Assessment of transient gene expression in plant tissues using the green fluorescent protein as a reference

Four different promoters (35S and enhanced 35S of the cauliflower mosaic virus, polyubiquitin of maize and actin1 of rice) were compared in a transient assay using maize leaves and particle bombardment. A gene encoding the jellyfish green fluorescent protein (GFP) driven by the 358 promoter was used as an internal standard to monitor the effectiveness of each bombardment. Normalisation of the transient expression assay using the GFP reference significantly reduced the variability between separate bombardments and allowed for a rapid and accurate evaluation of different promoters in microprojectile-bombarded leaves.

A protocol for the fluorometric quantification of mGFP5-ER and sGFP(S65T) in transgenic plants

The Green Fluorescent Protein (GFP) from Aequorea victor-in has begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a non-destructive manner, whereas detection of enzyme-based reporters often requires destruction of the plant tissue. The use of GFP as a reporter enables transgenic plant tissues to be screened in vivo at any growth stage. Quantification of GFP in transgenic plant extracts will increase the utility of GFP as a reporter protein. We report herein the quantification of a mGFP5-ER Variant in tobacco leaf extracts by UV excitation and a sGFP(S65T) variant in sugarcane leaf and callus extracts by blue light excitation using the BioRad VersaFluor(TM) Fluorometer System or the Labsystems Fluoroskan Ascent FL equipped with a narrow band emission filter (510 +/- 5 nm). The GFP concentration in transgenic plant extracts was determined from a GFP-standard series prepared in untransformed plant extract with concentrations ranging from 0.1 to 4 mu g/ml of purified rGFP. Levels of sgfp(S65T) expression, driven by the maize ubiquitin promoter, in sugarcane calli and leaves ranged up to 0.525 mu g and 2.11 mu g sGFP(S65T) per mg of extractable protein respectively. In tobacco leaves the expression of mgfPS-ER, driven by the cauliflower mosaic virus (CaMV) 35S promoter, ranged up to 7.05 mu g mGFP5-ER per mg extractable protein.

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.

In vivo and in vitro toxicities of pithraj and neem against rice green leafhopper (Nephotettix cincticeps Uhler)

Seed extracts of Aphanamixis polystachya Wall et Parker (pithraj) and Azadirachta indica A. Juss (neem) were evaluated for their in vivo and in vitro toxicity to Nephotettix cincticeps Uhler (rice green leafhopper). Crude extracts from both plants showed toxicity to leafhopper. Among them, the methanol extract of pithraj (MCX) was most toxic and showed 95% mortality effects at 72 h after treatment (HAT), followed by neem (74%). When LD50's were compared, it was found that the neem extract possessed the highest toxicity (LD50 16.59 μg/insect) at 72 HAT. Both the pithraj (MCX) and neem extracts showed their enzyme inhibition effectiveness against rice green leafhopper. The highest inhibition rate (IR) was caused by neem (60%) at the concentration of 2.0 mg/ml, followed by MCX (47%). The lowest IR50 value (0.97 mg/ml) was observed in neem at 30 min.

Occurrence and elimination of cyanobacterial toxins in two Australian drinking water treatment plants

In Australian freshwaters, Anabaena circinalis, Microcystis spp. and Cylindrospermopsis raciborskii are the dominant toxic cyanobacteria. Many of these Surface waters are used as drinking water resources. Therefore, the National Health and Medical Research Council of Australia set a guideline for MC-LR toxicity equivalents of 1.3 mug/l drinking, water. However, due to lack of adequate data, no guideline values for paralytic shellfish poisons (PSPs) (e.g. saxitoxins) or cylindrospermopsin (CYN) have been set. In this spot check. the concentration of microcystins (MCs), PSPs and CYN were determined by ADDA-ELISA, cPPA, HPLC-DAD and/or HPLC-MS/MS, respectively, in two water treatment plants in Queensland/Australia and compared to phytoplankton data collected by Queensland Health, Brisbane. Depending on the predominant cyanobacterial species in a bloom, concentrations of up to 8.0, 17.0 and 1.3 mug/l were found for MCs, PSPs and CYN, respectively. However, only traces (< 1.0 mug/l) of these toxins were detected in final water (final product of the drinking water treatment plant) and tap water (household sample). Despite the low concentrations of toxins detected in drinking water, a further reduction of cyanobacterial toxins is recommended to guarantee public safety. (C) 2004 Elsevier Ltd. All rights reserved.

Host recognition by a polyphagous lepidopteran (Helicoverpa armigera): primary host plants, host produced volatiles and neurosensory stimulation

An important question in the host-finding behaviour of a polyphagous insect is whether the insect recognizes a suite or template of chemicals that are common to many plants? To answer this question, headspace volatiles of a subset of commonly used host plants (pigeon pea, tobacco, cotton and bean) and nonhost plants (lantana and oleander) of Helicoverpa armigera Hubner (Lepidoptera: Noctuidae) are screened by gas chromatography (GC) linked to a mated female H. armigera electroantennograph (EAG). In the present study, pigeon pea is postulated to be a primary host plant of the insect, for comparison of the EAG responses across the test plants. EAG responses for pigeon pea volatiles are also compared between females of different physiological status (virgin and mated females) and the sexes. Eight electrophysiologically active compounds in pigeon pea headspace are identified in relatively high concentrations using GC linked to mass spectrometry (GC-MS). These comprised three green leaf volatiles [(2E)-hexenal, (3Z)-hexenylacetate and (3Z)-hexenyl-2-methylbutyrate] and five monoterpenes (alpha-pinene,beta-myrcene, limonene, E-beta-ocimene and linalool). Other tested host plants have a smaller subset of these electrophysiologically active compounds and even the nonhost plants contain some of these compounds, all at relatively lower concentrations than pigeon pea. The physiological status or sex of the moths has no effect on the responses for these identified compounds. The present study demonstrates how some host plants can be primary targets for moths that are searching for hosts whereas the other host plants are incidental or secondary targets.

Phosphorus uptake from green manures and phosphate fertilizers applied in an acid tropical soil

Quantifying the relative contribution of different phosphorus (P) sources to P uptake can lead to greater understanding of the mechanisms that increase available P in integrated P management systems. The P-32-P-33 double isotope labeling technique was used to determine the relative contribution of green manures (GMs) and P fertilizers to P uptake by Setaria grass (Setaria sphacelata) grown in an amended tropical acid soil (Bungor series) in a glasshouse study. The amendments were factorial combinations of GMs (Calopogonium caeruleum , Gliricidia sepium and Imperata cylindrica) and P fertilizers [phosphate rocks (PRs) from North Carolina (NCPR), China (CPR) and Algeria (APR), and triple superphosphate (TSP)]. Dry matter yield, P uptake, and P utilization from the amendments were monitored at 4, 8, and 15 weeks after establishment (WAE). The GMs alone or in combination with P fertilizers contributed less than 5% to total P uptake in this soil, but total P uptake into Setaria plants in the GM treatments was three to four times that of the P fertilizers because the GMs mobilized more soil P. Also, the GMs markedly increased fertilizer P utilization in the combined treatments, from 3% to 39% with CPR, from 6-9% to 19-48% with reactive PRs, and from 6% to 37% with TSP in this soil. Both P GM and the other decomposition products were probably involved in reducing soil P-retention capacity. Mobilization of soil P was most likely the result of the action of the other decomposition products. These results demonstrate the high potential of integrating GMs and PRs for managing P in tropical soils and the importance of the soil P mobilization capacity of the organic components. Even the low-quality Imperata GM enhanced the effectiveness of the reactive APR more than fourfold.

Lesions caused by cardiovascular flukes (Digenea : Spirorchidae) in stranded green turtles (Chelonia mydas)

Evidence of infection with spirorchid flukes (Digenea: Spirorchidae) was sought at necropsy of 96 stranded green turtles, Chelonia mydas, that were examined during the course of a survey of marine turtle mortality in southeastern Queensland, Australia. Three species of spirorchid (Hapalotrema mehrai, H. postorchis, and Neospirorchis schistosomatoides) were identified. Severe disease due to spirorchid fluke infection (spirorchidiasis) was implicated as the principal cause of mortality in 10 turtles (10%), and appeared to be one of multiple severe problems in an additional 29 turtles (30%). Although flukes were observed in only 45% of stranded C. mydas in this study, presumed spirorchid fluke infection was diagnosed in an additional 53% of turtles, based principally on characteristic necropsy lesions and to a lesser extent on the histopathological detection of spirorchid eggs. Characteristic necropsy lesions included miliary spirorchid egg granulomas, which were observed most readily on serosal surfaces, particularly of the small intestine. Cardiovascular lesions included mural endocarditis, arteritis, and thrombosis, frequently accompanied by aneurysm formation. Resolution of thrombi was observed to occur via a combination of granuloma formation about indigestible components (spirorchid fluke egg shells) and exteriorization through the vessel wall, which resulted in granulomatous nodules on the adventitial surface. Septic aortic thrombosis complicated by disseminated bacterial infection, observed in five turtles, was recorded for the first time. Egg granulomas were ubiquitous in turtle tissues throughout this study. Although they generally appeared to be mild or incidental lesions, they were occasionally associated with severe multifocal granulomatous pneumonia or meningitis.

The proportion of individual lucerne plants resistant to Phytophthora medicaginis and Colletotrichum trifolii in Australian cultivars

Phytophthora root rot (Phytophthora medicaginis) and colletotrichum crown rot (Colletotrichum trifoli) are the 2 most serious pathogens of lucerne in eastern Australia. Work reported in this paper shows that in glasshouse tests of the 11 most commonly grown Australian lucerne cultivars, the proportion of individual plants with resistance to both pathogens ranges from 0 (Hunter River and Aurora) through to a maximum of 19.8% (Sequel HR). Within 9 of the cultivars, the proportion of individual plants resistant to the 2 pathogens was <7%. Since these 2 diseases are known to cause serious losses in eastern Australia, the results indicate further improvement in lucerne production can be obtained by increasing the proportion of individual plants in a cultivar resistant to both pathogens. This would be best achieved by identifying dominant sources of resistance and incorporating this into on-going lucerne breeding programs.

Hapalotrema (Digenea : Spirorchidae) in the green turtle (Chelonia mydas) in Australia

Hapalotrema mehrai Rao, 1976 and Hapalotrema postorchis Rao, 1976 (Digenea: Spirorchidae) are redescribed from the heart and pulmonary arteries of the green turtle, Chelonia mydas, from Moreton Bay in south-eastern Queensland. Hapalotrema pambanensis Gupta and Mehrotra, 1981 from C. mydas in India is made a synonym of H. mehrai. Hapalotrema dorsopora Dailey, Fast and Balazs, 1993 from C. mydas from Hawaii was described with a dorsally opening uterine pore, but this is found to be the opening of Laurer's canal; therefore H. dorsopora is also made a synonym of H. mehrai. In addition to differences in the numbers of testes and general dimensions, H. mehrai and H. postorchis differ in the development of Laurer's canal and in the absence of a canalicular seminal receptacle in H. postorchis.

Photoinhibition in differently coloured juvenile leaves of Syzygium species

Photoinhibition, as measured by the dark-adapted chlorophyll a fluorescence ratio F-v/F-m, was assessed in Syzygium moorei, a species with dark green juvenile leaves, Syzygium corynanthum, which has light green juvenile leaves, and two species with pink-red juvenile leaves (Syzygium wilsonii and Syzygium luehmannii). All plants were glasshouse-grown (maximum PPFD 1500 mu mol m(-2) s(-1)) under optimum nutrition and water. When measured at midday, dark-adapted F-v/F-m ratios of juvenile leaves gradually increased in art species as percentage of full leaf expansion (% FLE) increased. Fluorescence measurement 3 h after sunset or pre-dawn also showed a developmental effect on F-v/F-m, with juvenile leaves of S, luehmannii and S. wilsonii showing much lower F-v/F-m at all stages of development. Dark-adapted F-v/F-m values in both juvenile and mature leaves generally never exceeded 0.8 at any stage in any of the species. Courses of F-v/F-m on sunny days showed greater diurnal photoinhibition in green juvenile (c, 50% FLE) leaves of S, moorei (24%) and S, corynanthum (36%) than in mature leaves of the previous flush in these species (<10%), Diurnal photoinhibition was statistically similar (18-24%) in pink-red juvenile and green mature leaves of S, luehmannii and S, wilsonii. Re-positioning juvenile leaves of S, wilsonii horizontally increased diurnal photoinhibition, Exposure of leaves to a standard mild photoinhibitory right treatment (30 min at 1000 mu mol m(-2) s(-1)) showed that juvenile leaves of air species had a lower percentage of high energy state quenching (qE) and a higher percentage of photoinhibitory quenching (ql) than mature leaves.