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Biochar produced from biomass based Empty Fruit Bunches (EFB) pyrolysis presents an alternative energy source. Biomass through the pelletization process will produce bio-pellets with higher density, while microwave pyrolysis achieves better energy efficiency and cost effectiveness. This two-step process will produce a product with a longer combustion duration and higher thermochemical conversion efficiency. This study aims to utilize EFB waste through a two-step process (pelletization and microwave pyrolysis) to produce biochar as an alternative renewable energy approaching commercial standards. EFB bio-pellets with a mass of 80, 100, and 120 g were pyrolyzed in a microwave pyrolysis reactor with the power of 450, 600, and 850 W, and in time variations of 20, 30, and 40 min. The results showed that 850 W power and 80 g mass were the optimal conditions to produce biochar with the highest heating value (26.73 MJ/kg), moisture content (0.48 %) volatile matter content (12.56 %), ash content (22.33 %), and fixed carbon (64.62 %). The SEM-EDX analysis indicated that the biochar product showed an increase in carbon (C) content up to 67.4 %. It is expected that combination of the pelletization process and microwave assisted pyrolysis could become a new alternative for producing biochar that could be commercialized. This research was carried out to address the problem of accumulating EFB in palm oil mills and generating renewable energy from waste.

Palm oil mill effluent (POME) has high levels of contaminants, one of which is Chemical Oxygen Demand (COD) of around 40,000–60,000 mg.L−1. The very high COD levels in POME are harmful in terms of toxicology and aesthetics to the environment. POME treatment by anaerobic digestion can be carried out through the Upflow Anaerobic Sludge Blanket-Hollow Centered Packed Bed (UASB-HCPB) fermentor to produce biogas, while the liquid phase will end up as effluent. However, these effluent not met the quality threshold so further treatment is required. Aerobic process can be address this issue by utilizing microalgae. Chlorella vulgaris is a microalgae that is widely used in wastewater treatment because of its resistance to pollutants and degradation effectiveness. The purpose of this research is to treat the effluent from the UASB-HCPB fermentor by utilizing Chlorella vulgaris in a fed-batch system. The novelty of this research is the utilization of microalgae of Chlorella vulgaris in photobioreactor for treating the effluent from the UASB-HCPB fermentor so that it meets the predetermined quality standards and safe to be discharged into the environment. In this study, the culturing process of Chlorella vulgaris was carried out in photobioreactor with variations of lighting intensity (12:12) and (24:0), with and without aeration. The cultivation process was carried out in the same of the photobioreactor with variation of fed-batch system with effluent addtion of 450 mL.day−1, 900 mL.2 day−1, and 1350 mL.3 day−1 with variation of agitation rate of 10 rpm, 20 rpm, 30 rpm, 50 rpm, and 100 rpm. Based on the experimental results in culturing process, it was found that the aeration and lighting process (24:0) increased the growth of Chlorella vulgaris best with an absorbance of 0.759 with the relationship between optical density and biomass dry weight with a value of (g.mL−1) = 0.008 OD + 0.0017 with R2 = 0.981. The best substrate addition in cultivation process was obtained with the addition of 900 mL.2 day−1 with absorbance 0.758 and specific growth 0.164 day−1. The best COD decomposition efficiency 87.5 % was obtained with the addition of 900 mL.2 day−1. The best agitation of 30 rpm was obtained with absorbance of 0.850 and COD decomposition efficiency of 93.75 % for 12 days cultivation. Total cost estimate offered of 11.1 IDR/L was obtained. This indicates that these conditions are effective for POME effluent treatment applications involving Chlorella vulgaris. The utilization of Chlorella vulgaris optimistically can be applied as a new low cost solution in the integration of sustainable POME treatment in the palm oil industry.

We have investigated the effectiveness of a new soybean fertilization technique, named crack fertilization, which involves the application of nodule bacteria on biochar to soybean roots through cracks formed between planting rows during midterm tillage. In the present study, the factors and timing of crack fertilization were investigated in an upland field converted from a paddy. The variables investigated were: 1) crack formation without the application of any agricultural materials, and 2) the application of biochar or 3) nodule bacteria on biochar into cracks. The treatment periods were: 1) before sowing, 2) during midterm tillage, and 3) during both periods. The combination of crack fertilization and reduced tillage was also tested. The method of crack fertilization that increased yield was the combination of crack formation and the application of biochar, and the most effective period for the treatment was before sowing in the reduced tillage field. Seed yields in conventional and reduced tillage fields were comparable in the upland field converted from the paddy. These results suggest that the application of biochar into cracks after scratching the soil surface to remove weeds before sowing is a practical method of increasing soybean yield in upland fields converted from paddies.

Methane production characteristics of anaerobic co-digestion of pig manure (PM) and fermented liquid feed (FLF) were investigated in a continuous digester under mesophilic conditions. The experiment followed three phases. PM alone was digested in phase I. In phases II and III, PM and FLF were mixed in a ratio of 95:5 and 90:10 (% v/v), respectively. The specific methane yields (SMYs) during phases I, II, and III were 238, 278, and 326.8 mLCH4·gVS−1-added, respectively. It was due to the effect of balancing the feedstock carbon-to-nitrogen ratio by adding FLF. This improvement can also be attributed to the readily biodegradable compounds in the FLF. The higher SMY obtained in this study showed a positive synergistic effect in the anaerobic co-digestion of PM and FLF. The results also indicate that adding the FLF positively affected and maintained a constant pH level, avoiding volatile fatty acid accumulation and ammonia inhibition in the anaerobic digestion (AD). Thus, this study provides valuable information regarding the usage of unused or wasted FLF as a co-substrate for the practical AD of PM. The production of fermented liquid additives such as FLF to improve the methane production from the AD of PM is a potential novel alternative to food waste recycling in Japan, besides compost and animal feeding.

Summary: Circumventing the emergence of fungicide-resistant strains is a crucial issue for robust disease management in agriculture. The agricultural fungicide ferimzone has been used for the control of rice diseases including rice blast. The emergence of ferimzone-resistant strains in rice fields has not been reported. Here, we identified the copper transport CoICT1 gene as the ferimzone sensitivity gene in Colletotrichum orbiculare and the rice blast fungus Magnaporthe oryzae. Genetic and cytological analyses showed that functional defects in the copper transport pathways, consisting of CoIct1 and P-type ATPase CoCcc2, led to the low sensitivity to ferimzone and the pathogenicity defect due to attenuated melanization in the appressorium. Importantly, the presence of CuSO4 induced high sensitivity to ferimzone even in the coict1 mutant. Our study shows that there is a trade-off relation between the sensitivity to ferimzone and fungal pathogenicity.

Anaerobic digestion is widely considered as an environmentally friendly technology for various organic waste including sewage sludge. Although the implementation of anaerobic digestion as an alternative treatment method for sewage sludge can be seen in many countries, its status in Malaysia is not clear. This study reviewed the current state of sewage sludge treatment in Malaysia and discussed the challenges to promote anaerobic digestion in sewage sludge treatment. Other than the common constraints faced, namely technical, political and economic, the characteristics of sewage sludge in Malaysia are considered to be a factor regarding feasibility. Anaerobic co-digestion is the simultaneous anaerobic digestion of two or more substrates which is a promising possible option to overcome the disadvantages of mono-digestion, and improve the economic viability due to higher methane production. There are a variety of biomasses as co-substrates in Malaysia. However, the anaerobic co-digestion of food waste and sewage sludge might be the most feasible method to overcome such constraints. Adding food waste as co-substrate is suggested as the possible approach to not only improve the process's performance but also help to handle the increasing volume of food waste in Malaysia. This study aims to highlight the potential as well as to provide a starting point for further studies regarding the treatment of sewage sludge using anaerobic digestion in Malaysia.

The combination of baffled air flotation and a membrane system for the treatment of palm oil mill effluent (POME) was studied. The POME was obtained from a palm oil factory in PTPN I Tanjong Seumantoh, Aceh, Indonesia. Operation variables and conditions, such as the hydraulic retention time and air flow rates, were varied to find the optimum process. The air flotation process is able to reduce the concentration of suspended solids and fats/ oils contained in the wastewater, which increases the performance of the membrane by reducing clogging. The results showed that this method was promising for POME treatment. The optimum organic removal efficiency of the air flotation pretreatment was obtained at HRT = 5 days and at an air flow rate of 11 L/min. The effluent was subsequently passed through an anaerobic membrane system to achieve the highest removal efficiency treatment. The removal efficiency of chemical oxygen demand (COD), total suspended solids (TSS), turbidity, mixed liquor suspended solids (MLSS), mixed liquor volatile suspended solids (MLVSS), and fats/oils after passing through the membrane system were 97%, 93.9%, 99.8%, 94.5%, 96.2%, and 99.9%, respectively. The results also showed that the pH could be neutralized to 6.18, while a dissolved oxygen (DO) level of 1.60 mg/L could be achieved. A high quality of effluent was obtained, which met the standards for POME effluent.

A field experiment was conducted to examine the possibility of introducing winter legumes, hairy vetch and faba bean, as green manures to turmeric cropping in a temperate region. Hairy vetch shoots were incorporated to determine the effect of N and P added as green manure. Higher values in plant height and number of leaves of turmeric were observed in the treatment with incorporation of hairy vetch than in that without incorporation (no-incorporation) throughout the growth periods. The differences in total amounts of N and P of turmeric between incorporation and no-incorporation treatments were the highest on 15 October, when the amount was increased by 8.0 g N and 1.1 g P m–2 compared with the no-incorporation treatment. From September to October, curcumin content rapidly increased with rhizome thickening, and gradually increased. We also quantified the N and P contribution from faba bean residues to the succeeding turmeric. The total amounts of N and P in turmeric cultivated after incorporating shoot and root residues into previously cultivated faba bean field were 2.5 g N and 1.0 g P m–2, respectively, larger than incorporating only roots. In previously fallow field, the incorporation of the shoot increased the total amount of N and P in tumeric by 4.5 g and 1.9 g m–2, respectively, compared with that without incorporation. In the second year after incorporation, growth and nutrient uptake of the turmeric crop did not significantly differ from those without incorporation. In the temperate region, these winter legumes would be used as basal organic matters for turmeric production.

The important role of auxin polar transport in the early growth stage of etiolated maize (Zea mays L. cv. Honey Bantam) seedlings was intensively studied. Rapid growth in the basal region of coleoptile and the apical region of mesocotyl was observed in etiolated maize seedlings in the dark. Indole-3-acetic acid (IAA) applied to the top of coleoptile substantially promoted the growth of coleoptile and mesocotyl dose-dependently, suggesting that endogenous IAA is responsible for the growth of coleoptile and mesocotyl in etiolated maize seedlings. Polar transport of radiolabeled IAA ([14C]IAA) in the coleoptile segment was significantly higher than that in the mesocotyl segment from etiolated maize seedlings. Auxin polar transport in the coleoptile and mesocotyl segments from etiolated maize seedlings increased with the growth of the seedlings from which the segments were excised. Maximum expression of the gene (ZmPIN1a) closely related to auxin polar transport was observed just prior to maximum levels of auxin polar transport in the coleoptile and mesocotyl segments. In addition, the growth rate was well correlated with the auxin polar transport and the expression of ZmPIN1a gene in coleoptile and mesocotyl of etiolated maize seedlings. These results strongly suggest that auxin polar transport regulated by ZmPIN1a protein is essentially required for the growth of coleoptile and mesocotyl in the early growth stage of etiolated maize seedlings.

Legume-grass mixed cropping may alleviate N starvation when incorporated as grass green manure which has a higher C/N ratio than legume manure. We focused on N and P absorption of oat in mixed cropping with hairy vetch, and investigated the effect of the mixedcropping green manure on the growth and nutrient uptake of the succeeding corn. The total N and total P contents of oat mixed-cropped with hairy vetch were higher than those of sole-cropped oat, and dry weight in addition to N and P contents of oat were greatly increased by cutting mixed-cropped hairy vetch before blooming. Sole cropping of hairy vetch was the best green manure to enhance the growth and nutrient uptake of the succeeding corn crop, but mixed cropping of oat with hairy vetch was also highly effective. Further investigations are needed to determine the long-term effect of mixed cropping green manure on N and P sequestration as soil organic matter.

A fiber reinforced plastic (FRP) pot, equipped with a slanted pipe through which water is supplied, has been used for assessment of rice herbicides. This pot may also be useful for controlling water table in upland conditions. In this experiment, early growths of soybean and morning glory grown under waterlogging and excess soil water conditions were evaluated. The waterlogging condition was attained by adjusting the top of the slanted pipe to soil surface level, and excess soil water condition (0.2 m3 m−3) by adjusting to 15 cm below the soil surface. The water content of the soil did not fluctuate during the treatment. Shoot growths of soybean and morning glory were poorer under waterlogging than excess soil water condition. The present experimental procedures using a FRP pot could be available for evaluation of wet damage of field crops grown under ill-drained field.

The behavior of heavy metals eluted from the wall of Ni/Fe-based alloy reactors after high-temperature and high-pressure water reaction were studied at temperatures ranging from 250 to 400oC. For this purpose, water and cysteic acid were heated in two reactor materials which are SUS 316 and Inconel 625. Under the tested conditions, the erratic behaviors of soluble metals eluted from the wall of Ni/Fe-based alloy in high temperature water were observed. Results showed that metals could be eluted even at a short contact time. The presence of air also promotes elution at sub-critical conditions. At sub-critical conditions, a significant amount of Cr was extracted from SUS 316, while only traces of Ni, Fe, Mo and Mn were eluted. In contrast, Ni was removed in significant amounts compared to Cr when Inconel 625 was tested. It was observed that eluted metals tend to increased under acidic conditions and most of those metals were over the limit of WHO guideline for drinking water. The results are significant both on the viewpoint of environmental regulation on disposal of wastes containing heavy metals, toxicity of resulting product and catalytic effect on a particular reaction.

Supercritical fluid extraction (SFE) was used in the analysis of bacterial respiratory quinone (RQ), bacterial phospholipid fatty acid (PLFA), and archaeal phospholipid ether lipid (PLEL) from anaerobically digested sludge. Bacterial RQ were determined using ultra performance liquid chromatography (UPLC). Determination of bacterial PLFA and archaeal PLEL was simultaneously performed using gas chromatography-mass spectrometry (GC-MS). The effects of pressure, temperature, and modifier concentration on the total amounts of RQ, PLFA, and PLEL were investigated by 23 experiments with five settings chosen for each variable. The optimal extraction conditions that were obtained through a multiple-response optimization included a pressure of 23.6 MPa, temperature of 77.6 °C, and 10.6% (v/v) of methanol as the modifier. Thirty nine components of microbial lipid biomarkers were identified in the anaerobically digested sludge. Overall, the SFE method proved to be more effective, rapid, and quantitative for simultaneously extracting bacterial and archaeal lipid biomarkers, compared to conventional organic solvent extraction. This work shows the potential application of SFE as a routine method for the comprehensive analysis of microbial community structures in environmental assessments using the lipid biomarkers profile.

Microbial community structure plays a significant role in environmental assessment and animal health management. The development of a superior analytical strategy for the characterization of microbial community structure is an ongoing challenge. In this study, we developed an effective supercritical fluid extraction (SFE) and ultra performance liquid chromatography (UPLC) method for the analysis of bacterial respiratory quinones (RQ) in environmental and biological samples. RQ profile analysis is one of the most widely used culture-independent tools for characterizing microbial community structure. A UPLC equipped with a photo diode array (PDA) detector was successfully applied to the simultaneous determination of ubiquinones (UQ) and menaquinones (MK) without tedious pretreatment. Supercritical carbon dioxide (scCO2) extraction with the solid-phase cartridge trap proved to be a more effective and rapid method for extracting respiratory quinones, compared to a conventional organic solvent extraction method. This methodology leads to a successful analytical procedure that involves a significant reduction in the complexity and sample preparation time. Application of the optimized methodology to characterize microbial communities based on the RQ profile was demonstrated for a variety of environmental samples (activated sludge, digested sludge, and compost) and biological samples (swine and Japanese quail feces).

The possibility of organic and amino acid production from the treatment of sludge and fish waste using water at sub critical conditions was investigated. The results indicated that at sub-critical conditions, where the ion product of water went through a maximum, the formation of organic acids was favorable. The presence of oxidant favored formation of acetic and formic acid. Other organic acids of significant amount were propionic, succinic and lactic acids. Depending on the type of wastes, formation of other organic acids was also possible. Knowing the organic acids obtained by hydrolysis and oxidation in sub-critical water of various wastes are useful in designing of applicable waste treatment process, complete degradation of organic wastes into volatile carbon and water, and also on the viewpoint of resource recovery. The production of lactic acid was discussed as well. The results indicated that temperature of 573 K, with the absence of oxidant, yield of lactic acid from fish waste was higher than sewage sludge. The maximum yield of total amino acids (137 mg/g-dry fish) from waste fish entrails was obtained at subcritical condition (T = 523 K, P = 4 MPa) at reaction time of 60 min by using the batch reactor. The amino acids obtained in this study were mainly alanine and glycine. Keywords: organic acids, amino acids, sub-critical water, hydrothermal, resources recovery

Enhancement of nitrogen fixation activities by a cultivation technique is one of the potential targets to improve soybean yield in Japan. A cultivation technique named “crack fertilization” is described and the nodulation control by this technique is analyzed by experiments in two fields with different soil and environmental conditions, and a root box experiment. Crack fertilization is a technique that utilizes the irregularly shaped soil cracks formed by subsoiling just before the flowering stage of soybean, and introduces root nodule bacteria, fertilizers, and so on to the deep subsoil layer. Production of ureide-form nitrogen at six weeks after the crack fertilization (only nodule bacteria application to the subsoil layer) was 1.4 times higher than the control in the Hikone field. Acetylene reduction activity at four or eight weeks after the crack fertilization (application of both nodule bacteria and low level of fertilizer to the subsoil layer) tended to be higher than in the control in both fields. In the root box experiment, nodule number was four times higher than that in the control at the lower portion of the root system, where modified crack fertilization treatment was conducted, and the acetylene reduction activity was increased significantly by the treatment. These results indicated that the soybean nodulation control, i.e., timing and position of nodulation, as well as the enhancement of nitrogen fixation activity could be achieved by this crack fertilization technique.

For rice cultivation in Japan, deep-flooding irrigation is used as a growth control method. To clarify the effects of deep-flooding cultivation under the different planting-pattern, we arranged conventional (Con, 22 hills m-2), narrow (Nar, 33 hills m-2) and very narrow (broadcast direct-seeding Model:BDSM, 100 hills m-2) planting plots under shallow-flooding (SF: 5 cm) and the deep-flooding (DF: 27 cm) conditions from active to maximum tillering stage and evaluated the growth, panicle weight yield, panicle components and community structure. DF was effective in controlling the weak tiller over the whole planting plots arranged. The panicle weight yield (m-2) was high in DF and high yield was kept even in BDSM. The panicle weight yield (m-2) increasesin DF was based on the panicle weight (panicle-1) increased by increasing secondary rachis-branches and their grains (Con and Nar), or on the panicle numbers (m-2) increase (BDSM). Shoot nitrogen content (tiller-1), reported to have the correlation with panicle components, was high in the Con and Nar in DF, and this might contribute to the increase in secondary rachis-branch grains. In BDSM in DF, the marked decrease in the percentage of productive tillers and serious lodging observed in SF were improved. The lodging resistance value in DF was higher than that in SF over the whole planting plots. The light transmission in DF was superior to that in SF in spite of the large LAI. Thus, the growth improving effect of DF was obvious over the wide planting pattern range.

Hairy vetch (Vicia villosa Roth) has the potentials for preventing soil erosion and suppressing weed growth as a winter cover crop. We evaluated the additional N supplied by this crop harvested at different growing stages to the succeeding sorghum in pot experiments. Hairy vetch was grown in 1/5000a Wagner pot, and shoots (S) and roots (R) were mixed separately or together (S+R) into the soil on 30 March, 18 April and 2 May. After incorporation of the plants, seeds of sorghum were sown in the pots. Dry weight and N content of hairy vetch increased throughout the growing period. The value of nitrogenase activity in root nodules peaked on 18 April and then drastically declined. When hairy vetch was harvested on 30 March, N content of sorghum in the S pots was definitely less than that in the R and S+R pots. When hairy vetch harvested on 2 May, however, the N content in the S pots was similar to that in the S+R pots, and it was significantly higher than that in the R pots. Although N input from hairy vetch was higher in the S pots than in the R pots, N uptake by sorghum was not reflected in those values. The belowground parts of hairy vetch may have a considerable effect on N uptake of sorghum when using this plant species as green manures.

Using symbiotically N2-fixing legumes as green manures is a way to supply N from the atmosphere to cropping ecosystems. Usually whole plants of the green manure are incorporated into soil; hence, the belowground parts as well as the aboveground parts would contribute to N transfer to succeeding crops. However, little is known about the contribution of the belowground parts alone. We assessed N transfer from belowground parts compared to whole plants of two legumes, Crotalaria spectabilis and Sesbania rostrata. Each of the legumes was grown approximately for 3 months in a 1/2000a Wagner pot filled with soil media, and then the roots alone (R) or shoot and root (S + R) were harvested and incorporated in the pots. Tendergreen mustard (Brassica rapa) as the succeeding crop was grown for 66 days in these pots without additional fertilizer. Although the amount of N in green manure in S + R pots was approximately 4-fold higher than that in R pots, differences in N uptake by tendergreen mustard between the S + R and R pots were smaller (1.7-fold for C. spectabilis and 2.3-fold for S. rostrata). This means that N recovery rate by tendergreen mustard was significantly higher in R than in S + R pots with either green manures. Differences in C/N ratio of the green manures could not likely explain the higher N recovery rate in R pots. Bioassay of the aqueous extracts from the green manure with lettuce seedlings suggested that growth inhibitory effects might be responsible for the lower recovery rate in S + R treatment.

Supercritical fluid extraction (SFE) was used to extract quinones from compost to monitor the microbial community dynamics during composting. The 0.3 g of dried compost was extracted using 3 mL min−1 of carbon dioxide (90%) and methanol (10%) at 45°C and 25 MPa for a 30 min extraction time. The extracted quinones were analysed using ultra performance liquid chromatography (UPLC) with 0.3 mL min−1 of methanol mobile phase for a 50 min chromatographic run time. A comparable detected amount of quinones was obtained using the developed method and an organic solvent extraction method, being 36.06 μmol kg−1 and 34.54 μmol kg−1, respectively. Significantly low value of dissimilarity index (D) between the two methods (0.05) indicated that the quinone profile obtained by both methods was considered identical. The developed method was then applied to determine the maturity of the compost by monitoring the change of quinone during composting. The UQ-9 and MK-7 were predominant quinones in the initial stage of composting. The diversity of quinone became more complex during the cooling and maturation stages. This study showed that SFE had successfully extracted quinones from a complex matrix with simplification and rapidity of the analysis that is beneficial for routine analysis.

The supercritical CO2 extraction of microbial quinones from activated sludge samples obtained from various activated sludge was investigated and compared to the conventional method using organic solvent extraction. The extraction was carried out in a supercritical fluid extraction (SFE) system in the temperature range of 25 to75 oC and the pressure up to 30 MPa. Different extraction conditions, such as the temperature, pressure, extraction time and modifier were employed to maximize the SFE efficiency. Significant amount of microbial quinones (ubiquinones and menaquinones) could be extracted rapidly with supercritical CO2. Results on the value of diversity and dissimilarity suggested that the SFE with supercritical CO2 extraction was a reliable technique for quinones extraction.

To evaluate the growth capacity and some chemical characteristics of two Crotalaria species, C. incana and C. pallida, used as green manure, we conducted a field experiment at Osaka, Japan. Both Crotalaria species exhibited vigorous vegetative growth, and leaf area was expanded in C. pallida and branching was promoted in C. incan. Top dry weight and nitrogen content of C. pallida were twice as high as those of C. incana at 87 and 120 days after transplanting (DAT), and the lower values in C. incana were attributed to drastic defoliation of lower leaves due to earlier anthesis from mid-July. Several indicators for rapid decomposition of the plants used as green manure, such as C/N ratio, lignin (L) content and L/N ratio at 56, 87 and 120 DAT, were significantly higher in C. pallida than in C. incana. However, these values might not be critical for nitrogen mineralization after incorporation of the materials into soil. To evaluate the qualitative traits of the two species as green manures, the materials were mixed with soil at a rate of 20 g fresh weight per pot, and seeds of wheat were sown at 10 and 30 days after mixing the green manure (DAM). The growth and nitrogen uptake of wheat grown on the soil mixed with C. pallida were inferior to those of wheat grown on the soil mixed with C. incana on both sowing dates, and the difference between the effectiveness of the two species as green manure was larger in the wheat sown on 10 DAM than on 30 DAM. The possible increase in nitrogen supply and growth inhibition by incorporation of these materials to wheat plants were discussed.

In Crotalaria juncea L., adventitious buds were formed in cotyledonary expiants cultured on 0.8% agar-solidified 1/2 MS basal medium containing B5 vitamins, 1.0 or 0.5 mg L–1 NAA, 5.0 or 10.0 mg L–1 BA and 3% sucrose. The frequency of adventitious bud formation was 30-45% in all combinations of NAA and BA. In histological observations, prominent mitotic figures were observed in several cells of the subepidermal palisade layers on the adaxial side of the expiants in contact with medium after 3 days of culture. Calli were formed within 6 days of culture. After 10 days of culture, numerous mature tracheary elements were produced at random in the proliferated regions, and cell divisions at the superficial region led to the formation of the meristematic structure. The shoot apex of the seedling produced numerous trichomes from superficial cells, but the adventitious bud formed on the cotyledon produced no trichomes. Initiation of the meristematic region in the expiant could be used as a target site for gene transfer experiments.

The effect of nitrate in the culture solution (0.7, 3.5, 7.0, and 14.0 mM N) on the growth, root nodule formation and nitrogenase activity of the peanut (cv. chico) inoculated with Bradyrhizobium sp. was examined at early developmental stages. The amount of nitrogen derived from fixation and its percentage to total nitrogen in the nitrate-fed plant were evaluated using 15N labeled nitrate. The dry weight and total nitrogen content of the plant at 40 days after transplanting (day 40) varied with the concentration of nitrate in the culture solution. Both of them were higher in the plants grown in the presence of nitrate at the concentrations above 0.7 mM. On the other hand, the number and fresh weight of nodules, and acetylene reduction activity per plant were the lowest in the presence of nitrate at 14.0 mM, relatively high at 0.7 mM, and the highest at 3.5 mM. The proportion of N derived from atmosphere to total nitrogen in the plants examined at days 18 and 33 was 23–31% in the presence of nitrate at 3.5 and 14.0 mM. However, the proportion examined at day 48 was as high as 57–58% at 3.5 mM, and it was only 22–24% at 14.0 mM.

The possibility of utilizing residual forest biomass to produce valuable chemicals was investigated. An outer layer of a bark of Hinoki (Chamaecyparis obutusa) tree, a Japanese cypress, was used as a sample material. Experiments were carried out in a semibatch reactor apparatus that allows the study of the effect of reaction temperatures in a single run. Analysis of the products by GC-MS reveals formation of useful compounds such as furfural, some aromatic compounds (1,3-di-tert-butyl benzene and 2,4-di-tert-butyl phenol), and fatty acids (myristic acid, palmitic acid and stearic acid). Keywords: fatty acids, furfural, high temperature water treatment, hinoki, residual biomass

A novel transgene silencing phenomenon was found in the ornamental plant, gentian (Gentiana triflora x G. scabra), in which the introduced Cauliflower mosaic virus (CaMV) 35S promoter region was strictly methylated, irrespective of the transgene copy number and integrated loci. Transgenic tobacco having the same vector did not show the silencing behavior. Not only unmodified, but also modified 35S promoters containing a 35S enhancer sequence were found to be highly methylated in the single copy transgenic gentian lines. The 35S core promoter (-90)-introduced transgenic lines showed a small degree of methylation, implying that the 35S enhancer sequence was involved in the methylation machinery. The rigorous silencing phenomenon enabled us to analyze methylation in a number of the transgenic lines in parallel, which led to the discovery of a consensus target region for de novo methylation, which comprised an asymmetric cytosine (CpHpH; H is A, C or T) sequence. Consequently, distinct footprints of de novo methylation were detected in each (modified) 35S promoter sequence, and the enhancer region (-148 to -85) was identified as a crucial target for de novo methylation. Electrophoretic mobility shift assay (EMSA) showed that complexes formed in gentian nuclear extract with the -149 to -124 and -107 to -83 region probes were distinct from those of tobacco nuclear extracts, suggesting that the complexes might contribute to de novo methylation. Our results provide insights into the phenomenon of sequence- and species- specific gene silencing in higher plants.