Development of a high-speed sensing and detection system for automatic removal of packages with leaky seals from a high-speed food processing and packaging line

Contamination of packaged foods due to micro-organisms entering through air leaks can cause serious public health issues and cost companies large amounts of money due to product recalls, consumer impact and subsequent loss of market share. The main source of contamination is leaks in packaging which allow air, moisture and microorganisms to enter the package. In the food processing and packaging industry worldwide, there is an increasing demand for cost effective state of the art inspection technologies that are capable of reliably detecting leaky seals and delivering products at six-sigma. The new technology will develop non-destructive testing technology using digital imaging and sensing combined with a differential vacuum technique to assess seal integrity of food packages on a high-speed production line. The cost of leaky packages in Australian food industries is estimated close to AUD $35 Million per year. Contamination of packaged foods due to micro-organisms entering through air leaks can cause serious public health issues and cost companies large sums of money due to product recalls, compensation claims and loss of market share. The main source of contamination is leaks in packaging which allow air, moisture and micro-organisms to enter the package. Flexible plastic packages are widely used, and are the least expensive form of retaining the quality of the product. These packets can be used to seal, and therefore maximise, the shelf life of both dry and moist products. The seals of food packages need to be airtight so that the food content is not contaminated due to contact with microorganisms that enter as a result of air leakage. Airtight seals also extend the shelf life of packaged foods, and manufacturers attempt to prevent food products with leaky seals being sold to consumers. There are many current NDT (non-destructive testing) methods of testing the seal of flexible packages best suited to random sampling, and for laboratory purposes. The three most commonly used methods are vacuum/pressure decay, bubble test, and helium leak detection. Although these methods can detect very fine leaks, they are limited by their high processing time and are not viable in a production line. Two nondestructive in-line packaging inspection machines are currently available and are discussed in the literature review. The detailed design and development of the High-Speed Sensing and Detection System (HSDS) is the fundamental requirement of this project and the future prototype and production unit. Successful laboratory testing was completed and a methodical design procedure was needed for a successful concept. The Mechanical tests confirmed the vacuum hypothesis and seal integrity with good consistent results. Electrically, the testing also provided solid results to enable the researcher to move the project forward with a certain amount of confidence. The laboratory design testing allowed the researcher to confirm theoretical assumptions before moving into the detailed design phase. Discussion on the development of the alternative concepts in both mechanical and electrical disciplines enables the researcher to make an informed decision. Each major mechanical and electrical component is detailed through the research and design process. The design procedure methodically works through the various major functions both from a mechanical and electrical perspective. It opens up alternative ideas for the major components that although are sometimes not practical in this application, show that the researcher has exhausted all engineering and functionality thoughts. Further concepts were then designed and developed for the entire HSDS unit based on previous practice and theory. In the future, it would be envisaged that both the Prototype and Production version of the HSDS would utilise standard industry available components, manufactured and distributed locally. Future research and testing of the prototype unit could result in a successful trial unit being incorporated in a working food processing production environment. Recommendations and future works are discussed, along with options in other food processing and packaging disciplines, and other areas in the non-food processing industry.

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.

Use of a zeolite synthesised from alkali treated kaolin as a K fertiliser : glasshouse experiments on leaching and uptake of K by wheat plants in sandy soil

Zeolite N, a zeolite referred to in earlier publications as MesoLite, is made by caustic reaction of kaolin at temperatures between 80 °C and 95 °C. This material has a very high cation exchange capacity (CEC ≈ 500 meq/100 g). Soil column leaching experiments have shown that K-zeolite N additions greatly reduce leaching of NH4+ fertilisers but the agronomic effectiveness of the retained K+ and NH4+ is unknown. To measure the bioavailability of K in this zeolite, wheat was grown in a glasshouse with K-zeolite N as the K fertiliser in highly-leached and non-leached pots for four weeks and compared with a soluble K fertiliser (KCl). The plants grown in non-leached pots and fertilised with K-zeolite N were slightly larger than those grown with KCl. The elemental compositions in the plants were similar except for Si being significantly more concentrated in the plants supplied with K-zeolite N. Thus K-zeolite N may be an effective K-fertiliser. Plants grown in highly-leached pots were significantly smaller than those grown in non-leached pots. Plants grown in highly-leached pots were severely K deficient as half of the K from both KCl and K-zeolite N was leached from the pots within three days.

Phenological changes in six Australian subalpine plants in response to experimental warming and year-to-year variation

The likely phenological responses of plants to climate warming can be measured through experimental manipulation of field sites, but results are rarely validated against year-to-year changes in climate. Here, we describe the response of 1-5 years of experimental warming on phenology (budding, flowering and seed maturation) of six common subalpine plant species in the Australian Alps using the International Tundra Experiment (ITEX) protocol.2. Phenological changes in some species (particularly the forb Craspedia jamesii) were detected in experimental plots within a year of warming, whereas changes in most other species (the forb Erigeron bellidioides, the shrub Asterolasia trymalioides and the graminoids Carex breviculmis and Poa hiemata) did not develop until after 2-4 years; thus, there appears to be a cumulative effect of warming for some species across multiple years.3. There was evidence of changes in the length of the period between flowering and seed maturity in one species (P. hiemata) that led to a similar timing of seed maturation, suggesting compensation.4. Year-to-year variation in phenology was greater than variation between warmed and control plots and could be related to differences in thawing degree days (particularly, for E. bellidioides) due to earlier timing of budding and other events under warmer conditions. However, in Carex breviculmis, there was no association between phenology and temperature changes across years.5. These findings indicate that, although phenological changes occurred earlier in response to warming in all six species, some species showed buffered rather than immediate responses.6. Synthesis. Warming in ITEX open-top chambers in the Australian Alps produced earlier budding, flowering and seed set in several alpine species. Species also altered the timing of these events, particularly budding, in response to year-to-year temperature variation. Some species responded immediately, whereas in others the cumulative effects of warming across several years were required before a response was detected.

Criminology and genetically modified food

Genetically modified or engineered foods are produced from rapidly expanding technologies that have sparked international debates and concerns about health and safety. These concerns focus on the potential dangers to human health, the risks of genetic pollution, and the demise of alternative farming techniques as well as biopiracy and economic exploitation by large private corporations. This article discusses the findings of the world's first Royal Commission on Genetic Modification conducted in New Zealand and reveals that there are potential social, ecological and economic risks created by genetically modified foods that require closer criminological scrutiny. As contemporary criminological discourses continue to push new boundaries in areas of crimes of the economy, environmental pollution, risk management, governance and globalization, the potential concerns posed by genetically modified foods creates fertile ground for criminological scholarship and activism.

Corporate social and environmental responsibility in relation to the agricultural sector and the food supply chain : a Case Study of Waitrose

Waitrose has a strong commitment to organic farming but also uses products from 'conventional' farms. At the production stage, Waitrose own-label products are fully traceable, GM-free and all suppliers undergo a detailed assessment programme based on current best practice. Crop suppliers to Waitrose operate an authenticity programme to certify that each assignment is GM-free and produce is screened for pesticide residues. Waitrose sources conventional crops grown from 'Integrated Crop Management Systems' (ICMS) using best horticultural practices. The 'Assured Product' scheme regulates all UK produce to ICMS standards and these audits are being extended worldwide. Business is withdrawn from suppliers who fail the audit. In relation to this, Waitrose has increased its Fairtrade range as in its view 'Buying these products provides direct additional benefit to workers in the developing countries where they are produced and assists marginal producers by giving them access to markets they would not otherwise have'. Currently, Waitrose is developing its own sustainable timber assessment criteria. For livestock, protocols are in place to ensure that animals are reared under the 'most natural conditions possible' and free range produce is offered where animals have access to open space although some produce is not from free-range animals. Waitrose also use a 'Hazards Analysis Critical Points' system to identify food safety hazards that occur at any stage from production to point of sale and to ensure that full measures are in place to control them. In addition, mechanisms have been implemented to reduce fuel use and hence reduce CO2 emissions in the transport of products and staff, and to increase the energy use efficiency of refrigeration systems which account for approximately 60% of Waitrose energy use.

Development of a transformation system for sorghum (Sorghum bicolor L.)

Sorghum (Sorghum bicolor (L.) Moench) is the world’s fifth major cereal crop and holds importance as a construction material, food and fodder source. More recently, the potential of this plant as a biofuel source has been noted. Despite its agronomic importance, the use of sorghum production is being constrained by both biotic and abiotic factors. These challenges could be addressed by the use of genetic engineering strategies to complement conventional breeding techniques. However, sorghum is one of the most recalcitrant crops for genetic modification with the lack of an efficient tissue culture system being amongst the chief reasons. Therefore, the aim of this study was to develop an efficient tissue culture system for establishing regenerable embryogenic cell lines, micropropagation and acclimatisation for Sorghum bicolor and use this to optimise parameters for genetic transformation via Agrobacterium-mediated transformation and microprojectile bombardment. Using five different sorghum cultivars, SA281, 296B, SC49, Wray and Rio, numerous parameters were investigated in an attempt to establish an efficient and reproducible tissue culture and transformation system. Using immature embryos (IEs) as explants, regenerable embryogenic cell lines (ECLs) could only be established from cultivars SA281 and 296B. Large amounts of phenolics were produced from IEs of cultivars, SC49, Wary and Rio, and these compounds severely hindered callus formation and development. Cultivar SA281 also produced phenolics during regeneration. Attempts to suppress the production of these compounds in cultivars SA281 and SC49 using activated charcoal, PVP, ascorbic acid, citric acid and liquid filter paper bridge methods were either ineffective or had a detrimental effect on embryogenic callus formation, development and regeneration. Immature embryos sourced during summer were found to be far more responsive in vitro than those sourced during winter. In an attempt to overcome this problem, IEs were sourced from sorghum grown under summer conditions in either a temperature controlled glasshouse or a growth chamber. However, the performance of these explants was still inferior to that of natural summer-sourced explants. Leaf whorls, mature embryos, shoot tips and leaf primordia were found to be unsuitable as explants for establishing ECLs in sorghum cultivars SA281 and 296B. Using the florets of immature inflorescences (IFs) as explants, however, ECLs were established and regenerated for these cultivars, as well as for cultivar Tx430, using callus induction media, SCIM, and regeneration media, VWRM. The best in vitro responses, from the largest possible sized IFs, were obtained using plants at the FL-2 stage (where the last fully opened leaf was two leaves away from the flag leaf). Immature inflorescences could be stored at 25oC for up to three days without affecting their in vitro responses. Compared to IEs, the IFs were more robust in tissue culture and showed responses which were season and growth condition independent. A micropropagation protocol for sorghum was developed in this study. The optimum plant growth regulator (PGR) combination for the micropropagation of in vitro regenerated plantlets was found to be 1.0 mg/L BAP in combination with 0.5 mg/L NAA. With this protocol, cultivars 296B and SA281 produced an average of 57 and 13 off-shoots per plantlet, respectively. The plantlets were successfully acclimatised and developed into phenotypically normal plants that set seeds. A simplified acclimatisation protocol for in vitro regenerated plantlets was also developed. This protocol involved deflasking in vitro plantlets with at least 2 fully-opened healthy leaves and at least 3 roots longer than 1.5 cm, washing the media from the roots with running tap water, planting in 100 mm pots and placing in plastic trays covered with a clear plastic bag in a plant growth chamber. After seven days, the corners of the plastic cover were opened and the bags were completely removed after 10 days. All plantlets were successfully acclimatised regardless of whether 1:1 perlite:potting mix, potting mix, UC mix or vermiculite were used as potting substrates. Parameters were optimised for Agrobacterium-mediated transformation (AMT) of cultivars SA281, 296B and Tx430. The optimal conditions were the use of Agrobacterium strain LBA4404 at an inoculum density of 0.5 OD600nm, heat shock at 43oC for 3 min, use of the surfactant Pluronic F-68 (0.02% w/v) in the inoculation media with a pH of 5.2 and a 3 day co-cultivation period in dark at 22oC. Using these parameters, high frequencies of transient GFP expression was observed in IEs precultured on callus initiation media for 1-7 days as well as in four weeks old IE- and IF-derived callus. Cultivar SA281 appeared very sensitive to Agrobacterium since all tissue turned necrotic within two weeks post-exposure. For cultivar 296B, GFP expression was observed up to 20 days post co-cultivation but no stably transformed plants were regenerated. Using cultivar Tx430, GFP was expressed for up to 50 days post co-cultivation. Although no stably transformed plants of this cultivar were regenerated, this was most likely due to the use of unsuitable regeneration media. Parameters were optimised for transformation by particle bombardment (PB) of cultivars SA281, 296B and Tx430. The optimal conditions were use of 3-7 days old IEs and 4 weeks old IF callus, 4 hour pre- and post-bombardment osmoticum treatment, use of 0.6 µm gold microparticles, helium pressure of 1500 kPa and target distance of 15 cm. Using these parameters for PB, transient GFP expression was observed for up to 14, 30 and 50 days for cultivars SA281, 296B and Tx430, respectively. Further, the use of PB resulted in less tissue necrosis compared to AMT for the respective cultivars. Despite the presence of transient GFP expression, no stably transformed plants were regenerated. The establishment of regenerable ECLs and the optimization of AMT and PB parameters in this study provides a platform for future efforts to develop an efficient transformation protocol for sorghum. The development of GM sorghum will be an important step towards improving its agronomic properties as well as its exploitation for biofuel production.

Food modelling systems to assess menus in well population groups : not all black and white

Food modelling systems such as the Core Foods and the Australian Guide to Healthy Eating are frequently used as nutritional assessment tools for menus in ‘well’ groups (such as boarding schools, prisons and mental health facilities), with the draft Foundation and Total Diets (FATD) the latest revision. The aim of this paper is to apply the FATD to an assessment of food provision in a long stay, ‘well’, group setting to determine its usefulness as a tool. A detailed menu review was conducted in a 1000 bed male prison, including verification of all recipes. Full diet histories were collected on 106 prisoners which included foods consumed from the menu and self funded snacks. Both the menu and diet histories were analysed according to core foods, with recipes used to assist in quantification of mixed dishes. Comparison was made of average core foods with Foundation Diet recommendations (FDR) for males. Results showed that the standard menu provided sufficient quantity for 8 of 13 FDRs, however was low in nuts, legumes, refined cereals and marginally low in fruits and orange vegetables. The average prisoner diet achieved 9 of 13 FDRs, notably with margarines and oils less than half and legumes one seventh of recommended. Overall, although the menu and prisoner diets could easily be assessed using the FDRs, it was not consistent with recommendations. In long stay settings other Nutrient Reference Values not modelled in the FATDS need consideration, in particular, Suggested Dietary Targets and professional judgement is required in interpretation.

Validation of a food behavior questionnaire for male blue collar workers

During nutrition intervention programs, some form of dietary assessment is usually necessary. This dietary assessment can be for: initial screening; development of appropriate programs and activities; or, evaluation. Established methods of dietary assessment are not always practical, nor cost effective in such interventions, therefore an abbreviated dietary assessment tool is needed. The Queensland Nutrition Project developed such a tool for male Blue Collar Workers, the Food Behaviour Questionnaire, consisting of 27 food behaviour related questions. This tool has been validated in a sample of 23 men, through full dietary assessment obtained via food frequency questionnaires and 24 hour dietary recalls. Those questions which correlated poorly with the full dietary assessment were deleted from the tool. In all, 13 questions was all that was required to distinguish between high and low dietary intakes of particular nutrients. Three questions when combined had correlations with refined sugar between 0.617 and 0.730 (p<0.005); four questions when combined had correlations with dietary fibre as percentage of energy of 0.45 (p<0.05); five questions when combined had a correlation with total fat of 0.499 (p<0.05); and, 4 questions when combined had a correlation with saturated fat of between 0.451 and 0.589 (p<0.05). A significant correlation could not be found for food behaviour questions with respect to dietary sodium. Correlations for fat as a function of energy could not be found.

Viruses of banana in East Africa

Bananas are one of the world's most important food crops, providing sustenance and income for millions of people in developing countries and supporting large export industries. Viruses are considered major constraints to banana production, germplasm multiplication and exchange, and to genetic improvement of banana through traditional breeding. In Africa, the two most important virus diseases are bunchy top, caused by Banana bunchy top virus (BBTV), and banana streak disease, caused by Banana streak virus (BSV). BBTV is a serious production constraint in a number of countries within/bordering East Africa, such as Burundi, Democratic Republic of Congo, Malawi, Mozambique, Rwanda and Zambia, but is not present in Kenya, Tanzania and Uganda. Additionally, epidemics of banana streak disease are occurring in Kenya and Uganda. The rapidly growing tissue culture (TC) industry within East Africa, aiming to provide planting material to banana farmers, has stimulated discussion about the need for virus indexing to certify planting material as virus-free. Diagnostic methods for BBTV and BSV have been reported and, for BBTV, PCR-based assays are reliable and relatively straightforward. However for BSV, high levels of serological and genetic variability and the presence of endogenous virus sequences within the banana genome complicate diagnosis. Uganda has been shown to contain the greatest diversity in BSV isolates found anywhere in the world. A broad-spectrum diagnostic test for BSV detection, which can discriminate between endogenous and episomal BSV sequences, is a priority. This PhD project aimed to establish diagnostic methods for banana viruses, with a particular focus on the development of novel methods for BSV detection, and to use these diagnostic methods for the detection and characterisation of banana viruses in East Africa. A novel rolling-circle amplification (RCA) method was developed for the detection of BSV. Using samples of Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV) from Australia, this method was shown to distinguish between endogenous and episomal BSV sequences in banana plants. The RCA assay was used to screen a collection of 56 banana samples from south-west Uganda for BSV. RCA detected at least five distinct BSV isolates in these samples, including BSOLV and Banana streak GF virus (BSGFV) as well as three BSV isolates (Banana streak Uganda-I, -L and -M virus) for which only partial sequences had been previously reported. These latter three BSV had only been detected using immuno-capture (IC)-PCR and thus were possible endogenous sequences. In addition to its ability to detect BSV, the RCA protocol was also demonstrated to detect other viruses within the family Caulimoviridae, including Sugar cane bacilliform virus, and Cauliflower mosaic virus. Using the novel RCA method, three distinct BSV isolates from both Kenya and Uganda were identified and characterised. The complete genome of these isolates was sequenced and annotated. All six isolates were shown to have a characteristic badnavirus genome organisation with three open reading frames (ORFs) and the large polyprotein encoded by ORF 3 was shown to contain conserved amino acid motifs for movement, aspartic protease, reverse transcriptase and ribonuclease H activities. As well, several sequences important for expression and replication of the virus genome were identified including the conserved tRNAmet primer binding site present in the intergenic region of all badnaviruses. Based on the International Committee on Taxonomy of Viruses (ICTV) guidelines for species demarcation in the genus Badnavirus, these six isolates were proposed as distinct species, and named Banana streak UA virus (BSUAV), Banana streak UI virus (BSUIV), Banana streak UL virus (BSULV), Banana streak UM virus (BSUMV), Banana streak CA virus (BSCAV) and Banana streak IM virus (BSIMV). Using PCR with species-specific primers designed to each isolate, a genotypically diverse collection of 12 virus-free banana cultivars were tested for the presence of endogenous sequences. For five of the BSV no amplification was observed in any cultivar tested, while for BSIMV, four positive samples were identified in cultivars with a B-genome component. During field visits to Kenya, Tanzania and Uganda, 143 samples were collected and assayed for BSV. PCR using nine sets of species-specific primers, and RCA, were compared for BSV detection. For five BSV species with no known endogenous counterpart (namely BSCAV, BSUAV, BSUIV, BSULV and BSUMV), PCR was used to detect 30 infections from the 143 samples. Using RCA, 96.4% of these samples were considered positive, with one additional sample detected using RCA which was not positive using PCR. For these five BSV, PCR and RCA were both useful for identifying infected samples, irrespective of the host cultivar genotype (Musa A- or B-genome components). For four additional BSV with known endogenous counterparts in the M. balbisiana genome (BSOLV, BSGFV, BSMYV and BSIMV), PCR was shown to detect 75 infections from the 143 samples. In 30 samples from cultivars with an A-only genome component there was 96.3% agreement between PCR positive samples and detection using RCA, again demonstrating either PCR or RCA are suitable methods for detection. However, in 45 samples from cultivars with some B-genome component, the level of agreement between PCR positive samples and RCA positive samples was 70.5%. This suggests that, in cultivars with some B-genome component, many infections were detected using PCR which were the result of amplification of endogenous sequences. In these latter cases, RCA or another method which discriminates between endogenous and episomal sequences, such as immuno-capture PCR, is needed to diagnose episomal BSV infection. Field visits were made to Malawi and Rwanda to collect local isolates of BBTV for validation of a PCR-based diagnostic assay. The presence of BBTV in samples of bananas with bunchy top disease was confirmed in 28 out of 39 samples from Malawi and all nine samples collected in Rwanda, using PCR and RCA. For three isolates, one from Malawi and two from Rwanda, the complete nucleotide sequences were determined and shown to have a similar genome organisation to previously published BBTV isolates. The two isolates from Rwanda had at least 98.1% nucleotide sequence identity between each of the six DNA components, while the similarity between isolates from Rwanda and Malawi was between 96.2% and 99.4% depending on the DNA component. At the amino acid level, similarities in the putative proteins encoded by DNA-R, -S, -M, - C and -N were found to range between 98.8% to 100%. In a phylogenetic analysis, the three East African isolates clustered together within the South Pacific subgroup of BBTV isolates. Nucleotide sequence comparison to isolates of BBTV from outside Africa identified India as the possible origin of East African isolates of BBTV.

In vitro breeding strategies in the development of Australian native plants

Plant tissue culture is a technique that exploits the ability of many plant cells to revert to a meristematic state. Although originally developed for botanical research, plant tissue culture has now evolved into important commercial practices and has become a significant research tool in agriculture, horticulture and in many other areas of plant sciences. Plant tissue culture is the sterile culture of plant cells, tissues, or organs under aseptic conditions leading to cell multiplication or regeneration or organs and whole plants. The steps required to develop reliable systems for plant regeneration and their application in plant biotechnology are reviewed in countless books. Some of the major landmarks in the evolution of in vitro techniques are summarised in Table 5.1. In this chapter the current applications of this technology to agriculture, horticulture, forestry and plant breeding are briefly described with specific examples from Australian plants when applicable.

Assessing the impact of a food supplement on the nutritional status and body composition of HIV-infected Zambian women on ARVs

Background Zambia is a sub-Saharan country with one of the highest prevalence rates of HIV, currently estimated at 14%. Poor nutritional status due to both protein-energy and micronutrient malnutrition has worsened this situation. In an attempt to address this combined problem, the government has instigated a number of strategies, including the provision of antiretroviral (ARV) treatment coupled with the promotion of good nutrition. High-energy protein supplement (HEPS) is particularly promoted; however, the impact of this food supplement on the nutritional status of people living with HIV/AIDS (PLHA) beyond weight gain has not been assessed. Techniques for the assessment of nutritional status utilising objective measures of body composition are not commonly available in Zambia. The aim of this study is therefore to assess the impact of a food supplement on nutritional status using a comprehensive anthropometric protocol including measures of skinfold thickness and circumferences, plus the criterion deuterium dilution technique to assess total body water (TBW) and derive fat-free mass (FFM) and fat mass (FM). Methods/Design This community-based controlled and longitudinal study aims to recruit 200 HIV-infected females commencing ARV treatment at two clinics in Lusaka, Zambia. Data will be collected at four time points: baseline, 4-month, 8-month and 12-month follow-up visits. Outcome measures to be assessed include body height and weight, body mass index (BMI), body composition, CD4, viral load and micronutrient status. Discussion This protocol describes a study that will provide a longitudinal assessment of the impact of a food supplement on the nutritional status of HIV-infected females initiating ARVs using a range of anthropometric and body composition assessment techniques.

Food insecurity among Australian children : potential determinants, health and developmental consequences

Children in food-insecure households may be at risk of poor health, developmental or behavioural problems. This study investigated the associations between food insecurity, potential determinants and health and developmental outcomes among children. Data on household food security, socio-demographic characteristics and children’s weight, health and behaviour were collected from households with children aged 3–17 years in socioeconomically disadvantaged suburbs by mail survey using proxy-parental reports (185 households). Data were analysed using logistic regression. Approximately one-in-three households (34%) were food insecure. Low household income was associated with an increased risk of food insecurity [odds ratio (OR), 16.20; 95% confidence interval (CI), 3.52–74.47]. Children with a parent born outside of Australia were less likely to experience food insecurity (OR, 0.42; 95% CI, 0.19–0.93). Children in food-insecure households were more likely to miss days from school or activities (OR, 3.52; 95% CI, 1.46–8.54) and were more likely to have borderline or atypical emotional symptoms (OR, 2.44; 95% CI, 1.11–5.38) or behavioural difficulties (OR, 2.35; 95% CI, 1.04–5.33). Food insecurity may be prevalent among socioeconomically disadvantaged households with children. The potential developmental consequences of food insecurity during childhood may result in serious adverse health and social implications.