Apoptosis-related genes confer resistance to Fusarium wilt in transgenic ‘Lady Finger’ bananas

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana (Musa spp.). Apart from resistant cultivars, there are no effective control measures for the disease. We investigated whether the transgenic expression of apoptosis-inhibition related genes in banana could be used to confer disease resistance. Embryogenic cell suspensions of the banana cultivar, ‘Lady Finger’, were stably transformed with animal genes that negatively regulate apoptosis, namely Bcl-xL, Ced-9 and Bcl-2 3’ UTR, and independently transformed plant lines were regenerated for testing. Following a 12 week exposure to Foc race 1 in small-plant glasshouse bioassays, seven transgenic lines (2 x Bcl-xL, 3 x Ced-9 and 2 x Bcl-2 3’ UTR) showed significantly less internal and external disease symptoms than the wild-type susceptible ‘Lady Finger’ banana plants used as positive controls. Of these, one Bcl-2 3’ UTR line showed resistance that was equivalent to that of wild-type Cavendish bananas that were included as resistant negative controls. Further, the resistance of this line continued for 23 weeks post-inoculation at which time the experiment was terminated. Using TUNEL assays, Foc race 1 was shown to induce apoptosis-like features in the roots of wild-type ‘Lady Finger’ plants consistent with a necrotrophic phase in the lifecycle of this pathogen. This was further supported by the observed reduction of these effects in the roots of the resistant Bcl-2 3’ UTR transgenic line. This is the first report on the generation of transgenic banana plants with resistance to Fusarium wilt.

Systematics of the Ustilago-Sporisorium-Macalpinomyces complex of smut fungi

Smut fungi are important pathogens of grasses, including the cultivated crops maize, sorghum and sugarcane. Typically, smut fungi infect the inflorescence of their host plants. Three genera of smut fungi (Ustilago, Sporisorium and Macalpinomyces) form a complex with overlapping morphological characters, making species placement problematic. For example, the newly described Macalpinomyces mackinlayi possesses a combination of morphological characters such that it cannot be unambiguously accommodated in any of the three genera. Previous attempts to define Ustilago, Sporisorium and Macalpinomyces using morphology and molecular phylogenetics have highlighted the polyphyletic nature of the genera, but have failed to produce a satisfactory taxonomic resolution. A detailed systematic study of 137 smut species in the Ustilago-Sporisorium- Macalpinomyces complex was completed in the current work. Morphological and DNA sequence data from five loci were assessed with maximum likelihood and Bayesian inference to reconstruct a phylogeny of the complex. The phylogenetic hypotheses generated were used to identify morphological synapomorphies, some of which had previously been dismissed as a useful way to delimit the complex. These synapomorphic characters are the basis for a revised taxonomic classification of the Ustilago-Sporisorium-Macalpinomyces complex, which takes into account their morphological diversity and coevolution with their grass hosts. The new classification is based on a redescription of the type genus Sporisorium, and the establishment of four genera, described from newly recognised monophyletic groups, to accommodate species expelled from Sporisorium. Over 150 taxonomic combinations have been proposed as an outcome of this investigation, which makes a rigorous and objective contribution to the fungal systematics of these important plant pathogens.

Using Bayesian methods for the estimation of uncertainty in complex statistical models

The research objectives of this thesis were to contribute to Bayesian statistical methodology by contributing to risk assessment statistical methodology, and to spatial and spatio-temporal methodology, by modelling error structures using complex hierarchical models. Specifically, I hoped to consider two applied areas, and use these applications as a springboard for developing new statistical methods as well as undertaking analyses which might give answers to particular applied questions. Thus, this thesis considers a series of models, firstly in the context of risk assessments for recycled water, and secondly in the context of water usage by crops. The research objective was to model error structures using hierarchical models in two problems, namely risk assessment analyses for wastewater, and secondly, in a four dimensional dataset, assessing differences between cropping systems over time and over three spatial dimensions. The aim was to use the simplicity and insight afforded by Bayesian networks to develop appropriate models for risk scenarios, and again to use Bayesian hierarchical models to explore the necessarily complex modelling of four dimensional agricultural data. The specific objectives of the research were to develop a method for the calculation of credible intervals for the point estimates of Bayesian networks; to develop a model structure to incorporate all the experimental uncertainty associated with various constants thereby allowing the calculation of more credible credible intervals for a risk assessment; to model a single day’s data from the agricultural dataset which satisfactorily captured the complexities of the data; to build a model for several days’ data, in order to consider how the full data might be modelled; and finally to build a model for the full four dimensional dataset and to consider the timevarying nature of the contrast of interest, having satisfactorily accounted for possible spatial and temporal autocorrelations. This work forms five papers, two of which have been published, with two submitted, and the final paper still in draft. The first two objectives were met by recasting the risk assessments as directed, acyclic graphs (DAGs). In the first case, we elicited uncertainty for the conditional probabilities needed by the Bayesian net, incorporated these into a corresponding DAG, and used Markov chain Monte Carlo (MCMC) to find credible intervals, for all the scenarios and outcomes of interest. In the second case, we incorporated the experimental data underlying the risk assessment constants into the DAG, and also treated some of that data as needing to be modelled as an ‘errors-invariables’ problem [Fuller, 1987]. This illustrated a simple method for the incorporation of experimental error into risk assessments. In considering one day of the three-dimensional agricultural data, it became clear that geostatistical models or conditional autoregressive (CAR) models over the three dimensions were not the best way to approach the data. Instead CAR models are used with neighbours only in the same depth layer. This gave flexibility to the model, allowing both the spatially structured and non-structured variances to differ at all depths. We call this model the CAR layered model. Given the experimental design, the fixed part of the model could have been modelled as a set of means by treatment and by depth, but doing so allows little insight into how the treatment effects vary with depth. Hence, a number of essentially non-parametric approaches were taken to see the effects of depth on treatment, with the model of choice incorporating an errors-in-variables approach for depth in addition to a non-parametric smooth. The statistical contribution here was the introduction of the CAR layered model, the applied contribution the analysis of moisture over depth and estimation of the contrast of interest together with its credible intervals. These models were fitted using WinBUGS [Lunn et al., 2000]. The work in the fifth paper deals with the fact that with large datasets, the use of WinBUGS becomes more problematic because of its highly correlated term by term updating. In this work, we introduce a Gibbs sampler with block updating for the CAR layered model. The Gibbs sampler was implemented by Chris Strickland using pyMCMC [Strickland, 2010]. This framework is then used to consider five days data, and we show that moisture in the soil for all the various treatments reaches levels particular to each treatment at a depth of 200 cm and thereafter stays constant, albeit with increasing variances with depth. In an analysis across three spatial dimensions and across time, there are many interactions of time and the spatial dimensions to be considered. Hence, we chose to use a daily model and to repeat the analysis at all time points, effectively creating an interaction model of time by the daily model. Such an approach allows great flexibility. However, this approach does not allow insight into the way in which the parameter of interest varies over time. Hence, a two-stage approach was also used, with estimates from the first-stage being analysed as a set of time series. We see this spatio-temporal interaction model as being a useful approach to data measured across three spatial dimensions and time, since it does not assume additivity of the random spatial or temporal effects.

Congo Red adsorption by ball-milled sugarcane bagasse

The adsorption of Congo Red (CR) by ball-milled sugarcane bagasse was evaluated in an aqueous batch system. CR adsorption capacity increased significantly with small changes in bagasse surface area. CR removal decreased with increasing solution pH from 5.0 to 10.0. Maximum adsorption capacity was 38.2 mg/g bagasse at a CR concentration of 500 mg/L. The equilibrium isotherm fitted the Freundlich model and the adsorption kinetics obeyed pseudo-second order equation. CR adsorption obeyed the intra-particle diffusion model very well with bagasse surface area in the range of 0.58–0.66 m2/g, whereas it was controlled by multi-adsorption stages with bagasse surface area in the range of 1.31–1.82 m2/g. Thermodynamic analysis indicated that the adsorption process is an exothermic and spontaneous process. Fourier transform infrared analysis of bagasse containing adsorbed CR indicated interactions between the carboxyl and hydroxyl groups of bagasse and CR function groups.

Characterisation of the TaNF-Y family of transcription factors in wheat

Light plays a unique role for plants as it is both a source of energy for growth and a signal for development. Light captured by the pigments in the light harvesting complexes is used to drive the synthesis of the chemical energy required for carbon assimilation. The light perceived by photoreceptors activates effectors, such as transcription factors (TFs), which modulate the expression of light-responsive genes. Recently, it has been speculated that increasing the photosynthetic rate could further improve the yield potential of three carbon (C3) crops such as wheat. However, little is currently known about the transcriptional regulation of photosynthesis genes, particularly in crop species. Nuclear factor Y (NF-Y) TF is a functionally diverse regulator of growth and development in the model plant species, with demonstrated roles in embryo development, stress response, flowering time and chloroplast biogenesis. Furthermore, a light-responsive NF-Y binding site (CCAAT-box) is present in the promoter of a spinach photosynthesis gene. As photosynthesis genes are co-regulated by light and co-regulated genes typically have similar regulatory elements in their promoters, it seems likely that other photosynthesis genes would also have light-responsive CCAAT-boxes. This provided the impetus to investigate the NF-Y TF in bread wheat. This thesis is focussed on wheat NF-Y members that have roles in light-mediated gene regulation with an emphasis on their involvement in the regulation of photosynthesis genes. NF-Y is a heterotrimeric complex, comprised of the three subunits NF-YA, NF-YB and NF-YC. Unlike the mammalian and yeast counterparts, each of the three subunits is encoded by multiple genes in Arabidopsis. The initial step taken in this study was the identification of the wheat NF-Y family (Chapter 3). A search of the current wheat nucleotide sequence databases identified 37 NF-Y genes (10 NF-YA, 11 NF-YB, 14 NF-YC & 2 Dr1). Phylogenetic analysis revealed that each of the three wheat NF-Y (TaNF-Y) subunit families could be divided into 4-5 clades based on their conserved core regions. Outside of the core regions, eleven motifs were identified to be conserved between Arabidopsis, rice and wheat NF-Y subunit members. The expression profiles of TaNF-Y genes were constructed using quantitative real-time polymerase chain reaction (RT-PCR). Some TaNF-Y subunit members had little variation in their transcript levels among the organs, while others displayed organ-predominant expression profiles, including those expressed mainly in the photosynthetic organs. To investigate their potential role in light-mediated gene regulation, the light responsiveness of the TaNF-Y genes were examined (Chapters 4 and 5). Two TaNF-YB and five TaNF-YC members were markedly upregulated by light in both the wheat leaves and seedling shoots. To identify the potential target genes of the light-upregulated NF-Y subunit members, a gene expression correlation analysis was conducted using publically available Affymetrix Wheat Genome Array datasets. This analysis revealed that the transcript expression levels of TaNF-YB3 and TaNF-YC11 were significantly correlated with those of photosynthesis genes. These correlated express profiles were also observed in the quantitative RT-PCR dataset from wheat plants grown under light and dark conditions. Sequence analysis of the promoters of these wheat photosynthesis genes revealed that they were enriched with potential NF-Y binding sites (CCAAT-box). The potential role of TaNF-YB3 in the regulation of photosynthetic genes was further investigated using a transgenic approach (Chapter 5). Transgenic wheat lines constitutively expressing TaNF-YB3 were found to have significantly increased expression levels of photosynthesis genes, including those encoding light harvesting chlorophyll a/b-binding proteins, photosystem I reaction centre subunits, a chloroplast ATP synthase subunit and glutamyl-tRNA reductase (GluTR). GluTR is a rate-limiting enzyme in the chlorophyll biosynthesis pathway. In association with the increased expression of the photosynthesis genes, the transgenic lines had a higher leaf chlorophyll content, increased photosynthetic rate and had a more rapid early growth rate compared to the wild-type wheat. In addition to its role in the regulation of photosynthesis genes, TaNF-YB3 overexpression lines flower on average 2-days earlier than the wild-type (Chapter 6). Quantitative RT-PCR analysis showed that there was a 13-fold increase in the expression level of the floral integrator, TaFT. The transcript levels of other downstream genes (TaFT2 and TaVRN1) were also increased in the transgenic lines. Furthermore, the transcript levels of TaNF-YB3 were significantly correlated with those of constans (CO), constans-like (COL) and timing of chlorophyll a/b-binding (CAB) expression 1 [TOC1; (CCT)] domain-containing proteins known to be involved in the regulation of flowering time. To summarise the key findings of this study, 37 NF-Y genes were identified in the crop species wheat. An in depth analysis of TaNF-Y gene expression profiles revealed that the potential role of some light-upregulated members was in the regulation of photosynthetic genes. The involvement of TaNF-YB3 in the regulation of photosynthesis genes was supported by data obtained from transgenic wheat lines with increased constitutive expression of TaNF-YB3. The overexpression of TaNF-YB3 in the transgenic lines revealed this NF-YB member is also involved in the fine-tuning of flowering time. These data suggest that the NF-Y TF plays an important role in light-mediated gene regulation in wheat.

Centrifuging rotary vacuum filter mud at Tully Mill

Disposal of mud and ash, particularly in wet weather conditions, is a significant expense for mills. This paper reports on one part of a process to pelletise mud and ash, aimed at making mud and ash more attractive to growers across entire mill districts. The full process is described in a separate paper. The part described in this paper involves re-constituting mud cake from the filter station at Tully Mill and processing it in a decanter centrifuge. The material produced by re-constituting and centrifuging is drier and made up of separate particles. The material needs to mix easily with boiler ash, and the mixture needs to be fed easily into a flue gas drier to be dried to low moisture. The results achieved with the particular characteristics of Tully Mill rotary vacuum filter cake are presented. It was found that an internal rotor with a 20º beach was not adequate to process re-constituted rotary vacuum filter mud. A rotor with a 10º beach worked much more successfully. A total of four tonnes of centrifuged mud with a moisture content ranging from 60% to 65% was produced. It was found that the torque, flocculant rate and dose rate had a statistically significant effect on the moisture content. Feed rate did not have a noticeable impact on the moisture content by itself but torque had a much larger impact on the moisture content at the low feed rate than at the high feed rate. These results indicated that the moisture content of the mud can most likely be reduced with low feed rate, low flocculant rate, high dose rate and high torque. One issue that is believed to affect the operation of a decanter centrifuge was the large quantity of long bagasse fibres in the rotary vacuum filter mud. It is likely that the long fibres limited the throughput of the centrifuge and the moisture achieved.

An additional step towards modelling the mechanical behaviour of sugar cane bagasse

A better understanding of the behaviour of prepared cane and bagasse, especially the ability to model the mechanical behaviour of bagasse as it is squeezed in a milling unit to extract juice, would help identify how to improve the current milling process; for example to reduce final bagasse moisture. Previous investigations have proven with certainty that juice flow through bagasse obeys Darcy’s permeability law, that the grip of the rough surface of the grooves on the bagasse can be represented by the Mohr- Coulomb failure criterion for soils, and that the internal mechanical behaviour of the bagasse can be represented by critical state behaviour similar to that of sand and clay. Current Finite Element Models (FEM) available in commercial software have adequate permeability models. However, commercial software does not contain an adequate mechanical model for bagasse. Progress has been made in the last ten years towards implementing a mechanical model for bagasse in finite element software code. This paper builds on that progress and carries out a further step towards obtaining an adequate material model. In particular, the prediction of volume change during shearing of normally consolidated final bagasse is addressed.

Flying Spore Trap Airborne Based Surveillance : towards a Biosecure Australia

The main limitations with existing fungal spore traps are that they are stationary and cannot be used in inaccessible or remote areas of Australia. This may result in delayed assessment, possible spread of harmful crop infestations and loss of crop yield and productivity. Fitted with the developed smart spore trap the UAV can fly, detect and monitor spores of plant pathogens in areas which previously were almost impossible to monitor. The technology will allow for earlier detection of emergency plant pests (EPPs) incursions by providing efficient and effective airborne surveillance, helping to protect Australia’s crops, pastures and the environment. The project is led by the Cooperative Research Centre for National Plant Biosecurity, with ARCAA/ QUT, CSIRO and the Queensland Government also providing resources. The prototype airplane was exhibited at the Innovation in Australia event December 7.

AtBAG7, an Arabidopsis Bcl-2-associated athanogene, resides in the endoplasmic reticulum and is involved in the unfolded protein response

The Bcl-2-associated athanogene (BAG) family is an evolutionarily conserved, multifunctional group of cochaperones that perform diverse cellular functions ranging from proliferation to growth arrest and cell death in yeast, in mammals, and, as recently observed, in plants. The Arabidopsis genome contains seven homologs of the BAG family, including four with domain organization similar to animal BAGs. In the present study we show that an Arabidopsis BAG, AtBAG7, is a uniquely localized endoplasmic reticulum (ER) BAG that is necessary for the proper maintenance of the unfolded protein response (UPR). AtBAG7was shown to interact directly in vivo with themolecular chaperone, AtBiP2, by bimolecular fluorescence complementation assays, and the interaction was confirmed by yeast two-hybrid assay. Treatment with an inducer of UPR, tunicamycin, resulted in accelerated cell death of AtBAG7-null mutants. Furthermore, AtBAG7 knockouts were sensitive to known ER stress stimuli, heat and cold. In these knockouts heat sensitivity was reverted successfully to the wild-type phenotype with the addition of the chemical chaperone, tauroursodexycholic acid (TUDCA). Real-time PCR of ER stress proteins indicated that the expression of the heat-shock protein, AtBiP3, is selectively up-regulated in AtBAG7-null mutants upon heat and cold stress. Our results reveal an unexpected diversity of the plant's BAG gene family and suggest that AtBAG7 is an essential component of the UPR during heat and cold tolerance, thus confirming the cytoprotective role of plant BAGs.

Plant programmed cell death : Can't live with it; Can't live without it

The decision of whether a cell should live or die is fundamental for the wellbeing of all organisms. Despite intense investigation into cell growth and proliferation, only recently has the essential and equally important idea that cells control/programme their own demise for proper maintenance of cellular homeostasis gained recognition. Furthermore, even though research into programmed cell death (PCD) has been an extremely active area of research there are significant gaps in our understanding of the process in plants. In this review, we discuss PCD during plant development and pathogenesis, and compare/contrast this with mammalian apoptosis. © 2008 Blackwell Publishing Ltd.

The undesirable acetylation of cellulose by the acetate ion of 1-ethyl-3-methylimidazolium acetate

The ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate ([C2mim]OAc) is considered to be an inert solvent of cellulose and lignocellulosic biomass. Acetylation (1.7 % mol, or DS 0.017) of cellulose after dissolution in [C2mim]OAc (150 °C for 20 min), is demonstrated by compositional analysis, FTIR analysis and 13C NMR spectroscopy (in [C2min]OAc with 13C enriched acetate). This acetylation, in the absence of added acylating agents, has not been reported before and may limit [C2mim]OAc utility in industrial scale biomass processing, even at this low extent. For example, cellulose acetylation may contribute to IL loss in processes where the IL is recovered and reused and inhibit enzyme saccharification of cellulose in lignocellulosic biofuel production processes based on saccharification and fermentation.

Defining the role of phytoene synthase in carotenoid accumulation of high provitamin A bananas

Vitamin A deficiency (VAD) is a serious problem in developing countries, affecting approximately 127 million children of preschool age and 7.2 million pregnant women each year. However, this deficiency is readily treated and prevented through adequate nutrition. This can potentially be achieved through genetically engineered biofortification of staple food crops to enhance provitamin A (pVA) carotenoid content. Bananas are the fourth most important food crop with an annual production of 100 million tonnes and are widely consumed in areas affected by VAD. However, the fruit pVA content of most widely consumed banana cultivars is low (~ 0.2 to 0.5 ìg/g dry weight). This includes cultivars such as the East African highland banana (EAHB), the staple crop in countries such as Uganda, where annual banana consumption is approximately 250 kg per person. This fact, in addition to the agronomic properties of staple banana cultivars such as vegetative reproduction and continuous cropping, make bananas an ideal target for pVA enhancement through genetic engineering. Interestingly, there are banana varieties known with high fruit pVA content (up to 27.8 ìg/g dry weight), although they are not widely consumed due to factors such as cultural preference and availability. The genes involved in carotenoid accumulation during banana fruit ripening have not been well studied and an understanding of the molecular basis for the differential capacity of bananas to accumulate carotenoids may impact on the effective production of genetically engineered high pVA bananas. The production of phytoene by the enzyme phytoene synthase (PSY) has been shown to be an important rate limiting determinant of pVA accumulation in crop systems such as maize and rice. Manipulation of this gene in rice has been used successfully to produce Golden Rice, which exhibits higher seed endosperm pVA levels than wild type plants. Therefore, it was hypothesised that differences between high and low pVA accumulating bananas could be due either to differences in PSY enzyme activity or factors regulating the expression of the psy gene. Therefore, the aim of this thesis was to investigate the role of PSY in accumulation of pVA in banana fruit of representative high (Asupina) and low (Cavendish) pVA banana cultivars by comparing the nucleic acid and encoded amino acid sequences of the banana psy genes, in vivo enzyme activity of PSY in rice callus and expression of PSY through analysis of promoter activity and mRNA levels. Initially, partial sequences of the psy coding region from five banana cultivars were obtained using reverse transcriptase (RT)-PCR with degenerate primers designed to conserved amino acids in the coding region of available psy sequences from other plants. Based on phylogenetic analysis and comparison to maize psy sequences, it was found that in banana, psy occurs as a gene family of at least three members (psy1, psy2a and psy2b). Subsequent analysis of the complete coding regions of these genes from Asupina and Cavendish suggested that they were all capable of producing functional proteins due to high conservation in the catalytic domain. However, inability to obtain the complete mRNA sequences of Cavendish psy2a, and isolation of two non-functional Cavendish psy2a coding region variants, suggested that psy2a expression may be impaired in Cavendish. Sequence analysis indicated that these Cavendish psy2a coding region variants may have resulted from alternate splicing. Evidence of alternate splicing was also observed in one Asupina psy1 coding region variant, which was predicted to produce a functional PSY1 isoform. The complete mRNA sequence of the psy2b coding regions could not be isolated from either cultivar. Interestingly, psy1 was cloned predominantly from leaf while psy2 was obtained preferentially from fruit, suggesting some level of tissue-specific expression. The Asupina and Cavendish psy1 and psy2a coding regions were subsequently expressed in rice callus and the activity of the enzymes compared in vivo through visual observation and quantitative measurement of carotenoid accumulation. The maize B73 psy1 coding region was included as a positive control. After several weeks on selection, regenerating calli showed a range of colours from white to dark orange representing various levels of carotenoid accumulation. These results confirmed that the banana psy coding regions were all capable of producing functional enzymes. No statistically significant differences in levels of activity were observed between banana PSYs, suggesting that differences in PSY activity were not responsible for differences in the fruit pVA content of Asupina and Cavendish. The psy1 and psy2a promoter sequences were isolated from Asupina and Cavendish gDNA using a PCR-based genome walking strategy. Interestingly, three Cavendish psy2a promoter clones of different sizes, representing possible allelic variants, were identified while only single promoter sequences were obtained for the other Asupina and Cavendish psy genes. Bioinformatic analysis of these sequences identified motifs that were previously characterised in the Arabidopsis psy promoter. Notably, an ATCTA motif associated with basal expression in Arabidopsis was identified in all promoters with the exception of two of the Cavendish psy2a promoter clones (Cpsy2apr2 and Cpsy2apr3). G1 and G2 motifs, linked to light-regulated responses in Arabidopsis, appeared to be differentially distributed between psy1 and psy2a promoters. In the untranscribed regulatory regions, the G1 motifs were found only in psy1 promoters, while the G2 motifs were found only in psy2a. Interestingly, both ATCTA and G2 motifs were identified in the 5’ UTRs of Asupina and Cavendish psy1. Consistent with other monocot promoters, introns were present in the Asupina and Cavendish psy1 5’ UTRs, while none were observed in the psy2a 5’ UTRs. Promoters were cloned into expression constructs, driving the â-glucuronidase (GUS) reporter gene. Transient expression of the Asupina and Cavendish psy1 and psy2a promoters in both Cavendish embryogenic cells and Cavendish fruit demonstrated that all promoters were active, except Cpsy2apr2 and Cpsy2apr3. The functional Cavendish psy2a promoter (Cpsy2apr1) appeared to have activity similar to the Asupina psy2a promoter. The activities of the Asupina and Cavendish psy1 promoters were similar to each other, and comparable to those of the functional psy2a promoters. Semi-quantitative PCR analysis of Asupina and Cavendish psy1 and psy2a transcripts showed that psy2a levels were high in green fruit and decreased during ripening, reinforcing the hypothesis that fruit pVA levels were largely dependent on levels of psy2a expression. Additionally, semi-quantitative PCR using intron-spanning primers indicated that high levels of unprocessed psy2a and psy2b mRNA were present in the ripe fruit of Cavendish but not in Asupina. This raised the possibility that differences in intron processing may influence pVA accumulation in Asupina and Cavendish. In this study the role of PSY in banana pVA accumulation was analysed at a number of different levels. Both mRNA accumulation and promoter activity of psy genes studied were very similar between Asupina and Cavendish. However, in several experiments there was evidence of cryptic or alternate splicing that differed in Cavendish compared to Asupina, although these differences were not conclusively linked to the differences in fruit pVA accumulation between Asupina and Cavendish. Therefore, other carotenoid biosynthetic genes or regulatory mechanisms may be involved in determining pVA levels in these cultivars. This study has contributed to an increased understanding of the role of PSY in the production of pVA carotenoids in banana fruit, corroborating the importance of this enzyme in regulating carotenoid production. Ultimately, this work may serve to inform future research into pVA accumulation in important crop varieties such as the EAHB and the discovery of avenues to improve such crops through genetic modification.

Hydrothermal syntheses of zeolite N from kaolin

Zeolite N, an EDI type framework structure with ideal chemical formula K12Al10Si10O40Cl2•5H2O, was produced from kaolin between 100oC and 200oC in a continuously stirred reactor using potassic and potassic+sodic liquors containing a range of anions. Reactions using liquors such as KOH, KOH + KX (where X = F, Cl, Br, I, NO3, NO2), K2X (where X=CO3), KOH + NaCl or NaOH + KCl were complete (>95% product) in less than two hours depending on the batch composition and temperature of reaction. With KOH and KCl in the reaction mixture and H2O/Al2O3~49, zeolite N was formed over a range of concentrations (1M < [KOH] < 18M) and reaction times (0.5h < t < 60h). At higher temperatures or higher KOH molarity, other potassic phases such as kalsilite or kaliophyllite formed. In general, temperature and KOH molarity defined the extent of zeolite N formation under these conditions. The introduction of sodic reagents to the starting mixture or use of one potassic reagent in the starting mixture reduced the stability field for zeolite N formation. Zeolite N was also formed using zeolite 4A as a source of Al and Si albeit for longer reaction times at a particular temperature when compared with kaolin as the source material.

Expression Pattern of the Alpha-Kafirin Promoter Coupled with a Signal Peptide from Sorghum bicolor L. Moench

Regulatory sequences with endosperm specificity are essential for foreign gene expression in the desired tissue for both grain quality improvement and molecular pharming. In this study, promoters of seed storage α-kafirin genes coupled with signal sequence (ss) were isolated from Sorghum bicolor L. Moench genomic DNA by PCR. The α-kafirin promoter (α-kaf) contains endosperm specificity-determining motifs, prolamin-box, the O2-box 1, CATC, and TATA boxes required for α-kafirin gene expression in sorghum seeds. The constructs pMB-Ubi-gfp and pMB-kaf-gfp were microprojectile bombarded into various sorghum and sweet corn explants. GFP expression was detected on all explants using the Ubi promoter but only in seeds for the α-kaf promoter. This shows that the α-kaf promoter isolated was functional and demonstrated seed-specific GFP expression. The constructs pMB-Ubi-ss-gfp and pMB-kaf-ss-gfp were also bombarded into the same explants. Detection of GFP expression showed that the signal peptide (SP)::GFP fusion can assemble and fold properly, preserving the fluorescent properties of GFP.

The composition of sugarcane juices dervied from burnt cane and whole green cane crop

There has been substantial interest within the Australian sugar industry in product diversification as a means to reduce its exposure to fluctuating raw sugar prices and in order to increase its commercial viability. In particular, the industry is looking at fibrous residues from sugarcane harvesting (trash) and from sugarcane milling (bagasse) for cogeneration and the production of biocommodities, as these are complementary to the core process of sugar production. A means of producing surplus residue (biomass) is to process whole sugarcane crop. In this paper, the composition of different juices derived from different harvesting methods, viz. burnt cane with all trash extracted (BE), green cane with half of the trash extracted (GE), and green cane (whole sugarcane crop) with trash unextracted (GU), were investigated and the results and comparison presented. The determination of electrical conductivity, inorganic composition, and organic acids indicate that both GU and GE cane juice contain a higher proportion of soluble inorganic ions and ionisable organic acids, compared to BE cane juice. It is important to note that there are considerably higher levels of Na ions and citric acid, but relatively low P levels in the GU samples. A higher level of reducing sugars was analysed in the GU samples than the BE samples due to the higher proportion of impurities found naturally in sugarcane tops and leaves. The purity of the first expressed juice (FEJ) of GU cane was on average higher than that of FEJ of BE cane. Results also show that GU juices appear to contain higher levels of proteins and polysaccharides, with no significant difference in starch levels.