School of Agriculture, Food and Ecosystem Sciences - Theses
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ItemUnderstanding and mitigating the impacts of major dietary changes on dairy cows( 2018)Four experiments were conducted to investigate the effects of major dietary changes on ruminal pH, ruminal fluid composition, eating behaviour, feed intake and milk production of dairy cows. The impacts of both diet composition and management strategies were evaluated. The initial experiment investigated the impact of early adaptation when instigating a complete dietary change from one forage to another at calving, as is common practice in Irish dairy farming. Three weeks prior to their expected calving date, 14 spring calving dairy cows were assigned to one of two treatments: pasture silage pre-partum followed by fresh cut perennial ryegrass (PRG) post-partum, or fresh PRG both pre and post-partum. There were no differences in dry matter intake (DMI), body condition score, energy balance or milk yield and composition between the treatments. The results of the initial experiment suggested that early adaptation to avoid a major dietary change at calving did not result in health or production benefits. This was speculated to be due to the similarities of the two diets, creating little challenge for the rumen to adapt. The second experiment focused on a more challenging dietary change, incorporating a large amount of concentrate into a forage-only diet. Thirty-two lactating dairy cows were initially fed 100% lucerne hay cubes, wheat was then gradually substituted in until it comprised 40% of total dry matter (DM) and lucerne hay cubes, the remainder. Wheat was substituted for lucerne cubes via one of four strategies, (1) in six small increments (each 6.7% of total DM) over 6 days; (2) in six small increments (each 6.7% of total DM) over 11 days; (3) in three large increments (each 13.3% of total DM) over 6 days; or (4) in three large increments (each 13.3% of total DM) over 11 days. The 6-day strategies are considered rapid for the dairy industry yet none of the treatments resulted in ruminal fluid pH values that would have compromised ruminal function, nor were there differences in DMI or energy corrected milk (ECM) yields. Furthermore, there were no differences between ruminal fluid volatile fatty acid (VFA), lactate or ammonia concentrations. It is speculated that the properties of the lucerne cubes helped the ruminal contents resist the pronounced declines in pH often seen with the fermentation of large amounts of wheat. These results suggested that changes to rumen function are driven not only by the characteristics of the concentrate being introduced but also by those of the forage. The third experiment aimed to investigate the role of forages in grain adaptation. Twenty-eight lactating dairy cows were fed either PRG hay or lucerne hay and wheat was gradually substituted for forage in three equal increments, over 6 or 11 days, until wheat made up 40% of DM (~ 8 kg DM/cow per day). The results varied significantly with forage type. Cows fed lucerne hay ate more, produced more ECM and had lower ruminal pH values. Furthermore, of the cows fed lucerne hay, those adapted to wheat in the shorter time frame (6 days) exhibited significantly lower mean ruminal pH values. Despite the ruminal pH of these cows declining to levels typically considered low, none of their other measured parameters indicated compromised fermentation or acidosis. Rather, it was these same cows that had the greatest ECM yields, producing an average of 1.5 kg ECM/cow per day more than their 11-day counterparts. The 6-day adaptation strategy allowed for a rapid increase in metabolisable energy, while the hay promoted adequate buffering within the rumen. No difference was seen between adaptation strategies when PRG hay was fed. This was due to the higher metabolisable energy concentration and lower intake of the PRG hay resulting in a less pronounced increase in metabolisable energy intake with the wheat substitution. The greater intakes of cows fed the lucerne hay likely contributed to their greater ECM yields and lower ruminal pH values. However, both forages allowed the rumen contents to resist the large declines in ruminal pH that are typically seen during rapid grain adaptation. The final experiment aimed to further evaluate the role that forage plays in ruminal, behavioural and production responses to the incorporation of large amounts of wheat grain into the diet. Sixteen dairy cows in early lactation were fed a forage only diet of either lucerne hay, PRG hay or one of two cultivars of fresh PRG pasture (cultivar Bealey or Base) for three weeks. The forage-only diet was then supplemented with crushed wheat grain at a rate of 8 kg DM/cow per day, with no adaptation period. Wheat comprised between 32 and 43% of total DMI and was fed over two meals, followed by forage, for one day only. Feeding fresh pasture resulted in lower ruminal pH values, with pH remaining below 6.0 for longer each day. Following supplementation of wheat, cows fed pasture exhibited ruminal fluid pH levels associated with sub-acute ruminal acidosis. Hay created a ruminal environment that was better able to cope with the influx of acid produced as wheat was digested. A combination of increased ruminating time and a decreased rate of fermentation are likely responsible for the higher ruminal fluid pH values. The ruminal environment of cows fed lucerne hay remained most stable throughout the grain challenge, with ruminal fluid spending the least amount of time below pH 6.0. Reducing the introductory time for concentrates into a dairy cow’s diet means an ability to rapidly increase the energy content of a diet, resulting in milk production benefits. However, this thesis highlights the importance of forage choice when determining introduction strategies. Traditional, gradual adaptation strategies must still be employed when feeding highly digestible fresh forages. However, more aggressive adaptation strategies can be implemented when hays are used as the base forage. In situations where high energy grains are substituted for a low energy, high fibre basal forage, rapid introduction can have milk production benefits over gradual strategies.
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ItemProspects for ammonium sulphate to improve nitrogen and sulphur efficiency in alkaline soils of South Eastern Australia( 2016)Both nitrogen (N) and sulphur (S) are essential nutrients for plants growth. The N is typically provided by urea and S by gypsum fertilizer. The urea-N efficiency is often low due to the potential loss pathways [i.e. ammonia (NH3) volatilization, nitrate (NO3-) leaching intensified by nitrification, and denitrification] in the alkaline soils. This study investigated the effects of fertilization using ammonium sulphate (AS) or urea+AS instead of the most common practice of urea+gypsum on cereal and oil seed production and efficiency of N and S under variable agro-climatic conditions. The study was conducted in two south eastern Australian alkaline soils (i.e. the Vertosols of Nurrabiel and Horsham sites in the Wimmera and the Calcarosols in Nyrraby and Walpeup site in the Mallee region) with wheat and canola crops. The improved efficiency of AS over urea+gypsum was rarely observed in wheat under the Vertosols, however, promising in canola under the Calcarosols. In a 28 day lab incubation study, for example, AS maintained more N in ammonium (NH4+) form and reduced NO3- production with low N loss compared to urea+gypsum in the Walpeup Calcarosol. In glasshouse study of 28-42 days also indicated improved apparent N and S recovery in canola under the Walpeup Calcarosol. In three years (2009-2011) of field observation, the results indicated that biomass, grain yield, N and S recovery varied with soil types, crop types and agro-climatic conditions. The impact of fertilizer type on N and S efficiency was more pronounced with canola in the Calcarosol rather than canola in the Vertosol and wheat in the Calcarosol or Vertosol. For instance, AS increased biomass (39%), grain yield (49%) and N (49%) and S (44%) uptake in canola crops only in the Nyrraby Calcarosol in 2009 compared to urea+gypsum. Moreover, compared to urea+gypsum, the AS or urea+AS increased recovery efficiency of N by 5-8% and S by 17% in canola grown in the Nyrraby and Walpeup Calcarosols. The improvement in N recovery was attributed to the rhizosphere acidification in the Calcarosol as AS reduced soil pH by 0.2-0.5 units compared to urea+gypsum. Moreover, the low S uptake from gypsum compared to AS was caused by the negative interference of calcium (Ca) with N and phosphorus (P) uptake in canola under the Walpeup Calcarosol. Furthermore, an incubation study also showed that AS or urea+AS fertilizer reduced nitrous oxide (N2O 9-11%) emissions compared to urea+gypsum in the Walpeup Calcarosol. However, the advantage of using AS and/ or urea+AS fertilizers over urea+gypsum was variable and limited in wheat and the Vertosols of south eastern Australia. Therefore, AS remained as crop-soil specific in the fertilizer management strategies.
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ItemTransgene integration patterns in wheat( 2005)Wheat is a major crop worldwide. Continual development of wheat varieties by plant breeders is driven by the increasing demand for high-quality grain for an expanding range of end-uses. Starch quality is a target for improvement, as its end-use suitability depends on its composition and properties. In this thesis, microparticle bombardment was used to introduce an antisense sequence to the starch debranching enzyme, isoamylase into wheat (Triticum aestivum L.) and its diploid wild relative (Aegilops tauschii Cosson). In maize, rice and Arabidopsis, reduced isoamylase activity results in the replacement of amylopectin with a more highly branched polymer known as phytoglycogen. Ae. tauschii is often used in breeding programs as a source of traits for the improvement of bread wheat, but this is the first report of transgenic Ae. tauschii plants. Expression of the bar selectable marker and inheritance in the next generation was demonstrated, although most of the transgenic plants displayed transgene silencing. None of the transgenic bread wheat or Ae. tauschii had altered starch composition. Transgene expression and stability are determined by the structure of the transgenic locus, homologies between transgene and endogenous DNA sequences and the nature of the surrounding genomic DNA. Gene silencing is commonly associated with the presence of multiple intact or truncated transgene copies, which may be arranged as direct or inverted repeats, and the presence of bacterial sequences originating from the transformation vector backbone. This is a common observation amongst transgenic plants obtained via direct DNA delivery methods. The mechanism of transgene integration needs to be understood to enable the development of improved transformation systems and this was addressed as the second objective of this thesis. The structures of the transgenic loci in the wheat and Ae. tauschii were examined to provide possible reasons for transgene silencing and to examine the nature of transgene integration. Characteristics of the transgenic loci structures and the junctions between transgene-transgene and transgene-genomic DNA sequences were consistent with integration by illegitimate recombination involving double-strand break (DSB) repair. The proximity of transgenic loci to retrotransposon sequence may also have contributed to transgene silencing, as retrotransposons are often preferentially inactivated by host defence systems. The results presented in this thesis highlight the problems and limitations of wheat transformation. Improvements to transformation systems are essential to enable the recovery of useful transgenic plants at high frequencies, so that wheat breeders may fully utilise this technology in the development of improved wheat varieties. Agrobacterium-mediated transformation is likely to become the favoured method of transformation as integrated transgenes are more likely to be stably expressed and inherited. Alternative methods for obtaining gene knockouts, such as TILLING, are also discussed, as the adoption of such methods may be essential to allow commercial release of new improved wheat varieties circumventing public concern regarding genetically modified organisms.
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ItemPredicting the grain protein concentration of wheat from non-destructive measurements of the crop at anthesis( 2005)Grain protein concentration is an important specification for wheat, which determines the quality grade and price received by growers. It is difficult to achieve target grain protein concentration in semi-arid southern Australia, because of the low and variable rainfall. Growers may benefit from being able to predict grain protein concentration before harvest, especially where there is a threshold or `window' requirement for a particular grade. Grain outside specifications could be forward sold into other grades while prices were good. Spatial predictions of grain protein concentration would allow the pattern of harvest to be managed to optimise profit. This thesis proposed a method for predicting grain protein concentration from non-destructive measurements of the crop (spikes, spikelets) at or after anthesis. The theoretical propositions underlying the method were then evaluated using data from nitrogen fertiliser experiments, data from the literature, and a simulation exercise. The proposed method was to estimate grain number from spike or spikelet number. Variance in grain number, together with the diminishing returns response of grain number to nitrogen, would then be used. to estimate maximum grain number. Maximum grain number would be linked to a unique `critical' grain protein concentration, from which grain protein concentration at other grain numbers could be estimated. Spike and spikelet number were counted throughout grain-filling in nitrogen fertiliser experiments to determine the importance of time of counting. The time of counting was important for absolute, but not relative spike and spikelet numbers: Spike and spikelet number varied, throughout grain-filling, but interactions with nitrogen treatments were rare. Inclusion of spikelets in counting was based on glume length, which interacted with time of counting. Spike death was frequently observed and occurred in proportion to post-anthesis growth, at 0.187(±0.018) spikes/g. The rate with respect to grain yield was similar, at 0.190(±0.038) spikes/g. An analysis of mass/number relationships between grain, spike and spikelet number, and crop and spike biomass at anthesis, showed that grain number was better related to spike biomass, and that spike and particularly spikelet number, were better related to crop biomass. Spikelet number changed at .a rate of between 6.6 and 9.3 spikelets/g biomass across 'a range of experiments; spike number changed at a rate between 0.14 and 0.62 spikes/g. The interrelationships showed grain number should be related to spikelets/spike, and proportion of crop biomass in the spike. The relationships, however, only existed in some experiments and were not universal. An alternative suggested by the analysis was use of spike number as a direct proxy for grain number (ie. assuming constant grains per spike). Spike number was tested as a proxy for grain number initially by analysing the components of variance of grain number across nitrogen, rotation and plant density experiments. Spike number was the main component of variance in grain number (59.8- 71.0% of log(variance)) in nitrogen experiments, with no significant covariance between spike number and grains per spike. Grains per spike and covariance were much greater components of variance in plant density experiments, and grains per spike and spike number were equal sources of variance in rotation experiments, with small positive covariance. Spike number would be an unbiased, but not perfect proxy for grain number when nitrogen was the main factor varying, but not for factors related to rotation or plant density. Spike number and crop biomass at anthesis were compared as estimators of grain number in nitrogen experiments, in an analysis of the nature of the responses to nitrogen fertiliser. Grain number as an estimator of grain yield was included in the analysis to understand the likely effect of using grain number rather than yield as a predictor of grain protein concentration. Crop biomass at anthesis, spike number and grain number all reached maxima at similar nitrogen fertiliser rates, but crop biomass at anthesis was a more precise estimator for the maximum rate required for grain number (RMSE of nitrogen for maximum, 2.4 kg N/ha vs. 26.4 kg N/ha). Grain number had a maximum consistently higher (+32.6±8.0 kg N/ha) than the maximum for yield. Once nitrogen fertiliser rates were corrected for the different maxima, grain number and yield had identical relative response rates to nitrogen. The response rates of crop biomass at anthesis and spike number were both related to the response rate of grain number by a power relationship with exponent 0.6. The lack of methods for anticipating phase differences caused by late nitrogen application and pre-anthesis water deficit will prevent exploitation of these relationships in all environments. The estimation of maximum spike number from its variance was simulated across the width of an air-seeder, using consistent variations in nitrogen fertiliser rate between tynes to drive variance in spike number. Nitrogen fertiliser was normally distributed. It was possible to extrapolate the variance/spike number relationship to estimate the maximum only where the slope of the relationship was negative. Slopes close to zero caused errors. of fitting, where the `signal' from the relationship was indistinguishable from the `noise' in estimating variance. This coincided with low (below 0.8) relative spike numbers and led to over-estimation of low relative spike numbers. Low spike number because of sub- or supra-optimal nitrogen could be distinguished by the second derivative of the fitted function, which was positive for supra-optimal nitrogen. There was no unique `critical' grain protein concentration (for maximum yield or grain number) in southeastern Australia, but there was a consistent relationship between `critical' grain protein concentration and grain weight. The relationship in terms of grain nitrogen content was a linear function of grain weight. The parameters also varied with genotype, and signed relative grain number, calculated as GRS=1-G/GMax for supraoptimal nitrogen, and GRS=G/GMax-1 for sub-optimal nitrogen, where G is grain number. The best estimation of grain nitrogen across genotypes was: Grain N (mg N/grain) = 0.317 + 1.00 x GRS + (0.0115 -0.0181 x GRS) x W, where W is grain weight in mg/grain. The root mean squared error of grain protein concentration estimated from this function was 0.91%. Grain weight would need to be estimated to estimate grain protein concentration. Errors due to grain weight had more effect at higher GRS, and at lower grain weight. The conclusion was that grain protein concentration may be predicted using crop biomass or spike number as a proxy for grain number. Predictions would be best in the absence of pre-anthesis water deficit or nitrogen applied after Zadoks 32. The predictions would be best for relative grain number greater than 0.8 at sub-optimal nitrogen, and for any relative grain number at supra-optimal nitrogen. A confidence interval could still be provided for grain protein concentration at lower relative grain numbers with sub-optimal nitrogen. Predictions would be most accurate if grain weight was reliably above 35 mg/grain.
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ItemA molecular genetic study of seed dormancy in aegilops tauschii and expression of sprouting resistance in common hexploid wheat( 2004)The wild wheat relative Aegilops tauschii, has been identified as a useful source of preharvest sprouting (PHS) resistance for hexaploid bread wheat. Seed dormancy, a major contributor to PHS resistance, was shown to be partly expressed in hexaploid wheat derived from direct hybridisation between Triticum aestivum and Ae. tauschii. The enhanced seed dormancy possessed by the Ae. tauschii derived direct-cross wheat lines was manifested by embryo and seedcoat related mechanisms. The embryo related mechanism could not confer full expression of dormancy without the presence of seedcoat related factors, suggesting that the two mechanisms may be independently inherited. The presence of seedcoat related dormancy however, was not associated with the red seedcoat phenotype, which has traditionally been associated with PHS resistance in wheat. Red pigmentation of the seedcoat is likely to be "involved in the extreme dormancy possessed by Ae. tauschii but does not preclude partial expression within a white seedcoat background. The ability of Ae. tauschii derived wheat lines to enhance seed dormancy may have potential economic benefit to breeding for PHS resistance in white wheat varieties. Presently, white wheat varieties grown in the sprouting susceptible regions of Australia possess inadequate protection, costing the industry up to $100M annually. Inheritance of seed dormancy in Ae. tauschii was found to be controlled by one or two major genes which were influenced by minor genes and/or environmental factors. These results are consistent with the findings of several previous reports. Inheritance was shown to be dominant at the F3 grain generation, consistent with the generally dominant nature of dormancy possessed by red seeded genotypes. However, preliminary assessment of individual F2 seeds indicated recessive control of dormancy. Because genes possessed by the maternal tissues of the seedcoat do not segregate until the F3 seed generation, the F2 recessive model may be indicative of separate genetic control for the embryo related dormancy mechanism(s). Based on the above inheritance information, a bulked segregant analysis approach was initially undertaken for the development of linked molecular markers for seed dormancy. One microsatellite marker on chromosome 1D produced polymorphism between resistant and susceptible DNA bulks. A mapping approach was subsequently undertaken, revealing two significant QTL mapping to chromosome 1D. The putative QTL for seed dormancy will relate to the embryo component of dormancy, as the trait data employed related to the F2 seed generation, which was segregating for embryo related genes. The D genome of hexaploid wheat presently possesses the fewest QTL for PHS resistance of the three contributing genomes. Within the D genome, chromosome 1D was poorly represented in the literature. As such, 4e. tauschii represents a potential to bolster numbers of QTL for sprouting resistance in hexaploid wheat. Given the homology between the D genomes of Ae. tauschii and T aestivum, the microsatellite markers identified, flanking the putative QTL, will likely be transferable to hexaploid bread wheat. Seed dormancy is strongly influenced by conditions during growth. As such, unambiguous selection through use of molecular markers will expedite the introgression of this economically important trait into elite wheat cultivars.
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ItemA study of factors influencing in vitro stability of nitrate reductase from wheat leaves( 1979)This review and the following chapters are concerned predominantly with the processes occurring in higher and lower plants which regulate the amount of NR present in vitro as controlled by degradation, and the level of activity of the existing enzyme. Those factors regulating the synthesis of NR will not be discussed in any detail but only mentioned where they also affect other mechanisms regulating NR. Nitrate reductase is unstable both in vivo and in vitro (101,193, 252). In vitro instability occurs since the isolation of enzymes and other cell components from plant tissue involves disruption of the plant cell. This results in mixing of substances which in situ were rigidly compartmentalized and is likely to result in the isolation of an enzyme which is modified from its native form. Factors present in plant cells which make plant proteins particularly unstable in vitro have been reviewed by Stahrran (216) and Pirie (157). They include vacuole acids, carbohydrates, hydrolytic and oxidative enzymes and phenolic components and their derivatives. In vivo variation in activity occurs in response to a number of other factors, including tissue age (103,129,166,243,264) and environment (15,72,82,1.03,129,261). Tissue age has been shown to influence the activity or stability of NR extracted from a number of species including corn (194,195,264), wheat (221), oats (194,195), tobacco (195) and barley (48). Nitrate reductase has been demonstrated in nearly all plant parts (16) and its ubiquitous presence suggested in higher plants (16,184). Nevertheless, due to the number of factors involved, detection of activity would only occur given suitable physiological and environmental conditions together with use of the correct extraction and assay procedure. Determining if the level of activity derived is an accurate estimate of the in situ activity is even more difficult. This has been attempted by correlating NR activity and grain or plant nitrogen (28, 36) . In vivo instability is indicated by the decline in NR activity with the onset of darkness, depletion of nitrate supply, and water or heat stress (11,82,121,168,235). Under appropriate conditions these factors could also affect the enzyme in vitro.
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