Endophytic bacterial flora in the stem tissue of a tropical maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) genotype: isolation,identification and enumeration

The genotypic diversity of indigenous bacterial endophytes within stem of tropical maize (Zea mays L.) was determined in field and greenhouse experiments. Strains were isolated from stem tissues of a tropical maize cultivar (PEHM-1) by trituration and surface disinfestation and their population dynamics was determined. Endophytes were found in most of the growing season at populations ranging from 1.36–6.12 × 10⁵ colony-forming units per gram fresh weight (c.f.u./gm fw) of stem. Analysis of these bacterial endophytes using Gas Chromatography—Fatty Acid Methyl Ester (GC-FAME) led to the identification of Bacillus pumilus, B. subtilis, Pseudomonas aeruginosa and P. fluorescens as the relatively more predominant group of bacterial species residing in maize stem. When the maize seedlings grown in a greenhouse were inoculated with these four isolates individually, their population densities decreased (1.6–3.1 × 10⁵ c.f.u./gm fw of stem) as compared to the field-grown maize (1.8–3.8 × 10⁵ c.f.u./gm fw of stem). The highest persistence, however, was recovered in the case of B. subtilis with a population density of 3.1 × 10⁵ c.f.u./gm fw of stem tissue on 28 days after emergence (DAE). This is the first report on population dynamics of bacterial endophytes from tropical maize and the results establish that symptomless populations of bacteria exist in the maize stem.     

Industrial wastewaters and dewatered sludge: rich nutrient source for production and formulation of biocontrol agent, <Emphasis Type="Italic">Trichoderma viride</Emphasis>

Axenic cultivation of biocontrol fungus Trichoderma viride was conducted on a synthetic medium and different wastewaters and wastewater sludges in shake flasks to search for a suitable raw material resulting in higher biocontrol activity. Soluble starch based synthetic medium, dewatered municipal sludge, cheese industry wastewater sludge, pre-treated and untreated pulp and paper industry wastewater and slaughter house wastewater (SHW) were tested for T. viride conidia and protease enzyme production. The maximum conidia production followed the order, soluble starch medium (>10⁹ c.f.u./mL), untreated pulp and paper industry wastewater (4.9 × 10⁷ c.f.u./mL) > cheese industry wastewater (1.88 × 10⁷ c.f.u./mL) ≈ SHW (1.63 × 10⁷ c.f.u./mL) > dewatered municipal sludge (3.5 × 10⁶ c.f.u./mL) > pre-treated pulp and paper industry wastewater (1.55 × 10⁶ c.f.u./mL). The protease activity of T. viride was particularly higher in slaughterhouse wastewater (2.14 IU/mL) and dewatered municipal sludge (1.94 IU/mL). The entomotoxicity of soluble starch based synthetic medium was lower (≈6090 SBU/μL) in contrast to other raw materials. The entomotoxicity inversely decreased with carbon to nitrogen ratio in the growth medium and the conidia concentration and protease activity also contributed to the entomotoxicity. The residual c.f.u./g formulation of T. viride conidia were up to approximately, 90% after 1 month at 4 ± 1 °C and about 70% after 6 months at 25 ± 1 °C. Thus, production of T. viride conidia would help in marketability of low cost biopesticide from the sludge and safe reduction of pollution load.     

Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture

Malaysia is the world’s leading producer of palm oil products that contribute US$ 7.5 billion in export revenues. Like any other agro-based industries, it generates waste that could be utilized as a source of organic nutrients for microalgae culture. Present investigation delves upon Isochrysis sp. culture in POME modified medium and its utilization as a supplement to Nanochloropsis sp. in rotifer cultures. The culture conditions were optimized using a 1 L photobioreactor (Temp: 23°C, illumination: 180 ∼ 200 μmol photons m⁻²s⁻¹, n = 6) and scaled up to 10 L outdoor system (Temp: 26–29°C, illumination: 50 ∼ 180 μmol photons m⁻²s⁻¹, n = 3). Algal growth rate in photobioreactor (μ = 0.0363 h⁻¹) was 55% higher compared to outdoor culture (μ = 0.0163 h⁻¹), but biomass production was 1.3 times higher in outdoor culture (Outdoor = 91.7 mg m⁻²d⁻¹; Photobioreactor = 69 mg m⁻²d⁻¹). Outdoor culture produced 18% higher lipid; while total fatty acids (FA) was not significantly affected by the change in culture systems as both cultures yield almost similar concentrations of fatty acids per gram of sample (photobioreactor = 119.17 mg g⁻¹; outdoor culture = 104.50 mg g⁻¹); however, outdoor cultured Isochrysis sp. had 26% more polyunsaturated fatty acids (PUFAs). Rotifers cultured in Isochrysis sp./ Nanochloropsis sp. (1:1, v/v) mixture gave similar growth rate as 100% Nanochoropsis sp. culture (μ = 0.40 d⁻¹), but had 45% higher counts of rotifers with eggs (t = 7, maximum). The Isochrysis sp. culture successfully lowered the nitrate (46%) and orthophosphate (83%) during outdoor culture.     

Distribution and morphology of the ampullary organs of the estuarine long-tailed catfish, <Emphasis Type="Italic">Euristhmus lepturus</Emphasis> (Plotosidae,Siluriformes)

Ampullary organs of Euristhmus lepturus occur in high densities along the head and in four parallel pathways along the trunk of the body. Large ampullary pores (125–130 μm) are easily distinguishable from other sensory epithelial pores due to the differences in size and the presence of a collar-like structure. Simple, singular ampullary organs of the head region consist of an ampullary pore connected to a long canal with a diameter of 115–175 μm before terminating as a simple ampulla with an external diameter of 390–480 μm. The ampullary canal is composed of 1–2 layers of flattened squamous epithelial cells, the basement membrane and an interlocking collagen sheath. The innermost cells lining the canal wall are adjoined via tight junctions and numerous desmosomes, as are those of the receptor and supportive cells. Canal wall tissue gives rise to a sensory epithelium containing between 242 and 285 total receptor cells, with an average diameter of 11.7 ± 5.3 μm, intermixed with medially nucleated supportive cells. Each receptor cell (21.38 ± 4.41 μm, height) has an apically positioned nucleus and a luminal surface covered with numerous microvilli. Neural terminals abut the basal region of receptor cells opposite multiple presynaptic bodies and dense mitochondria. Supportive cells extend from the ampullary lumen to the basement membrane, which is adjacent to the complex system of collagen fibres.     

Microbial population dynamics, especially stress tolerant Bacillus thuringiensis, in partially anaerobic rice field soils during post-harvest period of the Himalayan, island, brackish water and coastal habitats of India

Population ( × 10⁷ c.f.u./g dr. soil) of the aerobic (30.5–154.1) and anaerobic (5.9–91.4) heterotrophic, aerobic (24.0–56.0) and anaerobic (2.4–4.2) spore forming, Gram (-)ve (1.6–2.9), phosphate solubilizing (10–20), asymbiotic N₂-fixing (0.5–0.9), sulfur oxidizing (1.1–2.0), nitrifying (1.0–5.8) and denitrifying (12.1–18.7) bacteria; as well as, the actinomycetes (about 10⁴ c.f.u./g dr. soil) and fungi (about 10⁵ c.f.u./g dr. soil) were variable in the partially anaerobic, saline and drain out flooded rice soils during the post harvest period of the Himalayan, brackish water flooded, island and coastal habitats of India. The aerobic heterotrophic and spore forming bacteria were more than the anaerobic counterparts in the soils. Population (0.51–3.51 × 10⁶ c.f.u./g dr. soil) and crystal morphotype (spherical, bipyramidal and polymorphic) of the Bacillus thuringiensis (Bt) isolates of different soils were variable. Bt index was 0.002 at Mahe but 0.006 in other soils. The Bt isolates tolerated 5–12% NaCl. The osmolytes (mg/g dr. wt.) like the amino acids (0.38–99.45) and proline (0.38–0.80); and the antioxidative enzymes (units (U)/mg protein/min) viz. the catalase (0.17–5.59) and superoxide dismutase (0.35–74.46) were related with intrinsic osmotic stress tolerance of the Bt but they formed spores to overcome anoxic stress. Two Bt isolates were potent tolerant to both osmotic and anoxic stresses.     

Thermalkalibacillus uzonensis gen. nov. sp. nov, a novel aerobic alkali-tolerant thermophilic bacterium isolated from a hot spring in Uzon Caldera, Kamchatka

A novel thermophilic, alkali-tolerant, and CO-tolerant strain JW/WZ-YB58^T was isolated from green mat samples obtained from the Zarvarzin II hot spring in the Uzon Caldera, Kamchatka (Far East Russia). Cells were Gram-type and Gram stain-positive, strictly aerobic, 0.7–0.8 μm in width and 5.5–12 μm in length and produced terminal spherical spores of 1.2–1.6 μm in diameter with the mother cell swelling around 2 μm in diameter (drumstick-type morphology). Cells grew optimally at pH^25°C 8.2–8.4 and temperature 50–52°C and tolerated maximally 6% (w/v) NaCl. They were strict heterotrophs and could not use either CO or CO₂ (both with or without H₂) as sole carbon source, but tolerated up to 90% (v/v) CO in the headspace. The isolate grew on various complex substrates such as yeast extract, on carbohydrates, and organic acids, which included starch, d-galactose, d-mannose, glutamate, fumarate and acetate. Catalase reaction was negative. The membrane polar lipids were dominated by branched saturated fatty acids, which included iso-15:0 (24.5%), anteiso-15:0 (18.3%), iso-16:0 (9.9%), iso-17:0 (17.5%) and anteiso-17:0 (9.7%) as major constituents. The DNA G+C content of the strain is 45 mol%. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain JW/WZ-YB58^T is distantly (<93% similarity) related to members of Bacillaceae. On the basis of 16S rRNA gene sequence, physiological and phenotypic characteristics, the isolate JW/WZ-YB58^T (ATCC BAA-1258; DSM 17740) is proposed to be the type strain for the type species of the new taxa within the family Bacillaceae, Thermalkalibacillus uzoniensis gen. nov. sp. nov. The Genbank accession number for the 16S rRNA gene sequence is DQ221694.The Genbank accession number for the 16S rRNA gene sequence of strain JW/WZ-YB58^T is DQ221694.     

Evaluation of Azotobacter and Azospirillum as Biofertilizers in Aquaculture

Summary A preliminary comparative evaluation of the two commonly encountered free-living nitrogen fixers in the aquatic system, Azotobacter and Azospirillum was carried out in the laboratory for use as biofertilizers in aquaculture considering the importance of eco-friendly and econo-friendly productivity optimization of freshwater aquaculture. The ammonium–nitrogen levels in water media in Azotobacter treatment varied in the range 2.59–34.34 μg-N/l and was found to be significantly different from that of Azospirillum treatment (p < 0.05). The viable population of the respective nitrogen fixers as colony forming units (c.f.u.) in water media in charcoal-immobilized Azotobacter treatment ranged from 0.39 to 2.48 × 10³ c.f.u./ml and were significantly higher (p < 0.05) than the counterparts. The nitrogenase activity in the same treatment similarly remained higher, at 8.3–12.15 nmol of ethylene/ml water/h followed by the alginate-immobilized Azotobacter treatment which was at 7.2–11.40 nmol of ethylene/ml water/h compared to 5.8–7.8 and 4.65–4.83 in the respective Azospirillum-treated counterparts. Hence, better performance of Azotobacter sp. over Azospirillum sp., and of charcoal immobilization over alginate immobilization were observed.     

Mn<Superscript>2+</Superscript> enhances theanine-forming activity of recombinant glutamine synthetase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus subtilis glutamine synthetase (GS) was highly expressed (about 86% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-glnA, which was induced by 0.4 mM IPTG in LB medium, and maximal theanine-forming activity of the recombinant GS induced in LB is 6.4 U/mg at a series concentration (0–100 mM) of Mn²⁺ at optimal pH 7.5. In order to get GS with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9-A (details are described in “Materials and methods”) and 0.1% (w/v) lactose were selected as culture medium and inducer respectively. Recombinant GS was also highly expressed (84% of total protein) and totally soluble in M9-A and the specific activity of the recombinant GS is 6.2 U/mg which is approximate to that (6.4 U/mg) induced in LB in the presence of 10 mM Mn²⁺ at optimal pH 7.5. The activity is markedly higher activated by Mn²⁺ than that by other nine bivalent cations. Furthermore, M9-B (5 μM Mn²⁺ was added into M9-A) was used to culture the recombinant strain and theanine-forming activity of the recombinant GS induced in M9-B was improved 20% (up to 7.6 U/mg). Finally, theanine production experiment coupled with yeast fermentation system was carried out in a 1.0 ml reaction system with 0.1 mg crude GS from M9-B or M9-A, and the yield of theanine were 15.3 and 13.1 g/L by paper chromatography and HPLC, respectively.     

Submerged culture of a mycelial formulation of a bioherbicidal strain of <Emphasis Type="Italic">Myrothecium </Emphasis><Emphasis Type="Italic">verrucaria</Emphasis> with mitigated mycotoxin production

A mycelial formulation of the fungus Myrothecium verrucaria (IMI 361690) containing 0.20% Silwet L-77 surfactant was found to be highly efficacious in controlling the exotic invasive weed kudzu. The mycelium can be rapidly (48–72 h) produced in several media, including an inexpensive soy flour–corn meal medium. Mycelial yields were 2, 10, and 25 g dry weight l⁻¹ in Czapek-Dox, Richard’s V-8, and soy flour–corn meal media, respectively. Scale-up production in soy flour–corn meal medium using laboratory fermenters (10–25 l), resulted in a mycelial formulation that caused 90% mortality of naturally-occurring mature (0.9–1.0 m in height) kudzu within 48 h after application in field experiments. HPLC analyses revealed that the mycelium produced in this liquid culture contained no detectable amounts of the trichothecene mycotoxins roridin A and verrucarin A (limit of detection 2 μg ml⁻¹). This has resulted in a safer, yet effective bioherbicidal product. We anticipate that these findings should improve the probability of EPA registration and subsequent commercial development of this bioherbicide.     

Drought,warming and soil fertilization effects on leaf volatile terpene concentrations in <Emphasis Type="Italic">Pinus halepensis</Emphasis> and <Emphasis Type="Italic">Quercus ilex</Emphasis>

The changes in foliar concentrations of volatile terpenes in response to water stress, fertilization and temperature were analyzed in Pinus halepensis and Quercus ilex. The most abundant terpenes found in both species were α-pinene and Δ³-carene. β-Pinene and myrcene were also abundant in both species. P. halepensis concentrations were much greater than those of Q. ilex in agreement with the lack of storage in the latter species (15205.60 ± 1140.04 vs. 0.54 ± 0.08 μg g⁻¹ [d.m.]). The drought treatment (reduction to 1/3 of full watering) significantly increased the total terpene concentrations in both species (54% in P. halepensis and 119% in Q. ilex). The fertilization treatment (addition of either 250 kg N ha⁻¹ or 250 kg P ha⁻¹ or both) had no significant effects on terpene foliar concentrations. The terpene concentrations increased from 0.25 μg g⁻¹ [d.m.] at 30°C to 0.70 μg g⁻¹ [d.m.] at 40°C in Q. ilex (the non-storing species) and from 2,240 μg g⁻¹ [d.m.] at 30°C to 15,621 μg g⁻¹ [d.m.] at 40°C in P. halepensis (the storing species). Both species presented negative relationship between terpene concentrations and relative water contents (RWC). The results of this study show that higher terpene concentrations can be expected in the warmer and drier conditions predicted for the next decades in the Mediterranean region.     

Efficient regeneration of plantlets from callus and mesophyll derived protoplasts of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.

A protocol for plant regeneration from mesophyll and callus protoplasts of Robinia pseudoacacia L. was developed. For leaves from in vitro raised shoots, an enzyme combination of 2.0% cellulose and 0.3% macerozyme for a digestion period of 20 h resulted in the best yield of protoplasts (9.45 × 10⁵ protoplast/g fresh weight). Mesophyll-derived protoplasts started cell wall regeneration within 24 h of being embedded in Nagata and Takebe (NT) medium supplemented with 5 μM NAA and 1 μM BAP followed by the first cell division on day three of culture and micro-colony (32 cells) formation within day 7–10 in the same medium. However, using callus as the starting material, a combination of 2.0% cellulose and 1.0% macerozyme for a digestion period of 24 h gave the highest protoplast yield (3.2 × 10⁵ protoplast/g fresh weight). Cell wall regeneration in callus-derived protoplasts started within 24 h followed by the first cell division on the day three (96 h) and the appearance of microcolonies of more than 32 cells by the end of first week (144 h) of culture on solid WPM medium supplemented with 5 μM NAA and 1 μM BAP. Microcalli were visible to the naked eye after 45 days on solid WPM medium. Proliferation of macro-calli was successfully accomplished on solid Murashige and Skoog (MS) medium with 5 μM NAA and 5 μM BAP. Both mesophyll and callus protoplast-derived calli produced shoots on MS medium with 0.5 μM NAA and 1 μM BAP within 25–30 days and multiplied on MS medium with 1.25 μM BAP. Excised microshoots were dipped in 1–2 ml of 2.0 μM IBA for 24 h under dark aseptic conditions and transferred to double sterilized sand for rooting. The flasks containing sand were inoculated with Rhizobium for in vitro nodulation. Forty-five plants transferred to pots in the glasshouse established well.     

Detection of living <Emphasis Type="Italic">Salmonella</Emphasis> cells using bioluminescence

ATP-based bioluminescence using mutant firefly luciferase was combined with an immunochromatographic lateral flow test strip assay for Salmonella enteritidis detection. In this combination method, the Salmonella-antibody–gold complex captured at the test line on the test strip was lysed by heat-treatment, and the ATP released from the cells was measured using mutant luciferase. This method resulted in approximately 1,000 times higher sensitivity in the detection of Salmonella (i.e. 10³ c.f.u./ml) compared to immunochromatographic lateral flow assay.     

<Emphasis Type="Italic">Hirsutella proturicola</Emphasis> sp. nov. isolated from a proturan, <Emphasis Type="Italic">Baculentulus</Emphasis> densus (Protura,Hexapoda)

A new species of Hirsutella, H. proturicola, isolated from a subterranean proturan (Baculentulus densus; Protura, Hexapoda), is described and illustrated. Hirsutella proturicola is characterized by producing monoblastic phialides of 24–51.5 × 2.5–5 μm with a slightly roughened neck, fusiform and curved conidia of 9–18 × 2.5–4 μm that have a truncate base and a papillate projection often capped with sheath-like mucilage, and pluricellular, globose to subglobose chlamydospores of 21–48 × 21–41.5 μm. This species is morphologically and phylogenetically close to H. rostrata, an acaropathogenic species, but can be distinguished from the size of the phialides and the size and shape of the conidia.     

<Emphasis Type="Italic">Halomonas sediminis</Emphasis> sp. nov., a new halophilic bacterium isolated from salt-lake sediment in China

A novel Gram-negative, slightly halophilic, catalase- and oxidase-positive, obligately aerobic bacterium, strain YIM-C248^T, was isolated from a sediment sample collected from a salt-lake in the Qaidam Basin in Qinghai, north-west China. Cells were non-sporulating short rods, occurring singly or as doublets, motile with peritrichous flagella. Growth occurred with 1–15% (w/v) NaCl [optimum 2–4% (w/v) NaCl], at pH 6.0–10.0 (optimum pH 7.5) and at 4–35°C (optimum 25–30°C). The major cellular fatty acids were C_18:1 ω7c, C_12:0 3-OH, cyclo C_19:0 ω8c, C_16:0 and C_16:1. The predominant respiratory quinone was Q-9 and the genomic DNA G + C content was 58.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YIM-C248^T should be assigned to the genus Halomonas. The sequence similarities between the isolate and the type strains of members of the genus Halomonas were in the range of 92.5–97.5%. The combination of phylogenetic analysis, DNA–DNA hybridization data, phenotypic characteristics and chemotaxonomic differences supported the view that strain YIM-C248^T represents a new species of the genus Halomonas, for which the name Halomonas sediminis sp. nov. is proposed, with YIM-C248^T (=CCTCC AA 207031 = KCTC 22167) as the type strain. The GenBank/EMBL/DBBJ accession number for the 16S rRNA gene sequence of strain YIM-C248^T is EU135707.     

Physiological differences in the growth of <Emphasis Type="Italic">Sargassum horneri</Emphasis> between the germling and adult stages

The effects of temperature, irradiance, and daylength on Sargassum horneri growth were examined at the germling and adult stages to discern their physiological differences. Temperature–irradiance (10, 15, 20, 25, 30°C × 20, 40, 80 μmol photons m⁻²s⁻¹) and daylength (8, 12, 16, 24 h) experiments were carried out. The germlings and blades of S. horneri grew over a wide range of temperatures (10–25°C), irradiances (20–80 μmol photons m⁻²s⁻¹), and daylengths (8–24 h). At the optimal growth conditions, the relative growth rates (RGR) of the germlings were 21% day⁻¹ (25°C, 20 μmol photons m⁻²s⁻¹) and 13% day⁻¹ (8 h daylength). In contrast, the RGRs of the blade weights were 4% day⁻¹ (15°C, 20 μmol photons m⁻²s⁻¹) and 5% day⁻¹ (12 h daylength). Negative growth rates were found at 20 μmol photons m⁻²s⁻¹ of 20°C and 25°C treatments after 12 days. This phenomenon coincides with the necrosis of S. horneri blades in field populations. In conclusion, we found physiological differences between S. horneri germlings and adults with respect to daylength and temperature optima. The growth of S. horneri germlings could be enhanced at 25°C, 20 μmol photons m⁻²s⁻¹, and 8 h daylength for construction of Sargassum beds and restoration of barren areas.     

Studies on inositol-mediated expression of MAL gene encoding maltase and phospholipid biosynthesis in Schizosaccharomyces pombe

In this study, the effects of inositol addition on expression of the MAL gene encoding maltase and phosphatidylinositol (PI) biosynthesis in Schizosaccharomyces pombe (a naturally inositol-requiring strain) were examined. We found that specific maltase activity was at its maximum when the concentration of added inositol reached 6 μg ml⁻¹ in a synthetic medium containing 2.0% (w/v) glucose. When the concentration of added inositol was 1 μg ml⁻¹ in the medium, repression of MAL gene expression occurred at glucose concentration higher than 0.2% (w/v). However, when S. pombe was cultured in the synthetic medium containing 6 μg ml⁻¹, repression of maltase gene expression occurred only at initial glucose concentration above 1.0% (w/v). More mRNA encoding maltase was detected in the cells grown in the medium with 6 μg ml⁻¹ inositol than in those grown in the same medium with 1 μg ml⁻¹ inositol. These results demonstrate that higher inositol concentrations in the synthetic medium could derepress MAL gene expression in S. pombe. PI content of the yeast cells grown in the synthetic medium with 6 μg ml⁻¹ of inositol was higher than that of the yeast cells grown in the same medium with 1 μg ml⁻¹ of inositol. This means that PI may be involved in the derepression of MAL gene expression in S. pombe.     

Plant regeneration from mesophyll-and cell suspension-derived protoplasts of <Emphasis Type="Italic">Dianthus acicularis</Emphasis> and characterization of regenerated plants

Summary Plant regeneration systems from mesophyll- and cell suspension-derived protoplasts were established in Dianthus acicularis (2n=90), a species with resistance to Burkholderia caryophylli (Psedomonas caryophylli). Protoplasts were isolated from both leaves of in vitro-grown plants and cell suspension cultures established from the calluses originated from leaves of in vitro-grown plants. Protoplasts isolated from both sources showed about the same response to the type and concentration of cytokinins, and gave the highest frequencies of cell division and colony formation in 0.1% (w/v) Gelrite^?-solidified Murashige and Skoog (MS) medium supplemented with 0.5M glucose, 1.0mgl⁻¹ (4.53 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5 mg l⁻¹ (2.28 μM) zeatin. Numerous plantlets were regenerated after transfer of the colonies to 0.8% (w/v) agar-solidified half-strength MS medium supplemented with 0.5 mgl⁻¹ (2.28 μM) zeatin. Most plantlets exhibited normal phenotypes, but some showed variations, such as abnormal morphology with reduced chromosome number, precocious flowering, and vigorous growth with a tetraploid chromosome number. Possible mechanisms responsible for the observed somaclonal variation are discussed.     

Phenylpropanoid production in callus and cell suspension cultures of <Emphasis Type="Italic">Buddleja cordata</Emphasis> Kunth

Plant tissue cultures represent a potential source for producing secondary metabolites. In this work, Buddleja cordata tissue cultures were established in order to produce phenylpropanoids (verbascoside, linarin and hydroxycinnamic acids), as these metabolites are credited with therapeutic properties. Highest callus induction (76.4–84.3%) was obtained in five treatments containing 2,4-Dichlorophenoxyacetic acid (2,4-d: 0.45–9.05 μM) with Kinetin (KIN: 2.32, 4.65 μM), whereas highest root induction (79.6%) corresponded to the α-Naphthaleneacetic acid (9.05 μM) with KIN (2.32 μM) treatment. Verbascoside was the major phenylpropanoid produced in in vitro cultures (root, white and green callus) [66.24–86.26 mg g⁻¹ dry weight (DW)], while linarin and hydroxycinnamic acid production was low (0.95–3.01 mg g⁻¹ DW). Verbascoside and linarin production were improved in cell suspension culture (116 mg g⁻¹ DW and 8.12 mg g⁻¹ DW, respectively).     

Pigmented Nanoflagellates Grazing on <Emphasis Type="Italic">Synechococcus</Emphasis>: Seasonal Variations and Effect of Flagellate Size in the Coastal Ecosystem of Subtropical Western Pacific

We investigated seasonal variation of grazing impact of the pigmented nanoflagellates (PNF) with different sizes upon Synechococcus in the subtropical western Pacific coastal waters using grazing experiments with fluorescently labeled Synechococcus (FLS). For total PNF, conspicuous seasonal variations of ingestion rates on Synechococcus were found, and a functional response was observed. To further investigate the impact of different size groups, we separated the PNF into four categories (<3, 3–5, 5–10, and >10 μm). Our results indicated that the smallest PNF (<3 μm PNF) did not ingest FLS and was considered autotrophic. PNF of 3–5 μm in size made up most of the PNF community; however, their ingestion on Synechococcus was too low (0.1–1.9 Syn PNF⁻¹ h⁻¹) to support their growth, and they had to depend on other prey or photosynthesis to survive. The ingestion rate of the 3–5 μm group exhibited no significant seasonal variation; by contrast, the ingestion rates of 5–10 and >10 μm PNFs showed significant seasonal variation. During the warm season, 3–5 μm PNF were responsible for the grazing of 12% of Synechococcus production, 5–10 μm PNF for 48%, and >10 μm PNF for 2%. Taken together, our results demonstrate that the PNF of 3–10 μm consumed most Synechococcus during the warm season and exhibited a significant functional response to the increase in prey concentration.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Eucalyptus phylacis</Emphasis> L. Johnson and K. Hill., A critically endangered relict from Western Australia

Summary In vitro methods were applied to the only remaining plant of the Meelup Mallee (Eucalyptus phylacis), a critically endangered species from the southwest of Western Australia. Shoot explants were initiated into culture using a 1/2 MS [Murashige and Skoog basal medium (BM) for all experiments] liquid medium supplemented with 1% (w/v) activated charcoal, which was replenished twice daily, followed by transfer of explants to agar medium supplemented with 0.5 μM zeatin. Explants were cultured under low intensity lighting (PPFD of 5–10 μmol m⁻²s⁻¹) to minimize blackening of tissues, and some explants were induced to produce nodular green calluses in response to BM supplemented with 5 μM thidiazuron. Nodular green calluses were induced to form adventitious shoots following transfer to medium supplemented with 0.5 μM zeatin and 1 μM gibberellic acid, A₄ isomer (GA₄). Development of shoots was completed on 1 μM zeatin + 0.1 μM 6-benzylaminopurine (BA) in vented culture tubes. Regenerated shoots were sequentially cultured on medium containing 0.5 μM zeatin + 0.2 μM indoleacetic acid (IAA) followed by either 0.5 μM zeatin + 1μM GA₄ for shoot elongation or 1 μM zeatin + 0.5 μM IAA to optimize shoot growth. Rooted microshoots were produced after 4 weeks on 5 μM indolebutyric acid (IBA) and survived acclimatization and transfer to potting mixture.