Somatic embryogenesis in vitro cultures of Trifolium nigrescens Viv.

Celem niniejszej pracy była kompleksowa analiza systemu regeneracji w kulturze zarodków zygotycznych w stadium liścieniowym Trifolium nigrescens Viv. Opracowano optymalne warunki dla indukcji somatycznej embriogenezy (bezpośredniej i pośredniej), ustalono pochodzenie i budowę zarodków somatycznych wykorzystując techniki histochemiczne i skaningowy mikroskop elektronowy (SEM), dokonano analizy immunolokalizacji wybranych epitopów pektyn (JIM5, JIM7, LM5, LM6) oraz białek arabinogalaktanowych (AGPs) (JIM4, JIM8, JIM13, JIM16, LM2, MAC207) w eksplantacie i w trakcie rozwoju zarodków somatycznych, ustalono lokalizację transkryptu genu TnSERK1 na drodze hybrydyzacji in situ oraz porównano profile białkowe eksplantatów wyjściowych z eksplantatami z kultur in vitro. Na wszystkich zastosowanych pożywkach na bazie MS z dodatkiem różnych stężeń auksyny (0,1; 0,5 lub 4 mg/l NAA) przy stałej koncentracji cytokininy (2 mg/l 2iP) uzyskano bezpośrednią somatyczną embriogenezę w rejonie hipokotyla zarodka zygotycznego, przy czym na pożywce zawierającej 0,5 mg/l NAA oraz 2 mg/l 2iP najwyższy procent eksplantatów (48%) wytwarzał zarodki somatyczne, które rozwijały się w 68% w rośliny. Stężenie stosowanej auksyny miało wpływ na czas indukcji somatycznej embriogenezy; na pożywkach o niskich stężeniach NAA (0,1 lub 0,5 mg/l) zarodki pojawiały się po 7 dniach kultury natomiast przy stężeniu wyższym (4 mg/l) po 10 dniach. W toku prowadzenia kultury obserwowano kalus, z którego również różnicowały się somatyczne zarodki. Analiza histologiczna i obserwacje w SEM wykazały, że zarodki somatyczne miały pochodzenie wielokomórkowe i większość dojrzałych zarodków somatycznych była dwubiegunowa z wyraźnie zaznaczonymi dwoma liścieniami oraz merystemem korzenia. Niektóre somatyczne zarodki w stadium torpedy oraz komórki kalusa pokrywała charakterystyczna warstwa (ECMSN, extracellular matrix surface network). Pozytywny sygnał znakowania monoklonalnymi przeciwciałami JIM5 oraz JIM7 sugeruje, że w skład ECMSN wchodziły pektyny. Rozwojowi zarodków somatycznych towarzyszyło zmniejszenie różnorodności wykrywanych epitopów białek arabinogalaktanowych; żaden z epitopów lokalizowanych w eksplantacie wyjściowym (JIM8, JIM16, LM2, MAC207) nie był obecny w dojrzałych somatycznych zarodkach. Gen TnSERK1 ulegał ekspresji w komórkach somatycznych zarodków na różnym etapie rozwoju oraz w komórkach struktur zarodkopodobnych. Transkrypt tego genu nie był obecny w komórkach nieregenerujących. Porównanie profilu białkowego eksplantatów wyjściowych, eksplantatów z zarodkami somatycznymi oraz eksplantatów tylko kalusujących pozwoliło stwierdzić, że trzy białka obecne były w większej ilości w eksplantatach z somatycznymi zarodkami. Po raz pierwszy dokonano szczegółowego porównania udziału pektyn i białek arabinogalaktanowych w ścianach komórkowych zarodków zygotycznych i zarodków somatycznych; szeroko przedyskutowano rolę ściany komórkowej i specyficznej warstwy zewnątrzkomórkowej (ECMSN) w procesie różnicowania. Opracowanie protokołu wydajnego tworzenia zarodków somatycznych u gatunku ważnego ekonomicznie ma również duże znaczenie praktyczne jako metoda mikrorozmnażania ale też dostarczająca materiału do manipulacji genetycznych

Salinity-induced changes in plastoquinone pool redox state in halophytic Mesembryanthemum crystallinum L.

We have analyzed the effect of salinity on photosystem II (PSII) photochemistry and plastoquinone (PQ) pool in halophytic Mesembryanthemum crystallinum plants. Under prolonged salinity conditions (7 or 10 days of 0.4 M NaCl treatment) we noted an enlarged pool of open PSII reaction centers and increased energy conservation efficiency, as envisaged by parameters of the fast and slow kinetics of chlorophyll a fluorescence. Measurements of oxygen evolution, using 2,6-dichloro-1,4-benzoquinone as an electron acceptor, showed stimulation of the PSII activity due to salinity. In salt-acclimated plants (10 days of NaCl treatment), the improved PSII performance was associated with an increase in the size of the photochemically active PQ pool and the extent of its reduction. This was accompanied by a rise in the $NADP^{+}$/NADPH ratio. The presented data suggest that a redistribution of PQ molecules between photochemically active and non-active fractions and a change of the redox state of the photochemically active PQ pool indicate and regulate the acclimation of the photosynthetic apparatus to salinity

Morphohistological and flow cytometric analyses of somatic embryogenesis in Trifolium nigrescens Viv.

Microscopy and flow cytometry (FCM) were used to study somatic embryogenesis (SE) from zygotic embryos of Trifolium nigrescens Viv. to determine if there were any relationships between characteristics of somatic embryos (morphology, anatomy, genome size stability) and their regenerability. Embryoids were induced on Murashige and Skoog (MS) medium containing 4 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg l−1 N6-[2-isopentenyl]-adenine (2iP) either directly from hypocotyls or via an intervening callus, depending on the duration of culture. The morphology of somatic embryos varied from zygotic-like structures to abnormal structures including horn-shaped, polycotyledonary, and fused embryoids. The incidence of abnormalities was higher in callus cultures than in direct regeneration. Horn-shaped embryoids were the most frequent type of abnormal embryos. Only embryoids having zygotic-like morphology regenerated into plantlets. Histological observations revealed that the absence of shoot and root apical meristems along with parenchymatization of embryos were major obstacles to conversion of horn-shaped embryoids. The estimated 2C value for T. nigrescens was 0.9 pg. FCM analysis revealed differences in DNA content between embryoids induced via an intervening callus and those produced directly from explants. Individuals with species-specific as well as increased DNA content were detected among those zygotic-like embryos derived from callus, but all horn-shaped embryoids had increased genome sizes. The observed lack of differences in DNA content between zygotic-like and horn-shaped embryoids, from direct SE, indicated that these phenotypic abnormalities were of physiological origin. The mean DNA content of regenerants was species-specific, suggesting that only diploid embryoids were capable for regeneration into plantlets

Floral structure and pollen morphology of two zinc violets (Viola lutea ssp. calaminaria and V. lutea ssp. westfalica) indicate their taxonomic affinity to Viola lutea

Two zinc violets, the yellow form of the Aachen–Liège area and the blue morph of Blankenrode in western Westphalia, have very restricted occurrence on heavy metal waste heaps. Their taxonomic affinities have not been finally resolved. The flower micromorphological analysis presented here indicates that both zinc violets are closely related to the alpine Viola lutea, in line with our earlier published molecular data, but not with the conclusions of other authors. The zinc violets are classed at the rank of subspecies as V. lutea: ssp. calaminaria for the yellow zinc violet and ssp. westfalica for its blue counterpart. Although the violets examined (V. lutea, V. lutea ssp. calaminaria, V. lutea ssp. westfalica) are closely related, there is no evidence that V. lutea ssp. westfalica is a descendent of V. tricolor. Here we provide the most detailed information on generative organ structure in the four violets studied

Technika i technologia woskowych płaskorzeźb z XVIII-wiecznego warsztatu z Landshut w świetle badań „Madonny z Pochyloną Głową” ze zbiorów Muzeum Narodowego w Poznaniu

Technique and technology of making Bavarian wax sculptures in 18th century Landshut workshop.  A case study of the wax sculpture of Our Lady of the Bowed Head from the collection of National Museum in Poznań, Poland In 1930 National Museum in Poznań, Poland, acquired a polychrome wax relief of unrecognized artisan technology, unknown provenance and uncertain iconography, depicting a bust of the Virgin Mary emerging from dark clouds. This article presents the results of research undertaken over eighty years later, resulting in a stunning discovery of a long-lost piece from a highly specialized workshop in a small town of Landshut, Bavaria, Germany. In cooperation with German and Swiss specialists, researchers from Nicolaus Copernicus University in Toruń, Poland, determined a complex crafting technology of such artworks and classifid the analysed relief as one of its few remaining examples. Utilising available research methods, such as UV / IR colour imaging, X-ray diffraction, Fourier transform infrared spectroscopy and Gas Chromatography, followed by micro sampling and petrographic tests, it was possible to identify pigments and dyers used in polychrome layers as well as the composition of each particular wax layer, and inner structure of the object including armatures of wood and cork or spherical glass-blown balls used as eyes. The crucial part of the project were non-invasive analyses resulting in identifiation of rare pigments and dyes which were diffiult to identify with other means. These methods were combined with micro sampling, which led to discovery of ground minerals and fires in the polychrome layers. This paper focuses on the importance of the non-invasive analysis in the complicated process of diagnostics of artworks and emphasise their fundamental role in the overall conservation process

Blue light-dependent changes in loosely bound calcium in Arabidopsis mesophyll cells : an X-ray microanalysis study

Calcium is involved in the signal transduction pathway from phototropins, the blue light photoreceptor kinases which mediate chloroplast movements. The chloroplast accumulation response in low light is controlled by both phot1 and phot2, while only phot2 is involved in avoidance movement induced by strong light. Phototropins elevate cytosolic Ca(2+) after activation by blue light. In higher plants, both types of chloroplast responses depend on Ca(2+), and internal calcium stores seem to be crucial for these processes. Yet, the calcium signatures generated after the perception of blue light by phototropins are not well understood. To characterize the localization of calcium in Arabidopsis mesophyll cells, loosely bound (exchangeable) Ca(2+) was precipitated with potassium pyroantimonate and analyzed by transmission electron microscopy followed by energy-dispersive X-ray microanalysis. In dark-adapted wild-type Arabidopsis leaves, calcium precipitates were observed at the cell wall, where they formed spherical structures. After strong blue light irradiation, calcium at the apoplast prevailed, and bigger, multilayer precipitates were found. Spherical calcium precipitates were also detected at the tonoplast. After red light treatment as a control, the precipitates at the cell wall were smaller and less numerous. In the phot2 and phot1phot2 mutants, calcium patterns were different from those of wild-type plants. In both mutants, no elevation of calcium after blue light treatment was observed at the cell periphery (including the cell wall and a fragment of cytoplasm). This result confirms the involvement of phototropin2 in the regulation of Ca(2+) homeostasis in mesophyll cells

Znaczenie techniki aCGH w nowotworach mieloproliferacyjnych – przegląd literatury

Nowotwory mieloproliferacyjne (MPN) są klonalnymi chorobami komórek macierzystych szpiku, które charakteryzują się proliferacją jednej lub więcej linii mieloidalnych krwiotworzenia: granulocytowej, czerwonokrwinkowej i/lub megakariocytowej. Do MPN (-) zalicza się m.in. czerwienicę prawdziwą (PV), nadpłytkowość samoistną (ET) oraz pierwotną mielofibrozę (PMF). Nowotwory mieloproliferacyjne (-) należą do najmniej poznanych nowotworów układu krwiotwórczego pod względem nabytych zmian genetycznych. Badania nad MPN trwają od ponad 60 lat. Podobieństwo objawów klinicznych wskazuje na wspólną patogenezę PV, ET i PMF. Głównym celem prowadzonych badań jest wytypowanie markerów, które nie tylko pomogą w diagnostyce poszczególnych typów MPN czy w klasyfikacji chorych do grup ryzyka, ale mogłyby być także celem terapeutycznym. Głównym celem terapii jest przedłużenie życia pacjentów, w tym także zminimalizowanie prawdopodobieństwa transformacji w ostrą białaczkę. aCGH jest niewątpliwie techniką bardzo przydatną klinicznie, która, choć nie znalazła jeszcze rutynowego zastosowania w klinice hematoonkologii, to wykorzystywana jest w coraz większej liczbie badań, zwłaszcza do analizy nowotworów, w których istotne są aberracje niezrównoważone. Niniejsza praca przedstawia zastosowanie techniki aCGH w nowotworach mieloproliferacyjnych

A different pattern of production and scavenging of reactive oxygen species in halophytic Eutrema salsugineum (Thellungiella salsuginea) plants in comparison to Arabidopsis thaliana and its relation to salt stress signaling

Isolated thylakoids from halophytic Eutrema salsugineum (Thellungiella salsuginea) produces more H2O2 in comparison to glycophytic Arabidopsis thaliana. The first objective of this study was to verify whether this feature is relevant also to the intact chloroplasts and leaves. Enhanced H2O2 levels in chloroplasts and leaves of E. salsugineum were positively verified with several methods (electron microscopy, staining with Amplex Red and with diaminobenzidine). This effect was associated with a decreased ratio of O2.-/H2O2 in E.s in comparison to A.thaliana as detected by electron paramagnetic resonance (EPR) method. As a next step, we tested how this specific ROS signature of halophytic species affect the antioxidant status and down-stream components of ROS signaling. Comparison of enzymatic antioxidants revealed a decreased activity of ascorbate peroxidase (APX), enhanced activity of glutathione peroxidase (GPX), and the presence of thylakoid-bound forms of iron superoxide dismutase (FeSOD) and ascorbate peroxidase (APX) in E.s.. These cues were, however, independent from application of salt stress. The typical H2O2-dependent cellular responses, namely the levels of glucosinolates and stress-related hormones were determined. The total glucosinolate content in E.s water-treated leaves was higher than in A.t. and increased after salinity treatment. Treatment with salinity up-regulated all of tested stress hormones, their precursors and catabolites (abscisic acid, dihydrophaseic acid, phaseic acid, 1-aminocyclopropane-1-carboxylic acid, salicylic acid, jasmonic acid, cis-(+)-12-oxo-phytodienoic acid and jasmonoyl-L-isoleucine) in A.t., whereas in E.s. only a stimulation in ethylene synthesis and abscisic acid catabolism was noted. Obtained results suggest that constitutively enhanced H2O2 generation in chloroplasts of E.s. might be a crucial component of stress-prepardeness of this halophytic species. It shapes a very efficient antioxidant protection (in which glucosinolates might play a specific role) and a fine tuning of hormonal signaling to suppress the cell death program directed by jasmonate pathway

Usefulness and limitations of pollen characters in environmental studies based on Viola L. species (sect. Melanium Ging.)

The aim of these studies was to determine the effect of environmental pollution on pollen development. Pollen heteromorphism (the presence of pollen morphs differing in aperture number in one flower of a plant), pollen viability (stainability) and pollen grain size in European metallophytes from sect. Melanium Ging. (Viola L., Violaceae) were analyzed by SEM and histochemical staining.Plants’ tolerance to heavy metals is positively correlated with their pollen viability, which should be termed stainability as it depends on the staining method applied and is not correlated with pollen germination. Abortive pollen can be produced as an effect of heavy metals but also may result from hybridization, a very common phenomenon in pansies. Pollen stainability in hybrids can be high (even exceeding 70%) or low (barely above 20%), and stainable pollen grains can differ greatly in size (from very small to giant), indicating a cytological imbalance resulting from disturbed meiosis. The number of pollen apertures is an adaptive character in facultative metallophytes. Plants from a metallicolous population produced a wider range of aperture number (3, 4, 5) than plants from a non-metallicolous population, which developed only 4- and 5-aperturate pollen. Three-aperturate longer-lived pollen are favored in the harsh conditions of a metal-polluted environment