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. / Thesis (M.Sc.)-University of Natal, Durban, 1992.

Sugarcane is an important crop for South Africa. It provides employment and valuable foreign / Thesis (M.Sc.Eng.)-University of KwaZulu-Natal, 2007

TPC is a 16 000 hectare estate located in Moshi, Tanzania and is currently planted under 8 800 hectares of sugarcane and produces over 60 000 tons of sugar per annum. The influence of volcanic parent material and volcanic ash over TPC, together with the alluvial nature of many of the soils, has imparted a unique combination of soil mineralogical, physical and chemical properties. Furthermore, irrigation with poor quality water has led to sodicity problems on the estate. Understanding the mineralogy and sodicity effects on soil hydraulic properties across the estate can lead to better irrigation management where it is important to prevent the build-up of salts due to over-irrigation. In response to this need, a study was carried out with the aim of characterising the mineralogical, physical and chemical properties in the five management areas of the estate (North, East, West, South and Kahe), in order to determine the relationships between various measured parameters. A total of 70 fields across TPC, as well as four sites outside the estate and two ash layers, were chosen for sampling. Undisturbed soil cores and bulk samples were collected from the A and B horizons from 45 of these fields and the four sites outside. Selected fields were sampled at more than one site to assess field variability, and where cane growth was patchy selected fields were sampled in a patch of poorly growing cane and an adjacent patch of better cane growth. Bulk soil samples were collected from the remaining fields and the two ash layers. Double ring infiltration measurements were carried out on 25 of the selected fields. X-ray diffraction, transmission electron microscopy and aluminium, iron and silica extractions were carried out to determine the mineralogy. Physical and chemical measurements included water retentivity, saturated hydraulic conductivity, bulk density, particle size distribution, organic carbon, pH (H2O), electrical conductivity, water soluble and exchangeable cations (Ca, Mg, K and Na), cation exchange capacity and clay specific surface area. The particle size distribution showed that the soils were mainly loams and sandy loams. Organic carbon values were generally greater in the A horizon compared to the B horizon and varied between 0.4 and 2.5 % in the topsoil and 0.3 and 2.1 % (with the exception of field 11 which had an organic carbon of 4.0 %) in the subsoil. X-ray diffraction patterns of sand and silt fractions were dominated by sanidine while clay patterns were weak and had high backgrounds and very broad peaks, suggesting the presence of poorly ordered material in the clay fraction. The Al and Fe extraction methods and electron micrographs indicated that this poorly ordered material was allophane. However, the dominant clay mineral across the estate was halloysite, in both tubular and spheroidal form, as well as very small (<< 0.5 μm) kaolinite particles. There was also gibbsite in some of the samples analysed. The combination of allophane, halloysite, kaolinite and gibbsite indicated that the primary volcanic minerals have weathered to various degrees across the estate. This is reflected in the alluvial nature of the soils where less weathered material has been periodically deposited onto older, more weathered material over some parts of the estate. The south and west areas had a slightly higher Alo + ½ Feo ratio than the other areas in both the topsoil (1.07 and 0.95, respectively) and the subsoil (1.16 and 1.06, respectively), a possible consequence of less weathered alluvial material that was deposited in these areas. Although the concentration of allophane was low (< 5 %), even in the south and west areas, its presence greatly increased the clay specific surface area (up to 145.94 m2 g-1) and consequently had a significant influence on the soil physical and chemical properties. Water retention across TPC was high, particularly at the lower matric potentials (between 0.13 and 0.45, and 0.09 and 0.24 m3 m-3 at -33 kPa and -1500 kPa, respectively). The high water retention is a result of allophane which gives the soils a high adsorption capacity and a porosity that is dominated by micro-pores. Generally, the south area had the highest water retention at the various measured matric potentials which corresponds to the higher allophane content. Variability in water retentivity across areas and within fields limited further interpretation and correlation with the mineralogical results. Infiltration rate was lowest in the south (60.85 mm hr-1) and highest in the Kahe area (171.20 mm hr-1). The main factor influencing the final infiltration rate was the concentration of sodium in the soil, with higher concentrations causing soil dispersion and blockage of soil pores. Clay dispersion has led to the development of calcareous surface crusts and reduced porosity, thus reducing the infiltration rate. Sodium concentration in the soil is likely to have had a dominating effect over the mineralogical composition of the soil. Poor cane growth in the south and west areas corresponded to higher pH (up to 10.32), electrical conductivity (up to 614 mS m-1), sodium absorption ratio (up to 20.63) and water soluble and exchangeable sodium (up to 53.20 mmolc l-1 and 14.87 cmolc kg-1 soil, respectively) in these areas. The soils are thus more dispersive and the combination of sodicity and allophane has resulted in “fluffy” soils with small particles clogging soil pores and thus surface crusts have formed easily. The combined effect of mineralogy and sodicity in the south is further complicated by the presence of perched water tables. High adsorption capacities and the dominance of micro-pores allow the occurrence of significant capillary rise which brings salts to the soil surface, further exacerbating the sodicity problem. Therefore, over-irrigation should be avoided where soils are prone to sodicity from a combination of irrigation with poor quality water, perched water tables and strong capillary rise action. Fields which are currently experiencing the negative effects of high sodicity, require irrigation with good quality water and adequate sub-surface drainage to ensure the leaching of salts. Further studies with specific focus on the south and west areas would be beneficial in accounting for the variability and in drawing correlations between the mineralogy and sodium content of the soils with the other measured properties. Fields which are prone to increased sodicity through over-irrigation with poor quality water, have strong capillary rise from perched water tables and which require remediation through sub-surface drains can thus be distinguished and the factors influencing sugarcane growth can be more clearly understood. Growth depends on the combination of these soil’s unique mineralogy and sodium content and the influence they have on the infiltration rate, adsorption capacity, micro-porosity and capillary rise from the water table. For future work, water movement modelling to predict saturated and unsaturated flow, as well as in situ measures of unsaturated flow, will lead to further understanding of the soil hydraulic properties and aid in improved irrigation management. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Typescript. / Thesis (Ph. D.)--University of Hawaii, 1962. / Bibliography: leaves 135-148. / xii, 148 leaves 3 mount, col. ill., mount. diagrs., tables

Melomys burtoni and M. cervinipes naturally occur in habitats adjacent to sugarcane crops in north Queensland, have been trapped within sugarcane crops, and are potentially damaging to sugarcane crops. However, little is known about their biology and pest status in sugarcane crops and this information is needed by the industry for the development of a sustainable pest management programme for these rodents. Field studies were undertaken between Tully and Innisfail in far north Queensland, to determine the extent to which either or both Melomys species inhabit sugarcane crops and to examine the biology of Melomys within the crop. Field diagnostic approaches were developed which, when blind tested using molecular techniques, proved 100% accurate in-field discrimination of the two Melomys species. Based on field trapping, M. cervinipes proved to be rare in sugarcane and should not be regarded as a pest by the industry. In contrast, M. burtoni were recorded in significant numbers within cane, were found to feed on cane and, in crop stage 5 (canopy closure to harvest) were responsible for damage to ~5% of stalks. Melomys burtoni were found to colonise sugarcane at the later stages of crop development than the other major sugarcane rodent, Rattus sordidus. The highest proportion of M. burtoni reproduction and juvenile recruitment also occurs in the later stages of crop development. The late colonisation of the crop by M. burtoni means that the Integrated Pest Management (IPM) strategy already in place for R. sordidus is not directly transferable to M. burtoni. If an effective IPM strategy is to be developed, further research is required to examine the population dynamics and dispersal of M. burtoni populations between the crop and the adjacent habitats within the sugarcane production system of far north Queensland.

Sugarcane holds great potential as a biofactory for the tailored production of novel products of commercial value. In many cases however, the accumulation of an alien product within the cytoplasm interferes with essential cell metabolism. To avoid potential interference, targeting the accumulation of biofactory products into vacuoles may be beneficial. Vacuoles represent one endpoint of the plant endomembrane system where proteins destined for inclusion must contain appropriate targeting peptide signals. However, targeting peptide signals used previously to direct heterologous proteins to the vacuole have not yet been shown to function efficiently in sugarcane. The emphasis of the work described in this thesis was first to characterise the diversity of vacuole types in selected sugarcane tissues, and second to identify and test the function of putative vacuolar targeting signals identified in vacuolar proteins of sugarcane. In order to investigate vacuole diversity in sugarcane cells, a series of membrane-permeable fluorescent probes were used to assess both the acidity and proteolytic properties of vacuolar compartments. It is clear that even from early in development, large vacuoles filled most of the volume of storage parenchyma cells within the developing sugarcane stem. These vacuoles were intensely acidic and contained active aminopeptidases. In leaf cells, vacuoles labelled by chromogenic indicators and enzyme substrates appear much more diverse in pH and proteolytic intensity owing to the multiple functions that leaf cells participate in. As the predominant sugarcane vacuole in vegetative tissues appears to be proteolytic, sugarcane sequences showing homology to proteases and protease inhibitors in other plant species were aligned and compared to identify potential vacuolar targeting signals. This analysis revealed the presence of several candidate vacuolar targeting motifs which displayed high conservation across plant homologues. One such motif, represented by the sequence IRLPS, was identified in the N-terminal region of a legumain protein from sugarcane, which was homologous to known vacuolar processing enzymes in other species. To test the efficacy of the legumain targeting signal and to compare with other motifs, a series of GFP reporter constructs was synthesised and expressed in sugarcane. The sugarcane legumain vacuole targeting signal was particularly efficient at directing an otherwise secreted GFP fusion protein into a large acidic and proteolytic vacuole in sugarcane callus cells as well as in diverse plant species. In mature sugarcane transgenic plants, the stability of GFP fusion proteins in the vacuole appeared to be dependent on cell type, suggesting that the vacuolar environment can vary in its ability to degrade introduced proteins. The legumain vacuole targeting signal was further tested for its ability to direct an avidin protein and a fructosyltransferase enzyme into the lytic vacuole of transgenic sugarcane plants. Avidin, derived initially from chicken egg white, is a glycoprotein that displays a high affinity to the vitamin biotin. For this reason it has been investigated for use in sugarcane as a biocontrol agent against cane grub species. For the production of avidin in planta careful targeting to an appropriate subcellular location is required to avoid a detrimental depletion of available plant cell biotin reserves. When the legumain targeting signal was fused to avidin and expressed as a stably integrated transgene, the avidin protein was detected by immunoblotting but appeared to be proteolytically cleaved within the lytic vacuole in all sugarcane tissues analysed. These plants were phenotypically indistinguishable from controls, indicating that avidin did not appreciably deplete cellular biotin reserves while in transit through the endomembrane system. In contrast, when avidin was designed for either retention in the endoplasmic reticulum or for transit to a different type of vacuole using a heterologous targeting signal, stably transformed plants exhibited a biotin deficient phenotype. This suggests that the legumain vacuole targeting signal was efficient at directing heterologous proteins to a lytic type vacuole where they can be degraded and inactivated if susceptible to proteolysis. When the fructosyltransferase (ftf) gene from Streptococcus salivarius was stably transformed into sugarcane and directed into the lytic vacuole using the legumain vacuole targeting signal, no fructan product could be detected. The low pH and proteolytic environment of this vacuole together with low expression of this bacterial transgene most likely resulted in minimal Ftf activity. Taken together, evidence is presented that the legumain vacuolar targeting signal functions efficiently in directing transgene products such as GFP, avidin and a fructosyltransferase enzyme into a lytic type vacuole. This vacuole has been demonstrated to be both acidic and proteolytic and therefore strategies to improve the stability of heterologous proteins targeted to this vacuolar environment are required and may be specific to the product in question.

Copy of author's previously published article inserted. / Bibliography: leaves 180-195. / xii, 195 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigation of the contribution of polyphenol oxidase (PPO) and peroxidase (POD) to enzymic browning in sugarcane juice. / Thesis (Ph.D.)--University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 1995

Thesis (Ph. D.)--Brigham Young University. Dept. of History, 2005. / Includes bibliographical references (leaves 289-299).

Thesis (M.Ag.Sc.) -- Dept. of Soil and Water, Waite Agricultural Research Institute, University of Adelaide, 2002? / Bibliography: leaves 105-129.

Thesis (Ph.D.) - James Cook University, 2003. / Typescript (photocopy). Appendices: leaves 156-172. Bibliiography: leaves 149-155.