Long-Term Effect of Nitrate Application from Lower Part of Roots on Nodulation and N2 Fixation in Upper Part of Roots of Soybean (Glycine max (L.) Merr.) in Two-Layered Pot Experiment

The long-term effect of the concentration and duration of application of nitrate from the lower part of soybean roots on the nodulation and nitrogen fixation in the upper part of roots was investigated using a two-layered pot system separating the upper roots growing in a vermiculite medium and the lower roots growing in a nutrient solution. Continuous absence of nitrate (hereafter referred to as “0–0 treatment”), and continuous 1 mM (1–1 treatment) and 5 mM (5–5 treatment) nitrate treatments were imposed in the lower pot from transplanting to the beginning of the maturity stage. In addition, 5 mM nitrate was supplied partially from the beginning of the pod stage till the beginning of the maturity stage (0–5 treatment) or from transplanting till the beginning of the pod stage (5–0 treatment). The values of the total plant dry weight and seed dry weight were highest in the 5–5 treatment, intermediate in the 1–1, 5–0, 0–5 treatments, and lowest in the 0–0 treatment. The values of the nodule dry weight and nitrogen fixation activity (acetylene reduction activity) were lowest in the 5–5 treatment. The value of the nodule dry weight in the upper roots was highest in the plants subjected to the 1–1 treatment and exceeded that in the 0–0 treatment. Total nitrogen fixation activity of the upper nodules per plant at the beginning of the pod stage was also highest in the 1–1 treatment. These results indicated that long-term supply of a low level of nitrate from the lower roots could promote nodulation and nitrogen fixation in the upper part of roots. Withdrawal of 5 mM nitrate after the beginning of the pod stage (5–0 treatment) markedly enhanced nodule growth and ARA per plant in the upper roots at the beginning of the maturity stage when the values of both parameters decreased in the other treatments. The nitrate concentration in the nodules attached to the upper roots was low, including the 5–5 treatment regardless of the stages of growth. This indicated that the inhibitory effect of 5 mM nitrate or promotive effect of 1 mM nitrate supplied from the lower roots was not directly controlled by nitrate itself, but was mediated by some systemic regulation, possibly by the C or/and N requirement of the whole plant.     

Rapid and reversible nitrate inhibition of nodule growth and N2 fixation activity in soybean (Glycine max (L.) Merr.)

Nodulated soybean (Glycine max. (L) Merr. cv. Williams) plants were hydroponically cultured, and various combinations of 1-week culture with 5 or 0 mm nitrate were applied using 13-d-old soybean seedlings during three successive weeks. The treatments were designated as 0-0-0, 5-5-5, 5-5-0, 5-0-0, 5-0-5, 0-5-5, and 0-0-5, where the three sequential numbers denote the nitrate concentration (mm) applied in the first-second-third weeks. The size of the individual nodule was measured periodically using a slide caliper. All the plants were harvested after measurement of the acetylene reduction activity (ARA) at the end of the treatments. In the 0-0-0 treatment, the nodules grew continuously during the treatment period. Individual nodule growth was immediately suppressed after 5 mm nitrate supply. However, the nodule growth rapidly recovered by changing the 5 mm nitrate solution to a 0 mm nitrate solution in the 5-0-0 and 5-5-0 treatments. In the 5-0-5 treatment, nodule growth was completely inhibited in the first and the third weeks with 5 mm nitrate, but the nodule growth was enhanced in the second week with 0 mm nitrate. The nodule growth response to 5 mm nitrate was similar between small and large size nodules. After the 5-5-5, 5-0-5, 0-0-5, and 0-5-5 treatments, where the plants were cultured with 5 mm nitrate in the last third week, the ARA per plant was significantly lower compared with the 0-0-0 treatment. On the other hand, the ARA after the 5-0-0 and 5-5-0 treatments was relatively higher than that after the 0-0-0 treatment, possibly due to the higher photosynthate supply associated with the vigorous vegetative growth of the plants supplemented with nitrate nitrogen. It is concluded that both soybean nodule growth and N₂ fixation activity sensitively responded to the external nitrate level, and that these parameters were reversibly regulated by the current status of nitrate in the culture solution, possibly through sensing of the nitrate concentration in roots and / or nodules.     

Variation of nitrogen accumulation and yield in response to phosphorus nutrition of soybean (Glycine max L. Merr.)

Phosphorus (P) is essential macronutrient for soybean [Glycine max (L.) Merr.] growth and function. The objective of this study was to determine effect of phosphorus nutrition (including phosphorus nutrition level and interruption of phosphorus supply) on nitrogen accumulation, nodule nitrogen fixation and yield of soybean plants by ¹⁵N labeling with sand culture. The results showed that they all presented a single peak curve with improvement of phosphorus nutrition level, when phosphorus concentration of nutrient solution was about 31 mg/L, they all reached the maximum and effect of phosphorus nutrition level on nodule nitrogen fixation was lower than that on yield formation level. Interruption of phosphorus supply during soybean growth period, nitrogen accumulation and nodule nitrogen fixation were seriously inhibited, and yield was decreased significantly when interruption of phosphorus supply during V₃-R₁ and R₁-R₅ period, while interruption of phosphorus supply during R₅-R₇ period had no significant effect on nitrogen accumulation, nodule nitrogen fixation and yield. So soybean nitrogen metabolism and yield were sensitive to phosphorus nutrition in the V₃-R₅ period, those were not sensitive to phosphorus nutrition after R₅ period.     

Differences in numbers of nodules and lateral roots between soybean (Glycine max L. Merr.) cultivars,Kitamusume and Toyosuzu

Two cultivars of soybean (Glycine max L. Merr .), Kitamusume and Toyosuzu were grown with commercially-purchased granulated soil in a greenhouse. Kitamusume formed a larger number of nodules per g shoot dry weight and its nodules showed a smaller average diameter and average dry weight per nodule than Toyosuzu regardless of plant age or rhizobial strains (Bradyrhizobium japonicum (AI017, J5033, 646, J1B140), B. elkanii (USDA94), and Rhizobium fredii (MAFF210054)) inoculated at the rate of 10⁸-10⁹ cells per 3 L pot. These differences were observed in the nodules formed on both primary and lateral roots. With a lower inoculation dose of rhizobia (10² and 10⁵ cells per pot), Kitamusume formed a smaller number of larger nodules than Toyosuzu inoculated with 10⁸-10⁹ cells per pot. This result indicated that the number of nodules was the factor directly controlled by host, because the size of the nodules was not determined by the genetic background, but changed depending on the number of nodules formed. The number of the first order lateral roots of 21 d old Kitamusume was 1.49 times larger than that of Toyosuzu. The relationship between the number of nodules and the number of first order lateral roots of 14 cultivars showed a significant positive correlation. These results indicated that the formation of nodules and lateral roots may be similarly controlled by a certain factor in different cultivars.     

Nitrogen Fixation,Ureide, and Nitrate Accumulation Responses to Soybean Aphid Injury in Glycine max

There is little information available about soybean aphid (Aphis glycines Matsumura) effects on the physiology and mineral nutrition of soybean (Glycine max [L.] merr.). Controlled-environment studies were conducted to measure soybean aphid infestation effects on dry weight, nitrogen (N) fixation, ureide-N, and nitrate-N concentration and accumulation. Plants grown in perlite using –N nutrient solution culture were infested at the 3rd trifoliolate (V3) stage and measured for N fixation, nodule characteristics, and ureide-N concentration at the full pod (R4) stage. When compared to uninfested control plants, aphid infestation reduced total nodule volume per plant by 34%, nodule leghemoglobin per plant by 31%, plant N fixation rate by 80% and shoot ureide-N concentration by 20%. Soil-grown plants were infested at the first trifoliolate (V1) stage and shoots were measured for dry weight, nitrate-N, and ureide-N at the full bloom (R2) stage. Infestation reduced shoot dry weight by 63%, increased nitrate-N concentration by 75%, but did not significantly affect ureide-N concentration. Because nutrient concentration is a single-point measurement that results from the integration of two dynamic processes, nutrient accumulation and dry matter production, we conclude that aphid-induced reductions in N fixation, coupled with decreased dry weight accumulation, caused shoot ureide-N concentration to remain unchanged in aphid-injured plants when compared to uninfested plants. Because nitrate-N concentration was greater in aphid-damaged shoot tissue, we further conclude that nitrate-N accumulation was less sensitive to aphid injury than dry weight accumulation.     

Lead-Induced Histological and Ultrastructural Changes in the Leaves of Soybean (Glycine max (L.) Merr.)

The seedlings of the soybean ( Glycine max. (L.) Merr.) cv. Polan were investigated by subjecting them to water culture for a period of 14 d. To the Knop nutrient solution, lead was added as PbCl₂ at four concentrations: 0, 10, 20 and 40 mg dm⁻³. Observations of soybean leaf tissues were carried out by light microscopy, transmission electron microscopy and scanning electron microscopy. The Pb levels used in the present study reduced the area of cotyledons and leaf blades of the soybean plants. Pb-induced changes in the leaf epidermis structure involved a reduction in the cell size, more abundant wax coating, and an increase in the number of stomata and trichomes per unit area with simultaneous reduction in the size of the guard cells. The lead treatment resulted in the reduction in the thickness of the leaf blades, reduction in the area of xylem and phloem in the vascular bundles and in the diameter of the xylem vessels. Under Pb stress, the leaf mesophyll cells were characterized by the presence of altered chloroplasts with a reduced lamellar system and multidirectional pattern of the thylakoid system. Burst stroma of the thylakoid system and cracked chloroplast envelopes were also observed. The importance of the increase in the number of stomata and trichomes for plants under the metal stress was examined.     

Soybean (Glycine max [L.] Merr.) shoots systemically control arbuscule formation in mycorrhizal symbiosis

Abstract

The roots of soybean (Glycine max [L.] Merr.) establish symbiosis with nodule-inducing rhizobia and arbuscular mycorrhizal (AM) fungi. The existing nodules systemically suppress subsequent nodule formation, a phenomenon known as autoregulation. Grafting experiments revealed that some forms of autoregulation are controlled by the shoot. In the present study, we examined shoot-controlled regulation of AM fungal colonization using a reciprocal grafting technique. Ten-day-old seedlings of wild-type soybean cv. Enrei and its hypernodulating mutant En6500 were cut below the cotyledons and the shoots were grafted to self or reciprocal roots. Grafted seedlings were inoculated with Bradyrhizobium japonicum and Gigaspora rosea and grown in a glasshouse for 60 days. The arbuscule abundance of the En6500 (shoot)/En6500(root) graft was 1.5-fold higher than that of the Enrei/Enrei graft. In grafts between Enrei and En6500, an increased arbuscule abundance was detected only when En6500 was used as the shoot. The arbuscule abundance of Enrei/En6500 when Enrei was used as the shoot was comparable to that of Enrei/Enrei. The intensity of AM fungal colonization was lower in Enrei/En6500 than in the other grafting treatments. From the results obtained, we suggest that soybean shoots systemically control arbuscule formation in both AM symbiosis and nodule formation.     

Soybean (Glycine max [L.] Merr.) shoots systemically control arbuscule formation in mycorrhizal symbiosis

The roots of soybean ( Glycine max [L.] Merr.) establish symbiosis with nodule-inducing rhizobia and arbuscular mycorrhizal (AM) fungi. The existing nodules systemically suppress subsequent nodule formation, a phenomenon known as autoregulation. Grafting experiments revealed that some forms of autoregulation are controlled by the shoot. In the present study, we examined shoot-controlled regulation of AM fungal colonization using a reciprocal grafting technique. Ten-day-old seedlings of wild-type soybean cv. Enrei and its hypernodulating mutant En6500 were cut below the cotyledons and the shoots were grafted to self or reciprocal roots. Grafted seedlings were inoculated with Bradyrhizobium japonicum and Gigaspora rosea and grown in a glasshouse for 60 days. The arbuscule abundance of the En6500 (shoot)/En6500(root) graft was 1.5-fold higher than that of the Enrei/Enrei graft. In grafts between Enrei and En6500, an increased arbuscule abundance was detected only when En6500 was used as the shoot. The arbuscule abundance of Enrei/En6500 when Enrei was used as the shoot was comparable to that of Enrei/Enrei. The intensity of AM fungal colonization was lower in Enrei/En6500 than in the other grafting treatments. From the results obtained, we suggest that soybean shoots systemically control arbuscule formation in both AM symbiosis and nodule formation.     

AFLP Analysis of Genetic Diversity in Indian Soybean [Glycine max (L.) Merr.] Varieties

AFLP technique was used to assess genetic diversity in 72 soybean varieties under cultivation in India. Selected 12 AFLP primer pairs produced 1319 products of which 1257 were polymorphic (95%). Wide variations were observed for the number of amplification products, percent polymorphism and average polymorphism information content (PIC). The Jaccard's similarity indices (J) based on the AFLP profiles of the 72 soybean varieties were subjected to UPGMA cluster analysis. The dendrogram generated revealed four major clusters, which were strongly supported by the high bootstrap values obtained from analyses of 1000 bootstrap samples. In addition, the Mantel's test for cophenetic correlation with r = 0.955 indicated very good fit of the varieties to a group in the cluster analysis. Some correspondence between the clustering pattern and the pedigree, place of release or target area of the variety was observed. Overall moderately high genetic diversity was observed which appears to be due to the higher genetic diversity prevalent in 12 of the varieties included in three diverse clusters and was indicative of the need to include more diverse germplasm in the soybean improvement programs.     

Photoassimilate partitioning in hypernodulation mutant of soybean (Glycine max (L.) Merr.) NOD1-3 and its parent williams in relation to nitrate inhibition of nodule growth

Nodule growth of a hypernodulating soybean (Glycine max (L.) Merr.) mutant line NOD1-3 was compared to that of its wild-type parent cv. Williams from 14 to 18 days after planting (DAP) in the absence of nitrate treatment (hereafter referred to as “0 mM treatment”) or with 5 mM nitrate treatment. The growth rate determined by increase in the diameter of the nodules was relatively lower in the mutant NOD1-3 than that of the parent Williams under nitrogen-free conditions (0 mM nitrate). The inhibition of nodule growth by 5 mM nitrate started at 1 d after the onset of the nitrate treatment in Williams, while the inhibition did not occur before the application of the nitrate treatment for 2 d in NOD1-3. The nodule growth was completely inhibited after 2 d in Williams and after 3 d in NOD1-3 during the 5 mM nitrate treatment period. After 4 d of 5 mM nitrate treatment, the nodule dry weight decreased by 22% in NOD1-3 and by 58% in Williams, respectively. The treatment with 5 mM nitrate decreased the acetylene reduction activity (ARA) in NOD1-3 by 60% per plant and by 50% per nodule g DW and these parameters were less sensitive to the treatment than those in Williams in which the inhibition rate was 90% per plant and 80% per nodule g DW. These results indicate that NOD1-3 is partially nitrate-tolerant in terms of individual nodule growth as well as total nodule dry weight and Nz fixation activity. A whole shoot of Williams and NOD1-3 plants was exposed to ¹⁴CO₂ for 120 min followed by 0 or 5 mM nitrate treatment for 2 d, and the partitioning of the photoassimilates among the organs was analyzed. Under 0 mM nitrate treatment, the percentages of the distribution of ¹⁴C radioactivity between the nodules and roots were 63 and 37% in Williams and 89 and 11% in NOD1-3. Under the 5 mM nitrate conditions, the percentages of the distribution of ¹⁴C between the nodules and roots changed to 14 and 86% in Williams and 39 and 61% in NOD1-3, respectively. These results indicated that the hypernodulating mutant NOD1-3 supplied a larger amount of photoassimilates to the nodules than to the roots under nitrogen-free conditions, and that the nitrate depression of photoassimilate transport to the nodules was less sensitive than that of the parent line.     

Analysis of regulation site and manner of abundant nodulation in a soybean (Glycine max (L.) Merr.) cultivar,kitantusunte, using grafting techniques

To characterize the regulation site and manner of the abundant nodulation in the soybean (Glycine max (L.) Merr.) cv. Kitamusume, three grafting eperiments were carried out as follows: reciprocal wedge grafting and inter-cultivar approach grafting between Kitamusume and a normal nodulating cultivar, Toyosuzu, as well as wedge grafting of scions of the supernodulating mutant En6500 onto either Kitamusume or Toyosuzu rootstock. In the reciprocal wedge grafting, the number of nodules per shoot dry weight and average weight per nodule in the grafted plants were consistent with those exhibited by the genotype of their rootstocks. Approach grafting did not affect the number of nodules per shoot dry weight on either side of the inter-cultivar approachgrafted plant. Although grafting of the mutant scion resulted in the loss of the autoregulatory response from the roots of both cultivars, difference in the number of nodules per g shoot dry weight still remained between the two cultivars. These results suggested that the abundant nodulation in Kitamusume is controlled by the root in a non-systemic manner and is independent of the autoregulation mechanism.     

Influence of Mercury on Soybean Plants (Glycine max L.) at Low pH

The influence of Hg on soybean plants under different pH conditions and Hg concentrations was studied. Growth inhibition by Hg was higher in roots than the upper part of the plant, but was highly dependant on pH condition. Growth inhibition of roots was observed when Hg concentration was higher than 1 mg Hg L⁻¹ for pH 4.0 and 5 mg Hg L⁻¹ for pH 6.0. Using ²⁰³Hg as a radioactive tracer, the amount of Hg (1 mg Hg L⁻¹) uptake in root was found to be about 1.5 times higher at pH 4.0 than that at pH 6.0; suggesting that Hg when highly accumulated at the lower pH induced inhibition of root growth. Decreased amounts of Hg due to evaporation during the plant growth were very low, but were higher at pH 6.0 than that at pH 4.0. There was hardly any translocation of Hg from roots to the upper parts through the stem within 24 h.     

Positive correlation between the number of root nodule primordia and seed sugar secretion in soybean (Glycine max L.) seedlings inoculated with a low density of Bradyrhizobium japonicum

Sufficient rhizobium population in the rhizosphere of legume seedlings is required for early and enough setting of root nodules. Potential of seed and seedling root exudates for proliferation of Bradyrhizobium japonicum PNT119 was evaluated in the soybean cvs. Enrei and Tachinagaha. In both cultivars, seed exudates showed a higher potential than root exudates. In the seed exudates, a low-molecular high-polarity fraction including sugars and amino acids showed a higher potential than both high-molecular fraction and low-molecular low-polarity fraction. The correlations between the sugar or amino acid contents of seed exudates and the number of root nodule primordia at 7 d after sowing were investigated among 12 soybean cultivars. When seeds were inoculated with a low rhizobium density, a high correlation coefficient was detected between them. However, there was no positive correlation in the plants inoculated with a high rhizobium density. These results suggest that the amount of low molecular substances in the seed exudates determines the number of root nodule primordia through rhizobium proliferation around seed and young roots when the rhizobium density is the limiting factor. The genetic trait relating to seed coat secretion should be considered as a possible key factor contributing to adequate root nodulation in soybean seedlings cultivated in fields with a low rhizobium density.     

Influence of Mercury on Soybean Plants (Glycine max L.) at Low pH

The influence of Hg on soybean plants under different pH conditions and Hg concentrations was studied. Growth inhibition by Hg was higher in roots than the upper part of the plant, but was highly dependant on pH condition. Growth inhibition of roots was observed when Hg concentration was higher than 1 mg Hg L^?1 for pH 4.0 and 5 mg Hg L^?1 for pH 6.0. Using ²⁰³Hg as a radioactive tracer, the amount of Hg (1 mg Hg L^?1) uptake in root was found to be about 1.5 times higher at pH 4.0 than that at pH 6.0; suggesting that Hg when highly accumulated at the lower pH induced inhibition of root growth. Decreased amounts of Hg due to evaporation during the plant growth were very low, but were higher at pH 6.0 than that at pH 4.0. There was hardly any translocation of Hg from roots to the upper parts through the stem within 24 h.     

Impact of pyrochar and hydrochar on soybean (Glycine max L.) root nodulation and biological nitrogen fixation

The aim of this study was to identify effects of carbonized organic material (“biochar”) on soybean growth, root nodulation and biological nitrogen fixation, and to elucidate possible underlying mechanisms. Soybean (Glycine max L.) was grown in four arable soils amended with carbonized organic material produced from wood or maize as feedstocks, by pyrolysis (“pyrochar”) or hydrothermal carbonization (“hydrochar”). Nodulation by Bradyrhizobium , biological nitrogen fixation (BNF) assessed by ¹⁵N techniques, plant growth, nutrient uptake and changes in chemical soil properties after soil amendment were determined. Data were analyzed by means of a three way ANOVA on the factors soil, carbonization technique and feedstock. It turned out that soybean root nodulation and BNF was influenced by the carbonization technique used to prepare the soil amendment. Hydrochar, in average and across all soils, increased nodule dry matter and BNF by factors of 3.4 and 2.3, respectively, considerably more than pyrochar, which led to 1.8 and 1.2 fold increases, respectively. Nodule dry matter and BNF correlated positively with available soil sulfur and negatively with available soil nitrogen. Hydrochars provided more available sulfur than pyrochars, and hydrochars caused a decrease in nitrogen availability in the soil solution, thereby exerting a positive influence on nodulation and BNF. Pyrochar amendment increased soil pH but had no effect on nodulation and BNF. Plant growth was affected by the soil and by the feedstock used for the “biochar”, and increased slightly more in treatments with pyrochar and hydrochar made from maize, which was richer in nitrogen and potassium. The results show that carbonized organic materials, and specifically hydrochar, have the capacity to increase BNF in soils. We suggest that this enhancement in BNF in response to soil amendments with carbonized organic materials is due to an increase in available sulfur and a reduction of available soil nitrogen.     

Real-time imaging of nitrogen fixation in an intact soybean plant with nodules using 13N-labeled nitrogen gas

Real-time images of nitrogen fixation in an intact nodule of hydroponically cultured soybean ( Glycine max [L] Merr.) were obtained. In the present study, we developed a rapid method to produce and purify ¹³N-labeled radioactive nitrogen gas (half life: 9.97 min). ¹³N was produced from a ¹⁶O (p, α) ¹³N nuclear reaction. The target chamber was filled with CO₂ and irradiated for 10 min with protons at an energy of 18.3 MeV and an electric current of 5 μA, which was delivered from a cyclotron. All CO₂ in the collected gas was absorbed and removed with powdered soda-lime in a syringe and replaced with helium gas. The resulting gas was injected into gas chromatography and separated and a 35 mL fraction, including the peak of [¹³N]-nitrogen gas, was collected by monitoring the chromatogram. The obtained gas was mixed with 10 mL of O₂ and 5 mL of N₂ and used in the tracer experiment. The tracer gas was fed into the underground part of intact nodulated soybean plants and serial images of the distribution of ¹³N were obtained non-invasively using a positron-emitting tracer imaging system (PETIS). The rates of nitrogen fixation of the six test plants were estimated to be 0.17 ± 0.10 μmol N₂ h⁻¹ from the PETIS image data. The decreasing rates of assimilated nitrogen were also estimated to be 0.012 ± 0.011 μmol N₂ h⁻¹. In conclusion, we successfully observed nitrogen fixation in soybean plants with nodules non-invasively and quantitatively using [¹³N]N₂ and PETIS.     

Classification and Characteristic of Maturity Groups of Chinese Landraces of Soybean [Glycine max (L.) Merr.]

To link Chinese soybean classification with the world soybean maturity group system suggested by American researchers, 264 soybean landraces from China and 48 varieties of 13 soybean maturity groups from the United States (US) were tested under the natural and extended day-length at Nanjing, China. Based on comparing Chinese with the US soybeans in days to maturity, Chinese soybean landraces were classified into maturity group (MG) 000, 00, 0, I, II,..., IX, which only MG X might not exist in China. Chinese soybean landraces in each of MG 0, I, II and III revealed large variation in days to flowering and were almost grown in all eco-regions in China. These four maturity groups were further divided into eight sub-groups according to the variation in days to flowering and geographical distribution of soybeans of the same maturity group. The geographic distribution of soybean maturity groups in China was also presented in this paper. This study should be significant for soybean researchers in the world to understand the whole feature of Chinese soybean landraces germplasm.     

Growth characteristics,phytate contents,and coagulation properties of soymilk from a low-phytate Japanese soybean (Glycine max (L.) Merr.) line

The phytic acid (myo-inositol hexakisphosphate or InsP₆) content of seed crops is important to their nutritional quality. Since it represents 75?±?10% of the total seed phosphorus (P), phytic acid is also important regarding the management of P in agricultural production. A low-phytate F5 line, No. T-2-250-4-20, was selected from the progeny of a cross between the low-phytate soybean line CX1834 and the Japanese commercial cultivar Tanbakuro. This line and its parents were grown in a field nursery, and the growth characteristics, phytate accumulation, and processing suitability for tofu were evaluated. At full maturity, the weight of seeds per plant of line T-2-250-4-20 was 5.2- and 1.3-fold higher than that of CX1834 and Tanbakuro, respectively. The amount of phytate-phosphorus as a percentage of the total P content in seeds was 23% in line T-2-250-4-20-34, 30% in CX1834, and 69% in Tanbakuro. No significant difference was observed among the three cultivars/lines in their seed magnesium (Mg), potassium (K), crude protein, and sugar. However, the calcium (Ca), crude fat and ash contents in seeds of line T-2-250-4-20-34 and Tanbakuro was lowered compared to that of CX1834. The breaking stress of tofu was estimated employing a rheometer with a decreasing concentration of the coagulant magnesium chloride (MgCl₂), starting at 15.7?mmol?L^?1. In tofu made from Tanbakuro, the concentration of MgCl₂ required to achieve the maximum breaking stress was 12.6?mmol?L^?1; however, it was 9.5?mmol?L^?1 for tofu made from T-2-250-4-20-34 and CX1834. The tofu made from Tanbakuro was soft and broke at 6.3?mmol?L^?1 MgCl₂, but, in line T-2-250-4-20-34, harder tofu was made with lower MgCl₂ concentrations. No difference was observed among the cultivars/lines in the SDS-PAGE patterns of protein in soymilk. These results indicate that we have developed a low-phytate soybean with adequate productivity, and confirmed that tofu made from the low-phytate T-2-250-4-20-34 soybean becomes coagulated and harder at a lower MgCl₂ concentration than that from high-phytate soybean cultivars.