Basta tolerance as a selectable and screening marker for transgenic plants of Norway spruce

The bar gene conferring resistance to the herbicide Basta (containing phosphinothricin) was transferred to embryogenic cultures of Picea abies by particle bombardment and transformants were selected on Basta medium. In total, 83 9-month-old transgenic plants of Picea abies from six transformed sublines were analysed for continued tolerance to Basta. PCR analysis showed that the bar gene was present in all transformed plants but not in the control plants. Northern blot analysis showed differences in expression level among plants from the same subline as well as among sublines. A simple biotest for screening for Basta tolerance based on the colour change of detached needles induced by Basta was developed. The tolerance to Basta varied among the plants from different sublines. Needles from four of the sublines were resistant to 100 mg l⁻¹ phosphinothricin, a concentration inducing yellowing in control needles, while plants from the other two sublines were on average two to four times as resistant as untransformed control plants. The biotest enables rapid semi-quantitative monitoring for continued transgene expression in long-lived tree species. Received: 21 October 1999 / Revision received: 24 January 2000 / Accepted: 24 January 2000     

根癌农杆菌介导的芦荟遗传转化条件的研究

以美国库拉索芦荟(Aloe.arborescens)的横切薄层切片(transverse thin cell layer, tTCL)作为转化受体, 通过受体材料对抗生素的敏感性实验和Gus 基因瞬时表达率的研究, 找出了较适合的外植体转化条件。研究表明:芦荟对头雹霉素(cefotaxime) 和羧苄霉素(carbenicillin)不敏感, 而对卡那霉素(kanamycin)和潮霉素(hygromycin)敏感;用靠近顶芽的材料得到的横切薄层切片芽再生率高, 有较高的Gus 基因瞬时表达率;乙酰丁香酮(acetosyringone)在芦荟转化是不可缺少的, 对其转化有明显的促进作用。     

Agrobacterium-mediated stable transformation of cell suspension cultures of barley (Hordeum vulgare)

Barley (Hordeum vulgare L. cvs. Igri and Dissa) cell suspension cultures, which had been initiated from immature embryo-derived (IED) and microspore-derived (MSD) callus, were co-cultivated with various Agrobacterium tumefaciens strains. The T-DNA vectors contained visually-detectable marker genes (C1/Lc orgusA-intron), as reporters of transient T-DNA transfer, and also drug resistance genes (hph or bar) to facilitate selection of stably transformed cell lines. A set of normal binary vectors in a super-virulent Agrobacterium strain [EHA101(pBECKS)] and also a super-binary vector [LBA4404(pTOK233)] were used in this study. Cells of the suspension cultures which received T-DNA were able to proliferate under selection regimes and a number of hygromycin- or phosphinothricin-resistant barley callus lines were isolated which expressed a co-transferred gusA gene. To ensure homogeneity of the cell lines, prolonged tissue culture regimes were used but these resulted in a loss of the capacity to regenerate plants from the transgenic callus lines. The frequency of recovery of transformed callus lines ranged from 0.3% to 2.9%. Southern blot analyses of the transformed callus lines confirmed the presence of the marker genes and demonstrated them to be associated with DNA which was distinct from that of the original Agrobacterium plasmid. Furthermore, independent transgenic lines showed diverse patterns of hybridising bands. These data suggest that the T-DNA fragment was stably maintained through integration into the genomes of the barley cell lines. This revised version was published online in June 2006 with corrections to the Cover Date.     

Unexpected progeny obtained from glasshouse study of soil pH influence onAcaulospora laevis inoculum

Summary Potential growth stimulation of two hosts by acid-tolerantAcaulospora laevis was tested in a soil adjusted to 5 pH levels from 5.0 to 7.7. By wet-sieving methods, the field-source inoculum was essentially a pure culture ofA. laevis spores. Upon harvest of sweetgum a completely different spore-type was found in large quantities;A. laevis spores were relatively few to non-existent. The results dramatically underscore the need for caution against relying solely on pre-trial identifications of inoculum spores or presuming that apparently single-species cultures/inoculum will remain pure throughout an experiment.Contribution of the Oregon State University Agricultural Experiment Station in cooperation with the US Department of Agriculture, Agricultural Research Service, technical paper #7366 of the former.     

Evolution of sex comb from the primitive bristle pattern in drosophila is associated with modification in the developmental regulatory protein dachshund

Sex comb is a recently evolved male specific character confined to the Sophophoran group of Drosophila. Such innovations in phenotypes as Waddington proposed, are, outcome of “canalization” in developmental pathways that occur due to mutations creating “choice points” in genetic regulatory pathways. Our interest in the present study is to understand the shifts in genetic network, which has lead to the origin of sex comb from the basic bristle pattern that is seen in rest of the members of Drosophilidae. Here we have made a comparative analysis of expression of some of the key regulators of sex comb morphogenesis, between D. melanogaster and a group of selected species, which primitively lack sex comb. Sex combs reduced (Scr), dachshund (dac), and bric‐a‐brac (bab) gene expression were studied. We show that, primitive bristle pattern is marked by a strikingly down regulated expression of Sex combs reduced in the first tarsal segment of the prothoracic leg discs of male flies. Further a remarkable change with respect to Dachshund, an activator of sex combs reduced gene in the sex comb regulatory pathway, is seen. This is attributed to changes in DAC protein that might have taken place between the two groups of species. bric‐a‐brac does not reveal any significant expression modulation between the sex comb bearing and the primitive patterned species. Earlier works had shown that within the Sophophoran group, dynamic changes in SCR expression is responsible for the diversity seen in sex comb morphology, where as no such variation is witnessed with respect to DAC expression. Our findings have demonstrated that the scenario is different between the group primitively lacking sex comb and D. melanogaster wherein an obvious change in the protein has taken place. genesis 51:97–109, 2013, 2013. © 2012 Wiley Periodicals, Inc.     

Consumption rate of some proteinic diets affecting hypopharyngeal glands development in honeybee workers

The experiment was carried out under laboratory condition to study the consumption of some proteinic diets and their effect on hypopharyngeal glands (HPG) development during nursing period. The results showed that the bee bread and the pollen loads mixture with sugar (1:1) were more consumed by honeybee workers followed by Nectapol® and Yeast-Gluten mixture. The lowest consumption amount was recorded with traditional substitute.Clear differences were found in HPG development under feeding with different diets. The maximum development degree was observed when fed with bee bread followed by pollen loads and mixture from Yeast, Gluten and sugar (1:1:2). The acinal surface of HPG showed clear difference under feeding with difference diets. The largest area was recorded when honeybee workers fed on bee bread followed by Yeast-Gluten-sugar mixture (diet,4) and pollen loads(diet,2).     

The dependence of lipid asymmetry upon phosphatidylcholine acyl chain structure

Lipid asymmetry, the difference in inner and outer leaflet lipid composition, is an important feature of biomembranes. By utilizing our recently developed MβCD-catalyzed exchange method, the effect of lipid acyl chain structure upon the ability to form asymmetric membranes was investigated. Using this approach, SM was efficiently introduced into the outer leaflet of vesicles containing various phosphatidylcholines (PC), but whether the resulting vesicles were asymmetric (SM outside/PC inside) depended upon PC acyl chain structure. Vesicles exhibited asymmetry using PC with two monounsaturated chains of >14 carbons; PC with one saturated and one unsaturated chain; and PC with phytanoyl chains. Vesicles were most weakly asymmetric using PC with two 14 carbon monounsaturated chains or with two polyunsaturated chains. To define the origin of this behavior, transverse diffusion (flip-flop) of lipids in vesicles containing various PCs was compared. A correlation between asymmetry and transverse diffusion was observed, with slower transverse diffusion in vesicles containing PCs that supported lipid asymmetry. Thus, asymmetric vesicles can be prepared using a wide range of acyl chain structures, but fast transverse diffusion destroys lipid asymmetry. These properties may constrain acyl chain structure in asymmetric natural membranes to avoid short or overly polyunsaturated acyl chains.     

Tracking the migratory success of stocked European eels Anguilla anguilla in the Baltic Sea

To investigate the extent to which European silver eels Anguilla anguilla, originating from stocking programmes in the Baltic Sea tributaries, effectively contribute to the spawning stock, two hundred and seventy‐four formerly stocked A. anguilla. emigrating from the Schwentine River near Kiel, Germany, were tagged with T‐Bar anchor tags. A total of 29 Anguilla spp. were recaptured (c. 11%) up to 14 months after release. Stocking history of recaptured A. anguilla. was confirmed by otolith microchemistry. Recapture locations were concentrated around the outlet of the Baltic Sea (Danish Belt Sea) with 62% of all recaptures reported here or in the Kattegat. Recaptured Anguilla spp. showed a reduction in both L_T and mass (mean ± s.d . = ?1·5 ± 0·9 cm and ?125·3 ± 50·1 g) while average total fat content remained in the order of values previously reported as high enough to provide energy resources to allow successful completion of the spawning migration (mean ± s.d . = 28·4 ± 4·4%). The documented mean rate of travel (0·8 km day^?1), however, indicated a delay in the target‐oriented migration that might be interpreted as a delayed initial migration phase of orientation towards the exit of the Baltic Sea.     

The Structure of the Cytochrome P450cam–Putidaredoxin Complex Determined by Paramagnetic NMR Spectroscopy and Crystallography

Cytochrome P450cam catalyzes the hydroxylation of camphor in a complex process involving two electron transfers (ETs) from the iron-sulfur protein putidaredoxin. The enzymatic control of the successive steps of catalysis is critical for a highly efficient reaction. The injection of the successive electrons is part of the control system. To understand the molecular interactions between putidaredoxin and cytochrome P450cam, we determined the structure of the complex both in solution and in the crystal state. Paramagnetic NMR spectroscopy using lanthanide tags yielded 446 structural restraints that were used to determine the solution structure. An ensemble of 10 structures with an RMSD of 1.3 Å was obtained. The crystal structure of the complex was solved, showing a position of putidaredoxin that is identical with the one in the solution structure. The NMR data further demonstrate the presence of a minor state or set of states of the complex in solution, which is attributed to the presence of an encounter complex. The structure of the major state shows a small binding interface and a metal-to-metal distance of 16 Å, with two pathways that provide strong electronic coupling of the redox centers. The interpretation of these results is discussed in the context of ET. The structure indicates that the ET rate can be much faster than the reported value, suggesting that the process may be gated.