Genetic variation of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> isolates forming fumonisin B<Subscript>1</Subscript> and moniliformin

Thirty single-spore isolates of a toxigenic fungus, Fusarium oxysporum, were isolated from asparagus spears and identified by species-specific polymerase chain reaction (PCR) and translation elongation factor 1-α (TEF) sequence analysis. In the examined sets of F. oxysporum isolates, the DNA sequences of mating type genes (MAT) were identified. The distribution of MAT idiomorph may suggest that MAT1-2 is a predominant mating type in the F. oxysporum population. F. oxysporum is mainly recognised as a producer of moniliformin—the highly toxic secondary metabolite. Moniliformin content was determined by high-performance liquid chromatography (HPLC) analysis in the range 0.05–1,007.47 μg g⁻¹ (mean 115.93 μg g⁻¹) but, also, fumonisin B₁ was detected, in the concentration range 0.01–0.91 μg g⁻¹ (mean 0.19 μg g⁻¹). There was no association between mating types and the mycotoxins biosynthesis level. Additionally, a significant intra-species genetic diversity was revealed and molecular markers associated with toxins biosynthesis were identified.     

Seasonal changes in growth rate, carrageenan yield and lectin content in the red alga Kappaphycus alvarezii cultivated in Camranh Bay, Vietnam

The three color morphotypes of the red alga Kappaphycus alvarezii (brown, red and green) were cultured in Camranh Bay, Vietnam, using the fixed off-bottom monoline culture method to evaluate the growth rate, carrageenan yield, 3,6-anhydrogalactose, gel strength and lectin content. The brown morphotype was cultivated over a 12-month period; the red and green morphotypes were over a 6-month period. At the 60-day culture timepoint, the brown morphotype showed a higher growth rate (3.5–4.6% day⁻¹) from September to February, and lower growth rate (1.6–2.8% day⁻¹) from March to August. Significant (P < 0.05) differences in growth rate between culture months were found with the brown morphotype. High growth rates for the red (3.6–4.4% day⁻¹) and green (3.7–4.2% day⁻¹) morphotypes were obtained from September to February. The carrageenan yield, 3,6-anhydrogalactose and gel strength of the three morphotypes showed little variation, with the highest values obtained in November–December. At the 30-day sampling point, the brown morphotype had a higher lectin content (167–302 μg g⁻¹ dry alga) from August to March and a lower lectin content (23–104 μg g⁻¹ dry alga) from April to July. High lectin contents were recorded for the red (139–338 μg g⁻¹ dry alga) and green (124–259 μg g⁻¹ dry alga) morphotypes from September to February. This study shows that the different morphotypes of K. alvarezii can be grown in the tropical waters of the Camranh during the northeast monsoon, and part of the southwest monsoon, especially the brown morphotype, which can be grown during any season.     

Identification of QTL underlying isoflavone contents in soybean seeds among multiple environments

Soybean isoflavones are valued in certain medicines, cosmetics, foods and feeds. Selection for high-isoflavone content in seeds along with agronomic traits is a goal of many soybean breeders. The aim of the study was to identify the quantitative trait loci (QTL) underlying seed isoflavone content in soybean among seven environments in China. A cross was made between ‘Zhongdou 27’, a soybean cultivar with higher mean isoflavone content in the seven environments (daidzein, DZ, 1,865 μg g⁻¹; genistein, GT, 1,614 μg g⁻¹; glycitein, GC, 311 μg g⁻¹ and total isoflavone, TI, 3,791 μg g⁻¹) and ‘Jiunong 20’, a soybean cultivar with lower isoflavone content (DZ, 844 μg g⁻¹; GT, 1,046 μg g⁻¹; GC, 193 μg g⁻¹ and TI, 2,061 μg g⁻¹). Through single-seed-descent, 130 F₅-derived F₆ recombinant inbred lines were advanced. A total of 99 simple-sequence repeat markers were used to construct a genetic linkage map. Seed isoflavone contents were analyzed using high-performance liquid chromatography for multiple years and locations (Harbin in 2005, 2006 and 2007, Hulan in 2006 and 2007, and Suihua in 2006 and 2007). Three QTL were associated with DZ content, four with GT content, three with GC content, and five with TI content. For all QTL detected the beneficial allele was from Zhongdou 27. QTL were located on three (DZ), three (GC), four (GT) and five (TI) molecular linkage groups (LG). A novel QTL was detected with marker Satt144 on LG F that was associated with DZ (0.0014 > P > 0.0001, 5% < R ² < 11%; 254 < DZ < 552 μg g⁻¹), GT (0.0027 > P > 0.0001; 4% < R ² < 9%; 262 < GT < 391 μg g⁻¹), and TI (0.0011 > P > 0.0001; 4% < R ² < 15%; 195 < TI < 871 μg g⁻¹) across the various environments. A previously reported QTL on LG M detected by Satt540 was associated with TI across four environments and TI mean (0.0022 > P > 0.0001; 3% < R ² < 8%; 182 < TI < 334 μg g⁻¹) in China. Because both beneficial alleles were from Zhongdou 27, it was concluded that these two QTL would have the greatest potential value for marker-assisted selection for high-isoflavone content in soybean seed in China. G. Zeng, D. Li and Y. Han have equal contributions to the paper.     

Variations of soil enzyme activities in a temperate forest soil

Soil enzyme activities (dehydrogenase, urease, phosphatase, and arylsulfatase) in a temperate forest soil were determined in relation to landscape position and seasons. Overstory of the area is dominated by Quercus mongolica, Kalopanax pictus, Carpicus cordata, and Acer pseudo-sieboldianum. The activities were measured in three patches, namely a north-facing backslope, a ridge, and a south-facing backslope in autumn and spring over 2 years. In addition, spatially more detailed analysis for phosphatase was conducted before and after litterfall production in six patches. Dehydrogenase, urease, phosphatase, and arylsulfatase activities varied 1.8–18.5 μg INT-formazan g⁻¹ h⁻¹, 45.4–347.0 μg NH₄ ⁺ g⁻¹ h⁻¹, 0.9–4.5 mmol pNP g⁻¹ h⁻¹, and 0.7–2.6 mmol pNP g⁻¹ h⁻¹, respectively. In general, higher enzyme activities were found in the northern aspect than in the southern aspect. This variation appears to be related to differences in chemical properties (e.g., Fe, Al, and Mg) of soil as well as distribution of leaf litter. Two patterns were discernible in relation to seasonal variations. Dehydrogenase and urease exhibited a positive correlation with mean air temperature, suggesting that temperature would be a major controlling variable for those enzymes. In contrast, higher activities were detected in autumn for phosphatase and arylsulfatase activities, which appeared to be closely related to litter production and distribution. Overall results of this study indicate that soil enzyme activities in a forest floor are influenced by several variables such as temperature, nutrient availability, and input of leaf litter, which are closely related to landscape position.     

Antimicrobial activity of crude extracts from mangrove fungal endophytes

The aim of this work was to select endophytic fungi from mangrove plants that produced antimicrobial substances. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) or minimal fungicidal concentrations (MFC) of crude extracts from 150 isolates were determined against potential human pathogens by a colorimetric microdilution method. Ninety-two isolates (61.3%) produced inhibitory compounds. Most of the extracts (28–32%) inhibited Staphylococcus aureus (MIC/MBC 4–200/64–200 μg ml⁻¹). Only two extracts inhibited Pseudomonas aeruginosa (MIC/MBC 200/>200 μg ml⁻¹). 25.5 and 11.7% inhibited Microsporum gypseum and Cryptococcus neoformans (MIC/MFC 4–200/8–200 μg ml⁻¹ and 8–200/8–200 μg ml⁻¹, respectively), while 7.5% were active against Candida albicans (MIC/MFC 32–200/32–200 μg ml⁻¹). None of the extracts inhibited Escherichia coli. The most active fungal extracts were from six genera, Acremonium, Diaporthe, Hypoxylon, Pestalotiopsis, Phomopsis, and Xylaria as identified using morphological and molecular methods. Phomopsis sp. MA194 (GU592007, GU592018) isolated from Rhizophora apiculata showed the broadest antimicrobial spectrum with low MIC values of 8–32 μg ml⁻¹against Gram-positive bacteria, yeasts and M. gypseum. It was concluded that endophytic fungi from mangrove plants are diverse, many produce compounds with antimicrobial activity and could be suitable sources of new antimicrobial natural products.     

A study of the diatom-dominated microplankton summer assemblages in coastal waters from Terre Adélie to the Mertz Glacier, East Antarctica (139°E–145°E)

In January 2004 the microplankton community from the coastal waters of Terre Adélie and Georges V Land (139°E–145°E) was studied. Results showed a diatom-dominated bloom with chlorophyll a levels averaging 0.64 μg l⁻¹ at 5 m depth (range 0.21–1.57 μg l⁻¹). Three geographic assemblages of diatoms were identified, based on principal diatom taxa abundances. The stratified waters near the Mertz Glacier presented highest phytoplankton biomasses (0.28–1.57 μg Chl a l⁻¹ at 5 m) and diatom abundances (6,507–70,274 cells l⁻¹ at 5 m), but low diversity, dominated by Fragilariopsis spp. Lower biomasses (0.38–0.94 μg Chl a l⁻¹ at 5 m) and abundances (394–9,058 cells l⁻¹ at 5 m) were observed in the mixed waters around the Astrolabe Glacier with a diverse diatom community characterised by larger species Corethron pennatum and Rhizosolenia spp. Finally an intermediate zone between them over the shallower shelf waters of the Adélie Bank represented by Chaetoceros criophilus, where biomasses (0.21–0.35 μg Chl a l⁻¹ at 5 m) and abundances (1,190–5,431 cells l⁻¹ at 5 m) were lowest, coinciding with the presence of abundant herbivorous zooplankton.     

Bioremediation of Fungicides by Spent Mushroom Substrate and Its Associated Microflora

Experiments were conducted both under in vitro and in situ conditions to determine the biodegradation potential of button mushroom spent substrate (SMS) and its dominating microbes (fungi and bacteria) for carbendazim and mancozeb, the commonly used agricultural fungicides. During 6 days of incubation at 30 ± 2°C under broth culture conditions, highest degradation of carbendazim (17.45%) was recorded with B-1 bacterial isolate, while highest degradation of mancozeb (18.05%) was recorded with Trichoderma sp. In fungicide pre-mixed sterilized SMS, highest degradation of carbendazim (100.00–66.50 μg g⁻¹) was recorded with mixed inoculum of Trichoderma sp. and Aspergillus sp., whereas highest degradation of mancozeb (100.00–50.50 μg g⁻¹) was with mixed inoculum of Trichoderma sp., Aspergillus sp. and B–I bacterial isolate in 15 days of incubation at 30 ± 2°C. All these microbes both individually as well as in different combinations grew well and produced extracellular lignolytic enzymes on SMS, which helped in fungicides degradation. Under in situ conditions, among three different proportions of SMS (10, 20 and 30%, w/w) mixed with fungicide pre-mixed soil (100 μg g⁻¹ of soil), the degradation of carbendazim was highest in 30% SMS treatment, while for mancozeb it was in 20% SMS treatment. The residue levels of both fungicides decreased to half of their initial concentration after 1 month of SMS mixing.     

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.     

Composition and significance of picophytoplankton in Antarctic waters

Filter fractionated picophytoplankton from Antarctic coastal waters (summer 2001) represented only 7–33% of total phytoplankton, even though total stocks were low (average Chl a = 0.32 μg l⁻¹, range = 0.13–1.03 μg l⁻¹). Though all cells passed a 2 μm filter, electron microscopy revealed most cells were over 2 μm, principally Parmales, Phaeocystis sp., and small diatoms. CHEMTAX analysis of HPLC pigment data suggested type 8 haptophytes (e.g. Phaeocystis sp. plus Parmales and pelagophytes) contributed 7–58% of picoplanktonic chlorophyll a, type 6 haptophytes (e.g. coccolithophorids) 18–59%, diatoms 0–18% (mostly type 2 diatoms, e.g. Pseudonitzschia sp., 0–15%), prasinophytes 0–17%, with cell fragments of cryptophytes 0–40%, and dinoflagellates 0–11%. Only stocks of type 8 haptophytes and prasinophytes differed significantly due to successional changes. Zeaxanthin concentrations exceeded estimates from previous cyanobacterial counts and may derive from non-photosynthetic bacteria.     

Effect of nitrate and glucose addition on denitrification and nitric oxide reductase (<Emphasis Type="Italic">cnorB</Emphasis>) gene abundance and mRNA levels in <Emphasis Type="Italic">Pseudomonas mandelii</Emphasis> inoculated into anoxic soil

The effect of glucose addition (0 and 500 μg C g⁻¹ soil) and nitrate (NO₃) addition (0, 10, 50 and 500 μg NO₃–N g⁻¹ soil) on nitric oxide reductase (cnorB) gene abundance and mRNA levels, and cumulative denitrification were quantified over 48 h in anoxic soils inoculated with Pseudomonas mandelii. Addition of glucose-C significantly increased cnorB _p (P. mandelii and related species) mRNA levels and abundance compared with soil with no glucose added, averaged over time and NO₃ addition treatments. Without glucose addition, cnorB _p mRNA levels were higher when 500 μg NO₃–N g⁻¹ soil was added compared with other NO₃ additions. In treatments with glucose added, addition of 50 μg NO₃–N g⁻¹ soil resulted in higher cnorB _p mRNA levels than soil without NO₃ but was not different from the 10 and 500 μg NO₃–N g⁻¹ treatments. cnorB _p abundance in soils without glucose addition was significantly higher in soils with 500 μg NO₃–N g⁻¹ soil compared to lower N-treated soils. Conversely, addition of 500 μg NO₃–N g⁻¹ soil resulted in lower cnorB _p abundance compared with soil without N-addition. Over 48 h, cumulative denitrification in soils with 500 μg glucose-C g⁻¹ soil, and 50 or 500 μg NO₃–N g⁻¹ was higher than all other treatments. There was a positive correlation between cnorB _p abundance and cumulative denitrification, but only in soils without glucose addition. Glucose-treated soils generally had higher cnorB _p abundance and mRNA levels than soils without glucose added, however response of cnorB _p abundance and mRNA levels to NO₃ supply depended on carbon availability.     

Winter-time ecology in the Bothnian Bay, Baltic Sea: nutrients and algae in fast ice

Landfast ice algal communities were studied in the strongly riverine-influenced northernmost part of the Baltic Sea, the Bothnian Bay, during the winter-spring transition of 2004. The under-ice river plume, detected by its low salinity and elevated nutrient concentrations, was observed only at the station closest to the river mouth. The bottommost ice layer at this station was formed from the plume water (brine volume 0.71%). This was reflected by the low flagellate-dominated (93%) algal biomass in the bottom layer, which was one-fifth of the diatom-dominated (74%) surface-layer biomass of 88 μg C l⁻¹. Our results indicate that habitable space plays a controlling role for ice algae in the Bothnian Bay fast ice. Similarly to the water column in the Bothnian Bay, average dissolved inorganic N:P-ratios in the ice were high, varying between 12 and 265. The integrated chlorophyll a (0.1–2.2 mg m⁻²) and algal biomass in the ice (1–31 mg C m⁻²) correlated significantly (Spearman ρ = 0.79), with the highest values being measured close to the river mouth in March and during the melt season in April. Flagellates <20 μm generally dominated in both the ice and water columns in February–March. In April the main ice-algal biomass was composed of Melosira arctica and unidentified pennate diatoms, while in the water column Achnanthes taeniata, Scrippsiella hangoei and flagellates dominated. The photosynthetic efficiency (0.003–0.013 (μg C [μg chl a ⁻¹] h⁻¹)(μE m⁻²s⁻¹)⁻¹) and maximum capacity (0.18–1.11 μg C [μg chl a ⁻¹] h⁻¹) could not always be linked to the algal composition, but in the case of a clear diatom dominance, pennate species showed to be more dark-adapted than centric diatoms.     

Drift-ice and under-ice water communities in the Gulf of Bothnia (Baltic Sea)

The algal, protozoan and metazoan communities within different drift-ice types (newly formed, pancake and rafted ice) and in under-ice water were studied in the Gulf of Bothnia in March 2006. In ice, diatoms together with unidentified flagellates dominated the algal biomass (226 ± 154 μg ww l⁻¹) and rotifers the metazoan and protozoan biomass (32 ± 25 μg ww l⁻¹). The under-ice water communities were dominated by flagellates and ciliates, which resulted in lower biomasses (97 ± 25 and 21 ± 14 μg ww l⁻¹, respectively). The under-ice water and newly formed ice separated from all other samples to their own cluster in hierarchical cluster analysis. The most important discriminating factors, according to discriminant analysis, were chlorophyll-a, phosphate and silicate. The under-ice water/newly formed ice cluster was characterized by high nutrient and low chlorophyll-a values, while the opposite held true for the ice cluster. Increasing trends in chlorophyll-a concentration and biomass were observed with increasing ice thickness. Within the thick ice columns (>40 cm), the highest chlorophyll-a concentrations (6.6–22.2 μg l⁻¹) were in the bottom layers indicating photoacclimation of the sympagic community. The ice algal biomass showed additional peaks in the centric diatom-dominated surface layers coinciding with the highest photosynthetic efficiencies [0.019–0.032 μg C (μg Chl-a ⁻¹ h⁻¹) (μE m⁻² s⁻¹)⁻¹] and maximum photosynthetic capacities [0.43-1.29 μg C (μg Chl-a ⁻¹ h⁻¹)]. Rafting and snow-ice formation, determined from thin sections and stable oxygen isotopic composition, strongly influenced the physical, chemical and biological properties of the ice. Snow-ice formation provided the surface layers with nutrients and possibly habitable space, which seemed to have favored centric diatoms in our study.     

Abundance and composition of the summer phytoplankton community along a transect from the Barguzin River to the central basin of Lake Baikal

The abundance and composition of phytoplankton were investigated at six stations along a transect from the Barguzin River inflow to the central basin of Lake Baikal in August 2002 to clarify the effect of the river inflow on the phytoplankton community in the lake. The water temperature in the epilimnion was high near the shore at Station 1 (17.3°C), probably due to the higher temperature of the river water, and gradually decreased offshore at Station 6 (14.5°C). Thermal stratification developed at Stations 2–6, and a thermocline was observed at a 17–22-m depth at Stations 2–4 and an 8–12-m depth at Stations 5 and 6. The concentrations of nitrogen and phosphorus nutrients in the epilimnion at all stations were <1.0 μmol N l⁻¹ and <0.16 μmol P l⁻¹, respectively. Relatively high concentrations of nutrients (0.56–7.38 μmol N l⁻¹ and 0.03–0.28 μmol P l⁻¹) were detected in the deeper parts of the euphotic zone. Silicate was not exhausted at all stations (>20 μmol Si l⁻¹). The chlorophyll a (chl. a) concentration was high (>10 μg l⁻¹) near the shore at Station 1 and low (<3 μg l⁻¹) at five other stations. The <2 μm fraction of chl. a in Stations 2–6 ranged between 0.80 and 1.85 μg l⁻¹, and its contribution to total chl. a was high (>60%). In this fraction, picocyanobacteria were abundant at all stations and ranged between 5 × 10⁴ and 5 × 10⁵ cells ml⁻¹. In contrast, chl. a in the >2 μm fraction varied significantly (0.14–11.17 μg l⁻¹), and the highest value was observed at Station 1. In this fraction, the dominant phytoplankton was Aulacoseira and centric diatoms at Station 1 and Cryptomonas, Ankistrodesmus, Asterionella, and Nitzschia at Stations 2–6. The present study demonstrated the dominance of picophytoplankton in the pelagic zone, while higher abundance of phytoplankton dominated by diatoms was observed in the shallower littoral zone. These larger phytoplankters in the littoral zone probably depend on nutrients from the Barguzin River.     

Bioremoval of hexavalent chromium from water by a salt tolerant bacterium, Exiguobacterium sp. GS1

Pollution of terrestrial surfaces and aquatic systems by hexavalent chromium, Cr(VI), is a worldwide public health problem. A chromium resistant bacterial isolate identified as Exiguobacterium sp. GS1 by 16S rRNA gene sequencing displayed high rate of removal of Cr(VI) from water. Exiguobacterium sp. GS1 is 99% identical to Exiguobacterium acetylicum. The isolate significantly removed Cr(VI) at both high and low concentrations (1–200 μg mL⁻¹) within 12 h. The Michaelis–Menten K _m and V _max for Cr(VI) bioremoval were calculated to be 141.92 μg mL⁻¹ and 13.22 μg mL⁻¹ h⁻¹, respectively. Growth of Exiguobacterium sp. GS1 was indifferent at 1–75 μg mL⁻¹ Cr(VI) in 12 h. At initial concentration of 8,000 μg L⁻¹, Exiguobacterium sp. GS1 displayed rapid bioremoval of Cr(VI) with over 50% bioremoval in 3 h and 91% bioremoval in 8 h. Kinetic analysis of Cr(VI) bioremoval rate revealed zero-order in 8 h. Exiguobacterium sp. GS1 grew and significantly reduced Cr(VI) in cultures containing 1–9% salt indicating high salt tolerance. Similarly the isolate substantially reduced Cr(VI) over a wide range of temperature (18–45  °C) and initial pH (6.0–9.0). The T _opt and initial pH_opt were 35–40  °C and 7–8, respectively. Exiguobacterium sp. GS1 displayed a great potential for bioremediation of Cr(VI) in diverse complex environments.     

Factors influencing efficiency of shoot regeneration in <Emphasis Type="Italic">Ziziphus jujuba</Emphasis> Mill. ‘Huizao’

Several factors affecting the frequency of leaf regeneration of Ziziphus jujuba ‘Huizao’ were investigated in this study, including basal medium, plant growth regulators, leaf maturity, explant orientation, and additive chemicals. The results showed that the highest shoot regeneration frequency (82.25%) was obtained on woody plant medium supplemented with 2.27 μM thidiazuron, 1.07 μM α-naphthalene-acetic acid (NAA) and 2.94 μM silver nitrate after a 10 days incubation in darkness. This study also suggested an increased regeneration frequency of expanded young leaves which were taken from the mid-stem position, as well as from the explant abaxial surface contacting with the medium. In addition, hyperhydric of adventitious buds could be effectively reduced by adding 40 g l⁻¹ sucrose to the medium. A 95.56% rooting rate could be produced from shoots cultured on half-strength Murashige and Skoog (in Physiol Plant 15:473–497, 1962) medium plus 2.69 μM NAA.     

Inoculum size and auxin concentration influence the growth of adventitious roots and accumulation of ginsenosides in suspension cultures of ginseng ( Panax ginseng C.A. Meyer)

The effect of the root-inoculum size and axuin concentration on growth of adventitious roots and accumulation of ginsenosides were studied during suspension cultures of ginseng (Panax ginseng C.A. Meyer). Of the various concentrations of indole-3-butyric acid (IBA) and γ-naphthaleneacetic acid (NAA) used as supplementary growth regulators along with Murashige and Skoog medium, 25 μM IBA was found suitable for lateral root induction and growth, as well as accumulation of ginsenosides. Inoculum size of 5 g L⁻¹ was found suitable for optimal biomass (10.5 g L⁻¹ dry biomass) and ginsenosides (5.4 mg g⁻¹ DW) accumulation. Of the various length of root inocula tested (chopped to 1–3, 4–6, 7–10 mm and un-chopped), root inocula of 7–10 mm was found suitable for biomass and ginsenoside accumulation.     

Cost-benefit relationships in sclerophyllous leaves of the ‘Bana’ vegetation in the Amazon region

This study estimated the construction const (CC) and maintenance cost (MC) of leaf tissue on the basis of dry mass (CC_Mass, MC_Mass) and leaf area (CC_Area, MC_Area), as well as the maximum leaf gas exchange capacity, so as to examine leaf cost:benefit relationship in six dominant species of the ‘Bana’ vegetation. Minimum and maximum CC_Mass averaged 1.71 ± 0.03 and 1.78 ± 0.03 g glucose g⁻¹. The CC_Mass showed a statistically significant positive correlation with crude fibre, and tended to decline as leaves were larger. Thus, smaller leaves tended to be built out of a more expensive material than that found in species bearing larger leaves. The average CC_Area of the ‘Bana’ species was 376 ± 15 g glucose m⁻². A robust correlation was found between CC_Area with leaf dry mass to leaf area ratio, as well as with leaf thickness, but not with leaf density. MC_Mass (g glucose g⁻¹ day⁻¹) and MC_Area (g glucose m⁻² day⁻¹⁾ were positively correlated. Maximum and minimum MC_Mass increased significantly with protein and lipid content, respectively. Maximum carbon assimilation (A _max) was positively correlated with CC_Area. All the species operated at high stomatal conductance (g _s) and C _i/C _a which suggested low short-term water use efficiency. Potential nitrogen use efficiency (PNUE = A _max/N) averaged 35.4 ± 1.8 mmol CO₂ mol⁻¹ N. As the sclerophylly index (g crude fibre g⁻¹ protein) increased, the ratio of CC_Area to A _max increased significantly. This result suggests a trade-off between investments in an expensive resistant sclerophyllous leaf which should maximize carbon gain in the long term.     

Statistical medium optimization for enhanced azadirachtin production in hairy root culture of Azadirachta indica

Azadirachtin, a well-known biopesticide, is a secondary metabolite extracted from the seeds of Azadirachta indica. In the present study, azadirachtin was produced in hairy roots of A. indica, generated by Agrobacterium rhizogenes-mediated transformation of leaf explants. Liquid cultures of A. indica hairy roots were developed with a liquid-to-flask volume ratio of 0.15. The kinetics of growth and azadirachtin production were established in a basal plant growth medium containing MS medium major and minor salts, Gamborg’s medium vitamins, and 30 g l⁻¹ sucrose. The highest azadirachtin accumulation in the hairy roots (up to 3.3 mg g⁻¹) and azadirachtin production (∼44 mg l⁻¹) was obtained on Day 25 of the growth cycle, with a biomass production of 13.3 g l⁻¹ dry weight. To enhance the production of azadirachtin, a Plackett–Burman experimental design protocol was used to identify key medium nutrients and concentrations to support high root biomass production and azadirachtin accumulation in hairy roots. The optimal nutrients and concentrations were as follows: 40 g l⁻¹ sucrose, 0.19 g l⁻¹ potassium dihydrogen phosphate, 3.1 g l⁻¹ potassium nitrate, and 0.41 g l⁻¹ magnesium sulfate. Concentrations were determined by a central composite design protocol and verified in shake-flask cultivation. The optimized medium composition yielded a root biomass production of 14.2 g l⁻¹ and azadirachtin accumulation of 5.2 mg g⁻¹, which was equivalent to an overall azadirachtin production of 73.84 mg l⁻¹, 68% more than that obtained under non-optimized conditions.     

Enhanced extracellular production of trans-resveratrol in Vitis vinifera suspension cultured cells by using cyclodextrins and methyljasmonate

In this work, the effect of different inducing factors on trans-resveratrol extracellular production in Monastrell grapevine suspension cultured cells is evaluated. A detailed analysis provides the optimal concentrations of cyclodextrins, methyljasmonate and UV irradiation dosage, optimal cell density, elicitation time and sucrose content in the culture media. The results indicate that trans-resveratrol production decreases as the initial cell density increases for a constant elicitor concentration in Monastrell suspension cultured cells treated with cyclodextrins individually or in combination with methyljasmonate; the decrease observed in cell cultures elicited with cyclodextrins alone is far more drastic than those observed in the combined treatment. trans-Resveratrol extracellular production observed by the joint use of cyclodextrins and methyljasmonate (1,447.8 ± 60.4 μmol trans-resveratrol g⁻¹ dry weight) is lower when these chemical compounds are combined with UV light short exposure (669.9 ± 45.2 μmol trans-resveratrol g⁻¹ dry weight). Likewise, trans-resveratrol production is dependent on levels of sucrose in the elicitation medium with the maximal levels observed with 20 g l⁻¹ sucrose and the joint action of cyclodextrins and 100 μM methyljasmonate. The sucrose concentration did not seem to limit the process although it affects significantly the specific productivity since the lowest sucrose concentration is 10 g l⁻¹, the highest productivity is reached (100.7 ± 5.8 μmol trans-resveratrol g⁻¹ dry weight g⁻¹ sucrose) using cyclodextrins and 25 μM methyljasmonate.     

High-frequency shoot regeneration from leaf explants through organogenesis in bitter melon (<Emphasis Type="Italic">Momordica charantia</Emphasis> L.)

An efficient protocol for in vitro organogenesis was achieved from callus-derived immature and mature leaf explants of Momordica charantia, a very important vegetable and medicinal plant. Calluses were induced from immature leaf explants excised from in vitro (15-day-old seedlings) mature leaf explants of vivo plants (45 days old). The explants were grown on Murashige and Skoog (MS) medium with Gamborg (B5) vitamins containing 30 g l⁻¹ sucrose, 2.2 g l⁻¹ Gelrite, and 7.7 μM naphthalene acetic acid (NAA) with 2.2 μM thidiazuron (TDZ). Regeneration of adventitious shoots from callus (30–40 shoots per explant) was achieved on MS medium containing 5.5 μM TDZ, 2.2 μM NAA, and 3.3 μM silver nitrate (AgNO₃). The shoots (1.0 cm length) were excised from callus and elongated in MS medium fortified with 3.5 μM gibberellic acid (GA₃). The elongated shoots were rooted in MS medium supplemented with 4.0 μM indole 3-butyric acid (IBA). Rooted plants were acclimatized in the greenhouse and subsequently established in soil with a survival rate of 90%. This protocol yielded an average of 40 plants per leaf explant with a culture period of 98 days.