Expansion and chondrogenic potential of human articular chondrocytes on macroporous microcarriers

Regenerative medicine techniques are currently being investigated to replace damaged cartilage. Critical to the success of these techniques is the ability to expand the initial population of cells while minimising de-differentiation to allow for hyaline cartilage to form. Three-dimensional culture systems have been shown to enhance the differentiation of chondrocytes in comparison to two-dimensional culture systems. Additionally, bioreactor expansion on microcarriers can provide mechanical stimulation and reduce the amount of cellular manipulation during expansion. The aim of this study was to characterise the expansion of human chondrocytes on microcarriers and to determine their potential to form cartilaginous tissue in vitro. High-grade human articular cartilage was obtained from leg amputations with ethics approval. Chondrocytes were isolated by collagenase digestion and expanded in either monolayers (104 cells/cm2) or on CultiSpher-G microcarriers (104 cells/mg) for three weeks. Following expansion, monolayer cells were passaged and cells on microcarriers were either left intact or the cells were released with trypsin/EDTA. Pellets from these three groups were formed and cultured for three weeks to establish the chondrogenic differentiation potential of monolayer-expanded and microcarrier-expanded chondrocytes. Cell viability, proliferation, glycosaminoglycan (GAG) accumulation, and collagen synthesis were assessed. Histology and immunohistochemistry were also performed. Human chondrocytes remained viable and expanded on microcarriers 10.2±2.6 fold in three weeks. GAG content significantly increased with time, with the majority of GAG found in the medium. Collagen production per nanogram DNA increased marginally during expansion. Histology revealed that chondrocytes were randomly distributed on microcarrier surfaces yet most pores remained cell free. Critically, human chondrocytes expanded on microcarriers maintained their ability to redifferentiate in pellet culture, as demonstrated by Safranin-O and collagen II staining. These data confirm the feasibility of microcarriers for passage-free cultivation of human articular chondrocytes. However, cell expansion needs to be improved, perhaps through growth factor supplementation, for clinical utility. Recent data indicate that cell-laden microcarriers can be used to seed fresh microcarriers, thereby increasing the expansion factor while minimising enzymatic passage.

A site selection process for environmental offsetting purposes in Queensland

A method of selecting land in any region of Queensland for offsetting purposes is devised, employing uniform standards. The procedure first requires that any core natural asset lands, Crown environmental lands, prime urban and agricultural lands, and highly contentious sites in the region be eliminated from consideration. Other land is then sought that is located between existing large reservations and the centre of greatest potential regional development/disturbance. Using the criteria of rehabilitation (rather than preservation) plus proximity to those officially defined Regional Ecosystems that are most threatened, adjacent sites that are described as ‘Cleared’ are identified in terms of agricultural land capability. Class IV lands – defined as those ‘which may be safely used for occasional cultivation with careful management’,2 ‘where it is favourably located for special usage’,3 and where it is ‘helpful to those who are interested in industry or regional planning or in reconstruction’4 – are examined for their appropriate area, for current tenure and for any conditions such as Mining Leases that may exist. The positive impacts from offsets on adjoining lands can then be designed to be significant; examples are also offered in respect of riparian areas and of Marine Parks. Criteria against which to measure performance for trading purposes include functional lift, with other case studies about this matter reported separately in this issue. The procedure takes no account of demand side economics (financial additionality), which requires commercial rather than environmental analysis.

Essays on irrigation development, farm production and unaccounted costs : theory and empirical evidence

A review of the literature related to issues involved in irrigation induced agricultural development (IIAD) reveals that: (1) the magnitude, sensitivity and distribution of social welfare of IIAD is not fully analysed; (2) the impacts of excessive pesticide use on farmers’ health are not adequately explained; (3) no analysis estimates the relationship between farm level efficiency and overuse of agro-chemical inputs under imperfect markets; and (4) the method of incorporating groundwater extraction costs is misleading. This PhD thesis investigates these issues by using primary data, along with secondary data from Sri Lanka. The overall findings of the thesis can be summarised as follows. First, the thesis demonstrates that Sri Lanka has gained a positive welfare change as a result of introducing new irrigation technology. The change in the consumer surplus is Rs.48,236 million, while the change in the producer surplus is Rs. 14,274 millions between 1970 and 2006. The results also show that the long run benefits and costs of IIAD depend critically on the magnitude of the expansion of the irrigated area, as well as the competition faced by traditional farmers (agricultural crowding out effects). The traditional sector’s ability to compete with the modern sector depends on productivity improvements, reducing production costs and future structural changes (spillover effects). Second, the thesis findings on pesticides used for agriculture show that, on average, a farmer incurs a cost of approximately Rs. 590 to 800 per month during a typical cultivation period due to exposure to pesticides. It is shown that the value of average loss in earnings per farmer for the ‘hospitalised’ sample is Rs. 475 per month, while it is approximately Rs. 345 per month for the ‘general’ farmers group during a typical cultivation season. However, the average willingness to pay (WTP) to avoid exposure to pesticides is approximately Rs. 950 and Rs. 620 for ‘hospitalised’ and ‘general’ farmers’ samples respectively. The estimated percentage contribution for WTP due to health costs, lost earnings, mitigating expenditure, and disutility are 29, 50, 5 and 16 per cent respectively for hospitalised farmers, while they are 32, 55, 8 and 5 per cent respectively for ‘general’ farmers. It is also shown that given market imperfections for most agricultural inputs, farmers are overusing pesticides with the expectation of higher future returns. This has led to an increase in inefficiency in farming practices which is not understood by the farmers. Third, it is found that various groundwater depletion studies in the economics literature have provided misleading optimal water extraction quantity levels. This is due to a failure to incorporate all production costs in the relevant models. It is only by incorporating quality changes to quantity deterioration, that it is possible to derive socially optimal levels. Empirical results clearly show that the benefits per hectare per month considering both the avoidance costs of deepening agro-wells by five feet from the existing average, as well as the avoidance costs of maintaining the water salinity level at 1.8 (mmhos/Cm), is approximately Rs. 4,350 for farmers in the Anuradhapura district and Rs. 5,600 for farmers in the Matale district.

Expression pattern of Oct-4, Sox2, and c-Myc in the primary culture of human dental pulp derived cells

Dental pulp cells (DPCs) have shown promising potential in dental tissue repair and regeneration. However, during in vitro culture, these cells undergo replicative senescence and result in significant alteration in cell proliferation and differentiation. Recently, the transcription factors of Oct-4, Sox2, c-Myc, and Klf4 have been reported to play a regulatory role in the stem cell self-renewal process, namely cell reprogramming. Therefore, it is interesting to know whether the replicative senescence during the culture of dental pulp cells is related to the diminishing of the expression of these transcription factors. In this study, we investigated the expression of the reprogramming markers Oct-4, Sox2, and c-Myc in the in vitro explant cultured dental pulp tissues and explant cultured dental pulp cells (DPCs) at various passages by immunofluorescence staining and real-time polymerase chain reaction analysis. Our results demonstrated that Oct-4, Sox2, and c-Myc translocated from nucleus in the first 2 passages to cytoplasm after the third passage in explant cultured DPCs. The mRNA expression of Oct-4, Sox2, and c-Myc elevated significantly over the first 2 passages, peaked at second passage (P < .05), and then decreased along the number of passages afterwards (P < .05). For the first time we demonstrated that the expression of reprogramming markers Oct-4, Sox2, and c-Myc was detectable in the early passaged DPCs, and the sequential loss of these markers in the nucleus during DPC cultures might be related to the cell fate of dental pulp derived cells during the long-term in vitro cultivation under current culture conditions.

Religious information literacy : using information to learn in church community

What informs members of the church community as they learn? Do the ways people engage with information differ according to the circumstances in which they learn? Informed learning, or the ways in which people use information in the learning experience and the degree to which they are aware of that, has become a focus of contemporary information literacy research. This essay explores the nature of informed learning in the context of the church as a learning community. It is anticipated that insights resulting from this exploration may help church organisations, church leaders and lay people to consider how information can be used to grow faith, develop relationships, manage the church and respond to religious knowledge, which support the pursuit of spiritual wellness and the cultivation of lifelong learning. Information professionals within the church community and the broader information profession are encouraged to foster their awareness of the impact that engagement with information has in the learning experience and in the prioritising of lifelong learning in community contexts.

Occurrence of iron and associated bacterial populations in soils of a forested subtropical coastal catchment

Iron (Fe) biogeochemistry is potentially of environmental significance in plantation-forested, subtropical coastal ecosystems where soil disturbance and seasonal water logging may lead to elevation of Fe mobilization and associated water quality deterioration. Using wet-chemical extraction and laboratory cultivation, we examined the occurrence of Fe forms and associated bacterial populations in diverse soils of a representative subtropical Australian coastal catchment (Poona Creek). Total reactive Fe was abundant throughout 0e30 cm soil cores, consisting primarily of crystalline forms in well-drained sand soils and water-logged loam soils, whereas in water-logged, low clay soils, over half of total reactive Fe was present in poorly-crystalline forms due to organic and inorganic complexation, respectively. Forestry practices such as plantation clear-felling and replanting, seasonal water logging and mineral soil properties significantly impacted soil organic carbon (C), potentially-bioavailable Fe pools and densities of S-, but not Fe-, bacterial populations. Bacterial Fe(III) reduction and abiotic Fe(II) oxidation, as well as chemolithotrophic S oxidation and aerobic, heterotrophic respiration were integral to catchment terrestrial FeeC cycling. This work demonstrates bacterial involvement in terrestrial Fe cycling in a subtropical coastal circumneutral-pH ecosystem.

The role of soil characteristics on temperature sensitivity of soil organic matter

The uncertainty associated with how projected climate change will affect global C cycling could have a large impact on predictions of soil C stocks. The purpose of our study was to determine how various soil decomposition and chemistry characteristics relate to soil organic matter (SOM) temperature sensitivity. We accomplished this objective using long-term soil incubations at three temperatures (15, 25, and 35°C) and pyrolysis molecular beam mass spectrometry (py-MBMS) on 12 soils from 6 sites along a mean annual temperature (MAT) gradient (2–25.6°C). The Q10 values calculated from the CO2 respired during a long-term incubation using the Q10-q method showed decomposition of the more resistant fraction to be more temperature sensitive with a Q10-q of 1.95 ± 0.08 for the labile fraction and a Q10-q of 3.33 ± 0.04 for the more resistant fraction. We compared the fit of soil respiration data using a two-pool model (active and slow) with first-order kinetics with a three-pool model and found that the two and three-pool models statistically fit the data equally well. The three-pool model changed the size and rate constant for the more resistant pool. The size of the active pool in these soils, calculated using the two-pool model, increased with incubation temperature and ranged from 0.1 to 14.0% of initial soil organic C. Sites with an intermediate MAT and lowest C/N ratio had the largest active pool. Pyrolysis molecular beam mass spectrometry showed declines in carbohydrates with conversion from grassland to wheat cultivation and a greater amount of protected carbohydrates in allophanic soils which may have lead to differences found between the total amount of CO2 respired, the size of the active pool, and the Q10-q values of the soils.

Iron biogeochemistry and associated greenhouse gas evolution in a forested subtropical Australian coastal catchment : Poona Creek, Southeast Queensland

Iron (Fe) is the fourth most abundant element in the Earth’s crust. Excess Fe mobilization from terrestrial into aquatic systems is of concern for deterioration of water quality via biofouling and nuisance algal blooms in coastal and marine systems. Substantial Fe dissolution and transport involve alternate Fe(II) oxidation followed by Fe(III) reduction, with a diversity of Bacteria and Archaea acting as the key catalyst. Microbially-mediated Fe cycling is of global significance with regard to cycles of carbon (C), sulfur (S) and manganese (Mn). However, knowledge regarding microbial Fe cycling in circumneutral-pH habitats that prevail on Earth has been lacking until recently. In particular, little is known regarding microbial function in Fe cycling and associated Fe mobilization and greenhouse (CO2 and CH4, GHG) evolution in subtropical Australian coastal systems where microbial response to ambient variations such as seasonal flooding and land use changes is of concern. Using the plantation-forested Poona Creek catchment on the Fraser Coast of Southeast Queensland (SEQ), this research aimed to 1) study Fe cycling-associated bacterial populations in diverse terrestrial and aquatic habitats of a representative subtropical coastal circumneutral-pH (4–7) ecosystem; and 2) assess potential impacts of Pinus plantation forestry practices on microbially-mediated Fe mobilization, organic C mineralization and associated GHG evolution in coastal SEQ. A combination of wet-chemical extraction, undisturbed core microcosm, laboratory bacterial cultivation, microscopy and 16S rRNA-based molecular phylogenetic techniques were employed. The study area consisted primarily of loamy sands, with low organic C and dissolved nutrients. Total reactive Fe was abundant and evenly distributed within soil 0–30 cm profiles. Organic complexation primarily controlled Fe bioavailability and forms in well-drained plantation soils and water-logged, native riparian soils, whereas tidal flushing exerted a strong “seawater effect” in estuarine locations and formed a large proportion of inorganic Fe(III) complexes. There was a lack of Fe(II) sources across the catchment terrestrial system. Mature, first-rotation plantation clear-felling and second-rotation replanting significantly decreased organic matter and poorly crystalline Fe in well-drained soils, although variations in labile soil organic C fractions (dissolved organic C, DOC; and microbial biomass C, MBC) were minor. Both well-drained plantation soils and water-logged, native-vegetation soils were inhabited by a variety of cultivable, chemotrophic bacterial populations capable of C, Fe, S and Mn metabolism via lithotrophic or heterotrophic, (micro)aerobic or anaerobic pathways. Neutrophilic Fe(III)-reducing bacteria (FeRB) were most abundant, followed by aerobic, heterotrophic bacteria (heterotrophic plate count, HPC). Despite an abundance of FeRB, cultivable Fe(II)-oxidizing bacteria (FeOB) were absent in associated soils. A lack of links between cultivable Fe, S or Mn bacterial densities and relevant chemical measurements (except for HPC correlated with DOC) was likely due to complex biogeochemical interactions. Neither did variations in cultivable bacterial densities correlate with plantation forestry practices, despite total cultivable bacterial densities being significantly lower in estuarine soils when compared with well-drained plantation soils and water-logged, riparian native-vegetation soils. Given that bacterial Fe(III) reduction is the primary mechanism of Fe oxide dissolution in soils upon saturation, associated Fe mobilization involved several abiotic and biological processes. Abiotic oxidation of dissolved Fe(II) by Mn appeared to control Fe transport and inhibit Fe dissolution from mature, first-rotation plantation soils post-saturation. Such an effect was not observed in clear-felled and replanted soils associated with low SOM and potentially low Mn reactivity. Associated GHG evolution post-saturation mainly involved variable CO2 emissions, with low, but consistently increasing CH4 effluxes in mature, first-rotation plantation soil only. In comparison, water-logged soils in the riparian native-vegetation buffer zone functioned as an important GHG source, with high potentials for Fe mobilization and GHG, particularly CH4 emissions in riparian loam soils associated with high clay and crystalline Fe fractions. Active Fe–C cycling was unlikely to occur in lower-catchment estuarine soils associated with low cultivable bacterial densities and GHG effluxes. As a key component of bacterial Fe cycling, neutrophilic FeOB widely occurred in diverse aquatic, but not terrestrial, habitats of the catchment study area. Stalked and sheathed FeOB resembling Gallionella and Leptothrix were limited to microbial mat material deposited in surface fresh waters associated with a circumneutral-pH seep, and clay-rich soil within riparian buffer zones. Unicellular, Sideroxydans-related FeOB (96% sequence identity) were ubiquitous in surface and subsurface freshwater environments, with highest abundance in estuary-adjacent shallow coastal groundwater water associated with redox transition. The abundance of dissolved C and Fe in the groundwater-dependent system was associated with high numbers of cultivable anaerobic, heterotrophic FeRB, microaerophilic, putatively lithotrophic FeOB and aerobic, heterotrophic bacteria. This research represents the first study of microbial Fe cycling in diverse circumneutral-pH environments (terrestrial–aquatic, freshwater–estuarine, surface–subsurface) of a subtropical coastal ecosystem. It also represents the first study of its kind in the southern hemisphere. This work highlights the significance of bacterial Fe(III) reduction in terrestrial, and bacterial Fe(II) oxidation in aquatic catchment Fe cycling. Results indicate the risk of promotion of Fe mobilization due to plantation clear-felling and replanting, and GHG emissions associated with seasonal water-logging. Additional significant outcomes were also achieved. The first direct evidence for multiple biomineralization patterns of neutrophilic, microaerophilic, unicellular FeOB was presented. A putatively pure culture, which represents the first cultivable neutrophilic FeOB from the southern hemisphere, was obtained as representative FeOB ubiquitous in diverse catchment aquatic habitats.

A brief history of haemotopoietic stem cell Ex vivo expansion

Haematopoiesis is the process by which a hierarchy of mature and progenitor blood cells are formed. These cell populations are all derived from multipotent haematopoietic stem cells (HSC), which reside in the bone marrow ‘niche’ of adult humans. Over the lifetime of a healthy individual, this HSC population replenishes between 1010-1011 blood cells on a daily basis. Dysregulation of this system can lead to a number of haematopoietic diseases, including aplastic anaemias and leukaemias, which result in, or require for disease resolution, bone marrow cell depletion. In 1956, E. Donnall Thomas demonstrated that haematopoiesis could be restored by transplanting bone marrow-derived cells from one man into his identical twin brother, who was suffering from advanced leukaemia. His success drew significant interest in academic research and medicine communities, and 12 years later, the first successful allogeneic transplant was performed. To this day, HSCs remain the most studied and characterised stem cell population. In fact, HSCs are the only stem cell population routinely utilised in the clinic. As such, HSCs function as a model system both for the biological investigation of stem cells, as well as for their clinical application. Herein, we briefly review HSC transplantation, strategies for the ex vivo cultivation of HSCs, recent clinical outcomes, and their impact on the future direction of HSC transplantation therapy.

A study of environmental and management drivers of non-CO2 greenhouse gas emissions in Australian agro-ecosystems

Australian climate, soils and agricultural management practices are significantly different from those of the northern hemisphere nations. Consequently, experimental data on greenhouse gas production from European and North American agricultural soils and its interpretation are unlikely to be directly applicable to Australian systems. A programme of studies of non-CO2 greenhouse gas emissions from agriculture has been established that is designed to reduce uncertainty of non-CO2 greenhouse gas emissions in the Australian National Greenhouse Gas Inventory and provide outputs that will enable better on-farm management practices for reducing non-CO2 greenhouse gas emissions, particularly nitrous oxide. The systems being examined and their locations are irrigated pasture (Kyabram Victoria), irrigated cotton (Narrabri, NSW), irrigated maize (Griffith, NSW), rain-fed wheat (Rutherglen, Victoria) and rain-fed wheat (Cunderdin, WA). The field studies include treatments with and without fertilizer addition, stubble burning versus stubble retention, conventional cultivation versus direct drilling and crop rotation to determine emission factors and treatment possibilities for best management options. The data to date suggest that nitrous oxide emissions from nitrogen fertilizer, applied to irrigated dairy pastures and rain-fed winter wheat, appear much lower than the average of northern hemisphere grain and pasture studies. More variable emissions have been found in studies of irrigated cotton/vetch/wheat rotation and substantially higher emissions from irrigated maize.

Human corneal epithelial equivalents constructed on Bombyx mori silk fibroin membranes

Membranes prepared from a protein, fibroin, isolated from domesticated silkworm (Bombyx mori) silk, support the cultivation of human limbal epithelial (HLE) cells and thus display significant potential as biomaterials for ocular surface reconstruction. We presently extend this promising avenue of research by directly comparing the attachment, morphology and phenotype of primary HLE cell cultures grown on fibroin to that observed on donor amniotic membrane (AM), the current clinical standard substrate for HLE transplantation. Fibroin membranes measuring 6.3 ± 0.5 μm (mean ± sd) in thickness and permeable to FITC dextran of a molecular weight up to 70 kDa, were used. Attachment of HLE cells to fibroin was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. Nevertheless, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (K3/K12 expression) and displayed a comparable number and distribution of ΔNp63+ progenitor cells to that seen in cultures grown on AM. These results support the suitability of membranes constructed from Bombyx mori silk fibroin as substrata for HLE cultivation and encourage progression to studies of efficacy in preclinical models.

A dual-layer silk fibroin scaffold for reconstructing the human corneal limbus

Membranes prepared from Bombyx mori silk fibroin have shown potential as a substrate for human limbal epithelial (L-EC) and stromal cell cultivation. Here we present fibroin as a dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. We have compared the growth and phenotype of L-EC on non-porous versus porous fibroin membranes. Furthermore, we have compared the growth of limbal mesenchymal stromal cells (L-MSC) in either serum-supplemented medium or the MesenCult-XF® culture system within fibroin fibrous mats. The co-culture of L-EC and L-MSC in fibroin dual-layer constructs was also examined. L-EC on porous membranes displayed a squamous monolayer; in contrast, L-EC on non-porous fibroin appeared cuboidal and stratified. Both constructs maintained evidence of corneal phenotype (cytokeratin 3/12) and distribution of ΔNp63+ progenitor cells. L-MSC cultivated within fibroin fibrous mats in serum-supplemented medium contained less than 64% of cells expressing the characteristic MSC phenotype of CD73+CD90+CD105+ after two weeks, compared with over 81% in MesenCult-XF® medium. Dual-layer fibroin scaffolds consisting of L-EC and L-MSC maintained a similar phenotype as on the separate layers. These results support the feasibility of a 3D engineered limbus constructed from B. mori silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration.

Russian wheat aphids (Diuraphis noxia) in China : native range expansion or recent introduction?

In this study, we explore the population genetics of the Russian wheat aphid (RWA) (Diuraphis noxia), one of the world’s most invasive agricultural pests, in north-western China. We have analysed the data of 10 microsatellite loci and mitochondrial sequences from 27 populations sampled over 2 years in China. The results confirm that the RWAs are holocyclic in China with high genetic diversity indicating widespread sexual reproduction. Distinct differences in microsatellite genetic diversity and distribution revealed clear geographic isolation between RWA populations in northern and southern Xinjiang, China, with gene flow interrupted across extensive desert regions. Despite frequent grain transportation from north to south in this region, little evidence for RWA translocation as a result of human agricultural activities was found. Consequently, frequent gene flow among northern populations most likely resulted from natural dispersal, potentially facilitated by wind currents. We also found evidence for the longterm existence and expansion of RWAs in China, despite local opinion that it is an exotic species only present in China since 1975. Our estimated date of RWA expansion throughout China coincides with the debut of wheat domestication and cultivation practices in western Asia in the Holocene. We conclude that western China represents the limit of the far eastern native range of this species. This study is the most comprehensive molecular genetic investigation of the RWA in its native range undertaken to date and provides valuable insights into the history of the association of this aphid with domesticated cereals and wild grasses.

Effects of oxygen on zonal marker expression in human articular chondrocytes

Articular cartilage is organized in depth zones with phenotypically distinct subpopulations of chondrocytes that are exposed to different oxygen tensions. Despite growing evidence of the critical role for oxygen in chondrogenesis, little is known about its effect on chondrocytes from different zones. This study evaluates zonal marker expression of human articular chondrocytes from different zones under various oxygen tensions. Chondrocytes isolated from full-thickness, superficial, and middle/deep cartilage from knee replacement surgeries were expanded and redifferentiated under hypoxic (5% O 2) or normoxic (20% O 2) conditions. Differentiation under hypoxia increased expression of hypoxia-inducible factors 1alpha and 2alpha and accumulation of extracellular matrix, particularly in middle/deep chondrocytes, and favored re-expression of proteoglycan 4 by superficial chondrocytes compared with middle/deep cells. Zone-dependent expression of clusterin varied with culture duration. These results demonstrate that zonal chondrocytes retain important phenotypic differences during in vitro cultivation, and that these characteristics can be improved by altering the oxygen environment. However, transcript levels for pleiotrophin, cartilage intermediate layer protein, and collagen type X were similar between zones, challenging their reliability as zonal markers for tissue-engineered cartilage from osteoarthritis patients. Key factors including oxygen tension and cell source should be considered to prescribe zone-specific properties to tissue-engineered cartilage. © 2012, Mary Ann Liebert, Inc.

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.