Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1984. / Bibliography: leaves [171]-179. / Microfilm. / xv, 179 leaves, bound ill. 29 cm

An outline is given of the South African sugar industry, with particular emphasis on the unit operations which make up the industrial process for manufacturing sugar from cane. Current knowledge of the chemistry of soluble polysaccharides is reviewed and the structures of several polysaccharides, including starch, dextran, and pullulan, are discussed. It has been found that changes take place in the chemical composition of the juice in sugar cane (Saccharum officinarum) during post-harvest storage. With increasing storage time, there is a proportional decrease in the starch content of the juice, and a considerably larger proportional increase in the soluble polysaccharide content. The increased polysaccharide content was found to be due to a single glucan which, contrary to most previous publications on this subject, is definitely not a dextran. Following structural analysis, it has been established that the polysaccharide formed in stored cane had not been described before and the name "sarkaran" , derived from the Sanskrit word "Sarkara", meaning "sugar" is proposed for it. The polysaccharide was isolated from cane juice by precipitation with ethanol after the starch in the juice had been removed by centrifugation. The polysaccharide was purified by repeated dissolution in water and reprecipitation with ethanol. Analysis by gel chromatography resulted in a single symmetrical peak, indicating that the isolated polysaccharide is homogeneous. This was confirmed by hydrolysing fractions representing a section of the ascending and a section of the descending part of the peak of the chromatogram, using the enzyme pullulanase. Chromatographic separation and quantitative analysis of the isolated oligosaccharides showed that the compositions of the two enzymes digests were identical. Acid hydrolysis of the polysaccharide resulted in a single hexose. This was identified as glucose by paper chromatography, comparing the Rf value with that of pure glucose. Confirmation was obtained by comparing the osazone with that of glucose, using microscopic examination and determination of the melting points. Paper electrophoresis showed the molecule to be uncharged. Several techniques, both absolute and non absolute, were used to determine the molecular weight of the polysaccharide. A method involving viscosity determination indicated a molecular weight of 34 000 while a figure of 50 000 was obtained by gel chromatography on a Sephadex column, comparing the peak elution volume of the polysaccharide with that of dextrans of a defined molecular weight. Both these techniques are non absolute and yield rough estimates of the molecular weight. Osmometric measurement, an absolute method, showed the number average molecular weight to be 51 500. An absolute value for the weight average molecular weight of 250 000 was obtained by light scattering techniques. Data from the light scattering experiments were also used to determine a value of 200 - 250 A for the radius of gyration RG of the polysaccharide. End group analysis after exhaustive methylation resulted in a value of 24 000 for the number average molecular weight Mn. This indicates either that some degradation of the polysaccharide molecule occurs , during the methylation procedures or that there is a certain degree of association between individual molecules. Periodate oxidation showed that 32 percent of the glucosidic linkages are in ( 1 + 6 ) position. The polysaccharide was exhaustively methylated by several Haworth methylations followed by a number of Kuhn methylations. The fully methylated product was methanolysed and the methyl glucopyranosides analysed by gas liquid chromatography. The results were compared with those obtained from fully methylated starch and dextran. From the absence of disubstituted methyl derivatives in the methanolysate it was concluded that the polysaccharide is an unbranched glucan. From the quantities of Methyl 2,3,4,6 tetramethyl-O-Dglucopyranoside, Methyl 2,3,6, trimethyl-O-D-glucopyranoside and Methyl 2,3,4? trimethyl-0-D-glucopyranoside, it was concluded that the only linkages in the glucan are ( 1 + 4 ) and ( 1 + 6 ) and that these are present in the ratio 68:32. Enzymic hydrolysis, using pullulanase, was followed by paper chromatographic separation. Quantitative determination of the oligo-saccharides present in the enzyme digest resulted mainly in two oligosaccharides, maltotriose and maltotetraose, in nearly equal proportions. For this reason it was postulated that the polysaccharide is a maltotriose-maltotetraose polymer, and that the individual units are linked in ( I + 6 ) position, a linkage for which pullulanase is specific in certain configurations. The sequence of the maltotriose and maltotetraose units in the polymer has not been investigated further, although this could be carried out by partial acid hydrolysis, followed by isolation and identification of the various oligosaccharides formed. An alternate method for the determination of the sequence of the monomers is discussed. It was subsequently shown that the linkages in the polysaccharide are in the a configuration. The polysaccharide is highly dextra rotary and the magnitude of the rotation is comparable to that of other polysaccharides linked in a position, . such as starch and dextran. Infrared spectroscopy was used to confirm the configuration. The spectrogram of the polysaccharide contained an absorption peak at 840 cm-1 , which is typical of the a-anomeric absorption occurring, for example, in the IR spectrum of starch. The spectrogram exhibited no absorption peak at 891 cm-1 , the wavelength typical of the B-anomeric absorption in the IR spectrum of cellulose. In addition, it was found that all polysaccharides containing a ( 1 + 4 ) linkages show an absorption peak at 700 cm 1. This absorption peak was absent in all IR spectra obtained from various dextrans. This phenomenon has not been reported previously and it is suggested that the presence of this absorption peak in the IR spectrum of a glucan can be used to support the evidence of the presence of a( 1 + 4 ) linkages. It was not possible to correlate the formation of the polysaccharide with the occurrence of a specific micro organism. It is suggested that the formation of the polysaccharide is the result of enzymic reactions in the sugar cane after harvesting. The investigation of the composition of juices from deteriorated cane has not been confined to polysaccharides. Ethanol has been isolated from the juice of some samples of stored cane which had been burnt before harvesting. The ethanol was isolated by fractional distillation and identified by measurement of the boiling point. It was confirmed, by the formation of the molybdate-xanthate complex, that the product isolated was an alcohol. The identification was further confirmed by oxidising the ethanol to acetic acid and proving the identity of the acids by paper chromatography. It has been shown that, with the exception of two acids, the carboxylic acid composition of cane juice remains unaltered during post-harvest storage of the cane. The two exceptions , succinic and aconitic acids, were identified from their melting points and by specific spot tests. Ion exchange was used to isolate the acids from the juice. The eluate from the ion exchange column was concentrated and the acids separated by liquid-liquid chromatography, using a silica gel column. The levels of both aconitic and succinic acids were found to increase during the early period of storage but decreased again slowly thereafter. The percentage change was greater in the case of succinic acid, although aconitic acid was the most abundant carboxylic acid in the juice. Lactic acid was absent from the cane juices analysed. This is surprising, as lactic acid is a common product of the metabolism of carbohydrates by micro organisms. It is suggested that the changes in acid composition during the storage of harvested cane are caused by deactivation of enzymes of the Krebs cycle. Post-harvest deterioration of sugar cane can have serious consequences which can affect the whole Sugar Industry. Not only is crystallisable sugar lost but the products of the deterioration have adverse effects on factory processing and laboratory analysis. The problem, which will become more acute with the introduction of mechanical cane harvesting, can only be resolved through the cooperative efforts of all the parties concerned. / Thesis (Ph. D.)-University of Natal, Durban, 1973.

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1972. / Bibliography: leaves [160]-168. / xii, 168 l illus. tables

Chromatographic methods were used to determine the absolute values of cis-aconitic acid and trans-aconitic acid present in sugar cane factory processing streams and to determine the rates of isomerisation from the trans-aconitic acid to the cis-aconitic acid isomer. A reproducible, solid phase, ion exchange extraction method was developed to isolate the organic acids found in sugar factory process streams. The isolated acids were quantified using a dual ion-exclusion column, high performance liquid chromatographic method. To improve resolution of the acids, columns were maintained at different temperatures, whilst the combined use of both ultra-violet (UV) and refractive index detection proved useful in peak identification. Concentrations of the aconitic acid isomers were used to calculate the cis/trans aconitic acid isomer ratio occurring across the different processes found in a sugar cane factory. trans-Aconitic acid was found to be the predominant form present in the cane entering the factory. Analysis showed that isomerisation of the trans-aconitic acid to the cis-aconitic acid isomer occurred during processing. To understand and model this reaction, a reproducible experimental isomerisation method was developed making use of buffers to maintain pH conditions during experiments. A chromatographic analysis method, using ion-exclusion chromatography and UV detection, was developed to analyse the isomerisation reaction mixture. Chromatography was used in both an on-line and off-line mode for quantitation of the isomers. The method was used to study the isomerisation under conditions similar to those found in the factory. These included pH, temperature, ionic strength and the presence of monovalent and divalent cations found in sugar cane juices. It was shown that the isomerisation is a first order reversible reaction under the conditions studied. Temperature and pH were shown to be the important isomerisation variables. Temperature enhances the rate of isomerisation of the trans-aconitic isomer to the cis-aconitic acid isomer whilst pH affects the ultimate cis/trans aconitic acid ratio attained. Ionic strength was found to be a relatively unimportant factor. The presence of divalent and monovalent cations, at concentrations usually found in cane juice, was shown to have little effect on the rate of isomerisation. Activation parameters, including the activation energy (Ea), pre-exponential factor (log A), enthalpy (?H‡) and entropy of activation (?S‡), were calculated at each combination of buffer concentration and pH used in the experimental procedures. The values recorded are of a similar value to those reported for structurally similar compounds. cis-Aconitic acid was shown to undergo decarboxylation to itaconic acid. This occurred at low pH values and high temperatures. A detailed study was not undertaken since the conditions under which it occurs are considered extreme from the viewpoint of a sugar technologist. A model describing the equilibrium cis/trans aconitic acid isomer ratio was developed as a function of pH, temperature and time from the kinetic results. This was used to predict the equilibrium ratio for the aconitic acid isomers at the output of various processes in the sugar factory. Given the time, average pH and temperature the model can successfully predict the equilibrium ratio for the relevant process stream. / Thesis (M.Sc.)-University of Natal, Durban, 2000.

The soil being delivered with sugar cane consignments. from the cane fields to the factories, has been a recent cause for concern in the South African sugar industry. The soil impurities increase the wear of processing machinery reduce extraction efficiency and represents an unnecessary transport of material. The cost due to soil was estimated at R63 million (about US $8 million) over the 1996/97-season. The need to reduce costs, due to the unwanted soil component, has been given a high priority. Ashing is currently used by the sugar industry to estimate the amount of soil in cane. Although simple to implement, the method is destructive, requires long processing times and limited to small sample sizes. In fecent times, non-destructive techniques have become more prominent in industry. Hence, the decision to apply such techniques to the soil in cane-problem. This dissertation describes an experimental investigation into Dual-Energy Transmission (DuET) and X-ray lmaging for quantifying the amount of soil in cane. DuET can determine the relative concentrations of the components of a binary mixture by measuring the transmission of low- and high-energy gamma photons through the mixture. The principle of DuET was successfully demonstrated with aqueous solutions of ferric chloride. Experimentally-determined mass attenuation coefficients of water and ferric chloride were compared to theoretical values. DuET was then applied to dried, shredded sugar cane spiked with various amounts of soil. Results showed large variations in the predicted soil concentrations. These variations were attributed to radiation scatter and incomplete volume sampling by the radioactive source. However, new experimental arrangements are expected to improve the technique: initial test results are given of a sample holder that continuously rotates a sample up and down through the source-detector axis. An alternative approach to processmg DuET-spectra, using the discrete wavelet transform coupled with an artificial neural network, is also introduced. X-ray Imaging was the second technique investigated. A literature survey revealed that this technique had not previously been applied to the soil in cane-problem. The present work constituted an initial investigation to determine the feasibility of applying X-ray imaging to measure the amount of soil in cane. The soil/cane-samples, that were used for DuET, were imaged us ing a commercial mammography unit, and the resulting radiographs were analysed using image processing techniques. Although the results are promising, a more comprehensive investigation is foreseen. / Thesis (M.Sc.)-University of Natal, Durban, 2001.

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. / Thesis (Ph.D.)-University of Natal, Durban, 2000.

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. / Thesis (M.Sc.)-University of Natal, Durban, 2000.

The objective of the present study was to investigate the potential use of bulk segregant analysis (Michelmore et al., 1991) as a method to rapidly identify genetic markers linked to traits in sugarcane. Four bulked DNA samples were prepared from progeny of a sugarcane cross, AA157, based on segregation for the fibre trait. The bulks comprised five and ten individuals on either side of the fibre phenotypic extreme. The random amplified polymorphic DNA (RAPD) technique (Williams et ai., 1990) was used to screen for differences between the low and high fibre bulks. A total of 749 fragments were amplified in the bulks, eight of which were polymorphic. The segregation of the bulk specific polymorphism was analysed in 80 progeny of the AA157 cross; and seven were found to reproducibly segregate on a 1: 1 basis. This indicates that they are single dose fragments. A total of 79 polymorphisms were detected between the parents of the cross, indicating 10.5% variation in the genomic region sampled. Twenty two of the parental polymorphisms segregated as single dose fragments in the progeny of the cross AA157. Analyses of variance (ANOVAs), and multiple regression analyses, were used to ascertain linkage of the putative RAPD markers to fibre, and if linked, to determine the fibre variation ascribed respectively. Three RAPD fragments were found linked to the fibre trait. Fragments OPA17438 and OPC16889 (at the 99% significance level), and OPB1l464 (at the 95% significance level). These putative markers ascribed a total of 28.6% fibre variation in the 1993 season. The association of the RAPD markers with fibre in the different seasons (1992, 1993 and 1994) was investigated. Three RAPD markers were found linked to the fibre trait in each season, with a total of 5.5% and 31,4% fibre variation ascribed in the 1992 and 1994 seasons respectively. Marker OPA17438 was found to be linked to the fibre trait in all three seasons investigated, and marker OPC16889, was found linked to the fibre trait in the 1992 and 1993 seasons. Cross validation of the linkages of the RAPD markers to the fibre trait was carried out by a modified form of 'jacknifing' where the sample size was reduced to N-l0, and RAPD marker-fibre trait associations investigated as before. RAPD markers OPA17438 and OPC16889 were still consistent across the seasons, however marker OPA17438 was no longer linked to the fibre trait in the 1992 season. To investigate the genetic behaviour of RAPD based markers in sugarcane and the potential for their application in marker-assisted selection (MAS), two putative RAPD markers were converted to sequence characterised amplified regions (SCARs) (Paran and Michelmore, 1993). The RAPD fragments OPA17438, OPBl1464, and OPC16889 were excised from agarose gels, re-amplified and cloned into the pCR-Script SK (+) phagemid for sequencing. RAPD markers OPA17438 and OPB11 464 were converted to SCARs by using their sequences to design longer specific primers. A third SCAR marker, SAl1640, originally derived from sugarcane cDNA as a potential stem preferential expressed sequence tag, was included in the analysis to increase the sample size. All three SCAR markers segregated in a monomorphic fashion in the parents and progeny of the cross AA157. In addition, monomorphic length variants for markers, OPA17438 and OPB11 464 were detected with the SCAR amplification. All three SCARs segregated in a monomorphic fashion in different commercial varieties and bulks of S. officinarum and S. spontaneum, the progenitors of modern commercial varieties. The segregation analyses of the SCAR markers indicate that the RAPD polymorphism of marker SAl1640 was probably due to a point mutation or mismatch in the priming site. The segregation analyses of SCARs for the markers OPA17438 and OPB11464 indicate that their segregation in the RAPD analyses was due to an insertion mutation in the genetic locus. The combined results of the SCAR and RAPD segregation of markers OPA17438 and OPB11464 are indicative of preferential pairing in the cross AA157. Finally, to investigate the extent of linkage disequilibrium in a modern commercial variety, twenty two single dose RAPD fragments were investigated for their association with four traits in 53 progeny of cross AA157. The four traits investigated were fibre %cane, brix %cane, pol %cane and ers %cane over three seasons (1992, 1993 and 1994), at different ages of harvest (12, 8, and 9 months respectively). Seventeen linkages of RAPD markers to the four traits, over the three seasons, were detected. The phenotypic variation ascribed by the RAPD markers ranged from 7.6% fibre %cane variation explained by one marker in 1992, 29.6% fibre %cane (three markers) in the 1993 season to 10% (three markers) in 1994. A total of 14.1% brix %cane variation was ascribed by two markers in 1992, 9.6% (one marker) in 1993 and 16.3% (two markers) in the 1994 season. A total of 13.5% estimated recoverable sucrose %cane was ascribed by one marker in 1992, 12% (two markers) in 1993 and 15.3% (two markers) in the 1994 season. Two markers explained 17.2% pol %cane variation in 1992 and 25.4% in the 1994 season. Only four markers were detected across different environments, three of which were linked to fibre. These were OPA17438, OPB16618 and OPC16889, each linked to fibre in two seasons. RAPD marker OPB11 464 was linked to estimated recoverable sucrose %cane in two seasons. Two markers were found associated with different traits in a single season. RAPD marker OPB11 464 was found associated with brix %cane and estimated recoverable sucrose %cane in the 1993 season, and RAPD marker OPA17438 was found associated with all four traits in the 1994 season. / Thesis (Ph.D.)-University of Natal, Durban, 1998.

The biochemical basis for the regulation of sucrose accumulation is not fully understood. The present study was thus aimed at investigating aspects of 'coarse' (enzyme activity) and 'fine' (metabolite) control of glycolytic enzyme activity in relation to carbon partitioning in the developing stalk (internodes 3 to 10), and between varieties with significant differences in sucrose content. Particular emphasis was placed on studying pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90), since this enzyme has been implicated in sucrose metabolism in other plant species. Within the developing stalk, internodal maturation was associated with a redirection carbon from the insoluble matter and total respiration (C02 production and biosynthesis) to sucrose storage. Between varieties, with significant variation in sucrose content, there was an inverse relationship between hexose monophosphate partitioning into respiration and sucrose. The reduction in carbon flux to respiration was not associated with a decline in the extractable specific activity of PK, PFK and PFP. There was also no alteration in the regulation of PK, PFK and FBPase by change in the mass action ratios. Hexose monophosphate concentration declined approximately two to three-fold from internodes 3 to 9 and Fru-6-P concentration was within the lower Km or 80.5 range (Fru-6-P) of PFP and PFK, respectively (as reported from the literature) . Within the developing stalk, substrate limitation might have contributed to the decline in carbon partitioning to respiration. In sugarcane, the levels of PFP activity were controlled in part by PFP protein expression. 8ugarcane PFP polypeptide(s) are resolved as a single protein with a molecular mass of approximately 72 kO. PFP catalysed a reaction close to equilibrium in all intemodes investigated, and the concentration of Fru-2,6-P2 was shown to be in excess of the requirement to stimulate PFP activity. Carbon flux from the triose-P to hexose monophosphate pool was apparent in sugarcane, suggesting that PFP activity was functional in vivo. The developmental profile of specific PFP activity was not positively correlated to the increasing rate of sucrose accumulation in the top ten internodes of the developing stalk. Between different sugarcane varieties, specific PFP activity was shown to be inversely correlated to sucrose content. / Thesis (Ph.D.)-University of Natal, Durban, 1997.

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. / Thesis (M.Sc.)-University of Natal, Durban, 2003.