Plant Breeder's Rights: An Australian commentary December 2004

The aim of this manual is to provide a practical guide to the Plant Breeder's Rights Act 1994 (Cth). It is a resource of information about the legislation, its administration, and its operation. This commentary is intended to assist plant breeders, scientific researchers, and business managers who want to make a more effective use of the Plant Breeder's Rights Act 1994 (Cth) in the management and commercialisation of their intellectual property rights. It is also designed to enhance the understanding of the legislation among lawyers, patent attorneys, qualified persons, and policy-makers.

The new conquistadors: Patent law and expressed sequence tags

It's akin to the old Spanish, English and Portuguese explorers. They would take their boats until they found some edge of land, then they would go up and plant the flag of their king or queen. They didn't know what they'd discovered; how big it is, where it goes to - but they would claim it anyway. David Korn of the Association of American Medical Colleges This article analyses recent litigation over patent law and expressed sequence tags (ESTs). In the case of In re Fisher, the United States Court of Appeals for the Federal Circuit engaged in judicial consideration of the revised utility guidelines of the United States Patent and Trademark Office (USPTO). In this matter, the agricultural biotechnology company Monsanto sought to patent ESTs in maize plants. A patent examiner and the Board of Patent Appeals and Interferences had doubted whether the patent application was useful. Monsanto appealed against the rulings of the USPTO. A number of amicus curiae intervened in the matter in support of the USPTO - including Genentech, Affymetrix, Dow AgroSciences, Eli Lilly, the National Academy of Sciences, and the Association of American Medical Colleges. The majority of the Court of Appeals for the Federal Circuit supported the position of the USPTO, and rejected the patent application on the grounds of utility. The split decision highlighted institutional tensions over the appropriate thresholds for patent criteria - such as novelty, non-obviousness, and utility. The litigation raised larger questions about the definition of research tools, the incremental nature of scientific progress, and the role of patent law in innovation policy. The decision of In re Fisher will have significant ramifications for gene patents, in the wake of the human genome project. Arguably, the USPTO utility guidelines need to be reinforced by a tougher application of the standards of novelty and non-obviousness in respect of gene patents.

Combining transcriptome assemblies from multiple de novo assemblers in the allo-tetraploid plant Nicotiana benthamiana

Background Nicotiana benthamiana is an allo-tetraploid plant, which can be challenging for de novo transcriptome assemblies due to homeologous and duplicated gene copies. Transcripts generated from such genes can be distinct yet highly similar in sequence, with markedly differing expression levels. This can lead to unassembled, partially assembled or mis-assembled contigs. Due to the different properties of de novo assemblers, no one assembler with any one given parameter space can re-assemble all possible transcripts from a transcriptome. Results In an effort to maximise the diversity and completeness of de novo assembled transcripts, we utilised four de novo transcriptome assemblers, TransAbyss, Trinity, SOAPdenovo-Trans, and Oases, using a range of k-mer sizes and different input RNA-seq read counts. We complemented the parameter space biologically by using RNA from 10 plant tissues. We then combined the output of all assemblies into a large super-set of sequences. Using a method from the EvidentialGene pipeline, the combined assembly was reduced from 9.9 million de novo assembled transcripts to about 235,000 of which about 50,000 were classified as primary. Metrics such as average bit-scores, feature response curves and the ability to distinguish paralogous or homeologous transcripts, indicated that the EvidentialGene processed assembly was of high quality. Of 35 RNA silencing gene transcripts, 34 were identified as assembled to full length, whereas in a previous assembly using only one assembler, 9 of these were partially assembled. Conclusions To achieve a high quality transcriptome, it is advantageous to implement and combine the output from as many different de novo assemblers as possible. We have in essence taking the ‘best’ output from each assembler while minimising sequence redundancy. We have also shown that simultaneous assessment of a variety of metrics, not just focused on contig length, is necessary to gauge the quality of assemblies.

Development of multi-rotor localised surveillance using multi-spectral sensors for plant biosecurity

This report describes a proof of concept for multi-rotor localised surveillance using a multi-spectral sensor for plant biosecurity applications. A literature review was conducted on previous applications using airborne multispectral imaging for plant biosecurity purposes. A ready built platform was purchased and modified in order to fit and provide suitable clearance for a Tetracam Mini-MCA multispectral camera. The appropriate risk management documents were developed allowing the platform and the multi-spectral camera to be tested extensively. However, due to technical difficulties with the platform the Mini- MCA was not mounted to the platform. Once a suitable platform is developed, future extensions can be conducted into the suitability of the Mini-MCA for airborne surveillance of Australian crops.

Cytotoxic C20 diterpenoid alkaloids from the Australian endemic rainforest plant Anopterus macleayanus

In order to identify new anticancer compounds from nature, a prefractionated library derived from Australian endemic plants was generated and screened against the prostate cancer cell line LNCaP using a metabolic assay. Fractions from the seeds, leaves, and wood of Anopterus macleayanus showed cytotoxic activity and were subsequently investigated using a combination of bioassay-guided fractionation and mass-directed isolation. This led to the identification of four new diterpenoid alkaloids, 6α-acetoxyanopterine (1), 4′-hydroxy-6α-acetoxyanopterine (2), 4′-hydroxyanopterine (3), and 11α-benzoylanopterine (4), along with four known compounds, anopterine (5), 7β-hydroxyanopterine (6), 7β,4′-dihydroxyanopterine (7), and 7β-hydroxy-11α-benzoylanopterine (8); all compounds were purified as their trifluoroacetate salt. The chemical structures of 1–8 were elucidated after analysis of 1D/2D NMR and MS data. Compounds 1–8 were evaluated for cytotoxic activity against a panel of human prostate cancer cells (LNCaP, C4-2B, and DuCaP) and nonmalignant cell lines (BPH-1 and WPMY-1), using a live-cell imaging system and a metabolic assay. All compounds showed potent cytotoxicity with IC50 values of <400 nM; compound 1 was the most active natural product from this series, with an IC50 value of 3.1 nM toward the LNCaP cell line. The live-cell imaging assay on 1–8 showed a concentration- and time-dependent effect on the cell morphology and proliferation of LNCaP cells.

A multiscale study on the structural and mechanical properties of the luffa sponge from Luffa cylindrica plant

Cellular materials that are often observed in biological systems exhibit excellent mechanical properties at remarkably low densities. Luffa sponge is one of such materials with a complex interconnecting porous structure. In this paper, we studied the relationship between its structural and mechanical properties at different levels of its hierarchical organization from a single fiber to a segment of whole sponge. The tensile mechanical behaviors of three single fibers were examined by an Instron testing machine and the ultrastructure of a fractured single fiber was observed in a scanning electronic microscope. Moreover, the compressive mechanical behaviors of the foam-like blocks from different locations of the sponge were examined. The difference of the compressive stress-strain responses of four sets of segmental samples were also compared. The result shows that the single fiber is a porous composite material mainly consisting of cellulose fibrils and lignin/hemicellulose matrix, and its Young's modulus and strength are comparable to wood. The mechanical behavior of the block samples from the hoop wall is superior to that from the core part. Furthermore, it shows that the influence of the inner surface on the mechanical property of the segmental sample is stronger than that of the core part; in particular, the former's Young's modulus, strength and strain energy absorbed are about 1.6 times higher. The present work can improve our understanding of the structure-function relationship of the natural material, which may inspire fabrication of new biomimetic foams with desirable mechanical efficiency for further applications in anti-crushing devices and super-light sandwich panels.

Plant height affects Fusarium crown rot severity in wheat

Effects of plant height on Fusarium crown rot (FCR) disease severity were investigated using 12 pairs of near-isogenic lines (NILs) for six different reduced height (Rht) genes in wheat. The dwarf isolines all gave better FCR resistance when compared with their respective tall counterparts, although the Rht genes involved in these NILs are located on several different chromosomes. Treating plants with exogenous gibberellin increased FCR severity as well as seedling lengths in all of the isolines tested. Analysis of the expression of several defense genes with known correlation with resistance to FCR pathogens between the Rht isolines following FCR inoculation indicated that the better resistance of the dwarf isolines was not due to enhanced defense gene induction. These results suggested that the difference in FCR severity between the tall and dwarf isolines is likely due to their height difference per se or to some physiological and structural consequences of reduced height. Thus, caution should be taken when considering to exploit any FCR locus located near a height gene.

Land grabs, bio-piracy and the inversion of justice in Colombia

The possibility of commercially exploiting plant, animal and human genetic resources unlocked by biotechnology has given rise to a wide range of cultural, environmental, ethical and economic conflicts. While supporters describe this activity as bioprospecting, critics refer to it as biopiracy. According to this latter view, international legal agreements and treaties have disregarded opposition and legalized the possibility of appropriating genetic resources and their derivative products through the use of patents. The legal framework that permits the appropriation of natural genetic products in Colombia also criminalizes aspects of traditional ways of life and enables a legally approved but socially harmful land-grabbing process. The article describes these processes and impact in terms of the inversion of justice and the erosion of environmental sustainability.

Organosolv pretreatment of plant biomass for enhanced enzymatic saccharification

The combination of dwindling petroleum reserves and population growth make the development of renewable energy and chemical resources more pressing than ever before. Plant biomass is the most abundant renewable source for energy and chemicals. Enzymes can selectively convert the polysaccharides in plant biomass into simple sugars which can then be upgraded to liquid fuels and platform chemicals using biological and/or chemical processes. Pretreatment is essential for efficient enzymatic saccharification of plant biomass and this article provides an overview of how organic solvent (organosolv) pretreatments affect the structure and chemistry of plant biomass, and how these changes enhance enzymatic saccharification. A comparison between organosolv pretreatments utilizing broadly different classes of solvents (i.e., low boiling point, high boiling point, and biphasic) is presented, with a focus on solvent recovery and formation of by-products. The reaction mechanisms that give rise to these by-products are investigated and strategies to minimize by-product formation are suggested. Finally, process simulations of organosolv pretreatments are compared and contrasted, and discussed in the context of an industrial-scale plant biomass to fermentable sugar process.

Adventures in plant anatomy – not just a pigment of my imagination

An overview of teaching and research activities in the area of plant anatomy at QUT is provided. The current status of teaching of technical skills in plant anatomy is discussed briefly. Examples of applications of plant anatomy to a diverse range of fields are provided, including the crossover between art and science.

Fabrication and characterization of plasma polymer thin films from monoterpene alcohols for applications in organic electronics and biotechnology

After more than twenty years of basic and applied research, the use of nanotechnology in the design and manufacture of nanoscale materials is rapidly increasing, particularly in commercial applications that span from electronics across renewable energy areas, and biomedical devices. Novel polymers are attracting significant attention for they promise to provide a low−cost high−performance alternative to existing materials. Furthermore, these polymers have the potential to overcome limitations imposed by currently available materials thus enabling the development of new technologies and applications that are currently beyond our reach. This work focuses on the development of a range of new low−cost environmentally−friendly polymer materials for applications in areas of organic (flexible) electronics, optics, and biomaterials. The choice of the monomer reflects the environmentally−conscious focus of this project. Terpinen−4−ol is a major constituent of Australian grown Melaleuca alternifolia (tea tree) oil, attributed with the oil's antimicrobial and anti−inflammatory properties. Plasma polymerisation was chosen as a deposition technique for it requires minimal use of harmful chemicals and produces no hazardous by−products. Polymer thin films were fabricated under varied process conditions to attain materials with distinct physico−chemical, optoelectrical, biological and degradation characteristics. The resultant materials, named polyterpenol, were extensively characterised using a number of well−accepted and novel techniques, and their fundamental properties were defined. Polyterpenol films were demonstrated to be hydrocarbon rich, with variable content of oxygen moieties, primarily in the form of hydroxyl and carboxyl functionalities. The level of preservation of original monomer functionality was shown to be strongly dependent on the deposition energy, with higher applied power increasing the molecular fragmentation and substrate temperature. Polyterpenol water contact angle contact angle increased from 62.7° for the 10 W samples to 76.3° for the films deposited at 100 W. Polymers were determined to resist solubilisation by water, due to the extensive intermolecular and intramolecular hydrogen bonds present, and other solvents commonly employed in electronics and biomedical processing. Independent of deposition power, the surface topography of the polymers was shown to be smooth (Rq <0.5 nm), uniform and defect free. Hardness of polyterpenol coatings increased from 0.33 GPa for 10 W to 0.51 GPa for 100 W (at 500 μN load). Coatings deposited at higher input RF powers showed less mechanical deformation during nanoscratch testing, with no considerable damage, cracking or delamination observed. Independent of the substrate, the quality of film adhesion improved with RF power, suggesting these coatings are likely to be more stable and less susceptible to wear. Independent of fabrication conditions, polyterpenol thin films were optically transparent, with refractive index approximating that of glass. Refractive index increased slightly with deposition power, from 1.54 (10 W) to 1.56 (100 W) at 500 nm. The optical band gap values declined with increasing power, from 2.95 eV to 2.64 eV, placing the material within the range for semiconductors. Introduction of iodine impurity reduced the band gap of polyterpenol, from 2.8 eV to 1.64 eV, by extending the density of states more into the visible region of the electromagnetic spectrum. Doping decreased the transparency and increased the refractive index from 1.54 to 1.70 (at 500 nm). At optical frequencies, the real part of permittivity (k) was determined to be between 2.34 and 2.65, indicating a potential low-k material. These permittivity values were confirmed at microwave frequencies, where permittivity increased with input RF energy – from 2.32 to 2.53 (at 10 GHz ) and from 2.65 to 2.83 (at 20 GHz). At low frequencies, the dielectric constant was determined from current−voltage characteristics of Al−polyterpenol−Al devices. At frequencies below 100 kHz, the dielectric constant varied with RF power, from 3.86 to 4.42 at 1 kHz. For all samples, the resistivity was in order of 10⁸−10⁹ _m (at 6 V), confirming the insulating nature of polyterpenol material. In situ iodine doping was demonstrated to increase the conductivity of polyterpenol, from 5.05 × 10⁻⁸ S/cm to 1.20 × 10⁻⁶ S/cm (at 20 V). Exposed to ambient conditions over extended period of time, polyterpenol thin films were demonstrated to be optically, physically and chemically stable. The bulk of ageing occurred within first 150 h after deposition and was attributed to oxidation and volumetric relaxation. Thermal ageing studies indicated thermal stability increased for the films manufactured at higher RF powers, with degradation onset temperature associated with weight loss shifting from 150 ºC to 205 ºC for 10 W and 100 W polyterpenol, respectively. Annealing the films to 405 °C resulted in full dissociation of the polymer, with minimal residue. Given the outcomes of the fundamental characterisation, a number of potential applications for polyterpenol have been identified. Flexibility, tunable permittivity and loss tangent properties of polyterpenol suggest the material can be used as an insulating layer in plastic electronics. Implementation of polyterpenol as a surface modification of the gate insulator in pentacene-based Field Effect Transistor resulted in significant improvements, shifting the threshold voltage from + 20 V to –3 V, enhancing the effective mobility from 0.012 to 0.021 cm²/Vs, and improving the switching property of the device from 10⁷ to 10⁴. Polyterpenol was demonstrated to have a hole transport electron blocking property, with potential applications in many organic devices, such as organic light emitting diodes. Encapsulation of biomedical devices is also proposed, given that under favourable conditions, the original chemical and biological functionality of terpinen−4−ol molecule can be preserved. Films deposited at low RF power were shown to successfully prevent adhesion and retention of several important human pathogens, including P. aeruginosa, S. aureus, and S. epidermidis, whereas films deposited at higher RF power promoted bacterial cell adhesion and biofilm formation. Preliminary investigations into in vitro biocompatibility of polyterpenol demonstrated the coating to be non−toxic for several types of eukaryotic cells, including Balb/c mice macrophage and human monocyte type (HTP−1 non-adherent) cells. Applied to magnesium substrates, polyterpenol encapsulating layer significantly slowed down in vitro biodegradation of the metal, thus increasing the viability and growth of HTP−1 cells. Recently, applied to varied nanostructured titanium surfaces, polyterpenol thin films successfully reduced attachment, growth, and viability of P. aeruginosa and S. aureus.

The extremophile Nicotiana benthamiana has traded viral defence for early vigour

A single lineage of Nicotiana benthamiana is widely used as a model plant1 and has been instrumental in making revolutionary discoveries about RNA interference (RNAi), viral defence and vaccine production. It is peerless in its susceptibility to viruses and its amenability in transiently expressing transgenes2,3. These unparalleled characteristics have been associated both positively and negatively with a disruptive insertion in the RNA-dependent RNA polymerase 1 gene, Rdr14–6. For a plant so routinely used in research, the origin, diversity and evolution of the species, and the basis of its unusual abilities, have been relatively unexplored. Here, by comparison with wild accessions from across the spectrum of the species’ natural distribution, we show that the laboratory strain of N. benthamiana is an extremophile originating from a population that has retained a mutation in Rdr1 for ∼0.8 Myr and thereby traded its defence capacity for early vigour and survival in the extreme habitat of central Australia. Reconstituting Rdr1 activity in this isolate provided protection. Silencing the functional allele in a wild strain rendered it hypersusceptible and was associated with a doubling of seed size and enhanced early growth rate. These findings open the way to a deeper understanding of the delicate balance between protection and vigour.

Quarantine and surveillance strategies for plant pathogen detection and control

Incursions of plant pests and diseases pose serious threats to food security, agricultural productivity and the natural environment. One of the challenges in confidently delimiting and eradicating incursions is how to choose from an arsenal of surveillance and quarantine approaches in order to best control multiple dispersal pathways. Anthropogenic spread (propagules carried on humans or transported on produce or equipment) can be controlled with quarantine measures, which in turn can vary in intensity. In contrast, environmental spread processes are more difficult to control, but often have a temporal signal (e.g. seasonality) which can introduce both challenges and opportunities for surveillance and control. This leads to complex decisions regarding when, where and how to search. Recent modelling investigations of surveillance performance have optimised the output of simulation models, and found that a risk-weighted randomised search can perform close to optimally. However, exactly how quarantine and surveillance strategies should change to reflect different dispersal modes remains largely unaddressed. Here we develop a spatial simulation model of a plant fungal-pathogen incursion into an agricultural region, and its subsequent surveillance and control. We include structural differences in dispersal via the interplay of biological, environmental and anthropogenic connectivity between host sites (farms). Our objective was to gain broad insights into the relative roles played by different spread modes in propagating an invasion, and how incorporating knowledge of these spread risks may improve approaches to quarantine restrictions and surveillance. We find that broad heuristic rules for quarantine restrictions fail to contain the pathogen due to residual connectivity between sites, but surveillance measures enable early detection and successfully lead to suppression of the pathogen in all farms. Alternative surveillance strategies attain similar levels of performance by incorporating environmental or anthropogenic dispersal risk in the prioritisation of sites. Our model provides the basis to develop essential insights into the effectiveness of different surveillance and quarantine decisions for fungal pathogen control. Parameterised for authentic settings it will aid our understanding of how the extent and resolution of interventions should suitably reflect the spatial structure of dispersal processes.

The role of MicroRNAs in stress response in the resurrection plant Tripogon loliiformis

Research in this thesis focussed on the improvement of agricultural crops in increasing water use efficiency that impacts global crop productivity. The study identified key genetic regulatory mechanisms that the resurrection plant Tripogon loliiformis utilises to tolerate desiccation. Due to the conserved nature of the pathways involved, this information can be transferred for the enhancement of drought tolerance and water use efficiency in agricultural crops. Specifically this study used high throughput sequencing, microscopy and plant transformation to further the understanding of post-transcriptional regulatory mechanisms. It was shown that T. loliiformis uses microRNAs to regulate pro-survival autophagy pathways to tolerate desiccation.

Trehalose accumulation triggers autophagy during plant desiccation

Global climate change, increasingly erratic weather and a burgeoning global population are significant threats to the sustainability of future crop production. There is an urgent need for the development of robust measures that enable crops to withstand the uncertainty of climate change whilst still producing maximum yields. Resurrection plants possess the unique ability to withstand desiccation for prolonged periods, can be restored upon watering and represent great potential for the development of stress tolerant crops. Here, we describe the remarkable stress characteristics of Tripogon loliiformis, an uncharacterised resurrection grass and close relative of the economically important cereals, rice, sorghum, and maize. We show that T. loliiformis survives extreme environmental stress by implementing autophagy to prevent Programmed Cell Death. Notably, we identified a novel role for trehalose in the regulation of autophagy in T.loliiformis. Transcriptome, Gas Chromatography Mass Spectrometry, immunoblotting and confocal microscopy analyses directly linked the accumulation of trehalose with the onset of autophagy in dehydrating and desiccated T. loliiformis shoots. These results were supported in vitro with the observation of autophagosomes in trehalose treated T. loliiformis leaves; autophagosomes were not detected in untreated samples. Presumably, once induced, autophagy promotes desiccation tolerance in T.loliiformis , by removal of cellular toxins to suppress programmed cell death and the recycling of nutrients to delay the onset of senescence. These findings illustrate how resurrection plants manipulate sugar metabolism to promote desiccation tolerance and may provide candidate genes that are potentially useful for the development of stress tolerant crops.