Mesophyll Protoplast Culture and Plant Regeneration of Oriental Planetree (Platanus orientalis)

Large populations of mesophyll protoplasts were released from the leaves of 1.5–2 month old sterile seedlings, with a high protoplast yield (3.7× 10 6g-1FW) after protoplast purification. The purified protoplasts were cultured in a modified K8p liquid medium supplemented with 0.5 mg/L 2,4-D, 1 mg/L NAA and 0.5 mg/L BA. Higher density (1× 106/ml) in the initial culture of protoplasts is favourable to the division of cultured mesophyll protoplasts of this woody species among the densities tested. The protoplasts started to divide after 6 days of culture, and achieved 26.8% division frequency by 14 days. Sustained divisions resulted in mass production of cell colonies and small calli in 8 weeks. The calli further grew to 2–3mm on the gelrite-solidified K8 medium supplemented with 0.2 mg/L NAA aud 0.5 mg/L BA. Then, they were transferred onto the MSB proliferation medium with 0.1 mg/L NAA and 0.25 mg/L BA, where compact and cream-coloured calli were formed. Shoot formation was initiated on MSB differentiation medium coraming 0.5 mg/L IAA, 1 mg/L each of BA and ZT. It was observed that the frequency of shoot formation was about 28.7%. Whole plantlets were regenerated upon transferring 3 cm shoots to 1/2MS medium with 0.5mg/L IBA and 0.1mg/L BA, from which they were already transplanted into pots and grew well in the phytotron of Shanghai Institute of Plant Physiology.     

Concentrations and Distribution of Trace Metals in Water and Streambed Sediments of Orogodo River,Southern Nigeria

The distribution of Cd, Pb, Ni, Cr, Cu, Mn, Fe, and Zn in sediment and surface water, and some physico-chemical characteristics of Orogodo river sediments, were evaluated. The sediment pH ranged from 5.1–7.3; conductivity values ranged from 34.5 to 389.0 μScm^?1. Total nitrogen values ranged from 0.06–0.10%, NH₃-N values ranged from 0.25–0.44 mgkg^?1, percent total organic carbon ranged from 0.21–1.68%, and total phosphorus values ranged from 0.004–0.02% for dry and wet seasons. The sand fraction consists of 87–95%, silt fractions ranged from 0–2%, and clay fraction between 4–13%. The mean concentrations of metals (dry weight basis) in the streambed sediments ranged from 1.92–17.37 mgkg^?1 for Cu, 0.98–4.78 mgkg^?1 for Ni, 0.01–32.98 mgkg^?1 for Mn, 353.22–2045.64 mgkg^?1 for Fe, 69.96–100.16 mgkg^?1 for Zn, 0.21–1.32 mgkg^?1 for Cr, and Cd was less than 0.001 mgkg^?1 for wet and dry seasons. The mean concentrations of metals in the surface water ranged between 0.01–0.05–0.05 mg/L for Cu, nd-0.11 mg/L for Ni, 0.001–0.31 mg/L for Pb, 0.001–1.82 mg/L Mn, 0.01–3.52 mg/L for Fe, 0.16–0.61 mg/L for Zn, nd-0.007 mg/L for Cr, and <0.001 mg/L for Cd. Based on principal component analysis, two main sources of metals in the Orogodo River can be identified: (i) Cr, Cu, Pb, and Fe are mainly derived from industrial sources; (ii) Mn, Zn, and Ni associated with traffic activities. No element examined had a contamination/pollution index value greater than unity (pollution ranges). This implies that the multiple pollution indices obtained from the analysis showed that Orogodo River sediments were not polluted with heavy metals.     

Studies on Plant Regeneration from Mesophyll Protoplasts of Solanum tuberosum L.

Protoplasts from potato mesophyll of two strains (Solannum tuberosum L. cv. Xiao Yie Zi x Duo Zi Bia and Solanum tuberosum L. cv. Wu Meng 601) were induced to callus in culture medium of protoplasts. The callus derived from mesophyll protoplasts were transferred to MS medium with 2 mg/l ZT+0.1 mg/L IAA. Shoots regenerated from the callus were detected after 70 days of culture.The shoots which had grown to a height of 2–3 cm were transferred to MS medium with 0.05 mg/L NAA. Roots were coming out in a few days.Complete plantlets were achieved. Stern segments with 1–2 leaves were then transferred to a mixture of sterilized soil and grown, and produced tuber.     

Plantlet Regeneration from Leaf Protoplast cultures of Japanese Butterbur

Protoplasts were isolated by enzyme digestion from leaf of Japanese butterbur (Petasites japonicus). The enzyme incubation mixture consisted of 4% (W/V)cellulase RS, 2% (W/V) hemicellulase, 1% (W/V) pectinase-dissolved Y-23 and polygalacturonase in a solution of 0. 5 mol/L mannitol at pH 5.7 . In the basic medium of 1/4 MS inorganic salts and 1/2 MS vitamins supplemented with 2 mg/L NAA, 0. 2–0. 5 mg/L BA, 0. 5 mol/L mannitol and 10 g/L sucrose, the cells divided luxuriantly. Regenerated plantlets were formed from callus after bud induction and root initiation.     

Investigation of the antibacterial activity of a short cationic peptide against multidrug-resistant Klebsiella pneumoniae and Salmonella typhimurium strains and its cytotoxicity on eukaryotic cells

With the growing microbial resistance to conventional antimicrobial agents, the development of novel and alternative therapeutic strategies are vital. During recent years novel peptide antibiotics with broad spectrum activity against many Gram-positive and Gram-negative bacteria have been developed. In this study, antibacterial activity of CM11 peptide (WKLFKKILKVL-NH2), a short cecropin–melittin hybrid peptide, is evaluated against antibiotic-resistant strains of Klebsiella pneumoniae and Salmonella typhimurium as two important pathogenic bacteria. To appraise the antibacterial activity, minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and bactericidal killing assay were utilized with different concentrations (2–128 mg/L) of peptide. To evaluate cytotoxic effect of peptide, viability of RAJI, Hela, SP2/0, CHO, LNCAP cell lines and primary murine macrophage cells were also investigated with MTT assay in different concentrations (3–24 and 0.5–16 mg/L, respectively). MICs of K. pneumoniae and S. typhimurium isolates were in range of 8–16 and 4–16 mg/L, respectively. In bactericidal killing assay no colonies were observed at 2X MIC for K. pneumoniae and S. typhimurium isolates after 80–90 min, respectively. Despite the fact that CM11 reveals no significant cytotoxicity on RAJI, Hela, SP2/0, and CHO cell lines beneath 6 mg/L at first 24 and 48 h, the viability of LNCAP cells are about 50 % at 3 mg/L, which indicates strong cytotoxicity of the peptide. In addition, macrophage toxicity by MTT assay showed that LD₅₀ of CM11 peptide is 12 μM (16 mg/L) after 48 h while in this concentration after 24 h macrophage viability was about 70 %.     

Experimental evaluation of calcein and alizarin red S for immersion marking of silver carp Hypophthalmichthys molitrix (Valenciennes, 1844)

Calcein (CAL) and alizarin red S (ARS) at concentrations of 50–200 and 150–300 mg/L, respectively, were used for immersion marking of juvenile silver carp Hypophthalmichthys molitrix (79.65 ± 2.11 mm total length, mean ± SD). The marked fish were kept in separate tanks (0.012 individuals per litre, rearing temperature 18.4–25.7°C) for 360 days. Each experimental treatment group consisted of three replicates (16 individuals per replicate). Immersion for 24 h produced detectable marks in the sagittae, lateral line and non‐lateral line scales, and fin rays (dorsal, pectoral, ventral, anal, and caudal) at 360 days post‐marking. Acceptable marks in the sagittae were observed for CAL at concentrations of 150–200 mg/L and for ARS at concentrations of 200–300 mg/L. Fluorescent marks were poorly visible in all non‐lateral line scales from both the CAL‐ and ARS‐treated groups. Acceptable fluorescent marks in the lateral line scales and fin rays were detected for CAL at concentrations of 150–200 and 100–200 mg/L, respectively, and for ARS at concentrations of 200–300 and 150–300 mg/L, respectively. In particular, optimal marks were observed at the highest concentrations investigated in lateral line scales (200 mg/L CAL, 300 mg/L ARS) and fin rays (200 mg/L CAL, 200–300 mg/L ARS). There was no significant difference in the survival or growth of marked fish compared to controls throughout the experiment (P > 0.05).     

Studies on Induction of Pollen Plantlets from the Anther Cultures of Lily

Anthers with the filament of lily (Lilium davidii var. Willmottiae (Wilson) Roffill) were cultured on modified MS medium. Supplemented with different concentrations and compatible ratios of growth hormones (Z 2 mg/L,or 2,4-D 2 mg/L + KT 2mg/L, or 2,4-D 4mg/L+ 6 BA 2 mg/L). At this time the pollen grains in the anthers were at the late uninueleate stage. Anther cultures were incubated at 25—27 ℃, and illuminated with daylight of about 800–1200 lx. After 30 days, the calli or embryoids were produced from anthers. The frequency of the calli or embryoids induction was 8.89%. After transfer eventually to the differentiation medium, these calli or embryoids developed into plantlets in 70 days. Among the root tips of regenerated plantlets haploid, diploid and aneuploid cells were found, but the haploid cells were produced in about 86.4% of the root tips. It is quite evident that haploid plantlets are derived from the pollen grains.     

Relationship of protozoan biomass to phosphate and nitrate removal from activated sludge mixed liquor

The relationship between protozoan biomass concentration and phosphate and nitrate removal was investigated in mixed liquor using three different carbon sources as supplements. The study was carried out using three respective initial biomass concentrations in a shaking flask environment. Samples were taken every 24 h to determine phosphate, nitrate, dissolved oxygen and chemical oxygen demand. The results revealed a direct relationship between decreases in nutrient concentrations and increases in cell densities of the isolates. Between 24 and 96 h, the increases in the protozoan density corresponded to a phosphate decreases from initial ranges of 55.42–57.36 mg/L, 50.27–51.17 mg/L and 50.01–50.83 mg/L to final ranges of 2.46–11.90 mg/L, 0.61–11.80 mg/L and 1.29–13.89 mg/L, in the presence of Aspidisca, Trachelophyllum and Peranema, respectively. Nitrate concentrations were observed to decrease from initial ranges of 23.84–25.90 mg/L, 23.94–25.84 mg/L and 26.12–26.54 mg/L to final ranges of 0.11–6.32 mg/L, 0.16–5.60 mg/L and 0.24–9.04 mg/L, respectively. The study had revealed that an increase in cell density of the test isolates produces a corresponding increase in phosphate and nitrate removal.     

The combined effects of chlorine and ammonia on litter breakdown in outdoor experimental streams

The response of Potamogeton crispus L. breakdown to controlled doses of different levels of chlorine and chlorine + ammonia was investigated over two years in outdoor experimental streams. In 1985, downstream riffles of 2 streams were dosed (observed in-stream concentrations) at ca. 10 μg/L Total Residual Chlorine (TRC), one stream at 64 μg/L TRC and one stream at 230 μg/L TRC. Two control streams were not dosed and the upstream riffles of each stream served as within stream controls. In 1986, the downstream riffle of one stream was dosed at 70 μg/L TRC and a second stream was dosed at 200 μg/L TRC. Four streams were also dosed with 2.5 mg/L NH₃-N: one stream with no chlorine, one stream with ca. 10 μg/L TRC, one with 56 μg/L TRC, and one with 150 μg/L TRC. A seventh stream was dosed for 2 h at 2000 μg/L TRC and 2.5 mg/L ammonia and then allowed to recover (recovery stream). Each year, litter decomposition (degree day k values) was measured during two 35 day trials (Jun–Jul and Aug–Sep). In 1985, when streams were dosed with chlorine alone, decomposition was significantly reduced with the high (230 μg/L TRC) chlorine dose. Downstream decomposition was 27% (Jun–Jul) and 59% (Aug–Sep) of the upstream (control) rate. No other chlorine effects were found during this period. In Jun–Jul 1986, there was significantly lower decomposition in the downstream dosed sites of the 200 μg/L TRC alone stream, the 146 μg/L TRC + ammonia stream and the recovery stream; downstream decay rates were (respectively) 56%, 42% and 64% of the upstream control sites. No other up-down pairs were different in July 1986. In Aug–Sep, all three streams with chlorine + ammonia (6, 56 and 146 μg/L TRC + 2,5 mg/L ammonia) and the 70 μg/L TRC alone stream had significantly lower decomposition rates in the downstream dosed sites. For these streams, downstream decay rates ranged from 46% (high chlorine + ammonia) to 73% (low chlorine + ammonia) of the upstream control rates. No other up-down pairs were different during this trial. Up and downstream sites of the stream dosed with 2.5 mg/L ammonia alone were nearly identical for both trials (< 3% difference). These results indicate that TRC at less than 250 μg/L can significantly reduce litter decomposition and strongly suggest that addition of ammonia to chlorinated water can increase the toxic effect of chlorine. currently at the Department of Fisheries and Wildlife currently at the Department of Fisheries and Wildlife     

Somatic Embryogenesis and Plant Regeneration from Protoplasts of Cowpea (Vigna sinensis)

Immature cotyledons of cowpea (Vigna sinensis Endlo) were used for protoplast isolation. Enzyme solution for protoplast isolation contained 40% cellulase Onozuka R-10,0.30% Macerozyme R-10 and 2% hemicellulase. The purified protoplasts were cultured in Bs,MS or KM8p liquid medium in dark (25℃) at a density of 1 × 105–5 × 105/ml. The protoplasts started cell division in 3–5 days . Sustained cell divisions resulted ill formation of cell clusters and small calli,with cell division frequency reaching 23%–28% in MS medium . Calli of 2 mm in size were transferred onto MSB (MS salts+B5 vitamins) medium with 2 mg/L 2,4-D, 0. 5mg /L BA forfurther growth. Embryogenic calli appeared on this medium. After passage to fresh medium with the same composition, the embryogenic calli were transferred into MSB liquid medium to establish suspension culture. When the suspended calli were transferred back onto MSB agar medium with 0. 1 mg /L IAA, 0.5mg/L KT, 5% mannitol (cultured in light,2000 lx,12h/d), a lot of adventitious roots formed in 7–10 days, and then somatic embryos formed from the protoplast derived calli. But only a few embryoids developed further into the cotyledonary stage ,and the others died at globular, heart-shaped, or torpeto stage . Finally, some cotyledonary embryoids germinated and developed into plantlets or shoots with leaves.     

Anaerobic digestion of chicken manure: Influence of trace element supplementation

In this study, anaerobic digestion of nitrogen‐rich chicken (egg‐laying hen) manure at different trace element (TE) mix doses and different total ammonia nitrogen (TAN) concentrations was investigated in batch digestion experiments. With respect to nonsupplemented TE sets, addition of TE mixture containing 1 mg/L Ni, 1 mg/L Co, 0.2 mg/L Mo, 0.2 mg/L Se, 0.2 mg/L W, and 5 mg/L Fe at TAN concentrations of 3000 mg/L and 4000 mg/L, cumulative CH₄ production and CH₄ production rate improved by 7–8% and 5–6%, respectively. The results revealed that at a very high TAN concentration of 6000 mg/L, the effect of TE addition was significantly high and the cumulative CH₄ production and production rate were increased by 20 and 39.5%, respectively. Therefore, it is concluded that at elevated TAN concentrations the CH₄ production that was stimulated by TE supplementation was presumably occurred through syntrophic acetate oxidation.     

Effects of nitrogen,phosphorus, iron and silicon on growth of five species of marine benthic diatoms

The effects of nitrogen, phosphorus, iron and silicon on growth of five species of marine benthic diatoms, namely Navicula patrickae, Nitzschia panduriformis, Navicula thienemannii, Nitzschia longissima and Navicula atomus were studied by single factor experiments and the optimal concentration ratios of the four nutrient elements beneficial for diatoms growth were screened out separately using the L₉ (3⁴) orthogonal design. The results highlighted that nitrogen, phosphorus, iron and silicon all had highly significant effects on growth of five diatoms while the diatoms growth rates reached the highest averagely in the 2nd to the 6th culture day. In addition, the optimal concentrations (mg/L) of four nutrients suitable for diatoms growth were found higher than that in f/2 medium except that Nitzschia longissima had the same concentration of nitrogen as that in f/2 medium which is optimal for growth. Moreover, the optimal growth concentrations of four elements for five diatoms varied in the range of 12.36–74.16 mg/L for nitrogen, 1.70–3.98 mg/L for phosphorus, 2.00–4.00 mg/L for iron, 23.01–69.03 mg/L for silicon, respectively. By means of the orthogonal test of four nutrients for five benthic diatoms, the optimal concentration ratios N:P:Fe:Si (mg/L) were obtained as follows: 74.16:2.27:3.33:23.01 for N. patrickae; 37.08:3.98:4.00:11.50 for N. panduriformis; 49.44:3.98:3.33:34.51 for N. thienemannii; 12.36:1.70:4.00:11.50 for N. longissima; 74.16:2.27:4.00:69.03 for N. atomus.     

Toxic effects of some insecticides,herbicides, and plant essential oils against Tribolium confusum Jacquelin du val (Insecta: Coleoptera: Tenebrionidae)

Cereals are staple food for many countries and are grown on millions of hectares of land, but much of the harvest is wasted due to losses by pests. To minimize these losses, many pesticides are used which are damaging to the environment and human health. There are debates to get rid of these chemicals but they are still in use at large scale. An alternative control strategy for insect pests in storage houses is the use of botanicals. In this study, four plant essential oils, two plant extracts, two herbicides, and two insecticides were used against Tribolium confusum and the comparison of toxicity was made by calculating LC₅₀ and LT₅₀ values. LC₅₀ values were higher for abamectin (2.09–10.23 mg/L) and cypermethrin (3.41–11.78 mg/L) insecticides followed by neem essential oil (7.39–19.24 mg/L) and citrus extract (10.14–24.50 mg/L). However, LC₅₀ values were maximum in case of jaman plant extract (22.38–176.42 mg/L) followed by two herbicides, Logran (19.66–39.72 mg/L) and Topik (29.09–47.67 mg/L) However, LC₅₀ values were higher for topic herbicide (24.098 ppm) and jaman essential oil (16.383 ppm) after four days of treatment. Abamectin and cypermethrin insecticides, neem essential oil and citrus plant extract also killed adults of T. confusum quicker as compared other essential oils, extracts and herbicides. Results revealed that botanical formulations being environmentally safe could be used instead of highly hazardous pesticides for stored products’ pests. This study also elaborates the non-host toxicity of herbicides commonly applied in our agroecosystem.     

激素种类及其浓度对矮牵牛试管苗增殖及生根率的影响

以MS培养基为基本培养基,以矮牵牛试管苗为材料,并用不同浓度的细胞分裂素6-BA、KT、Ad分别与生长素NAA（0．10mg／L）进行配比试验,并用一定浓度的细胞分裂素6-BA、KT、Ad两两分别组合配比试验,探讨了不同浓度的细胞分裂素对矮牵牛试管苗的影响,以及两类生长素IBA和NAA对生根的影响。结果表明,适合于矮牵牛试管苗增殖的培养基有：（1）MS＋1．60mg／L 6-BA＋0．10mg／LNAA;（2）MS＋0．80mg／L 6-BA＋1．60mg／LKT＋0．10mg／L NAA;（3）MS＋0．80mg／L 6-BA＋0．20mg／LAd＋0．10mg／LNAA;（4）MS＋1．60mg／L KT＋0．20mg／L Ad＋0．10mg／L NAA。适合于矮牵牛试管苗生根的培养基有（1）1／2MS;（2）1／ZMS＋0．20mg／L1BA;（3）1／2MS＋0．20mg／LNAA。     

Acute toxicity of carbon dioxide to juvenile marine shrimp Litopenaeus vannamei (Boone 1931)

Elevated concentrations of dissolved carbon dioxide (CO₂) and reduced pH levels are observed during the culture and transportation of aquatic organisms. Studies on the toxicity effects of CO₂ in penaeid shrimp are scarce when compared to the amount of research in fish. The objective of the present study was to determine the lethal concentration and safety levels of CO₂ for juvenile white shrimp Litopenaeus vannamei. Juveniles (1.76 ± 0.36 g) were exposed for 96 h to one of six concentrations of dissolved CO₂ (14.5, 23.8, 59.0, 88.0, 115.0, and 175.0 mg/L) or a control condition (without the addition of CO₂), and their survival was monitored for 96 h. The LC₅₀ values with 95% confidence limits at 24, 48, 72, and 96 h were 130.05 (104.2–162.1), 77.2 (73.8–80.02), 69.65 (65.47–74.32), and 59.12 (53.08–66.07) mg/L of CO₂, respectively. The calculated safety level was 5.9 mg/L of CO₂, and the highest concentration that did not induce significantly higher mortality than that observed in controls (NOEC) was 23.8 mg/L of CO₂. We recommend that CO₂ levels should be kept below the safety level obtained in this study.     

Flow‐injection chemiluminescence method for the determination of naphazoline hydrochloride and oxymetazoline hydrochloride

A sensitive and simple flow‐injection chemiluminescence (FI‐CL) method, which was based on the CL intensity generated from the redoxreaction of potassium permanganate (KMnO₄)–formaldehyde in vitriol (H₂SO₄) medium, has been developed, validated and applied for the determination of naphazoline hydrochloride and oxymetazoline hydrochloride. Besides oxidants and sensitizers, the effect of the concentration of H₂SO₄, KMnO₄ and formaldehyde was investigated. Under the optimum conditions, the linear range was 1.0 × 10^?2–7.0 mg/L for naphazoline hydrochloride and 5.0 × 10^?2–10.0 mg/L for oxymetazoline hydrochloride. During seven repeated inter‐day and intra‐day precision tests of 0.1, 1.0 and 10.0 mg/L samples, the relative standard deviations all corresponded to reference values. The detection limit was 8.69 × 10^?3 mg/L for naphazoline hydrochloride and 3.47 × 10^?2 mg/L for oxymetazoline hydrochloride (signal‐to‐noise ratio ≤3). This method has been successfully implemented for the determination of naphazoline hydrochloride and oxymetazoline hydrochloride in pharmaceuticals. Copyright © 2009 John Wiley & Sons, Ltd.     

The effect of zinc on the increase in length of five species of intertidal fucales

The growth rate of Pelvetia canaliculata (L.) Dec. et Thur., Fucus spiralis L., F. vesiculosus L., F. serratus L., and Ascophyllum nodosum (L.) Le Jolis was measured in six different concentrations of zinc (0.025–14 mg/l) during a period of ten days. No significant growth response was observed at 0.1 mg/l of zinc. A significant reduction of growth during the first two to three days of exposure to 1.4 mg/l or more was followed by no or small further decline. Apices of A. nodosum from localities with different background load of zinc (0.1–0.5 mg/l and <0.01 mg/l) showed no difference in response to 2.6 mg/l of zinc.     

Somatic embryogenesis and plant regeneration from immature cotyledons of European chestnut (Castanea sativa Mill.)

Here, we established a protocol for induction of somatic embryogenesis and plant regeneration from immature cotyledons of open-pollinated seeds of European chestnut (Castanea sativa Mill.) cultivars ‘Osmano?lu’ and ‘Sar?a?lama’. Basal media, Murashige and Skoog medium (MS), Driver and Kuniyuki Walnut medium (DKW), and Woody Plant Medium (WPM) supplemented with l-glutamine or casein hydrolysate, with or without silver nitrate, agar or gelrite, and various plant growth regulator (PGR) combinations were tested in initial cultures for induction of somatic embryos. The effects of initial cultures on the percentage of somatic embryos and average number of embryos per cotyledon explant, subcultured monthly, were determined at the end of 4 mo. Interactions were observed among the different treatments for ‘Osmano?lu’ cultivar, with the highest rates of somatic embryogenesis (4.7–9.7%) being obtained in MS, DKW, or WPM basal media supplemented with (1) 6-benzyladenine (BA; 1 mg/L)?+?kinetin (KIN; 2 mg/L)?+?indole-3-butyric acid (IBA; 0.01 mg/L); (2) BA (1 mg/L)?+?1-phenyl-3-(1,2,3-thiadiazol-5-yl; TDZ 0.1 mg/L)?+?IBA (0.01 mg/L), and (3) KIN (2 mg/L)?+?TDZ (0.1 mg/L)?+?IBA (0.01 mg/L) PGR combinations plus l-glutamine or casein hydrolysate, with or without silver nitrate, and with either gelrite or agar. The highest percentages (12.0% and 11.2%) of somatic embryogenesis for ‘Sar?a?lama’ were obtained in DKW supplemented with PGR combinations of (1) BA (1 mg/L)?+?KIN (2 mg/L)?+?IBA (0.01 mg/L), (2) BA (1 mg/L)?+?TDZ (0.1 mg/L)?+?IBA (0.01 mg/L), respectively. The average number of somatic embryos ranged between 0 and 0.65 per explant for ‘Osmano?lu’ and between 0 and 0.49 per ‘Sar?a?lama’ explant. For germination of somatic embryos, root, shoot, and plantlet regeneration, different treatments included desiccation, cold and gibberellic acid (GA₃), and BA alone or in combination with auxins (IBA or α-naphthaleneacetic acid, NAA; 0.1 mg/L). The highest rate of somatic embryos regeneration (27.5%) occurred using MS basal media with half-strength microelements containing 0.1 mg/L BA?+?0.1 mg/L NAA, after treatments of desiccation, or desiccation plus cold or GA₃ (3 mg/L).     

Improvement of expression level of keratinase Sfp2 from Streptomyces fradiae by site-directed mutagenesis of its N-terminal pro-sequence

The keratinase Sfp2, produced by Streptomyces fradiae var. k11, is a serine alkaline protease first synthesized as pre-pro-mature precursor, of which the N-terminal propeptide must be autocatalytically cleaved on the C-terminal of P1 amino acid to produce mature enzyme. Single amino acid substitutions were introduced at positions ?1 and ?2 to improve the expression level of mature Sfp2. The specific activity of L(?1)F mutant (48935 U/mg) was nine times that of wild-type Sfp2, whereas the mutants L(?1)D, L(?1)G, L(?1)H, K(?2)E, and K(?2)L had 2–52 % of the specific activity of wild-type. The yield of mature Sfp2 of L(?1)F mutant was estimated to be 800 μg/mg total protein and 112 mg/l culture supernatant, nine and twice that of wild-type, respectively. The L(?1)F mutant exhibited similar enzymatic properties to wild-type.     

Efficient TDZ-induced regeneration from capitulum explants of Gerbera jamesonii Bolus ex Hooker F. - an ornamental plant with high aesthetic value

Gerbera jamesonii Bolus ex Hooker F. (Asteraceae), a most prominent ornamental plant ranking fifth in the world cut flower market has gained huge demand across the globe for its high aesthetic value. In the present study, an efficient plant regeneration protocol was developed using capitulum explants from the field grown mature plants of G. jamesonii. A successful surface sterilization procedure was established using a series of sterilants without affecting explant regenerability. The culture of capitulum explants on Pre-culture Induction Medium [(PIM), Murashige and Skoog (MS) medium fortified with 0.5?mg/L thidiazuron (TDZ) in combination with low levels of auxins] for a period of 2 weeks in dark conditions was crucial for inducing morphogenetic response. Upon transfer onto Shoot Regeneration Medium [(SRM), MS +4.0?mg/L 6-benzyladenine (BA) and 0.2?mg/L NAA], explants has significantly produced highest number of shoots (16?±?0.33). Incubation for 18–21?days on Shoot Multiplication and Elongation Medium [(SMEM), MS +2.0?mg/L BA +0.2?mg/L NAA] resulted in the highest number of multiple shoots (22.00?±?0.93) with increased mean shoot length (3.49?±?0.08). In vitro elongated shoots were rooted (95%) on ½ MS +1.0?mg/L indole-3-acetic acid (IAA) within 10–12?days and fully rooted plants were transferred to poly-house for hardening with 90% survival rate.