Direct somatic embryogenesis and encapsulation of somatic embryos for in vitro conservation of Bacopa monnieri (L.) Wettst

Direct somatic embryogenesis and shoot organogenesis were achieved from leaf explants excised from microshoots of Bacopa monnieri cultured on Murashige and Skoog medium containing N6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The maximum frequency of explants differentiated somatic embryos and shoot buds on MS medium supplemented with 12.5 µM BA and 1 µM 2,4-D. The frequency of explants differentiating somatic embryos decreased with increasing concentration of 2,4-D. Light and scanning electron microscopy revealed direct differentiation of somatic embryos and shoot buds from explants, and various developmental stages of the somatic embryos were observed. Somatic embryos and apical shoot tips were encapsulated in sodium alginate gel to produce synthetic seeds. The storage of synthetic seeds produced by encapsulation was studied at 4 and 25?°C (room temperature) for a period of 140 days. Encapsulated somatic embryos were found to retain viability after 140 days of storage at both temperatures, whereas encapsulated apical shoot buds failed to germinate even after 40 days when stored at 4?°C. The viability of synthetic seeds was higher when stored at 25?°C. All amplified markers scored by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) were monomorphic for all the plants produced from synthetic seeds following different periods of storage, thus establishing the clonal fidelity of propagated plantlets.     

Growth responses to temperature,salinity and nutrient variations,and biomass variation and phenology of Ahnfeltia plicata (Rhodophyta,Ahnfeltiales): a commercially interesting agarophyte from the Magellanic Region,Chile

Ahnfeltia plicata (Hudson) E.M. Fries (Rhodophyta, Ahnfeltiales) is one of the most commercially important agarophytes in the world for its production of agar that is high quality and low in sulfate content. In the Magellanic Region, A. plicata forms extensive beds with high biomass production, which could be commercially exploited for agar production. The purposes of this study were to determine the optimal conditions of temperature, salinity, and culture medium; to evaluate the effects of different types and concentrations of auxins and cytokinins on growth of red and yellow gametophytes; and to provide background information on ecological parameters of natural population of A. plicata. Temperatures of 5, 8, 15, and 23 °C were tested, and the interaction of salinity of 25 and 35 psu with Provasoli enriched medium in half (PES/2) and quarter strength (PES/4), and with von Stosch enriched medium in half (VSES/2) and quarter strength (VSES/4) was also conducted. Concentrations of 5.0 and 50.0 μM of two auxins (indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D)), and two cytokinins (isopentenyladenine (iP) and benzylaminopurine) were added to VSES medium and gelled with 0.5 % agar. Each treatment was tested with three replicates. Red gametophytes of A. plicata tolerate a range of temperature variation, from 5 to 23 °C, and the optimum temperature for growth was 15 °C. The highest growth rate was observed in salinity of 35 psu with half strength of von Stosch culture medium. Red and yellow gametophytes showed different responses to plant growth regulators, and red gametophytes were more sensitive than yellow ones to the addition of IAA and high concentration of iP. However, growth of red gametophytes of A. plicata was stimulated by 2,4-D. The differential sensitivity of red and yellow gametophytes to plant growth regulators suggests the need to test other types and concentrations of auxins and cytokinins.     

Biological and molecular responses of Chironomus riparius (Diptera,Chironomidae) to herbicide 2,4-D (2,4-dichlorophenoxyacetic acid)

2,4-Dichlorophenoxyacetic acid (2,4-D) is an agricultural contaminant found in rural ground water. It remains to be determined whether neither 2,4-D poses environmental risks, nor is the mechanism of toxicity known at the molecular level. To evaluate the potential ecological risk of 2,4-D, we assessed the biological parameters including the survival rate, adult sex ratio of emerged adults, and mouthpart deformities in Chironomus riparius after long-term exposure to 2,4-D. The larvae were treated with 0.1, 1 or, 10 μg L^? 1 of 2,4-D for short- and long-term exposure periods. The sex ratio was changed in C. riparius exposed to only 10 μg L^? 1 of 2,4-D, whereas mouthpart deformities were observed as significantly higher in C. riparius exposed to 0.1 μg L^? 1 of 2,4-D. Survival rates were not significantly affected by 2,4-D. Furthermore, we evaluated the molecular and biochemical responses of biomarker genes such as gene expression of heat shock proteins (HSPs), ferritins and glutathione S-transferases (GSTs) in C. riparius exposed to 2,4-D for 24 h. The expressions of HSP70, HSP40, HSP90 and GST levels in C. riparius were significantly increased after exposure to a 10 μg L^? 1 concentration of 2,4-D, whereas ferritin heavy and light chain gene expressions were significantly increased at all concentrations of 2,4-D exposure. Finally, these results may provide an important contribution to our understanding of the toxicology of 2,4-D herbicide in C. riparius. Moreover, the 2,4-D-mediated gene expressions may be generated by 2,4-D is the causative effects on most probable cause of the observed alterations. These biological, molecular and morphological parameters and the measured parameters can be used to monitor 2,4-D toxicity in an aquatic environment.     

Plasmid-mediated bioaugmentation of sequencing batch reactors for enhancement of 2,4-dichlorophenoxyacetic acid removal in wastewater using plasmid pJP4

Plasmid-mediated bioaugmentation was demonstrated using sequencing batch reactors (SBRs) for enhancing 2,4-dichlorophenoxyacetic acid (2,4-D) removal by introducing Cupriavidus necator JMP134 and Escherichia coli HB101 harboring 2,4-D-degrading plasmid pJP4. C. necator JMP134(pJP4) can mineralize and grow on 2,4-D, while E. coli HB101(pJP4) cannot assimilate 2,4-D because it lacks the chromosomal genes to degrade the intermediates. The SBR with C. necator JMP134(pJP4) showed 100 % removal against 200 mg/l of 2,4-D just after its introduction, after which 2,4-D removal dropped to 0 % on day 7 with the decline in viability of the introduced strain. The SBR with E. coli HB101(pJP4) showed low 2,4-D removal, i.e., below 10 %, until day 7. Transconjugant strains of Pseudomonas and Achromobacter isolated on day 7 could not grow on 2,4-D. Both SBRs started removing 2,4-D at 100 % after day 16 with the appearance of 2,4-D-degrading transconjugants belonging to Achromobacter, Burkholderia, Cupriavidus, and Pandoraea. After the influent 2,4-D concentration was increased to 500 mg/l on day 65, the SBR with E. coli HB101(pJP4) maintained stable 2,4-D removal of more than 95 %. Although the SBR with C. necator JMP134(pJP4) showed a temporal depression of 2,4-D removal of 65 % on day 76, almost 100 % removal was achieved thereafter. During this period, transconjugants isolated from both SBRs were mainly Achromobacter with high 2,4-D-degrading capability. In conclusion, plasmid-mediated bioaugmentation can enhance the degradation capability of activated sludge regardless of the survival of introduced strains and their 2,4-D degradation capacity.     

Physiological responses of Ostreopsis ovata to changes in N and P availability and temperature increase

Ostreopsis ovata is a benthic dinoflagellate that produces palytoxin and ovatoxins. Blooms of O. ovata causing human health problems and mortality of benthic fauna have been reported from many tropical and temperate marine waters. In the present study we examined the combined effects of temperature and different nutrient conditions on the biochemical composition, growth, toxicity and carbohydrate production of an O. ovata strain originating from the Tyrrhenian Sea. O. ovata cultures with N:P ratios of 1.6, 16 and 160 (N deficient, NP sufficient and P deficient, respectively) were grown at 20 °C and 30 °C. Biomass accumulation, growth rates, cell volumes, biochemical composition, cell toxicity and carbohydrate production in each treatment were studied. Results indicated that under nutrient sufficiency O. ovata biomass accumulation increased significantly compared to N and P deficiency and also that N limitation severely affected growth. The highest growth rates were recorded at 30 °C. Cellular contents and the atomic ratios of C, N and P were higher in the cells grown at 20 °C than in those grown at 30 °C. O. ovata cell volumes increased at 20 °C. N deficiency significantly increased cell toxicity. Toxicity per cell was higher at 20 °C, but per carbon was highest at 30 °C. The highest carbohydrate production was found in conditions of N deficiency and at the lower temperature.Our study suggests that temperature increases due to global warming and nutrient enrichment of coastal waters stimulate the proliferation of O. ovata, particularly for the strains that have become adapted to warm temperate waters.     

Combined effect of temperature and zinc on Caenorhabditis elegans wild type and daf-21 mutant strains

Heavy metal pollution in aquatic ecosystems is a far reaching environmental problem. The possible influences of heavy metal exposure and the potential harm to organisms when combined with other environmental stressors such as temperature have been largely unexplored. An aquatic toxicity test of Caenorhabditis elegans was performed to estimate the 24 h median lethal concentration (LC₅₀) of different zinc concentrations at different temperatures (15 °C, 20 °C, 25 °C, and 30 °C). We also examined the time course thermotolerance on wild type (N2) and daf-21 null (JT6130) adults exposed to 6.1 mM zinc at 37 °C. Hsp90 protein expression level in response to the combined effect of temperature and zinc toxicity was also investigated by both Western blots and ELISA. Our results show that C. elegans wild type nematodes exhibit severe lethal toxicity after a 24 h exposure to zinc at higher temperatures. In addition, the expression level of Hsp90 was highly inhibited in adult worms subjected to zinc stress. This toxicity assay at different temperatures provides insight into organism response to combined effects of temperature and zinc toxicity.     

Earthworm Egg Capsules as Vectors for the Environmental Introduction of Biodegradative Bacteria

Earthworm egg capsules (cocoons) may acquire bacteria from the environment in which they are produced. We found that Ralstonia eutropha (pJP4) can be recovered from Eisenia fetida cocoons formed in soil inoculated with this bacterium. Plasmid pJP4 contains the genes necessary for 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenol (2,4-DCP) degradation. In this study we determined that the presence of R. eutropha (pJP4) within the developing earthworm cocoon can influence the degradation and toxicity of 2,4-D and 2,4-DCP, respectively. The addition of cocoons containing R. eutropha (pJP4) at either low or high densities (10² or 10⁵ CFU per cocoon, respectively) initiated degradation of 2,4-D in nonsterile soil microcosms. Loss of 2,4-D was observed within the first week of incubation, and respiking the soil with 2,4-D showed depletion within 24 h. Microbial analysis of the soil revealed the presence of approximately 10⁴ CFU R. eutropha (pJP4) g⁻¹ of soil. The toxicity of 2,4-DCP to developing earthworms was tested by using cocoons with or without R. eutropha (pJP4). Results showed that cocoons containing R. eutropha (pJP4) were able to tolerate higher levels of 2,4-DCP. Our results indicate that the biodegradation of 2,4-DCP by R. eutropha (pJP4) within the cocoons may be the mechanism contributing to toxicity reduction. These results suggest that the microbiota may influence the survival of developing earthworms exposed to toxic chemicals. In addition, cocoons can be used as inoculants for the introduction into the environment of beneficial bacteria, such as strains with biodegradative capabilities.     

Seasonal acclimatization in metabolic rate of the fan-fingered gecko,Ptyodactylus hasselquistii (Reptilia: Gekkonidae)

The resting metabolic rate of the fan-fingered gecko Ptyodactylus hasselquistii of various body masses was determined in relation to ambient temperatures ranging from 20 to 35°C during winter and summer acclimatization. Oxygen consumption (ml g⁻¹ h⁻¹) decreased with increasing mass at each temperature. The intraspecific exponents of body mass in relation to metabolic rate ranged from 0.62 to 0.79. Winter-acclimatized geckos had significantly lower metabolic rates than summer-acclimatized geckos at different temperatures, especially at low temperature (20°C). The pattern of acclimatization exhibited by P. hasselquistii may conserve energy during inactivity in winter and make activity more easily achieved during active seasons.     

Effects of temperature on baseline susceptibility and stability of insecticide resistance against Plutella xylostella (Lepidoptera: Plutellidae) in the absence of selection pressure

Plutella xylostella L. (Lepidoptera: Plutellidae) is an important pest causing significant losses to vegetables worldwide. Insecticides resistance in P. xylostella is a serious issue for scientists since last 30 years. However, deltamethrin and Bt Cry1Ac are commonly used insecticides against P. xylostella but studies involving development of resistance in P. xylostella against these two insecticides at different temperatures are lacking. The current study was aimed to find out the toxicity of deltamethrin and Bt Cry1Ac, and resistance development in P. xylostella. Results showed that the positive correlation between the temperature and toxicities of deltamethrin and Bt Cry1Ac. The results indicated −0.051, −0.049, −0.047, and −0.046 folds of deltamethrin resistance at 15 °C, 20 °C, 25 °C, and 30 °C temperatures, respectively from 1^st to 12^th generations. The toxicity of Bt Cry1Ac after 24 h was 2.2 and 4.8 folds on 1^st generation at 20 °C and 25 °C temperatures, respectively compared to the toxicity recorded at 15 °C (non-overlapping of 95% confidence limits). Based on the results of this study, it is concluded that the temperature has a positive correlation with the toxicity of deltamethrin and Bt Cry1Ac against the larvae of P. xylostella. This study suggests that deltamethrin and Bt Cry1Ac can be included in the management program of P. xylostella on many vegetable crops. The baseline susceptibility data might be helpful to understand the resistance mechanisms in P. xylostella.     

Plantlet formation from cultured inflorescences of Dactylis glomerata L.

The objective of this study was to demonstrate plantlet formation from cultured mature inflorescences of field-grown orchardgrass (Dactylis glomerata L.) plants. Whole tillers were collected and maintained in the dark at 4°C for either 19 or 25 days before panicle sections were plated on a Linsmaier and Skoog (LS) or Schenk and Hildebrandt (SH) agar medium containing 2,4-D. Generally, better results for both cell proliferation and plantlet formation were obtained with 1) large explants (many florets) on 9.1 μM 2,4-D compared to small explants (few florets) on 1.0 μM 2,4-D, 2) SH rather than LS medium and 3) when tillers were pretreated at 4°C for 25 days rather than 19 days. Chromosome counts of basal leaf cells in 94 regenerated plants showed that no plants possessed the gametic chromosome number of n=14.     

Herbicides induce change in metabolic and genetic diversity of bacterial community from a cold oligotrophic lake

Pristine cold oligotrophic lakes show unique physical and chemical characteristics with permanent fluctuation in temperature and carbon source availability. Incorporation of organic toxic matters to these ecosystems could alter the bacterial community composition. Our goal was to assess the effects of simazine (Sz) and 2,4 dichlorophenoxyacetic acid (2,4-D) upon the metabolic and genetic diversity of the bacterial community in sediment samples from a pristine cold oligotrophic lake. Sediment samples were collected in winter and summer season, and microcosms were prepared using a ration 1:10 (sediments:water). The microcosms were supplemented with 0.1 mM 2,4-D or 0.5 mM Sz and incubated for 20 days at 10 °C. Metabolic diversity was evaluated by using the Biolog Ecoplate? system and genetic diversity by 16S rDNA amplification followed by denaturing gradient gel electrophoresis analysis. Total bacterial counts and live/dead ratio were determined by epifluorescence microscopy. The control microcosms showed no significant differences (P > 0.05) in both metabolic and genetic diversity between summer and winter samples. On the other hand, the addition of 2,4-D or Sz to microcosms induces statistical significant differences (P < 0.05) in metabolic and genetic diversity showing the prevalence of Actinobacteria group which are usually not detected in the sediments of these non-contaminated lacustrine systems. The obtained results suggest that contaminations of cold pristine lakes with organic toxic compounds of anthropic origin alter their homeostasis by inhibiting specific susceptible bacterial groups. The concomitant increase of usually low representative bacterial groups modifies the bacterial composition commonly found in this pristine lake.     

Tissue Culture of Maize I. Callus Induction and Growth

Callus induction and subculture was successful with mature embryos and stem sections of seedlings of Zea mays L. on Linsmaier and Skoog's medium modified to contain 4 mg/I of 2,4-D and 1 g/I of casamino acids. — 2,4-D was superior to NAA and IAA for both callus induction and growth. Callus subcultured on NAA formed abundant roots on agar-solidified media and numerous root-like primordia in liquid cultures. — Kinetin had no effect on callus induction in the presence of 2,4-D and neither kinetin nor gibberellic acid stimulated callus growth during subculture. — Callus grew equally well on the medium of Linsmaier and Skoog, that of Schenk and Hildebrandt, and the B-5 medium of Gamborg and Eveleigh containing 2% sucrose, 4 mg/I of 2,4-D and 1 g/I of casamino acids. — The callus grew more rapidly at 25°C than at 30°C or 35°C. Little difference was noted at any temperature in callus growth in alternating light (16 h) and dark (8 h) or continuous dark. — Sucrose was superior to glucose and maltose in both liquid and agar-solidified cultures. Lactose and galactose failed to support callus growth.     

Plant regeneration through somatic embryogenesis from immature and mature zygotic embryos of Musa acuminata ssp. burmannica

A simple and efficient protocol has been developed for in vitro regeneration of M. acuminata ssp. burmannica (AA) plants. Somatic embryos were produced when immature and mature zygotic embryo explants were cultured on Murashige and Skoog medium supplemented with plant growth regulators 2,4-dichlorophenoxyacetic acid; (2,4-D), picloram or benzyl adenine and indole acetic acid. In general, immature embryos responded better than mature embryos. Callus proliferation was highest in medium supplemented with 2,4-D (4.5???M). Subsequent transfer of callus to fresh medium produced rapidly proliferating embryogenic calli. Embryogenic calli were maintained in complete darkness for 15?d followed by cycles of 8?h dark and 16?h light, under white fluorescent lamps with a light intensity of 3,000?lm/m² and at temperature of 28?±?2°C. Regeneration of embryogenic calli into plantlets was higher for immature embryos (76.6%) than for mature embryos (50.6%). This plant regeneration protocol using mature or immature zygotic embryos, via somatic embryogenesis, has significant potential to improve germination efficiencies of hybrid progenies used in conventional breeding strategies. Furthermore, tests on seed storage showed that seed viability rapidly decline after harvesting and was negligible after 9?mo of storage. This indicates using freshly harvested seeds as explant material is necessary for maximizing the tissue culture response.     

Effect of 2,4-dichlorophenoxyacetic acid on the activity of o-methyltransferase in carrot cell culture

The activity of caffeic acid-O-methyltransferase (OMT) in carrot cells was greatly affected by the amount of 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented to the culture medium. The OMT fraction was purified by (NH₄)₂SO₄ followed by ultrafiltration and gel filtration or DEAE-Sephadex chromatography after cells were cultured in the medium containing [2-¹⁴C]-2,4-D. Thus, this purified fraction revealed high OMT activity and was still radioactive. The OMT activity was about eight-fold higher (or more) in cells cultured at 0.05 ppm 2,4-D than in those at 1.0 ppm 2,4-D. The ratio of radioactivity to OMT activity was about four-fold higher in cells cultured at 1.0 ppm 2,4-D than those at 0.05 ppm 2,4-D. On the other hand, the OMT fraction was separated into two radioactive protein fractions by DEAE-Sephadex chromatography. The radioactive fractions became Et₂O-soluble after HCl hydrolysis, but not after salt-urea treatment. From these results, it was concluded that 2,4-D is covalently bound to proteins in the OMT fraction. Such 2,4-D protein conjugates may play a role in the regulation of OMT activity.     

Transgenic tobacco plants overexpressing a cold-adaptive nitroreductase gene exhibited enhanced 2,4-dinitrotoluene detoxification rate at low temperature

Abstract

Plants encounter many environmental factors such as low and high temperatures during phytoremediation processes. In this study, our aim was to produce the transgenic tobacco plants by using a newly characterized bacterial nitroreductase, Ntr, which was active at a broad range temperature in order to detoxify 2,4-dinitrotoluene (2,4-DNT) at lower temperature. The presence of Ntr and its heterologous expression was verified in T1 transgenic plants and their growing ability were determined under toxic amount of 2,4-DNT (35?µM). Fresh weight and dry weight of transgenic plants were significantly higher than wild type (WT) under toxic 2,4-DNT at 22?°C, indicating higher growth capacity of the transgenics. Transgenic plants also showed a higher tolerance than WT when exposed to 2,4-DNT at 15?°C. Moreover, transformation rate of 2,4-DNT was gradually decreased through decreasing temperatures in WT media, however, it was increased through decreasing temperatures in transgenic plant TR3-25 media and it had the highest transformation rate (54%) of 2,4-DNT at 4?°C. Correlatively, 2,4-DNT treatment at 4?°C led to a significant decrease in H₂O₂ level in transgenic plants. Thus, transgenic plants overexpressing nitroreductase might have an important advantage for phytoremediation of toxic nitroaromatic compounds in field applications at low temperatures.     

Adaptation of Delftia acidovorans for degradation of 2,4-dichlorophenoxyacetate in a microfluidic porous medium

Delftia acidovorans MC1071 can productively degrade R-2-(2,4-dichlorophenoxy)propionate (R-2,4-DP) but not 2,4-dichlorophenoxyacetate (2,4-D) herbicides. This work demonstrates adaptation of MC1071 to degrade 2,4-D in a model two-dimensional porous medium (referred to here as a micromodel). Adaptation for 2,4-D degradation in the 2 cm-long micromodel occurred within 35 days of exposure to 2,4-D, as documented by substrate removal. The amount of 2,4-D degradation in the adapted cultures in two replicate micromodels (~10 and 20 % over 142 days) was higher than a theoretical maximum (4 %) predicted using published numerical simulation methods, assuming instantaneous biodegradation and a transverse dispersion coefficient obtained for the same pore structure without biomass present. This suggests that the presence of biomass enhances substrate mixing. Additional evidence for adaptation was provided by operation without R-2,4-DP, where degradation of 2,4-D slowly decreased over 20 days, but was restored almost immediately when R-2,4-DP was again provided. Compared to suspended growth systems, the micromodel system retained the ability to degrade 2,4-D longer in the absence of R-2,4-DP, suggesting slower responses and greater resilience to fluctuations in substrates might be expected in the soil environment than in a chemostat.     

Lethal temperatures of citrus blackflyAleurocanthus woglumi [Hom.: Aleyrodidae] and its parasite,Amitus hesperidum [Hym.: Platygasteridae]

Seasonally acclimatized citrus blackfly (CBF),Aleurocanthus woglumi Ashby, and its parasite,Amitus hesperidum Silv. were exposed to extreme temperatures for 3-hour periods and survivorship measured. In summer experiments, the LD₅₀ for CBF adults and 1st instar nymphs occurred between 40° and 45°C. The LD₅₀ forA. hesperidum adults occurred between 35° and 40°C and increased with decreased humidity, although some adults could continue to emerge from parasitized CBF up to 45°C. In winter experiments, the LD₅₀ for CBF eggs and adults occurred between ?5° and ?10°C and for other CBF stages between ?10° and ?15°C. The LD₅₀ for adultA. hesperidum, and survival and emergence of the parasites from CBF both occurred between ?10° and ?15°C. Comparing data from the 2 seasons shows that adults are the most temperature sensitive stage of CBF and the parasite is more temperature sensitive than CBF, especially at higher temperatures. Correlating lethal temperature data with field meteorological conditions shows that short-term temperature exposures cannot be expected to stop the potential spread of CBF orA. hesperidum through Florida.     

Effect of temperature on the functional response of Adalia bipunctata to Myzus persicae

The effect of temperature on the functional response of female adults of the two-spot ladybird, Adalia bipunctata L. (Coleoptera: Coccinellidae) was examined in petri dish arenas containing sweet pepper leaves infested with different densities of the green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae). The predator showed a type II functional response at three tested temperatures ranging from 19°C to 27°C. The theoretical maximum number of prey captured by the predator increased with temperature. Based on the random predator equation, the estimated attack rates ranged from 0.13 h^?1 at 19°C to 0.35 h^?1 at 27°C on a leaf area of 20–25 cm². There was no significant difference between the attack rates of the predator at 23°C and 27°C. Handling time significantly decreased as temperature increased from 19°C (0.39 h) to 27°C (0.24 h). This study shows that A. bipunctata displays high predation rates on M. persicae for a wide range of temperatures, indicating its potential for augmentative releases against this aphid pest. The limitations of the predictions generated by functional response experiments are discussed.     

Growth and toxigenicity of Fusaria of the sporotrichiella section as related to environmental factors and culture substrates

Isolates ofFusarium poae, F. sporotrichioides, F. sporotrichioides var.chlamydosporum andF. sporotrichioides var.tricinctum made their best growth on PDA substrates at 24 °C, but good growth was also made at 18 °C and 30 °C. At 35 °C growth made by theF. sporotrichioides var.chlamydosporum was quite good, and superior to that of the other fungi. Moderate growth was made by all fungi at 12 °C and byF. sporotrichioides var.tricinctum also at 6 °C, while growth of the other fungi at that temperature was slight. At low temperatures toxic isolates of all butF. sporotrichioides grew better than non-toxic isolates, and growth of all isolates usually was better in light than in darkness up to temperatures of 18 °C. F. poae andF. sporotrichioides produced highest toxicity on rabbit skins when grown at 5–8 °C,F. sporotrichioides var.tricinctum at 15–20 °C. Darkness always favoured toxin development at all temperatures. In a comparison of 3 liquid substrates, overall toxin production was stronger on a starch substrate than on Czapek's or carbohydrate-peptone substrates. Among grain substrates, barley gave highest overall toxicity, which was again favoured by darkness.F. poae isolates were most toxic when derived from soil,F. sporotrichioides isolates when derived from barley. Further tests with 8 liquid substrates confirmed thatF. poae andF. sporotrichioides produce stronger toxicity at 8 °C than at 25 °C, and substrates favoured toxin production at pH 5.6 more than at pH 3.8 or 7.2. At pH 5.6 the isolates induced marked changes in the pH level of the substrate on which they grew. No relation was found to exist between the vigour of growth made by any of these fungi under various environmental conditions and the severity of the toxiç reaction their extracts produced on rabbit skins.     

Plant regeneration from the embryogenic calli of five major Miscanthus species, the non-food biomass crops

With the growing shortage of oil, coal, and other traditional fossil fuels, scientists in various fields have been looking for new fuel sources to solve the energy crisis. The genus Miscanthus is an ideal biofuel crop due to its rapid vegetative growth and its potential for high biomass yields. Plant regeneration through somatic embryogenesis is a viable method to achieve large-scale production of plant biomass. Callus induction from immature inflorescences of five Miscanthus species was performed on two different media, and the relative rates of callus proliferation were calculated. The highest multiplication coefficient, 3.92, was obtained with M. sacchariflorus ssp. lutarioriparius when the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the medium was 4.0 mg/L, and this species also performed the best at the induction phase. The proliferation coefficient for M. floridulus increased to 3.19 when the concentration of 2,4-D was decreased from 4.0 to 2.0 mg/L. When the concentration of 2,4-D was 2.0 mg/L, the proliferation coefficient for M. sinensis was 2.47. In M. sacchariflorus, the proliferation coefficients were 2.89 and 2.49 for the lower and higher concentrations of 2,4-D, respectively. The multiplication coefficient of M. x giganteus was 2.60 on medium containing 4.0 mg/L 2,4-D. Three different regeneration media were tested to induce shoots in vitro. M. floridulus and M. sacchariflorus regenerated shoots at 100% frequency in three different regeneration media. The regeneration rate for M. sacchariflorus ssp. lutarioriparius reached 99.0% on medium containing 4.0 mg/L?N ⁶ -Benzylaminopurine (6-BA) and 0.1 mg/L α-Naphthaleneacetic acid (NAA). The best regeneration rate of M. sinensis was 35.2% using 2.0 mg/L 6-BA and 0.3 mg/L NAA, whereas the regeneration rate of M. x giganteus was 57.4% on medium supplemented with 3.0 mg/L 6-BA and 0.2 mg/L NAA. The in vitro-derived plantlets of all five species had 100% rooting rates on basal MS medium without supplementation. The survival rates of plantlets were above 90% for each species when subsequently grown outdoors.