School of Agriculture, Food and Ecosystem Sciences - Theses
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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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ItemThe effects of post-anthesis heat stress on wheat yield and quality( 1996)Post-anthesis temperature is a major determinant of wheat yield and quality. Post-anthesis temperatures in the moderately high range (ca 25-32C) are known to reduce grain yield but increase bread wheat quality, whereas very high (>32C) temperatures are known to significantly reduce both yield and quality. In Mediterranean and continental climates, such as Australia and the US., wheat is exposed to moderately high temperatures throughout most of the grain filling period, and very high temperatures may occur for an average 8% of grain growth. This thesis is primarily concerned with examining the effects of very high temperature on wheat yield and quality. Specifically, the study was designed to: 1) quantify the effects of short (3-5 day) periods of very high temperature on wheat yield and quality; and 2) determine the extent of genotypic variation in response of wheat yield and quality to very high temperature. Two varieties of wheat differing widely in heat tolerance were selected from 75 cultivars of wheat that were screened for tolerance to very high temperature. These two varieties (Oxley and Egret, heat sensitive and heat tolerant, respectively) were exposed to a variety of heat treatments in order to determine whether varietal differences in heat tolerance were maintained for heat treatments occurring at 3) different stages of grain growth and for 4) varying durations of heat stress. The 5) interaction of moderately high and very high temperatures was examined in order to determine whether cool temperatures following severe heat stress could alleviate the deleterious effects of very high temperature on yield and quality. In order to 6) examine the importance of acclimation to heat stress and to 7) establish a repeatable selection methodology, the impact of sudden increases to a high maximum temperature was compared with more gradual (6C h-1) rises to the same high temperature (40C). For each of the experiments 3 to 7 (above) results are presented for the effects of heat stress on: a) the accumulation of grain dry matter and water during grain growth; b) the accumulation during grain growth of total protein and its functionally-important fractions (SDS-soluble and SDS-insoluble polymer [glutenin], monomer [gliadin] and albumin/globulin), as determined by size-exclusion high-performance liquid chromatography and c) dough mixing behaviour using the 2-g mixograph. It is concluded that: 1) wheat genotypes vary widely in their responses of yield and quality to short periods of very high temperature; 2) the response to heat stress varies with the timing of stress: yield was reduced more by early than late-applied stress, whereas dough strength tended to decline most markedly in response to heat stress applied towards the end of grain filling; 3) both grain yield and dough strength declined linearly with increased duration of heat stress; 4) in a heat sensitive variety, moderately high and very high temperatures during grain filling each reduced grain yield and dough strength: cool temperatures following exposure to very high temperature did not reduce the effects of very high temperature on either yield or quality; 5) some varieties of wheat appear to acclimate rapidly to heat stress: a gradual (6C h-1) increase from ca 20-40C lessened the impact of heat stress on yield and quality when compared with a sudden increase over the same temperature range. These results are discussed with special reference to their implications for: 1) selecting and breeding for heat tolerance in wheat; 2) predictive modelling of the effects of very high temperature on wheat yield and particularly quality.
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ItemGenotype and environmental influences on phasic development in wheat (Triticum aestivum L.) and the expression of yield components, especially spikelet number per head( 1995)The variation in, and the influence of, certain environmental factors on preanthesis phases of development in wheat was examined with particular reference to the number of spikelets produced per head. When the pre-anthesis phase was divided into three phases; the Ieaf initiation, spikelet initiation and culm elongation phases, considerable cultivar variation was found in the durations and rates of the three phases. A cultivar was found that departed from the general negative correlation between rate and duration of spikelet initiation giving possible scope for breeding for increased spikelet number without altering the duration of spikelet initiation. Variation in the rate and duration of the three development phases was also found for a selection of diploid and tetraploid wheat. For these species, spikelet number was found to be more closely associated with the duration of spikelet initiation. This character could be of use in long-season wheat cultivars. When the effects of photoperiod and light intensity on wheat phasic development and spikelet number were compared, photoperiod was found to have more influence. The transfer of wheat cultivars between long and short photoperiods at double ridge and terminal spikelet determined that the rate of development was influenced by a "memory" effect; both prior and current photoperiods influenced the rate of development. It was also noted that initial exposure to long photoperiod could have a sustaining effect on wheat development. Subjecting wheat lines to increased temperature increased the durations of development, in terms of thermal time, indicating that the relationship was not linear. The durations of pre- and post-terminal spikelet phases were found to respond differently to temperature. A selection of 6 wheat cultivars, varying in time to anthesis were grown in the field and it was found that photoperiod responses exerted the major influence on the durations of development. Basic development responses and vernalisation were found to exert comparatively less influence on development. The importance of basic development responses were not discounted as a means for breeding wheat cultivars for specific environments.