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Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells

  • Jeon, Hye Lyun;Yi, Jung-Sun;Kim, Tae Sung;Oh, Youkyung;Lee, Hye Jeong;Lee, Minseong;Bang, Jin Seok;Ko, Kinarm;Ahn, Il Young;Ko, Kyungyuk;Kim, Joohwan;Park, Hye-Kyung;Lee, Jong Kwon;Sohn, Soo Jung
    • Toxicological Research
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    • v.33 no.2
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    • pp.107-118
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    • 2017
  • Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration ($IC_{50}$) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity.

Assessment of Quantitative Analysis Methods for Lung F-18-Fluorodeoxyglucose PET (폐 종양 FDG PET 영상의 다양한 추적자 역학 분석 방법 개발과 유용성 고찰)

  • Kim, Joon-Young;Choi, Yong;Choi, Joon-Young;Lee, Kyung-Han;Kim, Sang-Eun;Choe, Yearn-Seong;Kim, Yong-Jin;Kim, Byung-Tae
    • The Korean Journal of Nuclear Medicine
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    • v.32 no.4
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    • pp.332-343
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    • 1998
  • Purpose: The purpose of this study was to assess the diagnostic accuracy of various quantitation methods using F-18-fluorodeoxyglucose (FDG) in patients with malignant or benign lung lesion. Materials and Methods: 22 patients (13 malignant including 5 bronchoalverolar cell cancer; 9 benign lesions including 1 hamartoma and 8 active inflammation) were studied after overnight fasting. We performed dynamic PET imaging for 56 min after injection of 370 MBq (10 mCi) of FDG. Standardized uptake values normalized to patient's body weight and plasma glucose concentration (SUVglu) were calculated. The uptake rate constant of FDG and glucose metabolic rate were quantified using Patlak graphical analysis (Kpat and MRpat), three compartment-five parameter model (K5p, MR5p), and six parameter model taking into account heterogeneity of tumor tissue (K6p, MR6p). Areas under receiver operating characteristic curves (ROC) were calculated for each method. Results: There was no significant difference of rate constant or glucose metabolic rate measured by various quantitation methods between malignant and benign lesions. The area under ROC curve were 0.73 for SUVglu, 0.66 for Kpat, 0.77 for MRpat, 0.71 for K5p, 0.73 for MR5p, 0.70 for K6p, and 0.78 for MR6p. No significant difference of area under the ROC curve between these methods was observed except the area between Kpat vs. MRpat (p<0.05). Conclusion: Quantitative methods did not improve diagnostic accuracy in comparison with nonkinetic methods. However, the clinical utility of these methods needs to be evaluated further in patients with low pretest likelihood of active inflammation or bronchoalveolar cell carcinoma.

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Parametric Images of Standardized Uptake Values using P-18-FDG Attenuation Corrected Whole Body PET (F-18-FDG감쇠보정 전신 PET을 이용한 표준섭취계수 추정과 매개변수 영상의 구성)

  • Kim, Kyeong-Min;Kwark, Cheol-Eun;Lee, Dong-Soo;Jeong, Jae-Min;Chung, June-Key;Lee, Myung-Chul;Koh, Chang-Soon;Kim, Yong-Jin
    • The Korean Journal of Nuclear Medicine
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    • v.30 no.4
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    • pp.560-569
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    • 1996
  • Background and Purpose : Standardized uptake value(SUV) has been used as a quantitative index for differentiating benign and malignant tumors with F-18-FDG PET In this study, we produced whole body parametric images of SUV(WBPIS) by body weight normalization, and validated the values by comparison with SUV's calculated with regional scans. Subjects and Methods : Whole body scans were followed by regional scans sequentially on 23 patients. In whole body study, transmission and emission scans were acquired for 2 minutes and 6 minutes for each bed position, respectively. In regional study, transmission and emission scans were acquired for 20 minutes. Measured and segmented/ smoothed attenuation correction were applied using these 2 min transmission scans in whole body studies. The effects of attenuation correction on SUVs were evaluated quantitatively using F-18 filled cylindrical phantom. The mean and peak SUVs obtained from WBPIS were compared with SUVs of the regional scans. Results : In phantom studies, with any method of attenuation correction using regional or whole body studies of phantom, SUVs were nearly consistent. In whole body scan, SUV obtained using measured attenuation correction method was a little higher than SUV of regional scan. SUV obtained using segmented/smoothed attenuation correction method was a little lower. In patient studies, WBPIS using segmented/smoothed attenuation correction method was much smoother and more readable. SUVs of WBPIS obtained with both methods of attenuation correction were well correlated with SUVs of regional scans(r=0.9). SUVs of WBPIS with measured attenuation correction method were 5% lower than SUVs of regional scans. SUVs of WBPIS with segmented/smoothed attenuation correction method were 10% lower than SUVs of regional scans. The differences of SUVs of WBPIS by the two attenuation correction methods were relatively small compared with the possible differences derived from biological characteristics of tumors. Conclusion : We concluded that WBPIS could be useful in the quantification of tumor as well as in localization of whole body lesions, which were often outside the field of view in regional scan. WBPIS made using segmented/smoothed attenuation correction method could be used in clinical routines and SUVs from attenuation corrected F-18-FDG PET could be used interchangeably with SUVs of regional studies.

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Study on the biological activity of Artemisia iwayomogi KITAMURA (한인진(韓茵蔯)의 생리활성에 관한 연구)

  • Song, Young-Eun;Ryu, Ji-Sung;Chung, Ju-Ri;Kwak, Joon-Soo;Kim, Dae-Hyang;Kim, Bum-Suk;Rim, Chai-Woong
    • Korean Journal of Medicinal Crop Science
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    • v.9 no.2
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    • pp.116-123
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    • 2001
  • This study was carried out to investigate antioxidative, antimicrobial activity and the effect on hepatotoxicity in various extracts of Artemisia iwayomogi. The herb has been used widely for jaundice, hepatitis and liver cirrhosis in chinese medicine. Solid yield by various extraction solvents, 18.1%, was the highest in water extract. To find antioxidative activity in Artemisia iwayomogi was estimated radical scavenging effect by DPPH method in various extracts and change of the POV(peroxide value) of various extracts added in soybean oil during 20 days at $60^{\circ}C$. Radical scavenging effect by DPPH method was the most effective in methanol extract. Added 1,000ppm water extract and methanol extract in soybean oil, the POV of them, 46.8(meq/kg) and 50.8(meq/kg) were lower than that of control, 79.1(meq/kg), during 20 days storage. After antimicrobial activity of various extracts of Artemisia iwayomogi on bacteria was carried out by paper disc method, it found that the ethanol extract was the strongest activity on Vibrio parahaemolyticus. In vivo experiment was to investigate the effect of Artemisia iwayomogi water extract(AIWE) on hepatotoxicity by carbon tetrachloride$(CCl_4)$ in rats. The experiment groups were divided into five groups for recovery(for 3 days) and three groups for protection(for 10 days) in rat liver. The weights and morphological changes of liver and the body weight were examined in each groups. Compared with $CCl_4$ treatment groups$(CCl_4\;only)$, liver and liver/body(%) weights of AIWE pretreatment groups for 3 days and AIWE posttreatment groups for 10 days were declined. In macrography, fibrious exudates and swelling of liver were decreased in AIWE treatment groups. Accumulation of lipid droplets and necrosis of hepatocytes were also decreased in AIWE treatment groups in microscopically. In these results, AIWE seems to enhance hepato-protective and recoverable effect on $CCl_4$ induced hepatotoxicity in rats.

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Characterization of Catechol l,2-Dioxygenase Purified from the Benzoate Degrading Bacterium, Pseudomonas sp. NFQ-l Isolated from Dead Coal Pit Areas (폐광지역에서 분리한 Benzoate 분해세균 Pseudomonas sp. NEQ-1에서 정제된 Catechol 1,2-Dioxygenase의 특성)

  • Joo Jung-Soo;Yoon Kyung-Ha
    • Korean Journal of Microbiology
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    • v.40 no.4
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    • pp.275-281
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    • 2004
  • Our previous research has demonstrated that the bacterium, Pseudomonas sp. NFQ-l capable of utilizing quin­oline (2,3-benzopyridine) as the sole source of carbon, nitrogen, and energy was isolated and characterized [Yoon et ai. (2003) Kor. J. Biotechnol. Bioeng. 18(3):174-179]. In this study, we have found that Pseudomonas sp. NFQ-l could degrade quinoline as well as benzoate, and extended this work to characterize the catechol 1,2­dioxygenase (C1,2O) purified from the bacterium cultured in benzoate media. Initially, C1,2O has been purified by ammonium sulfate precipitation, gel permeation chromatography, and Source 15Q. After Source 15Q, puri­fication fold was increased to approximately 14.21 unit/mg. Molecular weight of C1,2O was about 33 kDa. Physicochemical characteristics (e.g., substrate specificity, Km, Vmax, pH, temperature and effect of inhibitors) of purified C1,2O were examined. C1,2O demonstrated the activity for catechol, 4-methylcatechol and 3-meth­ylcatechol as a substrate, respectively. The Km and Vmax value of C1,2O for catechol was 38.54 ${\mu}M$ and $25.10\;{\mu}mol{\cdot}min^{-1}{\cdot}mg^{-1}.$ The optimal temperature of C1,2O was $30^{\circ}C$ and the optimal pH was approximately 8.5. Metal ions such as $Ag^+,\;Hg^+,\;Ca^{2+},\;and\;Cu^{2+}$ show the inhibitory effect on the activity of C1,2O. N-terminal amino sequence of C1,2O was analyzed as ^1TVKISQSASIQKFFEEA^{17}.$ In this work, we found that the amino acid sequence of NFQ-l showed the sequence homology of 82, 71, 59 and $53\%$ compared with C1,2O from Pseudomonas aeruginosa PA0l, Pseudomonas arvilla C-1., P. putida KT2440 and Pseudomonas sp. CA10, respectively.

Applicability of Vegetation Index and SPAD Reading to Nondestructive Diagnosis of Rice Growth and Nitrogen Nutrition Status (식생지수와 SPAD를 이용한 벼 생육 및 질소영양상태의 비파괴적 진단 가능성 검토)

  • Kim Min-Ho;Shin Jin-Chul;Lee Byun-Woo
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.50 no.6
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    • pp.369-377
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    • 2005
  • Precise application of topdressing nitrogen (N) fertilizer is indispensible for securing high yield and good quality of rice and minimizing N losses to the environment as well. For precise N management, growth and nitrogen nutrition status (NNS) should be diagnosed rapidly and accurately. The objective of the study was to evaluate the applicability of vegetation index (VI) calculated from hyperspectral canopy reflectance measurement and SPAD reading to nondestructive in situ diagnosis of growth and NNS of rice. Canopy reflectance, SPAD read­ing, growth parameters, and NNS characteristics were measured from various N treatments to evaluate the relationships among them for two cropping seasons from 2001 to 2002. The correlation coefficient of VIs with variables of growth and NNS increased positively as rice canopy became more closed. Regardless of growth stages, VIs had significantly high correlations with LAI, shoot dry weight (DW), shoot N content and nitrogen nutrition index (NNI). Those correlation coefficients increased steadily before heading stage as rice grew up. However, tiller number and leaf N concentration showed significantly high correlations with VIs only at and after panicle initiation stage (PIS). Among the VIs, RVIgreen had significantly higher correlation with the measured parameters than the other VIs: it showed correlation coefficients greater than 0.8 with leaf and shoot N concentration and DW, and much higher coefficients greater than 0.9 with LAI, shoot N content, and NNI. At LAI of below 2.5, VIs had non-significant or low correlations with the growth and NNS indicators due to the background effects. SPAD reading had significantly high correlation with leaf N concentration and NNI at each growth stage. In addition, it had significant correlations with variables of growth and NNS at PIS and booting stage, particularly, at booting stage. Though SPAD reading had a significantly high correlation value at a given growth stage in each year, it showed very weak relationship with variables of growth and NNS when pooled across growth stages and years. In conclusion, RVIgreen was found to be the most reliable VI to estimate the growth and NNS of rice around at PIS, but SPAD reading had much limitations.

Effects of Hydrogen Peroxide on Germination and Early Growth of Sorghum (Sorghum bicolor) (과산화수소 처리가 수수의 발아 및 초기 생장에 미치는 효과)

  • Shim, Doobo;Song, Ki Eun;Park, Chan Young;Jeon, Seung Ho;Hwang, Jung Gyu;Kang, Eun-ju;Kim, Jong Cheol;Shim, Sangin
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.63 no.2
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    • pp.140-148
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    • 2018
  • As the global warming causing desertification increase, there is growing concern about damage of crops. It was to investigate how the treatment with hydrogen peroxide before leaf development affects the growth and yield of sorghum for minimizing a damage of crops to drought. The germination experiment was conducted at alternating temperature of $25^{\circ}C/20^{\circ}C$(12 hr/12 hr) under water stress condition of 0 ~ -0.20 MPa adjusted with PEG solution containing 0 and 10 mM $H_2O_2$. In order to know the effect of foliar application of hydrogen peroxide on the growth of sorghum, 10 mM hydrogen peroxide was treated to leaves at 3-leaf stage of sorghum growing in greenhouse conditions. Seed germination rate was increased by 20% in hydrogen peroxide treatment as compared to the Control. under water stress conditions (-0.15 ~ -0.20 MPa). The length of seedlings was also on the rise by the hydrogen peroxide treatment. In the greenhouse pot experiment, the morphological characteristics (plant height, stem diameter, leaf length, and leaf number) and physiological characteristics (chlorophyll content, chlorophyll fluorescence (Fv/Fm), stomatal conductance) were higher in the plants treated with hydrogen peroxide under the drought stress condition than those of plants of $H_2O$ treatment. Experiment conducted with the soil moisture gradient system showed that the foliar application of hydrogen peroxide increased photosynthetic ability of sorghum plant with respect to SPAD value and stomatal conductance and rooting capacity (root weight and root length) under drought condition. Generally, hydrogen peroxide treatment in sorghum increased the tolerance to drought stress and maintained better growth due to ameliorating oxidative stress.

Chemical and Functional Characteristics of Mechanically Deboned Chicken Meat and its Utilization in Processed Meat -I. Chemical and Functional Characteristics of Mechanically Deboned Chicken Meat- (기계발골가금육(機械拔骨家禽肉)의 특성(特性) 및 이용(利用)에 관(關)한 연구(硏究) -제(第) 1 보(報) : 기계발골가금육(機械拔骨家禽肉)의 특성(特性)-)

  • Ahn, Byung-Yoon;Kim, Jong-Won;Lee, Yu-Bang
    • Korean Journal of Food Science and Technology
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    • v.13 no.3
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    • pp.171-175
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    • 1981
  • Hand deboned and mechanically deboned chicken meat were produced from domestic broilers and spent layers. Meat yield, chemical composition, functional characteristics, storage stability and microbiogical properties were investigated. The results obtained were as follows: 1. 35% of carcass freight was recovered primarily as hand deboned chicken meat (HDM) and 45% secondarily as mechanically deboned chicken meat(MDM), total meat yield reaching 80% of carcass weight. 2. Moisture, protein, fat. ash and calcium content of MDM were 65, 12, 20, 1.7 and $0.2{\sim}0.4%$, respectively MDM was higher than HDM in fat, ash and calcium, but significantly lower in moisture and protein Total pigment content of MDM was 2.5 times higher than that of HDM, such high content being attributed to the increased inclusion of hemoglobin during the mechanical masceration of carcass in the deboning process. 3. The emulsifying capacity (ES) of MDM per g meat was only 70% that of HDM, but when ES was expressed on unit g of protein basis MDM showed even higher ES than HDM primarily due to the higher proportion of salt soluble protein fraction of MDM. 4. Since the TBA value of MDM increased rapidly after 4 weeks of frozen storage at $-20^{\circ}C$, the maximum possible storage period of MDM is estimated to be about 4 weeks. 5. Total microbial counts of MDM was approximately $1.8{\times}10\;cells/g$ showing no great difference from HDM or red meat.

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Current Wheat Quality Criteria and Inspection Systems of Major Wheat Producing Countries (밀 품질평가 현황과 검사제도)

  • 이춘기;남중현;강문석;구본철;김재철;박광근;박문웅;김용호
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.47
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    • pp.63-94
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    • 2002
  • On the purpose to suggest an advanced scheme in assessing the domestic wheat quality, this paper reviewed the inspection systems of wheat in major wheat producing countries as well as the quality criteria which are being used in wheat grading and classification. Most wheat producing countries are adopting both classifications of class and grade to provide an objective evaluation and an official certification to their wheat. There are two main purposes in the wheat classification. The first objectives of classification is to match the wheat with market requirements to maximize market opportunities and returns to growers. The second is to ensure that payments to glowers aye made on the basis of the quality and condition of the grain delivered. Wheat classes has been assigned based on the combination of cultivation area, seed-coat color, kernel and varietal characteristics that are distinctive. Most reputable wheat marketers also employ a similar approach, whereby varieties of a particular type are grouped together, designed by seed coat colour, grain hardness, physical dough properties, and sometimes more precise specification such as starch quality, all of which are genetically inherited characteristics. This classification in simplistic terms is the categorization of a wheat variety into a commercial type or style of wheat that is recognizable for its end use capabilities. All varieties registered in a class are required to have a similar end-use performance that the shipment be consistent in processing quality, cargo to cargo and year to year, Grain inspectors have historically determined wheat classes according to visual kernel characteristics associated with traditional wheat varieties. As well, any new wheat variety must not conflict with the visual distinguishability rule that is used to separate wheats of different classes. Some varieties may possess characteristics of two or more classes. Therefore, knowledge of distinct varietal characteristics is necessary in making class determinations. The grading system sets maximum tolerance levels for a range of characteristics that ensure functionality and freedom from deleterious factors. Tests for the grading of wheat include such factors as plumpness, soundness, cleanliness, purity of type and general condition. Plumpness is measured by test weight. Soundness is indicated by the absence or presence of musty, sour or commercially objectionable foreign odors and by the percentage of damaged kernels that ave present in the wheat. Cleanliness is measured by determining the presence of foreign material after dockage has been removed. Purity of class is measured by classification of wheats in the test sample and by limitation for admixtures of different classes of wheat. Moisture does not influence the numerical grade. However, it is determined on all shipments and reported on the official certificate. U.S. wheat is divided into eight classes based on color, kernel Hardness and varietal characteristics. The classes are Durum, Hard Red Spring, Hard Red Winter, Soft Red Winter, Hard White, soft White, Unclassed and Mixed. Among them, Hard Red Spring wheat, Durum wheat, and Soft White wheat are further divided into three subclasses, respectively. Each class or subclass is divided into five U.S. numerical grades and U.S. Sample grade. Special grades are provided to emphasize special qualities or conditions affecting the value of wheat and are added to and made a part of the grade designation. Canadian wheat is also divided into fourteen classes based on cultivation area, color, kernel hardness and varietal characteristics. The classes have 2-5 numerical grades, a feed grade and sample grades depending on class and grading tolerance. The Canadian grading system is based mainly on visual evaluation, and it works based on the kernel visual distinguishability concept. The Australian wheat is classified based on geographical and quality differentiation. The wheat grown in Australia is predominantly white grained. There are commonly up to 20 different segregations of wheat in a given season. Each variety grown is assigned a category and a growing areas. The state governments in Australia, in cooperation with the Australian Wheat Board(AWB), issue receival standards and dockage schedules annually that list grade specifications and tolerances for Australian wheat. AWB is managing "Golden Rewards" which is designed to provide pricing accuracy and market signals for Australia's grain growers. Continuous payment scales for protein content from 6 to 16% and screenings levels from 0 to 10% based on varietal classification are presented by the Golden Rewards, and the active payment scales and prices can change with market movements.movements.

Annul Variation of Soil Properties and Yield of Soybean in Paddy Field (콩 논 재배시 연차별 토양특성 및 생산력변화)

  • Kim, Min-Tae;Seo, Jong-Ho;Cho, Hyeoun-Suk;Seong, Ki-Yeong;Lee, Jong-Ki;Eom, Sun-Pyo;Jeon, Weon-Tai;Lee, Jang-Yong
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.52 no.4
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    • pp.370-374
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    • 2007
  • This study was carried out to investigate the soil chemical and physical properties by annul variation and yield properties according to cropping rotation system of paddy field which was cultivated rice and soybean of each 1 year by turns, each 2 year by turns, 1 year of rice and 3 years of soybean and 4 years of soybean continuously. This study was conducted for 3 years from 2002 to 2004 in National Institute of Crop Science(NISC), Korea. Study was controlled by Shinpaldal 2 of midmatured species, sow a field with soybean by $60{\times}15cm$ in 27 May. Chemical fertilizer was spread in the field by N 3.0 kg, $P_2O_5 $ 3.0 kg, $K_2O$ 3.4 kg per 10a. This experiment was carried for increase the degree of self sufficiency of soybean from detect the better cropping system in paddy field. The results of the study were as follows; Soil prosity was increased 17.4% in each one year rotation and 21.8% in the sector of cultivated soybean for four years. Results was indicated that $5{\sim}9cm$ of stem length, $0.5{\sim}1.0ea$ of branch number and $3.5{\sim}7.0$ of SPAD value was decreased in the sector of cultivated soybean for $3{\sim}4$ years compared to $1{\sim}2$ year cultivated sector. The soybean yield was reduced 12.9%(222 kg/10a) in the 2 year cultivated sector and 21%(201 kg/10a) in the 3 year cultivated sector compare with paddy-upland switching cultivation(255 kg/10a). Root nodule weight and number was tend to decrease according to the increasing duration of cultivated soybean.