The manipulation of apoptosis-related genes to generate resistance to Fusarium wilt and water stress in banana

Bananas are susceptible to a diverse range of biotic and abiotic stresses, many of which cause serious production constraints worldwide. One of the most destructive banana diseases is Fusarium wilt caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc). No effective control strategy currently exists for this disease which threatens global banana production. Although disease resistance exists in some wild bananas, attempts to introduce resistance into commercially acceptable bananas by conventional breeding have been hampered by low fertility, long generation times and association of poor agronomical traits with resistance genes. With the advent of reliable banana transformation protocols, molecular breeding is now regarded as a viable alternative strategy to generate disease-resistant banana plants. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi. Further, the transgenic plants showed increased resistance to a range of abiotic stresses. In this thesis, the use of anti-apoptosis genes to generate transgenic banana plants with resistance to Fusarium wilt was investigated. Since water stress is an important abiotic constraint to banana production, the resistance of the transgenic plants to water stress was also examined. Embryogenic cell suspensions (ECS) of two commercially important banana cultivars, Grand Naine (GN) and Lady Finger (LF), were transformed using Agrobacterium with the anti-apoptosis genes, Bcl-xL, Bcl-xL G138A, Ced-9 and Bcl- 2 3’ UTR. An interesting, and potentially important, outcome was that the use of anti-apoptosis genes resulted in up to a 50-fold increase in Agrobacterium-mediated transformation efficiency of both LF and GN cells over vector controls. Regenerated plants were subjected to a complete molecular characterisation in order to detect the presence of the transgene (PCR), transcript (RT-PCR) and gene product (Western blot) and to determine the gene copy number (Southern blot). A total of 36 independently-transformed GN lines (8 x Bcl-xL, 5 x Bcl-xL G138A, 15 x Ced-9 and 8 x Bcl-2 3’ UTR) and 41 independently-transformed LF lines (8 x Bcl-xL, 7 x BclxL G138A, 13 x Ced-9 and 13 x Bcl-2 3’ UTR) were identified. The 41 transgenic LF lines were multiplied and clones from each line were acclimatised and grown under glasshouse conditions for 8 weeks to allow monitoring for phenotypic abnormalities. Plants derived from 3 x Bcl-xL, 2 x Ced-9 and 5 x Bcl-2 3’ UTR lines displayed a variety of aberrant phenotypes. However, all but one of these abnormalities were off-types commonly observed in tissue-cultured, non-transgenic banana plants and were therefore unlikely to be transgene-related. Prior to determining the resistance of the transgenic plants to Foc race 1, the apoptotic effects of the fungus on both wild-type and Bcl-2 3’ UTR-transgenic LF banana cells were investigated using rapid in vitro root assays. The results from these assays showed that apoptotic-like cell death was elicited in wild-type banana root cells as early as 6 hours post-exposure to fungal spores. In contrast, these effects were attenuated in the root cells of Bcl-2 3’ UTR-transgenic lines that were exposed to fungal spores. Thirty eight of the 41 transgenic LF lines were subsequently assessed for resistance to Foc race 1 in small-plant glasshouse bioassays. To overcome inconsistencies in rating the internal (vascular discolouration) disease symptoms, a MatLab-based computer program was developed to accurately and reliably assess the level of vascular discolouration in banana corms. Of the transgenic LF banana lines challenged with Foc race 1, 2 x Bcl-xL, 3 x Ced-9, 2 x Bcl-2 3’ UTR and 1 x Bcl-xL G138A-transgenic line were found to show significantly less external and internal symptoms than wild-type LF banana plants used as susceptible controls at 12 weeks post-inoculation. Of these lines, Bcl-2 3’ UTR-transgenic line #6 appeared most resistant, displaying very mild symptoms similar to the wild-type Cavendish banana plants that were included as resistant controls. This line remained resistant for up to 23 weeks post-inoculation. Since anti-apoptosis genes have been shown to confer resistance to various abiotic stresses in other crops, the ability of these genes to confer resistance against water stress in banana was also investigated. Clonal plants derived from each of the 38 transgenic LF banana plants were subjected to water stress for a total of 32 days. Several different lines of transgenic plants transformed with either Bcl-xL, Bcl-xL G138A, Ced-9 or Bcl-2 3’ UTR showed a delay in visual water stress symptoms compared with the wild-type control plants. These plants all began producing new growth from the pseudostem following daily rewatering for one month. In an attempt to determine whether the protective effect of anti-apoptosis genes in transgenic banana plants was linked with reactive oxygen species (ROS)-associated programmed cell death (PCD), the effect of the chloroplast-targeting, ROS-inducing herbicide, Paraquat, on wild-type and transgenic LF was investigated. When leaf discs from wild-type LF banana plants were exposed to 10 ìM Paraquat, complete decolourisation occurred after 48 hours which was confirmed to be associated with cell death and ROS production by trypan blue and 3,3-diaminobenzidine (DAB) staining, respectively. When leaf discs from the transgenic lines were exposed to Paraquat, those derived from some lines showed a delay in decolourisation, suggesting only a weak protective effect from the transgenes. Finally, the protective effect of anti-apoptosis genes against juglone, a ROS-inducing phytotoxin produced by the causal agent of black Sigatoka, Mycosphaerella fijiensis, was investigated. When leaf discs from wild-type LF banana plants were exposed to 25 ppm juglone, complete decolourisation occurred after 48 hours which was again confirmed to be associated with cell death and ROS production by trypan blue and DAB staining, respectively. Further, TdT-mediated dUTP nick-end labelling (TUNEL) assays on these discs suggested that the cell death was apoptotic. When leaf discs from the transgenic lines were exposed to juglone, discs from some lines showed a clear delay in decolourisation, suggesting a protective effect. Whether these plants are resistant to black Sigatoka is unknown and will require future glasshouse and field trials. The work presented in this thesis provides the first report of the use of anti-apoptosis genes as a strategy to confer resistance to Fusarium wilt and water stress in a nongraminaceous monocot, banana. Such a strategy may be exploited to generate resistance to necrotrophic pathogens and abiotic stresses in other economically important crop plants.

EMG biofeedback in the treatment of tinnitus : an experimental evaluation

26 tinnitus patients received either electromyogram (EMG) biofeedback with counterdemand instructions, EMG biofeedback with neutral demand instructions, or no treatment. Assessment was conducted on self-report measures of the distress associated with tinnitus, the loudness, annoyance and awareness of tinnitus, sleep-onset difficulties, depression, and anxiety. Audiological assessment of tinnitus was also conducted and EMG levels were measured (the latter only in the 2 treatment groups). No significant treatment effects were found on any of the measures. There was a significant decrease in the ratings of tinnitus awareness over the assessment occasions, but the degree of change was equivalent for treated and untreated groups. Results do not support the assertion that EMG biofeedback is an effective treatment for tinnitus.

An evaluation framework for educational reform projects for teacher quality improvements in developing countries : a case study of Egyptian education reform

The role of the evaluation for Official Development Assistance (ODA) enterprises including educational development has become critical after increasing “aid fatigue” experienced by the international community in the 1990s. To date, however, monitoring and evaluating outcomes of the projects has been limited to the project life. Consequently these have been mainly through the international aid agencies. Furthermore, the monitoring and evaluation led by international aid agencies have paid little attention to aspects of the sustainability of technical cooperation in educational development. To sustain the impact of technical cooperation, the reinforcement of evaluation has drawn increasing attention in light of the emerging modalities in international development. Therefore this research was inspired to investigate alternative evaluation frameworks for an educational reform project for teacher quality improvement that may increase possibilities for long term sustainability. Importantly, the new modalities in international development and educational issues provide new options. In addition, the research reviewed theoretical and practical issues surrounding evaluation in general, and highlighted the evaluation of education reform projects. The research reported explored via case studies, the evaluation processes employed by the Egyptian education reform projects implemented by the Japan International Cooperation Agency (JICA) and the United Nations Children’s Fund (UNICEF). The case studies used three data sources (archival and relevant documents, a survey questionnaire and interviews) to illuminate the contextually-embedded evaluation processes. The research found that process evaluation is a potential alternative method since it is likely to be locally institutionalised, which may yield long-term sustainability of the projects.

An evaluation of how well undergraduate property students are prepared for commencing their careers

The evolution of property education to adapt to the changing business environment requires changes to course content, methods of delivery and assessment. Many universities have a special interest in understanding how successfully students transition in and transition out of property programs. This study sought the views of final year students engaged in professional employment, employer representatives and representatives from professional associations to assess the preparedness of students to commence professional employment. The study showed that generally students possessed an appropriate level of technical and ‘soft skills’ to enter the professional realm with development required in some areas. All stakeholders agreed that the transition out of university was made more seamless through greater engagement with industry through field work and work experience from the intermediate stages of the property course.

An evaluation of viability of public private partnerships in social infrastructure procurement projects in Queensland, Australia

Public private partnerships (PPP) have been widely used as a method for public infrastructure project delivery not only locally and internationally, however the adoption of PPPs in social infrastructure procurement has still been very limited. The objective of this paper is to investigate the potential of implementation of current PPP framework in social affordable housing projects in South East Queensland. Data were collected from 22 interviewees with rich experiences in the industry. The findings of this study show that affordable housing investment have been considered by the industry practitioners as a risky business in comparison to other private rental housing investment. The main determents of the adoption of PPPs in social infrastructure project are the tenant-related factors, such as the inability of paying rent and the inability of caring the property. The study also suggests the importance of seeking strategic partnership with community-based organisation that has experiences in managing similar tenants’ profiles. Current PPP guideline is also viewed as inappropriate for the affordable housing projects, but the principle of VFM framework and risk allocation in PPPs still be applied to the affordable housing projects. This study helps to understand the viability of PPP in social housing procurement projects, and point out the importance of developing guideline for multi-stakeholder partnership and the expansion of the current VFM and PPPs guidelines.

Zeolite from alkali modified kaolin increases NH4+ retention by sandy soil : column experiments

Sandy soils have low water and nutrient retention capabilities so that zeolite soil amendments are used for high value land uses including turf and horticulture to reduce leaching losses of NH4+ fertilisers. MesoLite is a zeolitic material made by caustic treatment of kaolin at 80-95oC. It has a moderately low surface area (9-12m2/g) and very high cation exchange capacity (494 cmol(+)/kg). Laboratory column experiments showed that an addition of 0.4% MesoLite to a sandy soil greatly (90%) reduced leaching of added NH4+ compared to an unamended soil and MesoLite is 11 times more efficient in retaining NH4+ than natural zeolite. Furthermore, NH4+-MesoLite slowly releases NH4+ to soil solution and is likely to be an effective slow release fertiliser.

Empirical evaluation of a service analysis and design methodology

Service-orientation has gained widespread acceptance and is increasingly being employed as a paradigm for structuring both business and IT architectures. An earlier study of extant service analysis and design methodologies discovered a need for holistic approaches that equally account for both business and software services, which motivated the design of a new, consolidated service analysis and design methodology. A challenge in design-oriented research is to evaluate the utility of the newly created artefacts (here: the methodology), as they are often intended to become part of complex socio-technical systems. Therefore, after presenting a brief overview of the consolidated methodology, the paper discusses possible approaches for the “evaluate” phase of this design-science research process and presents the results of an empirical evaluation conducted in an Action Research study at one of Australia’s largest financial services providers.

Link-the-Wiki : performance evaluation based on frequent phrases

In this paper, we discuss our participation to the INEX 2008 Link-the-Wiki track. We utilized a sliding window based algorithm to extract the frequent terms and phrases. Using the extracted phrases and term as descriptive vectors, the anchors and relevant links (both incoming and outgoing) are recognized efficiently.

Behaviour of an in-service cast iron water reticulation pipe buried in expansive soil

This paper presents the measurements of strain and the subsequent stress analysis on an in-service cast iron water main buried in reactive soil. The results indicate that the pipe crown experienced predominantly tensile stresses during drying in summer and, subsequently, these stresses reduce, eventually leading to compressive stresses as the soil swells with increase in moisture content with the approach of winter. It is also evident that flexural movement caused by thermal stresses and soil pressure has led to downward bending of the pipe in summer and subsequent upward movement in winter. The limited data collected from pipe strains and strengths indicate that it is possible for pipe capacity to be exceeded by thermal and soil stresses leading to pipe failure, provided the pipe has undergone significant corrosion.

Field instrumentation of a water reticulation pipe buried in reactive soil

The loss of valuable water resources due to pipe failure has become a major problem in Australia, especially in areas under high level of water restrictions. Generally pipe failure occurs due to a combination of physical and environmental factors. Stresses induced by shrinking and swelling of reactive soils are one of the major factors affecting the performance of buried pipes. This paper presents the details of a field instrumentation undertaken to monitor the performance of an in-service water reticulation pipe buried in a reactive soil and subjected to seasonal climatic changes.

Design and evaluation of an image analysis platform for low-power, low-bandwidth camera networks

We describe the design and evaluation of a platform for networks of cameras in low-bandwidth, low-power sensor networks. In our work to date we have investigated two different DSP hardware/software platforms for undertaking the tasks of compression and object detection and tracking. We compare the relative merits of each of the hardware and software platforms in terms of both performance and energy consumption. Finally we discuss what we believe are the ongoing research questions for image processing in WSNs.

The evaluation of a biphasic osteochondral implant coupled with an electrospun membrane in a large animal model

Conventional clinical therapies are unable to resolve osteochondral defects adequately, hence tissue engineering solutions are sought to address the challenge. A biphasic implant which was seeded with Mesenchymal Stem Cells (MSC) and coupled with an electrospun membrane was evaluated as an alternative. This dual phase construct comprised of a Polycaprolactone (PCL) cartilage scaffold and a Polycaprolactone - Tri Calcium Phosphate (PCL - TCP) osseous matrix. Autologous MSC was seeded into the entire implant via fibrin and the construct was inserted into critically sized osteochondral defects located at the medial condyle and patellar groove of pigs. The defect was resurfaced with a PCL - collagen electrospun mesh that served as a substitute for periosteal flap in preventing cell leakage. Controls either without implanted MSC or resurfacing membrane were included. After 6 months, cartilaginous repair was observed with a low occurrence of fibrocartilage at the medial condyle. Osteochondral repair was promoted and host cartilage degeneration was arrested as shown by the superior Glycosaminoglycan (GAG) maintenance. This positive morphological outcome was supported by a higher relative Young's modulus which indicated functional cartilage restoration. Bone in growth and remodeling occurred in all groups with a higher degree of mineralization in the experimental group. Tissue repair was compromised in the absence of the implanted cells or the resurfacing membrane. Moreover healing was inferior at the patellar groove as compared to the medial condyle and this was attributed to the native biomechanical features.