Gm NAC15 overexpression in hairy roots enhances salt tolerance in soybean

The NAC （NAM, ATAF1/2 and CUC2） transcription factor family plays a key role in plant development and responses to abiotic stress. GmNAC15 （Glyma 15g40510.1）, a member of the NAC transcription factor family in soybean, was functionally characterized, especially with regard to its role in salt tolerance. In the present study, qRT-PCR （quantitative reverse transcription PCR） analysis indicated that GmNAC15 was induced by salt, drought, low temperature stress, and ABA treatment in roots and leaves. GmNAC15 overexpression in soybean （Glycine max） hairy roots enhanced salt tolerance. Transgenic hairy roots improved the survival of wild leaves; however, overexpression of GmNAC15 in hairy root couldn＇t influnce the expression level of GmNAC15 in leaf. GmNAC15 regulates the expression levels of genes responsive to salt stress. Altogether, these results provide experimental evidence of the positive effect of GmNAC15 on salt tolerance in soybean and the potential application of genetic manipulation to enhance the salt tolerance of important crops.

Soybean hairy roots produced in vitro by Agrobacterium rhizogenes-mediated transformation

Soybean is one of the world's most important oil and protein crops. Efficient transformation is a key factor for the improvement of soybean by genetic modification. We describe an optimized protocol for the Agrobacterium rhizogenes-mediated transformation of soybean and the induction of hairy root development in vitro. Cotyledons with 0.5-cm hypocotyls were cut from 5-day-old seedlings and used as explants. After infection and co-cultivation,hairy roots were produced in induction culture medium after 10–12 days. Using this method, 90%–99% of the infected explants of five different cultivars produced hairy roots within one month. Observations using reporter constructs showed that 30%–60% of the hairy roots induced were transformed. Based on high transformation efficiency and short transformation period, this method represents an efficient and rapid platform for study of soybean gene function.

Overexpression of GmBIN2,a soybean glycogen synthase kinase 3 gene,enhances tolerance to salt and drought in transgenic Arabidopsis and soybean hairy roots

Glycogen synthase kinase 3 （GSK3） is a kind of sedne/threonine kinase widely found in eukaryotes. Many plant GSK3 kinases play important roles in regulating stress responses. This study investigated BRASSINOSTEROID-INSENSITIVE 2 （GmBIN2） gene, a member of the GSK3 protein kinase family in soybean and an orthologue of Arabidopsis BIN2/AtSK21. GmBIN2 expression was increased by salt and drought stresses, but was not significantly affected by the ABA treatment. To examine the function of GrnBIN2, transgenic Arabidopsis and transgenic soybean hairy roots were generated. Overexpression of GmBIN2 in Arabidopsis resulted in increased germination rate and root length compared with wild-type plants under salt and mannitol treatments. Overexpression of GmBIN2 increased cellular Ca2~ content and reduced Na~ content, enhancing salt tolerance in transgenic Arabidopsis plants. In the soybean hairy root assay, overexpression of GmBIN2 in transgenic roots also showed significantly higher relative root growth rate than the control when subjected to salt and mannitol treatments. Measurement of physiological indicators, including proline content, superoxide dismutase （SOD） activity, and relative electrical conductivity, supported this conclusion. Furthermore, we also found that GmBIN2 could up-regulate the expression of some stress-related genes in transgenic Arabidopsis and soybean hairy roots. Overall, these results indicated that GmBIN2 improved tolerance to salt and drought in transgenic Arabidopsis and soybean hairy roots.

Effects of drought stress on root morphology and spatial distribution of soybean and adzuki bean

Due to global climate change,Korea is facing severe droughts that affect the planting and early vegetative periods of upland crops.Soybean and adzuki bean are important legume crops in Korea,so it is critical to understand their adaptations to water stress.This study investigated the changes in root morphological properties in soybean and adzuki bean and quantified the findings using fractal analysis.The experiment was performed at the National Institute of Crop Science in Miryang,Korea.Soybeans and adzuki beans were planted in test boxes and grown for 30 days.The boxes were filled with bed soil with various soil moisture treatments.Root images were obtained and scanned every two days,and the root properties were characterized by root length,depth and surface area,number of roots,and fractal parameters(fractal dimension and lacunarity).Root depth,length and surface area and the number of roots increased in both crops as the soil moisture content increased.The fractal dimension and lacunarity values increased as the soil moisture content increased.These results indicated that the greater the soil moisture,the more heterogeneous the root structure.Correlation analysis of the morphological properties and fractal parameters indicated that soybean and adzuki bean had different root structure developments.Both soybean and adzuki bean were sensitive to the amount of soil moisture in the early vegetative stage.Soybean required a soil moisture content greater than 70%of the field capacity to develop a full root structure,while adzuki bean required 100%of the field capacity.These results would be useful in understanding the responses of soybean and adzuki bean to water stress and managing irrigation during cultivation.

A LAMP-assay-based specific microbiota analysis reveals community dynamics and potential interactions of 13 major soybean root pathogens

Soybean root diseases are associated with numerous fungal and oomycete pathogens;however,the community dynamics and interactions of these pathogens are largely unknown.We performed 13 loop-mediated isothermal amplification(LAMP)assays that targeted specific soybean root pathogens,and traditional isolation assays.A total of 159 samples were collected from three locations in the Huang-Huai-Hai region of China at three soybean growth stages(30,60,and 90 days after planting)in 2016.In LAMP results,we found that pathogen communities differed slightly among locations,but changed dramatically between soybean growth stages.Phytophthora sojae,Rhizoctonia solani,and Fusarium oxysporum were most frequently detected at the early stage,whereas Phomopsis longicolla,Fusarium equiseti,and Fusarium virguliforme were most common in the later stages.Most samples(86%)contained two to six pathogen species.Interestingly,the less detectable species tended to exist in the samples containing more detected species,and some pathogens preferentially co-occurred in diseased tissue,including P.sojae–R.solani–F.oxysporum and F.virguliforme–Calonectria ilicicola,implying potential interactions during infection.The LAMP detection results were confirmed by traditional isolation methods.The isolated strains exhibited different virulence to soybean,further implying a beneficial interaction among some pathogens.

Effect of plough pans on the growth of soybean roots in the black-soil region of northeastern China

Plough pans （PPs）, common in the black-soil region of northeastern China, have significant effects on the root systems of crops. We conducted a field experiment to study the distribution of soybean roots under the influence of PPs. The soybean roots showed compensatory growth above the PP, with higher root length and weight in soil with a PP compared to those without a PP. Roots were heavier and longer in the 15-75 cm soil layer without a PP than with a PP. Soil porosity was lower in the PP and the soil below the PP, which likely decreased the oxygen content of the soil and induced more growth of roots above the PP. The PP is also likely to decrease infiltration of rain-water and hinder the migration of nitrate downward, which in turn increased the density and length of soybean root hairs, which, hence, promoted growth.

Resistance Evaluation of Soybean Germplasm from Huanghuai Region to Phytophthora Root Rot

The aim of the study was to establish a set of differential strains and to identify soybean resistant genes to Phytophthora root rot and then to apply those strains for analysis of the resistant genes Rps1a,Rps1c,and Rps1k that soybean cultivars or lines may carry.Virulence formula of 125 Phytophthora sojae isolates were determined using the hypocotyls inoculation technique,the strains,which includ 6 isolates with different virulence formulas,were applied to identify the resistance of 55 soybean cultivars or lines and resistant genes were analyzed using the gene postulating procedure.Eighteen reaction types occurred in 55 cultivars or lines and results of gene postulation indicated that 2 cultivars or lines probably carried gene Rps1c and no cultivar may carry genes Rps1a or Rps1k.A few of soybean cultivars or lines from Huanghuai Region carry Rps genes Rps1a,Rps1c and Rps1k and tend to infect by P.sojae,so resistant cultivars or lines need to be bred and popularized actively.

Model Construction and Visualization Simulation of Soybean Root System

This paper mainly focused on the growth law, model construction and visualization of a soybean root. A pot experiment was conducted in the laboratory to collect root system data and measure soybean root＇s length, diameter and number by excavating in different periods. On the basis of an in-depth analysis of root structure geometry, we analyzed the collected experimental data with logistic equations, and got the growth of soybean root equation, according to its morphological structure characteristics of self-similarity, and discussed the virtual modeling method on the soybean root based on L-system and in Visual c＋＋ Using OpenGL technology to achieve a soybean root system topology model and visualization simulation.

Impact of long-term chemical fertilizer and organic amendment to Fusarium root rot of soybean

Soil suppressiveness to Fusarium root rot of soybean had been observed in a black soil field after a long-term fertilization with nitrogen(N)and phosphorus(P)fertilizer combined with pig manure as organic amendment(NPM),rather than that with only nitrogen and phosphorus fertilizer(NP)or no fertilizer(NF).To determine the microbial role on this suppressiveness,fungal and bacterial community characteristics in NPM,NP and NF treatments were investigated by q PCR and DGGE.Compared with the similar bacterial community characteristics among 3 treatments,fungal community,especially Fusarium population size and community composition in NPM treatment were different with those of NP and NF groups.Based on the isolation and pathogenicity test,pathogenic F.oxysporum,F.graminearum,F.verticillioide and F.lateritium absolutely dominated Fusarium community in NF and NP groups.Nonpathogenic F.avenaceum,F.equiseti,F.culmorum,F.redolens,F.solani and F.tricinctum dominated Fusarium community in NPM group.Isolation rate of pathogenic Fusarium in NPM reduced from 100%to 38%in NF.These results suggested that the dominance of soil non-pathogenic Fusarium population induced by organic amendment might play an important role on suppressing Fusarium root rot in the tested field.

Evolution of Root Characters of Soybean Varieties Developed in Different Years

It was studied that the evolution of root characteristics among 42 soybean varieties developed in Heilongjiang and Jilin Province in different years. The results showed that there were differences on the root characteristics among soybean varieties. From 1950s to 1990s, root fresh weight, root volume, root surface, root dry weight, lateral root length of main root characters tendedly increased with the variable development

Hypoxia-Responsive Root Hydraulic Conductivity Influences Soybean Cultivar-Specific Waterlogging Tolerance

Excess soil moisture induces hypoxic conditions and causes waterlogging injury in soybean [Glycine max (L.) Merr.]. This study investigated the mechanism underlying the development of waterlogging injury. Nine Japanese soybean cultivars with varying degrees of waterlogging tolerance were grown in a hydroponic system for 14 days under hypoxic conditions. Shoot and root biomasses and root hydraulic conductivity were measured at an early vegetative stage for plants under control and hypoxic conditions. Root morphological traits and intramembrane aquaporin proteins were also analyzed. The tolerance of each cultivar to field waterlogging was based on biomass changes induced by the hypoxia treatment. Root hydraulic conductivity responses to hypoxia were associated with changes in total dry weight, leaf dry weight, and leaf area. The effects of hypoxic conditions on root hydraulic conductivity were also represented by the changes in root morphology, such as total root length, thick-root length, and number of root tips. Additionally, a 32.3 kDa aquaporin-like protein seemed to regulate root hydraulic conductivity. Our results from a hydroponic culture suggest that the soybean cultivar-specific responses to hypoxic conditions in the rhizosphere reflect fluctuations in hydraulic conductivity related to root morphological or qualitative changes.

Image-based root phenotyping for field-grown crops:An example under maize/soybean intercropping

Root architecture,which determines the water and nutrient uptake ability of crops,is highly plastic in response to soil environmental changes and different cultivation patterns.Root phenotyping for field-grown crops,especially topological trait extraction,is rarely performed.In this study,an image-based semi-automatic root phenotyping method for field-grown crops was developed.The method consisted of image acquisition,image denoising and segmentation,trait extraction and data analysis.Five global traits and 40 local traits were extracted with this method.A good consistency in 1st-order lateral root branching was observed between the visually counted values and the values extracted using the developed method,with R^(2)=0.97.Using the method,we found that the interspecific advantages for maize mainly occurred within 5 cm from the root base in the nodal roots of the 5th-7th nodes,and that the obvious inhibition of soybean was mostly reflected within 20 cm from the root base.Our study provides a novel approach with high-throughput and high-accuracy for field research on root morphology and branching features.It could be applied to the 3D reconstruction of field-grown root system architecture to improve the inputs to data-driven models(e.g.,OpenSimRoot)that simulate root growth,solute transport and water uptake.

Identification of Endophytic Bacillus BHL3501 from Wild Soybean and Its Inhibitory Effect against Soybean Cyst Nematode (Heterodera glycines Ichinohe)

[ Objective] The paper was to identify endophytic bacillus BHL3501 from wild soybean and its inhibitory effect against soybean cyst nematode. [ Meth- od] Through morphological characteristic, physiological and biochemical characteristics and 16S rDNA analysis, a strain of endophytic bacterium BHL3501 with ne- maticidal activity isolated and screened from the root of wild soybean was identified, the effects of BHL3501 metabolite solutions with different dilution multiples on egg hatching and activity of 2rd instar juvenile of soybean cyst nematode were also studied. [ Result ] BHL3501 strain was preliminarily identified to be Bacillus sp.. The relative inhibitory rate of its original fermentation broth on egg hatching of soybean cyst nematode after 24 h was 98.3% ; the relative inhibitory rate under 10- time diluted solution treatment was 79.5%, which had significant difference with sterile water control. The corrected mortality rate of 24 instar juvenile was 93.1% after treated by original fermentation broth for 24 h, the treatments of all diluted solutions had significant difference with sterile water control. [ Conclusion ] BHL3501 metabolites had strong inhibitory effect against egg hatching of soybean cyst nematode, which also had strong toxic effect on its 2nd instar juvenile.

Molecular regulatory mechanism of tooth root development

The root is crucial for the physiological function of the tooth, and a healthy root allows an artificial crown to function as required clinically. Tooth crown development has been studied intensively during the last few decades, but root development remains not well understood. Here we review the root development processes, including cell fate determination, induction of odontoblast and cementoblast differentiation, interaction of root epithelium and mesenchyme, and other molecular mechanisms. This review summarizes our current understanding of the signaling cascades and mechanisms involved in root development. It also sets the stage for de novo tooth regeneration.

Temporal and Spatial Dynamics of Bacterial Community in the Rhizosphere of Soybean Genotypes Grown in a Black Soil

The dynamics of rhizosphere microbial communities is important for plant health and productivity, and can be influenced by soil type, plant species or genotype, and plant growth stage. A pot experiment was carried out to examine the dynamics of microbial communities in the rhizosphere of two soybean genotypes grown in a black soil in Northeast China with a long history of soybean cultivation. The two soybean genotypes, Beifeng 11 and Hai 9731, differing in productivity were grown in a mixture of black soil and siliceous sand. The bacterial communities were compared at three zone locations including rhizoplane, rhizosphere, and bulk soil at the third node （V3）, early flowering （R1）, and early pod （R3） stages using polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DCGE） of 16S rDNA. The results of principal component analyses （PCA） showed that the bacterial community structure changed with growth stage. Spatially, the bacterial communities in the rhizoplane and rhizosphere were significantly different from those in the bulk soil. Nevertheless, the bacterial communities in the rhizoplane were distinct from those in the rhizosphere at the V3 stage, while no obvious differences were found at the R1 and R3 stages. For the two genotypes, the bacterial community structure was similar at the V3 stage, but differed at the R1 and R3 stages. In other words, some bacterial populations became dominant and some others recessive at the two later stages, which contributed to the variation of the bacterial community between the two genotypes. These results suggest that soybean plants can modify the rhizosphere bacterial communities in the black soil, and there existed genotype-specific bacterial populations in the rhizospbere, which may be related to soybean productivity.

Differences between soybean genotypes in physiological response to sequential soil drying and rewetting

Soybean genotypes show diverse physiological responses to drought, but specific physiological traits that can be used to evaluate drought tolerance have not been identified. In the present study we investigated physiological traits of soybean genotypes under progressive soil drying and rewetting, using a treatment mimicking field conditions.After a preliminary study with eight soybean genotypes, two drought-tolerant genotypes and one susceptible genotype were grown in the greenhouse and subjected to water restriction. Leaf expansion rate, gas exchange, water relation parameters, total chlorophyll(Chl), proline contents of leaves, and root xylem p H were monitored in a time course, and plant growth and root traits were measured at the end of the stress cycle. Drought-tolerant genotypes maintained higher leaf expansion rate, net photosynthetic rate(Pn), Chl content,instantaneous water use efficiency(WUEi), % relative water content(RWC), water potential(ψw), and turgor potential(ψp) during progressive soil drying and subsequent rewetting than the susceptible genotypes. By contrast, stomatal conductance(gs) and transpiration rate(Tr)of tolerant genotypes declined faster owing to dehydration and recovered more sharply after rehydration than the same parameters in susceptible ones. Water stress caused a significant increase in leaf proline level and root xylem sap p H of both genotypes but tolerant genotypes recovered to pre-stress levels more quickly after rehydration. Tolerant genotypes also produced longer roots with higher dry mass than susceptible genotypes. We conclude that rapid perception and adjustment in response to soil drying and rewetting as well as the maintenance of relatively high Pn, %RWC, and root growth constitute the mechanisms by which drought-tolerant soybean genotypes cope with water stress.

Determination of Organic Acids in Root Exudates by High Performance Liquid Chromatography: I. Development and Assessment of Chromatographic Conditions *1

Methods for determining nine low molecular weight organic acids in root exudates were developed by using reversed phase high performance liquid chromatography with UV (ultraviolet) detection at 214 nm. The mobile phase was 18 mmol L -1 kH 2PO 4 adjusted to pH 2.25 with phosphoric acid and the flow rate was 0.3 mL min -1 . The analytical column was a reversed phase silica based C 18 column (Shim pack CLC ODS). The root exudates were collected through submerging the whole root system into aerated deionized water for 2 hours. The filtered exudate solutions were concentrated to dryness by rotary evaporation at 40 °C, dissolved in 10 mL mobile phase. The chromatographic conditions of organic acid determination were analyzed. The results showed that there was a high selectivity and sensitivity in the organic acid determination by reversed phase high performance liquid chromatography. Coefficients of variation for organic acid determination were lower than 10% except lactic acid. The recoveries were consistently between 80.1% to 108.3%. Detection limits were approximately 0.05 to 4.5 mg L -1 for organic acids except succinic acid with the detection limit of 7.0 mg L -1 . Phosphorus deficiency may contribute to the release of organic acids in soybean root exudates especially malic, lactic and citric acids.

Conidia of one Fusarium solani isolate from a soybean-production field enable to be virulent to soybean and make soybean seedlings wilted

Fusarium is usually thought to cause soybean root rot, which results in a large quantity of annual yield loss in soybean production, by its secretions including Fusarium toxins and cell wall degrading enzymes, but not by the conidia themselves that do not underlie any virulence so far. Here we report that the conidia of one Fusarium solani isolate are able to be virulent to soybean and make soybean seedlings wilted alone. We isolated them from the wilted plants in a soybean-production field and molecularly identified 17 Fusarium isolates through phylogenetic analysis. Of them, except for one isolate that showed diversity of virulence to different soybeans （virulent to one soybean whereas avirulent to another soybean）, the others were all virulent to the two tested soybeans： both conidia cultures and secretions could make soybean seedlings wilted at 5 days post infection, and their virulence had dosage effects that only conidia cultures of at least 5×10^6 conidia mL-1 could show virulence to soybean; however, the sole conidia of the F. solani isolate #4 also exhibited virulence to soybean and could make soybean seedlings wilted. Finally, we developed the specific cleaved amplified polymorphic sequences （CAPS） markers to easily differentiate Fusarium isolates. The isolate #4 in this work will likely be used to investigate the new mechanism of virulence of Fusarium to soybean.

大豆根茎过渡区弯曲突变体M_(rstz)的鉴定与基因定位

植物根茎过渡区(root-stem transition zone,RSTZ)将根和茎相互连接,其发育形态决定了大豆的地上部株型和抗倒伏潜力。本研究通过EMS诱变获得一个RSTZ弯曲或旋转的大豆突变体M_(rstz),其形态特征能够稳定遗传,是探究大豆茎秆发育规律的特异材料。将该突变体和栽培大豆中黄13杂交构建重组自交系群体,对群体中直立和弯曲型后代的RSTZ进行解剖结构比较,发现弯曲型株系比直立型株系的维管形成层较宽、次生木质部细胞层数较多、细胞形状不规则,表明维管组织分化可能是导致RSTZ形态发生差异的重要因素之一。进一步对化学组分测定发现,木质素和粗纤维含量越高越不易弯曲。选取RIL群体中弯曲型和直立型2种极端株系进行BSA-Seq,采用SNP-index和InDel-index关联分析方法鉴定到调控RSTZ形态的关联区域Chr19:43030943~45849854,该区间共含有319个基因。结合生物信息学分析、基因注释信息和表达丰度分析筛选到7个候选基因,分别为Glyma.19G170200、Glyma.19G201500、Glyma.19G187800、Glyma.19G178200、Glyma.19G197000、Glyma.19G179100、Glyma.19G196900。其中,Glyma.19G187800、Glyma.19G178200和Glyma.19G196900在大豆驯化中潜在影响RSTZ形态建成。本研究为解析大豆RSTZ组织形成及其遗传基础提供了材料基础,并为挖掘调控大豆茎秆发育基因提供新的见解。

GC/GCMS analysis of the petroleum ether and dichloromethane extracts of Moringa oleifera roots

Objective:To explore the phytochemical constituents from petroleum ether and diehloromethane extracts of Moringa oleifera(M.oleufera)roots using GC/GC—MS.Methods:A total of 5.11 kg fresh and undried crashed root of M.oleifera were cut into small pieces and extracted with petroleum ether and diehloromethane(20 L.each) at room temperature for 2 d.The concentrated extracts were subjected to their GC—MS analysis.Results:The GC-MS analysis of the petroleum ether and diehloromethane extracts of M.oleifern roots,which showed promising biological activities,has resulted in the identification 102 compounds.These constituents belong to 15 classes of compounds including hydrocarbons,fatty acids,esters,alcohols,isolhioeyanate.thiocyanale,pyrazine,aromalics.alkamides.cyanides,steroids,halocompounds.urea and N-hydroxyimine derivatives,unsaturated alkenamides.alkyne and indole.GC/GC-MS studies on petroleum ether extraet of the roots revealed that it contained 39 compounds,belonging to nine classes.Cyclooctasulfur S8 has been isolated as a pure compound from the extract.The major compounds identified from petroleum ether extract were trans-13-clocosene(37.9%).nonacosane(32.6%).cycloartenol(28.6%) nonadecanoic acid(13.9%) and cyclooctasulfur S8(13.9%).Dichloromethane extract of the roots was composed of 63 compounds of which nasimizinol(58.8%) along with oleic acid(46.5%),N—benzyl-N-(7—cyanato heptanamide(38.3%),N—benzyl-N—(1—chlorononyl) amide(30.3%),bis[3—benzyl prop-2-ene]-1-one(19.5%) and N.N-dibeuzyl—2-ene pent 1.5—diamide(11.6%) were the main constituents.Conclusions:This study helps to predict the formula and structure of active molecules which can be used as drugs.This result also enhances the traditional usage of M.oleifera which possesses a number of bioactive compounds.