Potato leafroll virus (PLRV) is one of the most destructive potato viruses in South Africa. In order to establish resistance against PLRV in commercial potato cultivars, the coat protein (CP) gene of the virus was previously isolated, cloned and subcloned into the plant expression vector pBI121 in both the sense and antisense orientations (BURGER, unpublished results). The pBI121 constructs containing the PLRV-CP gene were subsequently transferred to Agrobacterium tumefaciens LBA 4404 in a triparental mating process with the helper plasmid pRK2013. Two A. tumefaciens- mediated transformation methods for potatoes were investigated, viz. vacuum infiltration and leaf disk transformation. In addition, optimal transformation and regeneration conditions were identified for potato cultivars Late Harvest and BP[1] In total, 27 transgenic potato lines containing the PLRV-CP, β-glucoronidase (GUS) and nptII (neomycin phosphotransferase II) trans genes were generated under kanamycin selection. Transgenic plants grown in the glasshouse appeared to be phenotypically normal, and no differences in ploidy level in comparison to non-transformed plants could be established. Stable transgene insertion into the genome of the transgenic plants was verified using PCR and Southern blot analysis. Expression of the GUS transgene was investigated using a fluorometric assay (JEFFERSON et al. 1987), and it was found that orientation of the inserted PLRV-CP gene upstream from the GUS gene had a direct influence on the levels of GUS expression. The expression of the PLRV-CP gene was analysed using DAS-ELISA and immunoblot detection. Coat protein could not be detected in either assay. RNA dot blots were used successfully to show PLRV-CP expression in transgenic potato plants at the mRNA level. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

For the expression of transgenes in plant cells, appropriate promoter sequences have to be introduced upstream of the gene to ensure efficient transcription. Tissue- or signal-responsive promoters are in high demand in practical plant biotechnology. The present study sought to characterise the activities of two promoters in sugarcane, namely the UBI (ubiquitin) promoter and the SUC-1 promoter (UBI linked in tandem to the cauliflower mosaic virus 35S promoter). It was hypothesised that the activity of UBI would be maintained or even increased under conditions of environmental stress, since it is well documented that ubiquitin is a stress-related protein. A further hypothesis was that SUC-1 might enhance overall gene expression since the CaMV 35S component is a constitutive promoter widely and successfully used in plant transformation. Plants of the sugarcane variety NC0310, containing the cry1A(c) (Bt) gene from Bacillus thuringiensis, were used as models in a system in which the plants were stressed by withholding water supply in a controlled manner. Since large numbers of clones of both transgenic and wild-type plants were needed for the water stress and expression experiments, three micropropagation techniques, namely, shoot tip-, callus- and node culture, were optimised and compared. The objective was to propagate genetically stable plants rapidly. Compared to shoot tip culture, node and callus culture proved slow and inefficient. Shoot tip culture was thus chosen as the most suitable for the regeneration of experimental material. Relative Water Content (RWC) determination, leaf elongation measurements and Infra Red Gas Analysis (IRGA) were compared in order to find the most appropriate method of measuring plant water status. In addition to being destructive, no observable differences were evident between the control (non-stressed) and water-stressed plants when using RWC as a measure. Results obtained from leaf elongation measurements compared favourably to the more sophisticated IRGA readings, showing that leaf elongation is as sensitive a measure of water stress. On the basis of preliminary studies with untransformed plants using the latter two techniques, water regimes for stress-induction in the final experiments were designed. Leaf elongation measurements, which are simple and non-destructive, were ultimately chosen to measure plant water status. In the final water stress experiment non-transgenic NCo310 and clonal populations of six transformants were used (three containing the UBI promoter; three the SUC-1 promoter). Exactly half of the plants of each type were stressed by withholding water supply, while the other half (controls) were watered manually twice a day. Leaf elongation measurements were made at the same time daily on the third youngest leaf of 6 plants from each population per treatment. At the same time, leaf samples were taken daily for molecular analysis. The stress regime led to marked differences in leaf elongation between control and water-stressed plants. In terms of physiological response (leaf rolling and senescing), plants containing the SUC-1 promoter appeared least affected. The reverse transcription-polymerase chain reaction (RT-PCR) and Northern hybridisation were used to assay UBI and SUC-1 activity. RT-PCR revealed that both promoters drove Bt gene expression in controls and experimentals throughout the stress period, although differences in signal intensity were not observed. The extent of expression occurring in each type of plant was revealed in Northern blots probed with two genic sequences (1) the transgene and (2) sugarcane EST ME42, homologous to heat shock protein 82 in rice. Individual transformants showed overall levels of transgene expression that were variable, possibly due to insert position in the plant genome, as well as variations in relation to the application of stress. SUC-1 seemed superior to UBI in terms of driving transgene expression under stressful environmental conditions, since UBI promoter activity appeared to decrease under stress, while SUC-1 promoter activity remained constant. In addition to the expected 2.0 kb Bt transcript, transcripts of smaller than expected size were also obtained, leading to the suggestion of premature polyadenylation signals in the coding region of the wild-type Bt234 gene. Upon inspection of the transgene sequence, a number of motifs rarely present in plant genes were observed, namely A/T rich sequences, ATTTA motifs and numerous potential polyadenylation sites. / Thesis (M.Sc.)-University of Natal, Durban, 1999.

The development of a genetic engineering system for soya bean (Glycine max L.) is described in this thesis. Routine tissue culture regeneration systems were developed for South African cultivars of soya bean despite the recalcitrant nature of this plant to in vitro manipulation. Regeneration of shoots was obtained when cotyledons were excised from seeds germinated for two days and cultured on B5 BA 20 medium containing 2 mg/I BA. The important problems of in vitro shoot elongation and rooting were overcome by culturing cotyledons in the dark for four weeks to produce shoots with unusually long stems. This was followed by one week of culture under conditions of high light intensity to obtain healthy green shoots which could be rooted , either in sorbarods or on solid Y2MS 30 medium. The use of a mist bed for the hardening off of rooted soya bean regenerants was essential for the recovery of fertile soya bean plants. Molecular techniques for the cloning of foreign genes into binary vectors suitable for plant genetic engineering were also studied and are described in the thesis. The Basta herbicide resistance gene, pat, was successfully cloned into the binary vector pBI121 which contains the [beta]-glucuronidase (GUS) reporter gene, uidA. The new construct, pB1121/Ac, was conjugated into various disarmed Agrobacterium tumefaciens strains and these strains, along with other binary vector-containing strains, were used to transform soya bean plant material. Although a protocol for the routine transformation of soya bean was not developed, transgenic soya bean material resistant to kanamycin and showing GUS activity was obtained. Transformation of wound sites on cotyledons was obtained in several experiments and transgenic shoots were regenerated from inoculated cotyledons. Only the A. tumefaciens strain C58C1 (pGV2260)(pJIT119) was able to transform cotyledonary cells of soya bean and, therefore, only kanamycin resistant soya bean shoots were produced. Transgenic soya bean plants resistant to the herbicide Basta were not produced due to the recalcitrant nature of the crop to genetic engineering. Transformation of the non-recalcitrant plant, tobacco, which is a model system for plant genetic engineering was achieved. The binary pat gene containing vector constructed in th is study, as well as vectors obtained from AgrEvo, were tested. The transgenic Basta resistant tobacco plants obtained were used to optimize assay systems for the analysis of transformed plant material containing the pat gene. These assay systems included the use of the polymerase chain reaction as well as digoxigenin-Iabelling of a DNA probe suitable for detection of the pat gene. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

Ornithine decarboxylase (ODC) is the rate determining enzyme in the biosynthesis of polyamines which are essential for cell growth. In chickens, significantly higher bioactivity is reported in broiler than in egg layer strains of chickens (Bulfield et al., 1988). To characterize the genetic differences in growth rates and ODC levels in chickens, an ODC cDNA and genomic gene were cloned and sequenced. Sequencing of ODC cDNA revealed that this clone (pODZ3: 2,052 bp) was not a full length of ODC cDNA and contained 2 putative introns. The open reading frame (introns deleted) coded for a protein of 404 amino acids which had about 85% amino acid identity with human ODC. Sequencing of genomic ODC clone (pODG2-8: 5098 bp) represented the 3$ prime$ end of ODC gene from downstream of intron 7. Northern blotting of chicken RNA probed with the insert of pODZ3 revealed 2 hybridizable messages of 1.6 and 2.1 kb, respectively. In addition, analysis of MspI restriction fragment length polymorphism (RFLP) using the 3$ prime$ end of ODC gene as a probe suggested that two MspI RFLPs present in the lean line of broiler chickens was related to selection of high lean body mass.

A hybrid vector, pBVl, has been constructed which is capable of regulating expression of cloned genes in both Escherichia coli and in cyanobacterium Synechocystis 6803. Vector pBVl contains the powerful rightward promoter of bacteriophage lambda to ensure that cloned genes are transcibed at a high level. In addition, pBV] also contains a gene, cI857, encoding the lambda temperature sensitive repressor protein.The CAT gene coding for chloramphenicol aceyltransferase (CmActase) was cloned into pBV1 downstream of the lambda regulatory features to make plasmid pTC1. The expression of the CAT gene was quantified spectrophotometrically following addition of chloramphenicol and a shift in the growth temperature of the cell culture. The specific activity of CmActase increased from 540 to 5400 units within 30 minutes. Thus, using the newly constructed hybrid vector, pBVl, temperature regulation of gene expression in E. coli was observed. / Department of Biology

A mouse embryo culture medium which would allow for in vitro development from 1-cell stage to blastocyst stage could offer many benefits for human research. Previous researchfrom our lab has demonstrated a mouse embryo culture medium named CZB seems to allow for in vivo-like conditions for development. Compared to other commonly used mouse embryo culture media, CZB medium promotes a higher frequency of 1 cell mouse embryos developing to blastocyst stage (Chatot et 1989). A key difference between CZB and other mouse embryo culture media is that CZB contains the amino acid glutamine metabolism is glutamate dehydrogenase (GDH). In order to determine if CZB cultured embryos follow in vivo-like patterns of gene expression for GDH, a quantitative competitive RT-PCR system was designed. A mutant GDH mRNA template was created which lacked a specific restriction enzyme site and was used as a competitive template in quantitative RT-PCR. This system was used to determine the amount of GDH mRNA present in in vivo grown blastocyst stage mouse embryos. It was determined that the amount of GDH mRNA present in in vivo blastocyst stage embryos was 282 fg/embryo. It is believed this system will also allow for quantitation of GDH mRNA in the earlier preimplantation stages of in vivo grown embryos, as well as the preimplantation stages 2-cell to blastocyst of CZB cultured embryos. / Department of Biology

The isolation and characterization of the gene(s) associated with and potentially responsible for the regulation of apomixis (asexual) and sexual reproduction in the grass species Tripsacum dactyloides is quintessential to agricultural advancement. Apomixis is the mechanism by which plants can produce seed without fertilization, where all progeny are genetically identical to the maternal parent. In natural populations of the genus Tripsacum, lower ploidy forms (i.e., diploid, 2n=36) reproduce sexually and the higher ploidy forms (triploid, 3n=54; tetraploid, 4n=72; etc.) reproduce asexually via apomixis. In order to gain a better understanding of sexual reproductive processes in plants, subtractive hybridization was performed on early and late female inflorescence gene products. This procedure allows for the recovery of gene products in the form of complimentary DNA, cDNA molecules, which correspond to messenger RNA (mRNA) present. These cDNA molecules were then used to extract unique sequence messages from the early developmental stage ovule tissue of diploid T. dactyloides. These cDNA molecules will allow for the direct isolation of the original form of the gene(s) from a large fragment library of the Tripsacum genome. Isolation and characterization of these gene(s) is of pivotal importance to our understanding of alternate modes of reproduction in Tripsacum. / Department of Biology

Genes responsible for differences in gene regulation and expression in normal cells and freezing tolerant cells were identified using two related wintergreen orchid species, Aplectrum hyemale and Tipularia discolor. Changes in gene expression observed in field-collected tissues obtained from different seasons were compared as were changes observed in plants subjected to cold shock in a laboratory environmental chamber. In order to clone these differentially expressed genes which may confer photosynthesis cold tolerance, the recently developed technique, mRNA differential display was employed. Using this process, mRNA was isolated from the tissue and reverse transcribed to cDNAs, which were amplified using specific anchored 3' primers and various random 5' primers. The 50-100 bands resulting from specific primers were compared on denaturing polyacrylamide gels. Bands differently expressed were excised from the gel and purified. In the future, if partial sequence analysis indicates they may code important regulatory proteins, they will be used as probes to obtain full-length genes from a cDNA library for further characterization. This study provides an opportunity not only to obtain important regulatory genes in plants, but also to understand more about temperature regulated gene expression in orchids. / Department of Biology

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