Agarose plating and a bead type culture technique enable and stimulate development of protoplast-derived colonies in a number of plant species

Two novel techniques improve division and colony formation from protoplasts:

1. Plating in agarose stimulates colony formation of protoplasts from a wide range of species. Protoplasts from Nicotiana tabacum developed to colonies from lower initial population densities in agarose than in agar or liquid. Protoplasts from Hyoscyamus muticus which do not divide in agar divided and formed colonies in agarose at higher efficiencies than in liquid medium.
2. Culture of gel embedded protoplasts in large volumes of liquid medium on a gyrotatory shaker (‘bead culture’) further improved plating efficiencies in some species (e.g. Lycopersicon esculentum and Crepis capillaris) and enabled sustained proliferation of protoplasts which had not previously developed beyond the few cell colony stage (Brassica rapa and a mutator gene variety of Petunia hybrida).

The combination of ‘agarose plating’ and ‘bead culture’ dramatically improved plating efficiencies of protoplasts in all species tested.     

Efficient use of reactive nitrogen for cultivation of bioenergy: less is more

A further increase in nitrogen (N) intensive biomass supplies to substitute fossil carbon sources implies inclusion of additional reactive nitrogen (Nr) into the biosphere. A Danish model study compared low‐intensity managed seminatural beech forest and a winter wheat system with respect to N losses and greenhouse gas (GHG) emissions. Losses of reactive N to air and groundwater per unit of energy produced were four to six times higher for the winter wheat system. The energy efficiency was an order of magnitude higher in the forest system, whereas the related GHG emission reduction by fossil coal substitution differed by <25%. The question is whether a low or a high intensity of cultivation yields the best overall ecosystem service performance? Given the detrimental effect of excess reactive N on natural ecosystems, we suggest that bioenergy production from unfertilized forest with seminatural structure and function should be preferred over N‐intensive crop production.     

Widespread hybridization between the Greater Spotted Eagle Aquila clanga and the Lesser Spotted Eagle Aquila pomarina (Aves: Accipitriformes) in Europe

Hybridization is a significant threat for endangered species and could potentially even lead to their extinction. This concern applies to the globally vulnerable Greater Spotted Eagle Aquila clanga, a species that co‐occurs, and potentially interbreeds, with the more common Lesser Spotted Eagle Aquila pomarina in a vast area of Eastern Europe. We applied single nucleotide polymorphism (SNP) and microsatellite markers in order to study hybridization and introgression in 14 European spotted eagle populations. We detected hybridization and/or introgression in all studied sympatric populations. In most regions, hybridization took place prevalently between A. pomarina males and A. clanga females, with introgression to the more common A. pomarina. However, such a pattern was not as obvious in regions where A. clanga is still numerous. In the course of 16 years of genetic monitoring of a mixed population in Estonia, we observed the abandonment of A. clanga breeding territories and the replacement of A. clanga pairs by A. pomarina, whereby on several occasions hybridization was an intermediate step before the disappearance of A. clanga. Although the total number of Estonian A. clanga × A. pomarina pairs was twice as high as that of A. clanga pairs, the number of pairs recorded yearly were approximately equal, which suggests a higher turnover rate in interbreeding pairs. This study shows that interspecific introgressive hybridization occurs rather frequently in a hybrid zone at least 1700‐km wide: it poses an additional threat for the vulnerable A. clanga, and may contribute to the extinction of its populations. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 725–736.     

Transgenic plants of Orchardgrass (Dactylis glomerata L.) from protoplasts

We have demonstrated the transfer and expression of a foreign chimeric gene in the grass species, Dactylis glomerata L. This species is a member of the Gramineae sub-family Pooideae, which includes the small grain cereals, from which transformed plants have not yet been obtained. A chimeric hygromycin-resistance gene was introduced into protoplasts isolated from an embryogenic suspension culture, using heat shock followed by electroporation or polyethylene glycol treatment. Cell colonies resistant to 20 g/ml hygromycin were selected in liquid medium using an agarose bead type culture system. Transformed calli were identified by Southern hybridization. Embryogenic callus was induced to regenerate plants and transformed plants were shown to contain the hygromycin resistance gene.Abbreviations 2,4-D 2,4-Dichloro-phenoxy-acetic acid - dicamba 3,6-dichloro-2-methoxy benzoic acid - PEG polyethylene glycol - SDS Sodium dodecyl sulfate - SH medium Medium of Schenk and Hildebrandt (1972)     

Genotyping of pantophysin I (Pan I) of Atlantic cod (Gadus morhua L.) by allele‐specific PCR

The two main allelic variants of the Atlantic cod (Gadus morhua L.) pantophysin I (Pan I) locus have different frequencies within different cod stocks. The Dra I polymorphism which distinguishes the two alleles can thus be used for discrimination of coastal and offshore cod populations. We present a new method for Pan I genotyping using fluorescent allele‐specific duplex polymerase chain reaction (PCR). This method is more rapid, reliable and cost‐effective than the previously published method and it is not affected by DNA source and quality. This improvement is important for studies demanding high throughput and accuracy of Pan I genotyping     

Evaluation of measurement uncertainties of an analytical method for the determination of aflatoxin M<Subscript>1</Subscript> in milk and milk powder

The mycotoxin aflatoxin M₁ (AfM₁) is a serious food safety hazard for which the European Commission has already established a maximum permissible level of 0.05 μg/kg AfM₁ in milk and products thereof. For control analysis laboratories are increasingly asked to submit full uncertainties of their analytical results.The evaluation of measurement uncertainties of an analytical method for the determination of AfM₁ in milk and milk powder on the basis of ‘in-house’ validation data in compliance with the ‘Guide to the Expression of Uncertainty in Measurement (GUM)’ [1] and the ‘EURACHEM Guide’ [2] is described. A similar approach will be used to assess the performance of methods employed by laboratories participating in the certification of reference materials for AfM₁ in milk powder.     

An invasive crayfish affects egg survival and the potential recovery of an endangered population of Arctic charr

1. Many fish stocks have declined, because of overharvesting, habitat destruction and introduced species. Despite efforts to rehabilitate some of these stocks, not all are responding or are recovering only slowly. 2. In freshwater systems, introduced crayfish are often problematic, and it has been suggested that their egg predation could reduce recruitment in depleted stocks of native fish. 3. Here, we report the results of a field experiment, using experimental cages, on the extent of predation on eggs of great Arctic charr (Salvelinus umbla) in Lake Vättern, Europe’s fifth largest lake. Here, the great Arctic charr has declined dramatically and is listed as critically endangered. 4. We were able to partition the total loss rate of eggs into background mortality, predation by introduced signal crayfish (Pacifastacus leniusculus) and predation by native fish. The mortality rate of charr eggs because of crayfish was estimated at more than five times that because of native fish. Of the total loss of eggs, 80% is believed to be caused by crayfish and 14% by fish, with 6% being natural background mortality. 5. In a worst case scenario, our data infer that only 25% of the original number of eggs would survive, compared with 75% in the absence of crayfish. This could impair recovery of the stock of the endangered great Arctic charr in Lake Vättern. 6. Contrary to earlier claims that crayfish predation on eggs of great Arctic charr is insignificant, our results indicate that crayfish predation may exceed fish predation and suggest that the abundance of signal crayfish on the spawning sites of great Arctic charr should be managed.     

TT232, A Novel Signal Transduction Inhibitory Compound in the Therapy of Cancer and Inflammatory Diseases

TT-232 is a structural analogue of somatostatin exhibiting strong and selective growth-inhibitory effects, inhibition of neurogenic inflammation, as well as general anti-inflammatory and analgesic potential without the wide-ranging endocrine side effects of the parent hormone and its “traditional” analogues. The anti-inflammatory action of TT-232 is mediated through the SSTR4 receptor, and its antitumor activity is mediated through the SSTR1 receptor and by the tumor-specific isoform of pyruvate kinase. Its mechanism of action is in line with a new era of molecular medicine called signal transduction therapy, where “false” intracellular or intercellular communication is inhibited or corrected without interfering with basic cell functions and machinery. TT232 has passed phase I clinical trials without toxicity and significant side effects, and phase II studies are running for oncological and anti-inflammatory indications, respectively. This compound has the perspective to become the first drug in molecularly targeted therapy of inflammation where a combined effect of anti-inflammatory, analgesic, and neurogenic inflammation-inhibiting activity can be achieved.