A Dominant Locus, qBSC-1, Controls β Subunit Content of Seed Storage Protein in Soybean(Glycine max(L.) Merri.)

Soybean seed storage protein is one of the most important plant vegetable proteins, and β subunit is of great significance to enhance soybean protein quality and processing property. F2 segregated population and residual heterozygous lines（RHL） derived from the cross between Yangyandou（low level of β subunit） and Zhonghuang 13（normal level of β subunit） were used for mapping of β subunit content. Our results showed that β subunit content was controlled by a single dominant locus, qBSC-1（β subunit content）, which was mapped to a region of 11.9 cM on chromosome 20 in F2 population of 85 individuals. This region was narrowed down to 2.5 cM between BARCSOYSSR_20_0997 and BARCSOYSSR_20_0910 in RHL with a larger population size of 246 individuals. There were 48 predicted genes within qBSC-1 region based on the reference genome（Glyma 1.0, Williams 82）, including the two copies of β subunit coding gene CG4. An InDel marker developed from a thymine（TT） insertion in one copy of CG4 promoter region in Yangyandou cosegregrated with BARCSOYSSR_20_0975 within qBSC-1 region, suggesting that this InDel marker maybe useful for marker-assisted selection（MAS）.

Generation of seed lipoxygenase-free soybean using CRISPR-Cas9

Beany flavor induced by three lipoxygenases(LOXs, including LOX1, LOX2, and LOX3)restricts human consumption of soybean. It is desirable to generate lipoxygenase-free new mutant lines to improve the eating quality of soybean oil and protein products. In this study, a pooled clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPRassociated protein 9(Cas9) strategy targeting three GmLox genes(GmLox1, GmLox2, and GmLox3) was applied and 60 T_0 positive transgenic plants were generated, carrying combinations of sg RNAs and mutations. Among them, GmLox-28 and GmLox-60 were gmlox1 gmlox2 gmlox3 triple mutants and GmLox-40 was a gmlox1 gmlox2 double mutant.Sequencing of T_1 mutant plants derived from GmLox-28, GmLox-60, and GmLox-40 showed that mutation in the GmLox gene was inherited by the next generation. Colorimetric assay revealed that plants carrying different combinations of mutations lost the corresponding lipoxygenase activities. Transgene-free mutants were obtained by screening the T_2 generation of lipoxygenase-free mutant lines(GmLox-28 and GmLox-60). These transgeneand lipoxygenase-free mutants could be used for soybean beany flavor reduction without restriction by regulatory frameworks governing transgenic organisms.

Changes of Plasma Membrane H^+-ATPase Activities of Glycine max Seeds by PEG Treatment

The soybean （Glycine max） Heihe No. 23 is sensitive to imbibitional chilling injury. Polyethylene glycol （PEG） treatment can improve chilling tolerance of soybean seeds to a certain extent. The changes of hydrolytic ATPase in plasma membranes and H^＋-pumping responses in soybean seeds were investigated during PEG treatments. Effects of exogenous calcium and exogenous ABA on the hydrolytic ATPase were also examined in order to understand the mechanism of chilling resistance. Highly purified plasma membranes were isolated by 6.0% aqueous two-phase partitioning from soybean seeds, as judged by the sensitivity of hydrolytic ATPase to sodium vanadate. PEG treatment resulted in a slight increase of the hydrolytic ATPase activity in 12 h. Then the activity decreased gradually, but still higher than the control. The H^＋-pumping activity increased steadily during PEG treatment. Exogenous calcium had both activating and inhibiting effects on the hydrolytic ATPase, but the activity was inhibited in soybean seeds treated with exogenous ABA. Results suggested that PEG treatment, not the exogenous calcium and ABA, up-regulated H^＋-ATPase activities in soybean seeds.

Effects of Osmoconditioning on Mitochondrial Respiration and Phosphorylation in Soybean Seeds

Cotyledon mitochondrion respiration and oxidative phosphorylation activity were studied in two groups of soybean seeds. One group was primed with polyethylene glycol (PEG) for different periods of time, and the other was unprimed (control), and both were then exposed to imbibition at low temperatures before their germination. The results indicated that when L-Malate (L-Mal) and á-Ketoglutarate (α-Kg) were used as substrates, the ADP-stimulated mitochondria respiration rates of control seeds were mark- edly higher than state Ⅲ respiration rates of primed seeds. However, the osmoconditioning pretreatment significantly enhanced the oxidative phosphorylation activity of cotyledon mitochondrion in 12 h. The oxidative phosphorylation activity of the mitochondrion of primed seeds was normal and the ADP/O value was consistent with the theoretical one. When reduced nicotinamide adenine dinu- cleotide (NADH) was used as the substrate, the mitochondria of control seeds still had oxidative phosphorylation activity, while ADP/O value was obviously lower than that of mitochondria of primed seeds. When Succinate (Succ) was used as the substrate, the oxidative phosphorylation activity of the primed seeds was normal after priming for 24 h. When different substrates were used, the emerging order of the oxidative phosphorylation activity of the primed seeds was NADH, α-Kg, Succ and in the last place L-Mal. The mechanism of soybean imbibitionl chilling injury and protective effect of PEG priming were discussed.

The Study of Alfalfa-Sinorhizobium meliloti Symbiosis Productivity under Different Water Conditions and the Influence of the Legume Seeds Lectin

The paper studies the effect of incubation of rhizobia with soybean seeds lectin on formation and functioning of alfalafa-Sinorhizobium meliloti symbiosis under the optimal water supply and drought conditions. It was shown that the addition of lectin to the inoculation compositions intensifies physiological processes in alfalfa plants： increases nitrogen fixing activity of symbiosis, nodule number on roots, as well as chlorophyll and carotenoid content in leaves, enhances plant growth during budding-fruiting period and reduces the negative effect of drought on alfalfa productivity.

Production of Biodiesel with Seed Soybean and Supercritical Ethanol

This paper presents a study of biodiesel production by a non-catalytical process. The innovation in this study is the use of novel materials for production: seed soybean (Glycine Max) “in natura” and ethanol in a supercritical state. To conduct the experiments, a bench reactor with a capacity of 150 mL, resistant to pressure of up to 300 bar and temperature of 350°C was developed. The fractional factorial experimental design () was used to evaluate the temperature, seed granulometry, molar ratio ethanol/oil and water percent of the mixture. The best yield observed was that of 94.07%, 10 minutes after the reactor entered a supercritical condition. Significant effects on seed granulometry, molar ratio ethanol, oil and temperature were verified. From the proposed process, biodiesel and toasted soybean seed were obtained. To purify the biodiesel sample it was necessary to use ultra-centrifugation to separate seed particles, and rotoevaporation to separate the fatty acid ethyl ester and unreacted ethanol. The chemical analyses were conducted directly by gas chromatography. The yield was calculated in accordance with concentrations obtained in the chromatographic analysis and seed mass of the experiment. Also checked was the presence of palmitate esters, stearate, oleate, linoleate and linolenate. By analyzing the ester composition it was possible to assess whether a good quality biodiesel was available. The roasted soybean seeds obtained after the reaction showed a calorific potential of 2203.17 kcal/kg and also be used as fuel.

Correlation Between Antioxidation and the Content of Total Phenolics and Anthocyanin in Black Soybean Accessions

The objective of this study was to investigate the correlations between antioxidations and the contents of the total phenolics and anthocyanin in 127 accessions of black soybean. A T-test, a fast clustering procedure, and a correlation coefficient analysis were used for experimentation. The variation ranges of the total antioxidant capacity （TAC）, the total phenolics, and anthocyanin contents in 127 black soybean accessions were 0.44-3.56, 7.05-74.82, and 0.22-1.87 mg g-l, respectively, displaying significant genotype differences. The major differences in TAC, the total phenolics, and the anthocyanin contents existed among various types of accessions from geographical regions. The differences between the accessions from black and yellow soybeans, spring and autumn, summer and autumn, Dongbeichun and Nanfangchun, Dongbeichun and Nanfangxia, Beifangchun and Nanfangchun, and Beifangchun and Nanfangxia were significant at 0.01 or 0.05 levels, respectively. The general tendency was that the TAC, the total phenolics, and the anthocyanin contents of Beifangchun accessions were higher than that of Dongbeichun ones, while that of Nanfangchun accessions were the worst. 127 black soybean accessions could be clustered into 6 clusters, which consisted of 3, 24, 20, 31, 37, and 12 accessions, respectively. The most significant （P 〈 0.01） correlations existed respectively between the TAC and the total showed that the total substances phenolics content, and the TAC and the anthocyanin content of black soybean. The results phenolics and anthocyanin in black soybean seed coat were the important antioxidation

Study on multi-size seed-metering device for vertical plate soybean precision planter

Aiming to solve the problem of small range of the appropriately sowing seeds existing in a vertical disc seed-metering device,the planter plate series with four sizes were developed according to the variety and size distribution of all soybeans in China.The structure and working principle of the vertical disc soybean seed-metering device were detailed,and the influence of the diameter of soybean on the working performance of the seed-metering device was analyzed through the software EDEM virtual simulation,so as to achieve the goal of covering the soybean seeds with all sizes by the minimum planter plate series as well as to obtain the most appropriate operating speed of each planter plate by optimization.For the planter plates with the hole diameter of 7,9,12,16 mm,the appropriate size ranges of sowing seeds are 4.5-6.0,6.0-8.0,8.0-10.5,10.5-13.0 mm,respectively,and the appropriate operating speeds are 9,8,7,6 km/h,respectively.The results show that this planter plate series can meet the requirements of seeding with all sizes of soybeans at the range of the most appropriate operating speed.The study method can provide a reference for design and optimization of precision planters.

Design and key parameter optimization of an agitated soybean seed metering device with horizontal seed filling

Since the low seed filling speed of mechanical seed metering devices reduces the low qualified rate of seed spacing during high-speed practices,it is significant to design agitated seed metering devices with horizontal seed filling that are suitable for high-speed practices.The combination of horizontal seed filling and agitated seed filling can accelerate the seed filling of mechanical seed metering devices,and improve the qualified rate of seed spacing during high-speed practices.In this study,theoretical analysis,discrete element method-based simulation and indoor bench test verification were conducted to investigate how key parameters of the agitated seed metering device with horizontal seed filling(angles,installation position and number of agitating plates,diameters of convex spoons)would affect the characteristics of soybean seed movement,seed number and seeding performance(qualified index,multiple index,missing seeding index)under different working speeds.Computer-based simulation,test design and regression analysis were combined to analyze the population moving rules and optimize the design parameters of seed metering devices.Based on the test scheme as designed,simulations were conducted on Fluent EDEM,and the optimal angle of the agitating plates was determined by analyzing the population migrating rules.Regression equations were established through the regression of test results,and used to find out the optimal design parameters(diameter of convex spoon,positions and number of agitating plates)of seed metering devices.Then the optimal parameter combination among different working conditions was determined that the angle,position and number of agitating plates were 30°,24.4 mm,and 13,respectively,and the diameter of convex spoon was 11.0 mm.With the optimal parameter combination and at the seeding speed of 12 km/h,the qualified index,multiple index and missing seeding index were 93.1%,2.1%and 4.8%,respectively.Under high-speed practices,the new seed metering device was not significantly different from the pneumatic seed metering device,but significantly outperformed the mechanical seed metering device.

Identification of diseases for soybean seeds by computer vision applying BP neural network

The use of computer vision for estimating quality in agriculture products has become wide spread in recent years and the composition,variety,or ripeness can be estimated.On the other hand,the appearance is one of the most worrying issues for producers due to its influence on quality.In this research,computer vision technology combined with BP artificial neural network(ANN)was developed to identify soybean frogeye,mildewed soybean,worm-eaten soybean and damaged soybean.Thirty-nine characteristic parameters from color,texture and shape characteristics were computed after preprocessing the acquired soybean images.The dimensionality of the characteristic parameters was reduced from 39 dimensionalities to 12 dimensionalities using the method of principal component analysis(PCA).MALAB software was used to build a prediction model according to 12 characteristic parameters.The identification accuracies of soybean frogeye,mildewed soybean,damaged soybean and worm-eaten soybean are 96%,95%,92%,and 92%,respectively.And the accuracy for heterogeneous soybean seeds with several diseases is 90%.The results show that the prediction model constructed by BP neural network can identify the diseases of soybean seeds.And it is useful to estimate appearance quality of soybean by computer vision applying BP neural network.

Osmopriming-Regulated Changes of Plasma Membrane Composition and Function were Inhibited by Phenylarsine Oxide in Soybean Seeds

The aim of the present work was to investigate the effects of osmoconditioning on chilling injury in chilling-sensitive soybean （Glycine max （L.） Merr. Zhonghuang No. 22） seeds during imbibition. Low temperatures reduced the germination rate and no seed germinated at 1 ℃. Osmoconditioning of seeds at 20℃ with a polyethylene glycol-8000 （PEG8000） solution at 1.5 MPa for 72 h followed by drying back to their initial moisture content （MC） reduced their chilling sensitivity. The phenylarsine oxide （PAO）, an inhibitor of protein tyrosinephosphatases, was used to investigate the possible involvement of phosphorylation-dephosphorylation of Tyr residues in the plasma membrane composition and function when seeds were osmoconditioned. The results showed the germination of osmoconditioned seeds decreased significantly when PAO was added in PEG solution after chilling treatment. PAO inhibited changes in composition of plasma membrane phospholipids and fatty acid induced by osmocondition, indicated that tyrosine protein phosphorylation is involved in the regulatory mechanisms of osmocondition-responsive chilling in soybean seeds. Western blot result further indicated that osmocondition treatment improved the activity of plasma membrane H^＋-ATPase after chilling treatment, but this effect was abolished by PAO. The possible regulation mechanism by Tyr protein phosphorylation is discussed.

Protein Precipitation to Remove Carbohydrates that Interfere in Protein-Bound Tryptophan Quantification in Soybean Seeds

Tryptophan is one of the nine essential amino acids in humans that can only be obtained through diets and supplements.It is a precursor to many biological processes,such as serotonin,melatonin,kynurenin,and niacin(nicotinamide)vitamin synthesis.The content of tryptophan in foods,such as soybean is an important indicator of nutritional value.Therefore,accurate quantification of tryptophan in soybean is crucial to soybean nutritional improvement.Quantification of soybean protein-bound amino acids first involves acid hydrolysis of total protein to liberate amino acids.However,tryptophan quantification following acid hydrolysis is difficult or impossible due to its reactions with soybean carbohydrates.Therefore,removal of carbohydrates from soy proteins prior to acid hydrolysis is necessary.In this study,we compared four common protein precipitation methods(i.e.,methanol,acetonitrile,acetone,and trichloroacetic acid(TCA)protein precipitation methods)to determine the best method to separate soy proteins from carbohydrates,and concluded that acetone provided the highest recovery of soy proteins.Tryptophan content in the precipitated proteins was determined after acid hydrolysis of the proteins using liquid chromatography-tandem mass spectrometry multiple reaction monitoring(LC-MS/MS-MRM).No significant difference in the tryptophan content was found among proteins precipitated with methanol,acetonitrile,and TCA,suggesting that these precipitated proteins have similar compositions.A slightly lower,but statistically significant tryptophan content was found in the acetonitrile-precipitated proteins,suggesting that these proteins contain slightly higher glycosylated proteins.