Initiation of embryogenic cell suspensions of taro (Colocasia esculenta var. esculenta) and plant regeneration

Embryogenic callus was initiated by culturing in vitro taro corm slices on agar-solidified half-strength MS medium containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) for 20 days followed by transfer to 1.0 mg/L thidiazuron (TDZ). Callus was subsequently proliferated on solid medium containing 1.0 mg/L TDZ, 0.5 mg/L 2,4- D and 800 mg/L glutamine before transfer to liquid medium containing the same components but with reduced glutamine (100 mg/L). After 3 months in liquid culture on an orbital shaker, cytoplasmically dense cell aggregates began to form. Somatic embryogenesis was induced by plating suspension cells onto solid media containing reduced levels of hormones (0.1 mg/L TDZ, 0.05 mg/L 2,4-D), high concentrations of sucrose (40–50 g/L) and biotin (1.0 mg/L). Embryo maturation and germination was then induced on media containing 0.05 mg/L benzyladenine (BA) and 0.1 mg/L indole-3-acetic acid (IAA). Histological studies of the developing embryos revealed the presence of typical shoot and root poles suggesting that these structures were true somatic embryos. The rate of somatic embryos formation was 500–3,000 per mL settledcell volume while approximately 60% of the embryos regenerated into plants.

Lung cancer stem cells: The root of resistance

In the absence of specific treatable mutations, platinum-based chemotherapy remains the gold standard of treatment for lung cancer patients. However, 5-year survival rates remain poor due to the development of resistance and eventual relapse. Resistance to conventional cytotoxic therapies presents a significant clinical challenge in the treatment of this disease. The cancer stem cell (CSC) hypothesis suggests that tumors are arranged in a hierarchical structure, with the presence of a small subset of stem-like cells that are responsible for tumor initiation and growth. This CSC population has a number of key properties such as the ability to asymmetrically divide, differentiate and self-renew, in addition to having increased intrinsic resistance to therapy. While cytotoxic chemotherapy kills the bulk of tumor cells, CSCs are spared and have the ability to recapitulate the heterogenic tumor mass. The identification of lung CSCs and their role in tumor biology and treatment resistance may lead to innovative targeted therapies that may ultimately improve clinical outcomes in lung cancer patients. This review will focus on lung CSC markers, their role in resistance and their relevance as targets for future therapies.

The transformation of banana with potential virus resistance genes

One approach to reducing the yield losses caused by banana viral diseases is the use of genetic engineering and pathogen-derived resistance strategies to generate resistant cultivars. The development of transgenic virus resistance requires an efficient banana transformation method, particularly for commercially important 'Cavendish' type cultivars such as 'Grand Nain'. Prior to this study, only two examples of the stable transformation of banana had been reported, both of which demonstrated the principle of transformation but did not characterise transgenic plants in terms of the efficiency at which individual transgenic lines were generated, relative activities of promoters in stably transformed plants, and the stability of transgene expression. The aim of this study was to develop more efficient transformation methods for banana, assess the activity of some commonly used and also novel promoters in stably transformed plants, and transform banana with genes that could potentially confer resistance to banana bunchy top nanovirus (BBTV) and banana bract mosaic potyvirus (BBrMV). A regeneration system using immature male flowers as the explant was established. The frequency of somatic embryogenesis in male flower explants was influenced by the season in which the inflorescences were harvested. Further, the media requirements of various banana cultivars in respect to the 2,4-D concentration in the initiation media also differed. Following the optimisation of these and other parameters, embryogenic cell suspensions of several banana (Musa spp.) cultivars including 'Grand Nain' (AAA), 'Williams' (AAA), 'SH-3362' (AA), 'Goldfinger' (AAAB) and 'Bluggoe' (ABB) were successfully generated. Highly efficient transformation methods were developed for both 'Bluggoe' and 'Grand Nain'; this is the first report of microprojectile bombardment transformation of the commercially important 'Grand Nain' cultivar. Following bombardment of embryogenic suspension cells, regeneration was monitored from single transfom1ed cells to whole plants using a reporter gene encoding the green fluorescent protein (gfp). Selection with kanamycin enabled the regeneration of a greater number of plants than with geneticin, while still preventing the regeneration of non-transformed plants. Southern hybridisation confirmed the neomycin phosphotransferase gene (npt II) was stably integrated into the banana genome and that multiple transgenic lines were derived from single bombardments. The activity, stability and tissue specificity of the cauliflower mosaic virus 358 (CaMV 35S) and maize polyubiquitin-1 (Ubi-1) promoters were examined. In stably transformed banana, the Ubi-1 promoter provided approximately six-fold higher p-glucuronidase (GUS) activity than the CaMV 35S promoter, and both promoters remained active in glasshouse grown plants for the six months they were observed. The intergenic regions ofBBTV DNA-I to -6 were isolated and fused to either the uidA (GUS) or gfjJ reporter genes to assess their promoter activities. BBTV promoter activity was detected in banana embryogenic cells using the gfp reporter gene. Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that generated by the maize Ubi-1 promoter. In transgenic banana plants, the activity of the BBTV DNA-6 promoter (BT6.1) was restricted to the phloem of leaves and roots, stomata and root meristems. The activity of the BT6.1 promoter was enhanced by the inclusion of intron-containing fragments derived from the maize Ubi-1, rice Act-1, and sugarcane rbcS 5' untranslated regions in GUS reporter gene constructs. In transient assays in banana, the rice Act-1 and maize Ubi-1 introns provided the most significant enhancement, increasing expression levels 300-fold and 100-fold, respectively. The sugarcane rbcS intron increased expression about 10-fold. In stably transformed banana plants, the maize Ubi-1 intron enhanced BT6.1 promoter activity to levels similar to that of the CaMV 35S promoter, but did not appear to alter the tissue specificity of the promoter. Both 'Grand Nain' and 'Bluggoe' were transformed with constructs that could potentially confer resistance to BBTV and BBrMV, including constructs containing BBTV DNA-1 major and internal genes, BBTV DNA-5 gene, and the BBrMV coat protein-coding region all under the control of the Ubi-1 promoter, while the BT6 promoter was used to drive the npt II selectable marker gene. At least 30 transgenic lines containing each construct were identified and replicates of each line are currently being generated by micropropagation in preparation for virus challenge.

Cultivar-specific effects of pathogen testing on storage root yield of sweet potato, Ipomoea batatas (L.) Lam.

The accumulation and perpetuation of viral pathogens over generations of clonal propagation in crop species such as sweet potato, Ipomoea batatas,inevitably result in a reduction in crop yield and quality. This study was conducted at Bundaberg, Australia to compare the productivity of field-derived and pathogen-tested (PT)clones of 14 sweet potato cultivars and the yield benefits of using healthy planting materials. The field-derived clonal materials were exposed to the endemic viruses, while the PT clones were subjected to thermotherapy and meristem-tip culture to eliminate viral pathogens. The plants were indexed for viruses using nitrocellulose membrane-enzyme-linked immunosorbent assay and graft-inoculations onto Ipomoea setosa. A net benefit of 38% in storage root yield was realised from using PT materials in this study.Conversely, in a similar study previously conducted at Kerevat, Papua New Guinea (PNG), a net deficit of 36% was realised. This reinforced our finding that the response to pathogen testing was cultivar dependent and that the PNG cultivars in these studies generally exhibited increased tolerance to the endemic viruses present at the respective trial sites as manifested in their lack of response from the use of PT clones. They may be useful sources for future resistance breeding efforts. Nonetheless, the potential economic gain from using PT stocks necessitates the use of pathogen testing on virus-susceptible commercial cultivars.

Development of the response coefficient-root function method for the transient vibration serviceability design of flat concrete floors

The vibration serviceability limit state is an important design consideration for two-way, suspended concrete floors that is not always well understood by many practicing structural engineers. Although the field of floor vibration has been extensively developed, at present there are no convenient design tools that deal with this problem. Results from this research have enabled the development of a much-needed, new method for assessing the vibration serviceability of flat, suspended concrete floors in buildings. This new method has been named, the Response Coefficient-Root Function (RCRF) method. Full-scale, laboratory tests have been conducted on a post-tensioned floor specimen at Queensland University of Technology’s structural laboratory. Special support brackets were fabricated to perform as frictionless, pinned connections at the corners of the specimen. A series of static and dynamic tests were performed in the laboratory to obtain basic material and dynamic properties of the specimen. Finite-element-models have been calibrated against data collected from laboratory experiments. Computational finite-element-analysis has been extended to investigate a variety of floor configurations. Field measurements of floors in existing buildings are in good agreement with computational studies. Results from this parametric investigation have led to the development of new approach for predicting the design frequencies and accelerations of flat, concrete floor structures. The RCRF method is convenient tool to assist structural engineers in the design for the vibration serviceability limit-state of in-situ concrete floor systems.

Ingroup and role identity influences on the initiation and maintenance of students' voluntary attendance at peer study sessions for statistics

Background: Although class attendance is linked to academic performance, questions remain about what determines students’ decisions to attend or miss class. Aims: In addition to the constructs of a common decision-making model, the theory of planned behaviour, the present study examined the influence of student role identity and university student (in-group) identification for predicting both the initiation and maintenance of students’ attendance at voluntary peer-assisted study sessions in a statistics subject. Sample: University students enrolled in a statistics subject were invited to complete a questionnaire at two time points across the academic semester. A total of 79 university students completed questionnaires at the first data collection point, with 46 students completing the questionnaire at the second data collection point. Method: Twice during the semester, students’ attitudes, subjective norm, perceived behavioural control, student role identity, in-group identification, and intention to attend study sessions were assessed via on-line questionnaires. Objective measures of class attendance records for each half-semester (or ‘term’) were obtained. Results: Across both terms, students’ attitudes predicted their attendance intentions, with intentions predicting class attendance. Earlier in the semester, in addition to perceived behavioural control, both student role identity and in-group identification predicted students’ attendance intentions, with only role identity influencing intentions later in the semester. Conclusions: These findings highlight the possible chronology that different identity influences have in determining students’ initial and maintained attendance at voluntary sessions designed to facilitate their learning.

Genetic evidence for the vital function of osterix in cementogenesis

To date, attempts to regenerate a complete tooth, including the critical periodontal tissues associated with the tooth root, have not been successful. Controversy still exists regarding the origin of the cell source for cellular cementum (epithelial or mesenchymal). This disagreement may be partially due to a lack of understanding of the events leading to the initiation and development of the tooth roots and supportive tissues, such as the cementum. Osterix (OSX) is a transcriptional factor essential for osteogenesis, but its role in cementogenesis has not been addressed. In the present study, we first documented a close relationship between the temporal- and spatial-expression pattern of OSX and the formation of cellular cementum. We then generated 3.6 Col 1-OSX transgenic mice, which displayed accelerated cementum formation vs. WT controls. Importantly, the conditional deletion of OSX in the mesenchymal cells with two different Cre systems (the 2.3 kb Col 1 and an inducible CAG-CreER) led to a sharp reduction in cellular cementum formation (including the cementum mass and mineral deposition rate) and gene expression of dentin matrix protein 1 (DMP1) by cementocytes. However, the deletion of the OSX gene after cellular cementum formed did not alter the properties of the mature cementum as evaluated by backscattered SEM and resin-cast SEM. Transient transfection of Osx in the cementoblasts in vitro significantly inhibited cell proliferation and increased cell differentiation and mineralization. Taken together, these data support 1) the mesenchymal origin of cellular cementum (from PDL progenitor cells); 2) the vital role of OSX in controlling the formation of cellular cementum; and 3) the limited remodeling of cellular cementum in adult mice.