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Studies on Genetic Transformation of Coffee by Using Electroporation and the Biolistic Method

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Studies on Genetic Transformation of Coffee by Using Electroporation and the Biolistic Method /üMc>r?oi, IÔÔO ;CO 2 0 OE£L . f.9.94 ÜB- Jos H.J. van Boxtel STUDIES ON GENETIC TRANSFORMATION OF COFFEE BY USING ELECTROPORATION AND THE BIOLISTIC METHOD Proefschrift ter verkrijging van de graad van doctor in de landbouw-en milieuwetenschappen op gezag van de rector magnificus, dr. C. M. Karssen, in het openbaar te verdedigen op woensdag 21 december 1994 des namiddags te vier uur in de Aula van de Landbouwuniversiteit te Wageningen Abstract Thepresen t study aimed simultaneously at animprovemen t ofcoffe e regeneration systems and at a definition of factors influencing the efficiency of direct gene transfer methods. The development of an improved regeneration system, based on high frequency somatic embryogenesis from leaf expiants, passing through multiplication of embryogénie callus in liquid medium, is described. This method can contribute to the obtaining of high protoplast yields and offers perspectives for use in genetic transformation systems of coffee. Several factors affecting protoplast isolation, electroporation and regeneration were studied. This system appeared to be appropriate for transient expression studiesbut , due to difficulties with protoplast regeneration, less promising for achieving stable expression. Further expression studies were performed using particle gun bombardment on different tissues of several coffee genotypes. Best results were obtained using in vitro cultured leaves of Coffea arabica and plasmids carrying the EFla-Al promoter of Arabidopsis thaliana. The effect of tungsten particles on callus induction and the fate of GUS-expressing cells after bombardement on leaves has been described, and the consequences for their use are discussed. Studies onfive selective agents showed best prospects of the herbicide glufosinate for detection of stably transformed coffee tissue. It was concluded that avoidance of polyphenolic oxidation, caused by tissue wounding, is of great importance for the development of a reliable genetic transformation method for coffee. CIP-GEGEVENS KONINKLIJKE BIBLIOTHEEK, DEN HAAG Boxtel, Jos H.J. van Studies on genetic transformation of coffee by using electroporation and the biolistic method/ Jos H.J. van Boxtel. - [S.l. : s.n.] + colour plates Thesis Wageningen. -Wit h ref. -Wit h summary in French and Dutch. BIBUOTHEFK ISBN 90-5485-315-8 LANDBOUWUNIVERSITEIT Subject headmgs: coffee ;geneti c transformation. n,A/~Evrtki^c».T WAGENINGE N omslagontwerp: YolandePerri n Prologue This thesis is the result of research carried out between May 1990 and January 1994 inth eBIOTRO P laboratory, whichi spar t ofth e "Centre deCoopératio n Internationale deRecherch e Agronomique pour leDéveloppement " (CIRAD) inMontpellier , France. During three years this work was financed by the CIRAD department "Cultures Pérennes" (CP). Further 6-months financial support was provided by the French ministery of Foreign Affairs. I am grateful to the association "Landbouw Export Bureau 1916-1918" for their financial support which amongst others, allowed the publication of this thesis. Besides my appreciation for these financial contributions, I would like to thank all personal contributions of people who where essential for the progress of this work and for my 4'/2yea r living in Montpellier. Evert Jacobsen was not always convinced of the feasibility of my adventurous plans. His conviction and trust on the long run gave me persistence and self- confidence. Evert, thanks a lot for being my promotor and for all the work related to it. Sans Bertus Eskes je n'aurais peut-être jamais effectué ma thèse en France. Merci Bertus pour ton soutien detou s les instants etpou r ton redoutable crayon rouge qui m'a fait souvent mal auxyeux , mais qui a fait naître des discussions fructueuses. Marc Berthouly a beaucoup contribué à ma thèse en apportant des milliers d'idées et enpartagean t son expérience acquise auCost a Rica. Jet eremerci e pour ton enthousiasme et ta bonne humeur. Je n'oublierai pas Magali Dufour qui m'a épaulée au début de ma thèsejusqu' à ce qu'elle parte pour le Costa Rica. Merci Jacques Schwendiman et Claude Teisson pour m'avoir accueilli dans le laboratoire BIOTROP et m'avoir ainsi accordé votre confiance. Je remercie toute l'équipe "caféier", indispensable à laréalisatio n de cetravail : Véronique Roussel, laprincess e de la serre, Claudine Verlhac, la technicienne laplu s rapide del'hémisphèr e nord, Lolita Triaire, l'oragan andalous del'histologie , et Cathy Carasco, la "technicopine" survoltée. MerciNicol eMichaux-Ferrière ,Jacque sEscoute ,Jean-Loui sNoyer ,Ange-Mari e Risterucci, Thierry Legavre, Daniel Bieysse etChristia n Cilaspou r votre disponibilité à répondre à mes questions histologiques, microscopiques, bio-molleuses, transformatiques, caféiques ou statistiques. Je remercie également les étudiantes Hélène Boetti et Alexandra Besson pour leurs contributions fructueuses. MerciValérie ,Florence , Corinnee tJanin epou r lesfeutres , scotche tpapie rbie n sûr, mais aussi pour votre disponibilité. Mafamill e ciradienne était grande:l'équip e dulaboratoir e de"Cultur e InVitro " m'a nourri, m'a abreuvé, m'a vêtu. Elle m'a grondé, àtort , m'a admiré, àraison . Un grand merci à Emmanuel, Hâna, Flop, Cath, Marc-Philippe, Ludo, Agnès, Jorge, Maria-Elena, Cecile, Daniel, Kilou, François, Floa, Flom, Aleth, Sophie, Dominique, Christian, Christophe et Lulu. Merci à Pascal, Hervé, Yolande, Laurence et Fred, les étudiants qui ont partagés mon bureau, pour avoir supporté quelquefois "l'Insupportable". Moi aussi,j e vous excuse. Pour les extraciradiens: merci Philippe Guerche (INRA, Versailles) pour tes conseils d'électroporation; merciNanc yWalke r (Sanofi, Toulouse)pou rm'avoi r initié aux bombardements; merci Rémy Pujol (CRIC, Montpellier) pour tes conseils en microscopie électronique etto n amitié endehor s de lavi e scientifique; merci Philippe Lashermes et Marie-Christine Combes (Orstom, Montpellier) pour m'avoir aidé dans la quête d'un caféier transgénique. Enfin, merci à ceux qui m'ont fait oublier le boulot quand il le fallait: Fabrice, Serge,Henri ,Jean-François , Xavier,Philippe ,"Chupa "Steph ,Thierr y "el djembéman", Véro "larieuse" ,Nat h mon professeur préférée envoil e ete n français, Jamel etCoco , lecoupl eroyal e dupimen t etd el'arachide . MerciRégi s "l'aigle deVercors" , bien que je tehaïsse s encorepou r lamarqu e du cadred eVT T sur mon épaule;merc i Marcpou r tes passes foireuses et les bières belges partagées; quant àto i Hervé, merci d'être un pôte en beton. Montpellier "l'inoubliable", au revoir et à bientôt. Ta chaleur profonde, le roulement nocturne desvague smoussante s etl'inlassabl e cricri descigale s sontgravé s dans ma memoire. C'est pour ça qu'ensemble avec Yolande, mon alterego, nous recréerons un petit montpellier dans la grisaille d'Amiens et puis de Norwich. Table of contents List of abbreviations Chapter 1. General introduction 1 Chapter 2. High frequency somatic embryogenesis from coffee leaves: factors influencing callus induction, and subsequent multiplication and regeneration in liquid medium 15 Chapter 3. Isolation of viable coffee protoplasts and preliminary outgrowth studies 35 Chapter 4. Transient expression studies of ß-glucuronidase in electroporated coffee protoplasts 45 Chapter 5. Transient expression of ß-glucuronidase following biolistic delivery of foreign DNA into coffee tissues 55 Chapter 6. Fate of GUS-expressing cells during biolistically-induced callus formation on Cqffea sp. leaves: a histological study 69 Chapter 7. Inhibitory effect of selective agents on callus development for selection of genetically transformed coffee tissues 83 Chapter 8. General discussion and future prospects 95 Summary 103 Résumé 106 Samenvating 110 References 114 Curriculum vitae 125 List of abbreviations BA 6-benzylaminopurine Bt Bacillus thuringiensis pCaMV-E35S cauliflower mosaic virus enhanced 35S promoter CBD coffee berry disease CIRAD Centre de coopération internationale en recherche agronomique pour le développement EDTA ethylenediaminetetraacetic acid EFla elongation factor la FDA fluorecein diacetate GGP greenhouse grown plants GUS ß-glucuronidase HFS E high frequency somatic embryogenesis IAA indole-3-acetic acid IBA indole-3-butyric acid IRCC Institut de recherches du café, du cacao et autres plantes stimulantes KIN kinetin LFSE low frequency somatic embryogenesis MES 2-(N-morpholino)-ethanesulfonic acid MR multiplication rate MS Murashige & Skoog MUG 4-methylumbelliferyl ß-D-glucuronide NBB naphthol blue black (protein specific coloration) OP open pollinated progeny PAS periodic acid - Schiff (Polysaccharid specific coloration) PCR poly chain reaction PCV packed cell volume PEG polyethylene glycol PVP polyvinylpyrrolidone rpm rotations per minute SCSE self controlled somatic embryogenesis SE somatic embryogenesis (in general) SEM somatic embryo-derived microcuttings TEM transmission electron microscopy UBQ ubiquitine X-Gluc 5-bromo-3-chloro-3-indolyl-ß-D-glucuronic acid 2,4-D 2,4-dichlorophenoxyacetic acid 2-iP 6-(y,y-dimetylallylamino)-purine STELLINGEN 1. Op basis van de waarneming dat de EFla-Al promoter goed tot expressie komt in de koffieplant, zou een selectiegen ontwikkeld kunnen worden dat optimaal functioneert in deze plant. Dit proefschrift. 2. Zowel regeneratie via somatische embryogenese, als transiente expressie van GUS-genen, geïntroduceerd m.b.v. een "partiele gun", zijn bij koffie genotype-afhankelijk Dit proefschrift. 3. Polyfenolische afweerstoffen in de koffieplant zijn niet alleen hinderlijk voor pathogenen, maar ook
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