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.

Aromatic l-amino acid decarboxylase : histochemical localization in rat kidney and lack of effect of dietary potassium or sodium loading on enzyme distribution

Utilizing a mono-specific antiserum produced in rabbits to hog kidney aromatic L-amino acid decarboxylase (AADC), the enzyme was localized in rat kidney by immunoperoxidase staining. AADC was located predominantly in the proximal convoluted tubules; there was also weak staining in the distal convoluted tubules and collecting ducts. An increase in dietary potassium or sodium intake produced no change in density or distribution of AADC staining in kidney. An assay of AADC enzyme activity showed no difference in cortex or medulla with chronic potassium loading. A change in distribution or activity of renal AADC does not explain the postulated dopaminergic modulation of renal function that occurs with potassium or sodium loading.

Book review: "English for telecoms and information technology" by T. Ricca and M. Duckworth; "English for legal professionals" by A. Frost; "English for the pharmaceutical industry" by M. Buchler, K. Jaehnig, G. Matzig, and T. Weindler; "English for cabin crews" by S. Ellis and L. Lansford; and "English for negotiating" by C. Lafond, S. Vine, and B. Welch.

This review examines five books in the Oxford Business English Express Series, including "English for telecoms and information technology" by T. Ricca and M. Duckworth; "English for legal professionals" by A. Frost; "English for the pharmaceutical industry" by M. Buchler, K. Jaehnig, G. Matzig, and T. Weindler; "English for cabin crews" by S. Ellis and L. Lansford; and "English for negotiating" by C. Lafond, S. Vine, and B. Welch.

Piecing together an integrative taxonomic puzzle: microsatellite, wing shape and aedeagus length analyses of Bactrocera dorsalis s.l. (Diptera: Tephritidae) find no evidence of multiple lineages in a proposed contact zone along the Thai/Malay Peninsula

Bactrocera dorsalis (Hendel) and B. papayae Drew & Hancock represent a closely related sibling species pair for which the biological species limits are unclear; i.e., it is uncertain if they are truely two biological species, or one biological species which has been incorrectly taxonomically split. The geographic ranges of the two taxa are thought to abut or overlap on or around the Isthmus of Kra, a recognised biogeographic barrier located on the narrowest portion of the Thai Peninsula. We collected fresh material of B. dorsalis sensu lato (i.e., B. dorsalis sensu stricto + B. papayae) in a north-south transect down the Thai Peninsula, from areas regarded as being exclusively B. dorsalis s.s., across the Kra Isthmus, and into regions regarded as exclusively B. papayae. We carried out microsatellite analyses and took measurements of male genitalia and wing shape. Both the latter morphological tests have been used previously to separate these two taxa. No significant population structuring was found in the microsatellite analysis and results were consistent with an interpretation of one, predominantly panmictic population. Both morphological datasets showed consistent, clinal variation along the transect, with no evidence for disjunction. No evidence in any tests supported historical vicariance driven by the Isthmus of Kra, and none of the three datasets supported the current taxonomy of two species. Rather, within and across the area of range overlap or abutment between the two species, only continuous morphological and genetic variation was recorded. Recognition that morphological traits previously used to separate these taxa are continuous, and that there is no genetic evidence for population segregation in the region of suspected species overlap, is consistent with a growing body of literature that reports no evidence of biological differentiation between these taxa.

Effect of a novel compound from Lycopodium obscurum L. on osteogenic activity of osteoblasts in vitro

We investigated the potential of an extract of Lycopodium obscurum L.; stigmastane-3-oxo-21-oic acid (SA), to enhance osteogensis of mouse osteoblastic MC3T3-E1 cells. SA at a concentration of 16 µM was found to have no significant effect upon the viability of the cells, thus concentrations of 8 µM and 16 µM of SA were used in all further experiments. Both concentrations of SA had an inhibitory affect upon alkaline phosphatase activity (ALP) after 8 days incubation, however, after 16 days activity was restored to control levels. However Alizarin red S staining showed increased levels of mineralization for both concentrations after 16 days culture. Real time PCR showed inhibition of genes Runx2 and Osterix genes responsible for the up-regulation of ALP. However early time point (8 days) up-regulation of bone matrix mineralization genes OPN and OCN, and late time point (16 days) up-regulation of both Jun-D and Fra-2 mRNA expression was significantly enhanced. These results suggest a potential me-chanism of SA in enhancing bone fracture healing is through the up-regulating bone matrix minera-lization.

Teaching and learning family and consumer sciences through K-W-L charts

While much of the control and many of the activities found in today’s classrooms have been placed in the hands of the learners and learning has become inquiry-based, there remains a need for teachers to use teaching tools that would facilitate this student-centered teaching process. This article identifies the K-W-L Chart as one such tool and follows a case study of four Kuwaiti ‘Family and Consumer Sciences��� teaching / learning events to evaluate their ability to enhance the learning outcomes of eight students. The research was designed from a qualitative, multi-tiered design approach and was assessed through a constant comparative method of data analysis of interview responses, classroom observations and worksheet-assessments. The results showed that the use of K-W-L Charts influenced the teachers and learners toward a more inquiry-based approach and facilitated a more student-centered and collaborative learning environment, raising the level of interest and the amount of personal input given by the students.

Limbal mesenchymal stromal cells (L-MSC) display immunosuppressive properties across donor and species boundaries

Purpose: We have evaluated the immunosuppressive properties of L-MSC with the view to using these cells in allogeneic cell therapies for corneal disorders. We hypothesized that L-MSC cultures would suppress T-cell activation, in a similar way to those established from human bone marrow (BM-MSC). Methods: MSC cultures were established from the limbal stroma of cadaveric donor eye tissue (up to 1 week postmortem) using either conventional serum-supplemented growth medium or a commercial serum-free medium optimized for bone marrow derived MSC (MesenCult-XF system). The MSC phenotype was examined by flow cytometry according to current and emerging markers for human MSC. Immunosuppressive properties were assessed using a mixed lymphocyte reaction (MLR) assay, whereby the white cell fraction from two immunologically incompatible blood donors are cultured together in direct contact with growth arrested MSC. T-cell activation (proliferation) was measured by uptake of tritiated thymidine. Human L-MSC were tested in parallel with human BM-MSC and rabbit L-MSC. Human and rabbit L-MSC were also tested for their ability to stimulate the growth of limbal epithelial (LE) cells in colony formation assays (for both human as well as rabbit LE cells). Results: L-MSC cultures were >95% negative for CD34, CD45 and HLA-DR and positive for CD73, CD90, CD105 and HLA-ABC. Modest levels (30%) of CD146 expression were observed for L-MSC cultures grown in serum-supplemented growth medium, but not those grown in MesenCult-XF. All MSC cultures derived from both human and rabbit tissue suppressed T-cell activation to varying degrees according to culture technique and species (MesenCult-XF >> serum-fed cultures, rabbit L-MSC >> human L-MSC). All L-MSC stimulated colony formation by LE cells irrespectively of the combination of cell species used. Conclusions: L-MSC display immunosuppressive qualities, in addition to their established non-immunogenic cell surface marker profile, and stimulate LE cell growth in vitro across species boundaries. These results support the potential use of allogeneic or even xenogeneic L-MSC in the treatment of corneal disorders.

Review essay -- Weber L and Pickering S (2011) Globalization and borders : death at the global frontier

The pertinence of this book cannot be overemphasised. The world’s refugee crisis has reached a two‐decade high with the United Nations recently announcing that ‘displacement is the new 21st century challenge’ (UNHCR 2013). The transnational movement of dislocated peoples fleeing conflict, persecution and poverty is a global responsibility requiring nation states to collaborate for humanitarian resolutions embedded in human rights. However, in times of human rights expansionism, and the relaxation of borders for maximising free‐trade and fiscal prosperity, the movement of people experiencing immense abuse and deprivation has witnessed an increase in draconian regulation within discourses of intolerance and deterrence. Weber and Pickering cogently and emphatically emphasise the human cost of inhumane and populist government immigration and border‐entry polices underpinned by ideologies of retribution, suspicion, and demonisation. It is a moving and engaging narrative: a book that exposes state prejudice and abuse, whilst advocating for the victims who undertake perilous journeys in search of safety from lives of violence and persecution. Moreover, it is a book that pushes ideological boundaries and seeks new criminological horizons, for which the authors must be sincerely congratulated. It is a text of innovation, inspired thinking and long lasting criminological value.

Mechanical and biological characterization of a porous poly-L-lactic acid-co-ε-caprolactone scaffold for tissue engineering

This article presents a method for making highly porous biodegradable scaffold that may ultimately be used for tissue engineering. Poly(L-lactic-co-1-caprolactone) acid (70:30) (PLCL) scaffold was produced using the solvent casting/leaching out method, which entails dissolving the polymer and adding a porogen that is then leached out by immersing the scaffold in distillated water. Tensile tests were performed for three types of scaffolds, namely pre-wetted, dried, and UV-irradiated scaffolds and their mechanical properties were measured. The prewetted PLCL scaffold possessed a modulus of elasticity 0.92+0.09 MPa, a tensile strength of 0.12+0.03 MPa and an ultimate strain of 23+5.3%. No significant differences in the modulus elasticity, tensile strength, nor ultimate strain were found between the pre-wetted, dried, and UV irradiated scaffolds. The PLCL scaffold was seeded by human fibroblasts in order to evaluate its biocompatibility by Alamar bluew assays. After 10 days of culture, the scaffolds showed good biocompatibility and allowed cell proliferation. However, the fibroblasts stayed essentially at the surface. This study shows the possibility to use the PLCL scaffold in dynamic mechanical conditions for tissue engineering

Aligned poly(L-lactic-co-ε-caprolactone) electrospun microfibers and knitted structure: a novel composite scaffold for ligament tissue engineering

We developed a novel technique involving knitting and electrospinning to fabricate a composite scaffold for ligament tissue engineering. Knitted structures were coated with poly(L-lactic-co-e-caprolactone) (PLCL) and then placed onto a rotating cylinder and a PLCL solution was electrospun onto the structure. Highly aligned 2-μm-diameter microfibers covered the space between the stitches and adhered to the knitted scaffolds. The stress–strain tensile curves exhibited an initial toe region similar to the tensile behavior of ligaments. Composite scaffolds had an elastic modulus (150 ± 14 MPa) similar to the modulus of human ligaments. Biological evaluation showed that cells proliferated on the composite scaffolds and they spontaneously orientated along the direction of microfiber alignment. The microfiber architecture also induced a high level of extracellular matrix secretion, which was characterized by immunostaining. We found that cells produced collagen type I and type III, two main components found in ligaments. After 14 days of culture, collagen type III started to form a fibrous network. We fabricated a composite scaffold having the mechanical properties of the knitted structure and the morphological properties of the aligned microfibers. It is difficult to seed a highly macroporous structure with cells, however the technique we developed enabled an easy cell seeding due to presence of the microfiber layer. Therefore, these scaffolds presented attractive properties for a future use in bioreactors for ligament tissue engineering.

Electrospinning and crosslinking of low-molecular-weight poly(trimethylene carbonate-co-L-lactide) as an elastomeric scaffold for vascular engineering

The growth of suitable tissue to replace natural blood vessels requires a degradable scaffold material that is processable into porous structures with appropriate mechanical and cell growth properties. This study investigates the fabrication of degradable, crosslinkable prepolymers of l-lactide-co-trimethylene carbonate into porous scaffolds by electrospinning. After crosslinking by γ-radiation, dimensionally stable scaffolds were obtained with up to 56% trimethylene carbonate incorporation. The fibrous mats showed Young’s moduli closely matching human arteries (0.4–0.8 MPa). Repeated cyclic extension yielded negligible change in mechanical properties, demonstrating the potential for use under dynamic physiological conditions. The scaffolds remained elastic and resilient at 30% strain after 84 days of degradation in phosphate buffer, while the modulus and ultimate stress and strain progressively decreased. The electrospun mats are mechanically superior to solid films of the same materials. In vitro, human mesenchymal stem cells adhered to and readily proliferated on the three-dimensional fiber network, demonstrating that these polymers may find use in growing artificial blood vessels in vivo.

Cross-linked poly(trimethylene carbonate-co-L-lactide) as a biodegradable, elastomeric scaffold for vascular engineering applications

A series of copolymers of trimethylene carbonate (TMC) and l-lactide (LLA) were synthesized and evaluated as scaffolds for the production of artificial blood vessels. The polymers were end-functionalized with acrylate, cast into films, and cross-linked using UV light. The mechanical, degradation, and biocompatibility properties were evaluated. High TMC polymers showed mechanical properties comparable to human arteries (Young’s moduli of 1.2–1.8 MPa and high elasticity with repeated cycling at 10% strain). Over 84 days degradation in PBS, the modulus and material strength decreased gradually. The polymers were nontoxic and showed good cell adhesion and proliferation over 7 days using human mesenchymal stem cells. When implanted into the rat peritoneal cavity, the polymers elicited formation of tissue capsules composed of myofibroblasts, resembling immature vascular smooth muscle cells. Thus, these polymers showed properties which were tunable and favorable for vascular tissue engineering, specifically, the growth of artificial blood vessels in vivo.

Voltammetric monitoring of gold nanoparticle formation facilitated by glycyl-L-tyrosine : relation to electronic spectra and transmission electron microscopy images

Voltammetric techniques have been introduced to monitor the formation of gold nanoparticles produced via the reaction of the amino acid glycyl-L-tyrosine with Au(III) (bromoaurate) in 0.05 M KOH conditions. The alkaline conditions facilitate amino acid binding to Au(III), inhibit the rate of reduction to Au(0), and provide an excellent supporting electrolyte for voltammetric studies. Data obtained revealed that a range of time-dependent gold solution species are involved in gold nanoparticle formation and that the order in which reagents are mixed is critical to the outcome. Concomitantly with voltammetric measurements, the properties of gold nanoparticles formed are probed by examination of electronic spectra in order to understand how the solution environment present during nanoparticle growth affects the final distribution of the nanoparticles. Images obtained by the ex situ transmission electron microscopy (TEM) technique enable the physical properties of the nanoparticles isolated in the solid state to be assessed. Use of this combination of in situ and ex situ techniques provides a versatile framework for elucidating the details of nanoparticle formation.