Are Early Somatic Embryos of the Norway Spruce (Picea abies (L.) Karst.) Organised?

Background

Somatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters.

Methodology/Principal Findings

The main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56^th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the ¹H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we investigated the effect of cluster orientation on the cultivation medium and the influence of the change of the cluster’s three-dimensional orientation on its development. Maintaining the same position when transferring ESEs into new cultivation medium seemed to be necessary because changes in the orientation significantly affected ESE growth.

Conclusions and Significance

This work illustrated the possible inner organisation of ESEs. The outer layer of ESEs is formed by individual somatic embryos with high metabolic activity (and with high demands for nutrients, oxygen and water), while an embryonal group is directed outside of the ESE cluster. Somatic embryos with depressed metabolic activity were localised in the inner regions, where these embryonic tissues probably have a very important transport function.     

Possible Involvement of PPAR-γ in the Anticonvulsant Effect of Aegle marmelos (L.) Correa

Aegle marmelos is well documented for antihyperglycemic effect and PPAR-γ activation has been suggested to be the molecular mechanism of its action. Also, the plant has been used in Ayurveda as a brain tonic and has been postulated to have antidepressant activities. The present study was designed to investigate the anticonvulsant effects of A. marmelos leaf extract (AME) in pentylenetetrazole and maximal electroshock induced convulsions; involvement of PPAR-γ, nitric oxide pathway and effect of chronic AME treatment on post-ictal depression. AME was administered at doses of 50, 100 and 200 mg kg^?1 in PTZ and MES model. Severity of convulsions was noted in both the models. Pretreatment with bisphenol A diglycidyl ether (BADGE) was used to study the involvement of PPAR-γ and l-arginine and N-nitro-l-arginine methyl ester hydrochloride (l-NAME) to study the involvement of nitric oxide (NO). Chronic treatment with AME interspersed with sub maximal doses of PTZ (50 mg kg^?1) on every fifth day up to 15 days was given to study post-ictal depression using forced swimming and actophotometer. AME showed significant increase in the onset time and decrease in the duration of convulsions in PTZ and MES models dose dependently. In MES a dose of 100 mg kg^?1 had effect comparable to phenytoin. Pretreatment with BADGE and l-arginine reversed the protective effect while l-NAME did not alter the protective effect, thereby indicating possible involvement of PPAR-γ and inhibition of NO. Chronic AME treatment ameliorated the post-seizure depression significantly as evidenced by increase in the locomotor activity and decrease in the immobility time.     

Regulation of α-galactosidase activity and the hydrolysis of galactomannan in the endosperm of the fenugreek (Trigonella foenum-graecum L.) seed

When endosperms were isolated from fenugreek seeds 5 h after sowing and incubated in a small volume of water, the development of α-galactosidase activity and the breakdown of the galactomannan storage polysaccharide were both inhibited relative to control endosperms incubated in larger volumes. The inhibition could be relieved by pre-washing the endosperms, and reimposed by the wash-liquors. If the endosperms were isolated 24 h after sowing, no inhibition was observed. Removal of the embryonic axis from germinating fenugreek seeds and from germinated seedlings also inhibited the development of α-galactosidase activity and galactomannan breakdown in the endosperms; the inhibition was more pronounced the earlier the axis was removed. Axis excision 5 h after sowing caused a delay in the onset of galactomannan breakdown and of the appearance of α-galactosidase activity in the endosperms. It also led to a decrease in the rates of galactomannan breakdown and α-galactosidase production. Axis excision 24 h after sowing caused only a slowing of the rates of galactomannan breakdown and α-galactosidase increase. The inhibition caused by axis removal at 5 h could be relieved partially by gibberellin (10^-4 M), benzyladenine (10^-5 M), mixtures of these and by the herbicide SAN 9789 [4-chloro-5-(methylamine)-2-(α,α,α-trifluoro-m-tolyl)-3-(2H)-pyridazinone]. These substances had no effect on the inhibition caused by axis-removal at 24 h. Excision of the cotyledons at 5 h-leaving the separated axis and the endosperm-also caused inhibition of galactomannan breakdown and α-galactosidase development. The results are consistent with the presence in the fenugreek seed endosperm of diffusible inhibitors of galactomannan mobilisation which are removed or inactivated during normal germination and early seedling development. They are also consistent with a role for the seedling axis in the control of galactomannan breakdown in the endosperm. Initially the axis appears to have a regulatory function (via gibberellins and/or cytokinins?) in determining the onset of α-galactosidase production in the endosperm. Thereafter its continued presence is necessary to ensure maximal rates of α-galactosidase production and galactomannan hydrolysis. The role of the axis may be initially to counteract the endogenous inhibitors in the endosperm and then to act as a sink for the galactomannan breakdown products released in the endosperm and taken up by the cotyledons.     

The Fine Structural Observation on the Development of Embryo and Endosperm in the Intergene- rfc Crosses of Fadish ♀ (Raphanus sativus L.) Cabbageo ♂ (Brassica oleracea L.)

The present report deals with the structural abnormalities and abortion of the endosperm, and the related abnormal development of the embryos in the intergeneric crosses of radish (Raphanus sativus L.) ♀ with cabbage (Brassica oleracea L.) ♂. The weak development of ER, the occurence of starch grains in chloroplasts, the curious distribution of chloroplasts around the nuclei, and the earlier formation of cellular endosperm are some primary structure abnormalities of endosperm and they may indicate the poor development, low metabolic activities, and precocious growth in the hybrid endosperm. The endosperm abortion, which starts from chalazal end endosperm, comprises the damage of both structure and function of ER membrane, and the disintegration of nuclei and organelles, or even death. The hybrid embryos degenerated very early in 2-cell stage or even zygote stage in case the endosperm Aborted early. In case of endosperm late-aborted, the hybrid embryos grew for quite a long period of time but become slow down as the endosperm showed abnormal development and started to abort. As the results, the embryos were smaller, the embryo propers and the suspensors showed no growth in step with one another, and the embryo cells appeared structural abnormalities, and finally degenerated and aborted.     

Cytology of the outer penis epithelium ofBuccinum undatum (L.) from the arctic region — an observation of the imposex phenomenon

TBT (tributyltin)-induced imposex is observed in femaleBuccinum undatum from the arctic region. The fine structure of the male penis epithelium was investigated and the results were compared between specimens from non-polluted (Spitsbergen, Norway) and highly-polluted (Brittany, France) areas. Differences in the arrangement and structure of the apical microvilli-border, the intercellular spaces, RER and SER content, as well as a high rate of parasitism in the tissue of individuals from Brittany can be detected between the species of both areas. The results indicate a higher degree of pathological changes in the penis epithelial cells of individuals from TBT-polluted areas than in those from Spitsbergen.     

ПРИСПОСОБЛЕНИЯ К ПЛАНИРОВАНИЮ ПЕРЕДНИХ КОНЕЧНОСТЕЙ У ЛЕТЯГИ(Pteromys volans L.)

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苹果蠹蛾Cydia pomonella(L.)

<正>苹果蠹蛾Cydia pomonella(L.)是世界重要的苹果害虫,原产于欧洲中南部,现在已经入侵世界5大洲71个国家。该入侵昆虫1953年首次报道在中国新疆发现,目前已入侵新疆、甘肃、宁夏、内蒙古、黑龙     

Isozyme polymorphism and genetic structure of the population of Sorbus torminalis (L.) Crantz from the Bytyń Forest (Poland)

Dormant buds collected from 35 wild service trees (Sorbus torminalis) in the Bytyń Forest were tested with horizontal gel electrophoresis to assess the genetic structure of the population. Among 16 investigated isozyme loci, seven loci (ADH-A, 6PGD-A, GDH-B, ME-A, SOD-A, PGM-A, PGM-B) proved to be polymorphic, whereas the other nine loci (SDH-A, SDH-B, DIA-C, DIA-D, FLE-A, FLE-B, GOT-B, IDH-A, IDH-B) were monomorphic. The number of alleles per polymorphic locus ranged from two to three, with a mean of 2.29. The average observed and expected heterozygosities were 0.2665 and 0.3462, respectively. The combined FIS value over all polymorphic loci was 0.2179, which reflects a substantial deficit of heterozygotes. Two polymorphic loci (SOD-A, PGM-A) were identified in S. torminalis for the first time.     

A review of the reproductive behavior of Polyphemus pediculus (L.) Müller (Crustacea: Branchiopoda)

The reproductive behavior of Polyphemus pediculus (L.) includes mating, laying of resting eggs and giving birth to juveniles. All these forms of behavior are performed in a population daily and simultaneously by means of individual and social actions. Individual behavior is manifested by certain sets of stereotyped movements which can be observed at any time of the day and at any location in a water body. Individual behavior has an episodic, stable character and low intensity. Social behavior is accomplished by specimens of similar physiological state who form transitory swarms. They form within the main continually existing main swarms of the population. Social behavior is rhythmic and of high intensity. Reproductive parthenogenetic swarms first form around dawn and later during the day: about noon in May–July and before sunset in Autumn. As swarms of parthenogenetic females disintegrate, swarms of old gamogenetic females form and mass laying of resting eggs begins. Young gamogenetic females and males swarm only when resting-egg-laying swarms disintegrate. Parthenogenetic reproduction peaks in northern Russia in May and August and persists until late September. Swarms of resting-egg-laying females form once a day around dawn, from May to September. Mating swarms form in summer in midmorning, at twilight and on moonlit nights. In Autumn, there is only one peak of mating, in midmorning. Intensity, duration and time of manifestation of different forms of social behavior change from spring to autumn. Location and character of their manifestation are predictable.     

Hormonal regulation of gene expression in the “slender” mutant of barley (Hordeum vulgare L.)

The slender mutant of barley resembles a normal barley plant treated with high doses of gibberellic acid (GA₃). Expression of GA₃-regulated and abscisic acid (ABA)-regulated mRNAs was studied in the endosperm and roots of mutant and wild-type (WT) plants.Production of -amylase (EC 3.2.1.1) by WT embryoless half-grains was dependent on the presence of GA₃, and was prevented by ABA. In contrast, -amylase was produced by half-grains of the slender mutant in the absence of added GA₃, although it was still reduced by ABA. The spectrum of -amylase mRNAs in slender embryoless half-grains incubated in the absence of added GA₃ was the same as in WT endosperm half-grains incubated in the presence of GA₃. These results indicate that the endosperm of the slender mutant exhibits similar properties to WT endosperm treated with GA₃.In roots the expression of an ABA-inducible mRNA was similar in slender and WT seedlings either treated with exogenous ABA or exposed to dehydration. This result, and the effect of ABA on -amylase production by the endosperm, indicate that the slender plants retain sensitivity to ABA.Abbreviations ABA abscisic acid - AMV avian myeloblastosis virus - GA gibberellin - GA₁ gibberellin A₁ - GA₃ gibberellic acid - WT wild-type     

Genetic structure of sable (Martes zibellina L.) in Eurasia—analysis of the mitochondrial lineages distribution

The phylogeography of the sable, which is a commercially valuable species, is extremely complicated and poorly investigated. Specifically, the effects of factors such as the range dynamics of the sable during the Pleistocene Epoch, the localization of glacial refugia, species distribution pattern in Holocene, and recent dramatic population decline, along with massive reacclimatization measures, on the species phylogeography remain unclear. Based on the sequence analysis of the control region of mitochondrial DNA from sables that inhabit different parts of the species range, a suggestion was made of the considerably high Pleistocene genetic diversity in sable, which was subsequently lost. The initial diversity of mitochondrial lineages is mostly preserved in the Urals, while in the eastern part of the range, it seems to have been depleted as early as before the last glacial maximum. On the other hand, the even greater depletion of the mitochondrial lineages observed in some populations of central Siberia can be associated with the dramatic population decline at the turn of the 20th century.     

Does body size difference in the leeches Glossiphonia complanata (L.) and Helobdella stagnalis (L.) contribute to co-existence?

The effect of predator and prey body size on the feeding success of the British lake-dwelling leeches Glossiphonia complanata and Helobdella stagnalis was examined in the laboratory, and any involvement of size difference between the leeches in allowing coexistence in the field assessed. G. complanata breeds in advance of H. stagnalis and maintains a body size advantage throughout their annual life-cycle. In experiments, conducted at 14 °C and a photoperiod of 16 hrs L: 8 hrs D, three size classes of leeches of each species were each exposed to each of three size classes of each of five prey species, viz. Tubifex sp., Chironomus sp., Asellus aquaticus, Lymnaea peregra and Potamopyrgus jenkinsi. For each prey species, three different types of experiments were performed: one leech exposed to four prey individuals; four leeches of the same species with sixteen prey; and two leeches of each species with sixteen prey. In the first experiment, all sizes of G. complanata were capable of feeding on all sizes of the prey types offered; the same was true for H. stagnalis with exceptions of feeding on large A. aquaticus and large L. peregra. For both species, but especially for G. complanata, there was a trend within each size class of leech for decreasing proportions of fed leeches with increasing prey size, and within each size class of prey for an increasing proportion of fed leeches with increasing leech size; however there were several exceptions to these trends. Both leeches fed extensively on Tubifex sp. but there were significant differences in the proportions feeding on other prey types; G. complanata fed more on A. aquaticus and the two snail species, and less on Chironomus, than H. stagnalis. The effect of increasing the number of leech individuals from one to four individuals, of the same or mixed species, had little effect on the proportion of leeches which had fed. It is concluded that large G. complanata will have access to large individuals of certain prey taxa denied H. stagnalis, which may lessen the intensity of interspecific competition.     

Inheritance and mapping of the ore gene controlling the quantity of β-carotene in cucumber (Cucumis sativus L.) endocarp

The metabolic precursor of vitamin A, ??-carotene, is essential for human health. The gene(s) controlling ??-carotene quantity (Q??C) has been introgressed from Xishuangbanna gourd (XIS, possessing ??-carotene; Cucumis sativus L. var. xishuangbannanesis Qi et Yuan; 2n?=?2x?=?14) into cultivated cucumber (no ??-carotene; Cucumis sativus L.). To determine the inheritance of Q??C in cucumber fruit endocarp, F1 progeny and a set of 124 F7 recombinant inbred lines (RILs) derived from the cultivated cucumber line CC3 and XIS line SWCC8 were evaluated for Q??C during 2009 and 2010 in Nanjing, China. Segregation analysis revealed that endocarp Q??C of greenhouse-grown fruit was controlled by a single recessive gene. Further, marker analysis indicated the gene controlling Q??C was linked to seven SSR markers on linkage group 3, where their order was SSR20710?CSSR19511?CSSR15419?CSSR07706?Core?CSSR23231?CSSR11633?CSSR20270. These markers and the putative candidate gene were mapped to cucumber chromosome 3DS. An evaluation of 30 genetically diverse cucumber lines indicated that marker SSR07706 has utility in further genetic analyses of the Q??C orange endocarp gene, designated ore. Moreover, the markers defined herein may have utility for marker-assisted selection directed towards the development of cucumber germplasm with high fruit ??-carotene content.     

The use of faecal markers for the delimitation of the European rabbit’s social territories (Oryctolagus cuniculus L.)

Olfactory signalling is widespread among mammal inter- and intra-specific communication systems. Social mammals use latrines (faecal accumulations) as a communication system, which address the needs of individuals both inside and outside the social group. Latrines are commonly used as olfactory and visual signals for the delimitation of territories, and the location of these signals may thus provide useful clues for the study of the marking animals’ behaviour and the delimitation of a spatial framework for these studies. In this work, we have tested the application of indigestible faecal markers in order to define territorial boundaries marked by latrines in wild rabbit social groups. Different combinations of baits and markers were first tested on wild rabbits kept in captive conditions, and the best combinations were then applied to rabbits in semi-natural conditions in order to test their feasibility for use with wild populations. As one possible application of the method, Minimum Convex Polygons and Kernel density estimators were used to analyse the distribution of the latrines of three wild rabbit social groups in semi-natural conditions. The results of this showed that the marked bait method is a useful tool for exploring the territorial behaviour of social mammals that use latrines for communication. It can also provide useful information for the definition of spatial frameworks based on social structures, and is fast, straightforward, easy, cheap, and does not require specialised training.     

Emission of hydrogen sulfide by twigs of coniferes — acomparison of Norway spruce (Picea abies (L.) Karst.), Scotch pine (Pinus sylvestris L.) and Blue spruce (Picea pungens Engelm.)

The emission of reduced volatile sulfur compounds from twigs of Norway spruce (Picea abies (L.) Karst.) was measured in the field by cryosampling and gaschromatographic analysis. Trees were growing in the Erzgebirge (E-Germany) at Oberbärenburg and at the Kahleberg and at a third stand in NW-Bavaria (S-Germany). Emission rates were also measured for Scotch pine (Pinus sylvestris L.) and Blue spruce (Picea pungens Engelm.) at the Kahleberg. Twigs still attached to the trees were enclosed in a flow-through gas exchange cuvette. H₂S was detected as the predominant reduced sulfur compound emitted from the twigs. The mean H₂S emission rate from twigs of Norway spruce varied between 0.04 pmol kg^-1 dw s^-1 at Würzburg and 6.21 pmol kg^-1 dw s^-1 at the Kahleberg. Comparing different species at the Kahleberg, the mean H₂S emission rate was almost the same from twigs of Norway spruce (6.2 pmol kg^-1 dw s^-1) and Blue Spruce trees (5.9 pmol kg^-1 dw s^-1) but it was approximately 18 times higher for Scotch pine (110 pmol kg^-1 dw s^-1). The percentage of SO₂-exclusion via H₂S-emission of the tree species investigated at the Kahleberg is calculated on the basis of data on SO₂ fluxes. It is very small for Norway spruce and Blue spruce. However, for Scotch pine, H₂S emission contributes about 10% to the detoxification of SO₂.     

A Bayesian analysis of gene flow from crops to their wild relatives: cultivated (Lactuca sativa L.) and prickly lettuce (L. serriola L.) and the recent expansion of L. serriola in Europe

Interspecific gene flow can lead to the formation of hybrid populations that have a competitive advantage over the parental populations, even for hybrids from a cross between crops and wild relatives. Wild prickly lettuce (Lactuca serriola) has recently expanded in Europe and hybridization with the related crop species (cultivated lettuce, L. sativa) has been hypothesized as one of the mechanisms behind this expansion. In a basically selfing species, such as lettuce, assessing hybridization in natural populations may not be straightforward. Therefore, we analysed a uniquely large data set of plants genotyped with SSR (simple sequence repeat) markers with two programs for Bayesian population genetic analysis, STRUCTURE and NewHybrids. The data set comprised 7738 plants, including a complete genebank collection, which provided a wide coverage of cultivated germplasm and a fair coverage of wild accessions, and a set of wild populations recently sampled across Europe. STRUCTURE analysis inferred the occurrence of hybrids at a level of 7% across Europe. NewHybrids indicated these hybrids to be advanced selfed generations of a hybridization event or of one backcross after such an event, which is according to expectations for a basically selfing species. These advanced selfed generations could not be detected effectively with crop‐specific alleles. In the northern part of Europe, where the expansion of L. serriola took place, the fewest putative hybrids were found. Therefore, we conclude that other mechanisms than crop/wild gene flow, such as an increase in disturbed habitats and/or climate warming, are more likely explanations for this expansion.     

Response of the gut neuroendocrine system of Leuciscus cephalus (L.) to the presence of Pomphorhynchus laevis Müller, 1776 (Acanthocephala)

Immunohistochemical tests were applied to sections of intestine of uninfected and Pomphorhynchus laevis Muller-infected chub, Leuciscus cephalus (L.) using 15 different antisera. Nerve cell bodies and fibres immunoreactive (IR) to the anti-bombesin, -Cholecystokinin-8 (CCK-8), -galanin, -Gastrin-Releasing Peptide (-GRP), -Nitric Oxide Synthase (-NOS), -Substance P (-SP), and -Vasoactive Intestinal Peptide (-VIP) sera were observed in the myenteric plexus of uninfected chub. The density of nerve components immunoreactive to these antisera was high in the intestine of the infected fish, especially near the site of attachment. Moreover, numerous nerve fibres, immunoreactive to anti-bombesin, -GRP, -galanin, -SP, and -VIP sera, were encountered in the connective tissue capsule surrounding the bulb and proboscis of P. laevis. The occurrence of P. laevis in the chub gut significantly increased the number of endocrine cells per intestinal fold immunoreactive to galanin, met-enkephalin and leu-enkephalin antisera. CCK-8, Neuropeptide Y and glucagon-like immunoreactive cells were less numerous in the intestine of infected chub. A large number of cells in the tunica propria-submucosa of L. cephalus infected with P. laevis were immunoreactive to anti-serotonin and -leu-enkephalin sera.     

Characterisation of the cDNA clones of two β-tubulin genes and their expression in the potato plant (Solanum tuberosum L.)

The cDNA clones of two potato -tubulin genes were isolated from a tuberising stolon tip library. Analysis of 20 positive clones showed that they represented one or another of two different but very similar -tubulin genes, designated TUBST1 and TUBST2. The expression pattern of -tubulin genes in the potato plant was investigated by RNA blot analysis and by RT-PCR. Southern analysis of potato genomic DNA with coding and non-coding -tubulin probes revealed that there are multiple -tubulin genes in the potato genome and that there is likely to be considerable divergence in the 3 non-coding sequences. Phylogenetic analysis of plant -tubulin genes is described.