School of Agriculture, Food and Ecosystem Sciences - Theses
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ItemNo Preview AvailableLinkage mapping and QTL analysis of ascochyta blight resistance in chickpea (Cicer arietinum)(University of Melbourne, 2003)Ascochyta blight is the most destructive foliar disease of chickpea worldwide. Resistance is available in the germplasm, and with a strong evidence of polygenic control, breeding is geared towards gene pyramiding for durable resistance via marker-assisted selection. This thesis generally aimed to map and analyze the quantitative trait loci (QTL) which condition ascochyta blight resistance (ABR) in an intraspecific Cicer arietinum genetic background. An intraspecific population of chickpea was used to create the first linkage map for the C. arietinum genome, and locate six QTL conditioning glasshouse and field resistance to ascochyta blight. Results from QTL analysis revealed a major genomic region that contained clusters of QTL (QTL 4, 5 and 6), and minor QTL specific for glasshouse (QTL 2 and 3) and field (QTL I) resistance. Multiple interval mapping revealed that resistance of chickpea to ascochyta blight was conferred by an epistatic interaction between the additive gene action of the major QTL and the dominance gene action of the other (minor) QTL. RGA markers PTOFENb212 and CLRRinv904 were found tightly-linked to the major ABR-QTL region. By sequence alignment, the RGA markers were confirmed to be the corresponding resistance gene orthologs (RGOs) in chickpea. Allele- and locus specific SCAR markers targeting the RGOs were developed and shown to be transferable to other resistance genotypes. The RGOs were also used as starting points to isolate the underlying major genes or cluster of genes for ABR. 5'-cDNA sequences of the major ABR genes, ArFEN and ArLRR, were then characterized. These are the first candidate gene sequences for ascochyta blight resistance identified in chickpea by a map-based candidate gene approach. Based on the homology alignment of ArFEN and ArLRR, and sequence information of the RGOs with published gene sequences, the R-genes underlying the major QTL region may be classified under the third group of R-genes that contains a serine-threonine protein kinase domain, with a leucine-rich repeat. Homology alignment of the candidate genes also revealed that the mechanism of resistance reaction conferred by the major ABR genes in chickpea was most likely that of a hypersensitive response.
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ItemNo Preview AvailableExpression of a recombinant mycobacterial antigen and characterization of fas-defective mutant strains in corynebacterium glutamicum and related species(University of Melbourne, 2003)The research undertaken in this thesis had the initial aim of investigating the utility of Corynebacterium glutamicum species as host for synthesis of foreign proteins of biotechnological importance, given that earlier research had shown that this industrially-important species, normally used for amino acid production, was able to express and secrete a variety of proteins from different bacterial sources. Of particular interest was the use of this species in making proteins that may be secreted or located at the cell surface, where their expression may have been important in antigenicity or other physiological functions: proteins made and secreted by related organisms, including pathogenic Mycobacterium species, were of particular interest because many pathogens cannot he cultured readily in the laboratory environment or exist as obligate intracellular parasites. It was also likely that the C. glutamicum and Mycobacterium species would share functionality in their cell surface proteins, an assumption based on their common synthesis of mycolic acids complex surface lipids that are involved in making cells from this group resistant to chemical and physical assault. The first part of the thesis describes a study on the synthesis and secretion of the 85 antigen complex, component of which are important immunogens in Mycobacterium and are secreted proteins with activity involved in the attachment of mycolic acids to the cell wall. During the course of this study, PCR amplification using primer sets based on the 85B antigen complex from M. bovis was followed up by sequencing the PCR products. Rather than corresponding to an 85B homologue, the DNA had high sequence similarity to a fatty acid synthase (FAS) gene previously found in Brevibacterium (now Corynebacterium) ammoniagenes. The subsequent focus of the thesis was on characterization of the presumptive fas gene in C. glutamicum in terms of gene inactivation and impact on lipid synthesis, for both fatty acids and mycolic acids. The first part of this project was to undertake controls for later experiments if the mycobacterial antigen 85 complex components (A, B and C) or any homologue of these were present in Corynebacterium glutamicum AS019 and its related strains, C. glutamicum ATCC13032, C. glutamicum CG2, C. lactofermentum BL1 and C. flavum BF4. This involved determining on the expression of a recombinant antigen 85 complex in C. glutamicum AS019 and related cell-surface mutants (MLB 133 and MLB 194). In order to detect the presence of the mycobacterial antigen 85 complex (A, B and C) or homologues, several experiments, including Polymerase Chain Reaction (PCR), Southern hybridization and Western blotting experiments, were carried out. The screening focused on the antigen 85B as this component is the most abundant of the antigen 85 complex in Mycobacterium species and there is considerable similarity between antigen 85 complex members at both nucleotide and amino acid sequence levels. Furthermore, antigen 85B was most extensively studied in the literature so that the materials needed to study this (such as PCR primers, antibody and probes for Southern hybridization experiments) were easier to make or obtain from other research groups. A PCR Mal 1/21 primer set was designed based on the most conservative regions of the antigen 85B from several species of Mycobacterium. Amplification produced several feint products from C. glutamicum AS019, C. lactofermentum BL1 and C. flavum BF4, which was an unexpected outcome. All the major PCR products were cloned into the pGEM�T vector and sequenced to determine similarity with the sequence of mycobacterial antigen 85 complex. However, the �0.8 kb PCR products from C. glutamicum AS019 and C. lactofermentum BL1 showed relatively high similarity (-70%) with a fatty acid synthase gene (fasA) of C. ammoniagenes and �60% with the fasA found in M. tuberculosis. Similar PCR products were found in all of the C. glutamicum strains tested. In Southern hybridization experiments, two different probes were used for more intensive analysis. One was the 0.8 kb PCR product produced by the Mal 1/21 primer set when pGEX-MPB59 (an expression vector which contains a mycobacterial antigen 85B gene) was used as template. The other was a 0.49 kb fragment produced by PstI/Xhol double-digestion of the above 0.8 kb PCR product. Relatively lower prehybridization/hybridization temperature (55�C) and longer expose time were employed to detect any loosely-bound signal, however, no specific signal was detected using gDNA from C. glutamicum AS019 and its related strains. In Western blotting experiments, anti-antigen 85B antibody raised in rabbits was used as the primary antibody to detect any signal from whole disrupted cells and culture filtrate of C. glutamicum AS019 and its related strains. No signal was found using culture filtrates concentrated 50 fold, however, a signal was found using disrupted cells of all strains. To determine the location of this protein, cell wall proteins were extracted from whole cells with 50 mM Tris buffer containing 2% Sodium Dodecyl Sulfate (SDS). No signal was found from cell wall extracts so that this protein may be located in the inner membrane or cytosol. The size of this protein was almost 40 kDa which is significantly bigger than the mycobacterial antigen 85 complex (B is 32 kDa and A and C are 30 kDa). Even though a signal was found from Western blotting experiments, it was concluded that there was no antigen 85 complex homologues present in C. glutamicum due to the negative results from PCR and Southern hybridization experiments. Several strains were then transformed with pCGL1055, which contains the antigen 85A gene, to determine the secretion level of recombinant antigen 85A in C. glutamicum AS019, C. glutamicum MLB133, C. glutamicum MLB194, C. lactofermentum BL1 and C. flavum BF4. Since pCGL1055 had the cspB promotor, two sets of primer were used to confirm the presence of the cspB gene (coding for PS2 protein in corynebacteria) in the strains used in this expression work. To quantify the amount of secreted recombinant antigen 85A, recombinant antigen 85B was prepared as the standard using Glutathione Sepharose 4B� affinity chromatography followed by thrombin cleavage and gel filtration chromatography using material prepared from E. coli TG2 transformed with pGEX-MPB59, the expression vector which contains the antigen 85B gene. C. glutamicum MLB 133 and MLB 194 showed relatively higher secretion level than the parent strain, C. glutamicum AS019, which suggests that the changes in the cell wall structure of the mutants caused better secretion of the foreign recombinant protein. C. flavum BF4 also showed relatively higher secretion levels than AS019. Volumetric and cellular productivity for the expressed recombinant antigen 85A from several corynebacterial strains were calculated to determine the efficiency of strains in secreting the protein into culture fluids. The amount of 85A protein secreted was expressed as the equivalent of 85B protein measured in parallel using anti-antigen 85B. In volumetric productivity from C. glutamicum strains AS019 (spontaneous Rif mutant of C. glutamicum ATCC 13059), MLB 133 and MLB 194 (the cell wall modified mutants of C. glutamicum ATCC 13059) was 16-17 ng/ml. However, C. lactofermentum BL1 and C. flavum BF4 expressed relatively higher amounts of antigen 85A (22-23 ng/ml) than the above strains. Cellular productivity was determined by calculating the amount of antigen 85A secreted from the given number of cells of each transformant by adjusting the optical density of each overnight cultured culture filtrate to 100 at 600nm. The cell wall modified mutants, C. glutamicum MLB 133 and C. glutamicum MLB194, showed higher efficacy (730-775 ng/ml) in expressing antigen 85A from the given number of cells than its wild-type strain, C. glutamicum AS019 (440 ng/ml). C. flavum BF4 showed almost the same efficiency (about 750 ng/ml) as the mutants. The new finding of a fas-gene homologue using PCR amplification prompted further studies aimed at characterizing its role in synthesis of fatty acids and mycolic acids in C. glutamicum. The inactivation of a gene by homologous recombination was used to characterize the fas-gene homologue, employing the 0.8 kb fragment found in the previous PCR experiments. The 0.65 kb fragment, the nested PCR product from the above 0.8 kb fragment, was amplified using the fas11/21-EcoR I-1 primer set. The amplified 0.65 kb fragment was digested with EcoR I and cloned into pECMA, a suicide vector that does not replicate in Corynebacterium strains without homologous recombination between the insert and recipient chromosomal DNA due to the lack of a replication origin on the vector. The newly constructed suicide vector, pECMAfas-AS019, was introduced into the donor strain, E. coli S17-1, a mobilizing strain constructed by the integration of self-transmissible (Tra') plasmid, RP4 2-Tc
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ItemNo Preview AvailableEvaluation of dual energy x-ray absorptiometry for predicting whole body and carcass composition of pigs(University of Melbourne, 2003)This dissertation aims to evaluate the dual energy X-ray absorptiometry (DXA) technique for measuring whole body and carcass composition of pigs. Section 1 provides the appropriate calibration for quantitatively accurate DXA measurements of live body and carcass composition of pigs of varying weight, as determined by chemical analysis and manual dissection. The DXA technology provided an accurate precise measurement of composition within experiments and when measurements were compared across experiments. The precision of DXA measurements were far better than weight and P2 backfat, particularly in the prediction of fat tissue, especially when measurements were compared across experiments. The DXA measurements were highly repeatable and measurement repeatability improved as animal size increased. The placement of the regional analysis grid influenced the repeatability of all measurements except for total weight, however this influence reduced with increasing animal size. It is recommended that the scan image be positioned in the arm region of the software regional analysis grid to measure whole body composition in pigs because it provides the most repeatable measure overall and a better measure for fat tissue when compared to chemical analysis and manual dissection. The DXA regional analysis software also provided an accurate and precise measurement of dissectible composition in the primal-cuts, ie. the ham, loin, belly and shoulder. However, care needs to be taken to ensure accurate and consistent delineation of sub-regions to the true position of primal-cuts in the DXA scan image to ensure a reliable measurement. Section 2 involved a series of experiments to evaluate the ability of the DXA technology in measuring the effects of various known and innovative body composition manipulators, all directly applicable to industry. Each experiment demonstrated the use of the developed calibration equations and the efficacy of the DXA technique. Overall, corrective equations were successfully applied to DXA estimates and largely corrected differences between raw DXA outputs and chemical values. The DXA measurements confirmed that diets deficient in protein lead to sub-optimal lean tissue and bone mineral growth with no effect on fat and were substantiated by chemical analysis. Serial DXA measurements in the same animal demonstrated the lifetime differences between contemporary genotype boars and barrows under different penning conditions. These data indicated that although boars have a lower propensity to deposit fat overall, under group penned conditions there is little incentive to produce boars in terms of body composition if slaughter weights were below about 80kg. However, in the late finisher period, boars did deposit more lean tissue than barrows, but this difference was reduced when animals were group-penned. The regional analysis software successfully demonstrated the effects of pST treatment on fat distribution within the animal, and was validated using manual dissection. A dose dependent decrease in belly, loin, ham and shoulder fat was also observed, although the proportionate decrease in belly fat was more pronounced than for the whole carcass and other primal cuts. Finally, the DXA technique was sensitive enough to detect the subtle body composition changes that were induced by supplemental dietary betaine. Dietary betaine improved growth rate and protein deposition, and consequently carcass lean tissue content, in pigs fed restricted energy and the effects were additive with those of pST. Lean tissue in the loin was particularly responsive. The energy spared by betaine was not enough to increase fat deposition, carcass fat or P2 backfat regardless of energy intake. However, dietary betaine did increase fat in the more energy responsive belly region under ad libitum conditions but not under a restricted intake typical of commercial conditions. Therefore, dietary betaine did alter the distribution of lean meat and fat within the animal. These data demonstrate that the DXA technology is a "viable" replacement for the standard methodologies in animal growth and body composition research experiments and has much potential for implementation into the pork industry for carcass grading and providing data for pig growth simulation models.
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ItemSubsoil physicochemical constraints and growth of cereals on alkaline soils in the Victorian Mallee( 2003)Alkaline soils used for dryland cropping across the semi-arid regions of south=eastern Australia typically have high levels of salinity (ECe), sodicity (ESP) and soluble boron (B) in the subsoil. The current research was undertaken to improve our knowledge of the physicochemical characteristics of these alkaline soils and assess what impact they have on the growth and water use of cereals. A survey of representative alkaline soils of the southern Mallee and Wimmera regions of Victoria, comprising 140 Calcarosol profiles and 10 Vertosol profiles, revealed high correlation between exchangeable Na+ and both ESP (r = 0.96) and B (r = 0.88). ESP and ECe (r = 0.71) and B and pH1:5 (r = 0.70) were also highly correlated. Readily measured properties (field texture, pH1:5, ECe and exchangeable Na+) were found to provide good estimates of ESP and boron in these soils. Overall, ESP was best defined as 1.47 + 2.68 x Na+ (r2 = 93.9) and boron by 0.34 +3.93 x Na+ (r2 = 76.7). A break point values for pH was defined such that soils with pH 1:5 < 8.1 have low levels of soil boron that are not potentially toxic to cereal growth. The growth and water use of the boron tolerant wheat cultivar, Frame, was monitored on these soils during the 1999-growing season. A descriptive model that explained 54% of the variation in grain yield (range: 1.3 - 6.1 Mg/ha) was established using ridge regression analysis, which is not confounded by the correlation that exists between the physicochemical factors (collinearity). The statistical analysis identified rainfall around anthesis, available soil water in the 0.10-0.40 m layer at sowing, nitrate in the 0-0.10 m layer and salinity and sodicity in the 0.60-1.00 m layer as significant explanatory variables for grain yield and water use. Subsoil salinity (ECe) and sodicity (ESP) were effective surrogates for extractability of water in the deep subsoil. It is estimated that crops can make use of water at depth if subsoils have ECe <8 dS/m and ESP <19%. Levels of soluble soil boron (reaching concentrations of up to 52 mg/kg), were not significantly correlated with root growth, water uptake or yield of wheat. It is proposed that the boron tolerance of cv. Frame rendered high boron as non-limiting and that the high Na+ in these soils had an overriding effect in driving variation in crop yield. The impact of boron tolerance, watering regime and soil disturbance on the growth and water use of near-isogenic lines of wheat and barley was assessed using large intact soil cores (0.3 m diam. x 1.0 m height), containing an alkaline soil (Calcarosol) sampled from the southern Mallee. Within the subsoil (0.80-0.90 m) ECes, ESP and B was 8.1 dS/m, 29% and 31 mg/kg respectively. In the shallower (0.61-0.71 m) layer respective levels were 7.2 dS/m, 22% and 29 mg/kg. Crop root growth between these layers decreased significantly with depth and no net water extraction occurred beyond 0.80 m, irrespective of crop boron tolerance. As the concentration of soil boron was equivalent across these layers, it was discounted from being a constraint to water extraction by crop. Rather, the increase in EC0 and more so ESP suggests these factors were constraining water use at depth. Grain yield per unit of applied water for wheat and barley grown under low water supply was 1.5 times that of their high water counterparts, suggesting inefficiencies in water capture by crops under the high water regime. Deep ripping had no effect on grain yield. Importantly these results showed boron tolerance provided little benefit to cereals on soil where constraints in addition to high boron exist. A glasshouse trial was conducted to assess how interactive effects of boron and salinity and cultivar affected the early growth of wheat using a range of soil salinity and soluble boron levels observed in the field survey. The three cultivars, Frame, BT Schomburgk and Schomburgk, varied significantly in tolerance to boron, with critical soil concentrations were estimated at 53, 32 and 27 mg/kg respectively in the absence of salinity. These 3 varieties did not differ in tolerance to salt in the absence of high boron, where cultivars equally tolerated ECe = 9 dS/m. Boron and Na+ in shoot tissue could not be used to define critical concentration for toxicity. This trial demonstrates the value Of using genetic variation for adaptation of wheat to high levels of boron, but appears not the case for salt tolerance here. This thesis confirmed that, due to the strong intercorrelation that exists between the physicochemical factors, salinity, sodicity and soluble boron, that these factors are likely to operate simultaneously to reduce cereal growth. Adequate boron tolerance currently appears to exist in commercial wheat cultivars i.e. Frame, enabling these varieties to withstand the high boron levels encountered in the alkaline cropping soils of north-western Victoria. High. levels of salinity and sodicity, however, are more likely to be constraining growth through osmotic, toxic and physical impediment. This thesis indicates the need for improve tolerance of cereals to salinity and the need for pyramiding tolerances for crops targeted to alkaline soils where constraints exist together.
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ItemThe Domestication and improvement of Kunzea pomifera (F.Muell.), muntries( 2003)Experiments were conducted to evaluate the vegetative propagation, morphological and molecular variation, and reproductive biology of the Australian native plant species Kunzea pomifera (F.Muell.), Muntries. The species produces an edible berry with potential commercial appeal, and the primary aim of the study was to determine the potential for its domestication and suitability for commercial production. Collections were made of the species natural populations across its geographical range in south-eastern South Australia and western Victoria. These collections were grown at a single location at Burnley College, Melbourne and were used as the basis for the studies that were considered important for evaluating the species potential for domestication. The capacity for vegetative propagation in K. pomifera was significantly improved with the exogenous application of IBA at concentrations of 1000, 3000 and 5000ppm compared to no auxin, within a temperature range 16-25C. Significant variation between accessions was found for percentage of rooted cuttings, number of roots and mean root length per rooted cutting, indicating a potential for maximising successful vegetative propagation in commercial cultivars. The production of K. pomifera by grafting, as a scion, on to each of K. ambigua and K. ericoides rootstocks was demonstrated, and was found to offer the potential for the rapid improvement of plant habit and increasing the edaphic tolerances of K. pomifera. The examination of the presence of topophysis in the cuttings of K. pomifera was examined and was found to be transitory and therefore did not appear to be of potential value for the physiological modification of growth habit in its domestication. Leaf morphology (area, length, width, elongation, and stomata, oil-gland and trichome densities) variation in Kunzea pomifera was studied in three natural populations. Significant variation was found within and between populations for all characters, indicating a high probability for such variation in other characters of use for the species domestication, such as fruit size and yield. Molecular variability was also evaluated in these three natural populations using the random amplified polymorphic DNA technique (RAPD). Molecular variation was greatest between individuals within populations, with little population differentiation evident between the three populations. Significant genetic variation between genotypes of K. pomifera indicates the likely variation for characters of potential use in the domestication and improvement of the species for commercial production. Pollen viability and longevity (at 4C 10% R.H.) and the onset and duration of stigma receptivity was evaluated in k pomifera. Significant variation in the germination level of pollen was found between genotypes, indicating the potential for breeding cultivars with high pollen viability. No significant correlation was found between germination percentage and storage time over 370 days, indicating that in vitro pollen germination capacity was not significantly affected by such storage. For K. pomifera, the optimum pollination period, for maximising the level of seed set per fruit was found to be ten days (a period from two to eleven days following anthesis). Such a long period of receptivity indicates that artificial pollination would be generally successful for producing hybrids within the species. K pomifera was found to have a very low level of self-compatibility, which was manifest as the prevention in the growth of 'self' pollen-tubes in the style or ovary. Considerable variation was found between genotypes of K. pomifera for the percentage fruit set after artificial hybridisation. This indicates a potential for breeding and selection to maximise yields in commercial cultivars. High interspecific cross-compatibility was found between K. pomifera (U) and K. ambigua (a) wherein seed set per pollinated flower was comparable with the mean value intraspecific crosses. A high level of incongruity, however, was observed between K pomifera (U) and K ericoides (a), which was manifest as the swelling of pollen-tube tips and cessation of growth in the style. Studies of molecular and morphological variation within and between natural populations of Kunzea pomifera (muntries) and its reproductive biology are discussed in terms of its domestication for commercial production, culminating in the proposal of an ideotype for its commercial production. The proposed ideotype for this species grown in southern Australia has a phenotype primarily of upright habit, tolerant of a wide range of soil types, reproductively precocious, regular bearing, with a capacity for high yields of glabrous fruit of consistent size and colour with few seeds and a high pulp:seed ratio, sugar and moisture content. Appropriate breeding and selection procedures are also considered, in terms of their relationship to the further refinement of the specified ideotype characters. It is proposed that such considerations are fundamental to improving the potential for domestication o f Kunzea pomifera.
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ItemApplication of non-conventional disinfection techniques to extend the shelf life of minimally processed foods( 2003)Minimally processed (MP) fruits and vegetables are a growing market segment in Australia. Chlorinated water is commonly used in the sanitising washing steps of minimal processing. However, there is a growing concern over the presence of organo-chemical residues, such as chloramines and trihalomethanes, together with other by-products derived from the use of chlorine. A number of alternative sanitising systems are available for water treatment, and one of these utilises the electrolytic generation of silver in the presence of hydrogen peroxide. In the current study, this system has been investigated for its potential application as an alternative sanitiser to wash horticultural produce including MP fruits and vegetables. The developed system (silver/hydrogen peroxide) was also tested to disinfect fruit juices and liquid milk. In a resting cell test system, electrochemically generated silver (5-5000ppb) and hydrogen peroxide (0.04-40ppm) showed a bactericidal effect on Pseudomonas fluorescens, a predominant bacterium on MP leafy salad products. This bactericidal effect was observed after treatment with silver or hydrogen peroxide separately and in combination. However, the combined treatment (silver and hydrogen peroxide) produced a more severe effect. Several trials have also investigated the use of silver nitrate as an alternative source of silver and the results showed a similar reduction in the microbial count. The minimum inhibitory concentration (MIC) of silver on 20 strains including species of Pseudomonas, Aeromonas, Enterobacter, Erwinia, Escherichia, Xanthomonas, Clavibacter and yeasts ranged from 100-150 ppb. Five silver resistant isolates were also developed from parent cultures by growth in successively higher concentrations of silver ions to investigate the mechanisms of silver resistance in the developed resistant strains. The tolerance of both parent and resistant strains was examined against silver, hydrogen peroxide, and other heavy metals, including copper, cadmium and zinc. The resistant isolates showed higher levels of resistance towards copper and cadmium, indicating a possible cross-resistance to these metals. Additionally, similar mechanism(s) of resistance may be present in the derived silver resistant isolates as those that have already been reported for copper and cadmium resistance in microorganisms. The silver resistance determinant recently reported by Silver and Gupta in 2000 was used to identify a similar silver resistance determinant in the derived silver resistant isolates using PCR techniques. The PCR product corresponding to silE gene was used to amplify Escherichia coli carrying a silver resistant plasmid pMG101 and the silver resistant strains. However, the specific silver resistant determinant was not found in the derived isolates, which may indicate that other mechanisms may be involved in the derived isolates. The enhanced bactericidal effect of silver in the presence of hydrogen peroxide was further evaluated as a sanitiser in the washing steps for minimally processed vegetables including, lettuce, broccoli and carrots. Results from both lab and pilot scale washing trials showed the potential use of silver/hydrogen peroxide to reduce the populations of Pseudomonads, Enterobacteriaceae and yeasts in minimally processed products during storage at 4 or 12 C. The application of the proposed system was also extended to fruit juices and milk as a possible alternative to thermal treatment. The effect of the combined silver and hydrogen peroxide treatment on spoilage yeast populations in fruit juices was found to be significant at approved concentrations of both disinfectants. Application of this technology in milk systems was not as promising due to the chelating effect of milk organic constituents on silver. The development and application of the silver/hydrogen peroxide disinfectant system to disinfect water and fruit juices, and wash fresh-cut produce will have potential applications in the food industry as a possible alternative to chlorination and thermal pasteurisation. The success of this novel technology requires appropriate regulatory measures and consumer acceptance.