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.

Not-for-profit ratios for financial resilience and internal accountability : a study of Australian international aid organisations

Not-for-profit (NFP) financial ratio research has focused primarily on organisational efficiency measurements for external stakeholders. Ratios that also capture information about stability, capacity (liquidity), gearing and sustainability, enable an assessment of financial resilience. They are thus valuable tools that can provide a framework of internal accountability between boards and management. The establishment of an Australian NFP regulator highlights the importance of NFP sustainability, and affirms the timeliness of this paper. We propose a suite of key financial ratios for use by NFP boards and management, and demonstrate its practical usefulness by applying the ratios to financial data from the 2009 reports of ACFID (Australian Council for International Development)-affiliated international aid organisations.

Taxing charities : reform without reason?

The proposal to tax the unrelated business income of charitable organisations was announced in the 2011 budget, but the course of events has overtaken the stated policy rationale. We identify and discuss the policy for the imposition of the new tax and demonstrate that the measure is unnecessary given a recent judgement by the High Court, establishment of a new charity regulator and a better understanding of the applicable tax theory.

Introduction

Australia is presently witnessing concerted federal government policy initiatives to reform many aspects of the Australian Third Sector with an initial emphasis on charity organisations. Policy initiatives include the establishment of a new federal regulator, the Australian Charities and Not-for-profits Commission (ACNC), a national online financial reporting register, a statutory definition of charity, a tightening of taxation regulation and exemption including an unrelated business income tax and national fundraising regulation. This ambitious policy programme has had a gestation over 15 years of government inquiries lamenting lax, inconsistent and complex state-based regulation, and seeking a coherent and modern regulatory environment for nonprofit organisations, which are increasingly being used by the state to deliver outsourced community services..

The cell surface glycoprotein CUB domain-containing protein 1 (CDCP1) contributes to epidermal growth factor receptor-mediated cell migration

Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.

Rail incident investigator training: Is there support for a standardised Australasian training framework?

Background: Historically rail organisations have been operating in silos and devising their own training agendas. However with the harmonisation of the Australian workplace health and safety legislation and the appointment of a national rail safety regulator in 2013, rail incident investigator experts are exploring the possibility of developing a unified approach to investigator training. Objectives: The Australian CRC for Rail Innovation commissioned a training needs analysis to identify if common training needs existed between organisations and to assess support for the development of a national competency framework for rail incident investigations. Method: Fifty-two industry experts were consulted to explore the possibility of the development of a standardised training framework. These experts were sourced from within 19 Australasian organisations, comprising Rail Operators and Regulators in Queensland, New South Wales, Victoria, Western Australia, South Australia and New Zealand. Results: Although some competency requirements appear to be organisation specific, the vast majority of reported training requirements were generic across the Australasian rail operators and regulators. Industry experts consistently reported strong support for the development of a national training framework. Significance: The identification of both generic training requirements across organisations and strong support for standardised training indicates that the rail industry is receptive to the development of a structured training framework. The development of an Australasian learning framework could: increase efficiency in course development and reduce costs; establish recognised career pathways; and facilitate consistency with regards to investigator training.

Incident investigation : on a collision course towards a national standardised approach

The investigation of rail incidents is a highly specialised and important area within the rail industry. Historically training for investigators has been disjointed, with no standard approach being applied consistently. Currently in Australia, rail incidents are investigated by the various rail operators and regulators of each State, with the more serious incidents investigated by the Australian Transport Safety Bureau (ATSB). However, it is hoped with the introduction of a National Safety Regulator for the industry, a standardised competency framework for rail incident investigators can be developed. Consequently, this will also lead to more standardised training across the industry for these specialised career paths. A previous scoping report published by the CRC for Rail Innovation highlighted a need within the industry for a standardised competency framework and training package. Based on the results of the scoping report, a comprehensive Training Needs Analysis for the rail industry was undertaken. This paper will examine potential barriers and facilitators that the industry may face when implementing this national training. Furthermore, based on the results of the Training Needs Analysis, differences and similarities in the needs of rail organisations as well as between operators and regulators will be examined.

A critical assessment of the Australian OHS accreditation system's "Body of Knowledge (BoK)"

In response to an increasing perception of poor OHS consultancy quality amongst the Australian public, regulator and OHS professionals, the Safety Institute Australia (SIA) was tasked by the Victorian government to establish an accreditation process for OHS professionals. The OHS accreditation board decided to base its accreditation on a core "body of knowledge" (BoK), against which applicants are assesssed. While the foundation and structure of the BoK is unclear, the BoK consists of a collection of essays from a variety of invited authors. The BoK comprises about 811 pages in 34 chapters, with significant redundancy and considerable subjective components. The SIA BoK is benchmarked against two international best-practices, the German "Core Definition, Object Catalog and Research Domains of Labour Science (Ergonomics)" (Luzcak, Volpert, Raeithel & Schwier, 1989)(100 pages) and the American "Core Competency Model" for the "Master's Degree in Public Health" (Association of Schools of Public Health, 2006) (21 pages). Both "core definition" and "core competency model" are on a comparative level to the BoK. While the German expert panel consisted of 14 eminent professors, the American panel consisted of 135 members, organized in 6 groups chaired by discipline leading academics. The Australian approach employed a broad approach, where 137 professionals, consultants, emerging academics and academics contributed to 8 workshops. Both the German and the American panels maintained an open communication amongst members and with the discipline community throughout the process, whereas SIA applied an open and directed peer-review process. Moreover, the German process involved an analysis of all congress content and journal publications in the scientific domain in a set timeframe, which were then systematically clustered. These results were further expanded by structured interviews with 38 professors in the discpline, grasping their research and teaching practice. The American workgroup however assumed core scientific areas, underlying the domain. Based upon the a-priori assumption, they then established well defined competencies across all areas using a modified Delphi process. Although the BoK attempts to explore the knowledge in the OHS domain without an imposed structure in a bottom-up approach, it does not result in a structured systematic of the science. We conclude that the outcome of the German, rigorous academic approach, and the US American democratic approach under unambiguous academic leadership both outperform the Australian advocacy group approach. This product was determined for both structure and content of the taxonomy delivered through the processes.

Accounting regulation for charities : international responses to IFRS adoption

This paper examines charity regulatory systems, including accounting standard setting, across five jurisdictions in varying stages of adoption of International Financial Reporting Standards, and identifies the challenges of this process. Design/methodology/approach Using a regulatory space approach, we rely on publicly available archival evidence from charity regulators and accounting standard setters in five common-law jurisdictions in advanced capitalist economies, all with vibrant charity sectors: United Kingdom, United States of America, Canada, Australia and New Zealand. Findings The study reveals the importance of co-operative interdependence and dialogue between charity regulators and accounting standard setters, indicating that jurisdictions with such inter-relationships will better manage the transition to IFRS. It also highlights the need for those jurisdictions with not-for-profit or charity-specific accounting standards to reconfigure those provisions as IFRSs are adopted. Research limitations/implications The study is limited to five jurisdictions, concentrating specifically on key charity regulators and accounting standard setters. Future research could widen the scope to other jurisdictions, or track changes in the jurisdictions longitudinally. Practical implications We provide a timely international perspective of charity regulation and accounting developments for regulators, accounting standard setters and charities, specifically of regulatory responses to IFRS adoption. Originality/value: The paper contributes fresh insights into the dynamics of charity accounting regulation in an international context by using regulatory space as an organising framework. While accounting regulation literature provides a rich interpretation of regulatory issues within the accounting arena, little attention has been paid to charity accounting regulation.

The Australian nonprofit sector legal and accounting almanac 2012

This is the fifth year that The Australian Centre for Philanthropy and Nonprofit Studies has published an Almanac summarising annual developments in the law relevant to Australia's nonprofit sector. The Almanac provides comprehensive summaries of legal cases in Australia and overseas, and changes to relevant legislation in all Australian jurisdictions, during 2012. It also includes articles outlining developments in taxation and charity law, and the regulation of nonprofit organisations. This edition naturally includes extensive discussion of the new national regulator, the Australian Charities and Not-for-profits Commission (ACNC), and the legal arrangements surrounding its establishment. There are also articles on matters of interest arising in New Zealand, the UK, and Canada.

A Critical Perspective of the Indonesian Institutional Framework for PPP Toll Roads

The development of toll roads in Indonesia started around 1978. Initially, the management and development of toll roads sat directly under the Government of Indonesia (GoI) being undertaken through PT JasaMarga, a state owned enterprise specifically established to provide toll roads. Due to the slow growth and low capability of toll roads to fulfil infrastructure needs in the first ten years of operation (only 2.688kms/year), GoI changed its strategy in 1989 to one of using private sector participation for roads delivery through a Public Private Partnership (PPP) scheme. In this latter period, PT JasaMarga had two roles, both as regulator on behalf of the private sector as well as being the operator. However, from 1989 to 2004 the growth rate of toll roads actually decreased further to 2.300kms/year. Facing this challenge of low growth rate of toll roads, in 2004GoI changed the toll road management system and the role of regulator was returned to the Government through the establishment of the Toll Road Regulatory Agency (BPJT). GoI also amended the institutional framework to strengthen the toll road management system. Despite the introduction of this new institutional framework, the growth of toll roads still showed insignificant change. This problem in toll road development has generated an urgent need for research into this issue. The aim of the research is to understand the performance of the new institutional framework in enhancing PPP procured toll road development. The methodology of the research was to undertake a questionnaire survey distributed to private sector respondents involved in toll road development. The results of this study show that there are several problems inherent in the institutional framework, but the most significant problem comes from the uncertainty of the function of the strategic executive body in the land expropriation process.

Cross-sectional study of a microsatellite marker in the low densitity lipoprotein receptor gene in obese normotensives

1. The low density lipoprotein receptor is an important regulator of serum cholesterol which may have implications for the development of both hypertension and obesity. In this study, genotypes for a low density lipoprotein receptor gene (LDLR) dinucleotide polymorphism were determined in both lean and obese normotensive populations. 2. In previous cross-sectional association studies an ApaLI and a HincII polymorphism for LDLR were shown to be associated with obesity in essential hypertensives. However, these polymorphisms did not show an association with obesity in normotensives. 3. In contrast, this study reports that preliminary results for an LDLR microsatellite marker, located more towards the 3' end of the gene, show a significant association with obesity in the normotensive population studied. These results indicate that LDLR could play an important role in the development of obesity, which might be independent of hypertension.

Cyclin A/CDK2 regulates multiple G2 phase pathways

The cell cycle is a carefully choreographed series of phases that when executed successfully will allow the complete replication of the genome and the equal division of the genome and other cellular content into two independent daughter cells. The inability of the cell to execute cell division successfully can result in either checkpoint activation to allow repair and/or apoptosis and/or mutations/errors that may or may not lead to tumourgenesis. Cyclin A/CDK2 is the primary cyclin/CDK regulating G2 phase progression of the cell cycle. Cyclin A/CDK2 activity peaks in G2 phase and its inhibition causes a G2 phase delay that we have termed 'the cyclin A/CDK2 dependent G2 delay'. Understanding the key pathways that are involved in the cyclin A/CDK2 dependent G2 delay has been the primary focus of this study. Characterising the cyclin A/CDK2 dependent G2 delay revealed accumulated levels of the inactive form of the mitotic regulator, cyclin B/CDK1. Surprisingly, there was also increased microtubule nucleation at the centrosomes, and the centrosomes stained for markers of cyclin B/CDK1 activity. Both microtubule nucleation at the centrosomes and phosphoprotein markers were lost with short-term treatment of CDK1/2 inhibition. Cyclin A/CDK2 localised at the centrosomes in late G2 phase after separation of the centrosomes but before the start of prophase. Thus G2 phase cyclin A/CDK2 controls the timing of entry into mitosis by controlling the subsequent activation of cyclin B/CDK1, but also has an unexpected role in coordinating the activation of cyclin B/CDK1 at the centrosome and in the nucleus. In addition to regulating the timing of cyclin B/CDK1 activation and entry into mitosis in the unperturbed cell cycle, cyclin A/CDK2 also was shown to have a role in G2 phase checkpoint recovery. Known G2 phase regulators were investigated to determine whether they had a role in imposing the cyclin A/ CDK2 dependent G2 delay. Examination of the critical G2 checkpoint arrest protein, Chk1, which also has a role during unperturbed G2/M phases revealed the presence of activated Chk1 in G2 phase, in a range of cell lines. Activated Chk1 levels were shown to accumulate in cyclin A/CDK2 depleted/inhibited cells. Further investigations revealed that Chk1, but not Chk2, depletion could reverse the cyclin A/CDK2 dependent G2 delay. It was confirmed that the accumulative activation of Chk1 was not a consequence of DNA damage induced by cyclin A depletion. The potential of cyclin A/CDK2 to regulate Chk1 revealed that the inhibitory phosphorylations, Ser286 and Ser301, were not directly catalysed by cyclin A/CDK2 in G2 phase to regulate mitotic entry. It appeared that the ability of cyclin A/CDK2 to regulate cyclin B/CDK1 activation impacted cyclin B/CDK1s phosphorylation of Chk1 on Ser286 and Ser301, thereby contributing to the delay in G2/M phase progression. Chk1 inhibition/depletion partially abrogated the cyclin A/CDK2 dependent G2 delay, and was less effective in abrogating G2 phase checkpoint suggesting that other cyclin A/CDK2 dependent mechanisms contributed to these roles of cyclin A/CDK2. In an attempt to identify these other contributing factors another G2/M phase regulator known to be regulated by cyclin A/CDK2, Cdh1 and its substrates Plk1 and Claspin were examined. Cdh1 levels were reduced in cyclin A/CDK2 depleted/inhibited cells although this had little effect on Plk1, a known Cdh1 substrate. However, the level of another substrate, Claspin, was increased. Cdh1 depletion mimicked the effect of cyclin A depletion but to a weaker extent and was sufficient at increasing Claspin levels similar to the increase caused by cyclin A depletion. Co-depletion of cyclin A and Claspin blocked the accumulation of activated Chk1 normally seen with cyclin A depletion alone. However Claspin depletion alone did not reduce the cyclin A/CDK2 dependent G2 delay but this is likely to be a result of inhibition of S phase roles of Claspin. Together, these data suggest that cyclin A/CDK2 regulates a number of different mechanisms that contribute to G2/M phase progression. Here it has been demonstrated that in normal G2/M progression and possibly to a lesser extent in G2 phase checkpoint recovery, cyclin A/CDK2 regulates the level of Cdh1 which in turn affects at least one of its substrates, Claspin, and consequently results in the increased level of activated Chk1 observed. However, the involvement of Cdh1 and Claspin alone does not explain the G2 phase delay observed with cyclin A/CDK2 depletion/inhibition. It is likely that other mechanisms, possibly including cyclin A/CDK2 regulation of Wee1 and FoxM1, as reported by others, combine with the mechanism described here to regulate normal G2/M phase progression and G2 phase checkpoint recovery. These findings support the critical role for cyclin A/CDK2 in regulating progression into mitosis and suggest that upstream regulators of cyclin A/CDK2 activation will also be critical controllers of this cell cycle transition. The pathways that work to co-ordinate cell cycle progression are very intricate and deciphering these pathways, required for normal cell cycle progression, is key to understanding tumour development. By understanding cell cycle regulatory pathways it will allow the identification of the pathway/s and their mechanism/s that become affected in tumourgenesis. This will lead to the development of better targeted therapies, inferring better efficacy with fewer side effects than commonly seen with the use of traditional therapies, such as chemotherapy. Furthermore, this has the potential to positively impact the development of personalised medicines and the customisation of healthcare.

The Australian corporate rescue provisions : how do they compare?

A notable feature of corporate legislative development in western countries for the past 30 years is the various mechanisms introduced to facilitate the survival of company structures facing insolvency. Australia’s corporate rescue version, called a “voluntary administration”, is now contained in Part 5.3A of the Corporations Act 2001 (Cth), although first introduced in 1993. The Australian provisions apply to all corporate entities and commence with a short moratorium followed by a meeting of creditors. At the creditors’ meeting a “rescue” plan called a deed of company arrangement may be entered into, or, alternatively the company may be liquidated. The voluntary administration provisions have become a significant part of Australia’s corporate insolvency landscape and are critical to the operation of corporate law outside of insolvency. Australia does not have a specialist bankruptcy court, rather it utilises the English approach where insolvency practitioners are accountants and appointed to the insolvent company as administrators. In Australia, insolvency practitioners must be registered with the Australian Securities and Investments Commission (“ASIC”), the corporate and securities regulator. A voluntary administration is usually commenced by the board of directors appointing an insolvency practitioner to the company. There exists no opportunity for a voluntary administration to commence at the creditors’ or court’s behest. This chapter seeks to address the comparative necessity of Australia’s corporate regime.

Polyalanine repeat polymorphism in RUNX2 is associated with site-specific fracture in post-menopausal females

Runt related transcription factor 2 (RUNX2) is a key regulator of osteoblast differentiation. Several variations within RUNX2 have been found to be associated with significant changes in BMD, which is a major risk factor for fracture. In this study we report that an 18bp deletion within the polyalanine tract (17A>11A) of RUNX2 is significantly associated with fracture. Carriers of the 11A allele were found to be nearly twice as likely to have sustained fracture. Within the fracture category, there was a significant tendency of 11A carriers to present with fractures of bones of intramembranous origin compared to bones of endochondral origin (p=0.005). In a population of random subjects, the 11A allele was associated with decreased levels of serum collagen cross links (CTx, p=0.01), suggesting decreased bone turnover. The transactivation function of the 11A allele was quantitatively decreased. Interestingly, we found no effect of the 11A allele on BMD at multiple skeletal sites, although these were not the sites where a relationship with fracture was most evident. These findings suggest that the 11A allele is a biologically relevant polymorphism that influences serum CTx and confers enhanced fracture risk in a site-selective manner related to intramembranous bone ossification.