Hiroyuki Daimon

Improved plant regeneration from cultured leaf segments in peanut (Arachis hypogaea L.) by limited exposure to thidiazuron

Bud primordia were induced from leaf segments, which were harvested from young seedlings of Spanish type peanut (Arachis hypogaea L. cv. Chico), on 0.8% agar-solidified medium containing Murashige and Skoog (MS) basal salts supplemented with B5 vitamins, 1 mg/l NAA and various cytokinins such as benzyladenine (BA), isopentenyladenine (2ip), kinetin (KIN), chloropyridylphenylurea (4PU), thidiazuron (TDZ), zeatin (ZTN) in different concentrations. Among the cytokinins tested, TDZ was found to be the most efficient for inducing bud primordia. However, continuous culture on TDZ-containing media induced abnormal development of these primordia, and they failed to grow into plantlets. Histological observations revealed that the malformation most often obtained was a shoot-like structure which lacked shoot apical meristem (SAM) and had disorganized vascular bundles. For normal shoot regeneration, it was necessary to limit the culture period of the explants on TDZ-containing medium to 7 days at 10 mg/l or 21 days at 1 mg/l and then transfer them onto plant growth regulator-free medium. The percentage of conversion from shoot buds to shoots was 34.7%. When shoots were removed from the explants and transferred onto basal medium containing 1 mg/l NAA, all regenerated shoots readily rooted and successfully acclimatized. All of the acclimatized plants produced viable seeds in the greenhouse condition.

Plant regeneration from hairy roots induced by infection with Agrobacterium rhizogenes in Crotalaria juncea L.

Abstract Hairy roots were induced from leaf segments of Crotalaria juncea, which is used as a green manure crop antagonistic to nematodes, by infection with a mikimopine type wild strain of Agrobacterium rhizogenes A13 (MAFF02-10266). These roots exhibited vigorous growth and abundant lateral branching on half-strength Murashige and Skoog (1/2MS) medium without phytohormones. The adventitious shoots were induced from 30% of root segments 3 months after transfer onto medium containing 3 mg/l benzyl adenine. These shoots produced roots 1 month after transfer onto 1.2% agar-solidified 1/2MS medium without phytohormones. Regenerated plants were successfully grown under greenhouse conditions. The transgenic nature of the regenerated plants was confirmed by Southern-blot analysis.

Strict de novo methylation of the 35S enhancer sequence in gentian.

A novel transgene silencing phenomenon was found in the ornamental plant, gentian (Gentiana triflora × 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.

Effect of Hairy Vetch Incorporated as Green Manure on Growth and N Uptake of Sorghum Crop

Abstract 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.

Role of Belowground Parts of Green Manure Legumes, Crotalaria spectabilis and Sesbania rostrata, in N Uptake by the Succeeding Tendergreen Mustard Plant

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.

Effects of Deep-Flooding Irrigation on Growth, Canopy Structure and Panicle Weight Yield Under Different Planting Patterns in Rice

Abstract 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.

Continuous application of biochar inoculated with root nodule bacteria to subsoil enhances yield of soybean by the nodulation control using crack fertilization technique.

Abstract Crack fertilization is a soybean cultivation technique for nodulation control in which midterm subsoiling is used to supply fertilizing materials to deep soil just before the flowering stage. This study examined the effects of fertilizing materials and the continuous application of nodulation control, on soybean yield enhancement in two field experiments. The survival of nodule bacteria in deep soil was also evaluated by a bioassay of nodule bacteria in a root box. When the nodule bacteria on biochar were continuously applied without any other chemical fertilizers for three successive years, seed weight was significantly heavier being up to 1.34 times that of the control. The application of nodulation control in the previous year but not in the experimental year did not have residual effects on seed weight. The enhancement of seed weight in a farm field converted from a paddy was much lower. This may be partly attributed to the midterm tillage practice, whichdestroys the crack structure after the nodulation control, together with soil water status and cultivar differences. Nodule growth and nitrogen fixation activities significantly increased in the soybean plants grown on the soil collected from the subsoil to which nodule bacteria on biochar had been applied the previous year. This suggests that nodule bacteria in the subsoil survived in the biochar habitat for at least a year after application. These results indicate that nodulation control by the crack fertilization technique leads to yield enhancement when nodule bacteria on biochar are continuously applied.

Evaluation of Mixed Cropping of Oat and Hairy Vetch as Green Manure for Succeeding Corn Production

Abstract 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.

Possibility of Introducing Winter Legumes, Hairy Vetch and Faba Bean, as Green Manures to Turmeric Cropping in Temperate Region

Abstract 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.

Alternative Experimental Method Using a FRP Pot for Evaluating Wet Damage in Soybean and Morning Glory Grown under Excess Soil Water Conditions

Abstract 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.