Weeks In Dark Research Articles

The interacting effect of prolonged darkness and temperature on photophysiological characteristics of three Antarctic phytoplankton species.

Photophysiological characteristics of the Southern Ocean phytoplankton species Phaeocystis antarctica, Geminigera cryophila, and Chaetoceros simplex were assessed during 7 weeks of darkness and subsequent recovery after darkness at 4 and 7°C. Chlorophyll a fluorescence and maximum quantum efficiency of PSII decreased during long darkness in a species-specific manner, whereas chlorophyll a concentration remained mostly unchanged. Phaeocystis antarctica showed the strongest decline in photosynthetic fitness during darkness, which coincided with a reduced capacity to recover after darkness, suggesting a loss of functional photosystem II (PSII) reaction centers. The diatom C. simplex at 4°C showed the strongest capacity to resume photosynthesis and active growth during 7 weeks of darkness. In all species, the maintenance of photosynthetic fitness during darkness was clearly temperature dependent as shown by the stronger decline of photosynthetic fitness at 7°C compared to 4°C. Although we lack direct evidence for this, we suggest that temperature-enhanced respiration rates cause stronger depletion of energy reserves that subsequently interferes with the maintenance of photosynthetic fitness during long darkness. Therefore, the current low temperatures in the coastal Southern Ocean may aid the maintenance of photosynthetic fitness during the austral winter. Further experiments should examine to what extent the species-specific differences in dark survival are relevant for future temperature scenarios for the coastal Southern Ocean.

First Report of Scab Disease Caused by Venturia effusa on Pecan in Anhui Province of China.

Pecan (Carya illinoinensis) is a world-famous nut tree which widely cultivated in China. Quanjiao County, located in Anhui province, is reputed to be the capital of pecan production in China. Since 2019, typical scab symptoms were observed on most pecan cultivars in orchards located in the regions of Quanjiao (32°5'7.08″ N, 118°16'2.91″ E). In April, dark brown to black lesions of scab could be observed on both the abaxial and adaxial surface of the lamina, and were often associated with the veins or midrib. In July, small, brownish, and circular lesions ranging from 1 to 2 mm in diameter were observed at the end of stems and shoulder of the fruit. In the surveyed orchards, disease incidence on the leaves reached more than 35%. While, according to the number of infected nut clusters, disease incidence ranged from 40 to 60% on the infected fruits. Using a sterilized scalpel, conidia were scraped from the surface of a single lesion from the infected leaves or fruits, and a dilute spore suspension was prepared in sterile distilled water, of which 100 microliters was spread on 1% water-agar plate (Bock et al. 2014). The conidia were incubated at 25°C for 48 h under fluorescent lights with a 12-hphotoperiod. Single germinated conidia were selected and transferred into potato dextrose agar (PDA) plate to obtain monospore isolates. From 2019 to 2020, more than 20 isolates were obtained from the infected leaves and fruits. Incubated at 24°C for 6 weeks in darkness on PDA, the colonies were gray-black with circular morphology and floccose texture, which were consistent with the characteristics of Venturia effusa described previously (Gottwald 1982). The conidia were pyriform to ellipsoid, zero to one septate, smooth, attenuated towards apex and base, base truncate, pale brown and 10.08 to 18.14 × 4.86 to 9.56 μm (n = 50) in size. To further identify the isolates, the regions of internal transcribed spacer (ITS), beta-tubulin 2 (TUB2) and translation elongation factor 1 alpha (EF1-a) were amplified and sequenced from genomic DNA for the three representative isolates (AH-81 and AH-82 from the infected leaves, and AH-41 from the infected fruits), respectively (White et al. 1990; Young et al. 2018; Bensch et al. 2006). Sequences of them were deposited in GenBank under nos. OP199056 to OP199058 (ITS), OP566581 to OP566583 (TUB2) and OP566578 to OP566580 (EF1-a). Multilocus phylogenetic analysis revealed that three isolates and V. effusa were clustered in the same clade, indicating high genetic similarity between these organisms. Their morphological and molecular characteristics were consistent with those for V. effusa. The pathogenicity of three isolates were tested on two-year-old container-grown pecan seedlings, which were grown in the nursery. The conidial suspension with a concentration of 5 × 105 conidia/ml was sprayed evenly on the surface of leaves of a healthy pecan seedling, and each isolate inoculated four pecan seedlings. The pathogenicity experiment was repeated three times. The plants inoculated with sterile water were used a negative control. The inoculated plants were enclosed in plastic bags for 2 days, and kept in the nursery greenhouse. Four weeks after inoculation, a similar symptom of scab was observed on leaves of cultivar Mahan, and V. effusa was isolated again from inoculated leaves with the frequency of 100% by the single-spore isolation, whereas no symptoms were observed on the control plants. To our knowledge, this is the first report of V. effusa as a scab pathogen on pecan in Anhui Province of China and underscores the need for monitoring this disease and developing disease control strategies to prevent severe reduction in the value of fruit.

In vitro callus and shoot initiation of Black cohosh (Actaea racemosa. L) pre-incubated in darkness treatments

A medicinal plant that is commonly overharvested in its native habitat, Black cohosh (Actaea racemosa L.) rhizomes are used to mitigate symptoms of menopause and are a common alternative to hormone replacement therapy. Developing an accurate protocol for initiation of Black cohosh in tissue culture is becoming a necessity. Wild harvesting practices are the main source of Black cohosh rhizomes processed by pharmaceutical companies and are causing a significant decrease of these wild plants, leading to an At-Risk status. Growing these plants in a plant tissue culture laboratory is more sustainable than current practices, and having an accurate protocol for initiation of these plants is crucial to this. Our lab has previously developed a protocol for initiation of Black cohosh and this research was meant to optimize this protocol. Leaf explants of Black cohosh were cultured in vitro to determine the optimal duration of time for initiation of callus and/or shoot under darkness, and appropriate plant growth regulators (PGR) for shoot growth. Leaf explants incubated under darkness for increasing amount of time (0 to 6 weeks of darkness) were analyzed at two stages of tissue development. Stage 1 involved initiation of leaf explant material under darkness (treatment) and measured the callus development in leaf explants; Stage 2 was induction of adventitious shoot growth and cell division and compared the effect of two PGRs, KT and BA, on shoot growth for each darkness treatment. Leaf explants were given a score using a culture chart that assigns a numerical score to leaf explants based on their physiological characteristics. These numerical scores took into account color of leaf explant, callus development, shoot growth, and presence of dead leaf tissue. Stage 2 measured the number of shoots produced per explant within culture containers between PGRs and the number of weeks in darkness. Overall trends indicated that mortality of leaf explants decreased as the duration of incubation under darkness increased with mortality of explants decreasing from 63.9% (T0 darkness treatment) to 2.8% (T6 darkness treatment). Culture scores of explants (including dead explants) was highest for T6 weeks of darkness (culture score 2.96 on average), and for culture scores without dead explants (culture score of 3.05). During the adventitious shoot growth stage, however, T5 weeks of darkness performed significantly higher (KT: 28 shoots/ BA: 17.3 shoots) than T6 weeks of darkness (KT: 12.9 shoots/ BA: 9.4) in Replication 2 for both plant growth regulators KT PGR performed better, on average, than BA PGR

First report of Fusarium fujikuroi causing root rot and seedling elongation of soybean in Indiana.

In summer 2020, 127 soybean (Glycine max (L.) Merr) seedlings (V1-V3 stage) showing reduced vigor or crown lesions were collected at Purdue's Agronomy Center for Research and Education in West Lafayette, Indiana. Root tissues from two seedlings with necrotic cotyledons and root rot were surface-sterilized and plated on dichloran-chloramphenicol-peptone agar (Andrews and Pitt 1986). Emerging hyphal tips were transferred to potato dextrose agar (PDA). Single-spore cultures were obtained and grown on PDA. Both isolates developed floccose white aerial mycelia with reddish-pink coloration in the media in 2 weeks on the benchtop. On carnation leaf agar, macroconidia formed on orange sporodochia within 2 weeks in darkness at 25C. Macroconidia were 3-5 septate, measuring 26 - 41 × 2.5 - 3.7 μm (avg. 34.8 × 3.2 μm, n=40). Microconidia were abundant in chains and false heads forming on both mono- and polyphialides, and measured 2.5 - 8.75 x 2.5 μm (avg. 5.9 × 2.5 μm, n=40). These characteristics were consistent with species descriptions of F. fujikuroi [Sawada] Wollenw. (teleomorph Gibberella fujikuroi) (Leslie and Summerell 2006). DNA was extracted from mycelium and the following genes were amplified and sequenced: the internal transcriber spacer (ITS) region using ITS1/ITS4 primers (White et al. 1990) (GenBank accessions MW463362/MW463363), the mitochondrial small subunit (mtSSU) rDNA using MS1/MS2 primers (White et al. 1990) (MW465310/MW465307), and the partial translation elongation factor 1-alpha (TEF1α) gene using 983F/1567R primers (Rehner and Buckley 205) (MW475297/MW475298). In GenBank BLAST searches, these sequences showed 100% identity to both F. proliferatum and F. fujikuroi. Species-specific forward primers Fuji1F and Proli1F were then used in combination with reverse primer TEF1R to amplify another region in the TEF1α gene (Amatulli et al. 2012). Proli1F/TEF1R primers failed under a variety of annealing temperatures while Fuji1F/TEF1R primers succeeded, and the products were sequenced (MW475299/MW475300). GenBank BLAST searches revealed 100% identity of both isolates to F. fujikuroi (MT448248.1). A pathogenicity test was conducted with isolate AC13 in the greenhouse following the protocol of (Ellis et al. 2013). Ten seeds (cv. Williams) each were used for inoculation and control, respectively, with one seed per cup. Root rot symptoms similar to those observed in the field were observed 14 days after planting on all inoculated plants but not on controls (VC stage). Infected plants showed symptoms of pre-emergence damping off, reddish-brown lesions on the tap and lateral roots, and root necrosis. Three plants also exhibited hyper-elongation of the stem (12.5, 11.1, and 18 cm, vs controls: avg. 6.8 cm, max. 8.5 cm, stdev 0.78 cm). F. fujikuroi was successfully reisolated from inoculated plants but not from controls and identified as described above. F. fujikuroi has been reported causing soybean root rot in China (Zhao et al. 2020), Korea (Choi et al. 2019), and the state of Kansas (Pedrozo et al. 2015). To our knowledge this is the first report of F. fujikuroi infecting soybeans in the state of Indiana. F. fujikuroi is known to cause elongated seedlings in rice (Leslie and Summerell 2006). Pedrozo et al. (2015) reported that F. fujikuroi isolated from soybean caused seedling elongation in rice but not in soybean. The increased distribution and new host symptomology observed here warrants heightened attention for the control of this pathogen.

First Report of Atypical Scab Caused by Venturia asperata on Apple in China.

Apple cv. 'Huangtaiping' (Malus pumila Mill.) is grown widely in northern China for the production of jellies, preserves, and cider. In 2018, atypical scab symptoms were observed on fruits of Huangtaiping in Heilongjiang Province of China. The disease incidence was estimated at approximately 0.4%. Symptoms were scab-like black spots (3 to 5 mm diam.) distinct from scab caused by Venturia inaequalis. Conidia were generally produced on lesions and using a modified microscope (Goh 1999), a single spore was picked up from each sample on water agar plate with a glass needle and then transferred to PDA amended with lactic acid (0.50 ml/L) and sulfate streptomycin (0.20 g/L). Fifteen isolates were obtained and incubated at 21°C for 6 weeks in darkness on PDA. The colonies on PDA were gray-black with circular morphology and floccose texture, which were similar with the characteristics of V. asperata described previously (Turan et al. 2019). The conidia were cylindrical to fusiform, 0 to 1 septate, yellowish and 19.7 (13.5 to 25.8) × 5.7 (3.6 to 6.9) μm (n = 10) in size, which were larger than previously described ones (Turan et al. 2019). DNA of three randomly selected isolates were extracted by a modified SDS method (Ping et al. 2004). The internal transcribed spacer (ITS) region of rDNA of the three selected isolates was amplified with the primers ITS4/ITS5 (White et al. 1990), sequenced and deposited in GenBank (MN958665, MN95866 and MN958667). BLAST analysis showed that the amplified sequences were identical and had 99.3% sequence identity with V. asperata (AF333447, MT459450 and MT459451), 95.4% sequence identity with V. cerasi (MK810963 and MK810964) and 94.3% sequence identity with V. carpophila (MN958609, MN958610 and MN958611). In addition, the complete large subunit ribosomal RNA gene (LSU) was amplified with the primers LROR/LR5 (Vilgalys and Hester 1990), sequenced and deposited in GenBank (MT845787, MT845788 and MT845789). BLAST analysis showed that the amplified sequences were identical and had 99.7% sequence identity with V. asperata (EF114711), 99.2% identity with V. carpophila (MT772296, MT845732 and MT845733 ) and 98% identity with V. cerasi (MK810848 and MK810849). Phylogenetic analysis based on concatenated ITS and LSU sequences showed that the tested isolates grouped with V. asperata strain 2349 in the same clade and the closest species with V. asperata was V. carpophila, followed by V. cerasi. In July 2019, pathogenicity of the isolate VAHLJ3-1-1 was evaluated on Huangtaiping. A conidia suspension with a concentration of 5×105/ml was sprayed evenly on the surface of six fruits. In order to maintain high humidity, inoculated fruits were wrapped with a plastic bag (a cotton ball with water was placed in the plastic bag) to maintain wetness for 3 days. Six fruits sprayed with water were used as a control. Four weeks after inoculation, similar symptom of atypical scab was observed on fruits of Huangtaiping, and V. asperata was isolated again from six inoculated fruits with reisolation frequency of 100% by the single spore isolation, while no symptom was observed on the control fruits. Based on the morphological and molecular identifications, the causal agent of atypical scab on Huangtaiping was identified as V. asperata. Apple scab is usually caused by V. inaequalis (Shen et al. 2020). However, apple scab has also been caused by V. asperata in Italy and France (Caffier et al. 2012; Turan et al. 2019). To the best of our knowledge, this is the first report of V. asperata associated with apple scab-like lesions in China. This information augments our knowledge of the spectrum of Venturia species associated with disease on apple fruit and will be a valuable foundation underpinning management strategies for this cultivar.

The investigation of stability of n-CdS/p-Cu2S solar cells prepared by cold substrate method

In this study, two different n-CdS/p-Cu2S solar cells were prepared by evaporating Cu at different substrate temperatures (200 K and 300 K) by vacuum evaporation method on a single crystal CdS semiconductor. Field emission scanning electron microscope images showed that the Cu layer obtained at a temperature of 200 K was composed of nanoparticles in accordance with the soliton growth mechanism. Cu film thickness was determined as 395 ∓ 0.76 nm at 300 K substrate temperature and 187 ∓ 0.45 nm at 200 K substrate temperature. The current–voltage (I–V) and capacitance–voltage (C–V) characteristics of the solar cells were examined for 12 weeks in dark and light environments. Open-circuit voltage (V oc), short-circuit current (I sc), maximum power (P max), filling factor and efficiency (η) were calculated from I–V measurements. For the prepared solar cells, the highest efficiency value was obtained in the 7th week (η= 0.1360) at 200 K substrate temperature, while it was obtained in the 5th week (η = 0.0384) at 300 K substrate temperature. From C–V measurements, donor density (N d) and barrier potential (V bi) were calculated. The solar cell produced at 200 K substrate temperature has higher donor density (1st week 2.99 × 1016 cm−3) and barrier potential values (12th week 0.411 V). At the end of the 12-week period, the deterioration rate of solar cells created at 200 K and 300 K substrate temperatures was 51% and 94%, respectively.

Taxon‐specific dark survival of diatoms and flagellates affects Arctic phytoplankton composition during the polar night and early spring

AbstractEffects of prolonged darkness on Arctic phytoplankton composition were investigated with lab experiments and a pigment time series in Kongsfjorden, Spitsbergen (78°55′N). Chlorophyll a (Chl a), pigment composition, particulate organic carbon, cell numbers, and photosynthetic characteristics were studied in Arctic diatoms (Thalassiosira antarctica, Thalassiosira nordenskioeldii) and flagellates (Rhodomonas sp., Micromonas sp.) during 8 weeks of darkness and subsequent recovery in irradiance. Loss of photosynthetic functionality after 2 weeks of darkness was reversible in all species when returned to irradiance. Diatoms were more resistant to prolonged darkness (> 2 weeks) compared to the flagellates, with lower decline rates of Chl a and maximum quantum yield of PSII. T. nordenskioeldii showed rapid growth during recovery throughout 8 weeks of dark incubation, whereas recovery of flagellates diminished within 4 weeks. Ratios of taxonomic marker pigments relative to Chl a of all species showed limited variation during 8 weeks of dark incubation. The experimentally observed enhanced dark survival of diatoms was in agreement with pigment observations during four polar nights (2013–2017) in Kongsfjorden, which showed increased relative diatom abundance during declining biomass (down to 0.02 mg Chl a m−3). Therefore, a period of prolonged darkness gives Arctic diatoms a head start during the early stages of the spring bloom. The taxon‐specific survival traits can influence the geographical distribution of diatoms and flagellates within the polar oceans and their phenology. Furthermore, the persistence of Chl a of nonviable phytoplankton during darkness might influence biomass estimates during the polar night.

<i>In Vitro</i> Regeneration of Coconut Plantlets from Immature Inflorescence

Clonal propagation of elite, disease-free coconut palms is a promising technique for producing uniform planting material with high yield and disease resistance. Over the past few decades cloning of coconut has been attempted in a number of laboratories worldwide; however, success has been limited. In the present study, immature inflorescences of 2–12 cm size were collected from West Coast Tall variety and the rachilla segments were cultured on four different media combinations in dark condition. White translucent outgrowths were maximum in Y3 medium supplemented with 4.54 μM 2,4-dichlorophenoxyacetic acid (92%) followed by medium 72 with 41.4 μM picloram, 61.8 μM putrescine and 4.54 μM thidiazuron (TDZ) (87%). After eight weeks in dark, shoot-like outgrowth was noticed more in Y3 III (65%) followed by Y3 I. After eight months dark incubation, the cultures were transferred to 1/2 Murashige and Skoog (MS) with two hormone combinations and high frequency of multiple shoot formation was noticed in 1/2 MS with 5.37 μM naphthalene acetic acid (NAA) and 4.44 μM 6-benzylaminopurine (BAP). Maximum shoot development was observed Y3 medium fortified with 5 μM 2-isopentenyl adenine (2ip) and 5 μM BAP. The individual shoots after development of 3–4 leaves were transferred to 1/2 Y3 medium supplemented with 5.37 μM NAA and 24.6 μM indole-3-butyric acid (IBA), and root initiation was observed in 39.28% plantlets. Start codon targeted (SCoT) profiling based on banding pattern of PCR-amplified products confirmed the clonal fidelity of in vitro regenerated coconut plantlets. The study indicates the possibility of developing an in vitro regeneration protocol for coconut using immature inflorescence explants.

In vitro tetraploid induction of Malus × domestica Borkh. using leaf or shoot explants

Abstract Triploids and tetraploids of Malus × domestica Borkh. are widely used in breeding programmes because they are characterised by lush growth, larger organs and greater resistance to biotic and abiotic stress. The aim of this study was to develop an in vitro method of apple polyploidisation using leaf and shoot explants of six cultivars. At first, the procedure of efficient in vitro shoot regeneration from leaves was optimised. The leaf regeneration capacity was generally increased by preculture of donor shoots with 4.5 μM thidiazuron (TDZ) compared to a standard preculture in the presence of 4.5 μM benzyladenine (BA). Therefore, for polyploidisation, the leaf explants were collected from four-week shoot cultures that were pretreated with TDZ and the shoot explants derived from standard four-week multiplication subculture. The explants were incubated for six days in darkness on induction medium containing 2.5 μM 1-naphthaleneacetic acid (NAA) and 4.5 μM TDZ or 18 μM BA (leaves) or and 0.5 μM indole-3-butyric acid (IBA) and 4.5 μM BA (shoots) and one antimitotic agent: colchicine, trifluralin, oryzalin or amiprophos methyl (APM). Subsequently, the explants were cultured for four weeks in darkness on the medium without antimitotic agents and then subcultured on multiplication medium containing BA over a 16-h photoperiod. From leaf explants, 58, 38 and 6 tetraploids were obtained for three out of six cultivars, ‘Pinova’, ‘Redchief’ and ‘Sander’, respectively. For leaf explants, the highest polyploidisation efficiency, approximately 20%, was recorded for colchicine at 125 and 250 mg l −1 . For shoot explants, tetraploids were detected for all the cultivars with the higher tetraploid numbers – 13, 26 and 27, for Co-op 32, ‘Free Redstar’ and ‘Redchief’, respectively – and a few tetraploids were obtained for other genotypes (‘Gala Must., ‘Sander’ and ‘Pinova’). For shoot explants, treatment with 10 mg l −1 APM resulted in the highest polyploidisation efficiency of 9.8%. Additionally, mixoploids were detected three times more than tetraploids when shoots were used for polyploidisation, compared to the sporadic occurrence of mixoploids when leaf explants were used.

Optimisation of a highly efficient shoot regeneration system using leaf explants of Chinese jujube (Ziziphus jujuba Mill.) by response surface methodology

ABSTRACTChinese jujube (Ziziphus jujuba Mill.) is a major fruit crop in Asia. In this study, response surface methodology (RSM) was successfully employed to establish a highly efficient in vitro propagation and regeneration system for the ‘Teapot’ jujube via shoot organogenesis. Among the tested factors, gibberellic acid (GA3) concentration showed the most significant positive effect. The pre-culture darkness timing and medium were also important factors for highly efficient shoot regeneration of the ‘Teapot’ jujube. The highest regeneration (> 75%) was achieved by 1 week in darkness and culture on wood plant medium (WPM) containing 0.25 mg·L−1 GA3, 0.5 mg·L−1 6-benzylaminopurine (BAP) and 0.1 mg·L−1 3-indoleacetic acid (IAA). In vitro-derived shoots rooted very well in the modified 1/2 Murashige and Skoog (MS) medium containing 0.4 mg·L−1 3-indolebutyric acid (IBA), resulting in a 100% rooting rate. These findings suggest that the RSM can be employed to optimise the protocols needed for successful in vitro plant regeneration of jujube cultivars, with potential applications in plant genetic transformation practices, polyploidy induction and germplasm preservation.

Temporal dynamics in photosynthetic activity of Spirodela polyrhiza turions during dormancy release and germination

Turions (vegetative propagules) of aquatic plants functionally resemble seeds but in fact they are overwintering buds having the ability to photosynthesize. They spend long periods in dormant state in the dim and cold bottom layer of natural waters during winter of temperate regions. In this study photosynthetic acclimation of Spirodela polyrhiza (L.) Schleiden (giant duckweed) turions to contrasting environments was assessed in laboratory experiments by means of chlorophyll fluorescence induction and O2-evolution methods and photosynthetic pigment content.Photosynthetic performance of turions had been monitored for 5 weeks in dark and cold (after-ripening) and then for 48h under continuous irradiation at room temperature (germination induction).Photosynthetic activity of turions displayed biphasic acclimation during after-ripening. A gradual decrease in O2-evolution, variable fluorescence and relative fluorescence decrease was observed during the first 3 weeks with a parallel increase in chlorophyll fluorescence parameters indicative of excitation energy dissipation. Most interestingly later, while turions were still kept in dark and cold, their photosynthetic activity increased again to the level of newly-formed turions. This temporal pattern suggests that changes in photosynthetic performance might be regulated in close connection with dormancy release of turions.After transferring after-ripened turions to warm and illuminated conditions their photosynthetic acclimation took place rapidly. All parameters reflecting photosynthetic efficiency of germinating turions reached or even exceeded the respective levels of newly formed control turions within 24–48h.

Structural stress responses and degradation of dictyosomes in algae analysed by TEM and FIB‐SEM tomography

Stress-induced physiological deficiencies in cells are reflected in structural, morphological and functional reactions of organelles. Although numerous investigations have focused on chloroplasts and mitochondria as main targets of different stressors in plant cells, there is insufficient information on the plant Golgi apparatus as stress sensor. By using the advantages of field emission scanning electron microscopy tomography in combination with classical ultrathin sectioning and transmission electron microscopic analyses, we provide structural evidence for common stress responses of the large and highly stable dictyosomes in the algal model system Micrasterias. Stress is induced by different metals such as manganese and lead, by starvation in 9 weeks of darkness or by inhibiting photosynthesis or glycolysis and by disturbing ionic homeostasis via KCl. For the first time a stress-induced degradation pathway of dictyosomes is described that does not follow "classical" autophagy but occurs by disintegration of cisternae into single membrane balls that seem to be finally absorbed by the endoplasmic reticulum (ER). Comparison of the morphological features that accompany dictyosomal degradation in Micrasterias to similar reactions observed during the same stress application in Nitella indicates an ubiquitous degradation process at least in algae. As the algae investigated belong to the closest relatives of higher land plants these results may also be relevant for understanding dictyosomal stress and degradation responses in the latter phylogenetic group. In addition, this study shows that two-dimensional transmission electron microscopy is insufficient for elucidating complex processes such as organelle degradation, and that information from three-dimensional reconstructions as provided by field emission scanning electron microscopy tomography is absolutely required for a comprehensive understanding of the phenomenon.

Immature Embryo Germination and Its Micropropagation of Ilex crenata Thunb.

To shorten Ilex seed germination time and speed up breeding cycles, immature embryos of Ilex crenata ‘Sky Pencil’ seedlings were removed from fruits at their heart-shape stage and cultured in vitro on Murashige and Skoog (MS) medium or Woody Plant Medium (WPM) with 3% sucrose and 0.65% agar. Cultures were incubated at 27 °C for 2 weeks in darkness and subsequently moved to a growth chamber with 14-hour photoperiod (115 μmol⋅m−2⋅s–1). Embryos began to germinate 2–3 weeks after culture. The highest germination rate was 91.67% under 1/4 MS medium. Embryos cultured on MS medium also had high germination rates and produced the longest seedlings to 8.02 mm. Nodal segments with one axillary bud taken from embryo germination seedlings were cultured on MS medium with various concentrations of cytokinins and auxins for micropropagation. Zeatin (ZT; 4-hydroxy-3-methyl-trans-2-butenylaminopurine) increased the number of shoots and shoot lengths significantly more than 6-benzylaminopurine (6-BA). The recommended ZT concentration should be 2.28 µM. Rooting induction could be established on 1/4 MS medium with various concentrations of indole-3-butyric acid (IBA) or 1-naphthaleneacetic acid (NAA). IBA at 4.14 µM produced the best rooting percentage (91.67%) and good-root quality. All rooted plantlets were transplanted into a mixture of peatmoss and perlite (1:1 v/v) and acclimatized in a mist system. The average survival rate was 88.8%. The rapid embryo germination protocol for Ilex crenata could save Ilex breeders at least 2 years compared with traditional seed germination.

Iridoid and phenylethanoid glycoside production and phenotypical changes in plants regenerated from hairy roots of Rehmannia glutinosa Libosch.

Spontaneous shoot regeneration was obtained from the hairy roots of Rehmannia glutinosa cultured in liquid woody plant medium for 4 weeks in darkness after induction by Agrobacterium rhizogenes strain A4. These shoots were proliferated on solid Murashige and Skoog (MS) medium in 300 mL glass jars and in liquid MS medium in a 5 L nutrient sprinkle bioreactor. These media were supplemented with 0.1 mg L−1 of indole-3-acetic acid (IAA) and 1.0 mg L−1 of 6-benzylaminopurine. All shoots became rooted on MS agar medium containing IAA or indole-3-butyric acid within 4 weeks. The plantlets were then transferred into soil and grew in a greenhouse. The transgenic nature of the plants was confirmed by polymerase chain reaction (PCR) and reverse-transcriptase PCR analyses. The pRi-regenerated transformants demonstrated a few phenotypical alterations. Production of six iridoid (catalpol, aucubin, loganin, catalposide, harpagide, harpagoside) and two phenylethanoid (verbascoside and isoverbascoside) glycosides was determined in shoots cultured in vitro and leaves and roots of 6- and 12-month-old plants by ultra high performance liquid chromatography. The shoot culture was able to synthesize low amounts of catalpol, verbascoside and isoverbascoside. Leaves and roots of pRi-regenerants accumulated similar (harpagoside, isoverbascoside), higher (catalposide, aucubin, harpagide, verbascoside) or lower (catalpol, loganin) levels of the metabolites in comparison with untransformed R. glutinosa plants and their content varied with the age of the plants and the organ analyzed. However, the increase in shoot and root biomass of transformed plants resulted in a significantly greater yield of all analyzed compounds.

Emissions of carbon dioxide, methane, and nitrous oxide from short- and long-term organic farming Andosols in central Japan

Here we have investigated the emission of carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) from long- and short-term organic farming Andosols in Matsumoto city of Nagano, in central Japan. We focus on three upland plots in Matsumoto, distinguished by how long they had each experienced continuous organic farming (OF)—in these three cases, since 1971, 2009, and 2010 (plots hereafter termed M39-OF, M1-OF, and M1F-OF, respectively). Since 2001, in M39-OF, mainly rye (Secale cereale L., as green manure) and soybeans [Glycine max (L.) Merril, as crop] were cultivated, in winter and summer respectively, without tillage, other fertilizers and agro-chemicals. In contrast, from 2001 to 2008 in M1-OF, and from 2001 to 2009 in M1F-OF, these plots underwent conventional farming of some vegetables with tillage, fertilizer and agro-chemicals. Soils sampled from M39-OF and M1-OF in August 2009 were incubated for 4 weeks in darkness at 25°C. During these 4 weeks, M39-OF emitted 8.0 times more CO2 and 274 times more N2O than M1-OF. Less than 2 µg carbon (C) kg–1 dry soil of CH4 was emitted from both soils. From February 2010 until January 2011, CO2, CH4, and N2O emission rates of M39-OF and M1F-OF were measured almost monthly, using a closed-chamber method. Annual CO2, CH4, and N2O emissions were 317, –1.7, and 27 g CO2-C equivalent m–2 in M39-OF, and 138, –0.2, and 21 g CO2-C equivalent m–2 in M1F-OF, respectively. The rye yield in M39-OF was 334 g C m–2. Soybeans in M39-OF and M1F-OF yielded 290 and 286 g C m–2, and withdrew 230 and 224 g C m–2, respectively. Greenhouse gas (GHG) balance was calculated at –52 and 97 g CO2-C equivalent m–2 in M39-OF and M1F-OF, respectively. Negative GHG balance indicated that M39-OF was acting as a GHG sink, with higher CH4 absorption than M1F-OF. Further, this beneficial function for global warming was thought to be based on its cultivation system, which had included green manure application since 2001. The difference in gas exchange between incubation and field experiments was considered a reason for the difference in N2O emission between incubation and field experiments.

Caspase Inhibition with XIAP as an Adjunct to AAV Vector Gene-Replacement Therapy: Improving Efficacy and Prolonging the Treatment Window

PurposeAAV-mediated gene therapy in the rd10 mouse, with retinal degeneration caused by mutation in the rod cyclic guanosine monophosphate phosphodiesterase β-subunit (PDEβ) gene, produces significant, but transient, rescue of photoreceptor structure and function. This study evaluates the ability of AAV-mediated delivery of X-linked inhibitor of apoptosis (XIAP) to enhance and prolong the efficacy of PDEβ gene-replacement therapy.Methods Rd10 mice were bred and housed in darkness. Two groups of animals were generated: Group 1 received sub-retinal AAV5-XIAP or AAV5-GFP at postnatal age (P) 4 or 21 days; Group 2 received sub-retinal AAV5-XIAP plus AAV5- PDEβ, AAV5-GFP plus AAV5- PDEβ, or AAV- PDEβ alone at age P4 or P21. Animals were maintained for an additional 4 weeks in darkness before being moved to a cyclic-light environment. A subset of animals from Group 1 received a second sub-retinal injection of AAV8-733-PDEβ two weeks after being moved to the light. Histology, immunohistochemistry, Western blots, and electroretinograms were performed at different times after moving to the light.ResultsInjection of AAV5-XIAP alone at P4 and 21 resulted in significant slowing of light-induced retinal degeneration, as measured by outer nuclear thickness and cell counts, but did not result in improved outer segment structure and rhodopsin localization. In contrast, co-injection of AAV5-XIAP and AAV5-PDEβ resulted in increased levels of rescue and decreased rates of retinal degeneration compared to treatment with AAV5-PDEβ alone. Mice treated with AAV5-XIAP at P4, but not P21, remained responsive to subsequent rescue by AAV8-733-PDEβ when injected two weeks after moving to a light-cycling environment.ConclusionsAdjunctive treatment with the anti-apoptotic gene XIAP confers additive protective effect to gene-replacement therapy with AAV5-PDEβ in the rd10 mouse. In addition, AAV5-XIAP, when given early, can increase the age at which gene-replacement therapy remains effective, thus effectively prolonging the window of opportunity for therapeutic intervention.

Detailed Investigation of Surface Passivation Methods for Lifetime Measurements on P-Type Silicon Wafers

The effect of five different common surface passivation techniques on the measured bulk lifetime values of multi- and monocrystalline p-type silicon wafers was investigated. Mono-[Czochralski (Cz) and floatzone (FZ)] and multicrystalline [mc and edge-defined film-fed growth (EFG)] silicon wafers were either deposited with a dielectric passivating layer of SiNx, Al2O3, or amorphous silicon (a-Si) or were passivated chemically with 0.08 M iodine-ethanol (IE) or 0.07 M quinhydrone-methanol (QM) solutions. The temporal stability of annealed and nonannealed Cz wafers that were passivated with QM and IE was tested. The lifetime values of EFG, mc, and FZ wafers that were subjected to repeated QM passivation and mc wafers that were subjected to IE passivation without a surface etching between passivations were found to decrease with each passivation. Lifetime values of a set of 11 mc wafers that were passivated with Al2O3 were found to decrease about 30% after a period of four weeks in darkness. The decrease was reversible by annealing the samples. The lifetime values of annealed Cz samples that were passivated with Al2O3 and a-Si were found to decrease by >;20% within 5 h of annealing. Subsequent tests on 200-Ω·cm FZ material did (for a-Si) and did not (for Al2O3) show surface passivation degradation over this time period. Neighboring mc wafers were passivated dielectrically or wet chemically with IE or QM and characterized with photoluminescence imaging. All mc wafers that were subjected to dielectric passivation methods that include annealing at 400 °C displayed a greater area of high lifetime values but fewer areas of very high lifetime values, providing visible evidence of internal gettering and/or defect redistribution.

In vitro explants regeneration of the grape Wink (Vitis vinifera L. Wink)

The effects of different hormones and their concentration combinations, different explant types, dark periods, on the regeneration of the grape ‘Wink’ was investigated using in vitro leaves, petioles, internodes and radicles. The results showed that of all the media prepared, the explants on following media produced the highest regeneration rate: 78.74±1.60% of the leave explants on the medium prepared with MS+18.20 μM,TDZ+0.49 μM IBA regenerated with a mean of 6.75±0.75 shoots per explant; 39.33±1.47% of the petioles on the medium prepared with MS+9.10 μM TDZ+0.49 μM IBA regenerated with a mean of 3.55±0.50 shoots per explant and 41.37±1.13% of internodes on the same medium regenerated with a mean of 4.74±0.64. However, radicles did not generate on any of the media. TDZ enhanced regeneration rate of leaves better than BA. From 0 week to 4 weeks in dark, leave explants displayed a higher rate of regeneration for 2 or 3 weeks. Adventitious shoots were rooted on 3/4 MS+1.73 μM IBA medium, and the rooted plantlets survived after acclimatization and they were transplanted to the greenhouse. Key words: Grape, explants, regeneration, hormones, adventitious shoots.

Effects of Incubation Temperature on Seed Germination of Mediterranean Plants with Different Geographical Distribution Ranges

Little is known about consequences of the increasing global temperatures on seed germination and even less about the seed germination response of species with different geographical distribution ranges. In the present work, the effect of incubation temperature on seed germination of 31 species from the Iberian Peninsula is analyzed in relation to their geographical distribution range. We tested whether plants with different geographical distribution ranges (Iberian Peninsula endemics, Mediterranean, widely distributed species), respond similarly to incubation temperature using cross-species analysis as well as taking into account phylogenetic relationships among species. Seeds were incubated at 10°C, 15°C, 20°C and 25°C for 6 weeks in darkness. Distribution range appeared as a determinant of germination response to the temperature treatment independent of phylogeny. Widely distributed species were less sensitive than endemic or Mediterranean species to incubation temperatures, which may represent an advantage to colonize new areas or to tolerate changes in the environment. Although their ability to avoid germination during the warm and dry season might be favorable for endemic species in some situations, our results also suggest that under a future of temperature increase imposed by climate change, endemic plant species may be more vulnerable than widely distributed species.

Adventitious shoot regeneration in a bioreactor system and EST-PCR based clonal fidelity in lowbush blueberry ( Vaccinium angustifolium Ait.)

Adventitious shoots were regenerated from leaf explants of three lowbush blueberry ( Vaccinium angustifolium Ait.) clones: ‘QB1’, ‘QB2’ and ‘PB1’ by culturing on a gelled basal medium (BM) with 2.3–4.5 μM thidiazuron (TDZ) for four weeks followed by in a bioreactor system containing the same liquid medium but with 1.2–2.3 μM TDZ for another four weeks. Young expanding basal leaf segments with the adaxial side touching the culture medium and maintained for two weeks in darkness, produced the best results. Callus development and shoot regeneration were genotype dependent. Adventitious shoots were elongated in the liquid BM with 1 μM zeatin and rooted on a three peat: two perlite (v/v) medium. Acclimatized plantlets were grown actively in the greenhouse with an apparent normal leaf and shoot morphology. Ten random ‘QB1’ regenerated plants were screened using 14 expressed sequence tag-polymerase chain reaction (EST-PCR) markers and showed similar monomorphic amplification profiles confirming clonal fidelity of in vitro-derived ‘QB1’ plants. Results obtained suggested the possibility of adventitious shoot regeneration and true-to-type lowbush blueberry micropropagation using a bioreactor system combined with gelled medium.