Brilliant cresyl blue has been used but little in botanical histology. The preparation and methods of use of the stain in squash technique are described. The technique is then used to follow the mitotic cycle in Allium cepa. It is shown to be an excellent morphological stain and it has moreover some promise of being useful in cytology. Some seventy photomicrographs demonstrate its possibilities. / Science, Faculty of / Botany, Department of / Zoology, Department of / Graduate

There is a need to breed for root systems architectures (RSAs) that optimise soil resource acquisition. This requires high resolution and high-throughput quantification of RSA in as natural an environment as possible. Current imaging techniques are limited by cost, reproducibility, throughput and complexity. This thesis describes (1) the construction of a low cost, high-resolution, root phenotyping platform that requires no sophisticated equipment which is adaptable to most laboratory and glasshouse environments and (2) its application to quantify environmental and temporal variation in RSA between genotypes of Brassica rapa L. The high resolution root phenotyping system (HRP) that was constructed employed 24 scanners and could screen up to 72 individual plants at any time, with the possibility of capturing thousands of root images daily depending on the operational number of scanners and scanning periodicity. Plants were supplied with a complete nutrient solution through the wick of a germination paper. Images of RSA were acquired automatically, over extended periods, using multiple scanners controlled by customised software. The RSA data was used to validate a mechanistic model and mixed effects models were used to describe the sources of variation in traits contributing to RSA. Plants were also grown in rhizoboxes and under varying concentrations of P ([P]ext). Broad-sense heritability (H2), was highest (≥ 0.70) for shoot biomass, length of primary roots (PRs), number of lateral roots (LRs). Coefficients of variation in RSA traits within a genotype were large and ranged between 5 and 103%. It was found that between 4 and 48 replicates were needed to detect a significant difference (95% CI, 50% difference between trait means). Significant differences were found between genotypes in root traits with strong positive correlations among RSA traits and between biomass and RSA traits. Principal component analyses identified 5 significant axes of variation, accounting for approximately 86 and 78% of the variation in the genotypes on paper and soil substrates, respectively. Cluster analysis of the genotypes produced 5 discrete groups. Genotypes with more or less shoot biomass or with bigger or smaller RSA could be distinguished. A density-based 2D model reproduced experimental results accurately by simulating PR length and total length of LRs. Mixed-effects statistical models demonstrated that root traits show temporal variations of various types with significant effects of genotype. All genotypes followed a similar growth pattern with time, but differed in their maximum total root length (TRL), primary root length (PRL) and LR growth. A 3-parameter logistic model satisfactorily described TRL and PRL when genotypes were grown on both paper and soil substrates. On paper substrate, TRL required only a single, random-effect parameter (asymptote), describing maximum TRL. On soil substrate, TRL required two random-effects parameters, asymptote and inflection, describing maximum TRL and time at which ½ of maximum TRL occurs, respectively. Primary root length on both paper and soil substrates required only a single, random-effect parameter, describing maximum PRL. The growth rate of LRs of all ages followed a quadratic function and required only a single, random-effect parameter, describing maximum growth rate. There was variation in specific RSA traits and plasticity in response to [P]ext among genotypes. Length of the apical un-branched zone of the PR increased with increasing [P]ext. Total root length, total LR length and number of LRs was positively correlated with total plant tissue P concentration at low [P]ext but not at high [P]ext. Paper substrate was more suitable for screening seedling root traits but root phenotypes must be validated in situ in the field or in soil media because some differences were evident between data observed on paper and soil substrates. Scanner-based phenotyping of RSA provides economical means of studying the mechanisms underlying the plant-soil interactions and can be used to quantify environmental and temporal variation in traits contributing to RSA. The HRP system can be extended to screen the large populations required for breeding for efficient resource acquisition. The necessity for high replication and time-consuming image analysis could however limit throughput in the phenotyping system.

Durum wheat (Triticum turgidum L. var durum) and spring barley (Hordeum vulgare L.) are the most widely grown crop species in the semi-arid to arid areas of the Mediterranean region. However, their average on-farm yields are relatively low, 1.95 and 2.60 t ha-1, respectively (FAO, 2007). Water is generally recognized as the most limiting factor for barley and durum wheat production in the Mediterranean, though it has been found, at least for some regions, that N fertilizer applications have been limiting (Passioura, 2002). Water in the Mediterranean is relatively scarce and predictions for 2025 show that water limitations for agricultural production in that region will intensify (IWMI, 2000). Nitrogen fertilizer represents a significant cost of production for the grower and may also have negative environmental impacts through nitrate leaching, use of fossil fuels for manufacture and application, and N2O emissions associated with denitrification. Reducing excessive N fertilizer inputs and increasing water productivity, whilst maintaining acceptable yields, will be aided by increases in uptake efficiency.

Rice (Oryza sativa L.) provides up to 50% of the total calories consumed in countries such as India, Madagascar and Nigeria. As a crop, rice can require significant fertiliser inputs to maintain the required yields. Additionally, climate change has increased the need for rice varieties with improved drought resistance, tolerance to pests and more efficient acquisition of nutrients from soil. One major fertiliser input for rice is phosphate; reducing phosphorus (P) fertiliser use would have environmental and economic implications. Root traits linked to P acquisition in crops include shallow root angle, lateral root proliferation and increases in root hair length and density. Two T-DNA knockout alleles with reduced gravitropic response, Osaux1-1 and Osaux1-2, were used to investigate the influence of shallow root angle on P uptake. OsAUX1 is a rice ortholog for the Arabidopsis thaliana gene AUX1, which controls lateral root growth and gravitropic response. The wildtype and mutant rice plants were grown in soil and non-destructively imaged using X-ray micro Computed Tomography (X-ray CT). In Chapter Three, visualisation of rice roots in soil using X-ray CT was optimised by determining the ideal soil moisture content that would produce the best images. Water in soils has a similar X- ray attenuation density to that of plant roots and can influence segmentation of roots from soil in X-ray CT images. It was found that soil at nominal field capacity (ca. 3 days of drainage) produced the best contrast between soil fractions (organic matter, minerals and pore space) and root material. In Chapter Four, the impact of X-ray dose on root growth was quantified because the experimental design included repeated scanning of the same sample (Chapter Five). It was found that even under repeated scanning, the X-ray doses involved in this work (ca. 15 Gy per sample) did not significantly affect the root architecture and overall plant growth in rice cultivars used. In Chapter Five, Osaux1-1 and Osaux1-2 retained the agravitropic phenotype that was observed on agar-based systems when plants were grown in loamy sand soil. However, when subject to various soil P concentrations and distributions (Chapter Six), Osaux1-1 had similar gravitropic response and P uptake as wildtype. It was unclear what role gravitropism and topsoil foraging played in P uptake for these rice cultivars, if any. OsAUX1 could be linked to P uptake as well as responses to soil P concentration and distribution. Under uniformly low soil P wildtype had a shallower root system distribution than Osaux1-1. Of most interest were the results when sufficient soil P was sequestered to the top 4 cm of the soil column and low P was maintained in the bottom 6 cm. Under these conditions, wildtype took up more overall P, had almost twice the biomass, twice the total root length and twice the surface area when compared to Osaux1-1. This provides evidence that OsAUX1 can be linked to adaptation to P stress and distribution of P in soil through control of fine root characteristics and not necessarily its impact on gravitropic response. Chapter Seven describes the investigation into the impact of OsAUX1 on sub-architectural effects of the root system that could influence P uptake. It was determined that OsAUX1 was involved in root hair density and elongation under varying P availability for agar grown plants. In comparison to wildtype, Osaux1-1 had significant variation in root hair phenotype that seemed unrelated to a P stress response. In flooded environments, root hairs influence the potential for root:soil contact that is integral to P uptake in rice paddies which have reduced soil conditions and mass water flow that can transport plant available soluble P. This reinforces the potential for an interaction between OsAUX1 and P uptake in paddy rice.

Pollen development is critical for plant reproduction. Numerous nuclear mutations affect the function of pollen resulting in male sterility. The myb26 mutant is one such male sterile mutant allele, which results in anther indehiscence. Five putative MYB26 interactive proteins were previously identified from screening an Arabidopsis stamen yeast-2-hybrid library with MYB26 as bait. These proteins include Y2H128, Y2H320, Y2560, Y2H620 and Y2H970. Transient expression of these proteins, except Y2H128 were studied in planta by infiltration of Nicotiana benthamiana leaves and all were found to be expressed in the nucleus and co-localised with MYB26. Förster Resonance Energy Transfer (FRET) was used to confirm the positive interaction between MYB26 and the Y2H320 protein. Analysis of the expression and possible function of the putative interactors was examined using SALK knockout T-DNA insertion mutants, RNAi and over-expression lines. SALK knockout lines of four Y2H genes were fully fertile and produced viable pollen despite no expression of the corresponding genes in the insertional mutants of Y2H320 and Y2H560. Independent silencing by RNAi of the other two genes, Y2H970 and Y2H128, also resulted in no alteration in plant phenotype. Transgenic plants over-expressing the Y2H genes also showed no differences in secondary thickening of anthers in endothecium compared to the wild type (Ler). Using a Prom320::GUS transgene, GUS expression was observed in the anthers, nectaries and stigmatic tissues; this pattern of Y2H320 expression corresponds to that seen for MYB26, confirming that interaction in planta is possible. The research also involved an analysis of additional four Arabidopsis male sterile mutants in the M2 and M4 generations. The phenotypes of these mutants were similar to that of the myb26 mutant, where viable pollen was evident, but anther dehiscence did not occur. These novel mutants were not rescued by Jasmonic Acid (JA) treatment. Allelism/complementation analyses indicated that the two mutants c20 and mss are alleles of myb26, whilst msak and c12 are novel mutations at different loci. Gene mapping of the MSAK gene indicated that it is located on chromosome 1. Further higher resolution genetic mapping with Simple Sequence Length Polymorphism (SSLP) molecular markers identified a closer linked marker (12.57 cM to MSAK), suggesting that the gene is located ~3.2 Mb from the start of chromosome 1. A possible candidate for MSAK gene located within the region 3.4-3.5 Mb is the transcription factor Transducin/WD40 repeat-like protein (At1g10580; located at 3.49 Mb), which is involved in pollen development. Further investigation of additional candidate genes for MSAK in the region of ~3.0-4.0 Mb of chromosome 1 that are related to male gametophyte, pollen development or belong to MYB superfamily identified a number of genes, one likely candidate is At1g10770 (located at ~3.59 Mb). Previous reports indicated that reduction of At1g10770 transcript resulted in pollen tube growth retardation, partial male sterility and reduced seed set.

Pollen development and release involves a number of important stages, which govern the success of fertilisation and thus indirectly crop yields. The secondary cell wall in the anther plays a pivotal role in anther dehiscence by offering mechanical strength required for opening and pollen release (Wilson et al. 2011). MYB26/MALE STERILE35(MS35) is a key regulator of the secondary thickening development in anther, mutation of this gene results in a failure of anther dehiscence and functional male sterility (Steiner-Lange et al. 2003; Yang et al. 2007). However, the regulatory network of MYB26 remains to be fully identified. To address this issue, the MYB26 direct targets and interactive proteins were investigated. Putative targets of MYB26 were selected, based on their expression patterns, from previously determined expression profiles of the ms35 mutant (Song, 2009). PKSP, a receptor-like cytoplasmic kinase (RLCK) was co-expressed with MYB26. Chromatin immunoprecipitation (ChIP)-PCR indicated that MYB26 may bind to the first intron of PKSP. The NAC SECONDARY WALL–PROMOTING FACTOR1 (NST1) and NST2 genes have been demonstrated as direct targets of MYB26 in ChIP-PCR. However, electrophoretic mobility shift assay (EMSA) did not show binding and retardation, possibly due to the requirement of additional proteins to facilitate MYB26 binding. MYB26 interactive proteins were investigated using the yeast two-hybrid system (C.Yang, Z.A.Wilson, unpublished data) and Förster resonance energy transfer (FRET) assays. Y2H560, a CHY-type/RING-type Zinc finger protein and Y2H320/TGA9, a bZIP transcription factor family protein interacted with MYB26 in plant cell nuclei. Y2H320/TGA9 has been shown to be functionally redundant with TGA10 in regulating anther development (Murmu et al. 2010). The tga9tga10 double mutant produced indehiscent anthers and was male sterile due to the developmental arrest of the adaxial anther lobes and abnormal tapetum and pollen development in the abaxial anther lobes. In summary, MYB26 appears to regulate endothecium development and secondary thickening formation and thus anther dehiscence probably through directly regulating the subfamily VII RLCK PKSP and the NAC transcription factors NST1, NST2 and by interacting with a CHY-type/RING-type Zinc finger protein Y2H560 and the bZIP transcription factor Y2H320/TGA9.

High temperature can have a serious impact on plant development; rising temperatures and environmental fluctuations mean that this is becoming an increasing problem for sustainable agriculture. Many studies have indicated that pollen development is very susceptible to high temperature (HT) stress, particularly during early development, and that the anther tapetum cell layer is extremely vulnerable, resulting in reduced fertility or complete male sterility (MS). In this project, Arabidopsis plants were stressed with 32°C HT during flowering and then assessed by microscopy for phenotypic changes to anther and pollen development, and subsequent reproductive development. The results indicate that the HT had a significant negative impact on plant reproduction, particularly during the stress treatment, with some recovery of fertility post HT. Samples of plant buds were divided into different growth stages and collected for analysis of fertility and for gene expression analysis. Several genes, which appear from available microarray data to be associated with HT stress and are also specifically expressed during tapetum development, were chosen to test for expression changes associated with temperature stress, both during and after HT stress. Phenotype analysis of insertional knockout mutants of these genes, both with and without HT stress, was used to assess their potential impact on resilience to temperature stress. Transcriptomic analysis of whole genome was conducted by RNA-seq in young (prior to polarized microspore stage) and old buds (from polarized microspore stage to pollen mitosis) isolated from HT-stressed and non-stressed Arabidopsis Ler plants. This has identified a set of HT specific genes that are differentially expressed in different HT period treated plants. The anther tapetum serves to regulate pollen development and is critical in the production of the pollen wall. It goes through a defined process of programmed cell death (PCD) to facilitate transfer of pollen wall materials onto the developing pollen grains. Disturbance of the timing or progression of this PCD process, for example by heat stress frequently results in male sterility. Four GFP reporter constructs that have been used as markers during ovule PCD analysis were tested for expression during pollen development and particularly focusing upon the stages of tapetal PCD. These reporter genes showed different stage specific expression during anther development. They have now been introgressed into a number of Arabidopsis male sterile mutants that show PCD-related defects, including ms1, ams and myb26 male sterile mutants. The F1 generation of these showed similar GFP expression to the parent plants, however the homozygous male sterile F2 generation plants appeared to show different patterns of GFP expression. Two of them (BFN1 and CEP1) are expressed in the anther tapetum during the stages of tapetum PCD. Expression analysis suggests that HT-stress affects the expression of BFN1 and CEP1, which may be linked to abnormal degeneration of the tapetum under HT-stress.

Post-embryonic root development is a plastic process by which plants are able to interface with the rhizosphere in order to provide anchorage, and increase surface area available for acquisition of nutrients and water. While root growth is governed primarily by auxin/cytokinin interactions, roots are able to sense the presence of surrounding nutrient deposits and changes to environmental conditions – directing growth accordingly – due to the action of signalling cascades. This thesis presents data pertaining to the characterisation of two signalling elements involved in governing root architecture; the small signalling peptide CLAVATA/EMBRYO SURROUNDING REGION 26 (CLE26), and the receptor kinase ARABIDOPSIS CRINKLY 4 (ACR4). Initially, the origins and evolutionary history of root architecture are explored, and an overview of signalling elements involved in root architectural development is provided, before discussing the potential benefits that manipulation of signalling events may allow in targeted crop improvement. To provide background on peptide signalling, the physiological and biochemical effects of small signalling peptides are discussed in view of the current literature, demonstrating the diverse range of developmental processes which are known to be regulated by these ligands and their known receptors. Following this, functional characterisation analyses indicate CLE26 as a novel, potent inhibitor of primary root growth and protophloem development in Arabidopsis, and is also shown to induce a similar effect upon exogenous application to several crop species. Furthermore, data is presented demonstrating the clear requirement for functional analysis during the development phase of creating antagonistic peptides, as a previously described antagonistic peptide technology was not applicable in all cases. Concluding the exploration of CLE26 signalling, a phosphoproteomics screen was conducted to probe further into CLE26 function, determining 23 putative effectors of CLE26 signalling, which are discussed in view of their potential to mediate CLE26 signalling, according to current literature. Next, the known roles of ACR4 and its orthologues are reviewed, demonstrating the importance of ACR4 signalling in many developmental processes, including regulation of asymmetric cell division during postembryonic root development. Although ACR4 activity is known to regulate asymmetric cell division in both columella stem cells and lateral root primordia, little is known about the downstream mediators of ACR4 signalling. In an attempt to fill this gap in knowledge, yeast 2-hybrid and co-immunoprecipitation approaches were employed. These two parallel proteomics screens together resulted in identification of 19 putative interactors of ACR4 signalling (PAIPs), which are discussed as potential mediators of ACR4 signalling in view of current literature. Of the identified PAIPs, three were further characterised by loss of function analysis, demonstrating that loss of PHOSPHOLIPASE Iγ2 and a PROTEIN OF UNKNOWN FUNCTION (At1g49840/UNK) was able to affect root architecture. Further in-silico characterisation of UNK reveals its similarity to soluble phospholipase receptors, which, alongside PLA-Iγ2, may potentially implicate ACR4 as a key player in a novel mechanism involved in regulation of bioactive lipid production.

This thesis presents evidence that pollen colour and odour may be used as cues in resource-location by pollen feeding insects; namely, the pollen beetle, Meligethes aeneus, a pest of cruciferous crops such as oilseed rape, Brassica napus (OSR), and the honey bee, Apis mellifera, an economically important generalist pollinator. Pollen beetle adults were attracted to both the colour and odour of OSR flowers in a wind tunnel, and linear track olfactometer studies showed that part of the attractive floral odour emanated from pollen. Beetles were attracted to OSR floral odour throughout their life cycle, indicating that 'innate' search images may be important in location of hosts upon which they can reproduce. Responses to floral odours from plants upon which the beetles do not reproduce varied according to the beetle's sex, life cycle phase and feeding history. There was some evidence for the use of pollen cues in oviposition, when oviposition incidence in male-fertile buds containing pollen and male-sterile buds without pollen were compared in the field. In feeding studies conducted in the laboratory, larvae displayed obligatory requirements for the resources from OSR flowers; they were unable to develop in field bean, Vida faba (FB) flowers. Although pollen consumption was not obligatory for larval survival and development, it reduced developmental time, and improved survival and 'fitness'. Restrained honey bees were able to learn the odours of OSR and FB pollens associatively, and could discriminate between them in the conditioned proboscis extension bioassay. This ability was confirmed in more natural conditions; free-flying bees discriminated between OSR and FB pollens on the basis of their odour, but showed a colour preference for the yellow OSR pollen over the grey pollen of FB. The relative importance of pollen as an attractive signal in OSR and FB is discussed.

A knowledge of root growth and the activity of separate members of the root system is necessary before a comprehensive understanding of plant water and nutrient uptake is possible. The literature describing the developmental characteristics of wheat root systems is first reviewed. Methods of examining root systems in the field are compared, and studies of the contribution of seminal and nodal roots, and the effects of soil environment are discussed. Finally, nutrient and water uptake are considered mainly from the literature concerned with soil processes supplying nutrients to the root surface. The literature survey highlights the scarcity of field studies of water and nutrient uptake compared to laboratory studies and the poor understanding of the ways in which soil water status affects root growth and activity. An experiment in which spring wheat was grown in soil columns in a controlled environment is reported. Water was withheld during growth and the consequences for root growth and nutrient and water uptake followed. Nodal root growth was also restricted but this treatment was largely inconclusive because of the limited time during which conditions comparable to those in the field could be maintained. It was decided from these experiments to work with a field crop; a major study of the micro-climate and growth of winter wheat was in progress, so it was appropriate to examine in detail the growth and functioning of the crop's root system. A number of experiments were set up but this thesis mainly describes the root growth, and associated nutrient and water uptake of the normal field-grown crop. Measurements of root dry weight and length, plant nutrient content and water use are reported in early sections, with subsequent calculations of nutrient and water inflow; the possible contribution of mass-flow to plant nutrient is considered. A pattern of nutrient inflow not previously reported was found and possible explanations are discussed. The influence of soil properties, root distribution and atmospheric conditions on water inflow are also examined. The work shows the importance of field studies in understanding root growth and activity, and puts forward a number of suggestions for future progress.