Expression and purification of an anti-Foot-and-mouth disease virus single chain variable antibody fragment in tobacco plants.

Low-cost recombinant antibodies could provide a new strategy to control Foot-and-mouth disease virus (FMDV) outbreaks by passive immunization of susceptible animals. In this study, a single chain variable antibody fragment (scFv) recognizing FMDV coat protein VP1 was expressed in transgenic tobacco plants. To enhance the accumulation of scFv protein, the codon-usage of a murine hybridoma-derived scFv gene was adjusted to mimic highly expressed tobacco genes and fused to an elastin-like polypeptide (ELP) tag. This scFv-ELP fusion accumulated up to 0.8% of total soluble leaf protein in transgenic tobacco. To recover scFv-ELP protein from the leaf extract, a simple and scalable purification strategy was established. Purified scFv-ELP fusion was cleaved to separate the scFv portion. Finally, it was shown that the purified scFv proteins retained their capacity to bind the FMDV in the absence or presence of ELP fusion.

Transgenic plants for animal health: plant-made vaccine antigens for animal infectious disease control.

A variety of plant species have been genetically modified to accumulate vaccine antigens for human and animal health and the first vaccine candidates are approaching the market. The regulatory burden for animal vaccines is less than that for human use and this has attracted the attention of researchers and companies, and investment in plant-made vaccines for animal infectious disease control is increasing. The dosage cost of vaccines for animal infectious diseases must be kept to a minimum, especially for non-lethal diseases that diminish animal welfare and growth, so efficient and economic production, storage and delivery are critical for commercialization. It has become clear that transgenic plants are an economic and efficient alternative to fermentation for large-scale production of vaccine antigens. The oral delivery of plant-made vaccines is particularly attractive since the expensive purification step can be avoided further reducing the cost per dose. This review covers the current status of plant-produced vaccines for the prevention of disease in animals and focuses on barriers to the development of such products and methods to overcome them.

Steviol glycoside biosynthesis.

Steviol glycosides are found in high concentrations in the leaves of the Paraguayan perennial herb Stevia rebaudiana and their intense sweetness, as well as high concentration in Stevia leaf tissue, has made them the subject of research interest for over 100 years. Steviol glycosides are diterpenoids whose biosynthetic pathways share four steps in common with gibberellic acid formation. The convergence of genomics and plant biochemistry has led to the rapid elucidation of the genes coding for the various enzymes in the biosynthetic pathway. Functional characterization of the enzymes coded for by those genes is on-going. The first committed step in the pathway is the synthesis of the aglycone steviol and the various glycosides found in the leaf tissue result from the elaboration of steviol by a number of glucosyltransferases.

Expressed sequence tags from the Yukon ecotype of Thellungiella reveal that gene expression in response to cold, drought and salinity shows little overlap.

Thellungiella salsuginea (also known as T. halophila) is a close relative of Arabidopsis that is very tolerant of drought, freezing, and salinity and may be an appropriate model to identify the molecular mechanisms underlying abiotic stress tolerance in plants. We produced 6578 ESTs, which represented 3628 unique genes (unigenes), from cDNA libraries of cold-, drought-, and salinity-stressed plants from the Yukon ecotype of Thellungiella. Among the unigenes, 94.1% encoded products that were most similar in amino acid sequence to Arabidopsis and 1.5% had no match with a member of the family Brassicaceae. Unigenes from the cold library were more similar to Arabidopsis sequences than either drought- or salinity-induced sequences, indicating that latter responses may be more divergent between Thellungiella and Arabidopsis. Analysis of gene ontology using the best matched Arabidopsis locus showed that the Thellungiella unigenes represented all biological processes and all cellular components, with the highest number of sequences attributed to the chloroplast and mitochondria. Only 140 of the unigenes were found in all three abiotic stress cDNA libraries. Of these common unigenes, 70% have no known function, which demonstrates that Thellungiella can be a rich resource of genetic information about environmental responses. Some of the ESTs in this collection have low sequence similarity with those in Genbank suggesting that they may encode functions that may contribute to Thellungiella's high degree of stress tolerance when compared with Arabidopsis. Moreover, Thellungiella is a closer relative of agriculturally important Brassica spp. than Arabidopsis, which may prove valuable in transferring information to crop improvement programs.

Leaf Ests from Stevia rebaudiana: a resource for gene discovery in diterpene synthesis.

Expressed sequence tags (ESTs) are providing a new approach to gene discovery in plant secondary metabolism. Stevia rebaudiana Bert. leaves produce high concentrations of diterpene steviol glycosides and should be a rich source of transcripts involved in diterpene synthesis. In order to create a resource for gene discovery and increase our understanding of steviol glycoside biosynthesis, we sequenced 5,548 ESTs from a S. rebaudiana leaf cDNA library. The EST collection was fully annotated based on database search results. ESTs involved in diterpene synthesis were identified using published sequences as electronic probes, by keyword searches of search results, and by differential representation. A significant portion of the ESTs were specific for standard leaf metabolic pathways; energy and primary metabolism represented 17.6% and 13.1% of total transcripts respectively. Diterpene metabolism in S. rebaudiana represented 1.1% of total transcripts. This study identified candidate genes for 70% of the known steps in the steviol glycoside pathway. One candidate, kaurene oxidase, was the 8th most abundant EST in the collection. Identification of many candidate genes specific to the I -deoxyxylulose 5-phosphate pathway suggests that the primary source of isopentenyl diphosphate, a precursor of geranylgeranyl diphosphate, is via the non-mevalonic acid pathway. The use of ESTs has greatly facilitated the identification of candidate genes and increased our understanding of diterpene metabolism.

Evaluation of Host Resistance and Soil Fumigation for the Management of Black Root Rot of Tobacco in Ontario.

Black root rot of tobacco, caused by the soilborne fungus Thielaviopsis basicola, is a serious problem in many tobacco (Nicotiana tabacum L.)-growing regions of the world. In Ontario, the disease is favored by cool, wet soil conditions and heavy textured or poorly drained soils. Yield loss can be severe under these conditions and fumigants containing chloropicrin are used extensively for controlling the disease. Usually, fumigants control the disease reasonably well, but they are costly and could cause a negative environmental impact. A 2-year study was conducted to evaluate the performance of resistant (AC Gayed) and moderately susceptible (Delgold) tobacco cultivars and soil fumigation to black root rot. T. basicola reduced yield of the susceptible Delgold cultivar. The interaction between genotype and fumigation was significant for most traits examined, indicating that the two genotypes responded differently. Orthogonal comparisons indicate that yield from nonfumigated AC Gayed was higher than that of nonfumigated Delgold. Yield of nonfumigated AC Gayed was also not significantly different from the yield of AC Gayed treated either with Vorlex Plus (1,3-dichloropropene+methyl isothiocyanate) or with Vorlex Plus CP (1,3-dichloropropene+methyl isothiocyanate+chloropicrin). In contrast, the yield of nonfumigated Delgold was lower than Delgold treated with Vorlex Plus CP.

Diterpene synthesis in Stevia rebaudiana: recruitment and up-regulation of key enzymes from the gibberellin biosynthetic pathway.

Stevia rebaudiana Bertoni leaves accumulate a mixture of at least eight different glycosides derived from the tetracyclic diterpene steviol. These natural products taste intensely sweet and have similar biosynthetic origins to those of gibberellic acid (GA). The initial steps leading to the formation of GA result from the two-step cyclization of geranylgeranyl diphosphate (GGDP) to (-)-kaurene via the action of two terpene cyclases (-)-copalyl diphosphate synthase (CPS) and (-)-kaurene synthase (KS). Steviol biosynthesis probably uses the same mechanism although the genes and enzymes from S. rebaudiana that are involved in the cyclization of GGDP have not been characterized. We have isolated both the CPS and KS genes from S. rebaudiana and found that recombinant CPS and KS were catalytically active, suggesting that the CPS and KS genes participate in steviol biosynthesis. The genes coding for CPS and KS are usually present in single copies in most plant species and their expression is normally low and limited to rapidly growing tissues. The KS gene has been duplicated in the S. rebaudiana genome and both the KS and CPS genes are highly expressed in mature leaves, a pattern opposite to that found with GA biosynthesis. This pattern may, at least in part, lead to temporal and spatial separation of GA and steviol biosynthesis and probably helps to prevent over-expression from interfering with normal GA metabolism. Our results show that CPS and KS are part of the steviol glycoside biosynthetic pathway and that Stevia rebaudiana has recruited two genes to secondary metabolism from a highly regulated pathway involved in hormone biosynthesis.

Type II fish antifreeze protein accumulation in transgenic tobacco does not confer frost resistance.

Type II fish antifreeze protein (AFP) is active in both freezing point depression and the inhibition of ice recrystallization. This extensively disulfide-bonded 14 kDa protein was targeted for accumulation in its pro- and mature forms in the cytosol and apoplast of transgenic tobacco plants. Type II AFP gene constructs under control of a duplicate cauliflower mosaic virus 35S promoter, both with and without a native plant transit peptide sequence, were introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. AFP did not accumulate in the cytosol of transgenic plants, but active AFP was present as 2% the total protein present in the apoplast. Plant-produced AFP was the same size as mature Type II AFP isolated from fish, and was comparable to wild-type AFP in thermal hysteresis activity and its effect on ice crystal morphology. Field trials conducted in late summer on R1 generation transgenic plants showed similar AFP accumulation in plants under field conditions at levels suitable for large-scale production: but no difference in frost resistance was observed between transgenic and wild-type plants during the onset of early fall frosts.

Influence of rewatering and time of sampling on solute accumulation of two Populus deltoides clones.

We investigated effects of rehydration and time of sampling on solute accumulation in plants of two greenhouse-grown Populus deltoides Bartr. clones (Ohio Red and Platte) subjected to multiple cycles of water stress. Osmotic potential of leaves at full turgor was measured at predawn on well-watered (control) and water-stressed (conditioned) plants that had been rewatered the previous afternoon. Water-soluble organic solutes (carbohydrates, phenolic glucosides and organic acids) were determined at both predawn and midday, before and after rewatering. Conditioning resulted in solute accumulation; however, rewatering of conditioned plants decreased the predawn concentration of glucose by 19-35% and the total solute concentration by 14-15%, relative to values before rewatering. There was a 52% increase in salicin concentration in response to rewatering in conditioned plants of the Platte clone. In conditioned plants of both clones, the concentration of glucose was generally lower at midday than at predawn (16-47%), whereas the concentration of sucrose was higher at midday than at predawn (46-133%). Time of sampling was an important factor in determining whether conditioning resulted in accumulation of glucose and fructose. Compared with control plants, there was a significant accumulation of glucose and fructose at predawn and either no accumulation or a significant reduction of these solutes at midday both before and after rewatering of conditioned plants. Sampling time also affected the amount of solute that accumulated in response to conditioning.

Genetic diversity in North American ginseng (Panax quinquefolius L.) grown in Ontario detected by RAPD analysis.

Genetic diversity within North American ginseng (Panax quinquefolius L.) grown in Ontario was investigated at the DNA level using the randomly amplified polymorphic DNA (RAPD) method via the polymerase chain reaction (PCR). A total of 420 random decamers were initially screened against DNA from four ginseng plants and 78.8% of them generated RAPD fragments. Thirty-six of the decamers that generated highly repeatable polymorphic RAPD markers were selected for further RAPD analysis of the ginseng population. With these primers, 352 discernible DNA fragments were produced from DNA of 48 ginseng plants, corresponding to an average of 9.8 fragments per primer, of which over 45% were polymorphic. The similarity coefficients among the DNA of ginseng plants analyzed were low, ranging from 0.149 to 0.605 with a mean of 0.412, indicating that a high degree of genetic diversity exists in the ginseng population. Lower levels of genetic diversity were detected among 3-year-old ginseng plants selected on the basis of greater plant height than among the plants randomly selected from the same subpopulation or over the whole population, suggesting that genetic factors at least partly contribute to morphological variation within the ginseng population and that visual selection can be effective in identifying the genetic differences. The significance of a high degree of genetic variation in the ginseng population on its potential for improvement by breeding is also discussed.

Organic solute accumulation and dehydration tolerance in three water-stressed Populus deltoides clones.

The relationship between osmotic potential, water-soluble organic solutes and electrolyte leakage was investigated in three greenhouse-grown eastern cottonwood (Populus deltoides Bartr.) clones. The Ohio Red (from southern Ohio) and Platte (from eastern Nebraska) clones were selected because they differ in dehydration tolerance, gas exchange and osmotic potential. A third clone, Wildcat (from western Nebraska) was included because of its origin in a very dry area. Plants were either watered daily (control) or watered every 4-6 days (preconditioned). A third group of plants was watered daily until it was water-stressed at the end of the experiment (nonconditioned). Leaves for osmotic potential and water-soluble solute determinations were sampled at predawn after plants were rewatered. All clones showed osmotic adjustment ranging from 0.23 to 0.48 MPa. Organic solutes contributed up to 48% of the total osmotic adjustment. The solutes that contributed at least -0.05 MPa each to leaf osmotic potential in any clone were sucrose, malic acid, glucose, fructose, myoinositol and salicin. Fructose and glucose concentrations were unchanged or declined in preconditioned Ohio Red and Platte but increased in Wildcat. The Ohio Red clone had the highest sucrose concentration in both well-watered and preconditioned plants, and a lower injury index (more membrane stability) than Wildcat in the preconditioned plants, whereas nonconditioned Platte displayed the greatest electrolyte leakage of all clones and treatments. Cell membrane stability was correlated to osmotic potential and sucrose concentration only in Ohio Red.

Field performance and heavy metal concentrations of transgenic flue-cured tobacco expressing a mammalian metallothionein-beta-glucuronidase gene fusion.

Cadmium (Cd) is a nonessential heavy metal that can cause acute and chronic illness in humans. Some plant species such as tobacco (Nicotiana tabacum L.) tend to accumulate high levels of Cd in leaf tissue, the consumed portion of the plant. Tissue-specific expression of mammalian metallothionein has been suggested as a means of partitioning Cd in nonconsumed portions of transgenic plants. The purpose of the experiment reported here was to evaluate Cd concentration and agronomic performance of four field-grown transgenic tobacco lines harbouring a metallothionein-beta-glucuronidase (MG) gene fusion driven by the constitutive 35S promoter of cauliflower mosaic virus. The trial was grown in a region of Canada known to have high background levels of Cd. The agronomic evaluation showed that some of the transgenic lines were equal to, while others performed more poorly than, the untransformed control for yield, days to flower, and leaf number. Gene expression measured by beta-glucuronidase activity showed that all of the transgenic lines expressed the MG gene in the upper portion of the plant. One line did not express the MG gene in the roots. Cd levels in the leaf tissue of transformed lines were not significantly different from the untransformed control.

An integrated approach to oilseed rape cultivar selection using phenotypic stability.

Various methods of evaluating phenotypic stability have been proposed; however, no single method can adequately describe cultivar performance. The objectives of this study were to integrate a number of methods of evaluating stability and to use this approach for cultivar selection. These objectives were considered in the context of the broad-based oilseed rape cultivar (Brassica napus spp. oleifera) evaluation system currently used in western Canada. Regression analysis was used to assess cultivar response to environments. Cluster analysis was used to assemble cultivars into groups with similar regression coefficients (b i ) and mean yield. Three parametric stability parameters, years within locations mean square (MS; Y/L), Shukla's stability variance (σ i (2) ), and Francis and Kannenberg's coefficient of variability (CV i ), were compared to determine which method would be most suitable for selection of oilseed rape cultivars from within clustered groups. Yield data from three cultivars and six breeding lines that had been tested for 2 years at 26 locations in the Western Canola Cooperative Test 'A' were used for all calculations. The cluster analysis was successful in identifying commercially acceptable breeding lines. The parameter MS i Y/L was considered to be more appropriate than either CV i or σ i (2) , because it measured only the unpredictable portion of the genotype x environment interaction and was independent of the other cultivars in the test. The use of cluster analysis to group entries with similar b i values and mean yields, followed by selection for stability within groups, was advocated.

Rapid field measurement of the assimilation rate versus internal CO(2) concentration relationship in green ash (Fraxinus pennsylvanica Marsh.): the influence of light intensity.

Assimilation rate (A) versus intercellular CO(2) concentration (C(i)) relationships for leaflets of five-year-old green ash (Fraxinus pennsylvanica Marsh.) trees were computed from gas exchange measurements obtained in the field with a closed-circuit, portable photosynthesis measurement system comprising an LI-6200 gas analyzer and an LI-6000 computer, (Li-Cor, Inc., Lincoln, Nebraska, USA). Observations were made over a range of light intensities achieved by attenuating direct sunlight with neutral density filters, and over a range of ambient CO(2) concentrations achieved by breathing into the assimilation chamber and then lowering the CO(2) concentration to the desired level with the LI-6200's soda-lime scrubber. Boundary layer conductance was determined by use of a leaf replica made of moist filter paper. Typically, A-C(i) curves at four light intensities were obtained in three to four hours. The initial slope (when A = 0) of the A-C(i) curve obtained at a light intensity of 1750 micromol m(-2) s(-1) (full sunlight) was similar to that obtained at a light intensity of 840 micromol m(-2) s(-1). However, when light intensity was reduced further (to 370 and 160 micromol m(-2) s(-1)), the initial slope of the A-C(i) curve also decreased, indicating that at these light intensities, assimilation was limited by photochemical energy supply, as well as CO(2) concentration.

[A so-called atypical adenoma of the thyroid in an endemic struma area]. / Zur Problematik des sogenannten atypischen Adenoms der Schilddrüse im Strumaendemiegebiet.

In view of the benign clinical course of the atypical adenoma and the high diagnostic accuracy of our frozen sections we feel that subtotal strumectomy with in toto removal of the adenoma and closely spaced follow-ups constitutes an adequate treatment of this thyroid neoplasm. In our opinion primary lobectomy means unnecessarily exposing a disproportionately high percentage of patients to the higher complication risk of this more radical surgery. From 1977 to 1982 there were 47 cases of atypical adenoma among our patients. The ratio men: women was 1:3.7, the mean age of the patients was 44 years. The patients underwent subtotal strumectomy. In the period under review "atypical adenoma" diagnosed on the basis of frozen sections turned out to be carcinoma in 5 instances. The follow-up period of the 47 patients that underwent surgery ranges from 1 1/2 to 7 years. None of the patients has had tumor regrowth so far.

[Sonographic findings in autonomous adenoma of the thyroid gland in a goiter-endemic region]. / Der sonographische Befund beim autonomen Adenom der Schilddrüse im Strumaendemiegebiet.

Ultrasound scanning was carried out in 84 patients with autonomous nodules and 67 patients with inactive nodules. The echo pattern was correlated to the macro- and microscopic findings as well as to the function of the nodules. The autonomous nodules showed a low echogenicity in 57%, a normal parenchyma-identical echogenicity in 10% and a high echogenicity in 33%. In comparison the echo pattern of the inactive nodules showed low level echoes in 31%, normal in 36% and a high level echoes in 31%. Only one third of the nodules showed a homogeneous echo pattern, one third showed liquid degeneration and one third a mixed echo pattern. There is no correlation between the echo pattern of the autonomous nodules and the metabolic function of the nodules. 65 patients have been operated (37 autonomous and 28 inactive nodules). The macroscopic findings showed bleeding and cysts in most of the echo-poor nodules, also those with homogeneous structures. In the histological findings microfollicular structures were found in both high- and low-echogenicity nodules (18/16%). Macrofollicular changes and mixed forms were found mainly in nodules with parenchyma-identical and high echogenicity (58/48%). Ultrasound is very helpful in the morphological diagnosis of thyroid nodules, but there is no significant correlation to metabolic activity or to microscopic structures of the nodules in the thyroid gland.

[Antibiotic prevention in gynecology. Concentrations of ceftizoxime in the serum, myometrium, endometrium and fallopian tubes]. / Antibiotikaprophylaxe in der Gynäkologie. Konzentrationen von Ceftizoxim in Serum, Myometrium, Endometrium und Tuben.

34 patients received an intravenous bolus injection of 2 g ceftizoxime over 5 min at various times before abdominal or vaginal hysterectomy. Ceftizoxime concentrations in myometrium were substantially higher than in salpinges and endometrium. Tissue concentrations of 4 micrograms/g could be maintained for at least 3 hours. The ceftizoxime tissue concentrations attained in myometrium, endometrium and salpinges justify therapeutic studies and controlled prospective clinical trials on the prevention of postoperative wound infections with this antibiotic.

[Clinical value of sonography in the diagnosis of thyroid diseases]. / Klinische Wertigkeit der Sonographie in der Schilddrüsendiagnostik.

In 63 patients echography of thyroid was performed additionally to case history, palpation, scintigraphy and hormone tests for evaluating clinical significance of this method. The benefit of this technique is rapid measurement of thyroid size, demonstration of nodules in palpable diffuse goiters and differentiating of solid or cystic nodules of the thyroid. For diagnosis of autonomous areas in the thyroid scintigraphy remains the method of choice. Also there is no correlation of ultrasound findings and thyroid function. In routine diagnostic procedure of thyroid disease echography may replace scintigraphy only in diffuse goiter and if radionuclide imaging is not possible. Nevertheless ultrasonic evaluation of the thyroid is an important additional method in diagnosis of thyroid diseases.

Concentrations of ceftriaxone in serum and gynecological tissues.

31 patients received an intravenous 15-min injection of 2 g ceftriaxone at various times before abdominal or vaginal hysterectomy. Ceftriaxone concentrations in salpinges were significantly higher than in myometrium and endometrium. Tissue concentrations of 20 micrograms/g could be maintained for at least 5 h. Due to its high tissue concentrations maintained for the whole operative procedure, ceftriaxone does seem to be one of the antibiotics of choice for single-dose prophylaxis of postoperative infections in gynecology.