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.

The synthesis of novel isoindoline nitroxides bearing water-solubilising functionality

A range of novel tetramethyl- and tetraethylisoindolinenitroxides, possessing aryl-linked carboxylic acids, amines, alcohols and phosphonic acids were prepared. Notably, the chemistry established for the aromatic dibromination of the tetramethylisoindolines was not easily transferred to the corresponding tetraethylisoindoline system. Instead, various tetraethylisoindoline analogues were accessed by the oxidation of methyl groups attached to the aromatic ring to give the carboxylic acids. The increased steric bulk of the tetraethyl structures should limit bio-reduction and these compounds may have potential as antioxidants.

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.

Retention and degradation of organics in Rundle waste shale/retort water mixtures

Batch, column and field lysimeter studies have been conducted to evaluate the concept of codisposal of retort water with Rundle (Queensland, Australia) waste shales. The batch studies indicated that degradation of a significant proportion of the total organic load occurs if the mixture is seeded with soil or compost. These results are compared with those from laboratory column studies and from the field lysimeter at the Rundle site. G.c.-m.s. analysis of some of the eluants indicated that significant degradation of the base-neutral fraction occurs even if no soil seed is added, and that degradation of this fraction was higher under anaerobic conditions.

Modelling water droplet movement on a leaf surface

The central aim for the research undertaken in this PhD thesis is the development of a model for simulating water droplet movement on a leaf surface and to compare the model behavior with experimental observations. A series of five papers has been presented to explain systematically the way in which this droplet modelling work has been realised. Knowing the path of the droplet on the leaf surface is important for understanding how a droplet of water, pesticide, or nutrient will be absorbed through the leaf surface. An important aspect of the research is the generation of a leaf surface representation that acts as the foundation of the droplet model. Initially a laser scanner is used to capture the surface characteristics for two types of leaves in the form of a large scattered data set. After the identification of the leaf surface boundary, a set of internal points is chosen over which a triangulation of the surface is constructed. We present a novel hybrid approach for leaf surface fitting on this triangulation that combines Clough-Tocher (CT) and radial basis function (RBF) methods to achieve a surface with a continuously turning normal. The accuracy of the hybrid technique is assessed using numerical experimentation. The hybrid CT-RBF method is shown to give good representations of Frangipani and Anthurium leaves. Such leaf models facilitate an understanding of plant development and permit the modelling of the interaction of plants with their environment. The motion of a droplet traversing this virtual leaf surface is affected by various forces including gravity, friction and resistance between the surface and the droplet. The innovation of our model is the use of thin-film theory in the context of droplet movement to determine the thickness of the droplet as it moves on the surface. Experimental verification shows that the droplet model captures reality quite well and produces realistic droplet motion on the leaf surface. Most importantly, we observed that the simulated droplet motion follows the contours of the surface and spreads as a thin film. In the future, the model may be applied to determine the path of a droplet of pesticide along a leaf surface before it falls from or comes to a standstill on the surface. It will also be used to study the paths of many droplets of water or pesticide moving and colliding on the surface.

Layered titanate nanofibers as efficient adsorbents for removal of toxic radioactive and heavy metal ions from water

Titanate nanofibers with two formulas, Na2Ti3O7 and Na1.5H0.5Ti3O7, respectively, exhibit ideal properties for removal of radioactive and heavy metal ions in wastewater, such as Sr2+ , Ba2+ (as substitute of 226Ra2+), and Pb2+ ions. These nanofibers can be fabricated readily by a reaction between titania and caustic soda and have structures in which TiO6 octahedra join each other to form layers with negative charges; the sodium cations exist within the interlayer regions and are exchangeable. They can selectively adsorb the bivalent radioactive ions and heavy metal ions from water through ion exchange process. More importantly, such sorption finally induces considerable deformation of the layer structure, resulting in permanent entrapment of the toxic bivalent cations in the fibers so that the toxic ions can be safely deposited. This study highlights that nanoparticles of inorganic ion exchangers with layered structure are potential materials for efficient removal of the toxic ions from contaminated water.

PDMS spreading morphological patterns on substrates of different hydrophilicity in air vacuum and water

In paper has been to investigate the morphological patterns and kinetics of PDMS spreading on silicon wafer using combination of techniques like ellipsometry, atomic force microscope (AFM), scanning electron microscope (SEM) and optical microscopy. A macroscopic silicone oil drops as well as PDMS water based emulsions were studied after deposition on a flat surface of silicon wafer in air, water and vacuum. our own measurements using an imaging ellipsometer, which also clearly shows the presence of a precursor film. The diffusion constant of this film, measured with a 60 000 cS PDMS sample spreading on a hydrophilic silicon wafer, is Df = 1.4  10-11 m2/s. Regardless of their size, density and method of deposition, droplets on both types of wafer (hydrophilic and hydrophobic) flatten out over a period of many hours, up to 3 days. During this process neighbouring droplets may coalesce, but there is strong evidence that some of the PDMS from the droplets migrates into a thin, continuous film that covers the surface in between droplets. The thin film appears to be ubiquitous if there has been any deposition of PDMS. However, this statement needs further verification. One question is whether the film forms immediately after forced drying, or whether in some or all cases it only forms by spreading from isolated droplets as they slowly flatten out.

Hydrogen bonding in the water stabilized structure of the modified unsymmetrically substituted viologen chromophore, N-ethyl-N'-(2-phosphonomethyl)-4,4'-bipyridylium dichloride

The crystal structure of the modified unsymmetrically N, N'-substituted viologen chromophore, N-ethyl- N'-(2-phosphonoethyl)-4, 4'-bipyridinium dichloride 0.75 hydrate. (1) has been determined. Crystals are triclinic, space group P-1 with Z = 2 in a cell with a = 7.2550(1), b = 13.2038(5), c = 18.5752(7) Å, α = 86.495(3), β = 83.527(2), γ = 88.921(2)o. The two independent but pseudo-symmetrically related cations in the asymmetric unit form one-dimensional hydrogen-bonded chains through short homomeric phosphonic acid O-H...O links [2.455(4), 2.464(4)A] while two of the chloride anions are similarly strongly linked to phosphonic acid groups [O-H…Cl, 2.889(4), 2.896(4)Å]. The other two chloride anions together with the two water molecules of solvation (one with partial occupancy) form unusual cyclic hydrogen-bonded bis(Cl...water) dianion units which lie between the layers of bipyridylium rings of the cation chain structures with which they are weakly associated.

Can't see the water for the trees

The work was both conceived and constructed in-situ within Gnombup Swamp a seasonal water body at Bremer Bay, Western Australia. The work interacts with site-specific conditions including wind patterns and a datum of seasonal water levels marks. The work is the result of collaboration between soil scientist Paula Deegan and Ian Weir. The installation was documented with a series of 30 still digital photographs, later animated in Microsoft Powerpoint.

Water sensitive urban design : landscape planning and design to improve water quality in Shijiazhaung and Yueyang

Water quality issues are heavily dependent on land development and management decisions within river and lake catchments or watersheds. Economic benefits of urbanisation may be short‐ lived without cleaner environmental outcomes. However, whole‐of‐catchment thinking is not, as yet, as frequent a consideration in urban planning and development in China as it is in many other countries. Water is predominantly seen as a resource to be ‘owned’ by different jurisdictions and allocated to numerous users, both within a catchment and between catchments. An alternative to this approach is to think of water in the same way as other commodities that must be kept moving through a complex transport system. Water must ultimately arrive at particular destinations in the biosphere, although it travels across a broad landscape and may be held up temporarily at certain places along the way. While water extraction can be heavily controlled, water pollution is far more difficult to regulate. Both have significant impacts on water availability and flows both now and in the future. As Chinese cities strive to improve economic conditions for their citizens, new centres are being rebuilt and environmental valued