Improved vectors for Agrobacterium tumefaciens-mediated transformation of monocot plants

A series of improved vectors have been constructed that are suitable for use in Agrobacterium tumefaciens-mediated monocot transformation. These binary vectors have several useful features, including the selectable marker genes bar (phosphinothricin resistance) or hph (hygromycin resistance) driven by either the cauliflower mosaic virus (CaMV) 35S promoter or the maize ubiquitin promoter, a high-copy-number replication origin that allows reliable mini-prep DNA isolation from Escherichia coli, and a polylinker sequence into which target genes can be easily inserted. A significant improvement has been made to the hph gene by the introduction of an intron into its coding region. The presence of the intron abolishes hph expression in A. tumefaciens, rendering the bacterium susceptible to the selective agent hygromycin B. The use of such an intron-hph vector thus enables the antibiotic in plant culture media to function as both a selective agent for transformed tissue and as a contraselective agent for A. tumefaciens growth, thus minimising the overgrowth of A. tumefaciens on plant tissues during transformation. Furthermore, the intron appears to be correctly spliced in plant cells and significantly enhances hph expression in transformed rice tissue. In our experiments, the use of the intron-hph vector increased the frequency of rice transformation and has enabled the production of transgenic barley.

Agrobacterium tumefaciens-mediated transformation of an elite Australian barley cultivar with virus resistance and reporter genes

Efficient transformation of barley cv. Schooner was achieved using Agrobacterium delivery, hygromycin or bialaphos selection and embryogenic callus. Using this system, transgenic plants were generated that contained either the green fluorescent protein gene, or transgenes derived from barley yellow dwarf (BYDV) and cereal yellow dwarf (CYDV) viruses. Many of these plants contained 1-3 transgene copies that were inherited in a simple Mendelian manner. Some plants containing BYDV and/or CYDV derived transgenes showed reduced virus symptoms and rates of viral replication when challenged with the appropriate virus. The ability to transform Schooner is a significant advance for the Australian barley industry, as this elite malting variety is, and has for the last 15 years been, the most widely grown barley variety in eastern Australia.

Marker gene elimination from transgenic barley, using co-transformation with adjacent 'twin T-DNAs' on a standard Agrobacterium transformation vector

We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes.

Agrobacterium-mediated transformation of Australian rice cultivars Jarrah and Amaroo using modified promoters and selectable markers

We report the first successful Agrobacterium-mediated transformation of Australian elite rice cultivars, Jarrah and Amaroo, using binary vectors with our improved promoters and selectable markers. Calli derived from mature embryos were used as target tissues. The binary vectors contained hph (encoding hygromycin resistance) or bar (encoding herbicide resistance) as the selectable marker gene and uidA (gus) or sgfpS65T as the reporter gene driven by different promoters. Use of Agrobacterium strain AGL1 carrying derivatives of an improved binary vector pWBVec8, wherein the CaMV35S driven hph gene is interrupted by the castor bean catalase 1 intron, produced a 4-fold higher number of independent transgenic lines compared to that produced with the use of strain EHA101 carrying the binary vector pIG121-Hm wherein the CaMV35S driven hph is intronless. The Ubiquitin promoter produced 30-fold higher β-glucuronidase (GUS) activity (derivatives of binary vector pWBVec8) in transgenic plants than the CaMV35S promoter (pIG121-Hm). The two modified SCSV promoters produced GUS activity comparable to that produced by the Ubiquitin promoter. Progeny analysis (R1) for hygromycin resistance and GUS activity with selected lines showed both Mendelian and non-Mendelian segregation. Lines showing very high levels of GUS activity in T0 showed a reduced level of GUS activity in their T1 progeny, while lines with moderate levels of GUS activity showed increased levels in T1 progeny. Stable heritable green fluorescent protein (GFP) expression was also observed in few transgenic plants produced with the binary vector pTO134 which had the CaMV35S promoter-driven selectable marker gene bar and a modified CaMV35S promoter-driven reporter gene sgfpS65T.

Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens

In binary vectors, the antibiotic resistance gene used for selection of transformed plant cells is also usually expressed in the transforming Agrobacterium cells. This expression gives the bacterium antibiotic resistance, an unnecessary advantage on selective medium containing the antibiotic. Insertion of a castor bean catalase-1 (CAT-1) gene intron or a Parasponia andersonii haemoglobin gene intron into the coding region of the selectable marker gene, hph, completely abolished the expression of the gene in Agrobacterium, rendering it susceptible to hygromycin B. Use of these modified binary vectors minimized the overgrowth of Agrobacterium during plant transformation. Both of the introns were correctly spliced in plant cells and significantly enhanced hph gene expression in transformed rice tissue. The presence of these introns in the hph coding sequence not only maintained the selection efficiency of the hph gene, but with the CAT-1 intron also substantially increased the frequency of rice transformation. Transgenic lines with an intron-hph gene generally contained fewer gene copies and produced substantially more mRNA of the predicted size. Our results also indicate that transgenic plants with many copies of the transgene were more likely to show gene silencing than plants with 1-3 copies.

Comparison of promoters and selectable marker genes for use in Indica rice transformation

The effectiveness of different promoters for use in Indica rice transformation was compared. Plasmids encoding the Escherichia coli uidA (gus) gene under the control of CaMV 35S, Emu, Act1 or Ubi1 promoters were delivered into cell suspension cultures by particle bombardment. Transient gene expression, 48 h after delivery, was greatest from plasmids utilising the constitutive promoters, Act1 and Ubi1. Gene expression in stably transformed tissue was examined by bombarding embryogenic Indica rice calli with a pUbil-gus plasmid and a plasmid containing either the selectable marker gene, hph, which confers hygromycin resistance, or bar, which confers resistance to the herbicide phosphinothricin (BASTA) each under the control of the CaMV 35S, Emu, Act1 or the Ubi1 promoters. The bombarded calli were placed on the appropriate selection media and stained for GUS activity at 1 day, 3 weeks and 5 weeks after shooting. Callus bombarded with the pUbi1-hph or the pEmu-hph constructs gave a dramatic increase in the size of the GUS staining areas with time. No such increase in the size of GUS staining areas was observed in calli co-bombarded with pUbi1-gus and any of the bar containing constructs. Co-bombardment of calli with either the pEmu-hph or pUbi1-hph construct and a virus minor coat protein (cp) gene construct resulted in many fertile transgenic Indica rice plants, containing one to eight copies of both the hph and cp genes. These genes were stably inherited by the T 1 generation.

Modal flexibility method for structural damage detection in suspension bridges

Suspension bridges meet the steadily growing demand for lighter and longer bridges in today’s infrastructure systems. These bridges are designed to have long life spans, but with age, their main cables and hangers could suffer from corrosion and fatigue. There is a need for a simple and reliable procedure to detect and locate such damage, so that appropriate retrofitting can be carried out to prevent bridge failure. Damage in a structure causes changes in its properties (mass, damping and stiffness) which in turn will cause changes in its vibration characteristics (natural frequencies, modal damping and mode shapes). Methods based on modal flexibility, which depends on both the natural frequencies and mode shapes, have the potential for damage detection. They have been applied successfully to beam and plate elements, trusses and simple structures in reinforced concrete and steel. However very limited applications for damage detection in suspension bridges have been identified to date. This paper examines the potential of modal flexibility methods for damage detection and localization of a suspension bridge under different damage scenarios in the main cables and hangers using numerical simulation techniques. Validated finite element model (FEM) of a suspension bridge is used to acquire mass normalized mode shape vectors and natural frequencies at intact and damaged states. Damage scenarios will be simulated in the validated FE models by varying stiffness of the damaged structural members. The capability of damage index based on modal flexibility to detect and locate damage is evaluated. Results confirm that modal flexibility based methods have the ability to successfully identify damage in suspension bridge main cables and hangers.

Effects of wireless sensor network uncertainties on output-only modal-based damage identification

The use of Wireless Sensor Networks (WSNs) for Structural Health Monitoring (SHM) has become a promising approach due to many advantages such as low cost, fast and flexible deployment. However, inherent technical issues such as data synchronization error and data loss have prevented these distinct systems from being extensively used. Recently, several SHM-oriented WSNs have been proposed and believed to be able to overcome a large number of technical uncertainties. Nevertheless, there is limited research examining effects of uncertainties of generic WSN platform and verifying the capability of SHM-oriented WSNs, particularly on demanding SHM applications like modal analysis and damage identification of real civil structures. This article first reviews the major technical uncertainties of both generic and SHM-oriented WSN platforms and efforts of SHM research community to cope with them. Then, effects of the most inherent WSN uncertainty on the first level of a common Output-only Modal-based Damage Identification (OMDI) approach are intensively investigated. Experimental accelerations collected by a wired sensory system on a benchmark civil structure are initially used as clean data before being contaminated with different levels of data pollutants to simulate practical uncertainties in both WSN platforms. Statistical analyses are comprehensively employed in order to uncover the distribution pattern of the uncertainty influence on the OMDI approach. The result of this research shows that uncertainties of generic WSNs can cause serious impact for level 1 OMDI methods utilizing mode shapes. It also proves that SHM-WSN can substantially lessen the impact and obtain truly structural information without having used costly computation solutions.

On Chinese Modal Particle A (啊) : A Pragmatic and Semantic Study

Chinese modal particles feature prominently in Chinese people’s daily use of the language, but their pragmatic and semantic functions are elusive as commonly recognised by Chinese linguists and teachers of Chinese as a foreign language. This book originates from an extensive and intensive empirical study of the Chinese modal particle a (啊), one of the most frequently used modal particles in Mandarin Chinese. In order to capture all the uses and the underlying meanings of the particle, the author transcribed the first 20 episodes, about 20 hours in length, of the popular Chinese TV drama series Kewang ‘Expectations’, which yielded a corpus data of more than 142’000 Chinese characters with a total of 1829 instances of the particle all used in meaningful communicative situations. Within its context of use, every single occurrence of the particle was analysed in terms of its pragmatic and semantic contributions to the hosting utterance. Upon this basis the core meanings were identified which were seen as constituting the modal nature of the particle.

The transformation of Sweden from a haven for online pirates to the hope of the digital music industry [Musikbranschens föregångsland]

1974 was the year when the Swedish pop group ABBA won the Eurovision Song Contest in Brighton and when Blue Swede reached number one on the Billboard Hot 100 in the US. Although Swedish pop music gained some international success even prior to 1974, this year is often considered as the beginning of an era in which Swedish pop music had great success around the world. With brands such as ABBA, Europe, Roxette, The Cardigans, Ace of Base, In Flames, Robyn, Avicii, Swedish House Mafia and music producers Stig Andersson, Ola Håkansson, Dag Volle, Max Martin, Andreas Carlsson, Jorgen Elofsson and several others have the myth of the Swedish music miracle kept alive for nearly more than four decades. Swedish music looks to continue reap success around the world, but since the millennium, Sweden's relationship with music has been more focused on relatively controversial Internet-based services for music distribution developed by Swedish entrepreneurs and engineers rather than on successful musicians and composers. This chapter focusses on the music industry in Sweden. The chapter will discuss the development of the Internet services mentioned above and their impact on the production, distribution and consumption of recorded music. Ample space will be given in particular to Spotify, the music service that quickly has fundamentally changed the music industry in Sweden. The chapter will also present how the music industry's three sectors - recorded music, music licensing and live music - interact and evolve in Sweden.

Gas-Phase Transformation of Phosphatidylcholine Cations to Structurally Informative Anions via Ion/Ion Chemistry

Gas-phase transformation of synthetic phosphatidylcholine (PC) monocations to structurally informative anions is demonstrated via ion/ion reactions with doubly deprotonated 1,4-phenylenedipropionic acid (PDPA). Two synthetic PC isomers, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC16:0/18:1) and 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (PC18:1/16:0), were subjected to this ion/ion chemistry. The product of the ion/ion reaction is a negatively charged complex, \[PC + PDPA - H](-). Collisional activation of the long-lived complex causes transfer of a proton and methyl cation to PDPA, generating \[PC - CH3](-). Subsequent collisional activation of the demethylated PC anions produces abundant fatty acid carboxylate anions and low-abundance acyl neutral losses as free acids and ketenes. Product ion spectra of \[PC - CH3](-) suggest favorable cleavage at the sn-2 position over the sn-1 due to distinct differences in the relative abundances. In contrast, collisional activation of PC cations is absent of abundant fatty acid chain-related product ions and typically indicates only the lipid class via formation of the phosphocholine cation. A solution phase method to produce the gas-phase adducted PC anion is also demonstrated. Product ion spectra derived from the solution phase method are similar to the results generated via ion/ion chemistry. This work demonstrates a gas-phase means to increase structural characterization of phosphatidylcholines via ion/ion chemistry. Grant Number ARC/CE0561607, ARC/DP120102922

Global characteristics of school transformation in China

In many ways, China’s education system is quite different from systems of education in the West. Rich descriptions of school transformation, however, have revealed that the factors that fuelled transformation in schools in China are also evident in schools in Australia, England, Finland, Wales and the United States. This paper draws on an international project that examined how secondary schools from six countries achieved success by developing and drawing on their resources, referred to as four forms of capital: financial, intellectual, social and spiritual. It describes how five secondary schools in Chongqing, Western China, viewed each form of capital and how the four forms of capital were strengthened and aligned through outstanding governance to support the success of all students. The case is made that, although some aspects of the forms of capital found in schools in China may be viewed differently, the approaches adopted by these schools share a number of common elements with approaches to school transformation identified in Western schools. It is argued that these common elements from a range of international settings constitute a rich evidence base for understanding school transformation and for new insights in governance and leadership.

Health monitoring of short-and medium-spen bridges using an improved modal strain energy method

Increasing the importance and use of infrastructures such as bridges, demands more effective structural health monitoring (SHM) systems. SHM has well addressed the damage detection issues through several methods such as modal strain energy (MSE). Many of the available MSE methods either have been validated for limited type of structures such as beams or their performance is not satisfactory. Therefore, it requires a further improvement and validation of them for different types of structures. In this study, an MSE method was mathematically improved to precisely quantify the structural damage at an early stage of formation. Initially, the MSE equation was accurately formulated considering the damaged stiffness and then it was used for derivation of a more accurate sensitivity matrix. Verification of the improved method was done through two plane structures: a steel truss bridge and a concrete frame bridge models that demonstrate the framework of a short- and medium-span of bridge samples. Two damage scenarios including single- and multiple-damage were considered to occur in each structure. Then, for each structure, both intact and damaged, modal analysis was performed using STRAND7. Effects of up to 5 per cent noise were also comprised. The simulated mode shapes and natural frequencies derived were then imported to a MATLAB code. The results indicate that the improved method converges fast and performs well in agreement with numerical assumptions with few computational cycles. In presence of some noise level, it performs quite well too. The findings of this study can be numerically extended to 2D infrastructures particularly short- and medium-span bridges to detect the damage and quantify it more accurately. The method is capable of providing a proper SHM that facilitates timely maintenance of bridges to minimise the possible loss of lives and properties.

Mechanism of the α-to-β phase transformation in the LaNi5–H2 system

High-energy synchrotron in situ X-ray powder diffraction has been used to elucidate the mechanism of the hydriding phase transformation in a LaNi5 model hydrogen storage intermetallic in real time. The transformation proceeds at 10 °C via the transient growth of an interfacial phase, the γ phase, with lattice parameters intermediate between those of the α (dilute solid solution) and β (concentrated hydride) phases. The γ phase forms to partially accommodate the 24% change in unit cell volume between the α and β phases during hydriding and dehydriding. The α, γ and β phases coexist at the nanoscopic level.

Genetics and the transformation of the personal

The shared nature of genetic information presents new challenges for legal understandings of the self. Within traditional legal discourses the individual is conceptualised as separate and autonomous. In contrast, the genetic individual is understood as inherently relational. This paper analyses the transformation of our understandings of the personal. The transformative processes are assessed through discussion of the changing meanings of privacy in the context of genetic information within families; changing views over access to information about biological parentage by children conceived through assisted reproductive technology; preimplantation genetic diagnosis and the changing context of reproductive decisionmaking.