Direct DNA transfer using electric discharge particle acceleration (ACCELL™ technology)

Direct DNA transfer methods based on particle bombardment have revolutionized plant genetic engineering. Major agronomic crops previously considered recalcitrant to gene transfer have been engineered using variations of this technology. In many cases variety-independent and efficient transformation methods have been developed enabling application of molecular biology techniques to crop improvement. The focus of this article is the development and performance of electric discharge particle bombardment (ACCELL™) technology. Unique advantages of this methodology compared to alternative propulsion technologies are discussed in terms of the range of species and genotypes that have been engineered, and the high transformation frequencies for major agronomic crops that enabled the technology to move from the R&D phase to commercialization. Creation of transgenic soybeans, cotton, and rice will be used as examples to illustrate the development of variety-independent and efficient gene transfer methods for most of the major agronomic crops. To our knowledge, no other gene transfer method based on particle bombardment has resulted in variety-independent and practical generation of large numbers of independently-derived crop plants. ACCELL™ technology is currently being utilized for the routine transfer of valuable genes into elite germplasm of soybean, cotton, bean, rice, corn, peanut and woody species.     

Adult emergence phenology in checkerspot butterflies: the effects of macroclimate,topoclimate, and population history

The prediction of adult emergence times in insect populations can be greatly complicated by microclimatic gradients, especially in circumstances where distributions of juveniles along those gradients vary from year to year. To investigate adult emergence patterns in topographically heterogeneous habitats, we built a model of postdiapause development of the Bay checkerspot butterfly, Euphydryas editha bayensis. The model uses slope-specific insolation as the rate-controlling variable, and accounts for both solar exposure of the habitat and cloud cover. Instar-specific larval mass gains per unit of insolation were determined from mark-recapture experiments. A small correction for daily low temperatures was used to calibrate the model to five years of field data on larval mass. The model predicted mean mass of 90% of larval samples within 4 clear days over a 70–120 day growing season. The magnitude of spatial variation in emergence times across habitat slopes is greater than annual variation in emergence times due to yearly weather conditions. Historical variation (yearly shifts in larval distributions across slopes) is an important determinant of mean population emergence dates. All of these factors need to be considered in understanding adult emergence phenology in this butterfly and in other insects inhabiting heterogeneous thermal environments. Such an understanding can be useful in managing insect populations for both pest control and conservation.     

Just how important are structural elements as habitat components? Indications from a declining lycaenid butterfly with priority conservation status

Recently, it has been suggested that habitats for insect herbivores have been too narrowly defined, often on the basis of larval hostplants; in particular, non-consumable resources (called utilities; structural elements) have been ignored. Here, the importance of utility resources for roosting and mate location has been examined in the silver-studded blue butterfly Plebejus argus (L.) (Lycaenidae) on the Great Ormes Head, North Wales, UK. The methods included using dedicated surveys and correspondence analysis applied to behavioural observations in relation to vegetation structure on a transect through a key patch for this metapopulation model species. A substantial and significant bias in roosting (97%) and mating (75%) is found to occur outside hostplant areas on shrubs and rank bunched grasses and forbs. Population density is higher in shrubby areas and shrubs are increasingly occupied during the afternoons and night, during the late flight season and in cloudy, cool and windy weather. These findings suggest that shrubs are a valuable habitat component for this butterfly, at least at this coastal location, and important for their conservation. As scrub growth is inimical to calcicolous grassland, population status will depend on a fine balance between shrub and hostplant cover dependent on grazing and browsing by the indigenous goat, sheep and rabbit populations as well as on controlled cutting and burning. Opportunities exist for increasing population size and distribution on the headland but this will need to be managed carefully. There are also implications for metapopulation dynamics studies; the status of shrubs neighbouring host plant areas switches from that of barriers to resources and refuges.     

Responses of embryogenic mango cultures and seedling bioassays to a partially purified phytotoxin produced by a mango leaf isolate of Colletotrichum gloeosporioides penz

Summary Colletotrichum gloeosporioides Penz., the causal agent of mango anthracnose, produces a phytotoxin in vitro. The partially purified phytotoxin, presumably colletotrichin, caused anthracnose-like symptoms on young mango leaves, was toxic to embryogenic suspension cultures of two mango cultivars, ‘Hindi’ and ‘Carabao,’ and inhibited in vitro seed germination of two nonhosts, lettuce and tobacco. There were linear relationships between concentration of the partially purified phytotoxin and mortality of mango embryogenic cultures. Embryogenic cultures grown in the presence of the partially purified phytotoxin showed significantly lower growth rates than the controls. Similarly, embryogenic cultures grown in the presence of 40% (vol/vol) fungal culture filtrate showed significantly lower growth rates than unchallenged controls. Medium containing 40% (vol/vol) Czapek-Dox fungal broth did not reduce growth of embryogenic cultures, indicating the production of phytotoxin in vitro. The results suggest that either fungal culture filtrate or purified phytotoxin can be used as in vitro selection agents to screen for resistance to this fungus.     

Effect of differential gastric evacuation and multispecies prey items on estimates of daily energy intake in juvenile chinook salmon

Synopsis The caloric density of stomach contents in juvenile chinook salmon,Oncorhynchus tshawytscha, was not affected by gastric evacuation, suggesting a constant caloric density of stomach contents during evacuation. Differences in the caloric density of prey consumed did affect caloric density of stomach contents over a 24-h period. Consumption of the amphipodCorophium sp. was associated with reduced caloric densities of stomach contents. During periods whenCorophium contributed more than 4% of the stomach contents, average caloric density declined from 5.56 to 5.33 kcal g^–1. Despite this difference, estimates of daily energy intake of juvenile chinook salmon were only 3%, greater when developed from the mean caloric density of stomach contents excludingCorophium.     

Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato

Resistance to antibiotics mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, regulatory agencies and consumer concerns favor these to be eliminated from food crops. Several excision systems exist but none have been optimized or shown to be functional for clonally propagated crops. The excision of the nptII gene conferring resistance to kanamycin has been achieved here using a gene construct based on a heat-inducible cre gene producing a recombinase that eliminates cre and nptII genes flanked by two loxP sites. First-generation regenerants with the Cre-loxP system were obtained by selection on kanamycin media. Following a heat treatment, second generation regenerants were screened for excision by PCR using nptII, cre, and T-DNA borders primers. Excision efficiency appeared to be at 4.7% depending on the heat treatment. The footprint of the excision was shown by sequencing between T-DNA borders to correspond to a perfect recombination event. Selectable marker-free sprouts were also obtained from tubers of transgenic events when submitted to similar heat treatment at 4% frequency. Spontaneous excision was not observed out of 196 regenerants from untreated transgenic explants. Biosafety concerns are minimized because the expression of cre gene driven by the hsp70 promoter of Drosophila melanogaster was remarkably low even under heat activation and no functional loxP site were found in published Solanum sequence database. A new plant transformation vector pCIP54/55 was developed including a multiple cloning site and the self-excision system which should be a useful tool not only for marker genes in potato but for any gene or sequence removal in any plant.     

Exploring the role of galectin 3 in kidney function: a genetic approach

Galectin 3 belongs to a family of glycoconjugate-binding proteinsthat participate in cellular homeostasis by modulating cellgrowth, adhesion, and signaling. We studied adult galectin 3null mutant (Gal 3–/–) and wild-type (WT) mice togain insights into the role of galectin 3 in the kidney. Byimmunofluorescence, galectin 3 was found in collecting duct(CD) principal and intercalated cells in some regions of thekidney, as well as in the thick ascending limbs at lower levels.Compared to WT mice, Gal 3–/– mice had ~11% fewerglomeruli (p < 0.04), associated with kidney hypertrophy(p < 0.006). In clearance experiments, urinary chloride excretionwas found to be higher in Gal 3–/– than in WT mice(p < 0.04), but there was no difference in urinary bicarbonateexcretion, in glomerular filtration, or urinary flow rates.Under chronic low sodium diet, Gal 3–/– mice hadlower extracellular fluid (ECF) volume than WT mice (p <0.05). Plasma aldosterone concentration was higher in Gal 3–/–than in WT mice (p < 0.04), which probably caused the observedincrease in -epithelial sodium channel (-ENaC) protein abundancein the mutant mice (p < 0.001). Chronic high sodium dietresulted paradoxically in lower blood pressure (p < 0.01)in Gal 3–/– than in WT. We conclude that Gal 3–/–mice have mild renal chloride loss, which causes chronic ECFvolume contraction and reduced blood pressure levels.     

A new approach to quantify trabecular resorption adjacent to cemented knee arthroplasty

A new micro-computed tomography (μCT) image processing approach to estimate the loss of cement-bone interlock was developed using the concept that PMMA cement flows and cures around trabeculae during the total knee arthroplasty procedure. The initial mold shape of PMMA cement was used to estimate the amount of interdigitated bone at the time of implantation and following in vivo service using enbloc human postmortem retrievals. Laboratory prepared specimens, where there would be no biological bone resorption, were used as controls to validate the approach and estimate errors. The image processing technique consisted of identifying bone and cement from the μCT scan set, dilation of the cement to identify the cement cavity space, and Boolean operations to identify the different components of the interdigitated cement-bone regions. For laboratory prepared specimens, there were small errors in the estimated resorbed bone volume fraction (reBVfr=0.11 ± 0.09) and loss in contact area fraction (CAfr=0.06 ± 0.15). These values would be zero if there were no error in the method. For the postmortem specimens, the resorbed volume fraction (reBVfr=0.85 ± 0.16) was large, meaning that only 15% of the cement mold shape was still filled with bone. The loss of contact area fraction (CAfr=0.84 ± 0.17) was similarly large. This new approach provides a convenient method to visualize and quantify trabecular bone loss from interdigitated regions from postmortem retrievals. The technique also illustrates for the first time that there are dramatic changes in how bone is fixed to cement following in vivo service.     

Basement membrane chondroitin sulfate proteoglycans: localization in adult rat tissues

Heparan sulfate proteoglycans have been described as the major proteoglycan component of basement membranes. However, previous investigators have also provided evidence for the presence of chondroitin sulfate glycosaminoglycan in these structures. Recently we described the production and characterization of core protein-specific monoclonal antibodies (MAb) against a chondroitin sulfate proteoglycan (CSPG) present in Reichert's membrane, a transient extra-embryonic structure of rodents. This CSPG was also demonstrated to be present in adult rat kidney. We report here the tissue distribution of epitopes recognized by these MAb. The ubiquitous presence of these epitopes in the basement membranes of nearly all adult rat tissues demonstrates that at least one CSPG is a constituent of most basement membranes, and by virtue of its unique distribution is distinct from other chondroitin and dermatan sulfate proteoglycans previously described.