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.

Evaluation of fibroin-based scaffolds for ocular tissue reconstruction

The epithelium of the corneolimbus contains stem cells for regenerating the corneal epithelium. Diseases and injuries affecting the limbus can lead to a condition known as limbal stem cell deficiency (LSCD), which results in loss of the corneal epithelium, and subsequent chronic inflammation and scarring of the ocular surface. Advances in the treatment of LSCD have been achieved through use of cultured human limbal epithelial (HLE) grafts to restore epithelial stem cells of the ocular surface. These epithelial grafts are usually produced by the ex vivo expansion of HLE cells on human donor amniotic membrane (AM), but this is not without limitations. Although AM is the most widely accepted substratum for HLE transplantation, donor variation, risk of disease transfer, and rising costs have led to the search for alternative biomaterials to improve the surgical outcome of LSCD. Recent studies have demonstrated that Bombyx mori silk fibroin (hereafter referred to as fibroin) membranes support the growth of primary HLE cells, and thus this thesis aims to explore the possibility of using fibroin as a biomaterial for ocular surface reconstruction. Optimistically, the grafted sheets of cultured epithelium would provide a replenishing source of epithelial progenitor cells for maintaining the corneal epithelium, however, the HLE cells lose their progenitor cell characteristics once removed from their niche. More severe ocular surface injuries, which result in stromal scarring, damage the epithelial stem cell niche, which subsequently leads to poor corneal re-epithelialisation post-grafting. An ideal solution to repairing the corneal limbus would therefore be to grow and transplant HLE cells on a biomaterial that also provides a means for replacing underlying stromal cells required to better simulate the normal stem cell niche. The recent discovery of limbal mesenchymal stromal cells (L-MSC) provides a possibility for stromal repair and regeneration, and therefore, this thesis presents the use of fibroin as a possible biomaterial to support a three dimensional tissue engineered corneolimbus with both an HLE and underlying L-MSC layer. Investigation into optimal scaffold design is necessary, including adequate separation of epithelial and stromal layers, as well as direct cell-cell contact. Firstly, the attachment, morphology and phenotype of HLE cells grown on fibroin were directly compared to that observed on donor AM, the current clinical standard substrate for HLE transplantation. The production, transparency, and permeability of fibroin membranes were also evaluated in this part of the study. Results revealed that fibroin membranes could be routinely produced using a custom-made film casting table and were found to be transparent and permeable. Attachment of HLE cells to fibroin after 4 hours in serum-free medium was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. While HLE cultured on AM displayed superior stratification, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (cytokeratin pair 3/12 expression; CK3/12) and displayed a comparable number and distribution of ÄNp63+ progenitor cells to that seen in cultures grown on AM. These results confirm the suitability of membranes constructed from silk fibroin as a possible substrate for HLE cultivation. One of the most important aspects in corneolimbal tissue engineering is to consider the reconstruction of the limbal stem cell niche to help form the natural limbus in situ. MSC with similar properties to bone marrow derived-MSC (BM-MSC) have recently been grown from the limbus of the human cornea. This thesis evaluated methods for culturing L-MSC and limbal keratocytes using various serum-free media. The phenotype of resulting cultures was examined using photography, flow cytometry for CD34 (keratocyte marker), CD45 (bone marrow-derived cell marker), CD73, CD90, CD105 (collectively MSC markers), CD141 (epithelial/vascular endothelial marker), and CD271 (neuronal marker), immunocytochemistry (alpha-smooth muscle actin; á-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis), and co-culture experiments with HLE cells. While all techniques supported to varying degrees establishment of keratocyte and L-MSC cultures, sustained growth and serial propagation was only achieved in serum-supplemented medium or the MesenCult-XF„¥ culture system (Stem Cell Technologies). Cultures established in MesenCult-XF„¥ grew faster than those grown in serum-supplemented medium and retained a more optimal MSC phenotype. L-MSC cultivated in MesenCult-XFR were also positive for CD141, rarely expressed £\-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of L-MSC established in MesenCult-XF„¥ medium. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker £GNp63, along with the corneal differentiation marker CK3/12. Our findings conclude that MesenCult-XFR is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells. Following on from the findings of the previous two parts, silk fibroin was tested as a novel dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. In this section, the growth and phenotype of HLE cells on non-porous versus porous fibroin membranes was compared. Furthermore, the growth of L-MSC in either serum-supplemented medium or the MesenCult-XFR culture system within fibroin fibrous mats was investigated. Lastly, the co-culture of HLE and L-MSC in serum-supplemented medium on and within fibroin dual-layer constructs was also examined. HLE on porous membranes displayed a flattened and squamous monolayer; in contrast, HLE on non-porous fibroin appeared cuboidal and stratified closer in appearance to a normal corneal epithelium. Both constructs maintained CK3/12 expression and distribution of £GNp63+ progenitor cells. Dual-layer fibroin scaffolds consisting of HLE cells and L-MSC maintained a similar phenotype as on the single layers alone. Overall, the present study proposed to create a three dimensional limbal tissue substitute of HLE cells and L-MSC together, ultimately for safe and beneficial transplantation back into the human eye. The results show that HLE and L-MSC can be cultivated separately and together whilst maintaining a clinically feasible phenotype containing a majority of progenitor cells. In addition, L-MSC were able to be cultivated routinely in the MesenCult-XF® culture system while maintaining a high purity for the MSC characteristic phenotype. However, as a serum-free culture medium was not found to sustain growth of both HLE and L-MSC, the combination scaffold was created in serum-supplemented medium, indicating that further refinement of this cultured limbal scaffold is required. This thesis has also demonstrated a potential novel marker for L-MSC, and has generated knowledge which may impact on the understanding of stromal-epithelial interactions. These results support the feasibility of a dual-layer tissue engineered corneolimbus constructed from silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration. Further refinement of this technology should explore the potential benefits of using epithelial-stromal co-cultures with MesenCult-XF® derived L-MSC. Subsequent investigations into the effects of long-term culture on the phenotype and behaviour of the cells in the dual-layer scaffolds are also required. While this project demonstrated the feasibility in vitro for the production of a dual-layer tissue engineered corneolimbus, further studies are required to test the efficacy of the limbal scaffold in vivo. Future in vivo studies are essential to fully understand the integration and degradation of silk fibroin biomaterials in the cornea over time. Subsequent experiments should also investigate the use of both AM and silk fibroin with epithelial and stromal cell co-cultures in an animal model of LSCD. The outcomes of this project have provided a foundation for research into corneolimbal reconstruction using biomaterials and offer a stepping stone for future studies into corneolimbal tissue engineering.

Optimisation of Banana streak virus (BSV) diagnostic assays

Bananas are one of the world�fs most important crops, serving as a staple food and an important source of income for millions of people in the subtropics. Pests and diseases are a major constraint to banana production. To prevent the spread of pests and disease, farmers are encouraged to use disease�] and insect�]free planting material obtained by micropropagation. This option, however, does not always exclude viruses and concern remains on the quality of planting material. Therefore, there is a demand for effective and reliable virus indexing procedures for tissue culture (TC) material. Reliable diagnostic tests are currently available for all of the economically important viruses of bananas with the exception of Banana streak viruses (BSV, Caulimoviridae, Badnavirus). Development of a reliable diagnostic test for BSV is complicated by the significant serological and genetic variation reported for BSV isolates, and the presence of endogenous BSV (eBSV). Current PCR�] and serological�]based diagnostic methods for BSV may not detect all species of BSV, and PCR�]based methods may give false positives because of the presence of eBSV. Rolling circle amplification (RCA) has been reported as a technique to detect BSV which can also discriminate between episomal and endogenous BSV sequences. However, the method is too expensive for large scale screening of samples in developing countries, and little information is available regarding its sensitivity. Therefore the development of reliable PCR�]based assays is still considered the most appropriate option for large scale screening of banana plants for BSV. This MSc project aimed to refine and optimise the protocols for BSV detection, with a particular focus on developing reliable PCR�]based diagnostics Initially, the appropriateness and reliability of PCR and RCA as diagnostic tests for BSV detection were assessed by testing 45 field samples of banana collected from nine districts in the Eastern region of Uganda in February 2010. This research was also aimed at investigating the diversity of BSV in eastern Uganda, identifying the BSV species present and characterising any new BSV species. Out of the 45 samples tested, 38 and 40 samples were considered positive by PCR and RCA, respectively. Six different species of BSV, namely Banana streak IM virus (BSIMV), Banana streak MY virus (BSMYV), Banana streak OL virus (BSOLV), Banana streak UA virus (BSUAV), Banana streak UL virus (BSULV), Banana streak UM virus (BSUMV), were detected by PCR and confirmed by RCA and sequencing. No new species were detected, but this was the first report of BSMYV in Uganda. Although RCA was demonstrated to be suitable for broad�]range detection of BSV, it proved time�]consuming and laborious for identification in field samples. Due to the disadvantages associated with RCA, attempts were made to develop a reliable PCR�]based assay for the specific detection of episomal BSOLV, Banana streak GF virus (BSGFV), BSMYV and BSIMV. For BSOLV and BSGFV, the integrated sequences exist in rearranged, repeated and partially inverted portions at their site of integration. Therefore, for these two viruses, primers sets were designed by mapping previously published sequences of their endogenous counterparts onto published sequences of the episomal genomes. For BSOLV, two primer sets were designed while, for BSGFV, a single primer set was designed. The episomalspecificity of these primer sets was assessed by testing 106 plant samples collected during surveys in Kenya and Uganda, and 33 leaf samples from a wide range of banana cultivars maintained in TC at the Maroochy Research Station of the Department of Employment, Economic Development and Innovation (DEEDI), Queensland. All of these samples had previously been tested for episomal BSV by RCA and for both BSOLV and BSGFV by PCR using published primer sets. The outcome from these analyses was that the newly designed primer sets for BSOLV and BSGFV were able to distinguish between episomal BSV and eBSV in most cultivars with some B�]genome component. In some samples, however, amplification was observed using the putative episomal�]specific primer sets where episomal BSV was not identified using RCA. This may reflect a difference in the sensitivity of PCR compared to RCA, or possibly the presence of an eBSV sequence of different conformation. Since the sequences of the respective eBSV for BSMYV and BSIMV in the M. balbisiana genome are not available, a series of random primer combinations were tested in an attempt to find potential episomal�]specific primer sets for BSMYV and BSIMV. Of an initial 20 primer combinations screened for BSMYV detection on a small number of control samples, 11 primers sets appeared to be episomal�]specific. However, subsequent testing of two of these primer combinations on a larger number of control samples resulted in some inconsistent results which will require further investigation. Testing of the 25 primer combinations for episomal�]specific detection of BSIMV on a number of control samples showed that none were able to discriminate between episomal and endogenous BSIMV. The final component of this research project was the development of an infectious clone of a BSV endemic in Australia, namely BSMYV. This was considered important to enable the generation of large amounts of diseased plant material needed for further research. A terminally redundant fragment (.1.3 �~ BSMYV genome) was cloned and transformed into Agrobacterium tumefaciens strain AGL1, and used to inoculate 12 healthy banana plants of the cultivars Cavendish (Williams) by three different methods. At 12 weeks post�]inoculation, (i) four of the five banana plants inoculated by corm injection showed characteristic BSV symptoms while the remaining plant was wilting/dying, (ii) three of the five banana plants inoculated by needle�]pricking of the stem showed BSV symptoms, one plant was symptomless while the remaining had died and (iii) both banana plants inoculated by leaf infiltration were symptomless. When banana leaf samples were tested for BSMYV by PCR and RCA, BSMYV was confirmed in all banana plants showing symptoms including those were wilting and/or dying. The results from this research have provided several avenues for further research. By completely sequencing all variants of eBSOLV and eBSGFV and fully sequencing the eBSIMV and eBSMYV regions, episomal BSV�]specific primer sets for all eBSVs could potentially be designed that could avoid all integrants of that particular BSV species. Furthermore, the development of an infectious BSV clone will enable large numbers of BSVinfected plants to be generated for the further testing of the sensitivity of RCA compared to other more established assays such as PCR. The development of infectious clones also opens the possibility for virus induced gene silencing studies in banana.

Effects of oxygen and culture system on in vitro propagation and redifferentiation of osteoarthritic human articular chondrocytes

Regenerative medicine-based approaches for the repair of damaged cartilage rely on the ability to propagate cells while promoting their chondrogenic potential. Thus, conditions for cell expansion should be optimized through careful environmental control. Appropriate oxygen tension and cell expansion substrates and controllable bioreactor systems are probably critical for expansion and subsequent tissue formation during chondrogenic differentiation. We therefore evaluated the effects of oxygen and microcarrier culture on the expansion and subsequent differentiation of human osteoarthritic chondrocytes. Freshly isolated chondrocytes were expanded on tissue culture plastic or CultiSpher-G microcarriers under hypoxic or normoxic conditions (5% or 20% oxygen partial pressure, respectively) followed by cell phenotype analysis with flow cytometry. Cells were redifferentiated in micromass pellet cultures over 4 weeks, under either hypoxia or normoxia. Chondrocytes cultured on tissue culture plastic proliferated faster, expressed higher levels of cell surface markers CD44 and CD105 and demonstrated stronger staining for proteoglycans and collagen type II in pellet cultures compared with microcarrier-cultivated cells. Pellet wet weight, glycosaminoglycan content and expression of chondrogenic genes were significantly increased in cells differentiated under hypoxia. Hypoxia-inducible factor-3alpha mRNA was up-regulated in these cultures in response to low oxygen tension. These data confirm the beneficial influence of reduced oxygen on ex vivo chondrogenesis. However, hypoxia during cell expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential. Further improvements in cell culture conditions are therefore required before chondrocytes from osteoarthritic and aged patients can become a useful cell source for cartilage regeneration.

Generating transgenic banana (cv. Sukali Ndizi) resistant to Fusarium Wilt

Banana is one of the world’s most popular fruit crops and Sukali Ndizi is the most popular dessert banana in the East African region. Like other banana cultivars, Sukali Ndizi is threatened by several constraints, of which the Fusarium wilt disease is the most destructive. Fusarium wilt is caused by a soil-borne fungus, Fusarium oxysporum f.sp. cubense (Foc). No effective control strategy currently exists for this disease and although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by conventional breeding is difficult because of low fertility. Considering that conventional breeding generates hybrids with additional undesirable traits, transformation is the most suitable way of introducing resistance in the banana genome. The success of this strategy depends on the availability of genes for genetic transformation. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi, including Foc race 1 in banana cultivar Lady Finger. This thesis explores the potential of a plant-codon optimised nematode anti-apoptosis gene (Mced9) to provide resistance against Foc race 1 in dessert banana cultivar Sukali Ndizi. Agrobacterium-mediated transformation was used to transform embryogenic cell suspension of Sukali Ndizi with plant expression vector pYC11, harbouring maize ubiquitin promoter driven Mced9 gene and nptII as a plant selection marker. A total of 42 independently transformed lines were regenerated and characterized. The transgenic lines were multiplied, infected and evaluated for resistance to Foc race 1 in a small pot bioassay. The pathogenicity of the Ugandan Foc race 1 isolate used for infection was pre-determined and the spore concentration was standardised for consistent infection and symptom development. This process involved challenging tissue culture plants of Sukali Ndizi, a Foc race 1 susceptible cultivar and Nakinyika, an East African Highland cultivar known to be resistant to Foc race 1, with Fusarium inoculum and observing external and internal disease symptom development. Rhizome discolouration symptoms were the best indicators of Fusarium wilt with yellowing being an early sign of disease. Three transgenic lines were found to show significantly less disease severities compared to the wild-type control plants after 13 weeks of infection, indicating that Mced9 has the potential to provide tolerance to Fusarium wilt in Sukali Ndizi.

Strategies for improved disease resistance in micro-propagated bananas

Bananas (Musa sp) are one of the most important food crops in the world and provide a staple food and source of income in many households especially in Africa. Diseases are a major constraint to production with bunchy top, caused by Banana bunchy top virus (BBTV) generally considered the most important virus disease of bananas worldwide. Of the fungal diseases, Fusarium wilt, caused by the Fusarium oxysporum f.sp cubense (Foc), and black Sigatoka, caused by Mycosphaerella fijiensis, are arguably two of the most important and cause significant yield losses. The low fertility of commercially important banana cultivars has hampered efforts to generate disease resistance using conventional breeding. Possible alternative strategies to generate or increase disease resistance are through genetic engineering or by manipulation of the innate plant defence mechanisms, namely systemic acquired resistance (SAR). The first research component of this thesis describes attempts to generate BBTV-resistant banana plants using a genetic modification approach. The second research component of the thesis focused on the identification of a potential marker gene associated with SAR in banana plants and a comparison of the expression levels of the marker gene in response to biotic and abiotic stresses, and chemical inducers. Previous research at QUT CTCB showed that replication of BBTV DNA components in banana embryogenic cell suspensions (ECS) was abolished following co-bombardment with 1.1mers of mutated BBTV DNA-R. BBTV DNA-R encodes the master replication protein (Rep) and is the only viral protein essential for BBTV replication. In this study, ECS of banana were stably transformed with the same constructs, each containing a different mutation in BBTV DNA-R, namely H41G, Y79F and K187M, to examine the effect on virus replication in stably transformed plants. Cells were also transformed with a construct containing a native BBTV Rep. A total of 16, 16, 11 and five lines of stably transformed banana plants containing the Y79F, H41G, K187M and native Rep constructs, respectively, were generated. Of these, up to nine replicates from Y79F lines, four H41G lines, seven K187M lines and three native Rep lines were inoculated with BBTV by exposure to viruliferous aphids in two separate experiments. At least one replicate from each of the nine Y79F lines developed typical bunchy top symptoms and all tested positive for BBTV using PCR. Of the four H41G lines tested, at least one replicate from three of the lines showed symptoms of bunchy top and tested positive using PCR. However, none of the five replicates of one H41G line (H41G-3) developed symptoms of bunchy top and none of the plants tested positive for BBTV using PCR. Of the seven K187M lines, at least one replicate of all lines except one (K187M-1) developed symptoms of bunchy top and tested positive for BBTV. Importantly, none of the four replicates of line K187M-1 showed symptoms or tested positive for BBTV. At least one replicate from each of the three native Rep lines developed symptoms and tested positive for BBTV. The H41G-3 and K187M-1 lines possibly represent the first transgenic banana plants generated using a mutated Rep strategy. The second research component of this thesis focused on the identification of SAR-associated genes in banana and their expression levels in response to biotic and abiotic stresses and chemical inducers. The impetus for this research was the observation that tissue-cultured (TC) banana plants were more susceptible to Fusarium wilt disease (and possibly bunchy top disease) than plants grown from field-derived suckers, possibly due to decreased levels of SAR gene expression in the former. In this study, the pathogenesis-related protein 1 (PR-1) gene was identified as a potential marker for SAR gene expression in banana. A quantitative real-time PCR assay was developed and optimised in order to determine the expression of PR-1, with polyubiquitin (Ubi-1) found to be the most suitable reference gene to enable relative quantification. The levels of PR-1 expression were subsequently compared in Lady Finger and Cavendish (cv. Williams) banana plants grown under three different environmental conditions, namely in the field, the glass house and in tissue-culture. PR-1 was shown to be expressed in both cultivars growing under different conditions. While PR-1 expression was highest in the field grown bananas and lowest in the TC bananas in Lady Finger cultivar, this was not the case in the Cavendish cultivar with glass house plants exhibiting the lowest PR-1 expression compared with tissue culture and field grown plants. The important outcomes of this work were the establishment of a qPCR-based assay to monitor PR-1 expression levels in banana and a preliminary assessment of the baseline PR-1 expression levels in two banana cultivars under three different growing conditions. After establishing the baseline PR-1 expression levels in Cavendish bananas, a study was done to determine whether PR-1 levels could be increased in these plants by exposure to known banana pathogens and non-pathogens, and a known chemical inducer of SAR. Cavendish banana plants were exposed to pathogenic Foc subtropical race 4 (FocSR4) and non-pathogenic Foc race 1 (Foc1), as well as two putative inducers of resistance, Fusarium lycopersici (Fol) and the chemical, acibenzolar-S-methyl (BION®). Tissue culture bananas were acclimatised under either glass house (TCS) or field (TCH) conditions and treatments were carried out in a randomised complete block design. PR-1 expression was determined using qPCR for both TCS and TCH samples for the period 12-72h post-exposure. Treatment of TCH plants using Foc1 and FocSR4 resulted in 120 and 80 times higher PR-1 expression than baseline levels, respectively. For TCS plants treated with Foc1, PR-1 expression was 30 times higher than baseline levels at 12h post-exposure, while TCS plants treated with FocSR4 showed the highest PR-1 expression (20 times higher than baseline levels) at 72h post-exposure. Interestingly, when TCS plants were treated with Fol there was a marked increase of PR-1 expression at 12 h and 48 h following treatment which was 4 and 8 times higher than the levels observed when TCS plants were treated with Foc1 and FocSR4, respectively. In contrast, when TCH plants were treated with Fol only a slight increase in PR-1 expression was observed at 12 h, which eventually returned to baseline levels. Exposure of both TCS and TCH plants to BION® resulted in no effect on PR-1 expression levels at any time-point. The major outcome of the SAR study was that the glass house acclimatised tissue culture bananas exhibited lower PR-1 gene expression compared to field acclimatised tissue culture plants and the identification of Fol as a good candidate for SAR induction in banana plants exhibiting low PR-1 levels. A number of outcomes that foster understanding of both pathogen-derived and plant innate resistance strategies in order to potentially improve banana resistance to diseases were explored in this study and include identification of potential inducers of systemic acquired resistance and a promising mutated Rep approach for BBTV resistance. The work presented in this thesis is the first report on the generation of potential BBTV resistant bananas using the mutated Rep approach. In addition, this is the first report on the status of SAR in banana grown under different conditions of exposure to the biotic and abiotic environment. Further, a robust qPCR assay for the study of gene expression using banana leaf samples was developed and a potential inducer of SAR in tissue culture bananas identified which could be harnessed to increase resistance in tissue culture bananas.

A rapid and simple method of assaying plants transformed with hygromycin or PPT resistance genes

A very simple leaf assay is described that rapidly and reliably identifies transgenic plants expressing the hygromycin resistance gene, hph or the phosphinothricin resistance gene, bar. Leaf tips were cut from plants propagated either in the glasshouse or in tissue culture and the cut surface embedded in solid medium containing the appropriate selective agent. Non-transgenic barley or rice leaf tips had noticeable symptoms of either bleaching or necrosis after three days on the medium and were completely bleached or necrotic after one week. Transgenic leaf tips remained green and healthy over this period. This gave unambiguous discrimination between transgenic and non-transgenic plants. The leaf assay was also effective for dicot plants tested (tobacco and peas).

Cytotoxicity testing of burn wound dressings, ointments and creams : a method using polycarbonate cell culture inserts on a cell culture system

We have developed a method to test the cytotoxicity of wound dressings, ointments, creams and gels used in our Burn Centre, by placing them on a permeable Nunc Polycarbonate cell culture insert, incubated with a monolayer of cells (HaCaTs and primary human keratinocytes). METHODS: We performed two different methods to determine the relative toxicity to cells. (1) Photo visualisation: The dressings or compounds were positioned on the insert's membrane which was placed onto the monolayer tissue culture plate. After 24 h the surviving adherent cells were stained with Toluidine Blue and photos of the plates were taken. The acellular area of non-adherent dead cells which had been washed off with buffer was measured as a percentage of the total area of the plate. (2) Cell count of surviving cells: After 24 h incubation with the test material, the remaining cells were detached with trypsin, spun down and counted in a Haemocytometer with Trypan Blue, which differentiates between live and dead cells. RESULTS: Seventeen products were tested. The least cytotoxic products were Melolite, White soft Paraffin and Chlorsig1% Ointment. Some cytotoxicity was shown with Jelonet, Mepitel((R)), PolyMem((R)), DuoDerm((R)) and Xeroform. The most cytotoxic products included those which contained silver or Chlorhexidine and Paraffin Cream a moisturizer which contains the preservative Chlorocresol. CONCLUSION: This in vitro cell culture insert method allows testing of agents without direct cell contact. It is easy and quick to perform, and should help the clinician to determine the relative cytotoxicity of various dressings and the optimal dressing for each individual wound.

Mechanical tension as a driver of connective tissue growth in vitro

We propose the progressive mechanical expansion of cell-derived tissue analogues as a novel, growth-based approach to in vitro tissue engineering. The prevailing approach to producing tissue in vitro is to culture cells in an exogenous “scaffold” that provides a basic structure and mechanical support. This necessarily pre-defines the final size of the implantable material, and specific signals must be provided to stimulate appropriate cell growth, differentiation and matrix formation. In contrast, surgical skin expansion, driven by increments of stretch, produces increasing quantities of tissue without trauma or inflammation. This suggests that connective tissue cells have the innate ability to produce growth in response to elevated tension. We posit that this capacity is maintained in vitro, and that order-of-magnitude growth may be similarly attained in self-assembling cultures of cells and their own extracellular matrix. The hypothesis that growth of connective tissue analogues can be induced by mechanical expansion in vitro may be divided into three components: (1) tension stimulates cell proliferation and extracellular matrix synthesis; (2) the corresponding volume increase will relax the tension imparted by a fixed displacement; (3) the repeated application of static stretch will produce sustained growth and a tissue structure adapted to the tensile loading. Connective tissues exist in a state of residual tension, which is actively maintained by resident cells such as fibroblasts. Studies in vitro and in vivo have demonstrated that cellular survival, reproduction, and matrix synthesis and degradation are regulated by the mechanical environment. Order-of-magnitude increases in both bone and skin volume have been achieved clinically through staged expansion protocols, demonstrating that tension-driven growth can be sustained over prolonged periods. Furthermore, cell-derived tissue analogues have demonstrated mechanically advantageous structural adaptation in response to applied loading. Together, these data suggest that a program of incremental stretch constitutes an appealing way to replicate tissue growth in cell culture, by harnessing the constituent cells’ innate mechanical responsiveness. In addition to offering a platform to study the growth and structural adaptation of connective tissues, tension-driven growth presents a novel approach to in vitro tissue engineering. Because the supporting structure is secreted and organised by the cells themselves, growth is not restricted by a “scaffold” of fixed size. This also minimises potential adverse reactions to exogenous materials upon implantation. Most importantly, we posit that the growth induced by progressive stretch will allow substantial volumes of connective tissue to be produced from relatively small initial cell numbers.

Nucleic acid hybridizations as radioactive tool for rapid detection of banana bunchy top virus

Banana bunchy top disease (BBTD) caused by banana bunchy top virus (BBTV) was radioactively detected by nucleic acid hybridization techniques. Results showed that, 32P-labelled insert of pBT338 was hybridized with nucleic acid extracts from BBTV-infected plants from Egypt and Australia but not with those from CMV-infected plants from Egypt. Results revealed that BBTV was greatly detected in midrib, roots, meristem, corm, leaves and pseudostem respectively. BBTV was also detected in symptomless young plants prepared from diseased plant materials grown under tissue culture conditions but was not present in those performed from healthy plant materials. The sensitivity of dot blot and Southern blot hybridizations for the detection of BBTV was also performed for the detection of BBTV.

A particle based model to simulate microscale morphological changes of plant tissues during drying

Fundamental understanding on microscopic physical changes of plant materials is vital to optimize product quality and processing techniques, particularly in food engineering. Although grid-based numerical modelling can assist in this regard, it becomes quite challenging to overcome the inherited complexities of these biological materials especially when such materials undergo critical processing conditions such as drying, where the cellular structure undergoes extreme deformations. In this context, a meshfree particle based model was developed which is fundamentally capable of handling extreme deformations of plant tissues during drying. The model is built by coupling a particle based meshfree technique: Smoothed Particle Hydrodynamics (SPH) and a Discrete Element Method (DEM). Plant cells were initiated as hexagons and aggregated to form a tissue which also accounts for the characteristics of the middle lamella. In each cell, SPH was used to model cell protoplasm and DEM was used to model the cell wall. Drying was incorporated by varying the moisture content, the turgor pressure, and cell wall contraction effects. Compared to the state of the art grid-based microscale plant tissue drying models, the proposed model can be used to simulate tissues under excessive moisture content reductions incorporating cell wall wrinkling. Also, compared to the state of the art SPH-DEM tissue models, the proposed model better replicates real tissues and the cell-cell interactions used ensure efficient computations. Model predictions showed good agreement both qualitatively and quantitatively with experimental findings on dried plant tissues. The proposed modelling approach is fundamentally flexible to study different cellular structures for their microscale morphological changes at dehydration.

Application of meshfree methods to numerically simulate microscale deformations of different plant food materials during drying

Plant food materials have a very high demand in the consumer market and therefore, improved food products and efficient processing techniques are concurrently being researched in food engineering. In this context, numerical modelling and simulation techniques have a very high potential to reveal fundamentals of the underlying mechanisms involved. However, numerical modelling of plant food materials during drying becomes quite challenging, mainly due to the complexity of the multiphase microstructure of the material, which undergoes excessive deformations during drying. In this regard, conventional grid-based modelling techniques have limited applicability due to their inflexible grid-based fundamental limitations. As a result, meshfree methods have recently been developed which offer a more adaptable approach to problem domains of this nature, due to their fundamental grid-free advantages. In this work, a recently developed meshfree based two-dimensional plant tissue model is used for a comparative study of microscale morphological changes of several food materials during drying. The model involves Smoothed Particle Hydrodynamics (SPH) and Discrete Element Method (DEM) to represent fluid and solid phases of the cellular structure. Simulation are conducted on apple, potato, carrot and grape tissues and the results are qualitatively and quantitatively compared and related with experimental findings obtained from the literature. The study revealed that cellular deformations are highly sensitive to cell dimensions, cell wall physical and mechanical properties, middle lamella properties and turgor pressure. In particular, the meshfree model is well capable of simulating critically dried tissues at lower moisture content and turgor pressure, which lead to cell wall wrinkling. The findings further highlighted the potential applicability of the meshfree approach to model large deformations of the plant tissue microstructure during drying, providing a distinct advantage over the state of the art grid-based approaches.

Developing a three-dimensional in-vitro cell culture model for studying breast and prostate cancer using starPEG-heparin hydrogels

Introduction Hydrogels prepared from poly(ethylene glycol) (PEG) and maleimide-functionalized heparin provide a potential matrix for use in developing three dimensional (3D) models. We have previously demonstrated that these hydrogels support the cultivation of human umbilical vein endothelial cells (HUVECs) (1). We extend this body of work to study the ability to create an extracellular matrix (ECM)-like model to study breast and prostate cancer cell growth in 3D. Also, we investigate the ability to produce a tri-culture mimicking tumour angiogenesis with cancer spheroids, HUVECs and mesenchymal stem cells (MSC). Materials and Methods The breast cancer cell lines, MCF-7 and MDA-MB-231, and prostate cancer cell lines, LNCaP and PC3, were seeded into starPEG-heparin hydrogels and grown for 14 Days to analyse the effects of varying hydrogel stiffness on spheroid development. Resulting hydrogel constructs were analyzed via Alamar Blue assays, light microscopy, and immunofluorescence staining for cytokeratin 8/18, Ki67 and E-Cadherin. Cancer cell lines were then pre-grown in hydrogels for 5-7 days and then re-seeded into starPEG-heparin hydrogels functionalised with RGD, SDF-1, bFGF and VEGF as spheroids with HUVECs and MSC and grown for 14 days as a tri-culture in Endothelial Cell Growth Medium (ECGM; Promocell). Cell lines were also seeded as a single cell suspension into the functionalised tri-culture system. Cultures were fixed in 4% paraformaldehyde and analysed via immunostaining for Von Willebrand Factor and CD31, as well as the above mentioned markers, and observed using confocal microscopy. Results Cultures prepared in MMP-cleavable starPEG-heparin hydrogels display spheroid formation in contrast to adherent growth on tissue culture plastic. Small differences were visualised in cancer spheroid growth between different gel stiffness across the range of cell lines. Cancer cell lines were able to be co-cultivated with HUVECs and MSC. HUVEC tube formation and cancer line spheroid formation occured after 3-4 days. Interaction was visualised between tumours and HUVECs via confocal microscopy. Slightly increased interaction was seen between cancer tumours and micro-vascular tubes when seeded as single cells compared with the pre-formed spheroid approach. Further studies intend to utilise cytokine gradients to further optimise the ECM environment of in situ tumour angiogenesis. Discussion and Conclusions Our results confirm the suitability of hydrogels constructed from starPEG-heparin for HUVECs and MSC co-cultivation with cancer cell lines to study cell-cell and cell-matrix interactions in a 3D environment. This represents a step forward in the development of 3D culture models to study the pathomechanisms of breast and prostate cancer. References 1. Tsurkan MV, Chwalek K, Prokoph S, Zieris A, Levental KR, Freudenberg U, Werner C. Advanced Materials. 25, 2606-10, 2013. Disclosures The authors declare no conflicts of interest

The distribution and spread of citrus canker in Emerald, Australia

Citrus canker is a disease of citrus and closely related species, caused by the bacterium Xanthomonas citri subsp. citri. This disease, previously exotic to Australia, was detected on a single farm [infested premise-1, (IP1). IP is the terminology used in official biosecurity protocols to describe a locality at which an exotic plant pest has been confirmed or is presumed to exist. IP are numbered sequentially as they are detected] in Emerald, Queensland in July 2004. During the following 10 months the disease was subsequently detected on two other farms (IP2 and IP3) within the same area and studies indicated the disease first occurred on IP1 and spread to IP2 and IP3. The oldest, naturally infected plant tissue observed on any of these farms indicated the disease was present on IP1 for several months before detection and established on IP2 and IP3 during the second quarter (i.e. autumn) 2004. Transect studies on some IP1 blocks showed disease incidences ranged between 52 and 100% (trees infected). This contrasted to very low disease incidence, less than 4% of trees within a block, on IP2 and IP3. The mechanisms proposed for disease spread within blocks include weather-assisted dispersal of the bacterium (e.g. wind-driven rain) and movement of contaminated farm equipment, in particular by pivot irrigator towers via mechanical damage in combination with abundant water. Spread between blocks on IP2 was attributed to movement of contaminated farm equipment and/or people. Epidemiology results suggest: (i) successive surveillance rounds increase the likelihood of disease detection; (ii) surveillance sensitivity is affected by tree size; and (iii) individual destruction zones (for the purpose of eradication) could be determined using disease incidence and severity data rather than a predefined set area.