Natural abundance of stable carbon and nitrogen isotopes in Cannabis sativa reflects growth conditions

Stable carbon and nitrogen isotope signatures (delta C-13 and delta N-15) of Cannabis sativa were assessed for their usefulness to trace seized Cannabis leaves to the country of origin and to source crops by determining how isotope signatures relate to plant growth conditions. The isotopic composition of Cannabis examined here covered nearly the entire range of values reported for terrestrial C-3 plants. The delta C-13 values of Cannabis from Australia, Papua New Guinea and Thailand ranged from -36 to -25 parts per thousand, and delta N-15 values ranged from -1.0 to 15.8 parts per thousand. The stable isotope content did not allow differentiation between Cannabis originating from the three countries, but delta C-13 values of plantation-grown Cannabis differed between well-watered plants (average delta C-13 of -30.0 parts per thousand) and plants that had received little irrigation (average delta C-13 of -26.4 parts per thousand). Cannabis grown under controlled conditions had delta C-13 values of -32.6 and -30.6 parts per thousand with high and low water supply, respectively. These results indicate that water availability determines leaf C-13 in plants grown under similar conditions of light, temperature and air humidity. The delta C-13 values also distinguished between indoor- and outdoor-grown Cannabis; indoor- grown plants had overall more negative delta C-13 values (average -31.8 parts per thousand) than outdoor-grown plants (average -27.9 parts per thousand). Contributing to the strong C-13-depletion of indoor- grown plants may be high relative humidity, poor ventilation and recycling of C-13-depleted respired CO2. Mineral fertilizers had mostly lower delta N-15 values (-0.2 to 2.2 parts per thousand) than manure-based fertilizers (7.6 to 22.7 parts per thousand). It was possible to link delta N-15 values of fertilizers associated with a crop site to soil and plant delta N-15 values. The strong relationship between soil, fertilizer, and plant delta N-15 suggests that Cannabis delta N-15 is determined by the isotopic composition of the nitrogen source. The distinct delta N-15 values measured in Cannabis crops make delta N-15 an excellent tool for matching seized Cannabis with a source crop. A case study is presented that demonstrates how delta C-13 and delta N-15 values can be used as a forensic tool.

Olfaction in the Queensland fruit fly, Bactrocera tryoni. II: Response spectra and temporal encoding characteristics of the carbon dioxide receptors

Single-unit electrophysiology was used to record the nerve impulses from the carbon dioxide receptors of female Queensland fruit flies, Bactrocera tryoni. The receptors responded to stimulation in a phasic-tonic manner and also had a period of inhibition of the nerve impulses after the end of stimulation, at high stimulus intensities. The cell responding to carbon dioxide was presented with a range of environmental odorants and found to respond to methyl butyrate and 2-butanone. The coding characteristics of the carbon dioxide cell and the ability to detect other odorants are discussed, with particular reference to the known behavior of the fly.

Power of the joint segregation analysis method for testing mixed major-gene and polygene inheritance models of quantitative traits

Understanding the genetic architecture of quantitative traits can greatly assist the design of strategies for their manipulation in plant-breeding programs. For a number of traits, genetic variation can be the result of segregation of a few major genes and many polygenes (minor genes). The joint segregation analysis (JSA) is a maximum-likelihood approach for fitting segregation models through the simultaneous use of phenotypic information from multiple generations. Our objective in this paper was to use computer simulation to quantify the power of the JSA method for testing the mixed-inheritance model for quantitative traits when it was applied to the six basic generations: both parents (P-1 and P-2), F-1, F-2, and both backcross generations (B-1 and B-2) derived from crossing the F-1 to each parent. A total of 1968 genetic model-experiment scenarios were considered in the simulation study to quantify the power of the method. Factors that interacted to influence the power of the JSA method to correctly detect genetic models were: (1) whether there were one or two major genes in combination with polygenes, (2) the heritability of the major genes and polygenes, (3) the level of dispersion of the major genes and polygenes between the two parents, and (4) the number of individuals examined in each generation (population size). The greatest levels of power were observed for the genetic models defined with simple inheritance; e.g., the power was greater than 90% for the one major gene model, regardless of the population size and major-gene heritability. Lower levels of power were observed for the genetic models with complex inheritance (major genes and polygenes), low heritability, small population sizes and a large dispersion of favourable genes among the two parents; e.g., the power was less than 5% for the two major-gene model with a heritability value of 0.3 and population sizes of 100 individuals. The JSA methodology was then applied to a previously studied sorghum data-set to investigate the genetic control of the putative drought resistance-trait osmotic adjustment in three crosses. The previous study concluded that there were two major genes segregating for osmotic adjustment in the three crosses. Application of the JSA method resulted in a change in the proposed genetic model. The presence of the two major genes was confirmed with the addition of an unspecified number of polygenes.

Breeding for low input conditions and consequences for participatory plant breeding: Examples from tropical maize and wheat

Participatory plant breeding (PPB) has been suggested as an effective alternative to formal plant breeding (FPB) as a breeding strategy for achieving productivity gains under low input conditions. With genetic progress through PPB and FPB being determined by the same genetic variables, the likelihood of success of PPB approaches applied in low input target conditions was analyzed using two case studies from FPB that have resulted in significant productivity gains under low input conditions: (1) breeding tropical maize for low input conditions by CIMMYT, and (2) breeding of spring wheat for the highly variable low input rainfed farming systems in Australia. In both cases, genetic improvement was an outcome of long-term investment in a sustained research effort aimed at understanding the detail of the important environmental constraints to productivity and the plant requirements for improved adaptation to the identified constraints, followed up by the design and continued evaluation of efficient breeding strategies. The breeding strategies used differed between the two case studies but were consistent in their attention to the key determinants of response to selection: (1) ensuring adequate sources of genetic variation and high selection pressures for the important traits at all stages of the breeding program, (2) use of experimental procedures to achieve high levels of heritability in the breeding trials, and (3) testing strategies that achieved a high genetic correlation between performance of germplasm in the breeding trials and under on-farm conditions. The implications of the outcomes from these FPB case studies for realizing the positive motivations for adopting PPB strategies are discussed with particular reference for low input target environment conditions.

Diversity of Phytophthora clandestina isolated from subterranean clover in southern australia: Analysis of virulence and RAPD profiles

The virulence spectrum of 112 isolates of Phytophthora clandestina collected from 56 sites in four subterranean clover-growing states in southern Australia was determined using differential cultivars of subterranean clover. Five races were detected, with race 0 in all states except New South Wales, race 1 in all states, race 2 only in Victoria, race 3 only in New South Wales, and race 4 in Victoria and Western Australia. The level of genotypic diversity among the different P. clandestina populations was investigated using five RAPD primers. Among 30 bands amplified, only two were polymorphic. This enabled identification of four multilocus RAPD genotypes. Three of the four genotypes occurred in all four states. Races 2 and 3 occurred with RAPD genotypes 1 and 2 only whereas races 0 and 1 occurred in all four multilocus RAPD genotypes. These results indicate that the pathogenicity spectrum of P. clandestina can change rapidly.

Lucerne biology and genetic improvement - an analysis of past activities and future goals in Australia

Breeding methodologies for cultivated lucerne (Medicago sativa L.), an autotetraploid, have changed little over the last 50 years, with reliance on polycross methods and recurrent phenotypic selection. There has been, however, an increase in our understanding of lucerne biology, in particular the genetic relationships between members of the M. sativa complex, as deduced by DNA analysis. Also, the differences in breeding behaviour and vigour of diploids versus autotetraploids, and the underlying genetic causes, are discussed in relation to lucerne improvement. Medicago falcata, a member of the M. sativa complex, has contributed substantially to lucerne improvement in North America, and its diverse genetics would appear to have been under-utilised in Australian programs over the last two decades, despite the reduced need for tolerance to freezing injury in Australian environments. Breeding of lucerne in Australia only commenced on a large scale in 1977, driven by an urgent need to introgress aphid resistance into adapted backgrounds. The release in the early 1980s of lucernes with multiple pest and disease resistance (aphids, Phytophthora, Colletotrichum) had a significant effect on increasing lucerne productivity and persistence in eastern Australia, with yield increases under high disease pressure of up to 300% being recorded over the predominant Australian cultivar, up to 1977, Hunter River. Since that period, irrigated lucerne yields have plateaued, highlighting the need to identify breeding objectives, technologies, and the germplasm that will create new opportunities for increasing performance. This review discusses major goals for lucerne improvement programs in Australia, and provides indications of the germplasm sources and technologies that are likely to deliver the desired outcomes.

Differentiation of peanut seedborne potyviruses and curcumoviruses by RT-PCR

Seedborne peanut viruses pose important constraints to peanut production and safe movement of germ plasm. They also pose a risk of accidental introduction into previously disease-free regions. We have developed reverse transcription-polymerase chain reaction (RT-PCR) assays based on identical cycling parameters which identified peanut stripe, Peanut mottle, Peanut stunt, and Cucumber mosaic viruses through production of specific DNA fragments of 234 bp, 327 bp, 390 bp, and 133 bp, respectively. Assay sensitivity in the picogram range was achieved. The two potyviruses and two cucumoviruses could be differentiated using duplex RT-PCR assays. These assays should be useful for testing peanut leaves or seeds for virus identification in epidemiological studies, seed testing or in post-entry quarantine.

Characterisation of Leucaena condensed tannins by size and protein precipitation capacity

The ability of differently sized condensed tannins from the genus Leucaena, a fodder tree-legume, to bind protein at different pH values was evaluated to characterise their potential biological effects. Two factors affecting the ability of condensed tannin to bind protein, its major biological activity, have been purported to be the condensed tannin size and the pH of the reaction environment. To test these hypotheses, the protein-precipitating capacities of condensed tannin extracted from four Leucaena genotypes, L leucocephala (UHK636), L pallida (CQ3439), L trichandra (CP146568) and L collinsii (OFI52/88), were assessed. Condensed tannin from L leucocephala had approximately 50% of the ability to precipitate protein on a gg(-1) basis than L pallida or L trichandra, while L collinsii gave no measurable ability to precipitate protein (reaction environment pH 5.0). Increasing or decreasing the pH of the reaction solution away from pH 5.0 (approximately the isoelectric point of the protein) reduced the ability of condensed tannin from all the species to precipitate protein, the decrease being higher at pH 2.5 than at pH 7.5. Condensed tannins from each Leucaena species were also separated by size exclusion chromatography, and the fractions examined for protein-precipitating capacity. In general, it was found that the larger-sized condensed tannin of the accessions L pallida and L trichandra could precipitate more protein than the smaller-sized condensed tannin. This pattern was not found for L leucocephala. (C) 2001 Society of Chemical Industry.

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the beta -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.

Significance of gaseous nitrogen loss from a tropical dairy pasture fertilised with urea

This paper reports a study in the wet tropics of Queensland on the fate of urea applied to a dairy pasture in the absence of grazing animals. A nitrogen balance was conducted in cylindrical plots with N-15-labelled urea, and ammonia volatilisation was determined using a mass balance micrometeorological method. The pasture plants took up 42% of the applied nitrogen in the 98 days between fertiliser application and harvest. At harvest 18% of the applied nitrogen was found in the soil, and 40% was lost from the plant-soil system. The micrometeorological study showed that 20% of the unrecovered nitrogen was lost by ammonia volatilisation. As there was no evidence for leaching or runoff losses it was concluded that the remaining 20% of the applied nitrogen was lost by denitrification. It is evident from these results that fertiliser nitrogen is not being used efficiently on dairy pastures, and that practices need to be changed to conserve fertiliser nitrogen and reduce contamination of the environment.

Traffic and residue cover effects on infiltration

Wheel traffic can lead to compaction and degradation of soil physical properties. This study, as part of a study of controlled traffic farming, assessed the impact of compaction from wheel traffic on soil that had not been trafficked for 5 years. A tractor of 40 kN rear axle weight was used to apply traffic at varying wheelslip on a clay soil with varying residue cover to simulate effects of traffic typical of grain production operations in the northern Australian grain belt. A rainfall simulator was used to determine infiltration characteristics. Wheel traffic significantly reduced time to ponding, steady infiltration rate, and total infiltration compared with non-wheeled soil, with or without residue cover. Non-wheeled soil had 4-5 times greater steady infiltration rate than wheeled soil, irrespective of residue cover. Wheelslip greater than 10% further reduced steady infiltration rate and total infiltration compared with that measured for self-propulsion wheeling (3% wheelslip) under residue-protected conditions. Where there was no compaction from wheel traffic, residue cover had a greater effect on infiltration capacity, with steady infiltration rate increasing proportionally with residue cover (R-2 = 0.98). Residue cover, however, had much less effect on infiltration when wheeling was imposed. These results demonstrated that the infiltration rate for the non-wheeled soil under a controlled traffic zero-till system was similar to that of virgin soil. However, when the soil was wheeled by a medium tractor wheel, infiltration rate was reduced to that of long-term cropped soil. These results suggest that wheel traffic, rather than tillage and cropping, might be the major factor governing infiltration. The exclusion of wheel traffic under a controlled traffic farming system, combined with conservation tillage, provides a way to enhance the sustainability of cropping this soil for improved infiltration, increased plant-available water, and reduced runoff-driven soil erosion.

Tillage and traffic effects on runoff

Traffic and tillage effects on runoff and crop performance on a heavy clay soil were investigated over a period of 4 years. Tillage treatments and the cropping program were representative of broadacre grain production practice in northern Australia, and a split-plot design used to isolate traffic effects. Treatments subject to zero, minimum, and stubble mulch tillage each comprised pairs of 90-m 2 plots, from which runoff was recorded. A 3-m-wide controlled traffic system allowed one of each pair to be maintained as a non-wheeled plot, while the total surface area of the other received a single annual wheeling treatment from a working 100-kW tractor. Rainfall/runoff hydrographs demonstrate that wheeling produced a large and consistent increase in runoff, whereas tillage produced a smaller increase. Treatment effects were greater on dry soil, but were still maintained in large and intense rainfall events on wet soil. Mean annual runoff from wheeled plots was 63 mm (44%) greater than that from controlled traffic plots, whereas runoff from stubble mulch tillage plots was 38 mm (24%) greater than that from zero tillage plots. Traffic and tillage effects appeared to be cumulative, so the mean annual runoff from wheeled stubble mulch tilled plots, representing conventional cropping practice, was more than 100 mm greater than that from controlled traffic zero tilled plots, representing best practice. This increased infiltration was reflected in an increased yield of 16% compared with wheeled stubble mulch. Minimum tilled plots demonstrated a characteristic midway between that of zero and stubble mulch tillage. The results confirm that unnecessary energy dissipation in the soil during the traction process that normally accompanies tillage has a major negative effect on infiltration and crop productivity. Controlled traffic farming systems appear to be the only practicable solution to this problem.

An easy and reliable method for establishment and maintenance of leaf and root cell cultures of Arabidopsis thaliana

Cell suspension cultures are useful for a wide range of biochemical and physiological studies, yet their production can be technically demanding and often unreliable. Here we describe a protocol for producing Arabidopsis cell suspension cultures that is reliable and easy to use.