Improved molecular tools for sugar cane biotechnology

Sugar cane is a major source of food and fuel worldwide. Biotechnology has the potential to improve economically-important traits in sugar cane as well as diversify sugar cane beyond traditional applications such as sucrose production. High levels of transgene expression are key to the success of improving crops through biotechnology. Here we describe new molecular tools that both expand and improve gene expression capabilities in sugar cane. We have identified promoters that can be used to drive high levels of gene expression in the leaf and stem of transgenic sugar cane. One of these promoters, derived from the Cestrum yellow leaf curling virus, drives levels of constitutive transgene expression that are significantly higher than those achieved by the historical benchmark maize polyubiquitin-1 (Zm-Ubi1) promoter. A second promoter, the maize phosphonenolpyruvate carboxylate promoter, was found to be a strong, leaf-preferred promoter that enables levels of expression comparable to Zm-Ubi1 in this organ. Transgene expression was increased approximately 50-fold by gene modification, which included optimising the codon usage of the coding sequence to better suit sugar cane. We also describe a novel dual transcriptional enhancer that increased gene expression from different promoters, boosting expression from Zm-Ubi1 over eightfold. These molecular tools will be extremely valuable for the improvement of sugar cane through biotechnology.

An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane

Chemically inducible gene switches can provide precise control over gene expression, enabling more specific analyses of gene function and expanding the plant biotechnology toolkit beyond traditional constitutive expression systems. The alc gene expression system is one of the most promising chemically inducible gene switches in plants because of its potential in both fundamental research and commercial biotechnology applications. However, there are no published reports demonstrating that this versatile gene switch is functional in transgenic monocotyledonous plants, which include some of the most important agricultural crops. We found that the original alc gene switch was ineffective in the monocotyledonous plant sugar cane, and describe a modified alc system that is functional in this globally significant crop. A promoter consisting of tandem copies of the ethanol receptor inverted repeat binding site, in combination with a minimal promoter sequence, was sufficient to give enhanced sensitivity and significantly higher levels of ethanol inducible gene expression. A longer CaMV 35S minimal promoter than was used in the original alc gene switch also substantially improved ethanol inducibility. Treating the roots with ethanol effectively induced the modified alc system in sugar cane leaves and stem, while an aerial spray was relatively ineffective. The extension of this chemically inducible gene expression system to sugar cane opens the door to new opportunities for basic research and crop biotechnology.

Moisture and tensile strength properties of starch-sugar cane nanofibre films

There is an increasing need for biodegradable, environmentally friendly plastics to replace the petroleum-based non-degradable plastics which litter and pollute the environment. Starch-based plastic film composites are becoming a popular alternative because of their low cost, biodegradability, the abundance of starch, and ease with which starch-based films can be chemically modified. This paper reports on the results of using sugar cane bagasse nanofibres to improve the physicochemical properties of starch-based polymers. The addition of bagasse nanofibre (2.5, 5, 10 or 20 wt%) to (modified) potato starch (‘Soluble starch’) reduced the moisture uptake by up to 17 % at 58 % relative humidity (RH). The film’s tensile strength and Young’s Modulus increased by up to 100 % and 200 % with 10 wt% and 20 wt% nanofibre respectively at 58% RH. The tensile strain reduced by up to 70 % at 20 wt% fibre loading. These results indicate that addition of sugar cane bagasse nanofibres significantly improved the properties of starch-based plastic films

Straminipilan protists : a study of their taxonomic position, morphology and abiotic stress-mediated gene expression

Thraustochytrids have become of considerable industrial and scientific interest in the past decade due to their health benefits. They have been proven to be the principle source in marine and estuarine fish diets with high percentage of long chain (LC) or polyunsaturated fatty acids (PUFA). Therefore, the oil extracted from fish for human document.forms[0].elements[13].select();consumption is rich in PUFA with high omega-3 fatty acid content. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) of all of the omega-3 fatty acids, are considered beneficial essential oils for humans with a wide range of health benefits. These include brain and neural development in infants, general wellbeing of adults and drug delivery through precursor molecules. They have become one of the most extensively studied organisms for industrial oil preparations as PUFA extraction from fish becomes less profitable. Many forms of these Thraustochytrid oils are being trialled for human consumption all over the world. In Australia, there has been little research performed on these organisms in the past ten years. A few Australian studies have been conducted in the form of comparative studies related to PUFA production within the related genera, but not focussed on their identification or cellular and genomic characterisation. Therefore, the main aim of this study was to investigate the morphological and genetic characteristics of Australian Thraustochytrids in order to aid in their identification and characterisation, as well as to better understand the effect of environmental conditions in the regulation of PUFA production. It was also noted that there was a knowledge gap in the preservation and total genomic DNA extraction of these organisms for the purposes of scientific research. The cryopreservation of these organisms for studies around the world follows existing generic methods. However, it is well understood that many of these generic methods attract not only high costs for chemicals, but also uses considerable storage space and other resources, all of which can be improved with new or modified approaches. In this context, a simple and inexpensive bead preservation method is described, without compromising the storage shelf life. We also describe, for the first time, the effects of culture age on the successful cryopreservation of Thraustochytrids. It was evident in the literature that DNA and RNA extractions for molecular and genetic studies of Thraustochytrids follow the classical phenol-chloroform extraction methods. It was also observed that modern protocols failed to avoid the use of phenol-chloroform rather than improving preparation and cell disruption. In order to provide a high quantity and quality DNA extraction, a modified protocol has been introduced that employs the use of modern commercial extraction kits and standard laboratory equipment. Thraustochytrids have been shown to be highly conserved in their 18S rDNA gene sequences, which is used as the current standard for identification. It was demonstrated that the 18S rDNA gene sequence limits the recognition of closely related genera or within the genera from each member. Therefore, it was proposed that another profile, such as a randomly amplified polymorphic DNA (RAPD) based profiling system, be tested for use in the characterisation of Thraustochytrids. The RAPD profiles were shown to provide a unique DNA fingerprint for each isolate and small variations in their genome were able to be detected. This method involved the use of a minimum number of standard arbitrary primers and with an increase in the number of different primers used, a very high discrimination between organisms could be achieved. However, the method was not suitable for taxonomic purposes because the results did not correlate with other taxonomic features such as morphology. Another knowledge gap was found with respect to Australian Thraustochytrid growth characteristics, in that these had not been recorded and published. In order to rectify this, a record of colony and microscopic features of 12 selected isolates was performed. The results of preliminary studies indicated that further microbiological and biochemical studies are needed for full characterisation of these organisms. This information is of great importance to bio-prospecting of new Thraustochytrids from Australian ecosystems and would allow for their accurate identification, and so permit the prediction of their PUFA capability by comparison with related genera/species. It was well recognized that environmental stress plays a role in the PUFA production and is mainly due to the reactive oxygen species as abiotic stress (Chiou et al., 2001; Okuyama et al., 2008; Shabala et al., 2009; Shabala et al., 2001). In this aspect, this study makes the first attempt towards better understanding of this phenomenon by way of the use of real-time PCR for the detection of environmental effects on the regulation of PUFA production. Three main environmental conditions including temperature, pH and oxygen availability were monitored as stress inducers. In summary, this study provides novel approaches for the preservation and handling of Thraustochytrids, their molecular biological features, taxonomy, characterisation and responses to environmental factors with respect to their oil production enzymes. The information produced from this study will prove to be vital for both industrial and scientific investigations in the future.

The combination of plant-expressed cellobiohydrolase and low dosages of cellulases for the hydrolysis of sugar cane bagasse

Background The expression of biomass-degrading enzymes (such as cellobiohydrolases) in transgenic plants has the potential to reduce the costs of biomass saccharification by providing a source of enzymes to supplement commercial cellulase mixtures. Cellobiohydrolases are the main enzymes in commercial cellulase mixtures. In the present study, a cellobiohydrolase was expressed in transgenic corn stover leaf and assessed as an additive for two commercial cellulase mixtures for the saccharification of pretreated sugar cane bagasse obtained by different processes. Results Recombinant cellobiohydrolase in the senescent leaves of transgenic corn was extracted using a simple buffer with no concentration step. The extract significantly enhanced the performance of Celluclast 1.5 L (a commercial cellulase mixture) by up to fourfold on sugar cane bagasse pretreated at the pilot scale using a dilute sulfuric acid steam explosion process compared to the commercial cellulase mixture on its own. Also, the extracts were able to enhance the performance of Cellic CTec2 (a commercial cellulase mixture) up to fourfold on a range of residues from sugar cane bagasse pretreated at the laboratory (using acidified ethylene carbonate/ethylene glycol, 1-butyl-3-methylimidazolium chloride, and ball-milling) and pilot (dilute sodium hydroxide and glycerol/hydrochloric acid steam explosion) scales. We have demonstrated using tap water as a solvent (under conditions that mimic an industrial process) extraction of about 90% recombinant cellobiohydrolase from senescent, transgenic corn stover leaf that had minimal tissue disruption. Conclusions The accumulation of recombinant cellobiohydrolase in senescent, transgenic corn stover leaf is a viable strategy to reduce the saccharification cost associated with the production of fermentable sugars from pretreated biomass. We envisage an industrial-scale process in which transgenic plants provide both fibre and biomass-degrading enzymes for pretreatment and enzymatic hydrolysis, respectively.

Calcium phosphate flocs and the clarification of sugar cane juice from whole of crop harvesting

Sugar cane biomass is one of the most viable feedstocks for the production of renewable fuels and chemicals. Therefore, processing the whole of crop (WC) (i.e., stalk and trash, instead of stalk only) will increase the amount of available biomass for this purpose. However, effective clarification of juice expressed from WC for raw sugar manufacture is a major challenge because of the amounts and types of non-sucrose impurities (e.g., polysaccharides, inorganics, proteins, etc.) present. Calcium phosphate flocs are important during sugar cane juice clarification because they are responsible for the removal of impurities. Therefore, to gain a better understanding of the role of calcium phosphate flocs during the juice clarification process,the effects of impurities on the physicochemical properties of calcium phosphate flocs were examined using small-angle laser light scattering technique, attenuated total reflectance Fourier transformed infrared spectroscopy, and X-ray powder diffraction. Results on synthetic sugar juice solutions showed that the presence of SiO2 and Na+ ions affected floc size and floc structure. Starch and phosphate ions did not affect the floc structure; however, the former reduced the floc size, whereas the latter increased the floc size. The study revealed that high levels of Na+ ions would negatively affect the clarification process the most, as they would reduce the amount of suspended particles trapped by the flocs. A complementary study on prepared WC juice using cold and cold/intermediate liming techniques was conducted. The study demonstrated that, in comparison to the one-stage (i.e., conventional) clarification process, a two-stage clarification process using cold liming removed more polysaccharides (≤19%),proteins (≤82%), phosphorus (≤53%), and SiO2 (≤23%) in WC juice but increased Ca2+ (≤136%) and sulfur (≤200%)

Development of molecular tools for the improvement of transgene expression in sugar cane

Biotechnology has the potential to improve sugar cane, one of the world's major crops for food and fuel. This research describes the detailed characterisation of introns and their potential for enhancing transgene expression in sugar cane via intron-mediated enhancement (IME). IME is a phenomenon whereby an intron enhances gene expression from a promoter. Current knowledge on the mechanism of IME or its potential for enhancing gene expression in sugar cane is limited. A better understanding of the factors responsible for IME will help develop new molecular tools that facilitate high levels of constitutive and tissue-specific gene expression in this crop.

Spatially based data delivery and recording sys-Aust Sugar Cane industry

Establish an internet platform where spatially referenced data can be viewed, entered and stored.

Improved sugar-cane farming systems

This project - Improved Sugarcane Farming Systems (BSS286) - was designed to build on the outcomes of phase 1 and 2 or the Sugar Yield Decline Joint Venture (STDJV). Thus its main focus was on issues that had been identified in the SYDJV that were not fully researched in the earlier programs and/or required further development.

Life-history of Neurospora intermedia in a sugar cane field

The life-history of Neurospora in nature has remained largely unknown. The present study attempts to remedy this. The following conclusions are based on observation of Neurospora on fire-scorched sugar cane in agricultural fields, and reconstruction experiments using a colour mutant to inoculate sugar cane burned in the laboratory. The fungus persists in soil as heat-resistant dormant ascospores. These are activated by a chemical(s) released into soil from the burnt substrate. The chief diffusible activator of ascospores is furfural and the germinating ascospores infect the scorched substrate. An invasive mycelium grows progressively upwards inside the juicy sugar cane and produces copious macroconidia externally through fire-induced openings formed in the plant tissue, or by the mechanical rupturing of the plant epidermal tissue by the mass of mycelium. The loose conidia are dispersed by wind and/or foraged by microfauna. It is suggested that the constant production of macroconidia, and their ready dispersal, serve a physiological role: to drain the substrate of minerals and soluble sugars, thereby creating nutritional conditions which stimulate sexual reproduction by the fungus. Sexual reproduction in the sugar-depleted cellulosic substrate occurs after macroconidiation has ceased totally and is favoured by the humid conditions prevailing during the monsoon rains. Profuse microconidiophores and protoperithecia are produced simultaneously in the pockets below the loosened epidermal tissue. Presumably protoperithecia are fertilized by microconidia which are possibly transmitted by nematodes active in the dead plant tissue. Mature perithecia release ascospores in situ which are passively liberated in the soil by the disintegration of the plant material and are, apparently, distributed by rain or irrigation water.

Silicon, the silver bullet for mitigating biotic and abiotic stress, and improving grain quality, in rice?

Adequate silicon fertilization greatly boosts rice yield and mitigates biotic and abiotic stress, and improves grain quality through lowering the content of cadmium and inorganic arsenic. This review on silicon dynamics in rice considers recent advances in our understanding of the role of silicon in rice, and the challenges of maintaining adequate silicon fertility within rice paddy systems. Silicon is increasingly considered as an element required for optimal plant performance, particularly in rice. Plants can survive with very low silicon under laboratory/glasshouse conditions, but this is highly artificial and, thus, silicon can be considered as essential for proper plant function in its environment. Silicon is incorporated into structural components of rice cell walls were it increases cell and tissue rigidity in the plant. Structural silicon provides physical protection to plants against microbial infection and insect attack as well as reducing the quality of the tissue to the predating organisms. The abiotic benefits are due to silicon's effect on overall organ strength. This helps protect against lodging, drought stress, high temperature (through efficient maintenance of transpiration), and photosynthesis by protecting against high UV. Furthermore, silicon also protects the plant from saline stress and against a range of toxic metal stresses (arsenic, cadmium, chromium, copper, nickel and zinc). Added to this, silicon application decreases grain concentrations of various human carcinogens, in particular arsenic, antimony and cadmium. As rice is efficient at stripping bioavailable silicon from the soil, recycling of silicon rich rice straw biomass or addition of inorganic silicon fertilizer, primarily obtained from iron and steel slag, needs careful management. Silicon in the soil may be lost if the silicon-cycle, traditionally achieved via composting of rice straw and returning it to the land, is being broken. As composting of rice straw and incorporation of composted or non-composted straw back to land are resource intensive activities, these activities are declining due to population shifts from the countryside to cities. Processes that accelerate rice straw composting, therefore, need to be identified to aid more efficient use of this resource. In addition, rice genetics may help address declining available silicon in paddy soils: for example by selecting for characteristics during breeding that lead to an increased ability of roots to access recalcitrant silicon sources from soil and/or via selection for traits that aid the maintenance of a high silicon status in shoots. Recent advances in understanding the genetic regulation of silicon uptake and transport by rice plants will aid these goals.

Advances on the production of polyhydroxyalkanoates (PHA) by mixed microbial cultures from sugar cane molasses

Dissertação para obtenção do Grau de Mestre em Biotecnologia