Improvement of Aconitum napellus micropropagation by liquid culture on floating membrane rafts

An efficient method was developed using floating membrane rafts (Liferaft) for the micropropagation of Aconitum napellus (Ranunculaceae), a cut flower crop with a low natural propagation rate. This was achieved by introducing shoot tips into culture on Murashige and Skoog's (1962) solid medium, or liquid medium-supported rafts, supplemented by different levels of benzyl adenine (BA). Optimum shoot proliferation on solid medium required 4mg/l BA, whereas for expiants supported on rafts optimal proliferation was achieved at 0.25mg/l BA. Maximum shoot proliferation was found using the floating rafts (propagation ratio of 4.2 per month), 45% higher than the maximum value on solid medium. A similar value could be obtained on solid medium after a period of 2 months. The optimal response to BA was similar for fresh weight gain and shoot length. Growth in a shallow layer of liquid in shake flasks gives a similar shoot multiplication rate to that on floating rafts; however, submerged leaves brown and die.Abbreviations BA 6-benzylaminopurine - GA₃ Gibberellic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - NAA naphthalene acetic acid     

The effects of NaCl on antioxidant enzyme activities in callus tissue of salt-tolerant and salt-sensitive cotton cultivars (Gossypium hirsutum L.)

Summary To determine NaCl effects on callus growth and antioxidant activity, callus of a salt-tolerant and a salt-sensitive cultivar of cotton was grown on media amended with 0, 75, and 150 mM NaCl. Callus of the salt-tolerant cultivar, Acala 1517-8 8, grown at 150 mM NaCl, showed significant increases in superoxide dismutase, catalase, ascorbate peroxidase, peroxidase and glutathione reductase activities compared to callus tissue grown at 0 mM NaCl. In contrast, callus tissue of the salt-sensitive cultivar, Deltapine 50, grown at 0, 75, and 150 mM NaCl, showed no difference in the activities of these enzymes. At the 150 mM NaCl treatment, peroxidase was the only antioxidant enzyme from Deltapine 50 with an activity as high as that observed in Acala 1517-88. The NaCl-induced increase in the activity of these enzymes in Acala 1517-88 indicates that callus tissue from the more salt-tolerant cultivar has a higher capacity for scavenging and dismutating superoxide, an increased ability to decompose H₂O₂, and a more active ascorbate-glutathione cycle when grown on media amended with NaCl.     

The use of rafts made of polymers with hydrophobic properties in organ culture--a new modification of Chen's raft method

A new modification of tissue culture technique implying the use of rafts of hydrophobic polymers is suggested. Polyethylene rafts appeared to be the most appropriate for tissue culture of chick pituitary. The rafts were prepared as disks with an opening in the center. Millipore filter carrying the explants was placed over the opening. The rafts had good buoyancy, remained on the surface of the culture media for 1-2 weeks and were reusable.     

Stimulation of in vitro root and shoot growth of potato by increasing sucrose concentration in the presence of fluridone,an inhibitor of abscisic acid synthesis

Fluridone, an inhibitor of abscisic acid (ABA) biosynthesis, strongly stimulated rooting of nodal stem segments of potato (Solanum tuberosum L.) cultivar Arran Banner cultured in darkness on tuberisation medium. Inclusion of 10^-6 M ABA in the culture medium prevented this rooting response, indicating that root proliferation in the presence of fluridone could be due to inhibition of ABA synthesis. The rooting response to fluridone (increased total root number and root fresh weight) was obtained only at high sucrose concentrations (0.175 and 0.234 M) and was demonstrated with two potato cultivars and two culture media; one which favoured tuberisation and one which did not. Shoot numbers were also increased, but to a lesser extent than root numbers, and total fresh weight of plant material per culture was greatly increased by inclusion of both fluridone (10^-6 or 10^-5 M) and 0.234 M sucrose in the culture medium. The role of sucrose was not simply osmotic because when the osmolarity of fluridone medium was increased using mixtures of mannitol and sucrose, no root proliferation occurred unless sucrose predominated in the mixture.     

Effects of free and chelated iron on in vitro androgenesis in barley and wheat

A suitable form of iron supplement in the induction medium was found to be important for further development of induced pollen embryos in barley and wheat cultivars (genotypes), especially those providing few green plants viain vitro androgenesis. Genotypes able to regenerate many green plants were less susceptible to the lack of iron in induction medium. Although Fe-EDTA was found to be a suitable form of iron in the induction medium, androgenesis was also induced on media containing non-chelated iron (Fe²⁺ and Fe³⁺ ions). EDTA alone without iron inhibited the androgenic response even in the wheat cv. Florida, a model cultivar for androgenesis in wheat. In all barley cultivars under study including cv. Igri, a model cultivar for androgenesis in barley, EDTA alone caused an almost total suppression of androgenesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Metalloendopeptidase EC3.4.24.15 is constitutively released from the exofacial leaflet of lipid rafts in GT1-7 cells

Metalloendopeptidase EC3.4.24.15 (EP24.15) is a physiologically important neuropeptide-degrading enzyme involved in the metabolism of multiple neuropeptides. The mechanism of release of EP24.15 from neuronal cells is multimodal, being both constitutive and stimulatable. Previous studies have characterized stimulated EP24.15 secretion, yet little is understood concerning constitutive release of the peptidase. Utilizing the mouse hypothalamic neuronal GT1-7 cell line, we demonstrate that EP24.15 exists within lipid rafts in the plasma membrane, and that the enzyme is localized to the exofacial leaflet of lipid rafts. Further, we have found that biotinylated EP24.15 on the extracellular surface is released into the cell media in a fashion similar to constitutive release. In addition, classical and non-classical secretion pathway inhibitors were employed to understand the release of EP24.15 into surrounding cell media. The non-classical secretion inhibitor glyburide, a blocker of ATP-sensitive K+ channels, decreased the amount of constitutively released EP24.15 in cell media of GT1-7 cells. With these data, we conclude that EP24.15 association with lipid rafts on the extracellular surface precedes constitutive release of the peptidase into the extracellular milieu for its action on neuropeptides.     

The influence of culture conditions on anther culture response of commercial varieties of Solanum tuberosum L.

The effect of media manipulatioss, temperature pretreatment, carbohydrate source, and seasonal variation on tetraploid potato anther cultures was investigated. The anther culture responses of three commercial Nordic potato varieties from Scandinavia and two from Germany were compared on different media manipulations. With most of the varieties, solid MS media gave better yields than other published media manipulations. Pretreatments at +6°C and at +30°C were studied on Pito and Danva varieties. The +6°C pretreatment and no pretreatment had the same effect on the anther culture response of cv. Pito, while with cv. Danva pretreatment at +6°C promoted embryogenesis. The +30°C pretreatment had no positive effect on anther culture response on either cultivar. The effect of maltose, melibiose and mannitol individually and in combination with sucrose were compared to normal sucrose medium in cv. Pito anther cultures. Anthers incubated on normal sucrose medium gave the highest embryoid and plant yields; the second highest plant yields were obtained on pure maltose medium. Strong seasonal variation was observed throughout the year in cv. Pito anther cultures. The percentage of anthers producing embryoids ranged from 15–20% during September and October to just 1–3% from February through May. The annual average embryoid production rate was 6.18%.     

The inhibition of infection thread development in the cultivar-specific interaction ofRhizobium and subterranean clover is not caused by a hypersensitive response

Summary The cultivar specific interaction ofTrifolium subterranean cv. Woogenellup andRhizobium leguminosarum bv.trifolii strain ANU 794 was examined to establish the basis for nodulation failure on this cultivar. Infections were initiated by strain ANU 794 on cv. Woogenellup. Root hair curling, the initiation of infection threads, and cortical cell divisions were evident on the tap root and appeared normal after microscopic observation. However, in most cases, the infection threads stayed confined to the root hairs. No evidence was found for a hypersensitive response by the plant. The progress of infections on the tap roots was different from that on the lateral roots. This was confirmed by the differential tap and lateral root nodulation patterns of the mutants derived from strain ANU 794, which show enhanced nodulation on cv. Woogenellup. On the lateral roots, cortical cell divisions progressed further than those on the tap root and formed macroscopically visible swellings, which could be divided into two morphological classes. In some cases infection threads developed into these primordia but successful nodules were not established. The inhibition of infection appeared to be manifested at two levels: first, on the tap roots in the root hairs, where many of the infection threads are contained and secondly, in the primordia induced on the lateral roots, where the infection threads sometimes penetrate further than the root hair cell but stop in the primordial cells. It appears that an essential factor or trigger in the communication between plant and bacteria is missing or altered, resulting in an array of primordia-structures, which cease to develop.Abbreviations bv biovar - cv cultivar - Fix⁺ nitrogen fixing - GUS -glucuronidase - Nod⁺ nodulating - HR hypersensitive response - Km kanamycin - LOSs lipo-oligosaccharides - Sm streptomycin - Sp spectinomycin - X-Gluc 5-bromo-4-chloro-3-indonyl--glucuronic acid     

Characterization of diazotrophic bacteria associated with maize: effect of plant genotype, ontogeny and nitrogen-supply

Summary The population size of diazotrophic bacteria naturally associated with the maize rhizosphere can be affected by biotic and environmental factors. In this work we have evaluated the effect of two genotypes of maize, with and without nitrogen fertilization, on the population dynamics and distribution of diazotrophic bacteria associated with maize plants over different plant ontogenic stages. The study was carried out in a field experiment with and without nitrogen fertilization, using two maize cultivars (Santa Helena 8447 and Santa Rosa 3063) previously selected from 32 maize cultivars for the lowest and highest response to nitrogen fertilization, respectively. Microbiological and molecular approaches were used to characterize the diazotrophic bacterial population structure. Bacterial population was assessed by the most probable number using semi-solid N-free media, and by DNA isolation from soil and plant tissue followed by amplification of nifH gene fragments using nested PCR, and by RFLP analysis using the restriction endonucleases TaqI and HaeIII. The dynamics of the diazotrophic bacterial population were affected by the ontogenic stage of the maize plants, but not by the cultivar type. Roots were the preferred site of colonization, independent of cultivar type and growth stage. During the first stage of maize growth, addition of nitrogen fertilizer negatively affected the diazotrophic bacterial population.     

Factors that affect plant regeneration from in vitro culture of immature seeds in four lentil cultivars

An efficient and simple method for plant regeneration from immature lentil seeds (Lens culinaris) is described. Immature seeds from 1 to 6 mm of four lentil cultivars were cultured in vitro on 10 different media. Culture media included different concentrations of N ⁶ -benzylaminopurine (BAP), alone or in combination with other phytohormones. After 4 weeks in culture, multiple shoot regeneration was observed using media with BAP. Immature seed size showed significant effect on shoot regeneration. Regenerated shoots (up to 4 shoots per explant on medium with Kinetin (KN) and from 5 to 20 on media with BAP) formed adventitious roots 30 days after transferring them to a medium containing 11.4 μM indole-3-acetic acid (IAA). The efficiency of the rooting medium varied depending upon the shoot-regeneration medium and the cultivar tested. The highest rooting percentage (88.9%) was obtained from regenerated shoots of the cultivar Verdina on a medium with 1 μM α-naphthaleneacetic acid (NAA). This revised version was published online in June 2006 with corrections to the Cover Date.     

Lipid raft connection between extrinsic and intrinsic apoptotic pathways

Apoptosis in mammalian cells is modulated by extrinsic and intrinsic signaling pathways through the formation of death receptor-mediated death-inducing signaling complex (DISC) and mitochondrial-derived apoptosome, respectively. We found by ultrastructural approaches that the antitumor drug edelfosine induced aggregates of lipid rafts containing Fas/CD95 receptor and Fas-associated death domain-containing protein in leukemic cells. Death receptors together with DISC and apoptosome constituents were recruited in rafts during edelfosine treatment in multiple myeloma cells. This apoptotic response involved caspases-8/-9/-10 that were translocated to rafts. Lipid raft disruption by cholesterol depletion inhibited loss of mitochondrial transmembrane potential, caspase activation and apoptosis, whereas cholesterol replenishment restored these responses. Our data indicate that rafts act as scaffolds where extrinsic and intrinsic apoptotic signaling pathways concentrate, forming clusters of apoptotic signaling molecule-enriched rafts (CASMER), which function as novel supramolecular entities in the triggering of apoptosis, and play an important role in edelfosine-induced apoptosis in blood cancer cells.     

Cultivar and cultivar x environment effects on the development of callus and polyhaploid plants from anther cultures of wheat

Summary Plants of three common wheat (Triticum aestivum L. em. Thell) cultivars and one randomly selected doubled-haploid line derived by anther culture from each of the three cultivars were each grown in three environments, a field environment, a greenhouse environment, and a growth chamber environment. Anthers containing largely miduninucleate to late uninucleate microspores were cultured and calli were induced to regenerate plants in order to assess the effects of cultivar, cultivar family (cultivar and corresponding doubled-haploid derivative), anther-donor plant environment, and cultivar X environment interaction on androgenic responses. Large differences in response were observed among cultivars as well as between cultivars and doubled-haploids. Differences between cultivar and doubled-haploid within cultivar family usually resulted from higher frequency of response in the cultivar, contrary to the hypothesis that anther culture per se constitutes a general selective device for superior androgenic responses. Also, in a second experiment, anther callusing frequency was greater in the cultivar Kitt than in any of five unique doubled-haploid lines derived from Kitt. Significant effects were also observed in the first experiment for the interactions of cultivar family X environment as well as doubled-haploid vs. cultivar X environment, although the effect of environment itself was less significant than these interactions.Contribution from the USDA, SEA, AR, Beltsville, Md, and the Department of Agronomy, University of Maryland, College Park, Md, as scientific article No. A-3413, contribution No. 6486     

Comparative responses of ovipositing Anopheles gambiae and Culex quinquefasciatus females to the presence of Culex egg rafts and larvae

Field observations have demonstrated that gravid Anopheles gambiae Giles s.s. (Diptera: Culicidae) are selective in their choice of oviposition sites. For example, immature stages of An. gambiae s.s. are rarely found in water that contains Culex quinquefasciatus Say immatures. The possibility that this may, in part at least, reflect a response by ovipositing An. gambiae s.s. females to volatile signals associated with Culex juveniles was evaluated by testing the response of An. gambiae s.s. females to varying densities of Cx. quinquefasciatus egg rafts and/or larvae in oviposition choice assays. For comparison, the oviposition choices of Cx. quinquefasciatus to conspecific egg rafts and/or larvae were similarly assayed. At a low density of Cx. quinquefasciatus egg rafts (1-15 egg rafts/100 mL water), An. gambiae s.s. females laid more eggs in the treatment water than in the control, with a maximum of twice as many in the treatment water at 5 egg rafts/100 mL water. At higher egg raft densities and in all treatments that included Cx. quinquefasciatus larvae, oviposition decreased significantly in the treatment dishes in a density-dependent manner. As previous studies have indicated, ovipositing Cx. quinquefasciatus females were attracted to and laid egg rafts in dishes containing conspecific egg rafts and, interestingly, also in dishes containing larvae.     

Effect of microspore stage and media on anther culture of peanut (Arachis hypogaea L.)

This study was designed to study the effects of stage of microspore development and culture medium on androgenic response in peanut (Arachis hypogaea L.). Anthers of various developmental stages were cultured for 7 days, then fixed and observed cytologically. Three sets of media, involving different basal media, growth regulators, sucrose levels and glutamine concentrations, were tested. In all experiments, the stage of development of the microspores at the time of culture was highly significant. The early uninucleate microspores stage was identified as producing the highest anther response rating. The effect of media was nonsignificant in all experiments. However, the stepwise modification of the media through the course of the study resulted in an almost 8 x increase in anther response rating. Numerically, the best media tested was N₆ basal medium with 1 mg 1^-1 NAA, 0.1 mg 1^-1 BA, 5.5% sucrose, and 3.5 g 1^-1 glutamine. While no haploids were obtained, four-nucleate cells were observed, indicating the potential in peanuts for an androgenic reponse.     

Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase

The phosphatidylinositol (PI) signaling pathway mediates norepinephrine (NE)- and endothelin-1 (ET-1)-stimulated vascular smooth muscle contraction through an inositol-trisphosphate-induced rise in intracellular calcium and diacylglycerol (DG) activation of protein kinase C (PKC). Subsequent activation of DG kinases (DGKs) metabolizes DG to phosphatidic acid (PA), potentially regulating PKC activity. Because precise regulation and spatial restriction of the PI pathway is necessary for specificity, we have investigated whether this occurs within caveolae/rafts, specialized plasma membrane microdomains implicated in vascular smooth muscle contraction. We show that components of the PI signaling cascade-phosphatidylinositol 4,5-bisphosphate (PIP(2)), PA, and DGK-theta are present in caveolae/rafts prepared from rat mesenteric small arteries. Stimulation with NE or ET-1 induced [(33)P]PIP(2) hydrolysis solely within caveolae/rafts. NE stimulated an increase in DGK activity in caveolae/rafts alone, whereas ET-1 activated DGK in caveolae/rafts and noncaveolae/rafts; however, [(33)P]PA increased in all fractions with both agonists. Previously, we reported that NE activated DGK-theta in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner; here, we describe PI3-kinase-dependent DGK activation and [(33)P]PA production in caveolae/rafts in response to NE but not ET-1. Additionally, PKB, a potential activator of DGK-theta, translocated to caveolae/rafts in response to NE but not ET-1, and PI3-kinase inhibition prevented this. Furthermore, PI3-kinase inhibition reduced the sensitivity of contraction to NE but not ET-1. Our study shows that caveolae/rafts are major sites of vasoconstrictor hormone activation of the PI pathway in intact small arteries and suggest a link between lipid signaling events within caveolae/rafts and contraction.     

Identification and cloning of a submergence-induced gene OsGGT (glycogenin glucosyltransferase) from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) by suppression subtractive hybridization

A submergence-induced gene, OsGGT, was cloned from 7-day submerged rice (Oryza sativa L. plants, FR13A (a submergence-tolerant cultivar, Indica), using suppression subtractive hybridization and both 5- and 3-rapid amplification of cDNA ends (RACE). The full-length OsGGT cDNA contains 1,273 bp with an open reading frame of 1,140 bp (17–1,156) that encodes 379 amino acids. Its deduced amino acid sequence is homologous with glycogenin glucosyltransferase. We found that the OsGGT gene is located in the 17,970–20,077 bp region of genome fragment AAAA01002475.1 of the Indica cultivar and in the 53,293–51,186 bp region of genome fragment AC037426.12 of chromosome 10 of the Japanica cultivar. A time-course study showed that OsGGT-gene expression increased in FR13A during submergence but decreased in IR42 (submergence-intolerant cultivar, Indica). The expression of the OsGGT gene in FR13A was induced by salicylic acid and benzyladenine. The accumulation of OsGGT mRNA in FR13A also increased in response to ethylene, gibberellin, abscisic acid, drought and salt treatment, but methyl jasmonate treatment and cold stress had no effect on expression. These results suggest that the OsGGT gene could be related to submergence stress and associated with a general defensive response to various environmental stresses.The nucleotide sequences of OsGGT cDNA has been submitted to GenBank DDBJ under accession numbers AB164463.     

Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.     

Genotypic differences in phosphorus efficiency of wheat

In an attempt to evaluate whether breeding and selection for high yielding capacity did change the P requirements of modern wheat cultivars, the response of two wheat cultivars to different levels of P supply was investigated. A traditional cultivar ("Peragis") and a modern cultivar ("Cosir") were cultivated in a C-loess low in available P and high in CaCO₃ in 120 cm high PVC pots. Shoot and root growth at different developmental stages was compared. The grain yield of the modern cultivar Cosir was higher at limiting and non-limiting P supply and, therefore, this cultivar can be considered as more P-efficient than the traditional cultivar. From the results it can be concluded that the main factors contributing to the higher P efficiency of the modern cultivar are (i) efficient use of assimilates for root growth characteristics which enhance P acquisition: smaller root diameter, and longer root hairs, (ii) efficient remobilization of P from vegetative plant organs to the grains, and (iii) lower P requirement for grain yield formation because of lower ear number per plant but higher grain number per ear.     

Micropropagation of a Local Olive Cultivar for Germplasm Preservation

In vitro shoot culture was applied to an Italian local cultivar Nebbiara of olive (Olea europaea L.) to preserve its endangered germplasm. This cultivar showed a notable difficulty for the in vitro establishment due to heavy pathogen contamination. Mercury chloride and sodium hypochloride in the sterilisation step and antibiotics in culture media allowed to overcome the problem. Proliferation of shoot apical bud on olive culture medium with 36 g dm^–3 mannitol and 4.56 M zeatin appeared very satisfactory. All the explants tested rooted during a subculture (1 month) preceeded by a 5-d long dark pre-treatment.