The Microbiology of Exposed Areas of Aquatic Habitats of Northern Ellesmere Island

It has been known for many years that the soils and waters of the North American Arctic contain a great variety of microbes, such as fungi, bacteria, actynomycetes, myxobacteria and algae. ... The present paper constitutes a report on the microflora in exposed areas of shorelines of ponds and tarns located near Hazen Camp (81 49, 71 18 W), northern Ellesmere Island. ... All of the samples with two exceptions, had pH values on the alkaline side. Their carbon-to-nitrogen ratios varied from 8 to 29, such values are not being outside the range for many soils. When the conductivity measurements were converted into salinity classes, 22 per cent of the pond samples (nos. 2, 23C, 32 and 41) were found to be slightly saline; 17 per cent (nos 20, 26 and 45) moderately saline; and the remainder (61 per cent) strongly saline. Wide variations in numbers of microorganisms were observed, as indicated in Table 1. Salt concentration, pH and carbon-to-nitrogen ratio had no effect on the numbers; and, except possibly in the case of Cytophaga, temperature of incubation appeared to have no effect on them either. Numbers of Cytophaga tended to decrease somewhat as the temperature decreased. Holding et al and Parinkina also found fluctuations in the microbial counts for tundra sites, and there was no evidence that temperature was responsible for them. In contrast the situation found in studies dealing with cool-temperature regions of Canada, the number of fungal genera in the samples from the Arctic now being discussed was low, only eleven being identified. A similar paucity of fungal genera was noted by the present author in his study of four permafrost soils from the Mackenzie Valley. In the same study, the dominant fungal genus was Chrysosporium, to which an average of 45 per cent of all isolates belonged; the next most populous was Penicillium, which accounted for 20 per cent, and then Mortierella with 16 per cent. Phialophora, Cladosporium and Phoma each accounted for 4 per cent ofthe total isolates, and sterile mycelia 3 per cent. The remaining four genera (Oidiodendron, Cephalosporium, Coniothyrium, Gliomastix) were isolated with a frequency of 2 per cent or less. These results are similar to those of Dowding and Widden who, after assembling data from 33 tundra sites, found the most widespread fungal genera to be the sterile forms Penicillium, Chrysosporium, Cladosporium and Mortierella. It was also observed in the course of the study that, as the incubation temperature decreased, Chrysosporium was recorded far more frequently. This genus was represented by a single species C. pannorum, which is often abundant in cold environments and appears to be an important colonizer of such habitats. Since there are many more environmental parameters affecting microbial activity and numbers than were examined in this investigation, it would be difficult to discuss the relationship of the results to the High Arctic ecosystem. Nevertheless, if one keeps in mind that a large proportion of tundra mycoflora are psychrophilic and most of the nutrients in dead plants are released by microbes to aid growth of future vegetation, it becomes obvious that the presence of viable microbes in these lake shorelines, situated approximately 600 miles south of the North Pole, is very important. They are undoubtedly not only active in supplying plant nutrients but are helping to transfer part of the decaying vegetation to produce "stable" hummus which take part in the formation of the underlying and surrounding weakly-developed soil profiles. They also play an additional role in providing food for larger organisms, for Whittaker found that in Arctic sites the population peak for mites, follows that for fungi

Development of Lepidium campestre into a new oil and catch crop

A growing world population and dwindling fossil oil reserves demand an increase in the world plant oil production. The possibility to increase the area of cultivated land is limited due to the shortage of arable land. Also, the possibility for increasing the oil content in the limited number of cultivated oil crops is restricted. New, high-yielding oil crops, which can be grown in areas where no other oil crops can grow and have less environmental impact, need to be developed. It is preferable that such new oil crops would allow us to tailor-make the oil composition in planta for food, fuel or industrial applications. In Sweden, the main oil crop cultivated is winter rapeseed, which, due to weak winter hardiness, only can be grown in southern Sweden. Developing a new winter-hardy oilseed crop would extend the plant oil production in Sweden and other cold climate regions. Lepidium campestre is a wild Brassica species. It is very winter hardy, high-yielding, has an upright stature and synchronous flowering. Moreover, it is biennial, and thus being suitable as a catch crop. However, it needs to be domesticated first so that it possesses all important agronomic traits necessary for being a successful agricultural crop. The aim of this thesis was to improve some properties of L. campestre by genetic engineering with focus on: the seed oil content, pod shatter, seed oil composition and wax ester production in the seed oil. In order to enable genetic engineering of this wild species, a well-functioning regeneration and transformation protocol was first developed, which has greatly facilitated the subsequent genetic improvements of the target traits of the species. Through RNAi-down-regulation of the FAD2 and FAE1 genes, transgenic lines with oxidative stable oil high in oleic acid were generated, indicating the potential of the species for being used for food oil purposes. Moreover, transgenic lines with increased seed oil content were developed by expressing either the AtWRI1 or AtHb2 or BvHb2 gene. Transgenic lines with pod shatter resistance were produced by RNAi down-regulation of the IND gene. Wax esters were produced in this species by expression of the jojoba wax synthesis genes, showing the potential of the species as a new platform for industrial oil production. These transgenic lines are valuable materials for further breeding of this species

Factors affecting Agrobacterium transformation in oat

Oat (Avena sativa L.) is one of the crops that have been cultivated by mankind for the longest time (Lásztity, 1998) and today it is an important and traditional agricultural crop in Northern Europe (Bräutigam et al., 2005). In Sweden, most of the oat is used as feed (Bräutigam et al., 2005), but with a superior amino acid composition of the oat proteins (Lásztity, 1998), a high content of desirable soluble fibers (β-glucans), essential vitamins and minerals (Sadiq Butt et al., 2008) and antioxidants (Ryan et al. 2007) the interest in using oats for human consumption has increased (Carlsson, Personal conversation). In comparison to other cereals, oat has a much higher content of lipids. The oil is interesting because its energy content is high, while its content of saturated fatty acids is relatively low. A disadvantage, which may prevent an increase in using oat as food, is the imbalance of Omega-6/Omega-3 fatty acids. The amount of Omega-6 fatty acids is much higher than the amount of Omega-3 fatty acids (Welch & Legett, 1997). The imbalance in the fatty acid composition has shown to be a possible factor behind the increasing number of cases of cardiovascular disease, cancer, diabetes, asthma, depression, obesity, autoimmune diseases and rheumatism in the western countries (Simopoulos, 2004). With the help of transformation, it is possible to increase the amount of Omega-3 fatty acids, and thus get a better Omega-6/Omega-3-balance in oat. Omega-3 fatty acids have also shown to be potential therapeutic agents for autoimmune and inflammatory diseases (Simopoulos, 2002), the main reason behind the increased interest in using oat as food. Transformation allows an increase in Omega-3 fatty acids in oats, but previous studies concerning transformation of Avena sativa L. has resulted in low transformation frequencies. Oat is a monocotyledonous crop, not a normal host for Agrobacterium; it is thus difficult to be infected. The conditions have to be optimal for a successful transformation to occur. The aim of this work has been to examine various factors affecting oat transformation, and to develop a functional transformation protocol. Explants from the hypocotyl of the oat cultivar Matilda were infected by Agrobacterium-mediated transformation. In total, 15 batches of transformation were carried out, in which different combinations of bacterial strains, vectors and media were tested. GUS- and GFP assays were conducted to confirm Agrobacterium infection of the explants. No GUS expression was achieved in the GUS assays, but no certain conclusion can be drawn from the result. Endogenous GUS-like activity is triggered by low pH-values, but a raised pH-value may not only suppress the expression of endogenous GUS, but also the expression of true GUS. The explants analyzed for GFP expression exhibited whitish-colored spots, but further cultivation and repeated assays of the explants is necessary to confirm GFP expression. Further trials are needed in order to obtain a well-functioning oat transformation protocol

Heat affected zones and oxidation marks in fiber laser-oxygen cutting of mild steel

The effect of cutting speed and sheet thickness on surface oxidation and heat affected zones (HAZs) has been investigated for laser-oxygen cutting of mild steel sheet with a fiber laser. Optical and scanning electron micrographs were used to determine the extent of surface oxidation and HAZ from plan and cross-sectional views, respectively. The HAZ is consistently wider at the bottom of the cut compared to the HAZ at the top of the cut. With increasing speed, the width of the HAZ at the top of the cut decreases whereas the HAZ width at the bottom of the cut generally increases. No simple, direct relationship between HAZ width and surface oxidation was seen. However, it is possible to state that in each case considered here, the HAZ would be completely removed if they are machined back by a depth equal to the extent of the surface oxidation. (C) 2011 Laser Institute of America

Leaf structure and localization of a transgene protein in barley

Barley is one of the most important cereals cultivated in the Nordic countries. Climate change brings warmer and moisture climate which favors fungal diseases. In the cropland barley can be seriously infested with hard fungus attack. Since it is important that the yield bears a high quality it is of great importance to find varieties more resistant to attacks. Pathogenesis-related (PR) proteins are stress proteins induced in the plant in response to infection and abiotic stress (van Loon, 1997). PRs are shown to have antimicrobial activity differing between bacteria, fungi and oomycetes (Tandrup Poulsen, 2001). Among the 17 PR-families (van Loon et al., 1994, 2006) PR-5 is one of the most abundant ones in barley (Tandrup Poulsen, 2001, van Loon et al., 2006). Studies have shown that PR-5 exists naturally in the ground tissue of leaves, but not in the epidermis (Gregersen et al., 1997). In the epidermal cell walls an epidermis-specific promoter for PR-5 has been placed in front of the gene encoding PR-5, this to achieve a more resistant cultivar of barley. Enhanced resistance has been demonstrated, but it is not confirmed that this resistance is due to expression of PR-5 in epidermis (Santén et al., unpublished, Tandrup Poulsen, 2001). In the modified lines, preliminary studies have shown irregular structure of epidermis (Santén et al., unpublished). The aim of the study was to establish whether there were structural differences between regular and modified barley, and to localize PR-5 in epidermis. Counterstaining was used to be able to study the leaf structure in microscope, and immunocytochemistry was used to localize PR-5. Results showed irregular cell structure in epidermis, and in a few cases even in the ground tissue, of modified barley. Due to failure in method, unspecific binding of the antibody visualizer occurred and no confident result could be established regarding the localization of PR-5. Nothing indicates an existence of PR-5 in epidermal cell walls of modified barley. This could be a result of a non-working method, undetectable levels of PR-5 or that the gene encoding PR-5 is expressed as mRNA but not translated to protein. Occurrence of PR-5 has been demonstrated in epidermal cells of infected material. This detection could be due to the fact that the gene needs an infection to be 6 expressed as the protein. More studies are necessary to establish whether the showed enhanced resistance is due to expression of PR-5 in epidermis

Establishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre)

Field cress (Lepidium campestre) is a potential oilseed crop that has been under domestication in recent decades. CRISPR/Cas9 is a powerful tool for rapid trait improvement and gene characterization and for generating transgene-free mutants using protoplast transfection system. However, protoplast regeneration remains challenging for many plant species. Here we report an efficient protoplast regeneration and transfection protocol for field cress. Important factors such as type of basal media, type/combination of plant growth regulators, and culture duration on different media were optimized. Among the basal media tested, Nitsch was the best for protoplast growth in MI and MII media. For cell wall formation during the early stage of protoplast growth, relatively high auxin concentrations (0.5 mg L−1 NAA and 2,4-D), without addition of cytokinin was preferred for maintaining protoplast viability. After cell wall formation, 1.1 mg L−1 TDZ combined with either 0.05 mg L−1 NAA or 2,4-D was found to efficiently promote protoplast growth. On solid shoot induction medium, 1.1 mg L−1 TDZ without any auxin resulted in over 80% shoot generation frequency. A longer culture duration in MI medium would inhibit protoplast growth, while a longer culture duration in MII medium significantly delayed shoot formation. Using this optimized protoplast regeneration protocol, we have established an efficient PEG-mediated transfection protocol using a vector harboring the GFP gene, with transfection efficiencies of 50–80%. This efficient protoplast protocol would facilitate further genetic improvement of field cress via genome editing, and be beneficial to development of protoplast regeneration protocols for related plant species

Downregulation of the INDEHISCENT Gene by RNAi Resulted in Desired Pod Shatter Reduction of Lepidium campestre in Subsequent Generations

Wild species field cress (Lepidium campestre) has favorable agronomic traits, making it a good candidate for future development as an oil and catch crop. However, the species is very prone to pod shatter, resulting in severe yield losses. This is one of the important agronomic traits that needs to be improved in order to make this species economically viable. In this study, we cloned the L. campestre INDEHISCENT (LcIND) gene and prepared two LcIND-RNAi constructs with the IND promoter (long 400 bp and short 200 bp) from Arabidopsis. A number of stable transgenic lines were developed and evaluated in terms of pod shatter resistance. The majority of the transgenic lines showed increased resistance to pod shatter compared to the wild type, and this resistance was maintained in four subsequent generations. The downregulation of the LcIND gene by RNAi in the transgenic lines was confirmed by qRT-PCR analysis on T-3 lines. Southern blot analysis showed that most of the analyzed lines had a single-copy integration of the transgene, which is desirable for further use. Our results show that it is possible to generate stable transgenic lines with desirable pod shatter resistance by downregulating the LcIND gene using RNAi in field cress, and thus speeding up the domestication process of this wild species

Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.)

Difficulty in protoplast regeneration is a major obstacle to apply the CRISPR/Cas9 gene editing technique effectively in research and breeding of rapeseed (Brassica napus L.). The present study describes for the first time a rapid and efficient protocol for the isolation, regeneration and transfection of protoplasts of rapeseed cv. Kumily, and its application in gene editing. Protoplasts isolated from leaves of 3-4 weeks old were cultured in MI and MII liquid media for cell wall formation and cell division, followed by subculture on shoot induction medium and shoot regeneration medium for shoot production. Different basal media, types and combinations of plant growth regulators, and protoplast culture duration on each type of media were investigated in relation to protoplast regeneration. The results showed that relatively high concentrations of NAA (0.5 mg l(-1)) and 2,4-D (0.5 mg l(-1)) in the MI medium were essential for protoplasts to form cell walls and maintain cell divisions, and thereafter auxin should be reduced for callus formation and shoot induction. For shoot regeneration, relatively high concentrations of cytokinin were required, and among all the combinations tested, 2.2 mg l(-1) TDZ in combination with auxin 0.5 mg l(-1) NAA gave the best result with up to 45% shoot regeneration. Our results also showed the duration of protoplast culture on different media was critical, as longer culture durations would significantly reduce the shoot regeneration frequency. In addition, we have optimized the transfection protocol for rapeseed. Using this optimized protocol, we have successfully edited the BnGTR genes controlling glucosinolate transport in rapeseed with a high mutation frequency

Overexpression and Down-Regulation of Barley Lipoxygenase<i> LOX2.2 </i>Affects Jasmonate-Regulated Genes and Aphid Fecundity

Aphids are pests on many crops and depend on plant phloem sap as their food source. In an attempt to find factors improving plant resistance against aphids, we studied the effects of overexpression and down-regulation of the lipoxygenase gene LOX2.2 in barley (Hordeum vulgare L.) on the performance of two aphid species. A specialist, bird cherry-oat aphid (Rhopalosiphum padi L.) and a generalist, green peach aphid (Myzus persicae Sulzer) were studied. LOX2.2 overexpressing lines showed up-regulation of some other jasmonic acid (JA)-regulated genes, and antisense lines showed down-regulation of such genes. Overexpression or suppression of LOX2.2 did not affect aphid settling or the life span on the plants, but in short term fecundity tests, overexpressing plants supported lower aphid numbers and antisense plants higher aphid numbers. The amounts and composition of released volatile organic compounds did not differ between control and LOX2.2 overexpressing lines. Up-regulation of genes was similar for both aphid species. The results suggest that LOX2.2 plays a role in the activation of JA-mediated responses and indicates the involvement of LOX2.2 in basic defense responses