Browsing by Author "Huckett, Barbara Isobel."

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Animal model studies on the antelope schistosomes, Schistosoma margrebowiei and S. leiperi, with particular reference to their proposed role in limiting the distribution of human intestinal schistosomiasis.
(1992) Dettman, Charles David.; Schutte, Chris H. J.; Huckett, Barbara Isobel.
It has been postulated that the absence of human and cattle schistosomiasis in parts of southern Africa where lechwe antelope (Kobus leche) occur is a consequence of an immunologically-mediated protection induced by repeated exposure to the cercariae of Schistosoma margrebowiei and S. Leiperi, which are common parasites of these animals. The aim of the studies described was the development of animal models in which to investigate this hypothesis. The infection characteristics of the antelope schistosomes in BALB/c mice and Mastomys Coucha were assessed. Both schistosome species reached full patency in these hosts, although S. Margrebowiei infections deteriorated rapidly in M.Coucha. While they differed markedly in terms of egg production rates and preferred sites of tissue egg deposition, both species caused severe hepatosplenomegaly and portal hypertension in the mouse model. Modulation of the granulomatous responses to ova in the tissues was demonstrated. Mice harbouring mature antelope schistosome infections displayed strong partial resistance to challenge infections with both homologous parasites and the human schistosome, S. Mansoni. However, the failure of challenge parasites to become established was considered to be due largely to changes in the portal-hepatic vasculature resulting from egg-induced immunopathology. Resistance to S. Mansoni challenge did not develop in mice infected with radiation-attenuated cercariae of the antelope schistosomes. The suitability of rats and guinea pigs as alternative models was assessed. Worm recoveries from rats were low and there was no evidence of egg-deposition. Worm yields from the guinea pig were relatively high, but sexual development was poor and short-lived. Since excretion of S. Margrebowiei eggs has occasionally been reported from humans, and since the guinea pig supports full sexual maturation of S. Mansoni, this animal appeared to provide a particularly appropriate model for the present investigation. However, repeated exposure of guinea pigs to cercariae of the antelope schistosomes, over a period of 24 weeks, failed to induce significant resistance to S. Mansoni challenge infection. The need for further experimental and field studies is discussed. An area in the Okavango Delta (Ngamiland, Botswana) has been identified as a possible site for field work.
The application of microsatellites to sugarcane parentage determination and varietal identification.
(2002) Hack, Simon Matthew.; Huckett, Barbara Isobel.; Butterfield, Michael.
The use of microsatellite markers has matured and become commonplace for plant genome analyses and is now poised for widespread practical application in sugarcane. Sequence Tagged Microsatellite Site (STMS) amplification is the most prevalent microsatellite-based approach and involves the amplification of a microsatellite by designing primers that flank and hence define the microsatellite site, revealing variation in the length of repeat motifs between individuals. Twenty-six microsatellite primer pairs received from the International Sugarcane Microsatellite Consortium (ISMC) were evaluated and the STMS protocol was optimised to ensure robust and reproducible results. The objectives of this study were to use STMS for sugarcane parentage analysis and fingerprinting. Previously, Restriction Fragment Length Polymorphism (RFLP) marker data had suggested that the parentage of a genetic mapping population, sugarcane cross AA40 (N18 x CP57/614), was incorrect. Based on the assertion that the incorrect parentage was as a result of either mislabelling at planting or at seed collection, microsatellite parentage analysis was carried out on eight potential parent pairs (13 cultivars). A total of 75 markers were scored with non-parental bands (12 on average) being observed for all of the potential parent pairs and none could be identified as the true AA40 parents. It has been suggested in other plant species that PCR artefacts could give rise to non-parental bands and to investigate this the marker data of single parent DNA reactions and pooled parent pair DNA reactions or 'synthetic offspring' were compared. The results suggested that either a certain percentage of non-parental bands, perhaps 10% (maximum value observed), should be tolerated in microsatellite parentage analysis or a marker should only be considered to be discriminating for parentage if it is absent in both the parents and the pooled parent pair amplifications. Fingerprinting of 20 cultivars using 14 microsatellite primer pairs was conducted to evaluate the potential of the STMS approach for sugarcane varietal identification. It was found that only two microsatellite primer pairs were required to discriminate between all 20 cultivars with a theoretical number of non-differentiated pairs of cultivars (XK) of only 0.03. This estimator was used to determine the approximate number of microsatellites necessary for large-scale sugarcane fingerprinting.
Characterisation and role of sugarcane invertase with special reference to neutral invertase.
(2000) Vorster, Darren James.; Botha, Frikkie Coenraad.; Huckett, Barbara Isobel.
The relationship between extractable invertase activities and sucrose accumulation in the sugarcane (Saccharum spp. hybrids) culm and in vivo invertase mediated sucrose hydrolysis was investigated to determine the significance of invertases in sucrose utilisation and turnover. In vitro activities were determined by assaying the soluble acid invertase (SAI), cell wall bound acid invertase (CWA) and neutral invertase (NI) from internodes three to ten in mature sugarcane plants of cultivar NCo376. Extractable activities were verified by immunoblotting. In vivo invertase mediated sucrose hydrolysis was investigated in tissue discs prepared from mature culm tissue of the same cultivar. Sugarcane NI had a higher specific activity than SAI (apoplastic and vacuolar) in the sucrose accumulating region of the sugarcane culm. CWA was also present in significant quantities in both immature and mature tissue. Sugarcane NI was partially purified from mature sugarcane culm tissue to remove any potential competing activity. The enzyme is non-glycosylated and exhibits catalytic activity as a monomer, dimer and tetramer. Most of the activity elutes as a monomer of native Mr ca 60 kDa. The enzyme displays typical hyperbolic saturation kinetics for sucrose hydrolysis. It has a Km of 9.8 mM for sucrose and a pH optimum of 7.2. An Arrhenius plot shows the energy of activation of the enzyme for sucrose to be 62.5 kJ.mol-1 below 30°C and -11.6 kJ.mol-1 above 30°C. Sugarcane NI is inhibited by its products, with fructose being a more effective inhibitor than glucose. Sugarcane NI is significantly inhibited by HgCI2, AgNO-3, ZnCI2, CuSO4 and CoCI2 but not by CaCI2, MgCI2 or MnCI2. Sugarcane NI showed no significant hydrolysis of cellobiose or trehalose. When radiolabelled fructose was fed to sugarcane internodal tissue, label appeared in glucose which demonstrates that invertase mediated hydrolysis of sucrose occurs. A combination of continuous feeding and pulse chase experiments was used to investigate the in vivo contribution of the invertases and the compartmentation of sugars. Sucrose is synthesised at a rate greater than the rate of breakdown at all stages of maturity in sugarcane culm tissue. The turnover time of the total cytosolic label pool is longer for internode three than internode six. A higher vacuolar:cytosolic sugar molar ratio than previously assumed is indicated. Developmentally, the greatest change in carbon allocation occurs from internodes three to six. The main competing pools are the insoluble and neutral fractions. As the tissue matures, less carbon is allocated to the insoluble and more to the neutral fraction. The neutral fraction consists mainly of sucrose, glucose and fructose. The compartmented nature of sugarcane storage parenchyma carbohydrate metabolism results in a system that is complex and difficult to investigate. A computer based metabolic flux model was developed to aid in the interpretation of timecourse labelling studies. A significant obstacle was the global optimization of the model, while maintaining physiologically meaningful flux parameters. Once the vacuolar:cytosolic molar ratio was increased, the model was able to describe the internode three and six labelling profiles. The model results were in agreement with experimental observation. An increase in the rate of sucrose accumulation was observed with tissue maturation. Only the internode three glucokinase activity was greater than the experimentally determined limit. The rate was however physiologically feasible and may reflect the underestimation of the in vivo rate. SAI and NI contributed to sucrose hydrolysis in internode three but not in internode six. The rates in internode six were set to fixed low values to enable the model to fit the experimental data. This does not however preclude low levels of in vivo SAI and NI activity, which would prove significant over a longer time period. The flow of label through the individual pools, which comprise the experimentally measured composite pools could be observed. This provides insight into the sucrose moiety label ratio, SPS:SuSy sucrose synthesis ratio, and the rate of 14CO2 release. The model provides a framework for the investigation and interpretation of timecourse labelling studies of sugarcane storage parenchyma.
Comparision of two promoters driving transgene expression in water-stressed sugarcane.
(1999) Cassim, Tasmien Nadine.; Huckett, Barbara Isobel.; Botha, Frikkie Coenraad.; Watt, Maria Paula Mousaco Deoliveira.
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.
Development of in vitro culture and gene transfer techniques in sugarcane (Saccharum species hybrids).
(1992) Snyman, Sandra Jane.; Huckett, Barbara Isobel.; Watt, Maria Paula Mousaco Deoliveira.
In vitro cell and tissue culture systems were developed for sugarcane in order to utilise current transformation techniques to introduce genes to South African sugarcane varieties, which would be difficult, if not impossible to achieve in conventional breeding programmes. Embryogenic calli were initiated in the dark from stem explants of sugarcane varieties NCo376 and N13, on a MS medium containing sucrose (20-50 g/l), 2,4-D (2-4 mg/l), casein (1 g/l), inositol (100 mg/l) and agar (9g/l). After 2 months the somatic embryos were cultured in a light/dark photoperiod for a further 2 months. The best combination of sucrose and 2,4-D for callus initiation, and subsequent plant regeneration, was 20 g/l and 2 mg/l, respectively. Plant yields ranged from 16 to 36 plants per gram fresh weight callus, and the yields were not significantly increased by the addition of activated charcoal to the regeneration medium. When plantlets reached a height of 10 cm, they were transferred to autoclaved soil in pots, hardened-off and placed in the glasshouse. Suspension cultures were initiated from friable NCo376 calli in liquid MS medium shaken at 100 rev/min in the dark at 27°C, and were subcultured every 3-7 days. Protoplasts from various sources (leaf, calli and suspension cultures) were obtained after enzymatic digestion in cellulase (20-30 g/l), macerozyme (0,2 g/l), hemicellulase (5 g/l), and sorbitol (0,55 M) in a calcium and magnesium salt solution. Protoplasts cultured for 48 h resulted in a loss in viability of 84%. The potential of the seed as a recipient for direct gene uptake was investigated, as this eliminated the need for in vitro culture and plant regeneration. Uptake of [3H] pBR322 DNA by seeds was demonstrated, and seeds with the testa removed exhibited higher initial uptake rates than those with intact seed coats. However, transient expression, using the GUS reporter gene (coding for bacterial B-glucuronidase) carried on plasmid pBI221, could not be conclusively shown using the histochemical GUS assay, due to GUS activity generated by either microbial contamination or endogenous plant GUS activity. Neither microwaving to eradicate contaminants nor the addition of methanol (20%) to the GUS incubation buffer were successful in overcoming positive results observed in control seeds. An alternative approach to sugarcane transformation, using PEG-mediated DNA uptake and subsequent transient expression of GUS by protoplasts was investigated, but microbial contamination was a persistant problem and no positive results were observed. Further examination and elimination of endogenous contamination is required before transformation studies can be continued.
Development of novel antibacterial and antiviral transgene vectors and techniques for their application and analysis in sugarcane.
(2002) Pepper, Timothy Bryan.; Watt, Maria Paula Mousaco Deoliveira.; Rutherford, Richard Stuart.; Huckett, Barbara Isobel.
Sugarcane is challenged by a number of phytopathogenic bacteria and viruses that are best managed by the development of resistant varieties. Genetic engineering is a promising strategy in such breeding efforts, as it allows novel mechanisms of resistance not available in any parent germplasm to be introduced into the crop. DNA sequences encoding cystatin from papaya (Carica papaya), and pleurocidin from the winter flounder (Pleuronectes americanus) were envisaged as transgenes in this work due to their theoretical potential to increase sugarcane resistance to viruses and pathogenic or opportunistic bacteria, respectively. Cystatin is a cysteine proteinase inhibitor. Cysteine proteinases are used by potyviruses to cleave the polyprotein gene product, an essential step in the viral life cycle. Constitutive expression of cystatin may therefore lend the host plant resistance to a range of potyviruses, including the economically important pathogen sugarcane mosaic virus (SCMV). Pleurocidin is an amphipathic, α-helical, cationic peptide, with broadspectrum anti-bacterial activity at physiological pH. By binding to the cell membranes of both Gram positive and Gram negative bacteria, pleurocidin disrupts the membrane potential, causing it to become more permeable, especially to cations, leading to death of the bacterial cell. Initial microbiological bioassays showed that pleurocidin has inhibitory and bactericidal effects on the organisms which cause leaf scald (Xanthomonas albilineans), gumming disease (Xanthomonas campestris pv. vasculorum) and post-harvest sucrose conversion in sugarcane, as well as inhibitory effects against Leifsonia xyli ssp. xyli, which causes ratoon stunting disease (RSD). For transformation vector construction, the cystatin and pleurocidin coding sequences were altered so that their start codons were in the most favourable consensus context for expression in monocotyledonous plants. In the case of pleurocidin, an extracellular peroxidase signal sequence was attached. The prepared sequences were spliced into the vector pUBI510 in which the gene of interest is driven by the CaMV 35S promoter linked in tandem to a derivative of the maize ubiquitin promoter. The constructs generated were named pUBI510-cys3 and pUBI510-pleur08 respectively. The plasmid structures were confirmed using restriction endonuclease analysis and DNA sequencing. Since the transformation of sugarcane is known to be inefficient, two routes of morphogenesis for the production of somatic embryos were compared in the transformation procedure. These were (1) indirect embryo production via callus and (2) the direct and indirect production of embryos from transverse sections of leaf roll. Field grown sugarcane varieties N12 and NCo376 were the source of explant material. Plasmids pUBI510-cys3 and pUBI510-pleuro8 were respectively co-delivered by microprojectile bombardment with the antibiotic resistance selection plasmid pUBIKN containing the neomycin phosphotransferase gene (npt-II). Cultures were maintained in the dark on selection medium containing various concentrations of the antibiotic geneticin (G418) for several weeks before being allowed to regenerate in the light. Plantlets coming through selection were hardened off in the glasshouse when approximately 100mm high. Primer pairs for amplification of the cystatin insert were designed in various ways. The primer pair which ultimately proved most useful was designed to be complementary to the 5' and 3' ends of the papaya cystatin nucleotide sequence. Primer Premier analysis of a sorghum cystatin sequence provided additional possible primers. A further pair for potential future use was devised based on complementarity to conserved regions on maize cystatins 1 and 2, sorghum, rice, and papaya cystatins. The nucleotide sequence was constructed using the most common monocotyledon codon permutations for each amino acid. Pleurocidin primers were designed to be complementary to 5' and 3' regions of the nucleotide sequence encoding the pleurocidin pre-pro-protein. PCR and RT-PCR protocols for the detection of transgenes and transcript production in putative transgenic plants were developed using these primers. No plants survived selection via the callus route, although some were regenerated via direct embryogenesis. Putative transformed plants were analysed using PCR to test for the presence of integrated transgenes and Southern hybridization to determine transgene copy number. Both types of transgene were reproducibly detectable by PCR in DNA from some immature plants, but results were negative in DNA from those same plants when mature. Southern hybridization analysis detected the cystatin transgene in DNA from immature plants but no transgenes were detected in up to 20 µg DNA from mature plants. Single copy constructions of the transgenes in backgrounds of non-transformed DNA were detectable by both PCR and Southern hybridization analysis. Overall, PCR, RT-PCR and Southern hybridization results indicated that the plants regenerated fell into two categories: non-transformed plants that had survived selection (escapes) and chimaeric individuals with a component of both transformed and non-transformed cells, in which the transgene had probably become diluted during plant development under non-selective conditions. A method for extracting leaf exudates was tested, in conjunction with a cysteine proteinase assay to detect the presence of cystatin transgenes in the intracellular spaces of sugarcane leaves of confirmed transformants. Although it could not be applied within the scope of this project, this assay will prove useful in future work.
DNA restriction fragment length polymorphisms in the identification of clonal variants of eucalyptus.
(1993) Coulson, Mornay.; Huckett, Barbara Isobel.; Watt, Maria Paula Mousaco Deoliveira.
The technique of restriction fragment length polymorphism (RFLP) analysis, of chloroplastic and genomic DNA, was investigated as a means of identifying eucalypt species and cultivars which are morphologically indistinguishable from one another. In order to resolve chloroplast DNA (cpDNA) RFLPs, a method was developed to extract high yields of intact chloroplasts from Eucalyptus grandis S/N M6. Starch contamination was reduced by incubation of saplings in the dark for 48 h prior to extraction and watering with a solution containing 370 mM Na-phosphate and 296 mM KN03. Optimal chloroplast yields (25 ug chlorophyll/g fresh mass) were obtained by chopping leaf material, using a vertical homogenizer, in a buffer containing 350 mM sorbitol, 50 mM tris-HCL and 5 mM EDTA, 0.1 % (w/v) bovine serum albumin, 0.15 % (w/v) 2-mercaptoethanol, 2 mM L-ascorbic acid and 1 mM MgCI2 followed by washing of leaf pieces in a buffer containing only sorbitol, tris-HCL and EDTA. When these chloroplasts were used in an "in-organelle" DNA digestion procedure, polymorphisms were observed between the cpDNA profiles resolved for E. grandis S/N M6 and that of an outgroup species (spinach). However, the developed chloroplast extraction technique could not be used to obtain chloroplasts from various other eucalypt species, probably as a result of variability in the material at an ultrastructural or biochemical level. For the analysis of genomic DNA RFLPs, a DNA extraction procedure was optimized for use with various eucalypt species and cultivars. This included the development of a purifcation technique during which DNA was ammonium acetate-ethanol precipitated and subjected to mini-dialysis. Following Dra I restriction of DNA, the extract was electrophoresed and Southern blotted onto both nylon and nitrocellulose membranes. These were probed with a Hind-III restricted sample of the multilocus plasmid probe pV47-2. This probe was labelled using 32p as well as a non-radioactive labelling substance digoxygenin (DIG). Hybridization conditions, including the composition of the hybridization buffer, were optimized for use with these labels, and DNA RFLPs (fingerprints) were resolved for the eucalypt species E. grandis and E. macarthurii and cultivars of E. grandis (S/N M6, TAG 5 and TAG 14). An average of 8.5 bands were detected with 32p and 5.0 fragments with DIG. All the species and cultivars fingerprinted with the 32P-label could be distinguished from one another. However, as a result of the reduced sensitivity of the DIG system, two of the E. grandis cultivars, S/N M6 and TAG 5, could not be differentiated. It is concluded that the latter system would be most suitable for incorporation into a routine eucalypt screening programme, although it is suggested that the colourimetric detection assay, used in this study to resolve DNA bands, be replaced by a more sensitive one.
Effects of nitrogen nutrition on salt stressed Nicotiana tabacum var. Samsum in vitro.
(1992) Sweby, Deborah Lee.; Watt, Maria Paula Mousaco Deoliveira.; Huckett, Barbara Isobel.
The responses of Nicotiana tabacum L. var. Samsun to alterations in the nitrogen (N) supply under saline conditions in vitro were monitored. The aim was to test the hypothesis that nitrate-nitrogen supplementation to salt stressed plants alleviates the deleterious effects of salt on plant growth. Due to its capacity to be maintained under stringent environmental conditions, in vitro shoot cultures were chosen as the system of study. Nicotiana tabacum plantlets regenerated from callus in vitro were excised and rooted on solid MS culture medium containing a range of concentrations of NaCI (0 - 180 mM) and N (0 - 120 mM, as NO3--N, NH4+-N or a combination). A variety of parameters of root and shoot growth, nutrient utilisation and nitrogen metabolism were assessed over a 35 d period. Plant growth on 40 mM NO3--N + 20 mM NH4+-N (standard MS nutrients) was inhibited by the presence of salt, with root growth being more adversely affected by salt than stem growth. Root emergence was delayed from 6 d (0 mM NaCI) to 15 d (180 mM NaCI). Similar suppression of growth for all parameters, except root mass and leaf chlorophyll content, was observed when NaCI was replaced with mannitol at equivalent osmolalities. Root mass and leaf chlorophyll were significantly improved in plantlets supplied with mannitol. The time of root emergence was unaffected by mannitol supply, with all roots emerging after 10 d in culture. Plantlet growth on NH4+-N only (0 - 60 mM) was severely inhibited, even in the absence of NaCI, and was inferior to growth on NO3--N. Nitrate additions to salt stressed plantlets could not match growth in control (0 mM NaCI) plantlets. When plantlets were cultured on NO3--N only (0 mM, 30 mM, 60 mM, 120 mM), the increase in nitrate supply up to 60 mM resulted in a small improvement in growth on 90 mM NaCI, but had almost no effect on growth at 180 mM NaCl. A nitrate supply of 120 mM led to growth inhibition in all parameters, even in the absence of NaCl. Plantlet growth on isosmotic concentrations of mannitol in the presence of 0 - 120 mM NO3--N essentially mimicked that of NaCI, except for leaf chlorophyll content which was improved on mannitol at all NO3-·N levels. Nitrate uptake (measured as depletion from growth medium) by plantlets grown on 0 - 180 mM NaCI was positively correlated to availability of nitrate but negatively correlated to NaCI supply. Similar results were obtained for a mannitol supply except nitrate uptake was enhanced significantly on mannitol compared to NaCl. Sodium and chloride uptake appeared unaffected by nitrate concentration. Leaf protein content responded favourably to an increase in the NO3--N supply up to 60 mM and, in particular, appeared to be stimulated in the presence of 180 mM NaCl. Nitrate reductase (NR) activity was found to be inhibited drastically by salt and NO3--N supplementation to the salt medium had no effect on enzyme activity. A reduction in leaf total RNA content was recorded with an increase in NaCI concentration from 0 - 180 mM. A positive response to an increase in the NO3--N supply from 30 mM to 60 mM was detected in the presence of NaCl. Attempts were made to assess the levels of mRNA for NR in response to the various NaCl and N regimes. The plasmid pBMCI02010 containing a NR cDNA insert was isolated and purified and used in both radioactive and non-radioactive RNA slot blot hybridisation procedures. However, due to problems of non-specific binding of the probe, no quantification of the levels of NR mRNA in response to the various treatments could be made. Nitrate supplementation to plantlets of Nicotiana tabacum growing in vitro did not appear to ameliorate the effects of salinity stress, such that growth of plantlets in the presence of NaCI was always inferior to that in the absence of NaCl. As a large portion of growth inhibition was found in this study to be a result of osmotic rather than ionic effects of salt, it is questioned whether a nitrate supply would have an ameliorating effect on plant growth under field conditions.
Evaluation of sugarcane varieties for resistance to ratoon stunting disease.
(2003) McFarlane, Sharon Anne.; Huckett, Barbara Isobel.; Bailey, Roger.
Ratoon stunting disease (RSD), caused by the bacterium Leifsonia xyli subsp xyli, is well established in most sugarcane growing regions of the world and is considered to cause more yield losses worldwide than any other sugarcane disease (Hughes, 1974). In South Africa, field trials have demonstrated that yield reductions under rainfed conditions can exceed 40% in highly susceptible varieties (Bailey and Bechet, 1986). When cane is grown under irrigation, yield losses are less noticeable but still significant in many varieties (Bailey and Bechet, 1995). It is estimated that RSD currently results in a one percent reduction in industrial production in South Africa and between 10 and 20% in other African countries where South African varieties are grown (Bailey and McFarlane, 1999; Rutherford et al., 2003). For many years, the reaction of different sugarcane varieties to RSD has been based on large, replicated yield loss trials grown over a number of years under rainfed and irrigated conditions. Although these trials provide valuable information, they are time-consuming and require large areas of uniform land. They are therefore not suitable for incorporation into a routine disease screening programme in which large numbers of genotypes are assessed for their reactions to the important diseases occurring in the industry. As a result, the susceptibility of new commercial varieties to RSD is only known several years after release to the growers. The main objective of this study was to establish a suitable method to reliably evaluate sugarcane genotypes for RSD resistance as part of the plant breeding and selection programme. Emphasis was placed on the use of the tissue blot immunoassay (TBlA) developed by Harrison and Davis (1988) and modified by Davis et al (1994), in relation to the more traditional methods of variety assessment, such as the rate of spread of RSD in the field at harvest and yield loss trials. Although the immunoassay protocol was not altered, slight modifications to the blotting procedure resulted in clearer blots that were easier to interpret. Internode position and the age of the cane were shown to have a marked effect on the extent of colonisation and ultimately the RSD resistance rating. A trial investigating the effect of the extent of colonisation on the rate of spread of RSD at harvest was conducted and showed that the relationship between spread and colonisation was highly significant. This indicated that RSD spread more rapidly through varieties such as N14 and N22 that supported high populations of L. xyli subsp xyli. The control plots in the same trial provided useful information on the extent of colonisation in the twelve varieties planted. In another trial, the effect of RSD on the yield components of six commercially grown varieties was investigated and TBIA was also conducted to compare the two methods of variety assessment. The relationship between yield loss and the extent of colonisation was significant in both the plant and first ratoon crops. TBIA produced consistent results and the ranking of the six varieties was virtually identical, despite the different growing conditions during the two crop cycles. In an attempt to screen large numbers of genotypes under controlled glasshouse conditions, .TBIA was also tested on RSD-infected sugarcane transplants (seedlings). The results of this trial were variable and could not be reliably used as a screening tool. Based on the findings of this study, TBIA has now been adopted as a quicker and cheaper alternative to immunofluorescence microscopy for diagnosing RSD in sugarcane transplants. More importantly, TBIA has been accepted as a method of screening genotypes routinely for resistance to RSD and the first screening trial was planted in November 2002. It will now be possible to inform sugarcane growers of the RSD status of the new varieties as they are released, enabling them to make more informed decisions on how to manage each variety. This information will also be valuable when selecting parents in the crossing programme, with a long term view of improving the general resistance of commercially grown varieties to RSD. This should ultimately result in a substantial reduction in RSD levels in the industry.
Gene transfer by receptor-mediated endocytosis : stable expression of NEO following insulin-directed entry into HepG2 cells.
(1989) Huckett, Barbara Isobel.; Hawtrey, Arthur O.; Ariatti, Mario.
Evidence is presented for DNA delivery to cultured HepG2 hepatoma cells via the endocytotic pathway, under the direction of insulin, in a soluble system of transfection leading to stable gene expression. Serum albumin treated at pH 5.5 and 20°C for 48-60h with the water-soluble carbodiimide N-ethyl-N'(3-dimethylaminopropyl) carbodiimide hydrochloride has been found to produce positively charged N-acylurea albumin capable of binding different types of DNA in a reaction which is at least partially electrostatic in nature (Huckett et al, 1986). N-Acylurea albumin, synthesised at an albumin to carbodiimide mole ratio of 1 : 500, resulting in the attachment of 27 Nacylurea moieties per albumin molecule, was covalently conjugated to insulin by glutaraldehyde cross-linkage in order to produce a macromolecule, insulin-[N-acylurea albumin], with the facilities f or both DNA transport and receptor binding. The resultant conjugate, purified by gel filtration through Sephadex G-100, was characterised in terms of molecular size, charge properties and insulin content by polyacrylamide gel electrophoresis, agarose gel electrophoresis and immuno-dotblotting respectively. The conjugated protein was shown by gel band shift and nitrocellulose filter binding assays to bind DNA non-specifically in a reversible reaction which occurs rapidly, is dependent upon protein concentration and the ionic strength of the medium, and involves at least two types of intermolecular interaction. Furthermore, the conjugate was shown by competitive displacement of [ 125I ]insulin to bind specifically and particularly avidly to the HepG2 insulin receptor. When the expression vectors ptkNEO and pAL-8 which incorporate the neo gene were complexed to the conjugate in an in vitro transfection procedure using HepG2 cells, G418 resistant clones developed at frequencies of 2.0 - 5.5 X 10-5, possibly dependent upon vector promoter. Subsequently, a 923bp PstI fragment within the neD sequence was identified by Southern transfer in genomic DNA extracted from transfected cell populations which had been grown on a G418 regime through several subculture passages over a period of 44 days.
The metabolic fate of sucrose in intact sugarcane internodal tissue.
(2000) McDonald, Zac.; Botha, Frikkie Coenraad.; Huckett, Barbara Isobel.
The study was aimed at determining the metabolic fate of sucrose in intact sugarcane internodal tissue. Three aspects of the fate of sucrose in storage tissue of whole plants formed the main focus of the work. These were the rate of sucrose accumulation in the developing culm, the characterisation of partitioning of carbon into different cellular organic fractions in the developing culm and the occurrence of sucrose turnover in both immature and mature stem tissues. Specific attention was paid to confirming the occurrence of sucrose turnover in both immature and mature internodal tissue. This sucrose turnover has been described previously in both tissue slices and cell suspension cultures. However, certain results from previous work at the whole plant level have indicated that sucrose turnover does not occur in mature internodal tissue. Radiolabeled carbon dioxide (14CO2) was fed to leaf 6 of sugarcane culms of a high sucrose storing variety (Saccharum spp. hybrid cv. Nco376). All plants were of similar age (12 months) and were grown under similar conditions. The movement and metabolic fate of radiolabeled sucrose was determined at four time points, (6 hours, 24 hours, 7 days and 6 weeks) during a 6 week period. The metabolic fate of sucrose was determined in internodes number 3, number 6 and number 9. Internode 3 was found to have a relatively high hexose sugar content of 42 mg glc&fruc fw g-1 and a low sucrose content of 14 mg suc fw g-1. In contrast the sucrose content of internode 9 was much higher at 157 mg suc fw g-1 and the hexose sugar content much lower at 4.3 mg glc&fruc fw g-1. Based on previous work, the sugar content of internode 3 and internode 9 are characteristic of immature and mature tissues respectively. Internode 6 occupies an intermediary position between internode 3 and 6 with its sucrose content higher than its hexose sugar content, but with the hexose sugar content still being notable at 15 mg glc&fruc fw g-1. Although the metabolic fate of sucrose within sink tissue was the focal point of the study, the experimental design also allowed for certain aspects of sucrose production in the source to be investigated. The average photosynthetic rate for leaf 6 in full sunlight was estimated at 48 mg CO2 dm-2 s -1. During photosynthesis, only 30% of the fixed carbon was partitioned into the storage carbohydrate pool while the remaining 70% was partitioned into sucrose for immediate export from the leaf. This high rate of carbon fixation combined with a high rate of carbon export is characteristic of C4 plants such as sugarcane. On entering the culm, translocation of radiolabeled sucrose was predominantly basipetal with relatively little acropetal translocation. The majority of the radiolabeled carbon was found to be stored in mature internodes. No significant loss of radiolabeled carbon was observed in mature and elongating internodes over the study period. A 22% loss of total radiolabeled carbon was observed in immature internodes over the same period. This can probably be attributed to the higher rates of cellular respiration known to occur in immature tissues. There appear to be three phases of sucrose accumulation in the developing culm. Initially, the accumulation rate in rapidly growing tissue, as internode 3 develops into internode 6, is relatively low. This is followed by a rapid increase in the rate of sucrose accumulation during internode elongation, as internode 6 becomes internode 9. Finally, a decrease in the rate of sucrose accumulation is observed during late maturation, as internode 9 becomes internode 12. Determination of the sucrose content in internodes 3, 6 and 9 revealed that there is a notable increase in sucrose content during internode maturation. It is proposed that the higher sucrose content of mature tissue is not merely a consequence of the longer growth period of mature tissue, but is due to the increased rate of sucrose accumulation observed during internode elongation. Short-term (24 hours) analysis of carbon partitioning revealed that intemodal maturation was associated with a redirection of carbon from non-sucrose cellulal organic fractions to sucrose storage. In immature internodes only 20% of the total radiolabeled carbon was present in the sucrose pool 24 hours after feeding. In elongating internodes the figure increased to 54% while in mature internodes as much as 77% of the total radiolabeled carbon was retained in the sucrose pool. Concomitant with the increased carbon partitioning into stored sucrose down the developing culm is a decrease in carbon partitioning into the hexose sugar pool. In immature tissue, 42 % of the total radiolabel is present in the hexose sugar pool, while in mature tissue the percentage drops to 11%. This decrease is probably indicative of decreased levels of carbon cycling between the sucrose and hexose sugar pool as a result of internode maturation. Internode maturation was also found to be associated with a decrease in the amount of carbon in the water insoluble matter pool and the amino acid/ organic acid/ sugar phosphate pool. Thus, internode maturation is associated with a redirection of carbon from total respiration to sucrose storage. Long-term (6 weeks) analysis of carbon partitioning confirmed that sucrose storage in mature tissue is greater than that in immature tissue. From the 6 hour time point to the 6 week time point, an 87% reduction in the stored radiolabeled sucrose content was observed in immature internodes. During the same period only a 25% reduction in the stored radiolabeled sucrose was observed in mature internodes. Radiolabel loss from the radiolabeled sucrose pool in both mature and immature internodes was accounted for by relative radiolabel gains in other cellular organic fractions. At all time points during the study, and in all three tissues studied, radiolabel was found in the sucrose pool, the hexose sugars pool, the ionic pool and the water insoluble matter pool. The occurrence of radiolabel in the non-sucrose tissue constituents suggests that sucrose turnover is occurring in both immature, and mature internodal tissue.
Regulation of nitrate reductase during in vitro differentiation of nicotiana tabacum L. var. samsun.
(1993) Roberts, Michael Austin.; Cresswell, Christopher F.; Huckett, Barbara Isobel.; Watt, Maria Paula Mousaco Deoliveira.
The commencement of in vitro differentiation is mediated by genetic changes that result in selective expression of genes and a shift in metabolism. The role of nitrate reductase, a key enzyme of nitrate assimilation, during differentiation was examined in this study using an in vitro Nicotiana tabacum (tobacco) callus culture system. In particular, the effects of nitrogen and light/dark regimes on callus differentiation and nitrate reductase were investigated. Methodology required for the analysis of nitrate reductase regulation during in vitro tobacco callus differentiation was established. Optimised in vivo, in situ and in vitro nitrate reductase assays yielded similar values and patterns during tobacco callus culture development, and the in vivo assay was selected for nitrate reductase activity measurement during subsequent experiments. Western blot analysis of tobacco callus acetone-extracted protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a spinach polyclonal nitrate reductase antibody yielded major bands at 71 and 48 kD, with numerous minor bands. Extraction of callus protein in the presence of various protectants did not prevent cleavage of putative nitrate reductase polypeptide. Slot blot detection of nitrate reductase mRNA using a [32p]- labelled nitrate reductase cDNA probe isolated from the plasmid pBMC102010 was not possible due to non-specific binding to nitrocellulose filters. Northern blotting of RNA fractionated by agarose gel electrophoresis using a [32p]-labelled nitrate reductase cDNA probe identified a single mRNA species at 3.5 kb, the expected size of tobacco nitrate reductase mRNA. In vitro tobacco callus differentiation on 60 or 120 mM nitrogen regimes and under light/dark (16/8 h), continuous dark or continuous light treatments were comparable in terms of fresh weight, protein and nitrate uptake. Higher levels of in vivo nitrate reductase activity were observed prior to visible shoot primordia in all treatments, suggesting that the developmental status of callus mediated the regulation of nitrate reductase. Putative nitrate reductase protein levels were not correlated with in vivo nitrate reductase activity during initial stages of tobacco callus differentiation under various light treatments; nitrate reductase mRNA levels could not be ascertained. These results suggested that post-translational control mechanisms were involved in nitrate reductase regulation during in vitro tobacco callus differentiation.