Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Agrobacterium tumefaciens-mediated transformation technology has been used in a
variety of applications throughout the fields of cellular and molecular plant biology as well
as plant physiology. Research is conducted in order to extend this application range and
overcome some of the intrinsic limitations of the Agrobacterium transformation system.
Predominantly, these limitations can be attributed to the host range specificity of
A. tumefaciens, as well as adverse effects induced on explant tissue by active plant
defence mechanisms, triggered by the plant-pathogen-interaction. Typically, this active
defence mechanism culminates in the hypersensitive response (HR), characterised by
localised cell death and necrosis.
Not all Agrobacterium species, however, share the same host range and some have
evolved the ability to infect plant species not normally considered hosts of A. tumefaciens.
This host range specificity can be exploited to extend the application of existing
Agrobacterium transformation systems. In an attempt to establish an efficient
transformation system for Vitis vinifera which, has proven very difficult to transform with
A. tumefaciens, indigenous A. vitis strains have been evaluated as possible host-specific
transformation agents. Strains of Agrobacterium vitis should be suitable for this type of
endeavour, since they have evolved several unique characteristics directly linked to the
infection of their hosts. These include the ability to utilise, tartrate, a host abundant carbon
source, as well as the production of an acid polygalacturonase that could play a role
during the infection process. The proposition that the evolution of A. vitis is a fairly recent
event is also confirmed by the relatively little divergence observed between A. tumefaciens
and A. vitis.
In this study, a selection of A. vitis strains were evaluated in screenings designed to
accentuate desirable traits in strains such as good infectivity of grapevine material
(presumably an indicator of an efficient mechanism of gene transfer to be exploited in an
engineered transformation system) as well as a favourable reaction (causing no necrosis)
on grapevine somatic embryos. Two strains produced large tumours on grapevine cuttings
and caused little necrosis on the somatic embryos. Significant variation in infectivity as
well as callus necrosis was observed between the strains as well as in a genotype-specific
manner on the host material. This genotypic-specific effect of either host or pathogen could be an indication of the degree of specialisation developed by plant pathogens to
infect specific hosts. On the basis of these results, it was possible to select an A. vitis
strain for further biochemical and genetic characterisation.
Simple biochemical analysis classified the strain as an octopine strain. DNA-DNA
hybridisation techniques combined with a plasmid walking technique resulted in the partial
characterisation of the T-DNA of the selected A. vitis strain. A partial restriction enzyme
map of the T-DNA was constructed and the T-DNA and flanking areas were cloned.
Significant differences, most notably, the absence of a TB-area as well as the absence of
the agrocinopine (aes) gene from the 5' area of the T-DNA, were observed. Partial
sequencing data indicated the presence of at least four conserved T-DNA genes located
on the TA-DNA, as well as the presence of three bacterial insertion (IS-)elements flanking
the region. Two of these IS elements, both related to the IS 110 family of IS elements have
not yet been reported in A. vitis. In fact, these two elements seem to be the 5' and 3' ends
of a disrupted element and could therefore have played an evolutionary role in the
development of this strain.
This study provides fundamental background for the development of a more efficient
transformation system specific for grapevine, exploiting same of-the unique characteristics
of one of its pathogens, A. vitis. / AFRIKAANSE OPSOMMING: Agrobacterium tumefaciens-gebaseerde transformasiesisteme word in "n wye reeks van
toepassings in die velde van sellulêre- en molekulêre plantbiologie asook plantfisiologie
aangewend. Navorsing word voortdurend onderneem om die inherente beperkinge van
die Agrobacterium-transformasiesisteem te oorkom en sodoende die toepassingsveld van
die sisteem verder te verbreed. Die beperkinge tipies aan dié sisteem kan hoofsaaklik
toegeskryf word aan die gasheerspesifisteit van A. tumeteciens, asook die negatiewe
reaksies op eksplantmateriaal wat deur die plant se aktiewe verdedigingsmeganisme,
soos ontlok deur die plant-patogeen interaksie, veroorsaak word. Hierdie aktiewe
verdedigingsmeganisme lei gewoonlik tot In hipersensitiewe respons (HR) in die plant, wat
deur gelokaliseerde selafsterwing en nekrose gekenmerk word.
Alle Agrobacterium-spesies het egter nie almal dieselfde gasheerreeks nie en sommige
rasse het as gevolg van evolusionêre ontwikkelings die vermoë verkry om plantspesies
wat normaalweg buite die gasheerreeks van A. tumefaciens val, te infekteer. Hierdie tipe
gasheerspesifisiteit kan uitgebuit word om die toepassingsmoontlikhede van bestaande
Agrobacterium-transformasiesisteme te verbreed. In In poging om In effektiewe
transformasiesisteem vir Vitis vinifera, In moeilik transformeerbare gewas, te ontwikkel, is
inheemse rasse van Agrobacterium vitis ondersoek as moontlike gasheerspesifieke
transformasie-agente. Rasse van A. vitis behoort uiters geskik te wees vir so "n
toepassing, aangesien hulle verskeie unieke eienskappe, wat direk aan die infeksie van
die gasheer gekoppel is, vertoon. Van hierdie eienskappe is onder meer die vermoë om
tartraat, In koolstofbron volop in druifplante, te benut. A. vitis produseer verder ook In suur
poligalaktorunase wat vermoedelik In rol in die infeksieproses speel. Die voorstel dat die
evolusionêre ontwikkeling van A. vitis In redelike onlangse gebeurtenis is, word onderskryf
deur die betreklike homogenisiteit met A. tumefaciens.
In hierdie studie is "n groep A. vitis-rasse met behulp van siftingsprosedures wat
daarop gemik is om gesogte eienskappe in rasse uit te wys, beoordeel. Die vermoë om
druifplantmateriaal te infekteer (wat vermoedelik "n aanwyser van "n effektiewe meganisme
van geenoordraging is wat in "n gemanipuleerde transformasiesisteem benut kan word),
sowel as 'n gunstige reaksie (d.w.s geen nekrose) op druifplant somatiese embrio's is van
die gesogte eienskappe waarvoor gesoek word. Twee rasse het groot tumors op druifplant-stingelsegmente veroorsaak terwyl hulle bykans geen weefselskade op
somatiese embrio's geïnduseer het nie. Betekenisvolle verskille in infektiwiteit en in
kallusnekrose is tussen die rasse sowel as in 'n genotipe-spesifieke-verhouding
waargeneem. Hierdie genotipe-spesifieke effek, kenmerkend van óf die gasheer óf die
patogeen, kan aanduidend wees van die vlak van spesialisasie wat heers by die infeksie
van spesifieke gashere. Na aanleiding van bogenoemde resultate was dit moontlik om 'n
A. vitis-ras te selekteer wat verder aan biochemiese en genetiese analises onderwerp kon
word.
Eenvoudige biochemiese analises het dit moontlik gemaak om die ras as oktopien te
klassifiseer. DNA-DNA hibridisasietegnieke gekombineerd met 'n unieke plasmiedwandeltegniek
het gelei tot die gedeeltelike karakterisering van die geselekteerde A. vitisras.
In Gedeeltelike restriksie-ensiem (RE) kaart van die T-DNA kon gevolglik opgestel
word. Die T-DNA en die aangrensende gedeeltes is boonop gekloneer. Betekenisvolle
verskille, spesifiek die afwesigheid van In TB area, sowel as die afwesigheid van die
agrosinopien-sintasegeen (acs) aan die 51-kant van die T-DNA, is waargeneem.
Gedeeltelike basispaaropeenvolgingsdata het egter die teenwoordigheid van minstens vier
gekonserveerde T-DNA-gene, asook die teenwoordigheid van drie bakteriese
invoegingselemente (IS) aan weerskante van die area, geïdentifiseer. Twee van hierdie
elemente, wat beide homologie vertoon met die IS110 familie van IS elemente, is nog nie
vantevore in A. vitis aangetref nie. Dit wil boonop blyk of dié twee elemente die 51
- en 31
-
areas van In onderbroke element vorm, wat dus In moontlike aanduiding is van hul
potensiële rol in die evolusionêre ontwikkeling van die ras.
Hierdie studie verskaf basiese inligting wat daartoe kan lei dat 'n doeltreffender
transformasiesisteem spesifiek vir druifplante ontwikkel word deur van die unieke
kenmerke van een van sy patogene, A. vitis, uit te buit.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/51687 |
| Date | 03 1900 |
| Creators | Joubert, Dirk Albert, 1973- |
| Contributors | Pretorius, I. S., Vivier, M. A., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 117 p. : ill. |
| Rights | Stellenbosch University |
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