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.

Can rodents enhance germination rates in rainforest seeds?

The decline of large coevolved frugivorous species within fragmented habitats can have an effect on ecological processes, for example, seed dispersal and germination. It is therefore necessary for more resilient species to ensure essential processes are maintained within the system. This study investigates the influence of two rodent species, Melomys cervinipes (Fawn-footed Melomys) and Rattus fuscipes (Bush Rat), on the germination process of rainforest fruits. Both species are endemic to north Queensland rainforest and commonly found in fragmented habitats in high densities. We found in 85% of fruit species tested, rodent feeding increased seed germination rate by a factor of 3.5. Our results suggest that rodents can play a significant role in enhancing germination rates of fruits in the tropical rainforest of far north Queensland.

Pyrolysis decomposition of mahogony seeds (Swietenia macrophylla) for alternative fuel oil

A fixed bed pyrolysis has been designed and fabricated for obtaining liquid fuel from Mahogany seeds. The major components of the system are fixed bed pyrolysis reactor, liquid condenser and liquid collectors. The Mahogany seed in particle form is pyrolysed in an externally heated 10 cm diameter and 36 cm high fixed bed reactor with nitrogen as the carrier gas. The reactor is heated by means of a biomass source cylindrical heater from 450oC to 600oC. The products are oil, char and gas. The reactor bed temperature, running time and feed particle size are considered as process parameters. A maximum liquid yield of 54wt% of biomass feed is obtained with particle size of 1.18 mm at a reactor bed temperature of 5500C with a running time of 90 minutes. The oil is found to possess favorable flash point and reasonable density and viscosity. The higher calorific value is found to be 39.9 MJ/kg which is higher than other biomass derived pyrolysis oils.

Health and guardianship law : the decision of X v the Sydney Children's Hospitals Network [2013] NSWCA 320 : mature minors and the refusal of blood products

This recent decision of the New South Wales Court of Appeal considers the scope of the parens patriae jurisdiction in cases where the jurisdiction is invoked for the protection of a Gillick competent minor. As outlined below, in certain circumstances the law recognises that mature minors are able to make their own decisions concerning medical treatment. However, there have been a number of Commonwealth decisions which have addressed the issue of whether mature minors are able to refuse medical procedures in circumstances where refusal will result in the minor dying. Ultimately, this case confirms that the minor does not necessarily have a right to make autonomous decisions; the minor’s right to exercise his or her autonomous decision only exists when such decision accords with what is deemed to be in his or her best interests.

Seeds of cosmopolitan future? Young people and their aspirations for future mobility

Mobility across space is an exemplary characteristic of the global era and an important aspect of the cultural experience of many people in advanced industrialised nations. Mobility evokes powerful images that effectively counter any illusion of stationary life and provide a break from the insularity of the local and parochial. High levels of mobility are simultaneously a fact, a necessity and a cultural aspiration. In recent times, debates about mobility in social theory have considered the relationship between mobility and cosmopolitan culture and identities (Hannerz, 1990, Urry, 1990, 2000, Beck, 2006). Against this backdrop, this paper also draws on some of the more recent discussions about the emergence of globalised and cosmopolitan identities among young people (Beck and Beck-Gernsheim, 2009). Using data from a longitudinal study of young people in Queensland, this paper provides an insight into young people’s aspirations about future mobility. The data affirm Skeggs’ (2004) comment that mobility is an unequal resource, and demonstrate that aspirations of future mobility reflect numerous social, economic and cultural realities of young people’s lives. This inevitably leads us to problematise the established, and often abstract, nexus between cosmopolitanism and mobility in contemporary debates about cosmopolitanism.

Recombinant cellulase accumulation in the leaves of mature, vegetatively propagated transgenic sugarcane

The cost of enzymes that hydrolyse lignocellulosic substrates to fermentable sugars needs to be reduced to make cellulosic ethanol a cost-competitive liquid transport fuel. Sugarcane is a perennial crop and the successful integration of cellulase transgenes into the sugarcane production system requires that transgene expression is stable in the ratoon. Herein, we compared the accumulation of recombinant fungal cellobiohydrolase I (CBH I), fungal cellobiohydrolase II (CBH II), and bacterial endoglucanase (EG) in the leaves of mature, initial transgenic sugarcane plants and their mature ratoon. Mature ratoon events containing equivalent or elevated levels of active CBH I, CBH II, and EG in the leaves were identified. Further, we have demonstrated that recombinant fungal CBH I and CBH II can resist proteolysis during sugarcane leaf senescence, while bacterial EG cannot. These results demonstrate the stability of cellulase enzyme transgene expression in transgenic sugarcane and the utility of sugarcane as a biofactory crop for production of cellulases.

The alcohol culture of mature-aged women : similarities and differences across borders

Introduction: Relatively few attempts have been made to describe and understand women’s alcohol consumption beyond adolescence and young adulthood. In particular, there has been a lack of studies focusing on the alcohol culture that surrounds and guides mature-aged women’s drinking. As part of a larger cross-national comparison, the present study sought to address this gap by identifying the shared beliefs and values that impact on drinking outcomes among mature-aged women in Sweden and Australia. Method: The study was guided by an ethnographic methodology. To generate data, a series of semi-structured interview were conducted with 17 Australian (age = 45-57 years; M = 52.1, SD = 3.9) and 19 Swedish (age = 45-58 years; M = 52.2, SD = 4.8) women. All interviews were transcribed verbatim and thematically analysed. Results: With age, the focus of alcohol as a single purpose vehicle for intoxication had given way to a focus on the enjoyment and ritual of drinking itself; taste had become increasingly important and alcohol was strongly associated with pleasurable environments and experiences. The view of alcohol as a taste experience was particularly pronounced among the Swedish women, with alcohol (most commonly wine) often seen as inseparable from food. Among the Swedish women, this view of alcohol was also associated with a strong de-emphasis of the pharmacological effects of alcohol. In contrast, several Australian women understood and used alcohol as relief for anxiety and stress. Moderate drinking was linked to the social construction of both age and gender in the two samples, while heavy or abusive alcohol consumption was associated with strong proscriptive norms and stigma. Conclusions: Australian and Swedish women share a number of beliefs and values around alcohol, however, these findings also show unique country-level differences. Implications for drinking outcomes are discussed.

Biolistic transformation of elite indica rice (Oryza sativa L.) cultivars through semi-solid and liquid medium selection systems

Following microprojectile mediated delivery of a plasmid construct (pAHC-25) encoding bar (bialophos resistance) gene into five-day-old scutellar calli derived from mature embryos, the effectiveness of selection procedure for bar-gene expressing tissue was compared for two indica rice cultivars (IR-64 and Karnal Local). While IR-64 transformants could be selected through the generally used semi-solid selection medium, the same procedure was not effective in the basmati cultivar Karnal Local. In the latter case, while lower concentrations (2–4 mg 1−1) of the selective agent phosphinothricin (PPT) yielded only escapes, higher concentrations (6–8 mg l−1) inhibited proliferation of transformed as well as untransformed sectors. For Karnal Local, a liquid medium based selection system was successfully utilized for recovering transformed sectors and, eventually, regenerants. The study demonstrates the generation of transformants of two elite indica cultivars using the environment-independent system of mature embryos from seeds.

Exploring the influences of country-level factors on mature-aged women’s drink driving attitudes

The current study examined drink driving attitudes among mature-aged women in Sweden and Australia, two countries with a Blood Alcohol Concentration (BAC) limit of 0.02% and 0.05%, respectively. The study aimed to identify attitudes that might influence drink driving tendency among this group of women and further show how these attitudes vary across countries. Using an ethnographic approach, 15 mature-aged women (Sweden: mean age = 52.5years, SD = 4.8; Australia: mean age 52.2 years, SD = 3.4) were interviewed in each country. General patterns and themes from the data were developed using thematic analysis methods. The findings indicate that while women in both countries viewed drink driving negatively, the understanding of what the concept entailed differed between the two samples. The Swedish women appeared to cognitively separate alcohol consumption and driving, and consequently, drink driving was often spoken of as driving after any alcohol consumption. The Australian women’s understanding of drink driving was more closely related to the legal BAC limit. However, for some Australian women, a “Grey Zone” existed, which denoted driving with a BAC of just above the enforceable limit. While illegal, these instances were subjectively seen as similar to driving with a BAC of just under the legal limit and therefore not morally reprehensible. The practice of cognitively separating drinking from driving appeared to have implications for the tendency to drink and drive among the interviewed women. These findings are discussed in relation to current policy and legislation in Australia and the need for further research into mature-aged women’s drink driving is outlined.

Homology between jacalin and artocarpin from jackfruit (Artocarpus integrifolia) seeds. Partial sequence and preliminary crystallographic studies of artocarpin

Jacalin and artocarpin, the two lectins from jackfruit (Artocarpus integrifolia) seeds, have different physicochemical properties and carbohydrate-binding specificities. However, comparison of the partial amino-acid sequence of artocarpin with the known sequence of jacalin indicates close to 50% sequence identity. Artocarpin crystallizes in two forms, both monoclinic P2(1), with one and two tetramic molecules, respectively, in the asymmetric units of form I (a = 69.9, b = 73.7, c = 60.6 Angstrom and beta = 95.1 degrees) and form II (a = 87.6, b = 72.2, c = 92.6 Angstrom and beta = 101.1 degrees). Both the crystal structures have been solved by the molecular replacement method using the known structure of jacalin as the search model and ope of them partially refined, confirming that the two lectins are indeed homologous.

A soluble preparation from developing groundnut seeds (Arachis hypogaea) catalyzes de novo synthesis of long chain fatty acids

A 100,000 x g supernatant fraction prepared from developing groundnut seeds (30-35 days after flowering) catalyzed the synthesis of fatty acids from [l-14C]acetate at a rate of 120nmoles of acetate incorporated per hr per gram fresh weight of tissue. 90% of this incorporated label was associated with fatty acids. The major fatty acids formed were stearic- (77%) and palmitic acids (14%) with 4% of oleic acid. The fatty acid synthetase activity was stable when stored at 0-4 degrees C for at least fifteen days. It is concluded from these results that acetyl-coA carboxylase and all the enzymes of fatty acid synthetase from developing groundnut seeds are soluble.

Examining novice education: What can we learn from a compulsory program delivered to both mature-age and young pre-learner licence drivers?

This paper reports results from a qualitative evaluation of a compulsory pre-Learner driver education program within the Australian Capital Territory(ACT), Australia. Two methods were used to obtain feedback from those involved in the delivery of the program as well as those who participated in programs. The first, semi-structured interviews, was undertaken with class room teachers who run the program in their schools, group facilitators running the program with more mature-age students at private facilities (n = 15 in total), and former participants in both school-based and private-based versions of the program (n = 19). The second method used an on-line survey for students (n = 79). Results from both methods were consistent with each other, indicating that strengths of the program were perceived as being its interactive components and the high level of engagement of the target audience. There was strong support from young and mature-age students for the program to remain compulsory. However, consistent with other findings on novice driver education, mature-age participants identified that the program was less relevant to them. It may be that to have greater relevance to mature-age learners, content could address and challenge perceptions about behaviours other than intentional high-risk behaviours (e.g. low level speeding, fatigue) as well as encourage planning/strategies to avoid them. While a longer term, outcome focussed, evaluation of the pre-learner education program is needed, this study suggests that the program is well received by pre-licence drivers and that teachers and facilitators perceive it as both effective and beneficial.

Germination and hardseededness in desmanthus

The mechanisms and control of hardseededness in the 3 Australian cultivars of the genus Desmanthus were investigated in a series of experiments in which the effects of various seedsoftening treatments, particularly boiling water, were measured. Desmanthus seed is predominantly hard, only defective seeds being normally otherwise. As it has only very brief, early embryo dormancy, hardseededness is the only serious barrier to germination. Seed is most readily softened through rupture of the palisade at the lens (strophiole). The lens is of a typically mimosaceous type which is readily ruptured by immersion in boiling water or less readily by application of pressure to adjacent parts of the testa. Ruptures may consist only of separation of the palisade from underlying tissue, which alone does not confer permeability; mostly they also result in fractures to the palisade that then render seeds irreversibly permeable. The palisade becomes reflective as it separates, which allows the event to be witnessed at the moment of separation if suitable pressure is applied to the testa of an individual seed while it is viewed under magnification. Brief (4–10 seconds) immersion of highquality seed in boiling water consistently softened a high proportion of seeds without causing serious damage. Extending the duration of immersion led to a progressive increase in the proportion of seed deaths. Neither previous boiling water treatment nor scarification damage to the testa materially affected results of treatment, but immature and small seeds behaved differently, being more vulnerable to damage than mature seed, and less likely to undergo lens rupture. Adaptation of boiling water treatment to farm-scale seed handling was simple and reliable. Commercial treatment of seed by an alternative method suitable for greater bulks and consisting of passage through a rice-whitener was checked and found to be successful through a combination of gentle scarification and lens rupture, both attributable to the numerous minor impacts of the process. Percentage emergence of seedlings from soil in the greenhouse closely followed percentage laboratory germination, except when inferior seed grades were included in the comparison, when emergence was poor. Very little seed softened in soil. Already-permeable seed either germinated rapidly or died, while buried hard seed mostly remained hard and viable even more than a year after sowing.

Selective herbicide strategies for use in Australian desmanthus seed crops

Grass and broad-leaved weeds can reduce both yields and product marketability of desmanthus (Desmanthus virgatus) seed crops, even when cultural control strategies are used. Selective herbicides might economically control these weeds, but, prior to this study, the few herbicides tolerated by desmanthus did not control key weed contaminants of desmanthus seed crops. In this study, the tolerance of desmanthus cv. Marc to 55 herbicides used for selective weed control in other leguminous crops was assessed in 1 pot trial and 3 Queensland field trials. One field trial assessed the tolerance of desmanthus seedlings to combinations of the most promising pre-emergent and post emergent herbicides. The pre-emergent herbicides, imazaquin, imazethapyr, pendimethalin, oryzalin and trifluralin, gave useful weed control with very little crop damage. The post-emergent herbicides, haloxyfop, clethodim, propyzamide, carbetamide and dalapon, were safe for controlling grass weeds in desmanthus. Selective post-emergence control of broad-leaved weeds was achieved using bentazone, bromoxynil and imazethapyr. One trial investigated salvaging second-year desmanthus crops from mature perennial weeds, and atrazine, terbacil and hexazinone showed some potential in this role. Overall, our results show that desmanthus tolerates herbicides which collectively control a wide range of weeds encountered in Queensland. These, in combination with cultural weed control strategies, should control most weeds in desmanthus seed crops.

Damage potential of an introduced biological control agent Agonosoma trilineatum (F.) on bellyache bush (Jatropha gossypiifolia L.)

The seed-feeding jewel bug, Agonosoma trilineatum (F.), is an introduced biological control agent for bellyache bush, Jatropha gossypiifolia L. To quantify the damage potential of this agent, shadehouse experiments were conducted with individual bellyache bush plants exposed to a range of jewel bug densities (0, 6 or 24 jewel bugs/plant). The level of abortion of both immature and mature seed capsules and impacts on seed weight and seed viability were recorded in an initial short-term study. The ability of the jewel bug to survive and cause sustained damage was then investigated by measuring seed production, the survival of adults and nymph density across three 6-month cycles. The level of seed capsule abortion caused by the jewel bug was significantly affected by the maturity status of capsules and the density of insects present. Immature capsules were most susceptible and capsule abortion increased with jewel bug density. Similarly, on average, the insects reduced the viability of bellyache bush seeds by 79% and 89% at low and high densities, respectively. However, sustaining jewel bug populations for prolonged periods proved difficult. Adult survival at the end of three 6-month cycles averaged 11% and associated reductions in viable seed production ranged between 55% and 77%. These results suggest that the jewel bug has the potential to reduce the number of viable seeds entering the soil seed bank provided populations can be established and maintained at sufficiently high densities.