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Microprojectile-Mediated Transformation of Ornithogalum

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Microprojectile-Mediated Transformation of Ornithogalum --}II Microprojectile-mediated transformationtrans,formation of L \ ,I ,,' '\ ~,; , -..,' Ornithoga/umOrnitho.ga/um thyrsoides h~brid A2 a Susanna Magdalenaf de Villiers ty o Thesis presented for the degree of UniversiDoctor of Philosophy in the department of Microbiology, University of Cape Town August 1999 The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town Certification of Supervisors In terms of paragraph 9 of "General regulations for the degree of Ph.DPh .D." we , as supervisors of the candidate S.M.M. de VilliersVilliers,, certify that we approve the incorporation inin thisth is thesis of material that has been submitted for publication. Prof. J.A. Thomson Head of Department of Microbiology University of Cape Town a f ty o Dr. UniversiDO. KK.. Berger Research leader Biotechnology Division ARC-Roodeplaat Vegetable and Ornamental Plant Institite Acknowledgements My sincere thanks go to thethe following institutions and people: The Biotechnology Division of ARC-Roodeplaat, Hadeco, the USDA and UCT for financial support Dave Berger - supervisor,supervisor, fellow scientist, friend and "heavy" - an interesting combination which was an invaluable contribution to this study Johan Burger, who initiated this project because he did not want to see the 'tjienkerintjies'tjienkerintjies'' die of OrMV Professor Jennifer ThomsonThomson,, who invited me to collaborate with her at UCT on the Taxi technique My colleagues at Roodeplaat for many a shared laugh and their willingness to bounce ideas around to solve some "sticky" problems Chris Bornman, for providing insights into a worlda not quite as small as that of DNA Kathy Kamo,Kamo, for enthusiasmenthusiasm,, support fand friendship during my fellowship at the USDA and since My parents, Ben and Elsa Strydomty, o for always being there and for teaching me perseverance and a love for science Etienne,Etienne, for love,love, support and patience, especially with my computer moods and -pains. Also, for broadening my horizons and teaching me to dream big . Universi AbbreviationsAbbreviations ARCARC AgriculturalAgricultural ResearchResearch CouncilCouncil ARC-RoodeplaatARC-Roodeplaat ARC-RoodeplaatARC-Roodeplaat VegetableVegetable andand OrnamentalOrnamental PlantPlant InstituteInstitute BA 6-benzylaminopurine bar phosphinothricin acetyl transferasetransferase BME r.,-mercaptoethanol~-mercaptoethanol bp base pairs C Clone of T1 CaMV35S cauliflower mosaic virus 35S promoter CsCI cesiumchloride CSPD disodium3-(4-methoxyspiro{1 ,2-dioxetane-3,22-dioxetane-3,2'-(5'-chloro)'-(5'-chloro) tricyclo[3.3.1.1tricyclo[3.3.1.1 3,7]decan}-4-yl) phenyl phosphate CTAB cetyltrimethylammonium bromide °C degrees Celsius 2,4-0 2,4-dichlorophenoxyacetic acid Dicamba 3,6-dichloro-2-methoxybenzoic acid DIG digoxygenin DNA deoxyribonucleic acid dH20dH 20 distilled water a dNTP deoxynucleoside 5' triphosphate dsDNA double stranded DNAf EDTA ethylenediaminetetraacetate EtBr ethidium bromidety o EtOH ethanol g gram GTC guanidine thyocyanate GUSluidAGUS/uidA Universi~-glucuronidaser.,-glucuronidase h hour(s) hpt hygromycin phosphotransferase (hygromycin resistance) IAAIAA indoleindole 3-acetic acid M molar min minute(s) MS medium Murashige and Skoog medium MW molecular weight NAANAA a-naphtalene-acetica-naphtalene-acetic acidacid NaCINaCI sodiumsodium chloridechloride NaOAcNaOAc sodiumsodium acetateacetate NOS nopaline synthase NPTII neomycin phosphotransferase (kanamycin resistance) NT NOS terminatorterminator 00 optical density OrMV ornithogalum mosaic virus PAT/pat/bar phosphinothricin acetyl transferase PCR polymerase chain reaction PEG polyethylene glycol PIG particle inflow gun PPT phosphinothricin RNA ribonucleic acid rpm revolutions per minute RT room temperature RT-PCR reverse transcription PCR SDS sodium dodecyl sulphate sec second(s) SSC saline sodium citrate ssDNA single stranded DNA T Transgenic plant from the UCT study TAE Tris acetate EDTA (buffer) T-DNA transfertransfer-DNA-0 NA a TE Tris-EDTA (buffer) Ti-plasmid Tumour inducing plasmidf TNE Tris-NaCI-EDTA (buffer) U units of enzymety o UCT University of Cape Town USDA United States Department of Agriculture v volts X-Gluc Universi1-bromo-2-chloro-3-indolyl-r1-glucuronic acid List of figuresfigures Figure 1 Pathways by which plants can be regeneratedregenerated fromfrom leafleaf explants of Ornithoga/um thyrsoides.. 3.4 Figure 2 (a) Regeneration from leafleaf explants.. (b) Callus proliferation fromfrom leafleaf explants.. 33.10.10 Figure 3 Regeneration of plants fromfrom callus initiatedinitiated from leafleaf explants cultured on callus inductioninduction supplemented with (a) NAA and (b)(b) dicamba. 3.123.12 Figure 4 Micrographs of macroscopic developmental events inin the culture of Ornithoga/um thyrsoides leafleaf explant and callus tissues.tissues. 3.14 Figure 5 Light micrographs of glutaraldehyde-fixed and glycolmethacrylate­glycolmethacrylate- embedded tissues of Ornithoga/um. 33.14.14 Figure 6 Lethal concentrations of the selection agent (PPT) as determined for (a) leaf- and (b) callus explantsexplants.. 33.14.14 Figure 7 Gene constructs used for biolistic transformation of 0. thyrsoidesthyrsoides.. 4.64.6 Figure 8 Transient expression in callus explants of O.O. thyrsoides A2 as visualised by GUS staining.staining. a 4.8 Figure 9 Regeneration of putative transgenicf plants from callus bombarded at the (a) USDA and (b) UCT and cultured on selection medium.medium. 4.14 Figure 10 Standard curve that wasty oused to determine the protein concentrations of leaf samples analysed with PAT-ELISA. 5.65.6 Figure 11 Standard curve used to determine the amount of PAT enzyme in leaf extracts analysed with ELISA. 5.7 Figure 12 RegenerationUniversi from leaf explants of T1 and clones of it. 5.145.14 Figure 13 Plants that regenerated from callus tissue bombarded at the UCT that was cultured on selection medium for six months. 5.155.15 Figure 14 PCR analysis of ten plants obtained from experiments performed at the USDA. 5.15 Figure 15 PCR analyses of plants obtained from transformation experiments performed at the UCT. 5.155.15 Figure 16 ELISA results obtained with antibodies directed against the PAT enzyme. 5.165.16 Figure 17 Southern blot of genomic DNA fromfrom non-transformed plants spiked with various copies of thethe pat gene.. 5..18 Figure 18 DNA fromfrom greenhouse-grown clones of T1 ,, digested with restriction enzymes, resolved on an agarose gel and analysed by Southern hybridisation. 5..18 Figure 19 Southern blotblot of DNA fromfrom inin vitro cultured clones of T1 digested with restriction enzymes.enzymes. 5.18 Figure 20 Total RNA isolated from plants cultured inin vitro resolved on agarose gels. 5.205.20 Figure 21 Northern blots with total RNA isolated from T1 clones cultured in vitro and hybridised with pat and ubiquitin gene probes. 5.20 Figure 22 Transgenic clones of T1 cultivated in the greenhouse and analysed for phenotypic characteristics. 5.22 Figure 23 Optimisation of the leaf paint assay with Ignite® and analysis of transgenic T1 clones for herbicide resistance in the greenhouse. 5.22 Figure 24 Varying degrees of herbicide resistance displayed by eight T1 clones compared to non-transformed controls in the greenhouse and detail of individual painted leaves.a 55.22.22 f ty o Universi List of tables Table 1 Reaction off explants cultured on various regeneration media forfor six weeks.weeks. 3..9 Table 2 Reaction off leafleaf explants cultured on callus induction media forfor eight weekweekss. 3.9 Table 3 Reaction of callus that was initiated on dicamba and cultured regeneration medium for nine weeks.. 3.11 Table 4 Reaction of callus that was initiated on NAA and cultured on regeneration medium for nine weeksweeks.. 33.11.11 Table 5 Transient transformation frequencies for different bombardment conditions obtained with a Biorad® PDS 1000/He particle gun at the USDA. 4.10 Table 6 Optimisation of transient transformation frequencies for O. thyrsoides A2 at the UCT using pB1221pB1221. 44.11.11 Table 7 Bombardment of callus tissue for stable transformation at the USDA. 4.13 Table 8 Bombardment of callus tissues for stablea transformation at the UCTUCT.. f 4.13 Table 9 Bombardment of callus tissues for stable transformation at the UCT using the Taxi protocol.ty o 4.13 Table 10 PAT expression in putative transgenic plants analysed with ELISA. 5.17 Table 11 Leaf paint assay results of putative transgenic PPT-resistantPPT -resistant plants fromUniversi the USDA 5.21 Table 12 Leaf paint assay results from clones of T1 obtained from the UCT study 5.22 Table 13 Summary of molecular analyses performed on T1 and 17 clones from it. 5.26 TableTable ofof contentscontents AcknowledgementsAcknowledgements AbbreviationsAbbreviations List ofof figuresfigures List of tablestables Chapter 1: Summary 1. 1 Chapter 2: General introduction 2.1 OmithogalumOmithoga/um - a brief history 2.1 2.2 Economical importance 22.2.2 2.3 The need for a transformation system for Omithogalum 2.4 2.4 Plant transformation -- an overview 2.6 - Agrobacterium-mediated gene transfer 2.7 Direct gene transfer 2.9 Particle bombardment devices 22.10.10 Transient expression 22.11.11 The Taxi transformation system 2.12 2.5 Isolation of transgenic plants 22.13.13
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