Freeze–thaw processes and intense rainfall: the one-two punch for high sediment and nutrient loads from mid-Atlantic watersheds

Large runoff, sediment, and nutrient exports from watersheds could occur due to individual extreme climate events or a combination of multiple hydrologic and meteorological conditions. Using high-frequency hydrologic, sediment, and turbidity data we show that freeze–thaw episodes followed by intense winter (February) rainstorms can export very high concentrations and loads of suspended sediment and particulate organic carbon (POC) and nitrogen (PN) from mid-Atlantic watersheds in the US. Peak suspended sediment (> 5000 mg L^?1), POC (> 250 mg L^?1) and PN (> 15 mg L^?1) concentrations at our 12 and 79 ha forested watersheds for the February rainfall-runoff events were highest on record and the fluxes were comparable to those measured for tropical storms. Similar responses were observed for turbidity values (> 400 FNU) at larger USGS-monitored watersheds. Much of the sediments and particulate nutrients likely originated from erosion of stream bank sediments and/or channel storage. Currently, there is considerable uncertainty about the contribution of these sources to nonpoint source pollution, particularly, in watersheds with large legacy sediment deposits. Future climate projections indicate increased intensification of storm events and increased variability of winter temperatures. Freeze–thaw cycles coupled with winter rain events could increase erosion and transport of streambank sediments with detrimental consequences for water quality and health of downstream aquatic ecosystems.     

Agrobacterium-mediated transformation of Eruca sativa

An Agrobacterium tumefaciens—mediated transformation system was developed for Eruca sativa (eruca). Hypocotyl explants were co-cultivated with bacterial cells carrying a plasmid harboring a uidA:nptII fusion gene along a phosphinothricin acetyl transferase (PAT) gene cassette, for a period of 2 days. These were grown on a high cytokinin/auxin medium containing 5.0 mg l^?1 6-benzyladenine (BA), 1.0 mg l^?1 indole-3-acetic acid (IAA), and 0.1 mg l^?1 α-naphthaleneacetic acid (NAA). Explants were then transferred to a lower cytokinin/auxin medium containing 2.0 mg l^?1 BA and 0.1 mg l^?1 NAA along with 5.0 mg l^?1 silver nitrate and 300 mg l^?1 Timentin®. Upon transfer to a selection medium containing either 20 mg l^?1 kanamycin or 2 mg l^?1 L-phosphinothricin (L-ppt), shoot regenerants were observed. Expression of the transgenes in putative transformants was confirmed using a histochemical GUS assay. Presence of the PAT transgene in GUS-positive T0 plants was confirmed by Southern blot analysis. Moreover, spot tests of T1 seedlings were conducted using the L-ppt herbicide. A transformation frequency of 1.1% was obtained with more than 60% of transgenic lines containing single copies of the transgenes.     

Biogeochemical processes at the sediment–water interface, Bombah Broadwater, Myall Lakes

Myall Lakes has experienced algal blooms in recent years which threaten water quality. Biomarkers, benthic fluxes measured with chambers, and pore water metabolites were used to identify the nature and reactivity of organic matter (OM) in the sediments of Bombah Broadwater (BB), and the processes controlling sediment-nutrient release into the overlying waters. The OM in the sediments was principally from algal sources although terrestrial OM was found near the Myall River. Terrestrial faecal matter was identified in muddy sediments and was probably sourced via runoff from farm lands. The reactive OM which released nutrients into the overlying waters was from diatoms, dinoflagellates and probably cyanobacteria. Microcystis filaments were observed in surface sediments. OM degradation rates varied between 5.3 and 47.1 mmol m^?2 day^?1 (64–565 mg m^?2 day^?1), were highest in the muddy sediments and sulphate reduction rates accounted for 20–40% of the OM degraded. Diatoms, being heavy sink rapidly, and are an important vector to transport catchment N and P to sites of denitrification and P-trapping in the sediments. Denitrification rates (mean ～4 mmol N m^?2 day^?1), up to 7 mmol N m^?2 day^?1 (105 mg N m^?2 day^?1) were measured, and denitrification efficiencies were highest (mean = 86 ± 4%) in the sandy sediments (～20% of the area of BB), but lower in the muddy sediments (mean = 63 ± 15%). These differences probably result from higher OM loads and anaerobic respiration in muddy sediments. Most DIP (>70%) from OM degradation was not released into overlying waters but remained trapped in surface sediments. Biophysical (advective) processes were responsible for the measured metabolite (O₂, CO₂, DSi, DIN and DIP) fluxes across the sediment–water interface.     

A simple practical method for bulk and rapid extraction of free-living nematodes from marine and estuarine sediments

A simple, rapid method is described for the extraction of large numbers of free-living nematodes from estuarine sediments. This method does not physically or chemically alter or damage the nematodes, but instead relies on their downward movement through a filtering layer of double ply tissue paper and into aerated water-filled trays. Seven trials each with 10 trays kept at 25?°C for an initial period of 24 h yielded 3985 live nematodes l^?1 (± 511.5 standard deviation) of estuarine sediment, free of sediment and with minimal debris. Time effects were statistically significantly different, with the same 10 trays yielding another 1259 nematodes l^?1 (± 413.4) when kept for a second period of 24 h at the same temperature. Temperature effects were also significant, and 7 trials each with 10 trays kept for 24 h at 20–21?°C, produced a lower yield of 2160 nematodes l^?1 (± 532.7) of sediment. The method is expected to be of use in nematode extractions from both estuarine and marine sediments.     

Continuous in situ monitoring of sediment deposition in shallow benthic environments

Sedimentation is considered the most widespread contemporary, human-induced perturbation on reefs, and yet if the problems associated with its estimation using sediment traps are recognized, there have been few reliable measurements made over time frames relevant to the local organisms. This study describes the design, calibration and testing of an in situ optical backscatter sediment deposition sensor capable of measuring sedimentation over intervals of a few hours. The instrument has been reconfigured from an earlier version to include 15 measurement points instead of one, and to have a more rugose measuring surface with a microtopography similar to a coral. Laboratory tests of the instrument with different sediment types, colours, particle sizes and under different flow regimes gave similar accumulation estimates to SedPods, but lower estimates than sediment traps. At higher flow rates (9–17 cm s^?1), the deposition sensor and SedPods gave estimates >10× lower than trap accumulation rates. The instrument was deployed for 39 d in a highly turbid inshore area in the Great Barrier Reef. Sediment deposition varied by several orders of magnitude, occurring in either a relatively uniform (constant) pattern or a pulsed pattern characterized by short-term (4–6 h) periods of ‘enhanced’ deposition, occurring daily or twice daily and modulated by the tidal phase. For the whole deployment, which included several very high wind events and suspended sediment concentrations (SSCs) >100 mg L^?1, deposition rates averaged 19 ± 16 mg cm^?2 d^?1. For the first half of the deployment, where SSCs varied from <1 to 28 mg L^?1 which is more typical for the study area, the deposition rate averaged only 8 ± 5 mg cm^?2 d^?1. The capacity to measure sedimentation rates over a few hours is discussed in terms of examining the risk from sediment deposition associated with catchment run-off, natural wind/wave events and dredging activities.     

Longevity of simultaneous operation of aquaculture and mangrove forestry as explained in terms of water and sediment qualities

A silvofishery system (SFS) of 5.2 ha, simultaneously combining aquaculture (shrimp, crab, and fish) and forestry, was studied to understand how the water/sediment qualities had remained viable for 30 years. The long life of this SFS pond contrasts sharply with a short life of many conventional, intensively managed shrimp ponds (5 years on average). Total ammonia nitrogen in the SFS water (0.06 mg l^?1) was much lower than the Thai environmental safety standard for shrimp ponds (1.0 mg l^?1) and approximately 0.05 % of an average conventional, intensively managed shrimp pond. Total organic nitrogen of the pond sediment was 1.47 mg g^?1 which was almost half of conventional, intensively managed shrimp ponds. The flux study revealed that NH₄–N was the dominant form of nitrogen, with lesser amounts of NO₂–N and NO₃–N, and that NH₄–N was being released from the sediment into the water. Nitrogen loss from the pond, which was regarded as the denitrification rate, was estimated to be 71.5 mgN m^?2 d^?1, corresponded to 55 % of the total nitrogen input. As the average denitrification rate in a conventional, intensively managed shrimp pond is 13.4 %, the SFS was shown to be relatively efficient in removing accumulated nitrogen from the pond. Assuming accepted feed conversion rates, 3,340 kg of feed would have been necessary for the amount of fishery production recorded during 5 May 2005 and 22 March 2006. However, only 380 kg of trash fish was added, representing a saving of 2,960 kg of feed. Such a saving could be attributed to detritus from the mangrove trees that have been growing within the pond and algae encouraged to bloom by the shallow water depth. Therefore, it is suggested that the efficient nitrogen removal due to the high denitrification rate as well as the reduced feed input from mangrove detritus substitution, have contributed to maintaining favourable water and sediment qualities, resulting in the longevity of SFS pond.     

Plant regeneration of the arsenic hyperaccumulator <Emphasis Type="Italic">Pteris vittata</Emphasis> L<Emphasis Type="Italic">.</Emphasis> from spores and identification of its tolerance and accumulation of arsenic and copper

An in vitro plant regeneration system was established from the spores of Pteris vittata and identification of its tolerance, and accumulation of gametophytes and callous, to arsenic (As) and copper (Cu) was investigated. The highest frequency (100%) of callus formation was achieved from gametophyte explants treated with 0.5 mg l^?1 6-benzylaminopurine (6-BA) + 0.5 mg l^?1 gibberellin acid (GA). Furthermore, sporophytes were differentiated from the callus tissue derived from gametophyte explants on MS medium supplemented with 0.5 mg l^?1 6-BA, 0.5–1.0 mg l^?1 GA and additional 300 mg l^?1 lactalbumin hydrolysate (LH) for 4 weeks. The optimum combination of ½ MS + 1.0 mg l^?1 GA + 0.5 mg l^?1 6-BA + 300 mg l^?1 LH promoted sporophyte formation on 75 ± 10% of the callus. Every callus derived from gametophyte explants could achieve 3–4 sporophytes. The in vitro growth of gametophyte and callus was accelerated in the medium containing Na₃AsO₄ lower than 0.5 mM, but this growth was inhibited with 2 mM Na₃AsO₄. And with the increase of Na₃AsO₄ in the culture medium from 0 to 2 mM, the As accumulation in gametophytes and callus increased and achieved a level of 763.3 and 315.4 mg kg^?1, respectively. Gametophytes and calluses transplanted to culture medium, supplemented with different concentrations of CuSO₄, are similar to those in Na₃AsO₄, and the Cu accumulation in gametophytes could achieve 7,940 mg kg^?1 when gametophytes were subcultured in medium containing 3 mM CuSO₄. These results suggested that the high efficiency propagation system could be a useful and rapid means to identify other heavy metal tolerance and accumulation. Further, the regeneration ability of callus made it possible for genetic transformation of this fern.     

Hygromycin promotes somatic embryogenesis in spinach

Hygromycin (hyg) at low doses (0.5–1.0 mg l^?1) promoted somatic embryogenesis from apical sections of spinach lateral roots. The highest promoting effect on both the frequency of regeneration and the mean number of somatic embryos (SE) per explant was achieved at 0.5 mg l^?1 hyg. With increasing the concentration of hyg to 1 mg l^?1, the regeneration frequency decreased, while the mean SE number remained significantly higher than in control (hyg-free medium). Complete inhibition of SE regeneration started at 7.5 mg l^?1 hyg. Moreover, hyg efficiently promoted the process of secondary somatic embryogenesis. Compared to control, a 2.75-fold increase in the secondary somatic embryo (SSE) mean number was obtained at 0.5 mg l^?1 hyg, and the increment was still discernible at 1.0 and 2.5 mg l^?1 hyg. Both primary SE and SSE explants became completely necrotic at 12.5 mg l^?1 hyg. Since attempts with direct selection at 20 mg l^?1 hyg proved unsuccessful, the results obtained in this study suggest that a stepwise selection procedure is suitable, starting with selection at 0.5 mg l^?1 hyg, to exploit the promoting effect of low hyg doses on SE regeneration from transformed cells, then gradually increasing the hyg concentration to 20 mg l^?1 for final selection. Complete SE and SSE explant mortality at hyg above 12.5 mg l^?1 guarantees a low possibility of escape during the selection process. This study will be useful for increasing the efficiency of transgenic plant regeneration following genetic transformation in spinach.     

Effect of culture conditions, cytokinins, methyl jasmonate and salicylic acid on the biomass accumulation and production of withanolides in multiple shoot culture of Withania somnifera (L.) Dunal using liquid culture

The influence of cytokinins and culture conditions including medium volume, harvest time and elicitation with abiotic elicitors (SA/MeJ) have been studied for the optimal production of biomass and withanolides in the multiple shoot culture of Withania somnifera. Elicitation of shoot inoculum mass (2 g l^?l FW) with SA at 100 μM in the presence of 0.6 mg l^?l BA and 20 mg l^?l spermidine for 4 h exposure time at the 4th week in 20 ml liquid medium recorded higher withanolides production (withanolides A [8.48 mg g^?l DW], withanolides B [15.47 mg g^?l DW], withaferin A [29.55 mg g^?l DW] and withanone [23.44 mg g^?l DW]), which were 1.14 to 1.18-fold higher than elicitation with MeJ at 100 μM after 5 weeks of culture. SA-elicited cultures did not exhibit much variation in biomass accumulation when compared to control. This cytokinin induces and SA-elicited multiple shoot culture protocol provides a potential alternative for the optimal production of biomass and withanolides utilizing liquid culture.     

Sediment microstructure and resuspension behavior depend on each other

The critical shear stress of resuspension and rates of erosion for cohesive and loosely structured sediments must be obtained by direct measurements since there is no theoretical calculation. An in situ experiment on sediment resuspension was performed in a shallow lake (Langer See, NE Germany; area = 1.27 km², z_max = 3.8 m) in summer 2006 using a hydrodynamically calibrated erosion chamber (Ø 20 cm). Shear velocity (u*) was incrementally increased in 11 steps (0–2.19 cm s^?1) to initiate resuspension events. Entrainment rates (E) of suspended particulate matter (E_SPM), total P (E_TP), chlorophyll a (E_Chl a), and soluble reactive P (E_SRP) were determined by mass balance. Two subsequent critical u* (0.53 cm s^?1 and 1.48 cm s^?1) support the ‘two-layered bed’ model of a fluffy surface aggregate layer (freshly deposited phytodetritus prone to resuspension) and an underlying more consolidated biostabilised layer. Patterns in E_SPM (2–106 g m^?2 h^?1), E_TP (11–532 mg m^?2 h^?1), and E_Chl a (3–24 μg m^?2 h^?1) revealed a sediment surface maximum of TP and Chl a and their theoretical vertical logarithmic decrease within 4 mm sediment depth, the maximum thickness of sediment layer entrained. The advective E_SRP flux (17 mg m^?2 h^?1) was 43 times higher than the diffusive SRP flux (0.4 mg m^?2 h^?1). The TP and Chl a micro-profiles suggest that cohesive sediment bed formation is a function of both settling (fluff) and consolidation (biostabilisation). Thus, sediment microstructure and resuspension behavior depend on each other.     

Improved microspore embryogenesis induction and plantlet regeneration using putrescine,cefotaxime and vancomycin in Brassica napus L.

The effects of three periods of exposure (12, 24 and 48 h) to different levels of putrescine (0, 0.2, 0.5, 1.0, 2.0 and 5.0 mg l^?1), as well as three incubation periods (24, 48 and 72 h) to different levels of cefotaxime and vancomycin (0, 50, 100, 200 and 500 mg l^?1) on microspore embryogenesis of rapeseed cv. ‘Hyola 401’ were assessed. Microspore embryogenesis was enhanced about threefold compared with untreated culture following 48 h treatment with 0.2 mg l^?1 putrescine. Putrescine treatment at 0.5 mg l^?1 for 48 h effectively induced root formation and increased normal plantlet regeneration by 92 % when microspore-derived embryos (MDEs) were transferred to regeneration medium. The highest embryo yield (184.2 embryos Petri dish^?1) was possible when induction medium was supplemented with 50 mg l^?1 cefotaxime for 24 h and the highest normal regeneration was observed in cultures exposed to 50 and 100 mg l^?1 at all durations tested. More abnormal MDEs (76 and 82 %) were observed when microspores treated with 200 and 500 mg l^?1 cefotaxime many of which failed to regenerate normally and resulted in callusing. Vancomycin at 100 mg l^?1 during the 48 h exposure increased the number of MDEs (181.6 embryos Petri dish^?1) in contrast to untreated cultures (93.6 embryos Petri dish^?1) but, normal plantlet regeneration decreased as vancomycin level increased and high callusing (84 and 90 %) was observed with 200 and 500 mg l^?1 for 72 h. Microspore embryogenesis and plant regeneration could be improved by putrescine, cefotaxime and vancomycin when appropriate levels and durations of incubation were selected.     

In vitro plantlet regeneration and assessment of alkaloid contents from callus cultures of Ephedra foliata (Unth phog), a source of anti-asthmatic drugs

Ephedra foliata, (Gymnosperm) is a pharmaceutically important plant known for the last 5,000 years and has a number of medicinal properties. We describe here for the first time, a method for plant regeneration from callus established from axillary buds as explant, with the aim of optimizing alkaloids production in vitro. The tissue cultures initiated are being maintained for the last 3 years on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 0.5 mg l^?1 each of 2, 4-D and Kin. Maintained callus cultures exhibited regeneration potential and maximum number (23.5 ± 0.44 shoots per culture vessel) of shoots with an average height (4.94 ± 0.23 cm) was achieved on MS medium containing combination of 0.25 mg l^?1 each of Kin, BA and 0.1 mg l^?1 of NAA. About 84.9 % regenerated shoots were rooted under ex vitro conditions on Soilrite^®, if their base was treated with 500 mg l^?1 of IBA for 5 min. The rooted plantlets were successfully acclimatized under greenhouse conditions with ≈80 % survival rate. We analyzed alkaloid contents of tissue culture raised plants/callus as affected by the different concentrations and combination of two additives, i.e., l-phenylalanine and IBA. The alkaloid production was higher in the in vitro grown cultures than field-grown plants. Highest alkaloid content was recorded in callus culture on M₅ medium having 0.5 mg l^?1 each of 2, 4-D and Kin, 100 mg l^?1 l-phenylalanine and 5 mg l^?1 IBA. The present protocol may be applicable for the large-scale cultivation of E. foliata and selection of cell line having higher secondary metabolite contents of this pharmaceutically important threatened plant species.     

Effects of ascorbic acid,alpha-tocopherol,and glutathione on microspore embryogenesis in Brassica napus L.

The impact of culture conditions and addition of antioxidants to media on microspore embryogenesis in rapeseed (Brassica napus cv. ‘PF704’) was investigated. Different concentrations of ascorbic acid (0, 5, 10, 20, 50, 100, and 200 mg l^?1) and alpha (α)-tocopherol (0, 5, 10, 20, 50, 100, and 200 mg l^?1) were evaluated along with two temperature pretreatments (18 d at 30°C; 2 d at 32.5°C followed by 16 d at 30°C). In addition, combinations of reduced glutathione (0, 10, 50, and 100 mg l^?1) and ascorbic acid (5 and 10 mg l^?1) were tested. Microspore embryogenesis was significantly enhanced using 10 mg l^?1 ascorbic acid (334 embryos per Petri dish) compared with untreated cultures (184 embryos per Petri dish) at 30°C. α-Tocopherol (5 and 10 mg l^?1) enhanced (312 and 314 embryos per Petri dish, respectively) microspore embryogenesis relative to untreated cultures (213 embryos per Petri dish) at 30°C, although there were no significant differences among cultures treated with 5–50 mg l^?1 α-tocopherol. When 50 mg l^?1 α-tocopherol was combined with 5 or 10 mg l^?1 ascorbic acid, embryogenesis was significantly enhanced (308 and 328 embryos per Petri dish, respectively) relative to other ascorbic acid levels. Moreover, 10 mg l^?1 of reduced glutathione and 5 mg l^?l ascorbic acid enhanced microspore embryogenesis (335 embryos per Petri dish) compared to cultures without reduced glutathione (275 embryos per Petri dish). Microspore embryogenesis could be improved by adding ascorbic acid, α-tocopherol, and reduced glutathione when the appropriate combination and temperature pretreatment were selected.     

Plant regeneration via callus culture and subsequent in vitro flowering of Dendrobium huoshanense

A protocol for regenerating and subsequent in vitro flowering of an economical important and endangered medicinal orchid, Dendrobium huoshanense, was established mainly via indirect protocorm-like body (PLB) formation. A four-step method was developed to induce successful plant regeneration on 1/2 MS medium supplemented with suitable plant growth regulators (PGRs). Step 1 (callus induction): the root tip explants (1 cm long) were cultured at 1 mg l^?1 2,4-D + 1 mg l^?1 TDZ for 3 months. Step 2 (callus proliferation): the calli were subcultured with a 1-month interval at 1 mg l^?1 2,4-D + 1 mg l^?1 TDZ. Step 3 (PLB induction): the calli were cultured at 2 mg l^?1 NAA + 1 mg l^?1 BA for 2 months. Step 4 (plantlet conversion): the 2-month-old PLBs were cultured at 0.1 mg l^?1 IBA for 4 months. It took at least 6 months to produce well-rooted regenerated plantlets with an average of 3.2 roots and 3.6 leaves from the initial callus. The 6-month-old rooted plantlets were transferred onto PGR-free 1/2 MS medium for 6 months, and then potted with Sphagnum moss for acclimatization. After 2 month of culture, the survival rate was 100 %. The in vitro flowers were obtained on the 8-month-old plantlets at 1 mg l^?1 IBA, 5 mg l^?1 IBA and 0.1 mg l^?1 NAA, but the flowers showed a lack of the gynandrium. The abnormity was overcome by the aid of 5 mg l^?1 TDZ, and subsequently, the capsules formed without artificial pollination. This protocol provides the basis for further investigation on cell suspension, micropropagation, in vitro flowering and breeding programs in Dendrobium huoshanense.     

Re-flooding a Historically Drained Wetland Leads to Rapid Sediment Phosphorus Release

Wetland restoration provides many benefits, but re-flooding historically drained land can have unintended negative consequences, including phosphorus (P) release from sediments. To investigate the effects of re-flooding on P cycling, this study monitored a restoration in Michigan that back-flooded old drainage ditches and re-flooded former wetland soils. Immediately after re-flooding, previously exposed sediments released substantial amounts of P to the water column. Soluble reactive phosphorus (SRP) concentrations in re-flooded areas were as high as 750 μg P l^?1. At peak P concentrations, there were about 20 times more SRP and 14 times more total P in the surface water than in the much smaller flooded area that existed before re-flooding. Prolific growth of filamentous algae and duckweed was observed in subsequent summers. Sedimental analyses suggest that most of the P released originated from iron-bound fractions. The highest SRP concentrations occurred during the first year when surface water dissolved oxygen was low (<5.5 mg l^?1). Similarly low oxygen in the second year after flooding was not associated with such high P concentrations. After 1 year postflooding, SRP concentrations remained below 50 μg P l^?1 (but still high enough to produce eutrophic conditions) until the end of sampling about 15 months after re-flooding. When re-flooding historically drained soils, managers should consider the potential for sediment P release to jeopardize restoration goals and therefore should incorporate longer term monitoring of water quality into restoration plans. Knowledge of sediment P amounts and forms can indicate the potential for P release to overlying water.     

Establishment of long-term proliferating shoot cultures of elite <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. by controlling endophytic bacterial contamination

Leaf explants of the second or third node were collected from field-grown elite Jatropha curcas trees and incubated in Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) medium supplemented with growth regulators. Direct shoot organogenesis was induced when explants were incubated in a medium containing 0.5 mg l^?1 benzyladenine (BA) and 0.1 mg l^?1 indolebutyric acid (IBA). A maximum of seven shoot buds differentiated within 6 weeks of culture incubation. Indirect shoot organogenesis was obtained when explants were incubated in the medium supplemented with 0.5 mg l^?1 BA along with 1.0 mg l^?1 each of 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA). A pulse treatment of 0.5 mg l^?1 thidiazurone (TDZ) and 0.1 mg l^?1 IBA for 5 days was necessary for shoot organogenesis in green compact callus before subculture into 0.5 mg l^?1 BA and 0.1 mg l^?1 IBA containing medium. Leaf explants of J. curcas, collected from the field, contained endophytic bacterial contamination, which expressed itself after 2–3 subcultures. These bacteria were cultured and identified as Enterobacter ludwigii. After staining, these were found as gram-negative bacteria. Their sensitivity against different antibiotics has been tested by culturing them with different antibiotic stabs for 72 h. Finally, Augmentin® was found as the most effective and suitable antibiotic which not only controlled the bacteria within 2–3 subcultures but also supported the regeneration system and growth of the regenerated shoots and such cultures have been grown for a long-term of over 2 years without any contamination.     

Efficacious somatic embryogenesis and fertile plant recovery from shoot apex explants of onion (Allium cepa. L.)

An efficient somatic embryogenesis and regeneration system was developed for the first time in onion using shoot apex explants. These explants were used to initiate callus in Murashige and Skoog (MS) medium supplemented with 4.0 mg l^?1 2,4-dichlorophenoxyacetic acid. The induction frequency of primary callus in this medium was 85.3%. The primary calli were then transferred onto medium supplemented with 2.0 mg l^?1 2,4-dichlorophenoxyacetic acid. Following two biweekly subcultures, embryogenic callus formed. Inclusion of a low concentration of 6-benzylaminopurine in the subculture medium promoted the formation of embryogenic callus. The addition of 2.0 mg l^?1 glycine, 690 mg l^?1 proline, and 1.0 g l^?1 casein hydrolysate also increased the frequency of callus induction and embryogenic callus formation. The highest frequency of embryogenic callus (86.9%) and greatest number of somatic embryos (26.3 per callus) were obtained by the further addition of 8.0 mg l^?1 silver nitrate. Somatic embryos formed plantlets on regeneration medium supplemented with 1.5 mg l^?1 6-benzylaminopurine; addition of 2.0 mg l^?1 glycine to the regeneration medium promoted a high frequency of regeneration (78.1%) and plantlet formation (28.7 plants per callus). The regenerated plantlets were transferred to half-strength MS medium supplemented with 1.5 mg l^?1 indole-3-butyric acid for root development; the maximum frequency of root formation was 87.7% and the average number of roots was 7.6 per shoot. The regenerated plantlets were successfully grown to maturity after hardening in the soil. This is the first report of somatic embryogenesis and regeneration from shoot apex explants of onion.     

Plant regeneration from protoplasts in Indian local Coriandrum sativum L.: scanning electron microscopy and histological evidences for somatic embryogenesis

Coriandrum sativum L. is an annual herb belonging to the family Umbelliferae. It is used as a spice plant in Indian subcontinent and it has several medicinal applications as well. In this present article, an efficient plant regeneration protocol from protoplasts via somatic embryogenesis was established and is reported. This is the first ever protoplast isolation study in Indian local coriander in which plant regeneration was achieved. Hypocotyl-derived embryogenic callus was used as a source of protoplast. The embryogenic callus suspension was prepared by transferring tissues onto rotary-agitated liquid Murashige and Skoog, added with 1.0 mg l^?1 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l^?1 KIN (6-furfurylaminopurine). The suspension was digested with enzymatic solutions and a combination of cellulase (2.0 %), pectinase (1.0 %), macerozyme (0.02 %) and driselase (0.50 %) induced maximum yield of protoplasts (34.25 × 10⁵). In 1.0 mg l^?1 2,4-D + 1.0 mg l^?1 KIN containing medium, protoplasts divided well and formed maximum number of microcolonies (14.30/test tube). The protoplast callus (PC) biomass grew well in solid medium. The protoplast embryogenic callus was rich in protein, proline and sugar compared to non-embryogenic PC. The protoplast originated callus later differentiated into somatic embryos. The somatic embryo morphology, scanning electron microscopy and histology of embryo origin and development were investigated and discussed in details in this present communication. In 1.0 mg l^?1 2,4-D + 0.5 mg l^?1 BA (6-Benzyladenine), maximum number of embryos were formed on microcallus (26.6/callus mass). The embryo matured and germinated into plantlets at a low to moderate rate, highest (31.3 %) embryo germination was observed in 1.0 mg l^?1 BA + 0.5 mg l^?1 α-Naphthalene acetic acid added medium. The entire process of regeneration took about 4–5 months’ time for recovering plantlets from protoplasts.     

Carbon dioxide and methane emissions from interfluvial wetlands in the upper Negro River basin, Brazil

Extensive interfluvial wetlands occur in the upper Negro River basin (Brazil) and contain a mosaic of vegetation dominated by emergent grasses and sedges with patches of shrubs and palms. To characterize the release of carbon dioxide and methane from these habitats, diffusive and ebullitive emissions and transport through plant aerenchyma were measured monthly during 2005 in permanently and seasonally flooded areas. CO₂ emissions averaged 2193 mg C m^?2 day^?1. Methane was consumed in unflooded environments and emitted in flooded environments with average values of ?4.8 and 60 mg C m^?2 day^?1, respectively. Bubbles were emitted primarily during falling water periods when hydrostatic pressure at the sediment?Cwater interface declined. CO₂ and CH₄ emissions increased when dissolved O₂ decreased and vegetation was more abundant. Total area and seasonally varying flooded areas for two wetlands, located north and south of the Negro River, were determined through analysis of synthetic aperture radar and optical remotely sensed data. The combined areas of these two wetlands (3000 km²) emitted 1147 Gg C year^?1 as CO₂ and 31 Gg C year^?1 as CH₄. If these rates are extrapolated to the area occupied by hydromorphic soils in the upper Negro basin, 63 Tg C year^?1 of CO₂ and 1.7 Tg C year^?1 as CH₄ are estimated as the regional evasion to the atmosphere.     

Degradation of cationic surfactants using Pseudomonas putida A ATCC 12633 immobilized in calcium alginate beads

In this study, the degradation of tetradecyltrimethylammonium bromide (TTAB) by freely suspended and alginate-entrapped cells from the bacteria Pseudomonas putida (P. putida) A ATCC 12633 was investigated in batch cultures. The optimal conditions to prepare beads for achieving a higher TTAB degradation rate were investigated by changing the concentration of sodium alginate, pH, temperature, agitation rate and initial concentration of TTAB. The results show that the optimal embedding conditions of calcium alginate beads are 4 % w/v of sodium alginate content and 2 × 10⁸ cfu ml^?1 of P. putida A ATCC 12633 cells that had been previously grown in rich medium. The optimal degradation process was carried out in pH 7.4 buffered medium at 30 °C on a rotary shaker at 100 rpm. After 48 h of incubation, the free cells degraded 26 mg l^?1 of TTAB from an initial concentration of 50 mg l^?1 TTAB. When the initial TTAB concentration was increased to 100 mg l^?1, the free cells lost their degrading activity and were no longer viable. In contrast, when the cells were immobilized on alginate, they degraded 75 % of the TTAB after 24 h of incubation from an initial concentration of 330 mg l^?1 of TTAB. The immobilized cells can be stored at 4 °C for 25 days without loss of viability and can be reused without losing degrading capacity for three cycles.