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Plant Development and EvoDevo
Somatic Embryogenesis: 60 Years of Research Applied to Plant Cloning to Unravel Plant Totipotency View all 26 Articles
Munetaka Sugiyama
The University of Tokyo, Japan
Glória C. Pinto
University of Aveiro, Portugal
Tsuyoshi E. Maruyama
Forestry and Forest Products Research Institute, Japan
Front. Plant Sci., 20 December 2018 | https://doi.org/10.3389/fpls.2018.01898
Temperature and Water Availability During Maturation Affect the Cytokinins and Auxins Profile of Radiata Pine Somatic Embryos
Paloma Moncaleán1, Olatz García-Mendiguren1, Ondrej Novák2, Miroslav Strnad2, Tomás Goicoa3, María D. Ugarte3 and Itziar A. Montalbán1*
1Department of Forestry Science, NEIKER-Tecnalia, Arkaute, Spain
2Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Institute of Experimental Botany CAS, Palacký University Olomouc, Olomouc, Czechia
3Department of Statistics, Computer Science and Mathematics, Universidad Pública de Navarra, Pamplona, Spain
Somatic embryogenesis (SE) provides us a potent biotechnological tool to manipulate the physical and chemical conditions (water availability) along the process and to study their effect in the final success in terms of quantity of somatic embryos produced. In the last years, our research team has been focused on the study of different aspects of the SE in Pinus spp. One of the main aspects affecting SE is the composition of culture media; in this sense, phytohormones play one of the most crucial roles in this propagation system. Many studies in conifers have shown that different stages of SE and somatic embryo development are correlated with distinct endogenous phytohormone profiles under the stress conditions needed for the process (i.e., cytokinins play a regulatory role in stress signaling, which it is essential for radiata pine SE). Based on this knowledge, the aim of this study was to test the effect of different temperatures (18, 23, and 28°C) and gelling agent concentrations (8, 9, and 10 gL-1) during the maturation stage of Pinus radiata SE in maturation and germination rates. Parallel, phytohormone profile of somatic embryos developed was evaluated. In this sense, the highest gellan gum concentration led to significantly lower water availability. At this gellan gum concentration and 23°C a significantly higher number of somatic embryos was obtained and the overall success of the process increased with respect to other treatments assayed. The somatic embryos produced in these conditions showed the highest concentration of iP-type cytokinins and total ribosides. Although, the different conditions applied during maturation of somatic embryos led to different hormonal profiles, they did not affect the ex vitro survival of the resulting somatic plants, where no significant differences were observed.
The use of vegetative propagation in forestry is the fastest, most flexible and effective way to produce enough genetically improved material to meet future wood demands (Lelu-Walter et al., 2013). Somatic embryogenesis (SE) is currently considered one of the successful biotechnological techniques for mass propagation, enabling multi-varietal forestry (Park et al., 2016) and for cryopreservation of embryonal masses (EMs) from elite genotypes (Corredoira et al., 2006).
In the last years, several improvements in different aspects of Pinus spp. SE process, such as initiation (Montalbán et al., 2012), maturation (Montalbán et al., 2010) and germination (Montalbán and Moncaleán, 2018) have been carried out in our laboratory. Traditionally, the adjustments have been focused on modifications of basal medium components (Salajova and Salaj, 2005; Zhang et al., 2007), exposure to different types and concentrations of exogenous plant growth regulators (Choudhury et al., 2008) and the use of different initial explants (Hargreaves et al., 2017).
One of the main bottlenecks of the SE in conifers is the progression from immature embryogenic cultures into mature cotyledonary embryos able to develop well-growing plants (Harvengt, 2005). Several external stimuli, such as plant growth regulators (Ayil-Gutiérrez et al., 2013), osmotic agents (Kikuchi et al., 2006), nutritional components (Pěnčík et al., 2015), amongst others, have been recognized as essential factors in determining both the hormone biosynthesis and the developmental fate of explant cells (Grzyb et al., 2017). In conifers, maturation is stimulated by reduction of water availability by means of raising the osmoticum of the medium (Montalbán et al., 2010) or the concentration of gelling agent (Klimaszewska and Smith, 1997). Modifying gellan gum concentration in maturation media has been studied in a few species of angiosperms (Márquez-Martín et al., 2011) and gymnosperms (Morel et al., 2014); these authors reported an improvement in the development and maturation of somatic embryos (Se) of P. pinaster by increasing gellan gum concentration, pointing out that the effect of gellan gum concentration on plant response is provoked by changes of water availability in the culture medium. In our laboratory, we demonstrated in previous studies that the agar concentration and the temperature had a significant effect on initiation step of radiata pine SE; and in the case of temperature, in the subsequent phases of the process (García-Mendiguren et al., 2016). As far as we know, the effect of different temperatures in maturation step has not been tested in Pinus spp., only in some angiosperms (Wang et al., 2014).
The active cytokinin (CK) pool is regulated during development by biosynthesis, uptake from extracellular sources, metabolic interconversions, inactivation, degradation, and transport (Kakimoto, 2003). The relative abundance of different CKs can vary greatly between plant species, tissues and developmental stages, and depends on the environmental conditions (Frébort et al., 2011). Some studies have examined the effect of varying the culture atmosphere on endogenous CK levels after organogenesis in angiosperms (Jiménez and Bangerth, 2001; Moncaleán et al., 2003) and gymnosperms (Moncaleán et al., 2005; Montalbán et al., 2012). On the other hand, the role of indole-3- acetic acid (IAA) and CKs in the SE process have been analyzed in cotton (Zeng et al., 2007; Jin et al., 2014) and wheat (Hess and Carman, 1998; Jiménez and Bangerth, 2001) and fern (Grzyb et al., 2017). However, up to know a deep analysis of endogenous cytokinins and IAA concentration in SE process in pines has not been determined. In this sense, and as it has been described previously, IAA and other phytohormones have an important role in embryo differentiation events (Silveira et al., 2004; Vondrakova et al., 2018); thus, a better understanding of the phytohormone profile in somatic embryos under different environmental conditions could enable us to manipulate this stage of SE to increase its efficiency. Taking into account all the above mentioned studies, the main objective of this study was the evaluation of how the environment of cultures (gellan gum concentration and temperature) along maturation stage affects the success of radiata pine SE processes as well as to analyze its effect in CK and IAA endogenous levels.
Maturation Experiment
Ten embryogenic cell lines (ECLs) from four open-pollinated Pinus radiata D. Don trees were selected for the experiments. The trees belonged to a seed orchard established by Neiker-Tecnalia in Deba (Spain; latitude: 43°16′59′N, longitude: 2°17′59′W, elevation: 50 m). The ECLs were initiated and proliferated in clumps in standard conditions following Montalbán et al. (2012) (Figure 1).
Figure 1. Scheme of the experimental design of maturation experiment. Cultures were stored at three different temperatures: 18°C (circle), 23°C (square), or 28°C (hexagon) and at three different agar concentrations (inside circles, squares, and hexagons).
Maturation Procedure
The basal medium used in all phases of SE process was Embryo Development Medium (EDM, Walter et al., 2005). For initiation and proliferation 30 gL-1 sucrose, 4.5 μM 2,4-dichlorophenoxyacetic acid and 2.7 μM benzyladenine (BA) were added. As gelling agent, 3 gL-1 and 4.5 gL-1 Gelrite®were used for initiation and proliferation, respectively. The pH was adjusted to 5.7 prior to sterilization at 121°C for 20 min. After autoclaving, filter-sterilized solutions (pH 5.7) of the following amino acids (Walter et al., 2005) were added to partially cooled medium prior to dispensing into Petri dishes (90 mm × 20 mm): 550 mgL-1 L-glutamine, 525 mgL-1 L-asparagine, 175 mgL-1 L-arginine, 19.75 mgL-1 L-citrulline, 19 mgL-1 L-ornithine, 13.75 mgL-1 L-lysine, 10 mgL-1 L-alanine, and 8.75 mgL-1 L-proline. All chemical products were purchased from Duchefa.
Three weeks before the start of maturation experiment, EMs (Figure 2A) were suspended in liquid growth regulators-free basal medium in 50 mL centrifuge tubes; then the suspension was poured onto a filter paper disk (Whatman No. 2) in a Büchner funnel. The filter papers with 250 mg of EMs each were laid on proliferation media. Maturation of ECLs was carried out following the protocol described by Montalbán et al. (2010). Briefly, EMs were suspended in liquid growth regulators-free basal medium and filtered as described above. The filter papers with 80 mg of EMs each were laid on maturation media. The basal media used were the same as described before supplemented 60 gL-1 sucrose and the amino acid mixture used for initiation and proliferation of the EMs. Also, 60 μM abscisic acid (ABA, purchased from Olchemim) was added.
Figure 2. Pinus radiata embryonal masses (A), mature somatic embryos (B), germinated somatic planlets (C), and acclimatized somatic plants (D).
In order to increase or reduce the water availability of the medium, the amount of Gelrite®in the maturation medium was increased (10 gL-1) or reduced (8 gL-1), respectively, respect to the standard conditions (9 gL-1). The cultures were kept under different temperatures, 5°C above (28°C) and below (18°C) the standard (23°C). Thus, nine different treatments were tested, carrying out the other SE steps under standard conditions (Figure 1). Summarizing, the whole experiment comprised nine different treatments, 10 ECLs and four replicates per ECL and maturation condition (a total of 360 maturation plates). All the cultures were kept in darkness.
Germination Procedure
After 15–18 weeks, Se were transferred to germination medium. This medium was half strength macronutrients LP (Quoirin and Lepoivre, 1977, modified by Aitken-Christie et al., 1988) supplemented with 2 gL-1 activated charcoal (AC, Sigma-Aldrich) and 9.5 gL-1 Difco Agar granulated (Becton & Dickinson). In those ECLs and maturation conditions producing Se, three to four Petri dishes, and 20 Se per Petri dish, with the embryonic root caps pointing downward, were titled vertically at an angle of approximately 45°, and cultured at 23 ± 1°C under 16 h photoperiod at 100 μmolm-2s-1 provided by cool white fluorescent tubes (TFL 58 W/33; Philips, France). Plantlets were subcultured to culture vessels with medium of the same composition after 6 weeks.
After a germination period of 14–16 weeks, plantlets were transferred to sterile peat:perlite (7:3, v/v) and acclimatized in a greenhouse with environmental control where the humidity was progressively decreased from 99 to 70% during the first month.
Water Availability Determination
The water availability of maturation media (EDM) under different maturation conditions (three gellan gum concentrations and three temperatures) was determined by weighing and placing one autoclaved filter paper disk (Whatman No. 2) on the surface of the medium following Klimaszewska et al. (2000). The Petri dishes were incubated under the same conditions as those used for maturation for 15 weeks. Then, the filter paper disks were weighed and the amount of water (g) absorbed was calculated. Four replicates per maturation condition were used.
Extraction, Purification and Quantification of Endogenous Cytokinins
Somatic embryos (Figure 2B) obtained from EMs maturated at 18, 23, and 28°C in EDM medium solidified with 8, 9, and 10 gL-1 (Figure 1).
Quantitative Analysis of CKs and IAA by Liquid Chromatography-Single Quadrupole MS
The 41 CKs and auxins analyzed were the following: cis-Zeatin (cZ), cis-Zeatin riboside (cZR), cis-Zeatin O-glucoside (cZOG), cis-Zeatin-9-glucoside (cZ9G), cis-Zeatin riboside O-glucoside (cZROG), cis-Zeatin riboside-5′-monophosphate (cZMP), trans-Zeatin (tZ), trans-Zeatin riboside (tZR), trans-Zeatin O-glucoside (tZOG), trans-Zeatin-7-glucoside (tZ7G), trans-Zeatin-9-glucoside (tZ9G), trans-Zeatin riboside O-glucoside (tZROG), trans-Zeatin riboside-5′-monophosphate (tZMP), Dihydrozeatin (DHZ), Dihydrozeatin riboside (DHZR), Dihydrozeatin O-glucoside (DHZOG), Dihydrozeatin-7-glucoside (DHZ7G), Dihydrozeatin-9-glucoside (DHZ9G), Dihydrozeatin riboside O-glucoside (DHZROG), Dihydrozeatin riboside-5′-monophosphate (DHZMP), N6-Isopentenyladenine (iP), N6-Isopentenyladenosine (iPR), N6-Isopentenyladenine-7-glucoside (iP7G), N6-Isopentenyladenine-9-glucoside (iP9G), N6-Isopentenyladenosine-5′-monophosphate (iPMP), N6-Benzyladenine (BA), N6-Benzyladenosine (BAR), N6-Benzyladenine-9-glucoside (BA9G), N6-benzyladenosine-5′-monophosphate (BARMP), ortho-Topolin (oT), ortho-Topolin riboside (oTR), ortho-Topolin-9-glucoside (oT9G), meta-Topolin (mT), meta-Topolin riboside (mTR), meta-Topolin-9-glucoside (mT9G), para-Topolin (pT), para-Topolin riboside (pTR). Indol-3-acetic acid (IAA), 2-oxindole-3-acetic acid (oxIAA), IAA-aspartate (IAA-Asp), and IAA-glutamate (IAA-Glu).
Three replicates of 3 mg of Se were analyzed according to the protocol described by Svačinová et al. (2012) and Pěnčík et al. (2018) for cytokinin and auxin profiling, respectively, using miniaturized purification (pipette tip solid-phase extraction). Samples were extracted with the addition of stable isotope-labeled internal standards (each at 0.5 pmol per sample) and transferred after extraction onto Stage Tips and purified according to the aforementioned protocol, consisting of C18, SDB-RPS, and Cation-SR sorbents for cytokinins or C18 and SDB-XC sorbents for IAA and IAA conjugates. Eluates were collected into new clean microcentrifuge tubes and evaporated to dryness in a Speed-Vac concentrator and dissolved in 30 μl of mobile phase prior to UHPLC-MS/MS analyses.
Mass analysis was carried out using an Acquity UPLC®System (Waters, Milford, MA, United States), and a triple-quadrupole mass spectrometer XevoTM TQ-S MS (Waters MS Technologies, Manchester, United Kingdom). Quantification was achieved by monitoring protonated precursors and the appropriate product ions. Multiple reaction monitoring transitions as well as chromatographic and tandem mass spectrometry (MS) conditions were selected according to the method described by Novák et al. (2008, 2012). All MS data were processed using the MassLynxTM software with TargetLynxTM program (version 4.2. Waters, Milford, MA, United States), and compounds were quantified by standard isotope dilution analysis (Rittenberg and Foster, 1940).
Data Collection and Statistical Analysis
The ECLs subjected to different maturation conditions that had produced Se were registered and the percentage of maturation and the number of Se per gram of EM (fresh weight) were calculated. The number of somatic embryos from different maturation conditions that had germinated was recorded and germination percentages were calculated (Figure 2C). A logistic regression and the corresponding analysis of deviance were conducted to evaluate the effect of temperature and gellan gum concentration on different stages of plant conversion for P. radiata.
Then, the success rate (plants able to be planted in ex vitro conditions) was evaluated in plantlets coming from ECLs cultured under different maturation treatments and it was calculated with respect to standard conditions (23°C and 9 gL-1 gellan gum, considered as 100% for maturation rate, number of Se per gram of EM and germination percentage).
After 14 weeks under ex vitro conditions (Figure 2D), the survival was calculated. To evaluate the effect of the maturation treatments, an analysis of variance (ANOVA) was carried out.
For water availability analyses, a two-way analysis of variance (ANOVA) was carried out to assess the effect of temperature and gellan gum concentration on the water availability of the media. When significant differences between the levels of the two variables were found, the Tukey HSD post hoc test was carried out to find out which levels were statistically different.
Data for the different CK types and metabolites, for IAA contents and the ratio of IAA: active CKs were evaluated using a two-way ANOVA to identify differences between treatments. Multiple comparisons were performed using Tukey's post hoc test.
Detailed information on the statistical analyses performed is given as Supplementary Material.
Significant differences were found among the levels of gellan gum and temperature although the interaction between the tested variables was not significant. In this sense, the highest temperature analyzed (28°C) led to a significantly lower water availability than the other temperatures tested (Figure 3A). EDM medium supplemented with 10 gL-1 gelrite showed also significantly lower water availability than those supplemented with 8 or 9 gL-1 (Figure 3B).
Figure 3. Water availability (g) on EDM medium (A) stored at three different temperatures (18, 23, and 28°C), (B) supplemented with three different gellan gum concentrations (8, 9, and 10 g L-1). Different letters show significant differences at a significance level of α = 0.05. Pairwise comparisons have been assessed with the Tukey HSD post hoc test.
Maturation percentages were 100% in all treatments except in ECLs cultured at 23 and 28°C in culture media solidified with 8 gL-1 gelrite (90 and 60%, respectively) and in those cultured at 28°C with 10 gL-1 gelrite (80%, Figure 4).
Figure 4. Maturation percentages (%) in P. radiata for embryogenic cell lines cultured at three different temperatures (18, 23, and 28°C) and three gellan gum concentrations (8, 9, and 10 g L-1).
When the analysis of deviance for the number of P. radiata Se was evaluated, statistical differences were found for the different temperatures and gellan gum concentrations tested but no interaction between them was found. The Tukey HSD post hoc test revealed significant differences between the three temperatures tested; being 23 and 28°C the temperatures that produced the highest and the lowest number of Se (1036 and 374 Seg-1 EM, respectively, Figure 5A). Moreover, ECLs maturated in culture media with the lowest water availability (10 gL-1) produced a significantly higher number of somatic embryos (Figure 5B).
Figure 5. Number of P. radiata somatic embryos per gram of embryogenic tissue (fresh weight) maturated on EDM medium (A) at three different temperatures (18, 23, and 28°C), (B) supplemented with three different gellan gum concentrations (8, 9, and 10 g L-1). Different letters show significant differences at a significance level of α = 0.05. Pairwise comparisons have been assessed with the Tukey HSD post hoc test.
Regarding germination percentage, significant interaction between temperature, and gellan gum concentration at maturation was observed. The highest germination rates, above 90%, were obtained when EMs were maturated at 18 and 23°C, whereas maturation at 28°C led to lower germination rates, particularly when it was combined with 9 or 10 gL-1 gellan gum (74 and 64%, respectively, Figure 6).
Figure 6. Germination percentages (%) of P. radiata somatic embryos maturated on EDM medium supplemented with three different gellan gum concentrations (8, 9, and 10 g L-1) and stored at three different temperatures (18, 23, and 28°C). Different letters show significant differences at a significance level of α = 0.05. Pairwise comparisons have been assessed with the Tukey HSD post hoc test.
The effect of physical and chemical modification of culture media in the success of the conversion from EMs to plants is shown in Figure 7. An improvement in plant conversion was observed when the gellan gum concentration was increased at all temperatures tested. In this sense, 23°C and 10 gL-1 showed the best results improving by 132% the plant conversion process.
Figure 7. Success rate (%) in the process of plant conversion in P. radiata embryogenic cell lines maturated at three different temperatures (18, 23, and 28°C) and three gellan gum concentrations (8, 9, and 10 g L-1) with respect to standard maturation conditions (23°C and 9 gL-1 gellan gum, considered as 100% of success).
Somatic plants coming from different treatments during maturation stage did not show significant differences in ex vitro survival after 14 weeks growing in the greenhouse (Supplementary Material). However, highest rates were found in plants coming from EMs maturated at 28°C independently of the gellam gum concentration. Survival percentage increased parallel to the increment of temperature during the maturation stage.
Endogenous Phytohormone Analyses
Detailed information on the contents of the 41 CKs and auxins in all samples analyzed is provided as Supplementary Material.
Significant differences in CK bases were found among the levels of temperature, gellan gum (except for cZ) and the interaction between the tested variables. In all the Se analyzed the appearance of tZ was under the limit of detection. The concentration of cZ was the highest in Se maturated at the highest temperature with the standard concentration of gelrite (9 gL-1, Figure 8A). The levels of DHZ were significantly higher in somatic embryos cultured at 18 and 23°C with the lowest Gelrite concentration in the culture media (8 gL-1, Figure 8B). The iP concentration was the highest in Se developed in a culture media solidified with 10 gL-1 gelrite at 28°C (Figure 8C).
Figure 8. Endogenous levels (pmol g-1 DW) of the cytokinin bases in somatic embryos maturated at different temperatures in culture media solidified with different concentrations of gellan gum. cZ (A), DHZ (B), and iP (C) concentrations. Different letters indicate significant differences among treatments by Tukey's post hoc test (α = 0.05). Triangle (28°C), square continuous line (23°C), and square discontinuous line (18°C).
Significant differences in CK ribosides (Figure 9) were found among the levels of gellan gum but no significant differences were observed among the levels of temperature for tZR, cZR, and DHZR; however, a significant interaction between temperature and agar was observed for all CK ribosides analyzed. tZR levels increased significantly when the gelrite concentration increase from 9 to 10 in somatic embryos cultured at 18 and 23°C (Figure 9B). The highest DHZR concentration was found in Se cultured in presence of the lowest concentration of gelrite (8 gL-1) at 18°C (Figure 9C), the DHZR levels obtained this treatment did not present differences with those at standard temperature (23°C) regardless the gellan gum concentration. iPR levels were significantly higher in Se cultured at standard temperature in presence of the highest gelrite concentration (10 gL-1) (Figure 9D).
Figure 9. Endogenous levels (pmol g1 DW) of the cytokinin ribosides in somatic embryos maturated at different temperatures in culture media solidified with different concentrations of gellan gum. cis-zeatin riboside (cZR) (A), trans-zeatin riboside (tZR) (B), dihydrozeatin riboside (DHZR) (C), and N6-isopentenyladenosine (iPR) (D) concentrations. Different letters indicate significant differences among treatments by Tukey's post hoc test (α = 0.05). Triangle (28°C), square continuous line (23°C), and square discontinuous line (18°C).
The levels of tZRMP, cZRMP, DHZRMP, and iPRMP were under detection limit in all the samples analyzed. Also, tZOG, tZROG, cZROG, and DHZROG were under the limit of detection. Significant differences in CK O- glucosides were found among the levels of gellan gum and temperature (except for DHZOG), and for the interaction between the two variables. The CK O-glucosides were represented by a low amount of cZOG and DHZOG. In this sense, Se maturated at the highest temperature and gelling agent concentration showed the highest levels of cZOG (Figure 10A). On the contrary, DHZOG concentration was the highest in Se developed at the lowest temperature and gelrite concentration in the culture media (Figure 10B).
Figure 10. Endogenous levels (pmol g-1 DW) of the O-glucosides in somatic embryos maturated at different temperatures in culture media solidified with different concentrations of gellan gum. cis-zeatin O-glucoside (cZOG) (A), dihydrozeatin O-glucoside (DHZOG) (B) concentrations. Different letters indicate significant differences among treatments by Tukey's post hoc test (α = 0.05). Triangle (28°C), square continuous line (23°C), and square discontinuous line (18°C).
Regarding the isoprenoid CK type, significant differences in CK types were found among the levels of temperature (except for cZ), gellan gum and the interaction between the tested variables. The tZ-type CK concentration was the highest in Se developed at 18 and 23°C with the highest concentration of gelling agent and at 28°C with the standard concentration of gelrite (Figure 11A). The lowest cZ-type CK concentration was observed in treatments at 18 and 23°C with the standard gelrite concentration (Figure 11B). The concentration of DHZ-type CKs was significantly higher in Se developed at 18 and 23°C in culture media solidified with 8 gL-1 of gelrite (Figure 11C). On the contrary, iP-types endogenous concentration was significantly higher in Se cultured with the highest concentration of gelling agent (10 gL-1) at standard temperature (23°C) than in Se from other treatments (Figure 11D).
Figure 11. Endogenous levels (pmol g-1 DW) of different isoprenoid cytokinin types in somatic embryos maturated at different temperatures in culture media solidified with different concentrations of gellan gum. cZ-type (A), tZ-type (B), DHZ-type (C), and iP-type (D) concentrations. Different letters indicate significant differences among treatments by Tukey's post hoc test (α = 0.05). Triangle (28°C), square continuous line (23°C), and square discontinuous line (18°C).
Analyzing the results by groups, CK nucleotides and N-glucosides were under the limit of detection. Significant differences in CK functional groups were found among the levels of temperature, gellan gum and the interaction between the tested variables. A significantly higher concentration of bases was found in Se developed at 18 and 23°C in culture media solidified with the lowest gelrite concentration (Figure 12A). Ribosides was the CK group with the most remarkable presence, the highest values for this group were obtained in Se cultured at standard temperature (23°C) and the highest gelrite concentration (Figure 12B). O-glucosides were significantly higher in Se cultured at the highest temperature and gelrite concentration (Figure 12C).
Figure 12. Endogenous levels (pmol g-1 DW) of the cytokinin groups in somatic embryos maturated at different temperatures in culture media solidified with different concentrations of gellan gum. Cytokinin Bases (A), Ribosides (B), and O-glucosides (C) concentrations. Different letters indicate significant differences among treatments by Tukey's post hoc test (α = 0.05). Triangle (28°C), square continuous line (23°C), and square discontinuous line (18°C).
Figure 13 shows the relative abundance of isoprenoid CKs Se by treatment. CK ribosides are the predominant group of cytokinins respect to bases and O-glucosides and a decrease in the bases content is observed parallel to an increase of gelrite concentration and temperature. Although the presence of aromatic type CKs (N6-benzyladenine, meta-topolin, ortho-topolin, and para-topolin) and their metabolites was also analyzed, theirs levels were under the limit of detection.
Figure 13. Relative abundances (expressed in percentage) of endogenous isoprenoid cytokinins in somatic embryos maturated at different temperatures in culture media solidified with different concentrations of gellan gum. Bases (black bar), Ribosides (gray bar), and O-glucosides (white bar).
Taking a general look at the results presented, it was observed a similar trend for CK concentration in Se cultured at 18°C y 23°C when the agar concentration increased from 8 to 9 gL-1, except iPR, DHZR and cZOG. When considering the results by CK-type or CK-metabolite this trend was also observed in all cases. At 28°C, an increase of bases and ribosides concentration was observed from 8 to 9 gL-1; in the case of iP, iPR, and cZOG their concentration continued increasing significantly with gellan gum concentration (10 gL-1).
The IAA, 2-oxindole-3-acetic acid (oxIAA), and IAA-glutamate levels showed the highest levels in Se developed at the highest temperature with different concentrations of gelrite (Figures 14A–C). Moreover, Se cultured at 28°C in media with the highest gelrite concentration showed significantly higher levels of oxIAA and IAA-glutamate (Figures 14B,C). IAA-aspartate levels were under the limit of detection in all samples analyzed. When relation between IAA and active CKs (bases+ribosides) was evaluated, we could observe low values in Se developed at standard temperature (23°C) independently of the gelrite concentration in the culture media (Figure 15).
Figure 14. Endogenous levels (pmol g-1 DW) of indole-3-acetic acid (IAA) (A), IAA-glutamate (IAA-Glu) (B), 2-oxindole-3-acetic acid (oxIAA) (C) in somatic embryos IAA-glutamate (IAA-Glu), 2-oxindole-3-acetic acid (oxIAA) in somatic embryos maturated at different temperatures in culture media solidified with different concentrations of gellan gum. Different letters indicate significant differences among treatments by Tukey's post hoc test (α = 0.05). Triangle (28°C), square continuous line (23°C), and square discontinuous line (18°C).
Figure 15. Indole-3-acetic acid/Cytokinin bases and ribosides ration in somatic embryos maturated at different temperatures in culture media solidified with different concentrations of gellan gum. Triangle (28°C), square continuous line (23°C), and square discontinuous line (18°C).
The inductive conditions which allow differentiated somatic cells to develop into competent dedifferentiated cells can be achieved by the addition of specific phytohormones, and/or by exposing the tissue to different stress factors (Leljak-Levanič et al., 2016). Moreover, water relations between the embryo and its environment play a regulatory role in embryo development, particularly during maturation (from Márquez-Martín et al., 2011) and for this reason, the water availability of maturation media was determined in our experiments. Radiata pine somatic embryos cultured in EDM medium supplemented with 10 gL-1 and stored at 28°C grew in an environment with the lowest water availability in comparison to the other conditions assayed. To this respect, in Douglas-fir, a reduction in water availability of the culture media provoked an increase in Se quality (Lelu-Walter et al., 2018) probably because it has been described that the reduction in water availability leads to obtain embryos with low water content similar to their zygotic counterparts (Morel et al., 2014).
The culture of embryogenic calli in media with an increased gelling agent concentration is a common procedure for the production of high quality mature embryos (Krajňáková et al., 2009; Troch et al., 2009; Buendía-González et al., 2012). Our results in P. radiata are in agreement with those obtained in P. strobus where the best maturation results were obtained increasing gellan gum concentration due to a shift in the developmental program of the culture, from proliferation of cells and early somatic embryos to the production of embryos at advanced stages (Klimaszewska et al., 2000).
Respect to the number of embryos developed, the temperature and gellan gum concentration affected significantly the number of Se per gram of EM. In this sense the temperature (28°C) and the agar concentration (10 gL-1) that provoked the lowest water availability led to the lowest and the highest number of Se, respectively. For this reason, it seems that water availability was not related to the success of the process at this stage. In this sense, there are two studies suggesting that under sub-optimal water availability, plants may take to a premature drought stress response and arrest or drastically reduce growth due to the effect on source–sink relationships through preferentially inhibiting nutrient acquisition processes more and earlier than necessary (Skirycz et al., 2011). It is clear that the water availability registered in our experiments is not sub-optimal for the maturation of EMs. In our laboratory, the effect of different physical and chemical conditions at the initiation stage of P. radiata and P. halepensis (García-Mendiguren et al., 2016; Pereira et al., 2016) as well as in proliferation stage of P. halepensis (Pereira et al., 2017) was studied and we found that the highest temperature seemed to produce a selective pressure as pointed by Fehér (2015); In this regard, Bonga et al. (2010) suggested that reducing or increasing temperatures may improve initiation and proliferation since temperature stress may promote cellular reprogramming. Our results are in agreement with de Almeida et al. (2014) where temperature had a significant influence on the direct SE capacity in coffee.
Regarding gellan gum concentration, some reports suggested that increasing the concentration of a gelling agent from 4 to 8 gL-1 (Teyssier et al., 2011) or from 4 to 9 gL-1 (Morel et al., 2014) promoted the maturation of the Se of some conifers. In this sense, Garin et al. (2000) demonstrated that the phytagel concentration was critical for the maturation of Se of five tested ECLs of Pinus strobus. The increase of phytagel concentration in the maturation media allowed obtaining well-developed Se capable of germination in various species of pines (Klimaszewska et al., 2009) and hybrid larch (Lelu-Walter and Pâques, 2009) probably due to the action in endogenous ABA modulation in mature somatic embryos (Morel et al., 2014). In summary, our results agree with abovementioned studies pointing that increasing gelling agent concentration is a good strategy to improve the Se yield in radiata pine.
The highest germination rates (above 90%) were obtained in Se coming from EMs maturated at 18 and 23°C, whereas maturation at 28°C led to lower germination rates, particularly when it was combined with 9 or 10 gL-1 gellan gum. Our results are not in agreement to Prewein et al. (2004) when they described that desiccation of the embryos due to decreased water availability in maturation media improves the germination frequency, probably by reducing the endogenous ABA level or by changing the sensitivity to ABA (Jiménez, 2005). Unlike observed by Morel et al. (2014) in maritime pine, we found no differences in germination rates with increasing concentration of agar, however, this could be due to differences in the range of concentrations used by these authors (4 and 9 gL-1 Phytagel) and us (8, 9, and 10 gL-1 Gelrite).
An improvement in plant conversion when the gellan gum concentration was increased at all temperatures tested was observed. However, 23°C showed the best results improving by 132% the plant conversion process. Summarizing, increasing the gellan gum concentration in the maturation stage improved the SE process in all radiata pine cultures independently of the temperature of storage obtaining the best results when somatic embryos were submitted to maturation process at control temperature (23°C). So, higher temperature and gellan gum concentration applied at maturation stage did not result in an increment of the efficiency of SE process like it was observed when this stressful condition was applied at initiation stage (García-Mendiguren et al., 2016). Moreover, ex vitro survival did not show significant differences although the highest percentages were obtained in somatic plants coming from EMs maturated at 28°C.
There are only a few reports describing how the contents of different endogenous hormones are altered by different environmental conditions. The relative abundance of different CKs can vary greatly between plant species, tissues and developmental stages, and depends on the environmental conditions (Frébort et al., 2011). In our experiments, the prevailing isoforms of CKs were cis-isomers of Z and ZR according with Zur et al. (2015) in microspore embryogenesis in triticale. Based on studies with Arabidopsis, cis-isomers were regarded as CK derivatives without any or with low biological activity. Further studies with non-model plants, like pines, showed that cis-isomers can be the dominant form of CK in specific plant organs and/or stages of development (Emery et al., 1998: Vyroubalová et al., 2009; Kudo et al., 2012). To this respect, Gajdošová et al. (2011) proposed that cZ can be qualified as a regulator of CK responses in plants under growth-limiting conditions. Our results only showed significantly differences in cZ in Se cultured with 9 gL-1 of gelrite at the highest temperature (28°C) with the others analyzed.
Highest amount of bases, the active forms of cytokinins (Klemš et al., 2011), were found in Se developed under 18 and 23°C and low gellan gum concentration, treatments showing the highest water availability. So, our results are in agreement to other authors describing that a decrease in water availability could be related to the rapidly utilization of CK bases and tightly regulated by mechanisms that prevent their accumulation at high levels (Moncaleán et al., 2005; Stirk et al., 2005).
CK ribosides, mainly represented by cZR and iPR, were higher in the treatment that led to the highest somatic embryo production and success rate (23°C and 10 gL-1). In terms of metabolic groups, ribosides were the predominant group of cytokinins and its percentage respect to bases and O-glucosides increased in parallel with the decrease of water availability. Nevertheless, in peach seeds, phosphates and ribosides of the DHZ and iP-type are the predominant CKs and functions like embryo formation and growth, division of endosperm primary nucleus, endosperm cellularization and endosperm cell division have been attributed to them (Arnau et al., 1999). So, opposite to Pacheco de Freitas et al. (2016) active CK levels seem to be important during the later stages of embryo development and maturation showing large amounts of these phytohormones in treatments with high production of mature embryos.
CK O-glucosides (reversible forms of storage), particularly cZOG, were highest in those embryos coming from EMs maturated in the most stressful conditions (28°C and 10 gL-1 gelrite, lowest water availability). However, CK O-glucosides were found in minute amounts (below 4 pmolg-1 DW), for instance Montalbán et al. (2011) reported O-glucoside concentrations above 240 pmolg-1 DW in P. radiata vegetative buds.
Low content of IAA and iP were obtained in Se treated with the highest gelrite concentration at the standard temperature (23°C). In this sense, in cotton a high content of both groups of phytohormones were related to the redifferentiation stage that leads to embryogenesis induction (Zeng et al., 2007). Somleva et al. (1995) and Sáenz et al. (2003) have found an inverse relationship between cytokinins and the embryogenic response; in this sense, the treatment that provoked the highest success in the SE process (23°C, 10 gL-1 gelrite) showed a low level of IAA/bases+ribosides CKs relation. So it seems that IAA can play an important role in radiata pine SE maturation process; this conclusion is in agreement to the postulation that auxin metabolism (biosynthesis, conjugation, degradation), intercellular transport, and signaling is crucial in coordinating the morphogenesis and development of plant reproductive organs, including the formation of embryos (Benková et al., 2003; Bernardi et al., 2012). The accumulation of endogenous IAA in response to abiotic stress may have a mediating role during SE (Pěnčík et al., 2015; Nic-Can et al., 2016).
It seems that the differences found in specific CKs analyzed did not provoke changes in maturation rates. Our results suggest that none of phytohormones found acts alone in the acquisition of embryogenic maturation capacity; it seems that the dynamic and complementary actions of the auxin and cytokinin pathways regulate several developmental processes and their ability to crosstalk makes them ideal candidates for mediating stress-adaptation responses according with Bielach et al. (2017) as well as initiating various signal transduction pathways (Kohli et al., 2013). In this regard, comparing the endogenous levels of IAA, Z, ZR, iP, and iPR in several embryogenic habituated callus lines of Asparagus officinalis, no correlation between embryogenic potential and endogenous hormone levels could be found (Limanton-Grevet et al., 2000). These and other findings demonstrate that there is no direct correlation in endogenous hormone content and embryogenic competence of different genotypes, or between competent and non-competent genotypes, which suggests the involvement of additional, hormone-related internal mechanisms for embryogenic competence regulation and regeneration ability in vitro (Leljak-Levanič et al., 2016). However, from our results, it seems clear that low levels in the relation between IAA and CK bases+ribosides are related to highest success of SE process in radiata pine.
Further studies increasing the temperatures during shorter periods of time can help us to go further in the understanding of the mechanisms involved in SE in Pinus radiata as well as the role of phytohormones in this process.
PM and IM conceived and planned the experiments. PM, OG-M, and IM carried out maturation experiments. OG-M, ON, and MS carried out phytohormone analyses. IM, MU, and TG carried out the statistical analyses. All authors provided critical feedback and helped to shape the research, analyses, and manuscript.
This research was funded by MINECO (Spanish Government) project (AGL2016-76143-C4-3R), CYTED (P117RT0522), and DECO (Basque Government). The Ministry of Education, Youth and Sports of the Czechia provided support for this project via the National Program for Sustainability I (LO1204).
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Troch, V., Werbrouck, S., Geelen, D., and Van Labeke, M.-C. (2009). Optimization of horse chestnut (Aesculus hippocastanum L.) somatic embryo conversion. Plant Cell Tissue Organ Cult. 98, 115–123. doi: 10.1007/s11240-009-9544-8
Vondrakova, Z., Dobrev, P. I., Pesek, B., Fischerova, L., Vagner, M., and Motyka, V. (2018). Profiles of endogenous phytohormones over the course of norway spruce somatic embryogenesis. Front. Plant Sci. 9:1283. doi: 10.3389/fpls.2018.01283
Vyroubalová, S., Václavíková, K., Turecková, V., Novák, O., Smehilová, M., Hluska, T., et al. (2009). Characterization of new maize genes putatively involved in cytokinin metabolism and their expression during osmotic stress in relation to cytokinin levels. Plant Physiol. 151, 433–447. doi: 10.1104/pp.109.142489
Walter, C., Find, J. I., and Grace, L. J. (2005). "Somatic embryogenesis and genetic transformation in Pinus radiata," in Protocols for Somatic Embryogenesis in Woody Plants, eds S. M. Jain and P. K. Gupta (Dordrecht: Springer), 11–24. doi: 10.1007/1-4020-2985-3_2
Wang, H., Cai, J.-Z., Xia, H.-L., Li, X.-K., and Yang, S.-L. (2014). Factors influencing carrot embryogenesis and somatic embryo development. Chin. J. Pharm. Biotechnol. 21, 297–301.
Zeng, F., Zhang, X., Jin, S., Cheng, L., Liang, S., Hu, L., et al. (2007). Chromatin reorganization and endogenous auxin/cytokinin dynamic activity during somatic embryogenesis of cultured cotton cell. Plant Cell Tissue Organ Cult. 90, 63–70. doi: 10.1007/s11240-007-9253-0
Zhang, C. X., Li, Q., and Kong, L. (2007). Induction, development and maturation of somatic embryos in Bunge's pine (Pinus bungeana Zucc. ex Endl.). Plant Cell Tissue Organ Cult. 91, 273–280. doi: 10.1007/s11240-007-9294-4
Zur, I., Dubas, E., Krzewska, M., Waligórski, P., Dziuka, M., and Janowiak, F. (2015). Hormonal requirements for effective induction of microspore embryogenesis in triticale (×Triticosecale Wittm.) anther culture. Plant Cell Rep. 34, 47–62. doi: 10.1007/s00299-014-1686-4
Keywords: cytokinins, embryonal masses, embryogenic cell lines, indol-3-acetic acid, Pinus radiata, somatic embryogenesis
Citation: Moncaleán P, García-Mendiguren O, Novák O, Strnad M, Goicoa T, Ugarte MD and Montalbán IA (2018) Temperature and Water Availability During Maturation Affect the Cytokinins and Auxins Profile of Radiata Pine Somatic Embryos. Front. Plant Sci. 9:1898. doi: 10.3389/fpls.2018.01898
Received: 24 September 2018; Accepted: 07 December 2018;
Published: 20 December 2018.
Munetaka Sugiyama, The University of Tokyo, Japan
Glória Catarina Pinto, University of Aveiro, Portugal
Tsuyoshi E. Maruyama, Forestry and Forest Products Research Institute, Japan
Copyright © 2018 Moncaleán, García-Mendiguren, Novák, Strnad, Goicoa, Ugarte and Montalbán. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Itziar A. Montalbán, imontalban@neiker.eus


text:
[Enews] Otsego County Chamber Weekly Update
In a joint effort the Otsego County Chamber of Commerce and Citizen Voices are offering a Young Professionals Networking Mixer on June 5th at 6:00pm at Stella Luna Ristorante. Rsvp to Shelly Giangrant, Vice President Member Services at [email protected] or by calling (607) 432-4500 ext. 207
Join us for an Eye Opener Breakfast at maurices on Tuesday, June 17, 2014 at 8:30 am
Located in the Southside Mall in Oneonta, maurices offers fashion that is real, fresh and fun. Denim to dressy. Work week to weekend. Find the clothes that speak to you. Rsvp to Shelly Giangrant, Vice President Member Services at[email protected] or by calling (607) 432-4500 ext. 207
Springfield 4th of July Parade and "100 Years" Celebration July 4, 2014
The 100th Annual Springfield 4th of July Parade kicks off at 11:00 a.m. in Springfield Center. Follow the ultimate hometown parade to the Springfield Community Center for live music, great food, games, activities and displays including the Utica ZooMobile, "100 Years" exhibit by the Springfield Historical Society, the 3rd Annual 4th of July Quilt Show, and much more. Arrive early for free parking and shuttle service from the Glimmerglass Festival parking area on Rt. 80. To top off this historic celebration, head to Glimmerglass State Park for a free evening concert featuring singer/songwriter Jeff Walton followed by a display of fireworks, starting at 7:30 pm. For more information look for the free Springfield Parade "100 Years" commemorative booklet, find us on facebook, or email [email protected]. Springfield is the place to be on the 4th of July!
Contact person: Debra Miller, (315) 858-0304
FABULOUS FRIDAY STRAWBERRY SHORTCAKE
Friday June 6 will be the Greater Oneonta Historical Society's second annual Strawberry Shortcake Social. GOHS program chair Corinne Bresee Smith invites you to "Stop by the History Center between 5:00 and 8:00 PM and enjoy homemade shortcake topped with delicious strawberries and fresh whipped cream for a $2 donation." There will also be complimentary coffee and tea at this Fabulous Friday event.
At 7:00 PM the second of the five murals depicting past building occupants will be unveiled. The Jo-Ann Dress Shop mural & accompanying plaque will be uncovered at 7:00 PM. Jo-Ann Dress Shop, owned and operated by Antonia Chrones, occupied the first floor of the current Oneonta History Center from 1949 to 1968.
The History Center, located at 183 Main Street, will be open on June 6 from 4:30 to 8:30 PM. For more information, contact 432-0960 or [email protected]
Attention LANZA Customers! For the month of June, LANZA Shampoo and Conditioners are 2 for $39.00!!
*excludes Keratin Healing Oil Shampoo and Conditioner*
Keratin Healing Oil Shampoo and Conditioner is
2 for $59.00! This is a $20.00 savings!
Stock up now as the sale ends June 30th! Hope to see you soon!
Mane Street Cutters, 297 Main Street, Oneonta, NY 13820
The Wilber Park pool officially opens on Saturday, June 28.
At that time, the pool will be open 7 days a week. Open swim runs from 1:00-5:00 p.m. and then the pool will reopen for family swim from 6:00-7:30 p.m. Costs for non-residents are $1 child/$3 adult/$8 family. We will accept cash and credit cards.
For those wanting to visit the pool frequently, seasonal non-resident passes may also be purchased ($25 child/$75 adult/$125 family). City residents are free, however, they will need to pick up a city resident pass at the following locations and times. Seasonal non-resident passes can also be purchased here too.
At the Oneonta Family YMCA: May 27/29 5-7 p.m.
May 28/30 9-11 a.m.
At the Armory:
June 2-5 5-7 p.m.
June 7 10-noon
After June 7, passes may be picked up or purchased at the YMCA Monday through Friday from 6 a.m. – 9 p.m. When the pool opens on Saturday, June 28, passes will be available at the Wilber Park pool as well.
Copyright © 2014 The Otsego County Chamber of Commerce, All rights reserved.
Country Club Auto Group
Enrollment in preferred owner program – includes $200 towards your next purchase and $25 on next vehicle service. No restrictions …Read More

redpajama_set_name: RedPajamaCommonCrawl


See what's happening right now, view photos of the latest projects and join the conversation.
Inside information on what to do, where to go and local weather.
See a collection of some beautiful catches in spectacular locations.
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Search Results for: Barclays
Barclays says new FaceID sensor and 3D scanning planned for 2020 iPhone series
Jan 20, 2020, 1:33 pm EST
Apple's new 2020 iPhones will come with a new Face ID sensor for biometric facial recognition and its Pro series…
Barclays and Hitachi release updated biometric finger vein scanner
Nov 20, 2019, 10:52 am EST
Barclays and Hitachi have released an improved version of the Barclays Biometric Reader (BBR), a finger vein scanner that allows…
IDEMIA setting up IdentoGO retail location in the Barclays Center
Mar 8, 2018, 7:00 pm EST
Barclays Center and IDEMIA, formerly OT–Morpho, have announced a new partnership to promote and implement identity and security services from…
HSBC, Barclays join EU project aimed at testing digital ID standards for cross-border banking
Jul 12, 2017, 4:19 pm EDT
HSBC and Barclays have joined a new EU group comprised of private and public sector organizations testing the use of…
Barclays employs voice biometrics to authenticate phone banking customers
Aug 4, 2016, 6:26 pm EDT
Barclays has been testing voice biometrics technology since 2013 and this past weekend the bank announced that it is ready…
Barclays Bank launches online business portal to be used with Hitachi biometric reader
UK financial institution Barclays Bank has launched its online banking portal that can be used with Hitachi Europe's biometric reader…
Barclays joins Biometrics Institute, Biometrics 2014 conference sees record delegate numbers
The Biometrics Institute announced that Barclays Bank has joined the organization as the 163rd member. Additionally, Biometrics 2014 conference, "The…
Barclays using biometric technologies to "completely automate" branch operations
Oct 6, 2014, 5:07 pm EDT
Ashok Vaswani, CEO of retail and business banking at Barclays, said the British bank is currently developing new ways to…
Barclays introduces biometric finger vein reader for customers
Sep 8, 2014, 3:02 pm EDT
Barclays announced it has partnered with Hitachi Europe to launch its customer authentication solution, Barclays Biometric Reader, which is backed…
Barclays rolls out voice biometrics for telephone banking
Jun 23, 2014, 10:30 am EDT
British bank Barclays will soon be rolling out a voice recognition system, provided by Nuance Communications, which verifies customers based…
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On-demand webinar – Biometrics at the Edge
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After submitting this form, within 24 hrs we will contact you via email. Personal and Account details will be confirmed at this time. Account details can be changed during this discussion (so don't worry if you are not sure about disk space, email accounts etc). The total price will be confirmed before account setup.
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text:
Q: AppendRow() Limited Rows How can I save data from a form only up to line 15? (as in the picture)?
function addNovaLinha(dadosLinha) {
  const ss = SpreadsheetApp.getActiveSpreadsheet();
  const ws = ss.getSheetByName("resultados");
  const dataAtual = new Date();
  var limite = ss.getLastRow();

  ws.appendRow([dadosLinha.item, dadosLinha.qtd, dataAtual]);

  return true;
}

A: You need to add a condition that checks if the last row has already reached 15.
function addNovaLinha(dadosLinha) {
  const ss = SpreadsheetApp.getActiveSpreadsheet();
  const ws = ss.getSheetByName("resultados");
  const dataAtual = new Date();
  var limite = ss.getLastRow();

  // append row and return true ONLY IF last row is less than 15
  if(limite < 15) {
    ws.appendRow([dadosLinha.item, dadosLinha.qtd, dataAtual]);
    return true;
  }
}

A: thankful for help. I managed to solve. with the response from

redpajama_set_name: RedPajamaStackExchange


text:
Home Articles The Sun Nurses work together to help
Nurses work together to help
Moses-Ludington Hospital nurses appreciate National Nurses Week
by Lohr McKinstry
Nurse Karen Griffin prepares paperwork in the Moses-Ludington Hospital emergency department.
Photo by Lohr McKinstry
TICONDEROGA – Being a nurse requires dedication and training, and with National Nurses Week starting May 6, nurses at Moses-Ludington Hospital in Ticonderoga appreciate the recognition of their vital role in healthcare.
The salute to nurses originated by the American Nurses Associations ends on May 12, Florence Nightingale's birthday.
Karen Griffin has been a nurse for 40 years, coming to work at Inter-Lakes Health's Moses-Ludington Hospital in 2008.
"No day is typical," she said. "Some days we have sore throats and sprained ankles. Another day we'll see two people with chest pains in a row.
"It's unpredictable, and that's one of the things I like about the E.R."
Patients frequently arrive in unstable condition; they may be unconscious and key information such as medical history, underlying conditions and allergies is unavailable, nurses said.
Regardless, the patient's situation must be assessed and stabilizing treatment initiated quickly, nurses say.
Nurse Mary Hilder works in the emergency department Moses-Ludington Hospital.
Many patients can be treated and then released; but some require additional, specialized and immediate medical care.
For those patients, Moses-Ludington is in a unique position with a very unique and critical role: to assess and stabilize the patients for transport to a trauma center or other facility that can provide the specific type of care required, hospital spokeswoman Jane Hooper said.
Often, nurses can ride in the ambulance with the patient to assist en route.
Griffin said many patients arrive by car, with someone entering the E.R. to tell them they've just driven a patient into the parking lot.
"You have to be prepared for anything," she said.
Nurse Cheryl Meserve gets supplies in the Moses-Ludington emergency department. Meserve started as an E.R. technician as a high-school student, eventually attending nursing school.
Nurse Cheryl Meserve, with several years experience, said they often have to bring patients in from the parking lot themselves.
"Most people come in and say 'I have someone in the car,' We go out with a stretcher," she said.
The helipad at the hospital also gets frequent use, she said.
"We've had two (air transports) in one day," she said. "We see motor vehicle accidents, logging accidents, motorcycle accidents."
The Moses-Ludington emergency department's skilled nursing staff is poised to handle any situation at any moment, 24 hours a day, treating more than 6,500 patients each year, Hooper said.
She said the nurses at Moses-Ludington care for trauma patients, critical care patients, individuals involved in accidents, stroke victims, heart attack victims, those with broken bones and sick children in the middle of the night.
The staff is trained and prepared to handle it all – and sometimes, simultaneously.
"It can get hairy," Griffin said. "You prioritize. People work together in the E.R. You have to."
The E.R. has four beds and a stretcher and sometimes runs out of beds, although a coming reconstruction will solve that.
Nurses (from left) Mary Hilder and Karen Griffin assess a volunteer patient in the emergency room at Moses-Ludington.
"In the summer, we can move minor ailments to the (hospital) clinic," Griffin said.
Because they don't have constant intake, people can usually get in and out quickly, Griffin said, and they provide more personalized treatment.
"It's greatly appreciated by people," she said.
Meserve said being local makes their care more personal.
"You know the people who come in, or know their families," she said..
Their main concerns are space and staffing, but many nurses now go back and forth between Moses-Ludington and Elizabethtown Community Hospital.
"We're getting better space," Griffin said. "We'll have a new E.R. We'll be able to keep people up to 48 hours."
Once patients recover from things like cardiac ailments, they're back in the community.
"You see them around," Griffin said. "It's nice to be able to help them."
Both said one of the reasons they like working at Moses-Ludington is the people there.
"We have a great facility," Meserve said. "It's a great place to work. You get to know everybody and work with all departments."
Nurse Mary Hilder is new to the field, and has been learning from her co-workers.
"I've felt very welcomed here," she said. "Everything is very exciting. In any situation, someone has your back. I never feel like I'm alone."
She said she learns new skills every day in the E.R.
"There's always a skill to master," she said. "They're helping you, for sure."
The staff is accustomed to dealing with a variety of serious situations. In fact, the emergency department handles life-threatening, critical care and trauma on a regular basis, roughly 400 cases per year.
Nurses assess and manage patient care when there is limited time to treat someone.
"I do like it here," Hilder said. "It's awesome. And we're getting a new E.R."
Moses-Ludington is about to be transformed – the construction is set to begin within a few weeks, lasting roughly two years.
The emergency department will be dramatically improved, Hooper said. The changes:
• New ED will be roughly 4 times the size of the current ED
• Room to maneuver stretchers, room for nurses and other clinical staff to easily care for patients
• Easily accessible supply storage
• New equipment
• The ED will also have a 4-bed observation unit, so that clinical staff can monitor patients, as required.
Elizabethtown Community Hospital will manage and operate the services available at Moses-Ludington, which will become part of Elizabethtown Community Hospital.
Staff members from Elizabethtown Community Hospital and Moses-Ludington have been collaborating for a number of months.
ECH/MLH Chief Nursing Officer Julie Tromblee said they're working closely as it gets close to the start of construction on the hospital renovation project.
"We're making sure we're sharing common nursing practices (between hospitals), providing training, the latest equipment," she said. "We're here with support for them.
"They haven't had that extra support and everything they need. Systems have to align. It can be as simple as focusing on a common practice."
The two hospitals could share staff in the future, she said.
"The nursing staff at Moses-Ludington offers the community their collective skill, education, dedication and compassion every single day," she said. "As the organization is transformed, patients will certainly be more aware of the gorgeous new facility; but the exceptional clinical knowledge and unwavering compassion that the nurses at Moses-Ludington offer to patients will remain constant. It will simply be enhanced."
Hooper said Moses-Ludington's role is to provide initial, life-saving care that offers patients time to get to a trauma, heart, burn or stroke center.
"Patients with significant illness or injury have limited time to receive treatment – it's an amount of time known as the 'golden hour,'" she said. "Rural hospitals often serve to extend that hour by treating and stabilizing the patient, so that there is more time to get to a larger facility."
New York Moses Ludington Hospital National Nurses Week Elizabethtown Community Hospital Headline Story Julie Tromblee Jane Hooper Florence Nightingale American Nurses Association The Times of Ti Sun Karen Griffin, Crown Point Ticonderoga Inter-Lakes Health healthcare Mary Hilder Cheryl Meserve Nursing Lohr McKinstry


This has been a hard week in The World Of Courtney's Noveling Shenanigans.
I'm going to spare you the details. I've been writing about zombies, so the details are gory. And I know some of my darlingest inklings don't care to read about pus, blood, and guts. So all you need to know is that I've had a fairly miserable writing week. Actually, as a certain fantasy heroine would say, I've had a miserable writing tenday.
But. Friday and Saturday, I did something that I knew I needed to do and that a conversation with the writing group reinforced to me that I needed to do.
I took two days and finished writing my long synopsis.
And that, writerly friends and neighbors, has pretty much solved my penning problems. You see, since I hadn't fleshed out my synopsis yet, I kept running into spots where I didn't fully know what was supposed to happen next. I sort of knew, but it was the kind of knowing you have when you're at a family reunion, and across the buffet table of mashed potatoes and tuna casserole, you meet a third cousin six times removed whom your mom once told you about when you were a teenager asking about skeletons in the family closet.
Okay, so the thing with my story wasn't exactly like that, but it made a good visual, didn't it?
Anyway. My writing problem has been that the story images in my head have been way too fuzzy. When I sat down and made myself finish the long synopsis, I sharpened those images so that I'd know exactly which scenes needed to happen in each chapter. And after I finished up that chore, I allowed myself to get back to the story.
Wonder of wonders, my Monday was a 4,272-word day. I wrote a paragraph — I knew which dialogue had to follow. I crafted a conversation — I knew how it led into the next action sequence. I wrapped up a scene — I had no question as to which scene needed to come next. All day Monday, I danced through my story, and the movements were easy because I'd rehearsed them all weekend long.
Tomorrow is Thanksgiving, and I've already got family in town. My several-day lead is going to be non-existent 24 hours from now. But the good news is that if not for finishing that synopsis, I would be four days behind by tomorrow instead of right on target.
As of this moment, my word count is 41,076. That leaves 8,924 words until I win this thing. It's gonna happen, and it's gonna happen because I fell flat on my face over and over again rehearsing. When it comes time to take the stage for real, I'm gonna shine.
This was written by Courtney Cantrell. Posted on Wednesday, November 24, 2010, at 10:03 pm. Filed under For Fun. Tagged Aaron Pogue, NaNoWriMo, Prewriting, Storytelling, Word Count. Bookmark the permalink. Follow comments here with the RSS feed.	Both comments and trackbacks are currently closed.


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If you're like me, you spend your weekends catching up on a lot of housework you didn't do during the week. So, here is a place is a place to commiserate, ask for advice, or maybe brag about how clean you are. Talk about whatever you want!
Now that spring has come (or is coming…), what sort of outside cleaning up do you have to do? Do outdoor projects get in the way of your inside cleaning? How do you balance them?
Or, if you don't have an outdoor space, just talk about your current projects, successes, and challenges inside.


text:
from django.db import models
#from pharmacy import models as pharmacy_models
#from items import models as item_models

class sells(models.Model):
    pharmacy_id = models.ForeignKey('pharmacy.pharmacy',on_delete = models.CASCADE)
    item_id = models.ForeignKey('items.item',on_delete = models.CASCADE)
    quantity = models.IntegerField()
    price_per_item = models.FloatField()

redpajama_set_name: RedPajamaGithub


Q: Is "The Kindred" available on DVD?
Another "When science goes bad" themed b-movie film about a son who discovers the extent of his mothers experiments and the creature that lives within the bowels of her home.
Truth be told The Kindred has some good ideas, some of the practical effects are great and it's not entirely awful. Sadly on the flip side it's not very well made, for every decent sfx there is a poor one and the characters are instantly forgettable.
The Kindred is one of those very few films I'd like to see a reboot of, or at least modern sequel. The potential is there, it just needs a budget and a decent team to put all the pieces together.
In it's current form The Kindred is a barely passable effort that I'd advise only big fans of the genre give time to.
Had Japan made this film it would have been an entirely different movie, you know......the tentacles!


text:
Fed chair signals openness to rate cut by underlining risks
Jeanna Smialek The New York Times News Service
WASHINGTON — Federal Reserve Chairman Jerome Powell highlighted continuing risks to the U.S. economy in prepared testimony to lawmakers Wednesday, the latest signal that an interest-rate cut may come soon.
Fed officials said in June that they would monitor incoming data, including the impact of President Donald Trump's trade war, and act if needed to sustain what is now the longest economic expansion on record.
Since then, "it appears that uncertainties around trade tensions and concerns about the strength of the global economy continue to weigh on the U.S. economic outlook," Powell said in written remarks to be delivered before the House Financial Services Committee. "Inflation pressures remain muted."
Investors expect the Fed to cut rates at its meeting at the end of the month amid heightened trade tensions and slowing global growth. Trump has placed tariffs on $250 billion worth of imports from China, which has retaliated by imposing tariffs on U.S. goods, and it remains unclear whether or when the spat between the two nations will be resolved. Factory indexes around the world are weakening, and domestic price gains have been surprisingly tepid.
Powell did not explicitly say that an interest-rate cut was coming but did point to mounting economic concerns.
The Fed's baseline expectation is for a solid job market and gradually increasing inflation, but "uncertainties about the outlook have increased in recent months," Powell said. "Moreover, a number of government policy issues have yet to be resolved, including trade developments, the federal debt ceiling and Brexit" — a reference to Britain's continuing negotiations to leave the European Union.
The federal debt ceiling may need to be raised early this fall for the government to borrow more money.
The 17-member Fed policymaking committee split sharply in June over whether the central bank should cut rates this year, with eight officials projecting a cut before the end of the year and nine pointing to no change or a rate increase. Powell said at his post-meeting news conference that many of the officials who did not project a rate cut saw the case for one strengthening.


Mercury Lounge was conceived in order to bring our idea of Global Asian Cuisine to a larger audience, with a compliment of specialty drinks, a good selection of wine and exceptional and friendly service, build a strong reputation in the SOMA.
Mercury Lounge, with its unique combination of Asian Cuisine, unique specialty cocktails and a strong wine menu that compliments the food, strives to be a common destinations spot for dining and social gatherings.
Taking Global Asian Cuisine to a whole new level, Chef Dominic take pride in providing a culinary tour of Asia, without stopping too much in one area. His food represents many of the flavors of what Asia and especially the variety of what Southeast Asian cuisine has to offer.


In this series of blogs, the team behind Over the Alps will be introducing themselves. First up is Cash DeCuir.
My first memories of games are fuzzy: star-ships, a child in the wild west, Mickey Mouse running up a descending escalator. Clarity comes with 3D. The Christmas morning when Santa left a Nintendo 64 and a copy of Mario Kart beneath the tree; driving around Rainbow Road solely to take in its color.
I kept playing as I grew. On consoles, primarily. I tended toward action-adventure and roleplaying games. Whether it was well or poorly done, anything that allowed me to guide a character through an E rated story was magic to me.
My tastes expanded with my possibilities. When I finally broke into PC gaming, I lost days planning my digital war campaigns on physical maps with Risk pieces; or reading the mythology found in dense manuals and seeing how it played out in story and mechanics, while also researching for the real-life inspirations; or looking for groups to no avail, resigning myself to a single player run through a massively multiplayer world.
In the middle of all of this, I developed my love of writing. But it was only after I graduated college I connected that love to my passion for games. By a tremendous stroke of luck, I made a friend in my last year who played a browser game called Fallen London, made by Failbetter Games. If you're not familiar with the title, it's a text-heavy browser game set in a darkly comedic Victorian London.
I took to playing it myself. Some months later, Failbetter announced they were hiring a writer. The time that passed from then until I learned I had the job I measure not in months, but in writing tests.
And so, I stayed with the company for two years and eight months. In that time, I became a lead writer. I also found a friend in one of the company's developers, Sam Partridge. I remember our walking to work and discussing his idea, yet in its infancy, of a game called Over the Alps. We kept talking; it kept growing; and when he asked if I'd like to join as lead writer and designer, I gladly accepted.
But I fear that's all I can say for now, as I can only type to you in fleeting moments between projects. I hope you'll join me for future blogs, though, as I have so much more to share with you all. We'll talk about films, old RPG games, my American homeland and how it rends my heart, and goodness knows how much more.
Until then, I bid you all the best and all the rest. I hope I'll see you for the next one.