^ mustbe cbnsidelred as^ ;gen.^8;M?£i|#^l^^^e^.;:itjie. results of classical methods should not be underestimated. Great advances have been made, for example, in |he adaptation ot;.^^^-^Ameiit^'' *cerea^\tp. :]U3ie. .clbnatic con^nsJn deveiopjAg countrk tensive genetic work in thisiield isTorman E. Borlaug obtained the Nobel prize for peace in 1970, ;: - i One might restrict the term 'genetic engineering' to those methods using purified deoxyribpnucleic^acid (DNA) or a virus to alter the genetic malie-up of an organism.; However, we: thus neglect a number of extremely interesting unconventional genetic methods, such as somatic eell hybridization and the cultivation of haploid strains, which will certainly change our experimental approach to genetic problems in the near future. If we define genetic engineering to include only the un- conventional genetic methods, we must realize that what is unconven- tional today may be general practice in the future.
II. SURVEY OF CONVENTIONAL AND I'NV'.'N APPROACHES 2.1. The limitations of classical genetics In classical genetics offspring is produced by fertilizing egg cells with sperm cells through mating between female and male. The product is a fertilized egg or sygote which through cell division and cell differentia- tion develops into a new individual with a new combination of in- herited properties. Selection of special varieties is achieved by ingenious breeding schemes, which include in-breeding. The number of possible combinations cf genetic properties that can be brought together in a single organism is very much limited by the mating incompatibility between species as only closely related species can be crossed.
2.2. Handling eggs The incompatibility between species is not only due-to.-the inability to mate. Although fertilization can be brought about in vitro it does not necessarily make crosses between unrelated species possible. In vitro fertilization, possibly followed by re-implantation of the zygote, may facilitate crosses between related species. Artificial insemination, either performed in vivo orin vitro, speeds up the procedures;itis economically important not only in cattle-breeding but also in horticultufe.iThe in vitro cultivation of zygotes is also of importance when the foetus must be protected from microbial infection. This procedure must be con- sidered as a very early caesarian section. Manipulations with eggs were developed much further by Mintz (1967), who produced mosaic mice by mixing the cells of early-cleavage- stage eggs of white and black mice. The individual cells of the eggs did form an aggregate from which, after implantation in the uterus, a mosaic individual developed. More recently similar embryonic chimaeras have been obtained by Stern (1973) by'mixing' mouse arid rat cells, but no animals developed from such cell aggregates. It should be noted that genetics is not involved in these experiments; each cell in the aggregate keeps its genetic constitution. The experiment of Stern
8 is important because it should lead to a better understanding of the behaviour of individual cells and especially their migration in a de- veloping animal. The grafting of trees and other plants is a parallel •approach in the plant kingdom although here the host and scion tissues remain distinctly separate except in the area of the union. A more advanced mixing of tissues from different origin occurs, for example, in'the so-called periclinal chitnacra Cytisus adami:a Cytisus laburnum tree with the epidermis of Cytisus purpureus which results in smaller and darker leaves and purple-yellow flowers. In analogy the development of an apple variety with an epidermis resistant to fungi would represent an engineering success. The obvious 'real' genetic engineering experiment to carry out is to force fertilized or unfertilized eggs to take up isolated DNA and let them incorporate the foreign genetic information in their genome. This type of approach can not as yet be considered as very successful, as we shall see. •
2.3. Cloning of higher organisms When wild-type (heterozygous) male and female of the same species are crossed, all the individuals in the offspring are genetically different due to recombination events. A large number of almost identical in- dividuals can be obtained by in-breeding and these individuals are necessarily homozygous. Absolutely identical heterozygous organisms cannot be obtained in large numbers with conventional genetic tech- niques. With plants they can be obtained in large numbers using cuttings. Ail our potato varieties are genetically identical, highly heterozygous clones.For frogs a method has been""developed to^prbllu& a multitude of heterozygous identical individuals by transplantatipn of nuclei (Wood- land, Ford & Gursdn, l9J2). Fertilized eggs were used as recipient cells. Their nuclei were removed by micro-surgery and replaced by 'somatic* nuclei obtained from intestine epithelial cells. Identical mature animals were raised from thfe 'egg' cells with implanted nuclei, since each nucleus of the donor-body has the same genetic
•••complement.- 'r/.••••,••'•••• A:. '~S, :'~^;'•••-;•-• ^ - -••:•• • : : • • . In principle it should be possible to grow new mature organisms directly from explanted somatic cells. Every cell contains the complete genetic information for the organism, although different parts of the genome are phenotypically active in each type of differentiated cell. The major problem is to give the proper differentiation signals to start development, as happens in the fertilized egg. All the attempts with somatic cells from animals have been unsuccessful so far. In plants" it is now well known that explanted individual cells from a mature organism will develop into new plants provided they are cultured on the right media (see section 6.1).
2.4. Haploid and diploid organisms For genetic studies both haloid and diploid organisms have specific advantages. With haploid organisms, genetic exchanges induced'for ex- ample by radiation are more easily detected because a homologous gene cannot mask the effect. This is a major reason for the great; pop- ularity of bacterial systems; for genetic studies. On the other hand, mutations in vital functions cannot be introduced in haploid cells since they clie from^h 2.5. Somatic cell hybridization When an egg and a sperm cell fuse, azygote is formed which combines part of the genetic properties of both parents. Taking advantage of genetic cross-overs, genetic maps cart be constructed in which the dis- tance between genes is expressed as the chance that a cross-over between them occurs. In several organisms, however, genetic crosses cannot easily be studied. Among those is man who has a long generation time. In i960, Barski, Sorieul & Cornefert made the important discovery that cells from two mouse tumour lines can be fused into one cell. This fusion is not restricted to cells from the same species (Sell & Krooth, 1972). An important improvement of the fusion technique was de- veloped by Harris & Watkins (1965), who used inactivated Sendai virus to increase the fusion frequency. The observation by Weiss & Green 10 t human chromosomes are lost in man/mouse hybrids was another important discovery in somatic cell genetics. Random loss of chromosomes provides a mechanism by which segregation of genes can occur, which in the case of meiosis takes place in a very precise way. By simultaneously studying the loss of certain chromosomes and certain biochemically characterized functions, the location of these functions on a' distinct chromosome can be established. If the loss of chromosomes from hybrid cells could be prevented and if animal cells cultured in vitro could grow into whole organisms, genetic engineering would be a fact today. It should be appreciated that somatic plant cells can develop into flowering plants. In plants, how- ever, somatic hybridization, although it has been detected^ is lagging far behind in comparison with/that inthe mammalian systems (see section 6.5), - 3,6. Direct transformation by DNA In 1944, Avery, MacLeod & McCarty proved that the bacterium Diplococciis prietimoniae was able to take up purified DNA from, its en- vironment arid incorporate it into its genome. This was the first proof that DNA is the carrier of genetic information. Many scientists were rather reluctant in the beginning to accept the excellent evidence presented by Avery and co-workers, and it took several years before transformation by DNA was established for some other bacterial species.:; •, •;.•..." .•'.•• .••-.'. •-.'.. • :. ..;••-•:...-;. . '-.. ••••' : •,,• ..- •••;• - • Already in the 1950s it was claimed that DNA transformation could be observed with ducks^ which was followed by claims"for' Drbsqphila, plants and mammalian cellfstn vitro. The scientists who published-'these', results and who felt that their work was; not sufficiently appreciated could find comfort in the delay of appreciation that Avery et a/, ex- ; y perienced.';' '-.;• ;, ,v---^ i:: :4:;.-•;.-•.>; ;:;:-.:. ;> >: ,; . :../:-. V. ,_-..:_:.: -r^',':'-r '• •• Considering the complexity of the eukaryotic system one has to remain sceptical though some of the results of transformation work may have the benefit of the doubt. 2.J. Definition of genetic engineering After this survey of a few new approaches in genetics, we here define genetic'''engineering-', as-.genetic manipulations (by-passing the sexual cycle) "by which an individual having a new combination of inherited properties is established. 1 1 III. BACTERIAL GENETICS 3.1. The Escherichia coli system Our definition of genetic engineering also brings up the genetics of bacteria such as E. coli. For the following reasons it is justified to include a brief account of the features of the bacterial system in this paper: (i) Although the ways of exchanges of genetic material between bacterial cells exhibit unique properties, they have been models for unconventional genetic treatment of cells of higher organisms. (ii) Together with the manipulation of DNA in vitro (sec section IV) the bacterial system makes a processing of DNA molecules possible which will be useful for the genetic engineering of plants. (iii) Some people still consider bacteria to belong to the plant kingdom. It is not so much the taxonomy that counts, but the occurrence of inter- actions between plants; and bacteria) cells in nature (see section V). Bacterial DNA is an important donor material to think of when we try to incorporate a foreign DNA into a plant genome. The exchange of genetic material by E. coli can occur by three dis- tinct processes: conjugation, transduction and transformation. Trans- formation was the first process to be discovered although not in E. coli. After the initial discovery by Avery et al. (1944) with D. pneumoniae, several other bacterial species, e.g. B. subtilis and H. influenzae, were shown to be able to take up purified DNA (isolated from the same species) from the medium and to incorporate it in their genome. Only very recently (Cosloy & Oishi, 1973) transformation of E. coli was established beyond doubt in spite of the fact that several hundred workers with E. coli must have looked for it over the last 25 years. The very early observation of transformation of E. colt by Bovin & Vendreley (1946) could not be reproduced. The^secret of ET coli transformation appears to be the necessity for the loss of a gene function of the recipient cell. The gene involved is called rarflC and codes for an ATP-dependent nuclease which destroys externally added DNA. The loss of this func- tion results primarily in an inability to perform the recombination of DNA molecules. Secondary mutations in other genes (abcA or sbcB) restore this recombination ability without restoring the capacity to produce the ATP-dependent nuclease. Thus, in contrast to wild-type recBC+ E. coli, the recBCshe A otrecBC sbcB mutantsareable to take up purified DNA from the medium and to incorporate it in their genome. The story of E. coli transformation should warn us that similar 12 inhibitory genetic factors could hamper the transformation of cells of higher organisms. Although somatic cross-overs seem to occur in higher organisms, we believe it is our scanty knowledge of the molecular basis of genetic recombination in eukaryotic cells that accounts for the poor results obtained so far. Inter-species transformation in bacteria also gives poor results. The species specificity can be explained by the need for homology between recipient and donor DNA and by phenomena known as restriction and modification. It is generally accepted t. ,t recombination ,. :-veen DNA molecules requires a high degree of homology; the base FC;'I J ices of the two DNA molecules participating in the cross must be utmost identical to allow their pairing (synapsing), a process that precedes the actual formation of cross-overs between them. A new trick to overcome the lack of homo- logy will be discussed in section 3.3. Restriction is caused by the breakdown of the DNAs of even very closely related strains (E. coK B versus E. coli K 12) in each other's cytoplasms. The degradation is due to the action of very specific re- striction enzymes which recognize the unique base sequences in the foreign DNA. As we shall see later, these restriction enzymes have become tools for the processing of DNA in vitro (section IV). A foreign DNA escapes restriction when the unique base sequence which is recognized by the restriction nuclease is 'modified'. This modification is a very specific methylation of certain bases in the DNA. The specificity of restriction and modification is illustrated by the action of E. coli RII restriction endonuclease and modification methylase, recently described by Boyer et al. (1973). The restriction endonuclease produces two breaks in double strand DNA with the following sequence (molecule A): ( + ) strand: X—Y—X—Y—C—C—A—G—G--X—X—X—Y (->—: i-LUJJLIJJ-LUi (in which X—Y are arbitrary base pairs, G=C and A=T resp.guanine- cystosine and adenine-thymine base pairs). The endonuclease recog- and lt nizes the sequence SGTCC produces molecules with single stranded ends as follows: (molecule B) X—Y—X—Y I I 1 I Y—X—Y—X—G—G—T—C—C 13 (molecule G) Y—Y—Y—X I II ! C C A (I (1 V—V \r V The mcthylating enzyme adds in molecule A a methyl group to the . cytosine in the (+) strand immediately adjacent to the central adenine and to that in the ( —) strand immediately adjacent to the thymine, making the sequence 5 Me Y—C- C—A— G—G—X ! Ill 111 !! Ill ill I X—G—G-~T—C—C—Y • • ' v ••• •••• • 5 Me inaccessible to the action of the restriction riuclease. Nothing is known about restriction arid modification of eukaryotic DNA. The loss of human chromosomes observed in man/mouse hybrid cells (see section 2.5) has not been attributed to such actions on the molecular level.-.- ;://;;//:v:.-'V;;/ ^ '."."••"'"' y/J'^.C'"""'^ : '' .', - ..•' • •',•;'.;••?";. Trahsduction is the transfer of genetic material from one tacterium to another, mediated by a DNA virus. When viruses multiply in their) host, the final step is the^/enclosure of tiewly synthesize^ viral DNA by coat protein. Certain viruses make mistakes and pack into virus coats a bit of host DNA (defective virus particles). When such a pavticle infects another host, a small piece of DNA of the first host is transmitted to the second host and can subsequently be incorporated into its genome. Transduction is a widespread phenomenon in the bacterial world and transducing phages have been isolated for many species. It is not thr likely that^^ somethingr similar occurs with mammalian DNA viruses. Indeed, host cell PNA has been detected in polyoma virus particles. On a quantitative basis, however, it is unlikely that a transducing system can be made operational for eukaryotic cells. During virus assembly, the host DNA is chopped up in very small pieces and the chance that a certain piece of bacterial DNA is incorporated in a trans- ducing particle is low (io~6 per particle). The human genome is much larger than the E. coli genome and consequently the chance of in- corporation of a certain mammalian gene in a mammalian virus is ex- tremely low. It has been calculated that the isolation of a eukaryotic cell transduced by a virion would involve the screening of an infected 14 population in the order of io10 to ioia cells (N.N., Nature, editorial, 1971). For plants, many RNA viruses are known but very fuw DNA viruses. Nothing is known about the occurrence of plant DNA in defective virus particles. Specialized transduction is another form of transduction in bacteria, in which certain specific genes are transferred with a high frequency (see section 3.2). The process of bacterial conjugation has a remote resemblance to the sexual process in higher organisms. Male (donor) and female (recipient) cells are distinguished. In addition to a single large;genome (a DNA molecule of approximately 1 mm) the male cells contain a small circular DNA (plasmid) called F factor. In a strain carrying such an F factor, recombination can occur between the F factor DNA and the large chromosomal DNA and thus the F factor is incorporated into the genome. For the incorporation it does not show much preference for certain sites of the bacterial chromosome. No F-like factors have been described for eukaryotic cells. 3.2. Translocation: mixing genes in vivo From an E. colt strain with an inserted F factor, the latter can be released again spontaneously. When it is released, a small piece of chromosomal DNA may be attached to it. To distinguish such a plasmid from wild type it is indicated as F'. If, for instance, the lac operon of E. coli is attached to such a plasmid, it is indicated as F'lac+. In a partial diploid + like F'lac llac+) the F' factor can again insert itself in the chromosome of the bacterium and it shows a preference for insertion in the chromo- somal lac region. Since the part of the F' factor DNA carrying the lac+ genes is homologous with the corresponding part of the chromosome, recombination inside this region is likely to occur. If, however, the F'lac+ is transferred to an E. coli strain with a deletion for the lac region, no such recombination can occur and the F'lac+ shows no preference as to insertion. It may insert itself any- where and by certain selection techniques translocation of genes can be achieved. Translocation can also be brought about by lysogenic bacterial viruses. These (bacterio) phages insert, with a certain probability, their DNA into the bacterial genome. This phage DNA insertion resembles F factor insertion, except that almost all lysogenic phages (e.g. lambda, 15 , 0434) have a specific site for integration (the virus attachment site). The inserted phagc DXA is called prophage. The prophage can release itself again from the bacterial chromosome: tlic'phagc DNA will multiply autonomously at a much higher rate than the chromosomal DNA. It induces in the host the synthesis of virus specific proteins and ulti- mately a burst of new virus particles is produced. Occasionally the excision of the prophagc occurs inaccurately and a small.part of the bacterial genome that is adjacent to the inserted prophagc DNA, remains attached to it and replicates with it. Finally the DNA is incorporated into phagc heads and virus particles are produced which contain a small piece of specific bacterial genome. Upon the infection of another host, this part of the genome is transferred from one bacterial strain to another. If a transducing virus particle infects a strain in which the virus attachment site of the chomosome is deteted, the DNA can insert with low frequency elsewhere and translocation of the bacterial gene occurs. ~ "• Transducing particles are used not only to perform translocations but also for the mass production of certain bacterial genes; Purified trans- ducing particles, e.g. Afect or A^O/T, contain a relatively high amount of the corresponding lac and £«/ region of the bacterial chromosome and : this DNA can easily be extracted from these particles; Recently :Doy, Gresshoff &^Rplfe^ 1.972, 1973 a) claimed that upon massinfection of plant cells with such lambda transducing particles, bacterial genes were expressed in plant cells (see section 6.6). V 3.3. Mutatorphage Mu Phage Mu is a lysogenicvirusof E. cpli with the unusual property that it does not have one_attachmerit siteon the bacterial chromosome^but will insert itself with high frequency at any site (Taylor, 1963). If Mu inserts itself inside a structural gene, the coherence of the genetic message is broken, resulting in a mutations It is very tempting to consider Mu a bacterial equivalent ofa rrf^nimaliaritumour virus. It is generally accepted how that mammalian cells transformed by a DNA tumour virus contain in their genome at least part of the viral nucleotide sequences. Ifsuch viruseswere: inserting themselves into regulator genes of their hosts, the uncontrolled growth of tumour^ cells could simply be attributed^o resjultingm much more complex-, no mutation ma single gene has been observed to result from the infection ofmammalian-•-••cells by a virus. Further 16 studies on Mu have revealed other extremely interesting properties which keep the tumour _virologists alert.-For example,-Mu shows re- dundancy for certain DNA sequences, making internal cross-overs inside the Mu genome possible (Daniell, Boram & Abelson, 1973). Similar redundancy has also been observed for adeno virus. The genetic recombination induced by the insertion of Mu in chromosomes is of special interest (van de Putte & Gruythuyzen, 1972). When two un- related DNA molecules, between which no cross-over can occur because of the lack of homology, carry each an inserted Mu DNA sequence, genetic recombination can occur inside that sequence and the two un- related DNA molecules can be coupled or can exchange parts at either side of the inserted Mu DNA. De Graaff, Kreuning & van de Putte (1973) have transferred an E. coli F factor carrying Mu to a strain of Cilrobacter freundii also carrying a Mu prophage in its chromosome, which resulted in the incorporation of the E. coli F factor in the C. freundii chromosome. Thus Mu helps to overcome the lack of homology between unrelated DNA molecules. The limiting factor for general application of this trick is that there must first be a possibility to incorporate Mu itself into the relevant DNA molecule. With or- ganisms which do not have the proper receptor sites for phage Mu on their cell walls, the absorption ft~p must be by-passed, which in prin- ciple could be done by transformation (section 2.6 and 3.1). Naturally occurring interactions between bacteria and plants (see section V) may in the future be helpful in transferring a broad spectrum of bacterial genes to plants, that is if Mu can be transferred to those bacterial species or if Mu-like phages for these species can be found. IV. THE PROCESSING OF DNA IN VITRO 4.1. Enzymic fusion of genomes The restriction endonucleases (EC 3.1.4.32) of the type described in section 3.1 and DNA ligase (EC 6.5.1.1) are important enzymes used to fuse DNA molecules in vitro. The former enzyme produces pieces of DNA like the molecules B and C. Under appropriate conditions, two such fragments of DNA molecules from different sources with 'sticky' ends can join and covalent bonds can be introduced using DNA ligase. Several of such nucleases producing sticky ends have been de- scribed. An enzyme from H. influenzae cleaves the DNA of simian virus (SV40) into u fragments, another enzyme from H. parain- 17 fluensae cuts it into four fragments and the E. coli RI endonuclease introduces only one break per circular SV 40 molecule. To facilitate the hydrogen bond formation at sticky ends, another enzyme can be used: the terminal transferasc (EC 2.7.7,31). Starting with a 3'OH primer this enzyme joins deoxyribonucleosidetriphos- phates into a polydeoxynucleotide without the need for a template. Thus if a DNA of the configuration is incubated witlv: thymitline triphosphate and terminal transferase, the molecule is elongated at the 3'GH ends. When DNA from another source (R-Q)m is labelled with poly- deoxyadeninenucleotidc, the two DNAs can anneal through hydrogen bonding at the sticky ends. The remaining gaps in the annealed molecules can be filled by DNA polymerase (EC 2.7.I7.7) and eovalently closed by DNA ligase. The two original molecules are thus joined to each other by an AT-rich region. Following this procedure Jackson, Symons & Berg (1973) connected a complete SV 40 DNA molecule with a lambda DNA containing the galactose genes of E. coli to form one large closed circular molecule. ST. I ,3'OH N XI —T-T—T—T-T-T-T-T-T-.'jr .T_T-T-T-ly|v ST 3'OH ^ fx] —T—T—T—T—T—T—T—T—T—T^ ,T—T—T—T—I Yj v :A.-A-A-A—A-A-A-A—A-A Off n 5T 3'OH' 4.2. Synthesis and isolation of genes From a theoretical point of view, there seems to be no limitations to the in vitro joining of DNA molecules from various sources. Any bio- chemistry laboratory that can handle DNA and the appropriate enzymes can do it. If, however, we want to join specific genes, we first must isolate them from the large genomes in which they occur. One method, certainly not the easiest, is to synthesize the genes 18 synthetically. To do this, the base sequence must be known. This sequence could be deduced from the nucleotide sequence of the RNA produced from the gene or from the amino acid sequence of the protein coded for by the gene. The sequence of the nucleotides is known for only a few RNAs, including some of the tRNA molecules. Khorana and collaborators (Agarvval et al. 1970) have accomplished the gigantic task of synthesizing chemically the DNA sequence of yeast alanine - tRNA. If we have a mcsscnger-RNA of unknown nucleotide sequence, the corresponding DNA could, in principle, be produced by means of reverse-transcriptase. This DNA polymerase is found in mammalian RNA tumour viruses and uses RNA as a template to synthesize a polydeoxynucleotide copy (Baltimore, 1970). When purified messenger- RNA is available, we can also obtain its corresponding DNA in a dif- ferent way. \Vhen this RNA is mixed under appropriate conditions with denatured;DNAfrpin^the same: source^the;RNA will hybridize specifically with the region of the DNA of complementary base sequence. Shih & Martin (1973) immobilized the RNAon cellulose and performed hybridization on the column and subsequently recovered from it the selected^ DNA/RNA duplex. This heteroduplex has a large single strand region containing non-required DNA sequences. This contaminating DNA can be broken down by Neurospoffr crassaendo- nuclease (EC 3.1.4.21), an enzyme that is specific for single strand nucleic acid and that does not affect duplexes. From the remaining RNA/DNA hybrid, a DNA duplex can be made by DNA polymerase I. Methods for isolating small parts of a bacterial genome imply the isolation of F' plasmids or specialized transducing phages (see section 3.2)rButbesidesthe piece of ^^ contain some F factor-specific or virus-specific DNA, Shapiro et al. (1969) were the first to purify the genes of the lactose operon of E. coli from transducing lambda particles. They used two types of phage par- ticles, both containing the lactose operon but in opposite orientation. The rjhages had the following structures: A J a y z 0 p I R A' J' a' y z 0 p i' N' R' and A J i p 0 z y a N R A' J' i' p'o'z'y'a' N' R' 19 (ayzopi are lactose operon genes and functions, A, J, N and R phage functions; + strand sequence indicated as a, y, o etc.; — strand sequence as a', y't o', etc.). Upon denaturation and annealing, the following struc- ture was formed from the + strand o| the first phage and the - strand of the second phage DNA: ay zop a'y'z'o'p' This is a duplex structure for the lac operon with single-strand ends. The single-strand ends were removed by the iVc«roj/>orfl endonuclease and a DNA was left which carried the lac pperon only. It should be clear that the methods available to isolate genes are still limited. The possibilities are restricted to genes for which the tran- scription product can be obtained (tRNA and rRNA) arid to bacterial genes. 4.3. Genetic engineering of bacteria The combination of enzymic and genetic tricks which can be used in the E. coli system again focuses our attention on the bacteria. Especially E. coli with its transducing phages, F factors, and ability to be trans- formed by DNA, will be an Important intermediate in the processing of DNA. By the methods described in section 4.1, undefined pieces of 'foreign' DNA can be coupled to well-defined pieces of E. coli DNA (including F factor and lysogenic phage DNA). When these joint structures are taken up by competent (transformable) E^ coli cells, the E. coli moiety of the DNA may recognize the homologous region in the bacterial genome and recombirie with it. With a bit of luck, the foreign piece of DNA will go with the £» cxtli DNA and, if we: can select for the genetic property of the foreign D^FA; we should be able to accumulate E. coli cells carrying that specific piece of foreign DNA. By:F' transfer, genes for ribosomal proteins and for nitrogen fixation have been transferred from other bacteria into E. coli. As yet no gene transfer from animal or plant cells (or their viruses) to bacteria has 20 been performed. We are, however, reluctant to encourage such an approach without reserve. Some workers must be very near to trans- %rrmg animal virus genes into bacteria; for example. Jackson et_alf l(*972)> wn» nave prepared the joint SV 40-iambda DNA. It has recently) been pointed out at several meetings that one should not try to transfer eukaryotic virus DNA to a bacterium, because of the danger that such cells might escape from the laboratory and multiply beyond control in nature. It may some day be useful to transfer plant genes to bacteria, either because of economic interest (bacteria can grow faster than eukaryotic cells arid gene products may be produced at a higher rate), or in order to use the bacterial system for the processing of plant DNA. V. INFORMATION TRANSFER BY PHYTOPATHOGEKIC BACTERIA 5.1. The Agrobacterium system Many plant diseases are caused by bacteria, but in most cases the mechanism of the interaction with the hosts is poorly understood. In general the effect of the bacteria is detrimental to the plant because of the excretion of inhibitory substances or of cell-wall-digesting enzymes. The reaction of the plant sometimes includes abnormal growth, obviously brought about by a locally altered level of growth regulators (e.g. auxin, cytokinin, gibberellins). Symptoms can often be observed, associated with hyperauxiny that give rise to abnormal differentiation, e.g. adventitious root formation. Likewise strongly proliferating and hypertrophic tissues may arise from the imbalance of phytohormone metabolism. In a few diseases, transfer of genes from bacteria to plant cells may be involved, as for example in the forrhation of root nodules on leguminous plants and in the induction of crown galls in dicoty- ledons. Root nodules, which are centres of symbiotic fixation of nitrogen, result from the action of bacteria of the genus Rkizobium.The plant tumour crown-gallis inducedbyr Agrobdcieriumtumefaciem, v/hich also belongs to the family Rhizobiaceae. After wounding, which is found to be essential for the induction of tumour growth, ^4. iM?n«/aam cells penetrate into the intercellular spaces and into the injured cells which are filled with wound sap. In these places, the bacteria replidat^ arid interact with the adjacent plant cells; They never penetrate irito undamaged cells^ A bacterial attach- ment to the plant cell wall seems to be an obligatory initial stage in 21 tumour initiation (Lippincott & Lippincott, 1969; Schilperoort, J969). The time required to transform a 'conditioned' cell to a tumour cell was found to be at least 8 h at 25 °C (Lipctz, 1966). In Kalnnchoe maximal conditioning has been shown by Lipctz to occur prior to the first observed wound-stimulated cell divisions. The experiments of fiopp (igf)6) suggest that the period of DNA synthesis is of major importance in tumour initiation. This finding is of special interest with regard to studies showing the existence of base sequence similarity between DNA from A.tumefaciens cells and crown-gall DNA (Schilperoort etal, 1967). It is suggested that after bacterial attachment a large plasmid present only in virulent A. tumefaciens cells (Zaenen etal. 1974) is transferred to the plant cells during actual tumour induction (Schilperoort, Van Sit- tert & c ' '1, 1973). The bacterial genes come to expression in tumour cells as '•< *A and protein (Milo & Srivastava, 1969; Schilperoort et.aU 1969). All avirulcnt Wild-type strains tested do not contain the large plasmid, while the DNA homology studies of De Ley (1972) have shown that 100% homology exists between chromosomal DNAs of virulent and avirulent cells of the same taxonomic type. In sortie unknown way, certain functions of lysogenic A. tumefaciens phages (e.g. PS 8) might be involved in tumour initiation. Reports on the presence of phage activity in sterile homogenates of crown-gall callus cultures of different origin have been presented (Parsons & Beardsley, 1968). However, phage induction and the subsequent release of phages in infected wounds is probably not essential for tumour induction since there is no de- tectable difference in virulence between lysogenic strains and PS8-cured strains that do not produce phages at all (Ledeboer etal., unpublished results). It has been recently fdiind, however, that a PS8-cured strain still carries a defective phage in its plasmid. Together with the results showing the presence of PS 8-like DNA sequences in crown-gall DNA (Schilperoort et al. 1973), this suggests that this type of DNA, when integrated in a plasmid, may play a role in ^moiir induction. No definite conclusion about the significance of phage functions in the transformation process can as yet be made. It is worth while to in- vestigate whether the phage DNA sequences might function like the mutator phage Mu (see section 3.3) of E, coli, i.e. to facilitate the in- tegration of A tumefaciens plasmid into plant genomes. It should be noted that the plasmid of different wild type A. tumefaciens strains need not all contain the same phage, only the same function may be required. 22 5.2. The Rhizobium system Little is known about the genetic background of nitrogen fixation by Rhizobittm. It is not oven known if the genes involved are located in the leguminous plants or in the bacteria. No nitrogen fixation has been detected in pure cultures of Rhizubium. The infection process is entirely different from that of Agrobacterium. The Rhizobia invade roots by means of an infection thread separating the bacteria from adjacent cells. When this thread reaches the cortex, the bacteria enter the cortex cells and sometimes continue dividing. Nitrogen fixation seems to occur only after the membrane-encompassed bacteria within infected cortex cells have changed their morphology into bacteroids, though Rhisobium japonicum does not exhibit this morphological change (Bergersen & G6pdchild,;\.i973)^[r_he/-.nodule.Lpigment: leghemoglobin may be con- nected with the whole process since it is synthesized concomitantly with the first signs of nitrogen fixation (Schwinghamer, Evans & Dawson, 1970). Recently important data have been obtained indicating that the protein part of the pigment is coded by the legume genome (Dilworth, 1969) while theiheme part is produced by the bacteroids (Cutting & Schulman, 1969). It has been postulated that part of the rhizobial genome was transferred to the legume during the evolution of the symbiosis. Possibly a gene transfer, as suggested for tumour in- duction by A. tumefaciens cells, has taken place leaving behind bacteria that need the plant to be able to perform nitrogenfixation, A comparison with A. tumefaciens becomes even more interesting if we look at the data demonstrating that Rhizobium can form tumour-like structures on leguminous plants (MacGregor & Alexander, 1971). Most, but not all of these strains, still possess the capacity to form nodules on their specific hosts. The tumourigenic capacity does not seem to be very stable in some strains since it could be lost by subculturing the cells. Bacteria that lose their tumourigenic'activity retain their nodulatihg ability. It would be of great interest to see whether the loss of tumouri- genic capacity correlates with the loss of a plasmid and whether there exists sequence similarity between leguminous root DNA and a plasmid DNAV if this is presents If bacterial plasmids are involved in naturally occurring transfers, then in principle all kinds of bacterial genetic information can be in- tentionally introduced into plant genomes. A. tumefaciens cells, pro- vided with the desired; genetic information in its plasmid, might possibly 23 be used in future to achieve a reproducible and efficient transfer of genes. VI. THF QUALITIES OF THE PLANT SYSTEM 6.1. Gratcth and totipotency of plant cells The first successful cultivation of plant cells for prolonged periods in vitro was aclxieved in the early 1930s by White, Gauthurct and Nobecourl. Today consv Jerable progress has been made in this field of research (Vasii & Vasil, 1972). The discovery of the phytohormones auxin and cytokinih, which appear to be essential for growth and cell division, permits the use of well-defined synthetic media. Tissue explants of a wide variety of higher plants can be brought into culture on such media which are generally adapted to the specific requirements of each tissue type. The basic agar medium is composed of organic substances and inorganic salts supplemented with the appropriate phytohormones, A relatively homogeneous mass of cells, called callus, is obtained on this medium. The organic substances include sugars, vitamins and amino acids. Some- times less-defined mixtures of coconut milk, yeast extract and casein hydrolysates are added for vigorous growth, formation of embryoids, or to start cultures of plant varieties for which a defined medium is not yet known. Most tissue cultures are derived from dicotyls; in thepast few years monocotyledonous species have also been cultured success- fully.'' • • " •"'••; •<'^:;:-:-i-'C^:]::'^:-. '.^'-y": ^^:':- ' ^-;•''> ;:^" If segments of well-growing callus cultures are transferred into a liquid nutrient medium and are incubated on a gyrotory shaker, viable cell aggregates and single; cells will dissociate from the callus tissue. By subculturing the detached cells, one can establish a cell suspension culture. Such cultures selected for single cells and small cell aggregates can be plated and cloned by mixing the cells with a nutrient agar medium according to the technique developed by Bergmann (i960). The plating efficiency of cell suspensions is rather low. Differentiation and organogenesis ate under hormonal control and can be induced in undifferentiated callus cultures by changing the con- centration ratio of auxin and cytokinin. In this way it is possible to obtain at will either root or shoot formation. Even whole plants can be regenerated from callus cultures. The totipotency of higher plant cells (Schleiden, 1838 and Schwann, 1839) has clearly been demonstrated by the regeneration of adult (tobacco) plants from truly single cells 24 obtained from cell suspension cultures (Vasil &_Hildebrandt, J965). In this respect, plant cells in vitro offer unique experimental approaches to study cell functions and morphogenesis which up to now are not feasible with animal cells in culture. Moreover, the regeneration of whole plants has become an important means of vegetative propagation in horticulture and agriculture. During prolonged cultivation, however, callus cultures as well as suspension cultures give a variation in ploidy and show chromosome aberrations (Melchers, 1965; Sacristan, 1971). Such cultures generally lose their regeneration capacity. Moreover the regenerated plants may turn out to be incapable of sexual propagation. Therefore callus cultures which have been subcultured for oniy a limited number of times should be uscU in regeneration experiments andjas starting; material for cell suspensionculturesr 6.2. Cultivation of haploid plants Since the first description, more than half a century ago, of a haploid flowering plant (Blakeslee et al. 1922), haploid individuals of many more species have been detected and described. Even though they include important crop plants like wheat, rice, maize, tomato and tobacco, the practical application of these rare, sbrnaticaUy haploid, plants has been extremely limited. Many of them can be vegetatively propagated. Thus mutagenic treatment and easy identification and selection (also of re- cessive mutants) could have simplified and accelerated plant breeding. Once a wanted character is found in haploid material, homozygous and fertile plants can be produced by reversion to diploidy through col- chicine treatment (Blakeslee & Avery, 1937). Gulturing the tissue as a callus" alsorisomehowr favours the appearance of di- (and poly-) plbid lines. This has posed problems with the maintenance in culture of haploid cell lines, but recently the amino acid analogue parafluoro- phenylalanine has been claimed to selectively maintain the growth of haploid tobacco cells while inhibiting simultaneously the growth of diploid cells (Gupta & Carlson, 1972). Although the great importance of these haploid plants, not only for breeding purposes but also for fundamental plant physiology, has been shown on many Occasions (Melchers & Bergmann, 1958; Melchers, i960; Nei, 1963; Chase, 1964) botanists have been rather slow in ex- ploiting this new approach, though some use was made of these plants in the breeding of maize varieties. The situation has changed drastically, however, in the past 5 years. A new surge of interest in haploids has been 25 triggered by the development of techniques to grow haploid plants in large numbers through the special cultivation of anthers. The discovery that hnploki cmbryoids can originate in unripe anthers when cultivated on special nutrient media (Guha & Maheshwari, 1966), has initiated much imaginative and elegant work such as that of Nitscli and co- workers (e.g. Bourgin & Nitsch, 1967; Nitsch, 1969; Nitsch & Nitsch, 1969), eventually leading to completely controlled experimental andro- genesis in Nicotania. These techniques for the culture of anthers (and now also of free pollen) are being extended to other plants, including many which are important for food production: rice, cabbage, tomato. Haploid eel! and tissue cultures obtained in this way have been put to good use in combination with microbiological methods of mass screening. The first results of this approach yielded ceil lines of tobacco resistant to streptomycine (Binding, Binding & Straub, 1970; Maliga, Brcznovits & Marton, 1973), to DNA base analogues (Lescure, 1973) and to Pseudomonastoxin (Carlson, 1973 ft). The repeated pleas of Melchers (1972) for a coordinated effort in the production of large amounts of haploid crop plants for breeding purposes have finally re- sulted in the establishment of two special project groups in Germany (MPI fur Pflanzengenetik 'RosenhoF, Ladenburg) and similar groups in Denmark (Riso) and Canada (Ottawa). The rapid exchange of results in this field has been coordinated by Melchers (1973 b). 6.3. Plant protoplasts Mechanical methods for the isolation of naked plant cells have existed for nearly a century. However, only in the last decade have marked improvements in isolation and culture techniques made possible the important role which protoplasts are now playing in genetic* manipula- tion. The essential 'trick' in protoplast isolation is to plasmolyse the cells in a tissue by placing it in a rather concentrated salt, or sugar solution. The low osmotic potential of this solution causes water loss from the living cells, resulting in retraction of the protoplast from the cellulosic wall. The protoplast is drastically reduced in volume and assumes a more or less spherical shape. When these plasmolysed tissues are cut with a knife, there is a chance of disrupting the outer cell wall without damaging the protoplast inside it Of course the number.of intact protoplasts which can thus beisolated is rather small. The replace- ment of the damaging knife by crude but well-chosen enzyme mixtures has greatly stimulated the study of protoplasts (see the comprehensive 26 review by Cocking, 1972). Since 1968, when these crude enzyme prepara- tions became commercially available, the use of higher plant protoplasts has been even more widespread and in September 1972 the first International Symposium on Protoplasts and Fusion of Somatic Plant Cells was organized in Versailles by CNRS and INRA (Tempe, 1973) while an EMBO-workshop on Plant Cell Protoplasts and their Molec- ular Biological Implications was held in Tubingen. The enzymic method requires less osmotic shrinkage of the protoplasts, which, after all, is quite a traumatic treatment. It also renders many more plant species and tissue culture material amenable to protoplast isolation. But the major advantage is the huge number of protoplasts that can be obtained. That these populations of naked cells can be very homogeneous, even when they are derived from such differentiated organs as whole leaves, is illustrated by the method introduced by Takebe, Otsuki & Aoki (196S). They peel off the un^er-epidermi§ ^and then incubate the leaf pieces several times with a crude macerating enzyme. In this way tissue fractions are obtained which can then be separated in a population of viable palisade cells from spongy parenchyma cells and rhechanically damaged cells. Asecc/hd treatment with a crude cellulase liberates large numbers of spherical protoplasts. Protoplasts can be obtained and cultured under sterile conditions. As a useful alternative to isolation from intact plants (which always need intensive surface-sterilization) they can also be produced from already aseptic and more or less homogeneous callus culture material. The completely denned conditions of callus and suspension culture, as com- pared to the always variable physiological conditions found in gieen- house cultured plants, constitute another major advantage. Of course, merely maintaining plant protoplasts in oullure is not satisfactory and much effort has been put into the formulation of special media capable of inducing cell wall regeneration, cell division and differentiation in plated protoplasts. In the case of the very-well-studied tobacco and carrot cultures (Grambow et al. 1972) results were quickly obtained. Whole new tobacco plants, both from diploid (Takebe, Labib & Melchers, 1971) as well as from haploid (Nitsch & Ohyama, 1971) mesophyll protoplasts can now be grown (see Fig. 2). Recently this major achievement in culture technique was attained in a third species: Petunia hybrida (Durand, Potrykus & Donn, 1973). With their rigid cell walls taken away, plant protoplasts have been shown to take up a variety of particles and macromolecules. Complete 15 Schematic summary of a combined 'microbial technique' with conventional'crossings for plant breeding oS M - -h" T^S> rmbCr xx chromosom«: 48 = 'amphidiploid' tob- acco, « - haploid tobacco, i, Normal tobacco plant with petiolated leaves- 28 toxir>e); 10, n, only few ('resistant') colonies growing; 12, regenerating plants-, 13, 14, rooted plants diploidized with colchicine; 15, amphidiploid mutant) in this special case with the recessive gene for 'petiole winged' leaves) should be tested for resistance to the toxine. Reproduced from Haploids far Breeding by Mutation and Recombination, by G. Melchers (pub- lished by Joint FAO/IAEA Div. of Atomic Energy in Food and Agriculture, Vienna) with kind permission. Plants from protoplasts Fig. 2 (B) Photographs of stages 7-12 from (A), (a) Palisade cells isolated with pectinase from tobacoo mesophyll (note plasmolysis by mannitol), (6) proto- plasts after cellulasc-incubation (c. 150 x nat. size), (c) small group of callus cells from one protoplast (c. 150 x nat. size), (d) begger callus (as (c)), (e) Petri dish with calli from protoplasts {c. 0-55 x nat. size), (/) regeneration shoot apex from callus (i\ 70 x nat. size), (o-c) From coloured slides of Takebe; (d) liquid culture of Takebe, phot. Sacristan; {e,f) from cultures of Schilde- Rentschler; phot, (e), Rohfn. Reproduced from Melchers (1972) with kind permission. 29 Tobacco Mosaic Virus particles, as well as TMV-RNA (Aoki & Takebc, U)6Q) have been effective in the production of new virus in tobacco protoplasts.-Plant virolngists are taking advantage of the more or less ^synchronous infection observed »n the protoplast system. Even such foreign bodies as-pplystyrenc-latcx and whole bacteria (Duvey & 'Cocking, 1972) are apparently also engulfed, if added during the isola- tion procedures. It is not surprising therefore that also protein and various DMAs seem to be taken up by protoplasts, albeit in extremely small quantities and accompanied \ •/ rather intensive degradation. Although one would expect that plant cells once they have been liberated from their enclosing (enzyme-activity containing) cell walls would take up DNA more easily and with less dan?age than would intact cell suspensions, this assumed advantage of protoplasts remains as yet to be rigorously demonstrated. This is a difficult task, since adequate bio- chemical criteria for penetration of such 'adhesive' macromolecules as DNA and RNA are lacking. Especially when it is expected that only a very small fraction of the input will finally be taken up into the cells (Heyn & Schilperoort, 1973), adsorption phenomena may render the results unreliable. It is therefore of great importance that the phenotypic expression of exogenous generic material be studied concomitantly. 6.4. DNA transformation Long before the first attempts were made to make protoplasts take up foreign DNA, biochemical data had been accumulated on the penetra- tion of DNA into whole plant systems and on the subsequent fate of these exogenous informative molecules (see, for example, Ledoux, 1971; Hess, 1972). The ultimate goal of these studies is generally called trans- formation and by this we mean the transfer of biologically useful genetic information followed by its stabilization as inheritable material (whether integrated into the recipient chromosome or not). As is the case in bacteria, where such phenomena were discovered many years earlier and have been studied intensively ever since, transformation will be a rare event and will only take place under certain very special conditions. A multitude of factors, including size and origin of donor DNA molecules and such vague things as the' competence' of the recipient cells, influence the successful uptake and integration of DNA. It is quite evident that transformation even in bacteria is far from being completely understood (Tomasz, 1971). The data so far obtained in plant systems - in spite of many bold experiments - are difficult to interpret and should be 30 evaluated with great care. Some major pitfalls in the interpretation of biochemical experiments aiming at the transforrhstion of plant cells have been discussed by Hotta and Stern (in Ledoux, 1971; Hess, 1972). When considering biological evidence for transformation, the general mutagenic and physiological effects of DNA and of its breakdown products must be taken into account. Further, a direct relationship should be demonstrated between the biological observations and the precise genetic content of those donor DNA molecules actually inside the cells. Seen in this light, the inheritable recovery of cells from a nutri- tional deficiency can have significance, only if it is also shown that homologous DNA deleted for the studied character is incapable of in- ducing this recovery. In most cases such data are as yet missing!!;- The very interesting system of flower colour in Petunia has been frequently used in genetic studies] The colour of the flowers is de- termined by at least 20 genes (Wiering & de Vlaming, 1973). It is not only dependent on theanthocyanm variants present, but also on a number of co-pigments (flavonols) which themselves are colourless but which associate with the anthocyanins and change their colour. Variance in pH (5-3-6-z), whichisalso "genetically determined, can also influence the phenctype. In the apex of 8-day old seedlings, treated with DNA by Hess, two and probably three embryonic cell layers can be distin- guished Which develop independently from each other. By spontaneous (somatic) mutation in cells of one of these layers, mosaic flowers are produced With a frequency in the same range which has been claimed by Hess (1972) to occur in DNA treated plants (Bianchi & Walet- Foederer, 1974). i ; Transformation work with petunia does not necessarily have to be done with intact plants: the system of haploid protoplast isolation and regeneration to plant is completely available (Durand e£ al. 1973). Much can now be expected from transformation, studies not with whole plants in combination with complicated characters arising-only after the culture of adult individuals, but with haploid protoplasts (or truly single cells) which make it possible to use the powerful and well-tried methods of microbial genetics: mutagenic treatment of large numbers of individuals, massive plating and easy screening for mutants (Carlson, 1970) and transforrnants, followed by the eventual regeneration of complete* genetically altered, plants which could be reintegrated into the normal sexual reproduction cycle (Takebe et al 1971). The purposeful addition of a character such as resistance to a pathogen (isolated in the form of 31 DNA from a totally different species) to the cells of an otherwise successful variety of crop plant.' \v0ui3 iSeiin a iaost appreciated r broadening of thebase of genetic variability in plants. 6.5. Somatic cell hybridisation If we stretch the idea of the uptake of particles to the limit and place two complete protoplasts of different origin into such close contact that fusion takes place, we are dealing with somatic cell hybridization. This method of genetic alteration has attracted much interest and has given rise to many bold experiments ever since its discovery in animal cells by Barski (1969). That this is indeed an extremely reliable way of passing genetic information from cell to cell is highlighted by the fact that it is the essence of all sexual processes. Interspecific fusion of haploid cells is a form of alternative sex It is important though to realize that the study of artificial fusion of plant celts is only beginning, and that the results and insights obtained are; still lagging far behind those in the field of animal cell fusion. But systematic study of the numerous stages involved in the overall process of hybridization has started (Withers & Cocking, 1972). A crucial distinction, which has to be clearly made in all fusion studies, is the one between spontaneous and induced fusion. Spontaneous fusion may occur (even with a rather high frequency) without any inducing agent between cells which have never been completely separated from each other. This is the case for many cells in tissues such as leaf mesophyll, where the connexion be- tween cells are made via narrow plasma strands passing through the rigid cell walls (plasmodesmata). When protoplasts are produced by a one stepenzyme treatment, these connexions mayr expandarid allow complete mixing of the cell contents. This process rep^mbles very closely a reversal of cell division, even to the point of nuclear fusion. It is therefore clear that, even when cells of different species are involved, the production of multinucleate protoplast aggregates (heterokaryons) need not necessarily be the end of the fusion process. In the interspecific situation, starting with completely separated protoplasts, fusion has more or less to be induced, but such complicated agents as inactivated Sendai virus particles (widely ufsed in animal cell fusion) do not seem to be required. Various inofgani.i salts, of which sodium, nitrate is the most commonly employed, are sufficient in plant cells (Power, Cummins & Cocking, 1970; Keller & Melchers, 1974). A convenient way of showing the induced fusion of protoplasts is to take advantage of a visual 32 marker.such as vacuole colour. (Potrykus,-i97x)-Usefulgenetic-markers which would unambiguously establish the hybrid character of the fusion products, arc on the contrary particularly scarce. Hopefully, the isolation of haploid mutant cells wilt soon change this picture. The feasibility of genetic complementation at the cellular level (i.e. without nuclear fusion) has been shown with the aid of different muta- tions affecting the formation of chloroplasts (Giles, 1973): maize leaf protoplasts carrying such mutations developed normal chloroplasts after cellular fusion with normal protoplasts. In other experiments, isolated chloroplasts have been introduced into protoplasts of a maternally in-? herited alHno mutant of robacco, followed by the regeneration of a whole green plant. Apparently the foreign chloroplasts divided and were able to function under the direction of the wild-type nucleus.>. These examples clearly show the exciting possibilities of fusion studies also in the field of nuclear-cytoplasmic interactions and the genetics of subcellular differentiation. In the practical application of interspecific fusion products and in the completion of the parasexual cycle important technical difficulties are still to be solved: the induction of cell division followed by differentiation into hybrid plants, and the development of selective methods for the isolation of hybrid clones using nutritional dependencies. Only when we know how the produced hybrid cells can be selectively cultured, will we be able to exploit fully the special attribute of the somatic plant cell: its totipotency. Recently the feasibility of a parasexual cycle was shown in one successful fusion experiment using two different Nicotiana species (Carlson, Smith & Dearing, 1972). We are thus passing from the realm of near science-fiction to that of emerging reality. More tlian 107 protoplasts each of Nicotiana glauca and N. langsdoffiileaves wrefe processed by combining the techniques of protoplast plating and culture with those of sodium nitrate induced fusion (although this fusion method gives usually only low frequencies of fusion, with vacuolated mesophyll protoplasts). The phytohormone- containing medium (Nagata & Takebe, 1971) which was used for the plating of fusion products did fortunately not permit the growth into calli of cells of the parental species under the conditions used. In this experiment it was used in an effort to screen for the produced hybrid cells. In fact, a hybrid between these two species can also be obtained by sexual means; it has been the Object of intensive study because of its spontaneous tumorous growth capacities. Some of the 33 calli which were recovered from the screening were used for further tests and for 33 the differentiation into whole plants in order to confirm their hybrid character by comparison with the sexual hybrid. For three regenerated plants identity between" the somatic nnd sexual hybrids was established for six different characteristics including chromosome numbers and peroxidase isozyme patterns. With this lucky exercise in mind, all efforts should now bo directed towards the reproduction of this ex- periment in other laboratories and towards the development of genetic markers to make the selection of less easily identified hybrid cells possible. Chloroplasts being very si rnilar in widely different plant species, mutations involving this organelle (e.g. nuclear albino mutations) may possibly be used in a genera' selective system for hybrid cells (they would, for example, appear as green colonies). 6.6. Gene transfer and expression of foreign DNA in plant cells The ability of RNA-vims to penetrate into plant cells, to replicate there and to assemble into many new virus particles, is well known. It was, however, rather surprising that DNA from a bacterial virus can be modified in plant cells and come to expression in the form of coat protein with which it assembles into new infectious particles (Sander, 1967). It was claimed that the transcription and translation machinery of an eukaryotic cell can be successfully programmed with com- pletely 'strange' prokaryotic DNA. From the standpoint of genetic alteration, the next obvious experiment was to try and couple a gene to (transducing) phage DNA and see whether it would be 'transduced' and translated in eukaryotic cells. Using a culture of fibroblasts from a patient with a congenital enzyme deficiency and the DNA from phage lambda harbouring a wild-type bacterial gene for that enzyme, this experiment was successfullycarried out in 1971 (Merrill, Geier & Petricciani). No indications, however, were obtained on the mode of the preservation of the bacterial gene in these mammalian cells. Complete transducing phage particles also elicited the enzyme activity, but the efficiency of the phage was somewhat lower than that of bare DNA. Contrary to what one would expect, no protective or transport function can thus be inferred for the coat protein in this mammalian system. It remains to be seen what role, if atryv-the phage-specific RNAs, which are also produced, play in the correct expression of the bacterial gene. W\th many questions still open, the elegance of the scheme was too tempting and the first successful experiments of this sort have now been carried out by Doy et al. (1972, 1973 tf> b), who used the same trans- 34 10* \- Fig.3.(A)Inhibition of the growth of L. esculentum ANXJ-Hzy-1 by the phage $8osupF*. Callus inoculated with #8osupF+ (•), $80 (D), and no phage (T). Note that &80 had no observable effect. The phages were grown on E. coli Z32 [ 20 40 60 80 100 Time (d) (B) /7-Galactosidase activity of plant cells measured at various times aftsr initial inoculation of calluses with phage preperations. Calli were inoculated with a mixed phage preparation o£ VII. FURTHER CONSIDERATIONS AND CONCLUSIONS In genetic engineering, two major approaches can be outlined in broad terms:-the cellular and the molecular approach. The first involves the culturing of haploid organisms and the hybridization of somatic cells; the second involves the direct manipulation of the DNA. It is of little value to consider these approaches completely independently because several techniques are common to both, e.g. single cell techniques - including the handling of protoplasts- and the regeneration of whole plants from single cells or possibly haploid cells. It should be noted, however, that the cellular approach is bound to have a more limited scope than the molecular one, as the genes from organisms that are far apart on the evolutionary scale are unlikely to be interchangeabler So far we have no idea how much different plant cells can be and still establish a successful fusion, let alone whether or not whole plants can be regenerated from such fusion products. In this respect it is most important to know whether the chromosomes of one parent in inter- species plant hybrid cells are lost preferentially, as they are in mammalian hybrids. For the development of methods for the selection of hybrid cells from mixtures of parental cells we can draw upon the experience 36 »ccu!Tml^.tcu witli nisninialiHii cells nnci b^c^ris pect a steady progress in cellular methods in the near future. It is much more difficult to predict how fast and what progress will be made by the molecular approach. Despite the quite numerous claims that whole plants or multiccllular embryos have been transformed by DNA, we fail to see how these spectacular results really contribute to our understanding of the basic processes of the development of com- petence and the integration of DNA in a stable form. In fact we still have our doubts whether the claims of direct transformation arc justified by the experimental data so far presented. We certainly accept the idea that a piece of foreign DNA (even of bacterial origin) can be pheno- typically expressed in plant (or mammalian) cells. However,\it,has not yet been established whether a 'naked' (i.e. purified) DNA is in this respect more successful than an encapsulated DNA (e.g. a virus particle or an isolated chromosome). Nothing is known about the molecular mechanism of DNA integration into plant genomes and the transformation of whole plants because of the complexity of the system. The transformation of whole plants is unlikely to provide much useful information on the uptake and expression of foreign DNA by plant cells; We should focus our attention on the transformation of single cells in order to allow the scoring of a sufficiently large number of individuals and thus eliminate the problems surrounding spontaneous events and mosaics. Instead; of total genomes, the use of DNA, 'enriched' for certain markers or operons, possibly attached to readily recombining ('adhesive');DNAsequences wouldi also be of great advantage. In this respect the simulation of naturally occurring gene transfers (e.g. with crown-gall induction and possibly Rhizobium symbiosis) by the in- fection of protoplasts, must also be considered. When more details of such processes are available, these systems might be further exploited as a means of bringing other bacterial genes into the plant genome. Probably it is too early to consider in depth the benefits of the newly developing genetic methods; we can all too easily be carried away by pur imagination. It is sufficient to mention two possibilities of practical importance in plants: (i) the incorporation of genes for nitrogen nxation into plants and (2) the transfer of characters which determine resistance to pathogens into cells of otherwise successful varieties of crop plants. In our opinion the importance of genetic engineering for biology in general lies in its contribution to the studies of the processes of differentiation, morphogenesis and uncontrolled growth (tumour 37 induction). Besides the beneficial results countless useless monsters arc bound to be produced in the ttst-tube, raising the inevitable question of the ethical implications pertaining to this kind of research. We are unable to exclude beforehand the possibility of an accident producing, for example, a dangerous weed, just as bacterial studies involve the :risk of creating a potentially dangerous bacterium. However, the absence of a nervous system in members of the plant kingdom at least spares us the extremely serious conscientious considerations we must have when manipulating members of the animal kingdom. We gratefully acknowledge the critical reading of the manuscript by Dr C. Kitsch (Gif-sur-Yvette), Dr G. Mclchers (Tubingen), Dr F. Bian- chi (Amsterdam), Dr E. W. 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In Workshop oti Mechanisms and Prospects of Geneiic Exchange. Advances in the Bioscicnccs, no. 8. Pergamon Press. ZAENEN, I-, VAN LAREBEKE, N., TEUCHY, H. fi: SCHELL, J. (1974). Super- coiled circular DNA in crown-gall inducing Agrobacterium strains. J. molec. Biiil. (in press). 43 CHAPTER III RAPID AM) EFFICIENT ISOLATION OF HIGHLY p POLYMERIZKD PLANT DNA | R FRANSHEYN.ANKEK HERMANS S and ROBBERT A. SCHILI'EROORT | Department of Biochemistry, Leiden State University, i Wassenaarseweg 6-4. Leiden (The Sutherland*) ;) (•Received May I5tli. 1973) I (Revision received July 24th, 1973) f SUMMARY A small-scale method has been developed to isolate rapidly and efficiently pure DNA from plant sources. Mechanicaldisruption of lyopnilised material at a low temperature proved to be satisfactory to break plant cells. High salt concentrations, the use of pronase at 56° and filtration through agarose gel yielded pure, highly polymerized DNA within 5 h, without intensive shaking and precipitation steps. The efficiency of isolation ranged from 90 to 95%, even when very small samples were used containing around 1 Mg DNA. INTRODUCTION The main problems in the isolation of plant DNA are the breakage of the ceils, the frequently very low^yacuolaiLpH,andLthe removal of carbohydrates and proteins without too much degradation of the DNA due to hydrodynamic shear. Since we were not able to isolate pure, high molecular weight DNA when we used the existing methods, we developed a procedure suitable for our tobacco tissues '. Although it yields pure and highly polymerized DNA, this procedure is not suitable for small quantities of plant material and is rather time-consuming. In connection with the studies in our laboratory on plant protoplasts and organelles2'3, we developed a rapid and small-scale method to isolate pure DNA. MATERIALS AND METHODS \1) Plants were cultivated in a greenhouse. Tobacco (Nicotiana tabacum L., var. White Burley) normal and Crown gall tissues were cultured as described earlier4. : 44 (2) The plain material was lyophilized in polycarbonate centrifuge tubes immediately after harvesting and stored at—20° until use. After addition of Klassrbead.s-(aboutr-2-g-per-g dry-tissue; 0;5-mmdiameterrSuperbritc>r3M Com- pany) and a magnetic stirring bar, the dry material was cooled with liquid nitrogen and finely pulverized on a Vortex mixer at top speed for 10 to 20 M-c. Next, 10 nil pronase solution. 6,5 ml isolation buffer and DTT (1^4-di- tluuthreitol, Merck) to a final concentration of 0.1 mM ^vere added per g dry weight tissue. After gentle stirring, the mixture was warmed rapidly to 56° and incubated at this temperature for 3 h. Aftereooling,'cell debris was re- moved by eentrifugation at 5° in a Beckman J-21 centrifuge for 10 rain at 20 000 X g. The resulting pellet was washed twice with 10 ml wash buffer. The combined supernatants were then subjected to gel filtration according to Loeb and Chauveau \ We used 2.5 X 25 cm Sepharose 4B (Pharmacia) gel beds., run at about 0.5 to 1 ml per min, and collected 1.25-ml fractions, while following the UV absorption at 260 nm. The void volume fractions containing DNA were pooled and dialyzed against 0.01M phosphate buffer, pH 6.8. The DNA solution was concentrated, if necessary, by dialysis against the same buffer saturated with PEG 6000 (polyethylene glycol, Merck). (3) The pronase solution contained 8 mg Pronase E ( Merck) per ml and was brought to 1. M NaCl after inactivation of traces of DNAase by a modifica- tion of the procedure of Hotta and Basself1''. The solution was stored at —20°. The isolation buffer contained 10 mg/ml Sarkosyl (NL 97 from Ciba-Geigy), 5 mAf EDTA, 100 mM Tris-HCl, 6 mil-/ Na2 HPOa, 2 mM NaH: PO4 and 250 mM NaCl (pH 9.4). The same solution, but containing 1 M NaCl, was used as wash buffer. Both buffers were stored at 4°. Phenol (Merck) was redistilled and saturated with water containing 0.45 M NaCl and 0.045 M trisodium ci- trate, pH 7.3, and 8-hydroxyquinoline (Oxine, Fluka) was added to a con- centration of 0.1%. (4) Quantitative extraction of DNA was carried out according to Fleck and Begg7 omitting the lipid extraction. The amount of DNA was then de- termined with the diphenylamine reagent following Giles and Myers8. Poly- saccharides were assayed with the anthron reagent9. Protein was estimated with a modified microassay of Kihara and Kuno I0'1, using crystalline bovine serum albumin as a standard. Radioactivity in aqueous samples was measured by liquid scintillation counting in toluene/Triton X-100 (2:1) containing 0.4% PPO. Molecular weights of DNA were determined by band sedimentation analysis in 1 M NaCl according to Studier " . RESULTS Purity of the DNA Isolated DNA had in general the following UV absorption ratios: A230 nm/ = = •^260 nm 0;46 and A26o nmM2so nm 1-85. Crown gall DNA and DNA from Kalanchoe daigremontiana were some- times contaminated with polysaccharides resulting in an A230 /Ai60 ratio in the range of Q.5 to 0.6. Almost all those polysaeeharides (and also green pig- ments) could be removed with one volume of phenol. The phenol and water layers were gently mixejlb^ tenjnyomans ofthecantaimSyalvd separated by ;centrifugattQh-mJan MSESuper Klihor' table centrifuge. This procedure de- creased the DN A isqjatipnrefficieney by about 3—D'Y. That, this phenol treat- ment, after intensive pronase digestion, did not lead to selective loss of partic- ular DNA's. is'ind.eated by the fact that upon analysis in CsCl density gra- dients l!, a mitochond.-ial DMA band of buoyant density 1.706 g>cm "' was always detected. In the final DNA preparations, the polysaecharides never exceeded 0.-1 Mg glucose equivalents/^ig DNA, and were usually absent. Micro- assay of protein in the DNA preparations showed a maximum of 5 fig pro- tein, mg DNA. f RNA contamination was studied in more detail, since the method does not include an RXAase treatment. Normal tobacco tissue was labelled for 7 h -'• with [o'-i'H]undine (RadiochemicaHGentre, Amersham, 23 Ci/minole). RNA isolated u from this tissue had a specific activity of 8000 cpm/jug. DNA from the same tissue was isolated according to our procedure, and tested for radio- activity both before and after RNAase treatment' (since some radioactivity is also incorporated in plant DNA). Contamination with RNA thus determined amounted to 0.6% or less. It is possible that some of the larger (nuclear) RNA was degraded by endogenous RNAase during lysis. Elimination of most of. the RNA was due to precipitation with 2 M NaCl in the upper part of the Sepharose column. Passage over the column of two bed volumes of a low salt buffer effectively removed this RNA and other contaminants, thus preparing the column for reutilization. Properties of the isolated DNA Sedimentation coefficients ranged from 25 to 32 S, indicating molecular weights from 12 to 30 X 10* Daltons I4. Analysis of DNA in CsCl density gradients yielded the following densities (g-crrT3 ), based upon a buoyant density of Micrococcus lysodeikticus DNA of. 1.7310: normal tobacco tissue 1.6946; Crown gall tissue 1.6948; tobacco stem 1.6953 and tobacco leaves 1.6965. After preparative CsCl density gra- dient centrifugation 15 of normal tobacco tissue DNA, the bottom-side frac- tions are enriched (Fig. 1) in mitochondrial DNA lfl of density 1.7060 g-cm~J. Heat denaturation of DNA from normal tobacco tissue or pea stems (in 15 mAf NaCI—1.5 raM trisodiumcitrate) gave an increase in -A260 nm °f 36 to 40% and the Tm vvas 70.9 ± 0.5°. DNA isolation efficiency Quantitative extraction of DNA from normal tobacco tissue culture mate- rial yielded a value of 95 fig DNA/g fresh weight (equivalent to 45 mg dry weight). The methods of quantitative extraction and determination were checked using [Me-3H] fchymidine-labelled tissue ' and DNA isolated from it (specific activity 1440 cpm//ig). DNA content of labelled tissue, determined 46 Fig. 1. CsCl equilibrium density gradient analysis of pooled, high buoyant density fractions from a preparative CsCI centrifugation of normal tobacco tissue DNA. Densities (g-cm~3) of poaks from ten to right: main DNA 1.6946; mitochondrial DNA 1.7060 and marker (M. lys.) DNA 1.7310. using this specific activity, corresponded, to within 3%, with the value found using the diphenylamine assay. With the DNA isolation procedure 85 to 90 jug DNA could be obtained per g tissue. If necessary the isolation efficiency can be increased to nearly 100% by repeated washing of the tissue debris after lysis. That no (degraded) DNA was lost due to elution from the column after the void volume, was demonstrated by the elution profile (Fig. 2) of crude lysate from labelled tissue: no radioactivity eluted in the fractionation range " of the column. 60 SO Krsclion numtwr Fig. 2. Sepharose 4B gel filtration profile of crude lysate from [Me-3H]thymidine-labelled normal tobacco tissue. 47 DNA from other sources Our method has also successfully been used on pea stems and roots, Crcpis capillum stems and Kalanchoc daigremontiana leaves. Although developed " for small samples, it has been used with equal success on quantities of several hundred prams fresh weight. In this case the lyophilizod tissue was pulverized in a coffee-mill'. DISCUSSION The developed method combines strong DNAase inhibition with a low level of hydrodynamic shear and RNA removal without RNAase treatment. The action of DNAase was minimized by freezing of the tissues immediately after harvest, by high concentrations of pronase in a lysis buffer with EDTA and a high buffer capacity at pH 9. Furthermore, the dry and very cold tissue powder was quickly brought to the optimal temperature for pronase activity while being hydrated. By •"blind" pooling of the void volume fractions from a column with known elution characteristics, followed by concentration, DNA quantities around 1 (xg can be isolated. This feature of the procedure has been used in the isolation of DNA from nuclei, chlproplasts and mitochondria "' as well as from plant protoplasts2. l l9 In contrast to existing methods >'*« f the present one has the following advantages: (1) the near quantitative isolation of good quality DNA from both extremely small and rather large samples, (2) the ease and. more im- portantly, the speed of the manipulations, and (3) the inexpensiveness of the necessary equipment. ACKNOWLEDGEMENTS The authors express their gratitude to their colleagues Aad Hendriks, Hans Dons and Aat Ledeboer for valuable discussions and testing of the procedure during its development. REFERENCES 1 R.A. Schilperoort, Ph.D. Thesis, Leiden, 1969. 2 R.F. Heyn and R.A. Schilperoort, in Plant Protoplasts and somatic cell fusion, Coll. ; Intern, du CNRS No. 212, Paris, 1973, p. 385. 3 A.W. Hendriks, FEES Letters, 24 (1972) .101. 4E.M.J. Jaspars and H. Veldstra, Physiol. Plant., 18 (1965) 626. 5 J.E. Loeb and J. Chauveau, Biochim. Biophys. Ada, 182 (1969) 225. 6 Y. Hotta and A. Basse!, Proc. Nat I. Acad. Sci. (U.S.), 53 (1965) 356. 7 A. Fleck and D. Begg, Biochim. Biophys. Ada, 108 (1965) 333. 8 K.W. Giles and A. Myers, Nature, 206 (1965) 93. 9W.Z. Hassid and S. Abraham, in Methods in Enzymology, Vol. Ill, Academic Press. New York, 1957, p. 34. 48 10 H,K, Kihani and II. Kuno, Anal, ltiochem., 21 ( 19b8) 96. 11 HV Stuilii'i-,.7 Mul llml. 11 (196f>).'J73 1 •> N Sui'ok.i, J Mul Biot .3(1901) 31 H N.J Van Sitto.-l, I'll D Tltcsis. Leiden, 1972. 1 I J Kiuner and P. Doty,./ Mol liiul, 12 (19G5) 5 19 I'IWO Flainin. II.E Bond and H IS Burr, liioclum liiophys Aria. 129 (19G6) .'J 10. 16 AW Hoiulriks. I'll U VVICSIS. Lt-iden. 1073 17 A Pi-uiu'll and G Bminrdi, 49 CHAPTER IV THE USE OF PROTOPLASTS TO FOLLOW THE FATE OF ACROBACTERIUM TUMEFACIENS DNA ON INCUBATION WITH TOBACCO CELLS R F. HKYN ami H. A. SCHII.PHKOORT Ih-f'artmenl nj Mtjfhtmisttx, Vnner'.Uy nf Ltiitfit. The StthcrlittuU Tli.l>Anu.\ UK I'KOTDI'I.ASTKS I'ouu sfiVKK LK DKVKMU ms DMA x> Ajirohaclcrium Utmcfaciens I'ESIiANT r.'lSXI'HATHiN AVHC 11KS CELLULES UK TAB AC KE.SCME i. NtHis av^ns rtutlM' ic devenir dvi i >N A d'.-l. tnnu./mwus marque au -'-I' et nu Mi an emirs ilc 1'incubation ,t\<"t dcs cellules de tabac culln'Ccs on suspen.nion, a\-cc on snus I >[•! Al'"-di^\tram'. i Lc pi£tra!tt-inent au i-'ICAIi-dcxtr.iiu^ se traihiit par : rii 1 IH: trcs iit-ttc au^uu'nlit(ii*u ih'.1 lal^Mrpiton du I > N A dc luiut puids muh'-i ulairt* sur la parui. hi I no (Iiniiniitidii di liitit ivitc PNase inilniU' par le l.'N A. c) i:nc amelioration de la recuperation i\n 11XA isule des jirotoplastes. i/j I'no reduction marquee de la re1 utilisation des produits de degradation du DNA. 3. Nous avuns isoie des protoplastcs a partir ties cellules traitees, avec une efficrcitc de 10 a 15 %, pour distinguer la penetration de l'a(lsorptit.»n. .). L'analvsc par ccntrifugntion en gradient de densite montre SUMMARY 1. We have studied the fate of 'JIV3H labeled A. lame/acinus !>XA upon incubation with normal tobacco cells in suspension culture in the presence and absence of DKAE-dextran. z. Pretreatmont with DKAE-dextran results in : a) A markedly improved adsorption of high molecular weight DNA to the cell wall. /)) A lowering of the- DNA induced DNase activity. c) \n improved recovery of DNA isolated from protoplasts, d) A marked reduction of re-utilization of donor DNA breakdown products. 3. In order to distinguish penetration from adsorption in the overall uptake process, we isolated with 10-15 % efficiency protoplasts from DNA treated Cells. 4. Using CsCl equilibrium density gradient analysis we found that only 0.145 % of DNA taken up penetrated into the cell. 51 INIRODI CHON In I;,!- ifiirv <>i mir ~nii!n-> un the t>l,ti\t nuvp'i v rown <*>a]| — >i timmr inducod mi w MATERIALS AND METHODS 1. Culture methods. Normal callus tissue oiKicoticiaa cabacum L., var. White Burley was cultivated as described previously (Jospars and Veldstra, in6j,). Cell suspension cultures were derived from this callus culture. The cells were grown and subcultured under the same light conditions as callus tissues at 28 °C on a G 10 Gyratory sha'ter (New Brunswick Scientific Cornp., Inc.), set at 120 r. p.m. Narrow neck100 ml erlenmeyer flasks were used, containing 25 ml of the modified B medium of Jouanneau (1967) with 0.02 rng/1 kinetin, 0.2 mg/1 a-naphthaleneacetic acid and a four times higher phosphate concentration. Cells of A. lume/aciciis strain A 6 were cultured at 28 °C in 500 ml medium containing (g/1) : Bacto poptone (Difco) 10 and NaCl 5 ; pH 7.0. Overnight cultures were diluted with fresh medium and incubation was continued at 28 CC. When'the optical density at 660 nm had reached about 0.2, thymidine-methyl-3H (spec. act. 50.3 C/raM, radiochemical purity greater than 97 %, purchased from New England Nuclear) and 32P- phosphate (Philips-Duphar) were added up to concentrations of respectively 5 "and 30 [lC/ml. Incubation was stopped at an optical density of 0.7 by rapid cooling to o °C. 2. DNA treatment of tobacco cells. Tobacco cells from several suspension cultures were pooled, concentrated in a Sartorius nitration apparatus (SM 16 510) and washed intensively with about 150 ml of fresh culture medium. 52 Ilii" '.elU were resii-,|>einlii| in do mi fn-.li medium miitainiiu; I ooo-r sou utr radiolabelcd .1. limnia- ,,i]\ DNA .ind so up ml /-dcow th\ midim- ' ldK from Men k) followed In IMI uli.ition. under st.ind.ird iiliuie I'liiditi'iiis, in tl>e s.iitoiuis .ipp.ir.itus ( ells v.eie mi ub.ited with DNA and IdK lor z\ h"ur>, • In l)\ \ ' "iit.iiiiint; iiu-duim was nmo\ed .ind the i ells u.i-hed thru times with ,i tol.il of 1H0 ml fr>—h hum \itn ifiiiiiv.il uf .1 -null -~.ttnj.It fur total DN \ i-nl.ition the i ells uue used for prot'ipl.i-t isolation To establish tin1: iiitlueaie on DNA uptake of diethylaiiiinoethyl (DEAH)-dextran fl'liariacia, Swe- den ; made from dextran with M\V around 5 • TO'} some experiments included a preim ubaiion of the I'CII- with this polycation (100 ug/ml) for Jo inn in t«> ml medium (Howard ct al., i')/i). 3. Protoplast isolation. The fresh weight of tin: toban o cells was determined after harvesting by filtration, l'ectinase treat- ment was performed by sus|>e.iiding the cells (1 g fresli weight per 4 ml) in liquid medium (Jensen et at.. 1071 or Joiiiinnrau and Peaud-Lenoel, if/*-) coritaiuing as osmoticum 20 % (w/w) sucrose and further 0,005 "o potassium de.vtran sulphate 117,1 % sulphur, Meito Sangyo Co., Ltd., Nagoya, Japan) and 1 % Maceroi'Viiu" (All Japan Biochemical^ Co., Ltd., Nisiiinomiya, Japan), pH adjusted to 5.8. After incubation at 57 °C for 1 hour in a C, 24 Environmental Incubator shaker (New Brunawick Scientific Co., Inc.) set at So r. p. m., this premaceration is repeated once, followed by a combined pectinase and cellulase treat- ment using the same media (ijg fresli weight per 4 ml enzyme solution) containing 5 % Cellulase Onozuka .SS (1 500 units/g ; A. j. B., Nishinomiya), 3 % Macerozyme and 20 % (w/w) sucrose, pH adjusted to 5.5. This combined treatment takes about 5 to 6 hours at 37 °C with the shaker set at 40 r. p. m. Enzjme solutions were filter sterilized in a Sartorius filtration apparatus (type SM 16 510) provided with a cellulose acetate filter (SMn 106) and a glass fibre prefilter (SM 13 400). After these enzymatic treatments the still intact cells are thoroughly washed with medium '-'containing 20 % sucrose only in order to remove the s-nzymes. Protoplasts are tlien set free by gentle mechanical squashing. Small debris and free, intact and viable protoplasts are then separated from each other by passage of the -'suspension through a 77 a stainless steel filter and centrifugation of the filtrate (in 10 ml portions in graduated, coated glass tubes) for 3 mn at 2 500 r. p. in. in a MSE Super Minor table centrifuge with swing out rotor (about 550 x g at the meniscus). Fine debris is spun down, while free viable protoplasts float to the surface forming a thin band at the meniscus. From each 10 ml portion about r ml is carefully pipetted off (wide mouth pipet) containing almost all viable protoplasts. 4. DNA isolation. Bacterial DNA was isolated with a SDS-Pronase-phenol procedure as described earlier (Schilperoort, •-.-;.n)6g): DNA was handled under sterile conditions from the last extensive phenol treatment on and once more precipitated with 2 volumes ethanol 24 hours before incubation with tobacco cells. Molecular weights of about 107 \vere estimated-from the sedimentation constant. _ High molecular weight plant DNA was quantitatively isolated from small amounts of cells and proto- plasts by a method (Heyri et a?., manuscript in preparation) including the following steps :Tyophilization, lysis at elevated temperature in a Sarkosyl and Pronase containing buffer, purification of the lysate by gel filtration, concentration in pplyethyleneglycor6 000 and dialysis against 0.01 M potassium phosphate buffer pH 7. Prior to DNA isolation suspensions of purified protoplasts were a ncentrated by ultra filtra- tion using a Diaflo membrane XM-100 A in an Amicon model 52 cell (Amicon N. V., the Netherlands). 5. CsCl density gradient centrifugation. Giddients were'fornied^in 4.5 ml CsCl solutions with initial ren-active index 1.400 2 ;H 0.000 3 (Merck, suprapur) arid containing 0 01 M phosphate buffer, pH 7, by centrifugation for 62-66 hours at 25 °C in a Spinco 50 Ti rotor arid L-z centrifuge at 33 bop r. p. ni:(Fla.mm eial., Ig66). Tubecontentswere analysed for optical density (OD) at 260 nm by passage through a capillary flowcell (light pathL2 mm) in a Unicam SP r8oo spectropnotometer arid subsequently fractionated by collection of 55-60 three-drop-fractions 53 directlv into cuiiuting vials. During each run one or twu tubes containing A. luHufiuii'n\ and noitnal tob.ii'i"n DNA WITO .iNu Lfntrifugi'i.1 ty ii'Tvu ;j->i_n;[i'r<:nt;t!_s. 6. Radioactivity measurements. Fractions .from CsCl gradients were lirst diluted with I ml distilled water, brought t" o I N !I( I and incubated for 45 run at !>o UC in order to liydrolvse tl«: DNA. They were counted in ;i Philips Liquid *u m- tillatson Analyzer alter addition of 13 nil of toluene-Triton X-100 (2 : 1) containing c< 4 "„ lJl'O All otlu-r samples were spotted on Whatman CiV'/A filters, dried and counted in toluene-0.4 % PPO. 7. Assay of DNase activity. Sterile-filtered samples of used culture media were incubated at 30 °C with 3 ug Hi-labeled ,1, tume- fitciem DNA. At various times, 50 uJ samples were taken to measure radioactivity precipitable by 5 % (w/v) trichkinwetic arid using Whatman (iF/A filters. Values are expressed as percent of TCA precipitable radioactivity from input DNA. Integrity of donor DNA after incubation with tobacco cells was determined in OK- same way. RESULTS 1. Protoplast isolation. In our uptake experiments we used tobacco cells from suspension cultures (Fig. t. PI. I) as starting material tor protoplast isolation (Fig. 3. PI, I). The developed technique using the crude enzyme prepa- ration introduced by Takebe (Takebe ct«/., 1968) was also tried on callus tissue from which the suspension lines were derived, since it has been reported that tobacco callus tissues are generally refractive to efficient protoplast isolation (Motoyoshi, ro,/r; Sch/nitt«<«/., lt)/t). With our technique there was no great diffe- rence between the efficiencies starting from suspension cells or from callus tissue : ro-15 %. Isolation efficiency is expressed as % of the number of cells present in the starting material. This last, figure was routinely established using; total DNA extraction from a sample of each batch as a measure. Cytochemical determination of 'DNA content per cell nucleus of our tissue cultures — about iz pg (H. Dons, unpublished) — i» in good agreement With other reported values (Jensen el «/., 1964 ; Bendich and Fiiner, U171). Expressed as number of protoplasts isolated per gram fresh weight of tissue, yields ranged from u.2 to 0.5 • io'\ rendering subsequent biochemical analyses feasible. Final concentrations varied from io5 to lo6 purified protoplasts per ml (Fig. 2, PI. I). Viability of the floating protoplasts was checked using protoplasmic streaming and staining with neutral red as criteria. All the usual varieties of living protoplasts could he identified in the preparations (Fig. 3, Pi. I). Effective removal of the cell wall degrading enzymes before protoplast liberation forms a great advan- tage of the technique. -.- : Protoplasts can be directly uset :n cell'wall regeneration studies and protoplast culture without any further manipulations. - The single floatation step is needed only to remove the very fine debris and to obtain high concentra- tions of protoplasts, whereas in other procedures several repeated floatations are needed to dilute the enzymes (Schmitt et al., 1971 ; Chupeau and Morel, 1970). 2. Conditions of DNA treatment. Recently Bendich and Filn^r (1971) reported rather important DNA-ase and phosphatase activities in culture filtrates of the XD line of tobacco cells. In contrast to these findings, the DNA-ase activity in culture filtrates of our tobacco cells was mini- mal. We found evidence however, for the induction of DNase activity when DNA is added to tobacco cell cultures. Preliminary experiments using different amounts of DNA (up to 50 ug/vnl) showed that this phenomenon might be concentration dependent. When 3H-TdR-labeled A. tumefaciens DNA was first degraded by incubation with DNA-ase and then added to tobacco cell cultures for only 6 hours, radioactivity was incorporated in tobacco DNA as if the cells were bring labeled with pure 3H-TdR. 54 OD260nrri P •1.760 . 0.200- 1720 0120 -1,660 O.OiO 10 20 30 40 50 Fraction number l-i.i. .i. HflMilibiitiM! fluidity ^radit'iil protiif ot an artinoal mixture n; .-I. turn. 1J\A (I) and lobancu UNA (2j. Ill order to minimize re-utilization of degradation products at the precursor level for DNA synthesis, \vi; introduced relatively high amounts of TdR [o.i mM) into our incubation medium. With DNA concen- trations around 16 ug,/ml this TdR is enough to dilute 3H-TdR present in the donor DNA by a factor of 75_ooo. • . '. • .-.• • . . . ..- . .•-.'•;• !. The re-utilization of donor DNA degradation products is still further reduced by pretreatment of lhe cells with DEAErdextran as demonstrated by the experiments shown in figure 5 and 7. B. 0.03- 30- 0.01- I I0-, 0.5- -200--40 o 0.3 100--20 § o.i- 20 30 40 SO 60 Fraction number : FIG. 5. Kqiiilibritun density gradient analysis of DNA from non-DKAR dextran treated ccll«. A. Total isolation. B. After protoplast isolation 55 1 00 10 20 30 40 Fraction number v gnuii^ti: analysts uf l~)SA ir-yitx l>KAJ7-tie\*ran Ijeiiit'ii tc!U ; Tobacco cells with and without DKAK-atxtran pretrentment were incubated with double labeled .-1. tumefarienx DNA (•S2I-/3H ratio : 27.53 ; spec. act. ; 3iP 104 000 c. p. m./jjg, •'H 38S0. c. p. m./tiR) if re-utilisation of DNA degradation products had occurred, it would 4utve resulted in .newly' syuthH-M/td radiulabeU-d DXA with a ratio different front that of input DNA, This happens to a ..mall extent in the cast nf non-pretreated tobacco rplis only, DNA isolated ftom these cells and analyzed by CsC! density gradient U'ig- 5 A) showed a very small amount of radioactn, it\ banding at the position of tobacco DNA \c. p. m. ratio about 5). DNA isolated from protoplasts of prctrca- I ted cells (Fig. 7) does p.ot show any changK in the c. p. rn. ratio. \Vt found a large amount of high molecular weight DNA to be tightly bound to the DEAE-dextr.in pretreated cells (see below). This can explain why DNA breakdown by DNase in the medium is redur< d to such an extent. The adsorption of much DNA to the pietreated cells lowers the concentration of DXA in the medium. As a cou«>(|uence there will he much less induction of DNA-ase activity than in the ca-it of non-treated cells. After 24 huur&DNA treatmfent, culture filtrates of pretreated ceils contained'"•!sigmfi- caiitjy aiKireTCAprecipitabfe Tadioactivity (it % ot total radioactivity iii the filtrate) than the filtrates of non-pretreated cells (4 %).'.'..-rt •'••".".'• ::/-..-: ;r7.'i/" v~ ; " ^" ' :: ' Moreover we found th?t DEAE-dextfan prc-treattrient of tpliacco cells also .-.•influenced the DNA recovery from protoplasts -isolated from, these cells"': DNA could be isolated fjuantitatiyejy from these protoplKSts.Using the sairie isolation conditions a much loy.'& amount of DNA was recovered fvpm topla'its isolated from non-treated cells (Fig. 7 and 5 B). . 3. DNA uptake. The buoyant^ensitjes at:t.he host and donor pNA'sjn our system (1.694-6 g.cm~iftn&T./lj 7 g\cm 3 ± 0.000 3 respectively) are different enough to permit good separation in a CsCl gradient as shown by the OD pattern in figure 4. 56 Win i jii'it')|il.i-.t-.in iiul.it< il lioiii US \ ti> ,it< •!< li- .w i«'UiHi tiut a • un-iili r.ihli amount uf DN \ ii'lv (d-mhrd t.i tin ullu.il1 I- t hinm ittil > I.il.li I I Mia u >u. with .iiiim.il • i IK it i, kfinun th.it HI- \! di ulr.iu f.t< ilit.it/* .id*>>rfj[i"n to tlie nil surfeitC HI \ ird./ •It •! in . nk I>I s , .i—"• t ill' i MmviiVi'T, from ta!>U- 1 it i* r|rnr that after inlciisive ivrishin^ at the i.nd of the iinttbati. m pcrind, DNA iiwiciatrvl witli the i)ICAIv-dc\trit:i pr.:tre:iteil cells is tightly hound tn tin- cell wall. Of this DNA 2i ";,, w;t« i>i)!ate(i in a high molecular wight >t:tte us shnv.n hy tin: hand width of.!, ttiuwfacieus UNA in tin- tiital DNA piepanaiuti il-"ttr. t>\. VVhf-n this treatment was omitted iinly o.wN "„ of the tightly bound DNA was isolated in a high im.ilii iilar sveiglil stnte, iLltfiimgli ln;>t D.N'A was isolated (piantitatively in liotli cases from the same :niiinjut nf ci'li> f('(D patterns in I'ig. 3 A and (>). Thcrcfori.1 mo,* «>f the radioactivity tightly hound to ii.ijii-pretreati'd i ells n-prownts degraded DNA, while much more high molecular weight DNA U75 •:) 1- pri^ent mi prctreated i.ells. TA.UI.K 1 l/mlioadivity reiovtrrd from tobacco mils after 24 lipun expo&iirr to - Treat incut I Wash Ccltulase •i •- l>EAK-flcxtraii | j > -15 : - It !..\h-i cxtr.m j (in . I'liXETKATlON*. Tn the .t-otnl' process of DNA uptake we want to discriminate adsorption from actual penetration. Therefore protoplasts were isolated from large batclic both DEAE-dextran pretreated cells and non- pretn-ated cells. In the case of pretreatment (Fig. 7) high molcculac..weight donor DNA indeed penetrated iiiti- the i-ell-i. This .1. tuniifacicm, DNA, which succeeded in remaining intact in the tobacco cell's interior, .miutints to only 0.025 ",'o "f t'lc input. i£xprc-scd in a more significant way, this represent;. 0.145 % °f the high molecular weight DNA tightly bound to the cell walls. Thus if we define this tightly bound DNA together with the pent'trrAed DNA as DNA * taken up ;•, only 0.145 % of uptake is real penetration. Although only ' small amount of high molecular weight DNA was present in protoplasts purified (10:11 iion-prctr. .ited cells, the data shown in figure 5 B indicate that also in this case A. lumcfecicns DNA did penetrate into the cells. Here comparison with tightly bound DNA is however impossible. SPFCl'LATIONS A3OG7 TST Wlipn double labeled exogenous UNA is. integrated into the host cell genome we at least would expect the c. p. m, ratios of the isotopes, to be the same for the foreign DNA and the host DNA. Deviations of the c. p. m. ratio indicate the re-utilization of aiiior DNA breakdown products for DNA synthesis. The 32P/3H c. p. m. ratio of donor DNA used iii our experiments was 37.53 (S.E.M.: 0.30). The radio- labeled DNA banding at the position of tobacco DNA and isolated from DH-AJE-dextran treated cells (Fig. (1), has a "P/SH c. p. m. ratio of 20.28 (S. E.M. '.0.24). Since this ratio does not deviate from thec. p.m. ratio of donor DNA, one could be tempted to conclude that integration of donor DNA has taken place. 57 this doom DNA amount* to aUnit "i tin- tol>.u< <• I>N.A, win. h >-• oln i t....mm i>. ! lie re-ults ohtaiiwl attrr pruloj'la-l isolation {I'li; 7 ii"t on -in ii an .mvunt. If at all integration ha* t.iki-n i>!;uv. iitor.' "x^-iim - ,irv,ni:u!(al tt>;[•r<'\«" tlii>. hi our opinion,l!i<- donetDNA liamlmi; at a!i>ut tin- saturtli-n-iti- as'to'Kir >ml.l !«'iliir"lo a i-inili>l«w witti'DKAK-iir.xtr.ii)1. Dtscriminatibri bt-.twirn stiuli a comiUcx ami inlvwivittt ti'T DNA <;a:i be arliu.-vw.l !>v n.'rut_imug. tlivjw trnctions in an alkalir.v Cs(.! i;i,ii!ifiit. -70 \ A 03- t I -' \ I °a --50 \ j • 1 E 0 2. x i \ i ° \ U 4J Kqinllbnuiu ilrn»ity Kr.ulieitt ;inal>-si- nl UNA fr.mi i a;i-.t ' flU.; uft'-r LMSCUSSION Association of sc^ne polymerized P. aeruginosa DNA with a nuckar fraction from DNA treated tohacro cells has been recently reported (Bi.'ndicli and Filncr, 1971)- In these studies DNA was isolated from a crude nuclear fraction and it was found that approximately 0.5 % of the input DNA is present in the final DNA product in a partially degraded state. Using DEAE- dextran treatment of the cells, which increases both adsorption and penetration, we observed that only 0.025 % °f the input .-1. tumefacreiis DNA had penetrated into the cells. Although we used a different cell type;- we still believe that part of the differences hetweeft the results arises from the use we have made of protoplasts to measure DNA penetration; Contrary to nuclei, proto- plasts suffer from less contamination with exogenous DNA due to descrption and rcsoqitioh phenomena during isolation. At each step during the protoplast isolation procedure, part.of the adsorbed DNA is taken away from the re\]s, together with cell wall breakdown products by replacing the protoplast isolation medium. What is even more :.before liberation of the protoplasts from their cell walls the suspension of plasmolyzed cells is washed intensively in order to eliminate all free DNA and cell wall degrading enzymes. Only recently an adequate method for the isolation and purification of intact plant nuclei has been developed (Hendriks, 1972). By combining this technique with protoplast isolation a precise distinction can be achieved between DNA taken up into the cytoplasm and exogenous DNA already present in the nucleus. If in crown gall tumor induction exogenous DNA is integrated into the plant cell genome, we may use the isolation of protoplasts and nuclei as a promising technique to follow the fate of this DNA on its way to the nucleus. Experiments along these lines are in progress in our laboratory. 58 AC KNOWLEDGMEM S 1 h. •iiiil >r- MH.II t" th ml ilnir i. l!i I>:II« - I REFERENCES !'•! •-111111, V ) uml ri!M>, 1'.. H)7i. rpi.,1,,;,, i i'\i....-,i-.,u. us'A hv }'•;< •"".illniKS MIKI IOII.IK-O u>il-. sintittiioi «••>.. 13, njiyjit. :-i.\t v, \ I . i.j|- >m-(,r.f.! '-liu'ii n \i-.f M'iur- !i--|-*inMl>!'- !'.r ilniiur uiitmi-'ii I!! t r.iuii;:;iil. Am. I. lioltirty, 34, 2.U iU'i. i ,.• IT \l , V. in..! Mtli.1 I., ('.., i :;?i.. I llil.Tl..." '.'•• pi-.>l.jpl,l-,lf-s i!.- planli-. MipKHlMlLV' I p.nnii i!'- llh-.ll-. riiltivi's, 111 rilrn. <-'. h'. ,|,,ij. •>.-. f,.,u. >,.i.- )!. 270. •;(«!-,-.'trf.2. ' -I.I..M. K. I!. .!!:<( M:r, 1M >.i 1. I!. ! S.. 11,71, l-\ id.-iwi. Icj thr |ji, ^iic- .,1 I,,-,. -In i; c — | <•< iii< pr.iic-;iis in -I.Tili- trov.li a.iil lillii.ir ;i.ii«- I-I.I1I /'A; .K»'_, 48, i;',-l;,., !-- luinK, I K .Mid CJlK^tt^^.^. S<. A., l.jTI. \Mli;;rmr ,,t.(i iuili'.]l..i!.^|f .'.I *!i.-t'.r!liil|jij(l- ut (jlirf^l-H.-si^ in |.i:nH- tll!'-'l'''i witll .!;:i'.!w/ I ; iMil. U (... llnMi, II. i ,ir..i 111-kii, II. I... (.,H, Ifc-nMiv ^'.iiM-nt fnilu'liiiu.itinn "I UNA in ;i liioil iiii^lc rotor (JiWJIiBi. Hfpkv. AM. 129. iiu-iici. Hi -,i.nit:s. \. \\.. nj7i. rii (V<7 fc.-i..3il, •I1M•^i^. Ifv-ii «, K. "... I HAM-KI. U I. I". . ZdTlis, Jl., 1'iri Mri.ilmli.Mi • .1 HT-.irali'il I.Mi ..il-. I. I'li-n.ir.Hi™ o[ plmt.i-yiitlK-lif nllv .iniM- . »:IW (!•• in tuSi.b >n. 1'hnl My* it:!., 48, i; iv |..i VKM IN, |. I', .mil l'i vi n |,i '.n'yi.. I'., Kjfir. (- i.-l-»:in> i- .-I -\II|IIV-M- .].-- p|-..Ii:iili.> il- -u-peii-iuu- (i-ilul.iirr-. ill- t.ib.l. -rn-ibl.'- .1 l.i kiu.Huii-. I'kVMot. fJinnl . 20, SAI-S^O. 1 tri \r. .!., l-iO'i. Ciowii ^'.lll tuunvnj;rii.-.i^. II. K, !,-iti,>ji-. brturrn ui.tutil 1-tTilin^ nut! thi' nmn.:i^i-nif rrspnii^r. Cmur h' 115-154. ?:, '• ' .--. :-'••':••• '- "-.-.•••• .- -• ....'.-.:. VAS SITTKBT, N, .|,. 107:, investigations 011 plant tumors. Analysis of UNA and -UNA in the cruwn fjallttimfirci-ll.rliesi.i.. Leiden :• (Dutch \vitll Efish'sll ?iiuuni;!ry).- : -r . ^ '•-;•'- YAJKO, U. M. a'hil HF.c.rMAN; ti. !>., li)~l. Tiilndr iniliictiou liy .-I groliuderium\ htiurfneiens : ^pecinc transfer of li;ictt*rial ilecKyri- bonU'-lcic aoid to plutij ti^ue, J, HucUriol., lO8r973-o7o. : .._:.;--/. :;:';.•••.':". ,••'• DISCUSSION -. '•/_;,•.'•:. '•.-..'...... ".".. E. C COCKING. — I would ljke to ask why you have used cells and not isolated protoplasts in your studies on 1)NA uptaki! ? Many of your DNase problems could possibly be eliminated. Is; it possible to transform your cellsin the c,ulture with your DNTA? . •- R. F. HEVN, —• We are sttiaj'ing the initial stages of tumor induction in plants. Keal uptake is one of these stages and ha3;tobf distinguished rigorously from absorption to the cell wall. We do not think that isolation of protoplast, followed by the tedious reculturing of them into now callus, is necessary. It certainly does 110/ eliminate the DNA-ase problems, since this activity is excreted into the medium. We are trying to induce transformation using various DNA's isolated from A. 'tumefaciens. 59 \\ k \imu"H ll.»\<- \mi e.ur:eil mil 11\\\ I>N\ .ni.lnr K.VVUNA livlvrnlis.tlu £\ t-oiwi.1:'!! Ill'- t>\A ul A ^roi^u h-ri-^in and tin? K 1 II \ i \ i It il it i (i it II! I A. : I \ \ (I W ! N > m I | i "• t II ti r 1 11 | n I I uiii I lit HIM \ [I i I i II I Ir -\ K I I 1 N \ I i r 1 1 li »„ K 1 Hi VN Ut-...ill !.:-.• .i-, sur il" iU- filing ilv^li at the p-W! M.iK'-1 am! tlir pruto .! ,-s.ii.r: \s •-[,..!<•. I. »-i- uill isii|;itv v 111.- hiriliiT Uifr-1,1 l.i.N A ail.-r the i>.i*:..r_;(- 1 i'\K.''i with I'!•*AK-iSr-Mian t an [>n.-vriu plant I'NA ivuvu> that I >l-'. \i'. iit'xti.iis ciiu-r^ tlu1 i: vt*»pi;i,sni r U 1". HK\'S — It ti.nlii. indn'.l, i-i-ry wrll ilili! liiat DMM'.-ili-.MfjH (in uimhm.itiun with lUnmr iiy Inuli t'.imiHiiatuin- i.t tlnnur DNA. .ippar^ntly r«lm-i-il I", thi |>n--tr<-atmeni «nli I >!• \H-dc\ii,; FIG. I. Tobacco cells in suspension culture (bar equals 50(1). 60 ^O^frS'JH"' > ' ~,o ' FIG. -2, : .-.--•: Prutoplasti from tobicc.o suspension cells (bar equaK 100 n) TIG 3 Various types of protoplasts isolated from toliacco su pension cells (bar equals 61 CHAPTER V TOBACCO MESOPHYLL PROTOPLASTS ISOLATION, DNA SYNTHESIS AND PLANT REGENERATION INTRODUCTION Protoplasts of plant cells consist ,;f ordinary plant cells devoid of their rigid, mainly J.H>! ysaccharidp rontaini :ig cell wall. The last ten years, and even more so since the Versailles Meeting in 1972, work on these naked plant cells hat. prv^ressed very rapidly with special emphasis on their potentials as genetic systems (chapter II). A number of reviews have recently been pu- blished covering both, fundamental and applied aspects (2, 6, 9, II, ISt, }6, -i 3 ! . Tiie choice of '..'A: :".',>.; ••«•;••<•••;•.,"•. as experimental plant for our transforma- tion directed experiments was an obvious one. Much of the experience on crown gall accumulated over the last ten years in this Laboratory has been obtained from work with tobacco. Requirements for tissue-culture of tobacco are very well known and it was not surprising that work with this plant led to the first regeneration of a single pps to an intact plant. Moreover tobacco figures among the genetically well known plant species (47). As compared to suspension cultured cell clumps the leaf pps system has two major advantages: t) IK makes possible a simultaneous "inoculation" with fo- reign genetic material of all individuals as single, isolated entities, and 2) since for each isolation one goes back again to the intact plant, problems encountered with cultured material concerning changes in chromosome number and other genetic abnormalities are eliminated. In efforts directed towards tumor transformation using A.turn.DNA (e.g.42), the leaf pps seem to have still an additional advantage. In tumor formation a so-called "conditioned" state of the host cell is required (see for a recent literature ..review ref.13). This condition can be characterized as a stage in between afunctional» non-divi- ding cell and a dividing, meristematic cell. Action of A.turn, should take place just prior to the first cell division. Leaf pps correspond to such a description. In the expanded lep.i they were functionally specialized, non- dividing cells (dead-end cells)s but the removal of the cell wall and culture 62 • iii appropriate media can induce them to di_differentiate and to start divLding .gain. The rather synchronous nature of the onset of cell division is regarded . , .in .ulilitinn.il .ulv.mt,i',',f. Preparations "f le.if pns lur i r.in'%fi>rm.ition studies h <• to satisfy certain requirements: 1) they should umtain very large numbers of ,-is (e.g.50 x 10 ), .) they *; In HI li[ h.ivi- .i low pt rcent age of damaged pps f containing ing the medium with e.g. PNAase), j) they should be sterile and 4) the intact pps should be. viable enough to k'-ve reproducible plating efficiencies. We devoted much time lo the acquisition of sufficient experience in the isolation of tobacco meso- phyll pps (leading to the development of two techniques suitable for different qualities of leaf material). Viability and sterility of our pps preparations were demonstrated by plating experiments leading to the regeneration of intact plants. An indication for the synchronous onset of DNA synthesis was obtained. MATERIALS AND BASIC METHODS Plant maieria I iKeoiiana tabaaian L., var. White Burley has been used in all experiments reported here. Much of the work has been done with plants of this variety pro- pagated already for many years in this laboratory. In July 1974 we obtained from Dr. J.W.Watts seed of the White Burley "strain" used at the John Innes Institute, Norwich, England. It has completely different growth characteris- tics (greener, stronger, leaves more flat and lanceolate) and it is a better starting material for reproducible pps isolation when the "stripping" technique is used (53). After sowing at high density and germination in the dark, seedlings (ca. 20 mm high), are transferred to "Vacapots" (compartmentalised plastic trays) about two weeks after sowing. After 2 weeks the plants (at about 3 true-leaf stage) are potted in small pots (90 mm diameter), followed 2 to: 3 more weeks later by final . pottitig: in the early work pots of 130 mm diameter were used, later 220 mm. Commercial compost (Aalsmeerse molm), supplemented every two weeks with fertiliser (Pokon), is used throughout. All potted plants are water- ed from below, once a day. The greenhouse (nr.4) was maintained at about 24 and 60-70% relative humidity. From September to May additional light, for a total period of 16 h, was supplied by Philips high pressure mercury lamps (HLRG 400 W). The most commonly measured light intensities range from 3,000 to 10,000 lux, but on sunny days it may increase up to 45,000 lux in spite of thick layers of lime on the glass of the greenhouse roof. 63 A!! uper.uii-ns -.IIOMIO In j o i l ornu'.l .-.i-pt 11 .1! 1 v , pri t »T.ibl v in .) lauin.11 ** i i I li'W e.ihiiwc >r i 11 in iMTtu (r.invlcr r.--'m. IIMVI-,, briofU iitisod in running tap water-, ar'»-. «ti. . < -.sivrh n::r,t tsrj m : a) 0.5% (v/v) Juii iciiit »..'luri.':i (<-•. (;. li-fp.i 1 ' t^r J. win, b) 7Q* (v/v) etriana! for 0.5 - I rain. c s diluted sodium hypuch! cri t. e so hit ion, rotuaini.nj' again 0.3" Teejiul ;ts ;i weltir.t .i^oiit, iitr K) "in, d) t"hrcf i-iiaiiiccs %>f slcrilc distilled water. The solution UIKUT «.-.) is a Y", (v/v) dilution tram l.-ib grade SaOCl (H.D.H., r Pooif, England) givini', a t'itul louc. at available chlorine- of 0.3 - 0.43 (w/v) (16). Also household NaOCI ("Glufix", be KtMiix, Xuollf) al M (v/v) ran be ustd. It iimuins already a wetting agent and is more stable upon storage (U ), Ail itiese solutions are maintained in 2 I an toe Laved glass btakers, covered with a double layer or aluminium foil or a lar^e Petri dish cover. The leaves are handled vith long (0.2 m) sterile forceps. The cellulase containing enzyme tnixturf? "Onozuka" SS (lot nr. 223011) pre- 11 pared from ?>•:'•'":.: i;.i>.-;u i";. ; i r and the poiygalacturonar.e preparation Macero- .! zyaie (lot nr. 801088) from hhir.i:•;-UB up. (48) were obtained from All Japan Bio- chemicals Co., Ltd., Nishinomiya via its sales representative Kanematsu-Uosho GmbH, ^000 Dusseldorf, W.Germany. Before sterilization by filtration using a membrane of 0.45 pm pore size (SM 11106), the enzyme solutions were prefiltered under non-sterile conditions using the same type of filter in combination with a glass fibre filter in order to remove some particulate material without en- zyme activity. In some experiments "Driselase" (lot "hr.K34OI6, a gift from Kyowa Hakko Kogyo, Tokyo, Japan) was used; it has both pectiriase and (high) - cellulase activity and can be used at lower concentrations. Ronament P (pectin- glycosidase from Aspsvyillus) came from Rohtn GmbH; Darmstadt, W.Germany, and was a greatly appreciated gift of Dr. L.Schilde-Rentschler (Tubingen). Centrifugation All ccntrifugations were performed in a MSE Super Minor table centrifuge with a swing-out type rotor, accomodating four tubes. Round bottomed Beckman type JA 20 polycarbonate tubes with screw cap were used with an additional aluminium foil cover to facilitate aseptic handling. In order to diminish da- mage to the pps due to fast acceleration, the rotor was always allowed to at- .tin the preset speed very slowly (for the lvpii-.il 50 v. ;; (hi-; UIM-'. nlmur 2 id) .ind tlii- br.ike was not used .if lerw.irds . Alter i •».• Kit i •.!) .ind i.'.i'.hiiv, the pp.-, were always kept in t. lie culture medium •:" Ni^.it.i .in<] l.ikt-bi- ( J''t) rtlum-d ti> .is NT-medium. This is a modification of the basic tobacco tissue culture medium of Murasbige and Skoog (32) differing , IC;;IC!,J.2H.,0) x 0.5; (KILjPO, ) x << «nd (MgSO^. 7H?0) x 3.5. For plant regenera- tion purposes the ti'.i medium of Sacristan and Melchtirs (38) and the revised medium of Li nssnai er and Skoog (29) have been ust-d. Roots were induced on White's medium (54). J •'"-,"? •'* ~- '-*;*! ;.',* The hawnoeytometer used for pps counting was a double chamber model with modified Fuchs and Rosenthal rulings (0.2 mm depth) from Tamson, B.V., Zoeter- rneer. It was used in conjunction with a Leitz Orthoplan microscope at a mag- nification of 800 x. At least 300 pps per sample were screened to determine pps density and the approximate percentage of morphologically intact pps. '•'isofj I i-uncom, Kutei'ia Is Potassium dextran sulphate (Lot nr.RR-83I-SK-2) was obtained from Meito Sangyo Co., Ltd.(Nagbya, Japan). D-raannitol (29148) came from B.D.H.Chemicals, Ltd. (Poole, England). Soluene 100 (6OO3O2I) was from Packard Instruments Inter- nati->nal S.A.(Zurich, Switzerland). (Me- H)-thymidine(50 Ci/mmol, type TRK.418) came from The Radiochemical Centre (Amersham, England). Amphotericin B (Fungi- zonei cat.529) came from (?rand Island Biol.Comp., N.Y., U.S.A. and Carbenicil- line (Pyoperi) from Beecham Res.Lab.; 10 x concentrated stock solution was pre- pared in 0.7 M mannitol and stored in appropriate portions at -20 . Stainless steel '.'jewellers forceps (types'- 3 and 5) were manufactured by A. Dumont & Fils, Switzerland f: RESULTS -AND DISCUSSION Tr\ describe here all the trial and error type of experiments performed be- fore a satisfactory method of pps isolation had been found and the many dif- ficulties encountered in growing "pps-plants" of sufficient and especially reproducible quality, is impractical. Instead we will first describe in detail the two; methods, which we finally: adopted and will then give some general ,comments; on. the art of pps isolation, s^ • :; '.-'• ••"-.• ••'•--, .••--•-••-••' ••• - . .-65 in tin- i-n; visat ii li i ce > t i on of ri-1! walls, the main problem is to got the en;-v.-.u-- in ••.inl.i.l with their i insn 1 uh 1 o ) suhstr.ite. The problem has Bonor.illy ; i>r.:n -.rH,.-i w ; ; h b; shv. m.-nui.-i i- removal o! r. he lower ey ivO). Curtimj, thin slices roaibiiii'il with vjt.uiURi i uf i U.r.it; i on is ,i 1 SD: sat i sf _ac to; ry ai)ii is vorv cisv to perform under a.^ept i v;.ii nmdi lions, Ht'Ci:iitlv cuitinU ::T has hov-i; i;-,ei3 t1-*;. ti> pr;'ducil plii'tosyntlii? t: l c: 1 1 v active isot.ittHi oci' s 12.0, t',.'!! ti.sts i_'?i, in view oi the ditt\-reut ways vi enzyire petiotr.it ion wi »'i;r iWi- fiH-iluuis tin- "cutting" and "stripping" tt-chnicjut' re.specli vti 1 y, i«if surfac-.' sit-r i I i a«.'d laf is gvntls' driod botwevn two layers of sturili? i"i!t«-r p.ijHr, l-'sin^ a new r.izor bladi' (O.I mm, superf inc • the midrib is removed. Both le.i: h.ilve.-. .ir;' rn!lt.-d up lenglhwi «,• and are cut into thin slicqs (ca. I- J Kim • . during tlu-si' m.-ini pulat ions surgical Rloves (sccrilized with 70% (v/v) c111ajwl) iirf worn. The sliced leaf material is quickly transferred into a special polycarbonate jar, sterilized by autoclaving at 110 loir 20-30 min. It consists of the reci- pient vessel of Sartorius filtration apparatus SM 16 510 of which the (screw cap) cover Has been fitted with a stainless sr.t-el screen (ca.0.3 ran pore size); one of the three luer type openings is fitted with a small size filter assembly containing a membrane filter of O.I iitn pore size (SM 11309), the others as well as the central screw cap opening are closed. The jar contains already 50 ml of filter sterilized maceration solution containing (in 0.7 M raannitol, pll 5.8) 1% (w/v) Macerozyme and 0.5% (w/v) potassium-dextran-sulphate. The jar is brief- ly (0.5-1 min) evacuated: just enough for air bubbles to be released from the leaf material. When the vacuum is slowly released the leaf slices are infiltra- ted with the enzyme solution resulting in a darkening ofthe green color. Due to the floating of some material on the enzyme solution, this infiltration step has to be repeated once more after some shaking * The jar is placed for 0.5 h on a reciprocal shaker (72 excursions per min, 70 nan stroke, room temp.) and the released cell material (debris) discarded by decanting through the stainless steel filter and the central opening. With 40 ml fresh maceration solution shaking is continued for 1 h and the resulting cell suspension decanted as above into sterile centrifuge tubes followed by pelleting for 3 mir, at 100 x g. The supernatant is removed by suction and the cells are resuspended in the tube with 40 ml 0.7 M mannitol pH 5.6. A total of three bat- ches of cells are thus successively harvested and then combined. The approxima- 66 I ly _'O x 10 intiH'l, p 1 uaino I y Ki'd ci' 1 1 s isolated from one leaf are then i ncubated : h )d is I illul isi- sf 1 in I nn i.1" (w/\. ; Oim/u] ,i SS in 0.7 M mimiuol, pH J.J; ID ti>r 1 — 1 i h in ,i Imn/i>ni.i) 1 v pl.ni'd, tJ>'htly closed icnirifu^i tubi . By t ml nf this nit uli.it inn .ill tin- i »• 1 1 s .m innvtTtt'd inio pps ,ind .itt-.r r<-- , i) nt (In siipci n.it.int cn/vrat xilntion art- washed twite with ->0 ml 0.7 " ;.,i!iui to 1 pH 5.6 (SMU.'lining 10 mM CaCl.., and twice with 40 ml .NT-medium i ci'turifugat i on i mill -ii. 50 x g) . After surface sterilisation tin- Leaf is left in the laminar air flow cabi- net for 0.5 to 1 h to become prrtially flaccid, facilitating removal of the h'VL-r epidermis. The leaf should not become too flaccid because it may then hei/iime easily damaged in the next step. Using fine jewellers forceps (sterili- | /i-d by dipping in alcohol and flaming! and a flat support for the leaf, the lower epidermis between the main veins is gently pulled off. This peeling or stripping.is most successful if one inserts the forceps in a main vein and then pulls in a continuous, horizontal movement towards the edge of the leaf. With a piece of sterile filter paper the leaf is (gently) maintained in place | with the other hand. The success of this manipulation depends to some extent on a minimum amount of exercise and a judicious use of the leaf's vein system, which will diminish tearing of t«\e epidermis. Small pieces of epidermis re- maining on the leaf should be left there: danag« done to the ""esc/iyll ir try- ing to remove them is not balanced by an increase in yield. The most important determining factor, however, is the quality of the leaf. 2 Each time an area of some 20 cm has been stripped, it is cut using a scalpel, into 3 to 4 pieces, which are then placed, peeled side downwards, on the surface of a 0.7 M mannitol solution (ca.25 nil) maintained in a 14 cm glass Petri dish. Care should be taken to avoid folding of the edges of the pieces, since these regions will not be attacked by the enzymes. If the leaf is too flaccid this folding may cause serious problems. One Petri dish will generally accomodate about 1 g fresh weight of stripped leaf. The pieces are left floating on the osmoticum for 2 to 3 h to effect pre-plasmolysis. The osmoticum is then removed by suction from below the leaf pieces and is re- placed by 30 ml of a filter sterilized solution of 0.4% (w/v) Macerozyme and 0.8% (w/v) cellulase Onozuka SS dissolved in 0.7 M mannitol (pH 5.8). The stripped leaf pieces are then incubated in the dark overnight (17 h - 9 h next day) at 27° at 60 to 70% relative humidity. •••.••• .•••••••• • 67 After siuh an incubation nearly all tin- tells of the mesophyll are eon- verted into pps, but Chese remain :'>. it:'::, in the floating leaf matrix composed of i, undigested) upper epidermis and the network of smaller veins. Some gentle swirl ing of the I'otri dish releases the pps but leaves die undigested material intact. After tilting of the dish this floating debris is easily removed from the pps suspension using forceps. The pps are then allowed to settle out for 30 min. Tiie en::vine solution, containing some smaller debris, is removed by suction. The pipit should be handled in a near horizontal manner in order to leave the settled pps undisturbed. Subsequent washing of the pps is performed as dvsv-ribed under "cutting" technique. This various stages described are illu- strated in fig.V.l a-f. Ail authors active in the pps field stress the fact that the physiological rendition of the plants used is of utmost importance for ruproducibly success- ful pps isolation, but only a few describe them in detail. In fact it is very difficult to copy exactly the growth conditions found el$ewhere to be "optimal"; Moreover there is a tremendous variability between specific varieties and cul- tivars of widely used species such as Nicotianq tabaawn and Petunia hykvida (14, 18, 21). We have tried e.g. to imitate the conditions inducing very slow: growth of .'Jt:.:otiutia species as described by Chupeau et dl* (8) using seed pro- vided by them, but we completely failed to obtain the type of plant growth des- cribed. Sea^onai influence (18, 25, 37) was particularly pronounced during the sum- mer, when (probably due to a too high starch content) the percentage of pps damaged *uy centrifugat.ion increased remarkably. The use of a greenhouse with only artificial light (ca. 8,000 lux at mid plant level, from mercury lamps described above) has improved the reproducibilicy to some extent* but plants grown under such conditions are much" ".softer" than normally (causing stripping problems) and tend to yield pps that are. more difficult to pellet. Hibi et dL. (22) have recently highlighted the difficulty of controlling in a satisfactory way the physiological condit. ns of plant growth for periods up to 2 to 3 months. . Fig.V,l. Isolation of pps with the stripping: technique (next page). From left to right: materials needed for surface sterilization of the leaf; stripping of White Burley leaf; stripped leaf pieces in pre-plasmolyticum, ready to be incubated with filter sterilized enzyme mixture; epidermal debris pushed away after 5g overnight incubation; pps settled out and erizyme mixture re- moved; transfer to sterile centrifuge tube. 69 When we started our investigations the tobacco plants available were in bad; condition for pps isolation: they flowered prematurely, generally did not at- tain their normal size, had very wrinkled leaves and showed early yellowing of the lower leaves. The major improvements have been the use of larger pots, higher light intensities during autumn and winter, more greenhouse bench space per plant eliminating the artificial early elongation growth, a strict repot- ting scheme taking care of stagnating growth in too small pots, and USG of more fertilizer. The influence of root stunting, and nitrogen shortage on the enzy- matic release of tobacco mesophyll cells has recently1 been stressed again (7). The position on the stem of leaves to he used for pps isolation is of defi- nite irupoitance. -Very young or half expanded leaves will give pps in abundances t>ut they are very unstable. The leaves giving the highest yield of stable pps are just fully expanded (30-35 cm in length, weighing 10-16 g) and are general- ly in position 4 to 6 counting from the plant's apex (=0, including the very small leaves which are in a near vertical position). The leaves were excised before watering (late morning) and ware selected according to flatness of the lamina and homogeneous green color. In general each healthy plant will produce 3 to 4 good leaves. .. • •"';- Plasmolyiicum The plasmolyzing conditions used to stabilize plant pps liberated from the containment of their rigid cell walls are rather traumatic and greatly influ- ence cell metabolism (e.g. the tremendous increase in RNAase levels observed in tobacco, 28). The ability to divide after cell wall regeneration seems also to depend on a not too high concentration of osmoticum (Cocking, pers.cornm.). The osmoticum should be nonmetabolizable and nonpenetrable. Sucrose has therefore be';n used only rarely or for very short periods. The tv;o most em- ployed osmot.'.ca are mannitol and sorbitol.; We have tried both at concentra- tions from 0.'* to 0.8 M. Although sorbitol has the advantage of better solubi- lity in water Vaaking the use of concentrated stock solutions possible), we abandoned its use due to frequently encountered floatation problems, probably due to uptake of sorbitol reducing the density difference between pps aud os- nioticum. We would lose e.g. 20% of the isolated pps at each washing treat- ment. The same effect was sometimes observed at raaanitol concentrations of 0.5 or 0.6 M. We therefore stuck to the most commonly used concentration of 0.7 M. 70 Recently methods have been described using a combination of KC1 and MgSO, as osmotic stabilizers in the isolation (30) and culture (31) of tobacco pps. Although some cell divisions of essentially wall-less pps could be observed, this way of osmotic stabilization does not permit the synthesis., of a .rigid cell wall and subsequent plant regeneration. Some workers (8, 12, 18, 23) ad- vocate the addition of low concentrations of mineral salts from culture media to the enzyme mixtures, especially when incubating overnight. This would per- mit a better.stabilization of the pps and should increase their viability. We have used the medium of Cocking's school (which is the culture medium of Aoki and Takebe.A') with a !0 .times Inwer CaCl™ concentration), but did not. find any significant improvement of yield or morphological quality of the pps. Sponta- neous fusion C;i'O) which should also be diminished by the presence of these salts, can be reduced to negligible levels by pre-piasmolysis. Pre-plasmplysis ..A prolonged incubation of the leaf material in the osmoticum (with or with- out some shaking) in order to effect plasntolysis before the start of cell wall digestion, does not significantly improve pps yield. It does however decrease - ftorn about 20 to only 1% - the spontaneous fusion of pps occurring when the stripping technique and mixed enzymes are used (see also 4 and 18). With the two-step cutting technique spontaneous fusion (of adjacent cells) is obvious- ly impossible. At too high temperatures or too high cellulase concentrations some fusion will occur, however of those cells in close contact during eellu- lase treatment. Another aspect of pretreatment with osmoticum is that possibly toxic substances (like polyphenols) released during cutting or stripping (which also involves considerable damage to spongy parenchyma cells; can thus be wash- ed out before liberation of pps. This consideration has led some workers (4, and Gi].esr pers.coiam.) to cut tlie leaf material while immersed in osmbtiium. :& Enzymes. -. • - • -,,• '• •••••;,--'". ':L ~,'~ • '. v. '•: .'[''•••-'' ""'y ' .•} - '- ~ One should keep in mind that the commercial enzyme preparations (intended for. Indus trial application in beverages, soaps and pharmaceuticais) now widely used for pps isolation are very crude extracts containing e»g. nucleates and proteases. They have variable macerating activities towards plant cells, even from batch to batch of the same grade. The so-called standardized specific activities of various grades are determined using tests which have nothing to do with pps isolation. The reports in the literature about very definite con- 71 cer.trations of enzymes giving optimal results should therefore be interpreted very cautiously. Sinje three years All Japan Biochemical Co. markets a special, more expensive, "pps-grade" of their enzymes designated R-10. We have no ex- perience with these enzymes, buL they appear to be of consistently higher ac- tivity in pps isolation (8). We did perform some trials with the new prepara- tion "Driselase", which combines high pectinase with high cellulase activity and is thus a good choice for one step methods. Its activity in pps isolation was found to be higher, but care should be taken to use minimal concentrations and minimal incubation times, since the proteases present may start to attack the plasmalenvma of the isolated pps, resulting in clumping and bursting. Added at low concentrations to other enzymes it speeds up pps isolation and has thus been used successfully in the isolation of pps from tissue culture material (20, 24) retractile to digestion by many other enzymes when used alone. This illu- strates again that a particular mixture of various polysaccharide degrading enzymes is needed for the successful maceration of each particular type of cell ^ wall. ". ;. Pectin-glycosidase has been used (15, 41) to eliminate the sometimes tedious stripping of the lower epidermis. One hour incubation with this enzyme (at 0.1 mg/ml, not 10 mg/ml as erroneously printed in ref.41) digests the cutin suffi- ciently to obtain effective penetration of Macerozyme afterwards. We have tested this enzyme treatment followed by cell wall digestion as described under "cut- ting" technique (two-step method), to see if large unstripped leaf pieces could be used. The system works well, but reproducibility is variable and no time is saveJ in comparison with the two described techniques. Moreover this additional enzyme represents still another possible source of toxic substances. We there- fore abandoned its use. •• ~ :: : . rr \ . •: •"''.".•'..'.':'_ Desalting of the commercial enzyme preparations using gel filtration has been recommended (26, 40), but we have not found any increase in yield or intacthess of the pps when this slight purification was applied. dexzran sulphate . . ••'• The importance of this additive to enzyme mixtures has never been made very clear. It has been suggested (49) that Macerozyme solutions contain particular basic proteins that could be toxic to tobacco cells and that this polyanion ; could prevent damage to the pps by binding to chase proteins. Although we have no idea whether this explanation is correct, we did observe a slight increase in the yield of intact pps with the cutting technique, but (in accordance with 72 -.-::•}• Kassanis and White, 25) not with the stripping one (in which lower enzyme con- centrations are used). The usefulness of this expensive additive thus remains ..imbiguous. Many workers nowadays abandon its use. Hashing^pj':the\pps; .- ,...... One of the major advantages of.the/stripping technique'is that the upper^ epicarmis, with attached to it the major veins, remains floating on the sur- face of the enzyme solution and can easily be discarded with forceps. The smaller veins and about 90% of the enzyme solution can be pipetted off without cc-.ntrifugation. The first two washes with 0.7 M mannitol will lead to elimina- tion of broken pps, epidermal pps and chloroplasts, with an acceptable conco- mitant loss of some of the intact pps (about 5-10% per wash), but further mannitol washes will not increase the percentage of intact cells. Especially with a too advanced Driselase treatment the percentage of presumably intact cells floating in the supernatant after centrifugation may be higher than 50. This may indicate that the plasmalemma of these cells has been damaged resulting in some permeation of the osmoticum and subsequent decrease in the density dif- ference between pps and osmoticum. The washes with culture medium (containing considerable amounts of ions) only eliminate residual enzyme solution compounds, but do not further purify the pps preparation. Debris will also sediment and generally tends to clump somewhat. The observation of Binding (with Petunia pps, 4), that addition of high amounts of mineral salts to the washing solutions induced desaggregration of chloroplast clumps and broken pps resulting in a better purification, could thus not be confirmed with our tobacco pps. Movphologioal intactnese of pps The eviluatibn of the; quality of pps preparations is an as yet unresolved problem (&>. Before using freshly isolated pps for further experimentation one would, like ,to: have -'••&':.reliable.: estimate of the percentage of vital pps. Widholm has described the use offluoresceihdiacetate in-"vital staining" of cultured plant: cells^(55)i We have only limited experience with this dye, but found that it would indeed clearly, distinguish those pps that are absolutely dead. By microscopic observation (magnification 800 x) it is possible however to note also those'-cells' that are dying. Typical examples are- the pps that are extru- ding their central vacuo],e and those that have a random distribution of chloro- plasts. We counted as intact only those pps that are completely spherical and that exhibit the typical disposition of chloroplasts at the periphery, pressed ... . 73 against the plasma lemma (see cover illustration). Any important discontinuity of this chloroplast "ring" or bulging of Che pps was taken as a sign of ap- proaching death. With these criteria the percentage intact pps as determined by different observers on the same preparation agreed well. With the "cutting" and "stripping" technique this percentage was respectively 65-75 and 75-85. The rather intensive shaking used .with the "cutting" technique is probably the most important reason for this difference. Yields per gram fresh weight leaf mate- rial were also different : 1-2 x 10 (cutting) and 5-12 x 10 (stripping). It should be remembered however that all the major veins are included in the start- ing material of the first technique, while they are eliminated from the stripped pieces (as is the lower epidermis); moreover digestion of stripped material is complete, while this is certainly not the case with thin slices. Use cf ayitibiottjs When pps have to be cultured for only a limited period of time (as in meta- bolic studies (52, 39) or the determination of optimum conditions of DNA uptake," (chapter VII) it is not necessary to apply strictly aseptic techniques, which may become very tedious when e.g. large quantities of stripped leaf material are needed. In combination with the "stripping" technique we successfully controled infection for more than a week in pps suspension in liquid NT-medium using the combination of amphotericin B (antifungal agent) and carbenicillin as recommended by Watts and King (52). In that case we omitted all aseptic mani- pulations except at the end of the washing procedure and the distribution of pps samples over the culture vials. Materials and solutions were however sterilized and the antibiotics added both Co the (overnight) enzyme mixture and the final culture medium. Before the publication of ref.52 (which nicely illustrates the very high sensitivity to antibiotics of plant cells as com- pared to animal cells) we tested rifainpicirie.pimafucine and streptomycine, but these antibiotics proved to be very toxic to pps as indicated by a complete inhibition of H-thytfidine incorporation and the absence of any cell division. Onset of DNA synthesis Already in the first successful experiments, where sustained division could be induced in cells regenerated from tobacco mesophyll pps4 Nagata and Takebe (33) noticed a marked synchrony of the first division: no division up until the second day after isolation, but 80% division by the fourth day. Electron mi- croscopic observations (35) on tobacco pps cultured in NT-medium confirmed this synchronous onset of cell division and the transition to the statp- of callus- like meristematic cells. 74 On the level of DNA synthesis no data are available for cultured pps, ex- cept that Watts and King (51) noticed the complete absence of it for the first 24 h after isolation of pea leaf pps. Since the onset of DNA synthesis by the host cell may be of critical importance in DNA uptake and transformation ex- periments, we studied the incorporation of- H-thymidine by tobacco pps" up to the seventh day of culture in liquid NT-medium. In preliminary experiments we cultured (650 lux, 26°) the pps at a density of 200,000 per ml as 10 ml samples in 100 ml erlenmeyers. Microscopic observa- tion of the cultures confirmed the results of Nagata and Takebe. Before the first division a typical swelling of the pps took place and a redistribution of the chloroplasts away from the plasmalemma. In these first experiments label incorporation was measured after fractionation according to Schmidt and Thann- hauser (39, 44). Using label "pulses" of 24 h, a wave of active DNA synthesis was found around 50 h after isolation, with practically no incorporation during the first :24 h. Later we developed a more reproducible culture method : 2 ml samples at 50,000 pps/ml are incubated in sterile counting vials closed with a compressed paper plug. Series of samples coming from one isolation could thus be used. Light conditions were now according to Enzmann-Becker (15), i.e. about 400 lux for the first 48 h, later 2000 lux. Label incorporation was measured using the rapid method of Ferrari and Widholm (17), where a (v/v) 12:5:3 mixture of metha- nol, chloroform and water is used to wash away non-incorporated precursor from cells trapped on glass fibre filters. Although this method has been developed using C as label, it works equally well with H, if care is taken tc solu- bilize the cell material completely. To this end we incubated the washed pps filtersiyforv:3;;h.^in counting vials with 1 ml Sbluerie 100 containing 10% (v/v) Fig. V,2. Pulse labeling of tobacco mesophyll pps cul- tured in JT-medium (50,000 pps per ml; 16 h pulses; 10 2 4 6 uCi H-thymidine in 2 ml). Days after inoculation into medium 75 water, neutralized the mixture with 1 ml 1M Tris-HCl pH 7.5 and counted in the usual toluene-Triton X-100 cocktail.Some vigourous shaking of the vi.al on a vortex mixer will lead to the complete disintegration of the filter making dual label experiments possible. With this method we found a great variability in the onset of DHA synthesis for different PFS preparations. Using 8 or 16 h pulses the optimum 3H-thymidine incorporation activity was found anywhere from 40 to 100 h after isolation. In one particularly successful case (illustrated in fig.V,2) we found also a se- ' cond "wave" of DNA synthesis, which could possibly be correlated with the se- cond division cycle observed by Nagata and Takebe (33) on the seventh day. For the addition of foreign DNA to pps we concluded that the second day after iso- lation would be a good choice. Plant regeneration from White Bur'Ley pps A successful method for pps isolation must not only result in good yields, but more important the pps must be viable, capable of reforming a cell wall and; of development into a new callus. If genetic modification of pps is to be use- ful one has to be able to regenerate plants from these colonies. The first (1971) to achieve regeneration of a whole plant from tobacco (cultivar. Xanthi nc) pps were Takebe, Labib and Melchers (50). They used epidermal stripping and 3 two-step enzymatic pps isolation technique and a complex (coconut milk containing) medium for plating in agar according to Bergmann (3).'The same year Nagaca and Takebe (34) improved upon this method by adapting the basic Murashi- ge and Skoog medium (32) to the specific needs of pps. In order to be su^re that the White Burley pps isolated using our "cutting" technique could also be induced to form colonies and to regenerate into whole plants, we applied the Nagata-^Takebe technique to our pps. Pps suspended in liquid NT-medium (fig.V,3a) were mixed gently, but rather quickly with an equal volume of the same medium containing.1.2% (w/v) agar and kept just liquid at ca. 42 . Aliquots of 20 ml were poured into plastic Petri dishes of 9 cm dia- meter, where a thin homogeneous layer could be formed just prior to solidifica- tion of the agar. The pps density in the plates were varied, but best results 3 (20 to 30% plating efficiency) were obtained with:20 to 40 x 10 pps/ml, which is slightly higher than the plating densities reported by Nagata and Takebe (34). When plating density was either below 10 or atove 10 pps/ml no colo- def were formed, confirming earlier results (15, 34). Ths dishes, sealed with : Parafilm to avoid desiccation, were incubated at 26 with continuous illumina- tion from white fluorescent tubes (Philips type 33, 2000 lux). The thus plated pps, readily observable in the thin agar layer, were 76 dically examined under the light microscope. After regeneration of cell walls, cell division was initiated (starting the fourth day) in more than 50% of the pps (fig.V,3b), and after two to three weeks of culture compact cell masses were observed (fig.V,3c). Four to five weeks after plating the majority of Lhese cell masses, originating from individual pps, had grown into readily visible colonies of more or less khaki color, typical of White Burley callus cultures. This is in contrast to the results of Nagata and Takebe (34), who ob- tained light-green colored colonies. Most of the colonies reached sizes of 0.5 to 2 mm after six to seven weeks (fig,V,3d) and could then be transplanted onto media lacking the osmoticum. A binocular dissection microscope and a fine scal- pel were used to be sure to pick single, isolated colonies derived from one pps. In accordance with Nagata and Takebe (34) the colonies could be indefinitely subcultured as callus masses on Murashige and Skoog's medium with 1-naphtalene- acetic acid (3 mg/1) and 6-benzylaminopurine (1 mg/1). Such a culture is now maintained in our laboratory to serve as a source of cloned, phytohormohe de- pendent, normal tobacco tissue. When placed on B3 medium (38; containing 4 mg/1 indoleacetic acid and 2.56 mg/i 6-furfurylaminopurine : kinetin) the colonies readily differentiated shoots (fig.V,3e) after about three weeks. Regular ob- servation showed that such differentiation generally originated inside the. cal- lus masses, shoot primordia breaking through tiie outer cell layers of the callus clumps. As soon as they appeared the shoots turned green. No roots were formed on this medium. We wondered whether the organic additives in the B3 medium (pyridoxine-HCl, nicotinic acid and glycine) were necessary for shoot formation. Therefore some colonies were transplanted from NT-medium on B3 lacking these additives (29). No difference, in shooting w,±s ^served. The designation "shoot- ing medium" for B3 is thus only due to the particular phycohormone balance, which is known to play an important role in organogenesis (29, 46). Three weeks on shooting medium yielded plantlets (several per callus) which could be placed individually on White's-agar medium (54) in 250 ml glass bottles. Well developed root systems were produced on this medium within four weeks (fig.V,3f). Although pps from the tobacco variety White Burley have been used for studies of virus infection (25) anil antibiotics resistance (.52), this is the first time regeneration of intact plants from pps of this variety is reported. About 70% of the single pps derived colonies can thus be asepticaliy grown into normal plants with the aid of standard tissue culture media and within a period of two months. It is obvious that once a transformed or mutated colony has been selected, clones consisting of large numbers of individual, identical plants can thus be easily and quickly produced. 77 Fig.V,3. Plant regeneration from White Burley pps; a)freshly isolated pps in NT-medium (400 x), b)after one week culture embedded in agar medium (500 x), c)cell colony after three weeks culture (300 x), d)Petri dish with calli from pps, seven weeks after plating (1 x), e)shoot formation on B3 medium (1.5 x), fRegenerated tobacco plants on White's medium (0.5 x). 78 79 REFERENCES 1. Aoki, S. and Takebe, I., Virdojy 39(1969)439-448. 2. Bajaj, Y.P.S., F\L 'nyr:.\i 23(1974)633-649. 3. Bergmann, L., -r. j^'i.Thiisio:.. ^3(1960)841-851. 4. Binding, H., M.Pfi iKst'iphys,L'l. 74(i974)327-356. 5. Blaschek, W., Hess, D. and Hoffmann, F. , Z.Pflanzenphysiol. 72(1974\ 262-271. 6. Burgess, J., New Scientist 64(1974)242-247. 7. Cataldo, D.A. and Berlyn, G.P., Amer.J.Bot. 61(1974)957-9.63;. 8. Chupeau, V., Bourgin, J.P., Missonier, C, Dorion, N. and Morel, G., CR.Acad.5ai., Ser.D 278(1974)1565-1568. 9. Cocking, E.C., Annu.Reo.Plant Physiol. 23(1972)29-50. 10. Cocking, E.C., C -II. Intern.C.N.i"?. S. 212(1973)327-341...... -,. ,:. 11. Cocking, E.C., (1975) in..: Genstia Manipulations with Pl^xit Material (l.Ledoux, Ed.) Plenum Press, New York,p|311-327. 12. Cocking, E.C. and Peberdy, J.F. (Eds.)_(197%)'"The Use of Protoplasts from Fungi and Higher Plants as Genetic Systems - A. Practical Hand- book" , Dept.Botariy, Univ.Nottingham,England. 13. Dons, J.J.M.(19'/5) Thesis, State University of Leiden. 14. Durand, J., Potrykus, I. and Donn, G., Z.Pflanzenphysiol. 69(1973)26-34. 15. Enzmann-Becker, G., Z.Naturforsch. 28c(1973)470-47!. 16. Evans, P.K., Keates, A.G. and Cocking, E.C., Planta 104(1972) 178-181. 17. Ferrari, T.E. and Widholm, J.M., Anal.Biochem. 56(1973)346-352. 18. Frearson, E.M., Power, J.B. and Cocking, E.C., Developm.Biol. 33(1973) 130-137. 19. Gamborg, O.L., Constabel, F.,: Fowke, L., Kao, K.N., Ohyama, K., Kartha, K. and Pelcher, L., Can.J.Genet.Cyto/,. 16(1974)737-750. 20. Gamborg, O.L. and Wetter, L.R.(Eds.) (1975) "Plant Tissue Culture Methods" Natl.Res.Council Canada, Ottawa. 21. Hayward, C. and Power, J.B., Plant Sci.Lett. 4(1975)407-410. 22. Hibi, T., Rezelman, G. and Katnmen, A.van, Virology 64(1975)308-318. 23. Jensen, R.G., Francki, R.I.B. and Zaitlin, M., Plant Physiol, 48(S97.1) 9-13. 24. Kf->, K.N., Gamborg, O.L., Michayluk, M.R., Keller, W.A. and Miller, R.A., Coll.Interyi.C.N.R.S. 212(1973)207-213. 80 25. Kassanis, B. and Wliite, R.F., J.gen.Virol. 24(1974)447-452. 26. Keller, W.A., Harvey, B., Garaborg, O.L., Miller, R.A. and Eveleigh, D.E., Nature, 226(1970)280-282. 27. Keller, W.A. andMelchers, G., Z.Naturfor>soh. 28c(1973)737-741. 28r Lazar, G\, Borbely,C~, Udvardy, J., Premecz, G. and Farkas, G.L., Plant Sat.Lett. 1(1973)53-57. 29. Linsmaier, E.M. and Skoog, F,, Physiol.Plant. 18(1965)100-127. 30. Meyer,.Y., Pvotgplasma 31(1974)363-372. : 31. Meyer, Y. and Abel, W.O., Planta 123(1975)33-40. 32. Murashige, T. and Skoog, F., PhysioLPlant. 15(1962)473-497. 33. Nagata, T. and Takebe, I., Planta 92(1970)301-308. 34. Nagata, T. and Takebe, I., Planta 99(1971)12-20. 35. Nagata, T. and Yamaki, T., Z.Pflansenphysiol. 70(1973)452-459. 36. Nickell, L.G. and Heinz, D.J.(1973) in : Genes, Enzymes arid Populations •';'' (A.M. Srb:, "Ed.) vPlenum -Press, New York,p. 109-i 28. • 37. : Power, J.B. and. Cocking, E.C.j J.Exp.Bot, 21(1970)64-70. 38. Sacristan, M.D. and Melchers, G., Mol.gen.Genet. 105(1969)317-333. 39. . Sakai.E/ and.Takebe, I., Bioahim.Biophys.Acta 224(1970)531-540. 40.. Schenk, R.U./and; Hildebranat,: A.C.,; Crop Sci. 9(1969)629-631 . 41. Schilde-Rentschler, L., Z.Naturforsch. 27c(1972)208-209. ,42. Schilde-Rentschier, L., Coll.Intern.C.N.E.S. 212(1973)479-483. 43. Schiide-RentscHler, L., La. Recherche 6(1975)430-436. 44. Schmidt, G. and Thannliauser, S.J., J.Biol.Chem. 161(1945)83. 45. Scowcroft, W.R., Davey, M.R. and Power, J.B., Plant Sci.Lett. 1(1973) 451-456, 46. Skoog, F. And Miller, CO., Symp.Snc.Exp.Biol. 11(1957) 118-11 J. 47. Smithy H.H. 0975) in : Handbook of Genetics (R.C.King, Ed.)Plenum Press, New York,vol.2,p.281-314. 48. Suzuki, H., Abe, T., tirade, M., Nisizawa, K. and Kuroda, A., J.Ferment. Tech. 45(1967)73-85. 49. Takebe, I., Otsuki, Y. and Aoki, S., Plant Cell Physiol. 9(1968)115-124. 50. T.ikebe, i., Labib, G. andMelchers, C, Naturwiss. 58(1971)318-320. 51. Watts, J.W. and King, J.M., Coll.Inter*;..C.N.R.S. 212(1973) i 19-123. 52. Watts, J.W. and King, 3M., Planta 113(1573)271-277. 53. Watts, J.W., Motoyoshi, F.and King, J.M., Annals Bot. 38(1974)667-671. 54. White, P.R. in : Plant Tissue and Cell Culture (H.E.Street, Ed.) Botanical Monographs 11(1973)39-43. Blackwell Sci.Publ., Oxford. 55. Widholm, J.M., Slain Teah. 47(1972)189-194. CHAPTER VI ATTEMPTS TO DETECT EXPRESSION OF COLE1 DNA IN TOBACCO MESOPHYLL PROTOPLASTS INTRODUCTION Bacterial plasmids represent a class of small extrachromosomal DNA's which often determine characteristic gene products, like bacteriocins and enzymes which can inactivate antibiotics. The bactericidal action of bacteriocins is quite distinct from that of other low molecular weight antibiotics. They are proteins (or lipoprotein-carbohydrate complexes) exhibiting a high degree of antibiotic specificity, generally acting through adsorption to special receptor sites. Colicins are bacteriocins produced by certain strains of Enterobacteriaceae and are active against the same or closely related members of this family. The colicinogenic plasmid E! (ColEI DNA) is a relatively small plasmid (M.W. 4.2 x 10 D ; 2) that can be isolated as a covalently closed super- coiled circular molecule in relatively large quantities (4). Colicin El, its only well characterized (15) gene product (out of a maximum of 8-10), can be detected even in very low concentrations using a biological assay (10). This DNA, joining ease of isolation and low gene content to a sensitive test for one of its gene products, would therefore seem to be a good choice for studies of the possible expression of procaryot.ic exogenous DNA in eucaryotic cells. We have attempted to detect colicin El activity in tobaccapps after their exposure to CoIE 1 DNA and have established the limits of detection. Using competition hybridization with labeled ColEl-cRNA the detection limit of ColEl specific RNA has also been determined. Recently Behki and Lurquin (Mol, Belgium) have also tried to detect colicin El activity in tissue culture material of Arabidopsis thaliana after various treatments with purified ColEl DNA; they stopped their efforts for lack of any success (pers. comm.). 82 While the typescript for this thesis way in preparation, Goebel and Schiess (8) published their partly successful efforts to effectuate the replication, transcription and Translation i,f ColEl DNA in cultured mammalian cells. They could induce uptake of CcLEl DMA only by adding DEAE-dextran to the incubation mixture; 2 h incubation of 3 x 10 cells/ml with 2 pg/ml ColEl DNA gave an up- take of 2-8% of the input. Density shift experiments indicated that after It h culture part (40-60%) of the DNA that had been taken up may have been replica- ted in the nucleus. Direct hybridization of filter bound ColEl DNA with pulse labeled RNA isolated from logarithmically growing ColEl DNA-treated hamster cells indicated that some ColEl specific RNA was synthesized during the first two generations after uptake. Continued culture of the cells resulted only in extensive degradation of the foreign DNA, with a concomitant disappearance of detectable amounts of ColEl specific RNA. The crucial experiment, namely the detection of colicin El activity in extracts of ColEl DNA-treated cells, failed. These authors have now turned to other (unnamed) plasmids for their study of the replication and expression of foreign DNA's in eucaryotic systems. MATERIALS AND METHODS Strains E.coli JC4I1 thy" (ColEl) was obtained from Dr. D.B.Clewell (3, 4) by Dr. H.L.Heijneker (=LBE 1306). This E.coli K.12 strain is auxotrophic for methionine, leucine, histidine, arginine and thymine; it harbors the ColEl plasmid, which was transferred from E.coli K-30. Wildtype E.coli K12 (LBE !000) was obtained from Ms. M.A,E.Groothuis, and was used as indicator strain for colicin El activity assay. Media LB. medium is used for colicin assays and contains (per liter in distilled water): NaCl, 8 g; Difco tryptone, 10 g; Difco yeast extract, 5 g; pH 7. The minimal medium M 9 (12) containing; (per liter in distilled water) : Na2HP04, 6 g; kri^^P^.i g; NaCl, 0.5"g; NH^Cl, 1 g; CaCl^ZH^q, 14.7 mg; MgSO,.7H9O, 246 mg; glucose, 4 g; was supplemented wi^h Casamino Acids (Difco), 10 g; thiamine, 2.5 mg; and thymine, 20 mg; pH 7. Cell growth was at 37 C and was followed by measuring turbidity at 660 nm in a Vitatron colorimeter. Materials Sources were as follows: Brij 58 (a non-ionic detergent) from Serva (15232), Heidelberg, W.Germany; sodium deoxycholate (D0C.;43035) and ethidium bromide 83 (EtBr) both from B.O.H. Chemicals, Ltd., Poole, England; CsCl (Suprapur, Art. 2039) from E.Merck, Darmstadt. W.Germany; I,2-dimethoxyethane (8082) from J.T. Baker Chomicals, Devon tor, the Netherlands; mitoiaycin-C (M-01J03) from Sigma Chcm.Coinp. , St.I.ouis, U.S.A.;UKU- H)-thyraitiine (50 '".i/mffiol, type TRK.4I8), >UC-thymine (60 mCj./mrnol , type CFA. 182) and (5- H)-cytidine-5'-triphosphate (20 Ci/aanol, type TRK.339) from The Radiochemical Centre, Amersham, England. Pps were isolated from ^urfaie sterilized leaves of Che tobacco variety White Burley usinfi the aseptic "cutting" technique. Both this technique and the subsequent aseptic culture in NT-medium have been described in chapter V. ColEl DMA, needed for addition to pps, was prepared from a 600 ml culture uf E.coli JC4tl thy (C-vlEl> grown on a gyrstory shaVer in supplemented H9 me- dium. During the logarithmic growth phase, chlorampheni.ee 1 was iidde According to optical density criteria this DNA was relatively pure: 230 nm/ 260 nm = 0.50 and 260 ran/280 nm = 1.86. Total yield was about 1 mg DNA. Ana- lysis in a CsCl-EtBr gradient (0.1 mg EtBr per ml) showed that even after 1 month storage this DNA preparation consisted for 75% of covalently closed circular ColEl DNA. 84 For hybridization purposes C-ColEl DNA was prepared from a culture of JC411 thy (ColEI), where thymine in the medium had been replaced (after ad- dition of chloramphenieol) by 2- C-thymine (1 yCi/ml). After direct CsCl-EtBr ceht'ri'fugation of the cleared lysate, EtBr was removed from the fractions 14 containing supercoiled C-ColEI DNA by five"extractions with an equal volume of 20 SSC saturated isopropanol, overnight dialysis against 20 SSC followed by 0.1 SSC. Optical density ratios were 230 nm/260 nm = 0.40 and 200 nm/280 nm = 1.84, while the specific radioactivity amounted to 43,400 dpm/pg DNA. rurifixation of aoiiein El Isolation and purification of this protein from a mitomycin-C induced cul- ture of JC41I(ColEl) were carried out exactly according to the very well des- cribed procedures of Schwartz and Helinski (15), It involves salt extraction of extracellular, bound colicin from the cell surface without lysis, followed by ammoniumsulphate fractionation (40-60%) and ion exchange chromatography. The yield we obtained in a 1/10 scale preparation, as compared to the recipe, was slightly higher. The final protein solution was exhaustively dialyzed against distilled water and lyophilized in small portions. When testing the influence of pps lysates on colicin El activity, we made stock solutions in 0.9% NaCl. Hybridisation conditions 14 The C-ColEl DNA described above was fixed in very small quantities on cellulose nitrate filters by gravity filtration in 6 SSC, after heat denatura- tion in 0.1 SSC. ColEl-cRNA (unlabeled and JH-CTP-labeled) was prepared as previously described (14) using unlabeled ColEl DNA purified by CsCl-EtBr gra- dient centrifugation as a template. Hybridization was carried out in 1 ml 2SSC for 20 h at 66 according to our standard procedure (6) modified from Gillespie and Spiegelman (7). Each hybridization reaction was done at least in duplicate. Radioactivity was measured by liquid scintillation counting (using dual label settings) after complete solubilization of the filter. Incubation of a dry filter with 2 ml dimethoxyethane for 30 min, followed by addition of 9.5 ml scintillator cocktail (toluene:Triton X-100 23:7 (\ /) containing 0.4% 2,5-diphenyloxazole) and 0.5 ml water yields a completely clear and homogene- ous liquid with a very reproducible counting efficiency (20). The tobacco leafUNA used as an artificial pps-KNA "background" in the competition-hybridization experiments has been isolated by a method developed 85 for the isolation of high quality RNA from pps. It essentially consists of : (1> lysis with 0.52 SDS in thy RNA-lysis buffer of van Sittert (16), with ad- dition of Proteinase K (instead of phenol) to a final concentration of 0.45 ra^'tTil (,18) and 1 h incubation at room temperature; (2) removal of fine debris by low speed centrifugation and precipitation of high M.W. RNA with an equal volume of 4 M LiCl (I) for 20 h at 2 ; (3) dissolution of the pelleted pre- cipitate in O.I SSC and one (protein and color removing) extraction with an equal volume of Kirby-pheno1; (4) quick recovery of RNA at high purity by Sep'aadex G-75 chromatography in 0.1 SSC. The advantages of this method in- duce : speed, high yield and very good spectral purity; a disadvantage may be t'-.e rapid drop in the efficiency of LiCl precipi tat I- at concentrations of RNA in the lysate lower than 10 ug/ml. RESULTS AND DISCUSSION ry-.-yin F.I assay Purified colicin El protein wao used to test the biological assay (10) for use with pps lysates. This assay ior "drop test") is carried out by serial dilution of the solution to be tested in LB medium, and spotting a drop of each dilution on a LB agar (1.8%) plate freshly seeded with 10 indicator bacteria in 5 ml of soft LB agar (0.7%). The plates with only "bottom" agar can be stored at 4 after I h drying at 37 . After pouring the "top" agar (over the prewarmed bottom layer) the indicator plates can be used after 1 h drying at room temperature. The number of colicin-activity-units per ml is defined as the highest dilution which gives a clear zone of inhibition of growth of the indicator bacteria. In contrast to the specific activity (Units/mg protein) of purified colicin 5 • • -• -...-,..• El reported by Schwarz and Helinski (15): 1.5 x 10U/rag, that ofour prepara- tion was only-"about 0.12 x 10 U/mg, when first tested. Since strain YS40 (15) was not available, we used wild type E.coli K12 as indicator strain^ which could be less sensitive to colicin El. Alternatively our protein may have been more inactivated daring preparation than in the reported case. We would certainly not want to dilute the pps lysate eventually containing colicin El activity. Testing our colicin El we could detect 4 ng, using instead of just a drop a standard quantity of 0.010 ml delivered from an alcohol steri- lized Eppendorf microtip. Instead of attempting to use protein extracts or any other purification step, we considered that the highest possible concentration of eventual colicin El 86 would be attained by direct lysis of dense pps suspensions. We avoided the use of detergent and used osmotic shock (by suspending the pps pellet in a small volume of 0.9% NaCl) and ultrasonic treatment (MSE sonifier with microtip, at 20,000 Hz for 20 see). Pure colicin El is not inactivated by these manipula- tions. In the presence and absence of fixed amounts of colicin El, pps suspensions of increasing density were thus lysed and tested for their influence, on in- dicator strain growth. We went up to 40,000 lysed pps in a 0.010 ml spot and, without added colicin El, could not detect any inhibition of growth. The green spot of ppf. debris was just barely visible in the thick lawn of bacterial co- lonies. At this pps density, added colicin El activity was not influenced down to 40 ng; 20 ng could not any more completely inhibit bacterial growth. Com- pared with the pure coliciu Ei preparation, it would thus seem that activity is abolished by interaction with the pps lysate, possibly by the action of proteolytic enzymes or adsorption to the particulate pps debris. We tested this by spinning this debris down (5 min, 250 x g) and testing the supernatant clear lysate for activity in comparison with the original lysate. With 90 and 45 ng colicin El in the original lysate spot very good clear inhibition zones were obtained; after removal of particulate debris (from 4000 pps/0.010 ml) the 90 ng test was still positive, but the 45 ng one not. Also when 90 ng colicin El was lysed with 40,000 pps/0.010 ml and the debris spun down, the clear lysate showed barely any activity. When testing pps lycates for the possible presence of colicin El activity, after treating the pps with ColEl DNA, one may thus expect a lower detection limit of 40 ng, when debris is not spun down and when 0.010 ml is spotted on a lawn of 10 E.coli K12 bacteria in 5 ml LB soft agar. Attempts to detect expression of oolicin El -in pps Incubation of freshly isolated tobacco mesophyll pps with ColElDNA was carried out under the following conditions. Fps were aseptirally cultured at a density of 5 x 10 per ml in 2 ml NT medium (chapter V), as a thin layer in 50 mm diameter glass Petri dishes. ColEl DNA (in mannitol and consisting for 75% of intact circular DNA) was added to a final concentration of 10 pg/ml. The mixture was incubated for a total of six days. This long culture period was deemed necessary in order to assure, that those living cells that had taken up some biologically active DNA had the chance to synthesize some colicin El and could possibly devide and give more colicin El synthesizing cells, AS 87 bLancs dishes wilh pps only or UNA only were used. This hist mixture remained sterile and (by testing directly 0,010 ml) did not contain colicin EI activity. Tiu> pps were lysed and assayed as described above, but no activity above the de tec t i on limit I'ouhi be found. This kind of experiment lias been discontinued in view of the following two considerations : (I1") The detection limir. of our assay lies around 40 ng colicin El in lys.ite from 40,000 pps; since tho M.W. of colicin El is approximately 60,000 D, this means that • '• i ••': rp;~ should produce some 10 colicin El molecules; in highly successful infections of tobacco mesophyll pps with e.g. tobacco mosaic virus the average yield of progeny virus per pps was calculated to be 10 (17); i.e. in a naturally occurring plant disease this is the number of particles with which the iiick cells are finally packed. It is extremely un- like iy that the intricate and efficient system, with which plant viruses are able to force the metabolism of their host cells to produce new virus particles, can be imitated to any extent by purified ColEl DNA. it is also very unrealis- tic to presume that some biologically active ColEI DNA will end up in all of I'm; pps. Estimatitng the number of produced colicin El molecules per success- fully "infected" pps at 10 and taking the "infection" efficiency not to exceed 1%, the colicin El assay should be at least about 10 times more sensitive than the one used here before more of this kind of experiments would be justified. (2") While this work was in progress Schiess and Goebel (13) showed that even in E.coli the translation of ColEl specific mRNA into colicin El is a rather complicated process. According to these authors it probably requires a chro- mosomal (E.coli) factor. Trans'acion of such mRNA in eucaryotic cells would thus seem rather far-fetched. Corrrpetitiovi-hijb'cidizalion in the detection of ColEl specific RNA The examination of exogenous DNA expression at the transcription level is most difficult because of the very small amount of specific mRNA present in "infected" cells as compared to the various host cell RNA species. In order to be able to determine the eventual transcription of ColEl DNA after uptake by pps, we decided to test the use of competition-hybridization. This technique has proved to be very useful in the study of e.g. SV40 DNA transcription in transformed animal cells (11). It would eliminate the in vivo labeling of RNA (very inefficient in tobacco leaf cells) for each batch of DNA-treated pps and the subtraction of variable amounts of non-specifically bound counts, both needed in direct-hybridization studies. 88 RNA/DNA ratio 0 2 U ColE Fig.VI.l. Saturation of ColEl DNA (0.246 vg per filter) with ColEl H-cRNA (spec.act. 425,670 cpm/ug). Experiments were carried out to explore the nature of the reaction between ColEi H-cRNA and its homologous DNA. The results of increasing the concentra- tion ot'3H-cRNA (spec.act. 425,670 cpm/yg) in the 1 ml hybridization solution are illustrated in. fig.Vl.1. Since 0.246 yg ColEl DNA was baked onto the fil- ters inythese^experimentsy the expected radioactivity in the hybrid under con- ditions of saturatioh would be 52^350 cpm. This value is very closely approached by a 3H-cRNA concentration of 1,643 yg/ml. This result indicates that the RNA polymerase prepared in our group is capable of complete transcription of the DNA template. Conversion of the experimental values into a double-reciprocal plot results in points to which a straight line can be fitted. By extrapolation of this line to the ordinate, it has been assumed that the amount of hybrid formed at in- finite RNA concentration could be estimated. In our case this would give a 89 value of about 58,810 cpm. Comparison with our calculated value (not always possible in other syst.-.ns), reveals a net overestimation by the graphical method. The cause of this overestimation has been described by Young and Paul (19). In siu-fot viing competition experiments a ratio of H-cRNA/DNA of about 8 was considered adequate for saturation of the DNA. The fact that we could readily achieve near complete saturation of ColEl DNA with its cRNA was not compU-tely expected. While setting up the experiments we came across the report of Haa.s ,•;. ;.'. (9), in which the authors give sugges- tive evidence for the loss of complete hybrids from nitrocellulose filters when SV40 DNA is hybridized with on excess of its cRNA, At cRNA/DNA ratios of 5 - 10 the percentage of hybridization could only be made to fit the correct satu- ration curve, when the amount oi DNA lost from the filter was taken to be only the transcribed DNA strand and the values normalised accordingly. Although we did observe a small, normal loss of DNA, also occurring when incubating DNA filters without cRNA, we could correct for it in the normal way and obtain sa- tisfactory results at near saturation conditions. The effect observed by Haas 3 ' •'•'•' were mixed with a standard (saturating) amount of H-cRNA (350 ng), and hy- bridized together. The specific activity of the H-cRNA is thus lowered in proportion to the amount of unlabeled cRNA added. The radioactivity of the hybrid will be reduced in a predictable manner and permits the construction of a theoretical competition curve for comparison with actual experimental re- sults. Fig.VI.2. illustrates such an experiment, in a range where the theore- tical curve (drawn line) is nearly a straight line. Even at these very small dilutions of the H-cRNA the experimental values correspond closely to the 90 100-i Fig.VI,2. c o Competition of unlabeled ColEl cRNA D N in the hybridization of ColEl H-cRNA 14 with ColEl C DNA, in the presence 90H of 1^0 ug tobacco RNA, The drawn line c is the theoretical comretition curve. o u 01 o 0 0.1 . 0.2 competing cRNA/3H-cRNA calculated competition-curve. In terms of the detection of ColEl specific RNA among lf.rge quantities of total pps-RNA these results indicate that about 10 ng can be easily detected. Some improvement is possible by using cRNA of higher specific activity : e.g. with cRNA of 20 x 10 cpm/ug about 10 pg competing RNA would be detectable. About 3 x 10 pps can be handled per experiment yielding ca.150 yg of plant RNA. Detection of 10 pg of ColEl specific RNA would represent an average pro- duction of one such RNA molecule per pps. If the "infection" efficiency is 1%, this means that each successful infection must result in 100 ColEl specific RNA molecules. This seems to be a real possibility. But we have to be sure that enough DNA for such an RNA production enters metabolically active pps in a bio- logically active form. We therefore turned first to optimalizing the conditions for uptake of DNA in a "useful" form by tobacco mesophyll pps. 91 REFERENCES 1. Baltimore, P.. -'J.', .'.-. ;. 18(1966)421-428. 2. Baaaral, H. and Helinski, D.R., J .Mi'l.l'iot. 36(1.968)185-194-. 3. Clark, A..1. and fiargulies, A.I)., Pfa.uatI.A&IJ.SIM'-JJ.H.A. 53(1965) 451-450. 4. Clewell, D. B. , .r. >.--?r,: .>';',:/. I IO(I972)b67-67b. 5. Clewell, D.B. aad Helinski, D.R., FvocJiull..Aaad.S-•;'. :/.£,*. 62(1969) 1159-1166. 6. Dons, J.J.M.(1975) fhezis, State University of Leiden. 7. (iillespie, 0. and Spiegelman, 5., ,'.Mcl.Hicl. 12(1965)829-842. 8. Goebei, W. and Schiess, W., V, •; .;,•«.- ••>:. r;e-? >;.,;: t -. I 38( 1 975 )2 13-223, 9. Haas, H., Vogt, M. and Dulbeeeo, R., !Jv-.v,?.:\'at l./u-ad.Sci.U.S.A, 69(1972) 2160-2164. 10. Hershtnan, H.R. and Heiinski, D.R., J.Biol.Chem. 242(1967)5360-5368. 11. Martin, M.A. andAxelrod, D., Pvoc-.Hall.Aead.Sai.U.S.A. 64(1969) 120^-1210. 12. Miller, J.H.(1972) Experiments in Molecular Genetics, C.S.H.Lab., Cold Spring Harbor (N.Y.),p.431. 13. Schiess, W. and Goebei, M., FEBS Lett. 47(1974)356-359. 14. Schilperoort, R.A., Sittert, N.J.van, and Schell, J., Ear.J.Biochem, 330973)1-7. 15. Schwarz, S.A. and Helinski, D.R., J.Biol.Chem. 246(1971)6318-6327. 16. Sittert, N.J.van,(1972) Thesis, State University of Leiden. 17. Takebe, I. and Otsuki, Y., Proc.Nctl.Acad.Sci.U.S.A. 64(1969)843-848. 18. Wiegers, U. and Hilz, H., FEBS Lett. 23(1972)77-82. !. 19. Young, B.D. and Paul, J., Bioahem.J. 135(1973)573-576. 20. Zandvliet, G,M.(1974) Thesis, State University of Utrecht. 92 CHAPTER VII INTERACTION OF E. COLI DNA WITH TOBACCO MESOPHYLL PROTOPLASTS INTRODUCTION Although the use of pps as acceptor cells in transformation' studies has been widely advocated (27), studies on the interaction of D'TA with pps are very limited (17, 10, II) and none of them is at the moment very successful. Having established the length of time during which tobacco mesophyll pps do not synthesize DNA following their isolation (chapter V), it is important to know the extent of DNA uptake just before the ov.set of DNA synthesis (and possible integration) and to find optimal conditions for this uptake. We there- fore studied association of E.coli DSA with tobacco pps in order to establish whether Ohyama's results with pps from fast dividing suspension cultured cells also hold for pps from non-dividing mesophyll cells (first part of results). From our experiments with suspension cultured tobacco cells (chapter IV) we learned to be. very careful about the interpretation of "uptake" results *. adsorption phenomena play a very important role and may dp so also at the plasmalemma of naked pps.We therefore looked for a system enabling us to measure the amount of DNA "functionally" taken up into the pps in the presence of otftet?DNA, inerely sticking to the piasrnalemma. To this end one could consider the use of DNA of a bacterium with a well- tested transformation system, such as Bacillus subtilis, reisolate DNA from the treated pps and score for transformation activity, Although one would thus be able to distinguish between "useful" pieces of DNA and degradation products too. small to carry any intact genetic information, the problem of adsorption of DNA would not be solved however. In our opinion a better prospect for the solution of our dilemma (nc way o£ detecting transcription of the entered DNA and no clear cut procedure for washing off adhering DNA) might be expected from the use of a donor DNA with radiation damage. Establishing a certain amount of repair would indicate that some DNA has been taken up into the pps nucleus (or mitochondria and chloro- plasts). We used E.coli DNA with a large number of thymine containing pyrimi- dine. dim^rs, and tested whether the loss of dimers from DNA recovered from treated pps could give an indication for "real" uptake (second part of re- sults)-.. " ' ;";"-v"-' •; "••• '• :;"':;- •'•-.'• ' •''-.'; . •-'••' • "'•". ' " •" ••'. ." ••••;' ";:. ' •:."• :••••- '• ' :'^:* •..• • "•'" ''•"••• .•.'" ' 93 For the dimer assay r.o bc> useful at all a number of criteria, commented below, have to be nu't : tl) isolated tobacco raesophyll pps must ,,uisess functional enzymes capa'ile of eliminating dimers from DNA (by either dark repair or phor oreac t iv.J- t i on or Sot h ), i, 2) these en? vines must be active also on exogenous, foreign DNA, (j)) they must not become "saturated" with high amounts of exogenous DNA as corerared to their own DNA, and (•i) the amount, oi background DNA (adhering to the outside of the pps or taken up by aying pps.) should not exceed 951. FnoLoreactivation of UV-irradiated culmred tobacco cells (var. Xanthi) has been shown by Trosko and Mansour (28) to occur both at the level of growth inhibition as we 1 I as monomer 12ation of thymine-containing dimers in the DMA, The same authors stress the fact that, although photoreversal of many UV- inductd biological damages h,-JS Deen observed in hLgher pLants, they could not find evidence for excision repair. A number of other reports also seemed to indicate an absence of excision repair activity in higher plant cells. Very recently however Rowland (12) taking full advantage of the protoplast-systern, has shown that in pps isolated from cultured wild carrot cells pyrirnidine dimers can be excised in the dark. He also found that the extent of dimer excision de- pends on the initial number of dimers induced by the UV-dose. Previous failures to detect excision may thus be explained by too high UV-doses and their seconda- ry effects. We have assumed that Howland's results also hold for tobacco me- sophyll pps and have indeed obtained evidence for excision repair to occur in those pps. Concerning the second criterium it is Known that at least in mammalian cells UV-irradiated DNA from infecting virus can be repaired both by excision and: by photoreaetivation (see e.g. 30). In at least one series of experiments we have shown that exogenous bacterial DNA may be repaired in tobacco mesephyll pps. The importance of the third criterium is rather difficult to estimate. A large amount of exogenous irradiated DNA may be too much for the host cell's repair systems to cope with in a reasonable time period. Consequently the rate of repair of the incoming DNA may be so low that in the incubation period the relative loss of dimers is not detectable. No reliable measurement has as yet been made of the rate of repair in plant pps, but a very rough minimal deter- mination (12) suggests that it is comparable with rates reported for mammalian cells. It is probably sufficiently high to take care of the amount of dimers 94 introduced into the pps in our experiments. Tht' fourth criteriura is dictated by the accuracy of our dimer determina- tion, and might possibly be improved by the use oi two-dimensional chromato- graphy and even more by the preparation of a cleaned DNA extract before formic at' i d hydro lys i s . MATERIALS AND METHODS I Belation of protoplasts All experiments reported in this chapter have been performed with mesophyll pps of the tobacco variety White Burley, using the stripping technique des- cribed in chapter V. Since it was not our purpose to cultivate the pps for an extended period of time, the "antibiotics variant" was used. In order to re- cover from the isolation trauma, the pps were incubated overnight (about 16 hours) at a density of 2.10 per ml. Isolation of labeled E.aoli UNA An E.coli C culture (strain KMBL 2901, obtained from Dr.H.L.Heijneker) in Vogel-Bonner medium (29) supplemented with 0.1% yeast extract (Difco) was labeled during the logarithmic growth phase by continuous addition of (methyl- s' thymidine. After the initial lag period a 100 ml culture in a shaking (200 rpm) waterbath (37 ) was connected via a peristaltic pump to a vessel with 10 ml of additional medium containing 2 mCi tritiated thymidine, which was then slowly pumped into the shaking culture over a period of 1 to 1.5 generation times. This way ot adding the radioactive DNA-precursor resulted in a utilization of around 10% of added radioactivity. After chilling in ice, the cells were spun down at 5° in a Christ Zeta 20 centrifuge (heavy walled poly- carbonate tubes) at 9000 rpm.for 10 min. The subsequent DHA isolation procedure is a combination of the "cleared- lysate" method introduced by Clewell and Helinski (5) and methods in use for A.turn. DNA isolation (22), minimizing the duration of the isolation. The bac- terial pellet in the centrifuge tube was resuspended, in 12.5 ml of ice cold 25% (w/v) sucrose in 0.05 M Tris/HCl pH 8.0. Spheroplasts were produced (on ice) and gently lysed by successive addition (5 min.intervals) of ice cold ; 1) 1.75 ml freshly made lysozyme solution (10 mg/ml in 0.25 M Tris/HCl pH 8.0), 2) 6.50 ml 0.25 M. EDTA pH 8.0 and 3) 13.50'ml 2% (v/v) Triton-X-100 in 0.05 M Tris/HCl, 0.0625 M EDTA pH 8.0. 95 i'he very viscous lysate is left on ice for 30 nun with occasional very gentle mixing to lu'i\nsi« completely transparant. The high tnoleeu'fir weight DNA is then cleared frora the ly. -ite by centrifugation at 4 and 40,000 rptn for two hours us i n>i .i hi' Ti t .'tor and Bookman Spinco 1. 50 ultracontrifuge. I\\M non-viscous supernatant, is completely removed I torn the pellets, which are. then collected in a 100 ml Krlenmeijer flask with 12 ml O.i M NaCl, 0.1 M EIWA pi1; 8.5. After addition of 1 .2 ml I H Tris/HCl pH 9.0 containing 10.% (w/v) SDS and self digested Pronase E to a final cone, of 0,5 mg/tnl, the mixture is incubated overnight at 37° with mild shaking (100 rpm). KNAase stock solution (2 mu type A and 600 units type Tl per ml distilled water adjusted to pH 5.0 and held at !0Gv" for 10 min. to inactivate residual DNAase) is added to a final cone, of 0.1 mg/ml A and 30 units/ml Tl and incubation continued for one hour. The UNA is then sheared to a M.W. of approximately 20 x 10 D, by blending the solution in a Sorvall Omnimixer (50 ml stainless steel vessel) for 1 min. at position 4. After two phenol extractions the DNA is precipitated with ice cold 9bZ ethanol and spooled in one manipulation from the solution using the closed tip of a Pasteur pipette as a small glass rod. After evapo- ration of adhering ethanol, the DNA is dissolved in a small volume of sterile 0.1 SSC and extensively dialyzed against this buffer. The DNA soLution is stored at 4 in the presence of several drops of chloroform. The specific radio- activity of the DNA was variable, the maximum value obtained being 5.39 x 10 dpm/ug DNA. UV-irradiation of E.aoli DM ; Treatment of DNA with UV light (220-320 ran) results among others in the production of cyclobutyl pyrimidine dimers between adjacent pyrimidines of jne DNA strand (2). In preliminary experiments UV-irradiation was carried out with the DNA so- lution (20 ijg/ml) as a thin layer in a small glass tray under a Philips TUV 30 W tube. With this type of germidical lamp (emitting at least 90% of its _2 energy at 253.7 run) at the closest workable distance a dose rate of 52 erg.mm sec. could be obtained. Due to reconversion of the photoproduct at this wave length the amount of thymine containing pyrimidine dimers ( X()T/T% ) reached a maximum value of around 6% at doses exceeding 40,000 erg.nan . This is in agreement with data in the literature obtained using similar conditions (19, 25). Since the detection of repair of exogenous DNA against a substantial background of adsorbed DNA not entering the pps would benefit from as high an initial dimer content as possible, we decided to try another light source. 96 A high-pressure mercury lamp (HBO 1000 W, Osram) with forced air cooling was kindly provided by the section Photochemistry of the Dept. of Organic Chemistry of this university. In an effort to minimize reconversion of the desired photoproduct a cut-off filter solution was selected, reducing trans- mission of wavelengths below 263 run to /.ero while maintaining transmission of 270 nm at 60Z (10 ran filter depth). This filter solution contains 400 mg NaBr.2H 0 and i.2 mg Ag?SO per ml in water, No.4 page 17 of the thesis of Rappoldt (20). In view of its instability the absorption spectrum should :»e checked frequently. Preferably, a fresh filter is prepared for each irradia- tion, using stock solutions of the two salts. Both the DNA- and the filter- solution are kept in quartz cuvettes (10 ram depth) in a cuvette holder, the light source being focussed so as to give maximum intensity in the DNA cu- vette. Water-saturated nitrogen was bubbled through the DNA solution during irradiation. This gives sufficient mixing for the irradiation of 3 ml samples at DNA-concentrations ranging from 50 to 150 tig/ml. An X()T/T% of 17 to 18 could thus be attained after 3 hours of irradiation. Extrapolating from data in the literature (23, 18) this would mean that a total dose of approximately 5 -2 1.5 x 10 erg.mm has been applied. Kahn (13) recently published a method for the selective production of thymine-thymine dimers in DNA using long UV wavelengths and sensitization with silver nitrate. Such DNA could be useful in our experiments, since it would speed up the dimer assay. General incubation, washing and lysis conditions Protoplasts were generally used after overnight incubation in order to allow them to recover from the traumatic isolation treatment. The pps density was first increased to more than 2 x 10' per ml by centrifugation at 50 x g for 3 min and removal of excess medium. It has been claimed that this treat- ment just prior to nucleic acid addition enhances uptake (31). A desired •a quantity of E.coli H-DNA (in 0.1 SSC at a cone, of around 100 to 150 yg/ml) was then well mixed with the remaining supernatant medium avoiding osmotic shock to the pps. Following uniform suspension of the pps-pellet by gentle swirling and tilting of the tube, they were dispensed as 1 ml aliquots in sterile glass vessels (27 mm diameter counting vials stoppered with compressed paper plugs). When working with irradiated DNA all manipulations from the addition of DNA until lysis and freezing of the pps were performed under non-photoreactivating conditions (dark room with yellow light only; Philips TL color 16), except when photoreactivation was part of the pps-treatment. Incubation of the suspensions 97 was under tissue culture conditions: 28*" and 60% relative humidity, under 2000 lux from either yellow tubes or from Philips color 33 cool-white fluorescent light tubes. After tfi/mination nf incubation the pps were centrifuged at 50 x g for 3 min. The medium was removed by careful aspiration with a Pasteur pipette and the pps gently resuspended in first 1 ml of 0.7 M mannitol pH 5.7 and then the volume was adjusted to total of 5 ml. Care was taken that no" clumps re- mained. After removal of a total of three 5 ml washes, a DNAa.se treatment was applied, if necessary as indicated in the text: 5 min at 37 in a total volume of approximately I ml 0.7 M mannitol including 0.1 ml DNAa.se stock solution (1 ir.g/ml, in 0.7 M mannitol containing 0.03 M MgCl?). After addition of 4 ml 0.7 M mannitol and centrifugation, followed by one more 5 mi-wash (5th) the carefully drained pps-pellet was taken up in 0.9 ml water and quickly frozen. Thawing and 30 sec mixing on a Vortex mixer with 0.1 ml 10%(w/v) SDS in water completes pps-lysis. When the lysate has to be assayed for dimer content, an equal volume of 10% (w/v) ice-cold TCA is added. A precipitate is allowed to form at 4 for 20 min and is then collected by centrifugation at 2300 rpm at 4 (using an MSE Super Minor centrifuge). Dimer assay ( Xf)T/T %) This assay is carried out on ice cold 5% TCA-precipitable material according to the addaptation of van Sluis (24) of methods described by Carrier and Set- low(4). Hydrolysis The TCA precipitate is dissolved in 2 ml distilled water by 30 sec shaking on a vertex mixer. With a long size (230 mm) Pasteur pipette the material is quantitatively transferred to special hydrolysis tubes and lyophilized over- night, thus also removing the residual TCA. The dry material is suspended in 0.3 ml of 98% formic acid. Hydrolysis is performed by heating for 60 min at 175 in a temperature controlled solicone oil bath, equipped with a heavy, metal tube holder in view of explosion risks and placed under a hood. Due to much pressure built up during formic acid hydrolysis great care has to be taken during this step: 1) 180 mm long, thick walled (1.4 mm) Pyrex tu- bes, weighing appr.15 g and having a preformed constriction at 100 mm from the bottom, are used; 2) after deep-freezing in liquid nitrogen, the tube is eva- cuated and slowly heated at the top of the constriction with a gas-oxygen 98 torch until the glass becomes j.«st soft enough so as co seal the vial by the transformation of the constriction into a glass rod, without any pulling ; local overheating and pulling at the soft glass under evacuation may result in explosion of the vial upon heating at 175 ; 3) after hydrolysis, cooling to room temperature and deep-freezing, the vials are opened by scratching with a glass knife about halfway the vial and then hand breaking it in a con- trolled fashion: both ends in pieces of thick plastic tubing and this again wrapped in thick cloth; this procedure is preferred to touching the stratch with an overheated piece of glass. Formic acid is removed from the dark brown hydrolysate by heating to about 60 under vacuum in a flash evaporator. The dry material is then dissolved in 0.05 ml of 0.01 N HC1 containing 0.05 mg thymine as marker. Paper chromatography Formic acid hydrolysis of UV-irradiated DNA results in the release of free bases and acid-stable photoproducts. The cyclobutylpyrimidine dimers found are thymine-thymine, uracil-thymine, and uracil-uracil, the uracil coming from deamination of cytosine in cytosine containing dimers. Separation of thymine from thymine-containing dimers is achieved by paper chromatography using Whatman No. 1 paper strips(which should be kept away from moisture during storage). With the aid of a 0.05 ml capillary pipette and an air blower the material is applied in successive portions of 0.01 ml as a narrow streak of 20 mm on strips 30 mm wide and 500 mm long. The strips are developed for 16 hours by descending chromatography in the solvent of Smith (26) : n-butanol / acetic acid / water (v/v)80 /12 /30. The solvent was fresh- ly prepared for each run. Radioactivity assay On dried chromStograms the position of thymine is Iocat3d under IJV-light and the region from origin to just beyond thymine is cut into strips, 15 mm wide (x 30 inta) and usually 18 in number. Each strip,. cut into two squares, is placed in low potassium glass counting vials and the radioactive material eluted with 1 ml of water by shaking on a reciprocal shaker for 1 hour. After addition of 10 ml of scintillatqr cocktail (toluene-Trotin X-100 2:l(v/v), containing 6.4% 2,5-diphenyloxazole), shaking and cooling in the dark to 5 , the vials are shaken again for 15 sec on a vortex mixer in order to make the paper squares completely transparent and the liquid homogeneous. Radioactivity is assayed using a Nuclear Chicago Mark I type liquid scintillation counter, Counting efficiency is checked in each experiment using the external standard 99 channels ratio method and was found to be fairly constant: 15.5?. +_ 0.52. Ditner content is calculated by expressing the counts in the dimer region (approxima- tely R- 0.18 to 0.40, using th (Me-'H)-chymidine, specific radioactivity 50 Ci/mmol, type TRK.418, was obtained from The Radiochemical Centre (Amersham, England). Lysozytne (prod, no. L-6876) and poly-L-lysIne type V1I-B (approximate M.W. "10,000; No,P-2636) were both from Sigma Chem.Comp. (St.Louis, U.S.A.). Pronase F. (art.7433) and pancreatic DNAase, electrophoretical!y purified (art.2500!) came from E. Merck, Darmstadt, W.Germany. Trion X-100 (5548h - purified - Rohm & Haas) came from Koch-Light Lab.,Ltd..Colnbrook, England. SDS (sodiumdodecylsulphate; 20760) from Serva, Heidelberg, W.Germany, Pancreatic RNAase from Nutritional Biochem. Corp., Cleveland, U.S.A., Tl RNAase from Worthington Biochem.Corp., Freehold (N.J.), U.S.A. RESULTS AND DISCUSSION Conditions fov the association of DNA with pps The association, in a TCA-precipitable form, of non-irradiated E.coli H-DNA with tobacco mesophyll pps has been briefly studied in order to establish some starting conditions on which should be improved later on using functional up- take as criterium. General conditions are as described in Materials and Methods except when stated. TCA-precipitable material from the pps lysates is collected on What- man glass fiber filters (GF/C) and thoroughly washed with three times 10 ml of cold 5% (w/v) TCA. Residual green color is removed with one or two washes with a(v/v) 12:5:3 mixture of methanol, chloroform and water (7). Radioactivity is determined after solubilization with Soluene (chapter V). Pps density In a first -trial we used a DNA concentration about three times higher than is usual in pps infection with viral RNA in order to be able to detect any DNA association at all using radioactivity assay. For the same reason we refrained from the use of DNAase during washing of the pps. Increasing the pps density (fig.VII,!) a^so above that used with infection by virus or viral RNA (1 to 2 100 PPS density (per ml x10~B) Fig.VII.l. Effect of protoplast density on DNA association. E.coli 3H-DNA (5.6 ug/ml, 10.9 x 106 dpm) incubated 4 hours with pps in 1.4 ml; no DNAase treatment. x 10 per ml), we found a linear increase in DNA association up to densities which can just be handled practically. For all other experiments we used den- sities around 2 x 10 pps/ml. Without a DNAase treatment the association of DNA proved thus to be quite efficient. Also in comparison with the data of 14 Ohyama (17), obtained with E.coli C-DNA fed to pps isolated from suspension cultured Ammz visnaga cells, these levels of association seemed rather high. In later tests, using dimer containing DNA, the measurementvof realuptake could be made impossible if a too high level:of "background" DNAwould in some way be sticking to the outside of the pps. We therefore turned reluctant- ly to the use of DNAase, which - even during the: short period of its applica- tion - could also be taken up'by; the pps. State of associated DNA To get an impression of what kind of DNA is being washed off and enzymati- cally (5 min DNAase at 0.1 mg/ml and 37°) removed from the pps, we determined the TCA-precipitability of the material in the washing solutions, both after a normal 4 h incubation and after incubating overnight (18 h ; 2 x 10 pps mixed with 12 yg DNA). The results, shown in fig.VII,2, lead us to conclude that after the second wash the interstitial liquid in the pps-pellet has been cleared of material initially in the medium. The material in the third wash 101 generally is more than 904 TCA-precipitable and may already represent DNA being desorbed from the pps. After the DNAase treatment and wash,only one mor* wash is sufficient to obtain, in thi.- lysed pps-pellet, again ran re than '/u.; precipitabi 1 i ty. An 18 h incubation of the pps results i... ; . umplei.e break- down ot the DNA in the medium. The sateri.-U associated with the pps, however, is apparently protected from breakdown during incubation. Extensive breakdown oi DNA in the medium has recently also been reported (14) in studies on possi- ble DNA uptake usinfi Ch: ?>;;,• :m-.-K ;r r<-: > h:rf: mutant cells Unking the coll wall (i.e. alg.il pps). The intaclness ot the DNA associated with our tobacco mesuphy1 ! pps inrins a grea> contrast to Ohyama's results, where TCA-precipitable uptake was only 20% of total uptake. It is possible that pps isolated from actively dividing cells attack exogenous DNA more than not (yet) dividing mesophyll pps. DNA- association measured after DNAase treatment is generally 30-50Z lower than without it, but it still remains doubtful whether all DNA adsorbed to the out- 4 h 18h 80- -80 40- -40 0 0 M123D5P M123D5P Fig.VII,2. Percentage TCA-precipitable DNA in (numbered) washing solutions and lysed pps pellets. (2 x 10 pps mixed with 12 \ig DNA; M = incu- bation medium; D = DNAase wash; P = lysed pps-pellet) 102 Incubation time ( h) Fig.VII,3. Time course of DNA association with tobacco mesophyll protoplasts. E.coli 3H-DNA (10 pg, 2.94 x 106 dpm) mimixe: d with 2 x 106 pps in 1 ml; washing included DNAase treatment. side of the plasmelemma has really been removed. DNA adsorbed in a way com- parable to that occurring in DEAE-dextran-DNA complexes may be refractive to any DNAase treatment. The same may hold for DNA molecules sticking to mem- branes inside the cytoplasm: these also are most likely lost for functioning inside the ceil. Time course Looking at the time course of DNA association, we kept the DNA concentra- tion at 10 vg/ml and included DNAase treatment afterwards. From the results shown in fig.VII,3, we concluded that for practical purposes a 4 h incubation would be quite sufficient. One should keep in mind however that there is a great variability in the association of DNA with pps coming from different isolations. In other experiments e.g. DNA association was already at a maxi- mum after 1 h incubation and could not be distinguished from that after 2 or 4 h incubation. Moreover the so-called zero time samples (precooled before DNA addition and immediately put in ice again and then quickly washed in the cold taking a minimum of 35 min) do also contain associated DNA. Comparison 103 with the situation of pps infection with viral RNA, where infection takes only some minutes and is not significantly decreased by cooling to 0 (31), shows that those results with DNA should not be unexpected, even though no polyca- tions vwre used as is customary iu virus infection of pps. DNA concentration In the same experiment four remaining samples of the batch of pps were incubated for 4 hours with DNA at concentrations of 5, 15, 20 and 30 yg/ml. Association was practically linear vith concentration up to 20 ug/ml: Q.26, 0,57 and 0.73% of input, but the result with 30 ug/ml was identical to that with 20 ug/ml: 0.74%. It is not possible to draw any general conclusions from so few data. Concentrations much higher than 30 jJg/ml would noc seem to be very useful. 0hyama(17) found that DNA concentrations above 10 ug/ml did not increase association any more. Other considerations for not using too high DNA concentrations, were the possible toxic effects and the impracticality of ad- ding such large quantities of labeled DNA to very thick suspensions of pps. Incubation in 1 ml of 20 ug DNA having a M.W. oe about 20 x 10 D in the presence of 2 x 10 pps means that about 3 x 10 molecules of bacterial DNA are made available to each protoplast. This input level is of the same order of magnitude as in infection experiments of pps with viral RNA (21). Taking the value of 0.4% of input as an estimate of DNA really taken up into the cells, incubation of 20 v»g DNA with 2 x 10 equally absorbing pps (=approx. 22 iig plant DNA) would mean that each host cell is confronted with exogenous DNA equivalent to 0.36% of its own genome. Again this would seem to be quite reasonable. It remains to be seen however if indeed this DNA has really pene- trated into the cells. Effect of poiy-L-lysine on DNA association Polycations are well known to enhance the infectivity of viral nucleic acids with cultured animal cells and are almost always necessary 104 DEAE-dextran is extremely toxic to plant pps and actually lyses them at concentrations around ! ug/ml (9). Watts (31) has advocated the use of poly- L-lysine rather than poly-L-atnithine because it has a much lower toxicity and can be metabolized readily by the pps. Low toxicity is especially impor- tant wheu culture of the pps should eventually lead to callus formation afcer introduction of nucleic acids. We. therefore looked for DNA association with tobacco pps under conditions highly favourable for infection with virus RNA, Before mixing with the pps, DNA (at 70 yg/ml) was pre-incubated with poly-L-lysine (7 ug/ml) in 0.7 M mannitol buffered with 0.01 M sodium citrate at pH 5.2 and room temperature. After 4 hours incubation under standard conditions the pps were washed and DNAase treated. The results are summarized in table VII,I. Degradation of the DNA has indeed diminished, while association with pps increased. An indication was obtained that some precipitation of DNA from the medium must have occurred in the presence of poly-L-lysine. Testing the medium after incubation, only 79% of the DNA was present in the supernatant medium as com- pared to homogeneous distribution at the start of incubation, when the imput was checked. TABLE VII,1. Effeat of poly-L-lysine en DNA association with pps % TCA-prec. DNA in associated DNA addition medium after 4 h. ( % of input ) none 62.1 0.34 none 71.1 0.38 poly-L-lys. 84.7 0.48 poly-L-lys. 81.5 0.61 E.eoli 3H-DNA (16.7 ug, 8 x 106 dpm) mixed with 2 x 106 pps in 1 ml; poly-L-lys. added to 2 ug/ml. Repair of exogenous DNA The idea of using repair as a measure for DNA functionally taken up into plant pps is based on the assumption, that only those DNA molecules inserted in a non-degradative way in the plant cell's metabolism are worth looking for. All other molecules, whether sticking to the plasmalemma or to internal mem- branes or in pinocytotic vescicles are functionally lost. Repair of exogenous 105 DNA molecules also indicates that those molecules have migrated into the cell's nucleus or organdies and arc therefore in a good position to be transcribed or replicated. When wo -;t.irtod this work, nothing had been published on the assay of dimers in pps. We thereiore tested whether we needed to prepare more :>r less purified DNA or whether crude lysates of pps could be used facilitating the assay pro- cedure. The presence of TCA-preeipitable material other than DNA might have some influence on the chromatographic determination of the X()T/T%. In studies involving the in vivo induction of rather smsli numbers of dimers a correction is generally made using a parallel chromatograrc with unirradiated material. This is not possible in our system. We therefore eh- eked the reproducibility of the assay with various amounts of DNA and a constant, typical amount of lysate (from 2 x 10° pps). On the basis of the results, ^hov,n in :able VII,2, we. con-/ sider that any loss of dimers in excess of e.g. 51 (i.e.from 18.2 to 17.3) can be confidently attributed to repair. TABLE VII,2. :*:''luetue of pps Lysate OK the dirtier assay total rad. act. on XQT/T % chromatogram (cpm) (no lysate) 95,920 IS.2 101,530 18,2 (lysace added) 173,040 18.2 169,460 18.0 79,500 18.0 87,440 18.4 26,400 18.5 6,120 !8.4 Realizing that the omission of DNAase treatment will leave more DNA asso- ciated with the pps, and assuming that much of this extra DNA has not effec- tively entered the pps, we tested the dimer assay for its ability to discrimi- nate adsorbing DNA from DNA functionally taken up. Using pps from one isolation, we incubated either for 4 h under non-pho^reactivating conditions only, or 4 h under these conditions followed by an exposure to photoreactivating light for 14 more hours. The results of this experiment are shown in table VII,3 and in fig. VII,4, where the fraetionation of dimers and thymine in the first ex- periment is illustrated. Taking repair of the damaged DNA molecules either to be absent or to be complete, we used the percentage loss of dimer of the asso- ciated DNA to calculate the percentage (of input) DNA functionally taken up by the pps: "normalized uptake". The association percentages observed after 4 h 106 TABLE VII,3, Functional DNA uptake by tobacco neaophyll protoplasts Effects of incubation time and washing treatment incubation DNAase associated DNA XQT/T loss of ditner normalized uptake time wash ( % of input ) • ,( % ) ( % ) _{ % of input ) 4 h 0.80 7.2 58 0.46 A h Q.53 1.9 89 0.47 18 h 15.40 S2.8 25 3.85 IB h 10,60 11.2 34 3.60 E.eoli 3K-DNA (11.7 ug, 16.3 x 1Q6 dpra,X()T/T%=17.0) mixed with 1.9 x 106 pps in 1 ml. 1S510 „ 15- 1 THYMINE r r— X E a. DIMER ^•10- Q." "In /pe r > 5 u a SOLiVENT ORIGIN §R0Njr . ' •-• . .. • •-,'.-• —: ri .': ' • l——i_J 0 •.-•.: '• •••-. •' • .1 10 15 STRIP NUMBER Fig.VII,4. Paperchromatographic fractionation of formic acid hydrolyzed DNA from tobacco mesophyll protoplasts treated with UV-irradiated 3 - E.coli H-DNA (first experiment of table VII,3). agree well with those of the preliminary experiments, but those occurring af- ter 18 h are remarkably high. Although after such a long incubation the DNA in the medium was completely broken down, the associated DNA in the lysate was again for 97 and 82% TCA-precipitable. 107 Although the incorporation of H-thytnidine into the DNA of tobacco meso- phyll pps in liquid culture starts generally only 50 1) after isolation the remote possibility existed that this particular batch (used for analysis 34 h after isolation) had already started earlier and could thus account for the rather high normalized uptake of the 18 h samples. An additional sample vwi-th- out DNAase treatment) was therefore used for isolation of total BNA us the micro-isolation method (chapter- III). In this system ONA molecules with a H.W. of less than approximately 2 x 10 I) are retarted. The profile of radioactivi- ty eluted from the column after the void volume would thus reflect the M.W. heterogeneity of the associated DMA. No radioactivity could be detected in the void volume fractions, where in routine isolations all of the 20 ug of plant DNA from lysates of 2 x 10 pps were recovered. This observation is taken as evidence for the absence of both reuti1ization of donor-DNA-degradation- products and of integration of detectable amounts of donor DNA into the plant genome under our experimental conditions. Radioactivity started to elute from the column after about 4S% of the bed volume, while about 80% of the radio- activity came off in fractions where tRNA elutes from the column, a situat'on similar to the one encountered in DNA association studies with Chlatnydomonas veinkavdrl (14). Fractions between 56 and 63% of the bed volume were pooled, dialyzed and concentrated by flash evaporation. Analysis by sucrose gradient centrifugation (6) showed a broad spectrum of rather low M.W. species ranging from 10 to 8 x 10 D. After incubation for 18 h the material associated with the pps, though TCA precipitable, is thus very much degraded and can for only 10% or less be represented by DNA molecules large enough to accomodate one single gene. Although a high percentage of ditners in the donor DNA makes our assay less sensitive for high levels of DNA merely adhering to the. pps surface, it can also be argued that the introduction of so many dimers into the pps could very well saturate the there existing repair systems. This leads us to the following calculation. When 20 yg DNA (with X()T/T% of 18) is-offered to 2 x 106 pps and an average functional uptake of 0.4% of,the input takes place, the DNA entering one pps would then contain about 2.3 x 1.0 pyrimidine dimers (based on an ap- proximation of the relative frequencies of C()C, C()T and T()T dimers given by Setlow and Carrier (23). Comparison with the rough data for wild carrot pps exposed to UV-irradiation (8.4 x 10 pyrimidine dimers repaired in less than 24 h, 12), repair of such a level of dimers by excision repair or photo- reactivation or both should be quite possible, especially by pps which them- selves have not been exposed bo UV-light. 108 In addition to discriminating between DNA merely adsorbed to pps and DMA taken up into the pps and its metabolism, the diner assay also leaves out of consideration DNA, that after true uptake finds itself inside dying or dead pps, i.e. pps incapable of any amount of repair. It could thus be developed into a reliable measure of DNA entering" living :pps in a "useful" way. In conclusion it must be clearly stated, thats unfortunately, the results presented in table VII,3 are not what are generally called "typical results", but the outcome of one series of tests using one particular batch of pps. When trying to repeat the experiment (in a set-up where discrimination between photoreactivaticn and dark-repair should be possible and also including sam- ples treated with poly-L-lysine), we have been repeatedly unsuccessful. It is possible that the quality of the later batches of pps, isolated from not too good looking plants in late sprirtg, may be the causative factor. That would mean that - at this stage in the development of pps research - optimal con- ditions for functional DNA uptake should not be sought so much in the realm of;.incubation conditions, but rather in the conditions of plant growth and the art of pps isolation. A possibly more rewarding alternative would be to go to standardized and routinely cultured suspensions of cell clumps and iso- late pps- from them. The possible advantage of the synchronous start of DNA synthesis in mesophyll pps would thus be abandoned. ACtiKOVLEDGEMENTS I wish to thank G. Kalsbeek for excellent help in performing the experi- ments and, for valuable discussions. We are grateful to Dr. C.A.van Sluis for his expert advice on the dimer assays and his interest during the investigations. The ICnd helpand advice of Drs. H.Taraminga' and Dr. J.Cornelisse concerning UV- irradiation of DNA, carried out in their laboratory, is gratefully acknowledged. REFERENCES 1. Aoki. S, and Takebe, I., Virology 39(1969)439-448. 2. Beukers, R; and Berends, W., Bio'ckim.Biovhys.Acta 41(1960)550-551. 3. Burgess, J., Motoyoshi, F. and Fleming, E.N., Planta 111(1973)199-208. 4. Carrier, W.L. and Setlow, R.B. in: Methods in Ensymology (Grossman, L. and Mdldave, K.,Eds.) 21 D(1971)230-237, Academic Press., New York an.d London. 109 5, Clewell, D.B- and HeLinski, D.R., Proo.lkitl.Aead.Sai.U.S.A. 62(1969) 1 159-!166. 6, Dons. J.J.M.U975) 'Fiies's, State University of Leiden. 7, Ferrari, T.E. and Widholm, J.M., Anal.Bioahem. 56(1973)366-352. S. Graham, F.L. and Van der Eb, A.J., Virology 54(1973)536-539. 9. Hoffmann, F., Z.Pflansenphy$iol. 69(1973)249-261. 10. Hoffmann, F. and Hess, D., Z.Pflansenphijsiol. 69(1973)81-83. 11. HoU, F.B., Coll.Intern.C.N.R.S. (J.Tempe.Ed.) 212(1973)509-516, and (1974) in: Tissue Cult:a>e and Plant Science W?4 (H.E.Street, Ed,), Academic Press, London and New York, 301-327. 12. Howland, G.P., Nature 254(1975)160-161. 13. Kahn, M., Siapolymers 13(1974)669-675. 14. Lurquin, P.F. and Behki, R.M., Mutation Res. 29(1975)35-51. 15. Maes, R., Sedwick, W. and Vaheri, A., B-io •hzm.Biophys.A.aia 134(1967) 269-276. 16. Motoyoshi, F. and Hull, R., J.gen.Virol. 24(1974)89-99. 17. Ohyama, K., Gamborg, O.L. and Miller, R.A., Can.J.Bob. 50(1972)2077- 2080, and Coil.Intern.C.H.R.S.(J.Tempe, Ed.) 212(1973)423-428. 18. Rahn, R.0. and Hosszu, J.L., Photoohem.Photobiol. 8(1968)53-63. 19. Rabu, R.0. and Landry, L.C., BioaUm.Uiophijs.Acta 247(1971)197-206. 20. R^ppoldt, M.P. (1958) Thesis, State University of Leiden. 21. Sarkar, S., Upadhya, M.D. and Melchers, G., Mel.gen.Genet. 135(1974)1-9. 22. Schilperoort, R.A. (1969) Thesis, State University of Leiden. 23. Setlow, R.B. and Carrier, W.L., J.Mol.Biol. 17(1966)237-254. 24. Sluis, C.A. van, (1972) Thesis, State University of Leiden. 25. Small, G.D. and Sparks, R.B., Anal.Bioahem. 41(1971)116-125. 26. Smith, K.C., Photochem.Pkotobi.ol. 2(1963)503-^7. 27. Tempe, J.(Ed.) Coll.Intern.C.N.R.S. 212(1973):"Protoplastes et Fusion de Cellules Somatiques Vegetales", editions de lfI.N.R.A., Paris. 28. Trosko, J.E. and Mansour, V.H., Radiat.Res. 36(1968)333-343. 29. Vogel, H.J. and Bonner, D.M., J.Biol.Chem. 218(1956)97-106. 30. Wagner, E.K., Rice, M. and Sutherland, B.M., Nature 254(1975)627-628. 31. Watts, J.W., Cooper, D. and King, J.M. (1975) in: 2nd John Innes Symp. "Modification of the information cont&nt of -plant cells"(R.Markham et al.Eds.) North-Holland/American Elsevier, p.119^131. 110 CHAPTER VIII GENERAL DISCUSSION The main objective of our studies has been to determine how well cultured plant cells and protoplasts can be handled in transformation experiments. The numerous attempts to transform plant cells with purified DNA have produced only few positive results. An important reason for this lack of success ap- pears to be the fact that the technical problems involved in DNA "feeding" are not understood. Our experiments with intact tobacco cells from suspension culture clearly indicate that the amount of nucleic acid which remains ir- reversibly associated with plant cells is not a reliable measure of the amount that has actually entered the cells. Adsorption onto the cell surface is an important disturbing factor. The same probably holds for isolated pps. It is therefore necessary to go one step further and look for some kind of expres- sion of the DNA which is really inside the host cells. The repair of radiation damaged exogenous DNA may possibly prove to be a reliable criterium for real uptake of foreign DNA, but needs to be developed into an assay. In vitro induction of crown gall turner cells with A.turn.DNA still remains a most attractive system, since here at least we know something about the de- sired "end-product". As stated in chapter V the use of leaf pps may be advan- tageous, since these cells can be compared to "conditioned" cells and show a more or less synchronous onset of DNA synthesis. Using the carborundum rubbing technique (usual in plant virus research) we could readily induce tumors on tobacco leaves with A.turn.cells. They appeared to originate from a very small number of mesophyll cells anti could be easily brought into culture. Recently two attempts have been /made to transform tobacco mesophyll pps with total A. turn.DNA (2, 5). The freshly isolated pps were immediately inocu- lated using virus infection techniques s~id subsequently plated out in NT-medium without phytohormones. No positive results were,obtained. In the light of the findings of Scowcroft et at. (3) this is not surprising. They isolated pps from crown gall tissue culture material of various species (including tobacco) and showed that pps isolated, from these phytohormone independent tissues have an absolute requirement for low levels of these hormones during the first two weeks of culture. Once cell wall regeneration and the first cell division haw been initiated, it is possible to transfer colonies to a medium devoid of growth substances, and still maintain division* Another pitfall in the two men- Ill iioned transformation attempts is the use of total A.turn.DNA preparations. Certainly these preparations have been devoid of the large A.turn.plasmid, which is more and more implicated in tumor formation. Now that a rather sensitive assay for one of ihe plasmid expression products is available (presence of octopine or nopaline in tumor cells) and the isolation of size'able quantities of plasmid DNA has been worked out (A.M. Ledeboer, in preparation), it seems to be a worthwhile enterprise to feed this plasmid DNA to tobacco pps. The "inoculated" pps should then be cultured for some time in liquid medium with a low phytohormone content and the developed small colonies subsequently be plated out on medium lacking the hormones. Although this procedure could pos- sibly allow some very quickly "habituated" cells to grow also, identification of real transformation products is possible with the octopine assay, for which only minute quantities of biotnass are needed. One of the major problems of transformation studies with plant cells (of which the majority is never published) is the lack of well defined biochemical mutants. One of the best defined systems is 5-methyltryptophan resistance of diploid carrot and tobacco cells cultured in suspension. Apart from uptake mutants which are readily picked up, resistant cell lines with alterations in the enzyme anthranilate synthetase have been isolated providing a good expla- nation for the resistance mechanism. Attempts to transform normal cells with DNA isolated from the latter resistant ones remained without clesr-cut results (Widholm, pers.cotnm.). No study of the cause of resistance has been carried out for any of the few other cell lines resistant to e.g. streptomycin sul- phate, 5-bromodeoxyuridine and 8-azaguanine. Using among others suspension cultures derived from haploid Nicotiana sylvestris (a kind gift of Dr. G.Wen- zel, Ladenburg) a plant cell mutant selection programme has recently been set up in our laboratory, including enzyme studies to make possible an unambiguous identification of resistance mechanisms. This work should providr the much need- ed biochemical background for effective selection procedures. It is quite clear that the absence of adequate selection procedures is also the main bottle-neck in the field of fusion of somatic plant cells. The last few years a lot of observations (especially by the group of Gamborg, Saskatoon) have been made :. fusion products between pps of widely different species, u- sing as a "recognition" system one partner from cell culture origin and one from leaf mesophyll. In conjunction with the polyethyleneglycol fusion induction pro- cedure, it was possible to observe real fusion followed by some cell divisions in crosses between numerous agriculturally important species. But after some 112 cell proliferation these experiments have to be stopped since one loses track of the hybrids. But even if selection of hybrids will be possible, there is no a priori reason to believe that"It will: bepossible"to"raise new intact plants from thera. Cytoplasmic incompatibility and chromosome loss are important t. - peets which have not yet been well considered. Cocking's appraoch in this sense is the most cautious and involves the induced fusion of the closely related species Nicotiana tabacurn and Petunia parodii using media on which both parents will grow and regenerate intact plants. Selection can be applied using the naturally occurring differences in drug resistance of the parent species. One indication of total chromosome loss has been obtained by Cocking (pers. comra.) in one case of fusion between pps from crown gall tissue of Partheno- cissus triauspidata and from Petunia parodii mesophyll. A chimaeral tissue culture was derived from one hybrid product and subcultured for more than a year. Cytologlcal examination then showed that the resulting cells harbored only chromosomes from the Parthenocissus cells, while enzymological study re- vealed the presence of Petunia enzymes (e.g. peroxidase). Possibly some parts of the Petunia chromosomes had been incorporated in their Parthenocissus counterparts. The recent developments in the field of artificial DNA recombination make it possible to use tailor-made DNA molecules produced in large quantities in appropriate bacteria. The ColEl plasmid, with attached to it an E.coli gene coding for kanamycin resistance, has recently been used in DNA feeding studies with soybean and tobacco cell suspensions (1)- No data are as yet available on the eventual expression of the resistance gene, but it is clear that this ap- proach is very promising. Extention of our work on pure Co 1EJ DNA fed to tobac- co pps would seem warranted using such artificial hybrid molecules. Interest in using pps as acceptor cells for transformation studies is in- creasing arid very preliminary results with tobacco callus pps and oat mesophyll pps have been communicated (4). In an effort to protect DNA from degradation, and even more to get it taken up by a process analogous to fusion (which has now reached high efficiencies) Bacillus stibtilis DNA(together with some histon) has been "wrapped up" into vesicles made of soybean lecithin (White, pers. comm.). Acded to tobacco pps these DNA containing vesicles are taken up. Electron mi- croscopic study should eludicate the mechanism of uptake (endocytosis, fusion, or both in sequence). An advantage of this "packaging" approach is that after incubation the non absorbed vesicles can apparently be washed off easily. In that case reisolation of DNA and a bio-assay for transforming B.subtilis DNA 113 would give information on "useful" uptake. It should be pointed out however that in tlic ijsf ot endocytotic uptake, this approach may fail, sime endocy tot ic: vesicles art- general!v specialized in the i>f t ec t ive degradation of their contents. In conclusion it REFERENCES 1. Owens, L., Plant Physiol. 56S(1975)38. 2. Schilde-Rentschler, L., Coll.Intern.C.N.R.S. 212{1973)479-483. 3. Scowcroft, W.R., Davey, M.R. and Power, J.B., Plcp.fc Soi.Lett. 1(1973) 451-456. 4. Uchimiya, H. and Murashige, T., Plant Physiol. 56S(1975)38, and Galston-, A.W., et al., ibid. 5. Watts, J.W., Cooper, 0. and King, J.M. (1975) in : 2nd John Innes Symp. "Modification of the information content of plant cells" (R.Markham et al. , Eds.) North-Hoiland/American Elsevier, p.119-131. 114 DANKWOORD Het publiceren van een proefschrift geeft de auteur de ge- legenheid alien die hem bij de voorbereiding ervan behulpzaam zijn geweest te bedanken. Ik doe dat van ganser harte en wil daarbij speciaal Rob, Aad, Cees en mijn promoter H. Veldstra met name noemen. Niet alleen de bewoners van K52 en K31, maar alle ere- en aktieve leden van de crown-gall-clan hebben, ook bij nacht en ontij, door hun enthousiasme, raad en daad een sfeer van samen- werking geschapen, die geen enkele onderzoeker kan ontberen. Door de cechiische en administratieve staf van het BLLB werd steeds op prettige wijze zorg gedragen voor zaken zoals o.a. het persklaar maken van ruwe raanuscripten, het kweken van "protoplasten-planten", het vervaardigen van tekeningen en foto's, en het in stand en op pijl houden van apparatuur, ma- terialen en lab-kamer. In het kader van hun doctoraal opleiding hebben aan delen van het onderzoek aktief en met toewijding deelgenomen : Cees Ladage, Anke Burgers, Piet Klapwijk, Chiel Husmarm, Hanneke van der Kruit, Gijsbreght Kalsbeek en Dieuwke Tjepkema. Zeer erkentelijk ben ik de Nederlandse Organisatie voor zuiver-wetenschappelijk onderzoek Z.W.O. voor het toekennen van een beurs voor een korte studiereis naar Noord Amerika. 115 ih* v«r/v'»-k v .i :i \\y F ,' • \i 1 t t' i t J f r Wiskur.Uv on Na t uu r we l t n , \iippen v u 1 j; * h i e r o n der e c p. sort o v e r z i e 111 v a n ai i j n a v a A ••> m i s i • < •• t u d i e . iuni !^6J ei ndoxamnn gymnasium r aan hoc Crutius gymnas mr" tt Hi-lft. aujviscus 196J een jaar studio (Liberal Arts) alrt exchangc-stn Sinds September 1968 ben ik in dienst van de Rijksuniversiteit te Leiden verbonden aan het BLLB, en wel achcereenvolgens als student-, dottoraal-, hoofd-assistent en wetensch.ippel i jk medewerker. 16