Evaluation of extraction methodologies for corn kernel (Zea mays) DNA for detection of trace amounts of biotechnology-derived DNA

Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material requires pure, high-quality genomic DNA as template for subsequent amplification using the polymerase chain reaction (PCR). Six methodologies were evaluated for extracting DNA from ground corn kernels spiked with 0.1% (m/m) CBH351 (StarLink) corn. DNA preparations were evaluated for purity and fragment size. Extraction efficiency was determined. The alcohol dehydrogenase gene (adh1) and the CBH351 (cry9C, 35S promoter) genes in the genomic DNA were detected using PCR. DNA isolated by two of the methods proved unsuitable for performing PCR amplification. All other methods produced some DNA preparations that gave false negative PCR results. We observed that cornstarch, a primary component of corn kernels, was not an inhibitor of PCR, while acidic polysaccharides were. Our data suggest that amplification of an endogenous positive control gene, as an indicator for the absence of PCR inhibitors, is not always valid. This study points out aspects of DNA isolation that need to be considered when choosing a method for a particular plant/tissue type.     

Protein distribution in commercial wet- and dry-milled corn germ

To identify high-valued coproducts from commercially processed corn germ, it was necessary to determine the effect of processing conditions on corn germ proteins. We found that significantly less protein was extracted from commercial wet-milled as compared to dry-milled corn germ using Tris, sodium dodecyl sulfate (SDS) buffer containing 14 mM 2-mercaptoethanol at 100 degrees C for 10 min. SDS-polyacrylamide gel electrophoresis (PAGE) revealed a number of proteins with molecular masses ranging from approximately 10 to 66 kDa for the dry-milled corn germ as compared to only a few significant protein bands centered around 23 kDa in the wet-milled corn germ. The protein content of the wet- and dry-milled corn germ was approximately the same; however, nonprotein nitrogen values were significantly greater for the wet-milled than for the dry-milled germ. The distribution of fractionated germ protein freshly excised from the embryo of yellow dent corn kernels was more similar to that of dry-milled than wet-milled corn. SDS-PAGE of laboratory preparations of wet-milled corn germ more closely resembled commercial dry- than wet-milled corn germ, which could be attributed to limited microbial growth during steeping in the laboratory preparations.     

玉米高通量自动考种装置设计与试验

考种是玉米育种的重要环节,针对现有考种方法分别进行玉米果穗或玉米籽粒考种参数测量,难以满足玉米商业化育种过程大量样本自动考种需求的现状,该文设计了一种玉米高通量自动考种装置,主要包括玉米果穗考种单元、自动脱粒单元、玉米籽粒考种单元、后处理单元及系统控制单元,可实现玉米果穗考种、自动脱粒、籽粒考种、籽粒提升、籽粒封装、标签打印等全过程的自动化.依据设计方案进行原理样机试制及试验,结果表明:该装置可实现玉米穗质量、果穗长宽、穗行数、行粒数、秃尖区域、籽粒厚度、籽粒长、籽粒宽、穗粒数等考种参数的实时测量,且对果穗长、宽的平均测量精度分别为99.13％,99.25％、对籽粒数量的平均测量精度为99.39％.样机对单个玉米考种时耗时约为27.2 s,连续考种作业时处理速度达4穗/min.该研究为玉米高通量自动考种装备的实现提供了参考.     

Mycoflora and fumonisin contamination in Brazilian corn from sowing to harvest

The present study aimed to analyze the mycoflora and potential mycotoxin contamination of soil and corn samples collected at different plant maturity stages in Cap?o Bonito and Ribeir?o Preto, two regions of the State of S?o Paulo, Brazil. In addition, the data obtained were correlated with the occurrence of wind-dispersed fungi and the predominant climatic conditions of the two regions studied. Corn mycoflora profiles showed that Fusarium verticillioides prevailed in 35% of the samples from Cap?o Bonito and in 49% of the samples from Ribeir?o Preto. Examination of wind-dispersed fungi also revealed a high incidence of F. verticillioides. Soil mycoflora analyses showed that Penicilliumwas the most prevalent genus, although F. verticillioides was present in 55.5% of Cap?o Bonito's samples and in 26.7% of Ribeir?o Preto's samples. With respect to water activity, the corn kernels most contaminated with F. verticillioides had water activity levels of 0.70-0.80. HPLC analysis of fumonisins revealed that 88.5% of Cap?o Bonito's kernels were contaminated with fumonisin B(1) (FB(1)) (0.09-10.87 microg/g) and 53.8% with fumonisin B(2) (FB(2)) (0.05-0.52 microg/g); Ribeir?o Preto's kernels presented contamination levels of 93.5% for FB(1) (0.11-17.69 microg/g) and 61.3% for FB(2) (0.05-5.24 microg/g). No aflatoxins were detected by thin-layer chromatography in corn grains of either region. The concomitant occurrence of F. verticillioides and fumonisins in most of the field corn assayed demonstrates the importance of an effective control of cultivation throughout the plant maturity stages.     

Isoform patterns of chitinase and beta-1,3-glucanase in maturing corn kernels (Zea mays L.) associated with Aspergillus flavus milk stage infection

Isoform patterns of chitinase and beta-1,3-glucanase of maturing kernels of yellow dent corn (Pioneer 3394) infected with Aspergillus flavus at the milk stage were investigated through polyacrylamide gel electrophoresis (PAGE). Proteins on the sodium dodecyl sulfate (SDS) gel with an apparent molecular mass range of 23-46 kDa were differentially present in the kernels infected with both aflatoxin-producing and non-aflatoxin-producing strains of A. flavus. From in-gel (native PAGE) enzyme activity assays, three bands corresponding to chitinase isoforms and two bands corresponding to beta-1,3-glucanase isoforms were detected in the infected kernels. One chitinase isoform of 29 kDa was present only in the infected kernels, and another one of 28 kDa was present in both infected and noninfected kernels. They were judged to be acidic on the basis of their migration on an acrylamide isoelectric focusing (IEF) gel. For the beta-1,3-glucanase, one isoform of 35 kDa was present in both infected and noninfected kernels, but another one, a 33 kDa isoform, was present only in the infected kernels. Both acidic and basic beta-1,3-glucanase isoforms were detected in the IEF gel. The results of this study are the first to demonstrate patterns of enhanced or inducible proteins in maturing corn kernels in response to A. flavus infection at the milk stage. The results also indicate that only particular isoforms of the two hydrolytic enzymes are involved in the maturing corn kernels infected at the milk stage with A. flavus.     

Effect of Broken Corn Levels on Water Absorption and Steepwater Characteristics

Broken corn created by grounding sound corn kernels was added back at levels of 0, 4, 8, 12, or 16%, by weight, to whole kernels of three corresponding hybrids: FR27 × FRMo17 (a soft endosperm corn), FR618 × FR600 (amedium‐hard endosperm corn), and FR618 × LH123 (a hard endosperm corn). The samples had been dried from 28% moisture content to 15% moisture content either by using ambient air at ≈25°C or at 110°C. Samples were steeped for 36 hr at 52°C in 0.15% sulfur dioxide and 0.5% lactic acid steeping solution. The steepwater characteristics, such as water absorption, solids and protein content in the steepwater, and steepwater pH, were measured by periodic sampling and analyzed. Broken corn level has a significant effect on the amount of solids released during steeping and steepwater protein content for all samples. Both steepwater solids and protein content increased linearly as broken corn content increased. Corn drying temperature, kernel hardness, and interactions between drying temperature and kernel hardness has a significant effect on steepwater solids and protein content and steepwater pH in both broken and unbroken corn. Corn dried at low temperature released more soluble solids and protein into the steepwater than corn dried at high temperature. Soft endosperm and medium‐hard endosperm corn released more soluble solids and protein into the steepwater than hard endosperm corn. Soft endosperm corn resulted in a higher steepwater pH than medium‐hard and hard endosperm corn. No significant effect of broken corn content on final moisture content of steeped corn and steepwater pH was observed.     

Die Persistenz von Chlorcholinchlorid in Weizenpflanzen während der generativen Wachstumsphase und in lagernden Weizenkörnern

The persistance of chlorocholine chloride in wheat plants during the reproductive stage and in the kernels during storage In pot experiments with spring wheat the metabolization of the growth regulator chlorocholine chloride (CCC) has been studied during the reproductive stage. Also the persistance of ¹⁴C labelled CCC in wheat kernels has been examined during a period of one year. The following results were obtained:

• 1 The mobility of CCC in the plant was very low. Even when CCC was applied at a late stage (beginning of ear emergence), 98 to 99% of the applied ¹⁴C activity remained in the shoots and only 1 to 2% were translocated towards the ears. Also when CCC was twice applied the CCC content in the ears amounted only to 1 to 2 ppm.
• 2 Chlorocholine chloride was very stable in the plants and only a small percentage was converted to choline. Thus by far the main proportion of the applied ¹⁴C activity was recovered in the form of CCC and only 2 to 5% were found in the choline fraction. ¹⁴C activity in the other chemical fractions of the plant material was extremely low or zero.
• 3 In the choline fraction of the kernels the ¹⁴C activity amounted to 12% of the total ¹⁴C activity and thus was twice as high as in the straw. This relatively high 14C label in the choline fraction could be related to metabolic processes typical for grain growth. It is also possible that choline synthesized in the leaves is more easily translocated towards the kernels than chlorocholine chloride.
• 4 The mature kernels stored at room temperature did not show any metabolization of CCC during a period of one year. Neither the total ¹⁴C activity nor the content of CCC were altered significantly during this time.

     

Yield and Phytosterol Composition of Oil Extracted from Grain Sorghum and Its Wet‐Milled Fractions

Corn fiber contains an oil with high levels of three potential cholesterol‐lowering phytosterol compounds. Little information is available about the levels and types of phytosterols in sorghum. In this study, phytosterols were evaluated in grain sorhgum and its wet‐milled fractions and were compared with the phytosterols in corn. The study showed that sorghum kernels can provide a significant source of two phytosterol classes, free phytosterols (St) and fatty acyl phytosterol esters (St:E). Most of these phytosterols are concentrated in the wet‐milled fiber fraction followed by the germ fraction. In addition to phytosterols, other lipid classes such as wax esters and an aldehyde (50% C28 and 50% C30) are also present in the sorghum oil. Comparison of sorghum and corn kernels show that corn has 72–93% more phytosterols than sorghum.     

Iron deficiency on corn root mitochondrial adenosine triphosphatase activity

Abstract

Iron deficiency significantly reduced corn root mitochondrial ATPase (adenosine triphosphatase) activity. The root mitochondrial ATPase had two pH optima. In the presence of K and Mg ions, ferrous ion stimulated the ATPase activity especially when plants were Fe‐deficient.     

Corn Kernel Structural Integrity: Analysis Using Solvent and Heat Treatments

Most corn (Zea mays, L.) processing is accomplished by causing a structural change to the kernel. Associations between corn endosperm structural components were characterized using textural analysis after solvent and heat treating kernels. Intact Asgrow 405W and B73xMo17 kernels were incubated and treated at 20, 40, 55, and 90°C for 1, 24, and 48 hr in static air, in acetone, and in aqueous solutions of water, calcium chloride, sodium chloride, sodium bisulfite, lactic acid, lime, lye, ethanol urea, and sodium dodecyl sulfate (SDS). After treatment, kernels were compressed between flat platens. Acetone did not significantly soften endosperm structure. Ethanol reduced kernel fracturability by weakening cell‐to‐cell (wall) bonds, but ethanol did not effectively reduce kernel hardness. Water and aqueous solvents swelled and softened kernels by plasticizing structural components. Bisulfite and SDS softened kernels more than water only soaks because they denatured matrix proteins. Alkaline soaks reduced fracturability and softened the kernel by dissociating both cell‐to‐cell and intracellular (starch‐protein) bonds. Soaking for longer periods and at higher temperatures increased aqueous‐based solvent softening effect. Urea imbibition into the kernel and its softening effects were highly dependent on time and temperature of soak. Endosperm structural integrity is the governed by a combination of cell‐to‐cell bonds and intra‐cellular (starch‐protein) bonds. Reagents that denatured the endosperm matrix proteins and disrupted hydrogen bonds resulted in the greatest alterations to kernel structural integrity. Ultimately a better understanding of kernel structural integrity will lead to the development of improved hybrids and process technologies designed to facilitate desirable structural changes.     

Starch characterization and ethanol production of sorghum

This study aimed to characterize and compare the chemical structures, physical properties, and enzymatic hydrolysis rates of five sorghum starches (6B73, 6C21, 6C69, 7R34, and X789) with that of corn starch (B73). Sorghum kernels consisted of 68.7-70.6% starch, more than the B73 corn (67.4%). Sorghum starches displayed higher gelatinization temperatures (66.6-67.4 °C), greater gelatinization enthalpy changes (13.0-14.0 J/g), and greater percentages of retrogradation (60.7-69.1%), but slower enzymatic hydrolysis rates (83.8-87.8% at 48 h) than the B73 corn starch (61.7 °C, 10.1 J/g, 51.5%, and 88.5%, respectively). These differences could result from the sorghum amylopectins consisting of fewer short branch chains (DP 6-12) (12.8-14.0%) than the corn amylopectin (15.0%). The sorghum starches showed greater peak and breakdown viscosities but lower setback viscosities than the B73 corn starch, resulting from the lower amylose content of the sorghum starches. After 96 h of fermentation, most ground sorghums exhibited lower ethanol yields (30.5-31.8%) than the ground B73 corn (31.8%).     

Liquid chromatographic determination and stability of the Fusarium mycotoxin moniliformin in cereal grains

Moniliformin is a mycotoxin produced by Fusarium subglutinans and other Fusarium species. A rapid, liquid chromatographic method for its determination in corn and wheat is described. Samples are extracted in acetonitrile-water (95 + 5); following defatting with n-hexane, an aliquot of the extract is evaporated and cleaned up on small C18 and neutral alumina columns successively. Reverse-phase liquid chromatography (LC) is conducted on a C18 column with 10 or 15% methanol or acetonitrile in aqueous ion-pair reagent as mobile phase, with detection by ultraviolet absorption at 229 and 254 nm. Average recoveries of moniliformin (potassium salt) added to ground corn and wheat at levels of 0.05-1.0 micrograms/g were 80% (n = 20) and 85% (n = 12), respectively, and the limit of detection was ca 0.01-0.18 micrograms/g, depending on LC conditions. Analysis of 24 samples of wheat, 4 samples of rye, and 12 samples of corn showed moniliformin in only 2 corn samples (0.06 and 0.2 micrograms/g). Moniliformin was also detected in a sample of artificially damaged (slashed) corn (0.2 micrograms/g) and selected kernels of corn that were field-inoculated with F. subglutinans and F. moniliforme (50 micrograms/g and 0.5 micrograms/g, respectively). In stability studies, moniliformin (potassium salt, 1 microgram/g) in ground corn and ground wheat heated at 50, 100, and 150 degrees C for 0.5-2 h decomposed moderately, e.g., 55% remained in corn after 0.5 h at 100 degrees C.     

Desaturation of myristoyl-CoA to myristoleoyl-CoA by hen liver microsomal delta(9)-desaturase

The desaturation of myristoyl-CoA to myristoleoyl-CoA was measured in microsomal preparations of hen liver. The desaturation was maximal at pH 7.4. The enzymatic activity was linear with time up to 10 min and proportional to microsomal protein concentrations. The initial velocity was linear with substrate concentrations between 13 and up to 200 microM. A decrease in desaturation activity was observed at substrate concentrations greater than 266 microM. There was an absolute requirement for reduced pyridine nucleotide (NADH), while a maximum activity was observed at a myristoyl-CoA:NADH mole ratio of 1. Competitive inhibition studies of myristoyl-CoA desaturation suggest that the inhibitors, stearyl and oleyl-CoA, were more effective than palmitoyl-CoA. Free CoA did not inhibit the delta(9)-desaturase system. The desaturation of myristoyl-CoA was stimulated by bovine serum albumin and reduced by cytoplasmic proteins. The effect of cytoplasmic proteins on the enzymatic reaction was completely abolished by trypsin digestion and boiling for 30 min. On the basis of these data, it was concluded that 9,10-desaturation of acyl-CoA derivatives containing 14-18 carbon fatty acyl chains is catalyzed by the same enzyme.     

基于分级阈值和多级筛分的玉米果穗穗粒分割方法

为了有效克服果穗形状畸变和穗粒颜色差异对穗粒分割的影响,该文提出一种准确、鲁棒的玉米果穗穗粒分割方法。该方法利用果穗三维形状特征校正果穗径向畸变以最大程度恢复图像上果穗表面信息;采用分级阈值分割策略确定每颗穗粒最佳阈值范围,并利用穗粒几何特征实现穗粒初次筛分,消除穗粒间粘连效应;结合主成份分析和支持向量模型完成穗粒的二次筛分,生成果穗表面穗粒分布图。该方法整合了果穗径向畸变-分级阈值-穗粒多级筛分,实现果穗穗粒的精准分割,为玉米果穗自动化考种提供了基础方法。试验结果表明提出方法在穗粒分割准确性和鲁棒性上具有显著优势,平均计算效率达15 s/果穗。     

基于穗粒分布图的玉米果穗表型性状参数计算方法

玉米果穗表型性状是玉米育种、产量预测的重要参数,提出一种基于穗粒分布图的玉米果穗性状计算方法,全面解析玉米果穗和穗粒的几何、数量和颜色等表型性状。该文利用步进电机驱动果穗转动来获取果穗主要侧面图像,采用果穗畸变校正方法生成标准果穗图像序列,在像素尺度进行果穗轮廓分析,建立图像序列中果穗轮廓映射关系并生成果穗三维模型,在穗粒尺度拼接果穗整个表面的穗粒分布图,计算出果穗和穗粒的各项表型性状。试验结果表明,提出的表型性状计算方法对穗型及穗粒分布规则的玉米果穗具有较高检测精度,其中穗行数、行粒数、总粒数、果穗长和果穗粗的平均计算精度分别为98.231%、94.351%、96.921%、98.956%和98.165%。     

基于多相机成像的玉米果穗考种参数高通量自动提取方法

实现玉米果穗考种性状的准确、快速获取是提高玉米育种效率的关键环节。该文在前期设计的玉米高通量自动化考种装置基础上,提出了一种基于多相机的玉米果穗考种参数提取方法,通过4个等间隔均匀分布的摄像头同时获取果穗4个方向图像,针对每副图像分别经过背景去除、投影模型构建、籽粒跟踪、考种参数提取等处理,最后根据4副图像的处理结果,综合计算穗长、穗粗、平均粒厚、穗行数、行粒数、穗粒数等考种参数。在玉米高通量自动化考种装置的果穗考种模块上进行试验,结果表明,该文所提方法测得的穗长、穗粗、平均粒厚与人工方法测量值之间的决定系数R2分别为0.997 3、0.984和0.941 5,对穗行数、行粒数的测量精度分别为98.63%、95.35%,为玉米果穗考种参数提取提供了一种新思路,为高通量自动考种装置的实现奠定了基础。     

玉米果穗离散元模型构建与脱粒仿真验证

针对玉米脱粒离散元仿真中果穗模型难以表征籽粒分离和芯轴破碎的问题，该研究构建了玉米果穗聚合体离散元模型并进行脱粒仿真验证。基于玉米芯轴3层结构采用分层建模与网格划分方法建立玉米芯轴离散元模型，结合Plackett-Burman试验、最陡爬坡试验、Box-Behnken试验和仿真弯曲试验标定粘结参数；以马齿型玉米籽粒为原型，采用五球粘结的籽粒-芯轴连接方式建立玉米果穗聚合体离散元模型，仿真标定籽粒与芯轴的连接力；最后模拟梯形杆齿、圆头钉齿和纹杆块3种脱粒分离机构的玉米脱粒进程。结果表明：玉米芯轴弯曲破坏力和弯曲刚度仿真结果与实测平均值的相对误差分别为-0.12%和-0.14%，籽粒果柄轴向压缩力和径向压缩力仿真结果与实测平均值的偏差分别为-1.8和2.46 N，3种脱粒分离机构脱粒段仿真区域内籽粒平均法向接触力依次为12.50、12.32和8.03 N，3种脱粒元件对籽粒平均法向接触力的递减趋势与台架试验的籽粒破碎率变化一致，根据籽粒与脱粒元件接触合力的累积频率曲线确定籽粒破碎率的临界接触合力为550 N，仿真未脱净率依次为0.15%、0.37%、0.35%，较台架试验结果分别偏小0.07、偏高0.04和偏小0.25个百分点，沿滚筒轴向籽粒质量分布百分比曲线均表现为正偏态单峰分布，脱粒仿真试验的曲线峰值分别比台架试验高1.03、1.86和0.85个百分点，两者脱粒质量相近。该玉米果穗聚合体离散元模型参数标定准确，能够准确反映籽粒和芯轴的力学特性差异，可还原玉米脱粒分离过程，为后续脱粒分离机构的优化提供参考依据。     

Hydration Rates for Various Types of Mexican Maize Based on Single‐Kernel Measurements

Hydration kinetics for sound maize kernels in liquid water, determined by single‐kernel measurements for three different Mexican maize types, yielded water diffusion coefficients ordered as Celaya corn > Toluca corn > Palomero corn, at all temperatures examined. These diffusion coefficients are lower than those reported earlier for maize grains, possibly due to the fact that in the present study damaged kernels were rigorously excluded. The energies of activation determined from the Arrhenius plots were ordered as Palomero corn > Celaya corn = Toluca corn and were similar in value to those reported earlier for other maize types. Damage to the surface of the maize kernels during the hydration experiments occurs at a significant frequency. Even minor surface lacerations can strongly affect the rate of hydration of the kernels. Experiments with maize grains selectively varnished in various parts of their surface show that the entry of water into the kernels occurs predominantly through the pericarp, not through the tip cap, though the tip cap has a higher water inflow per unit area.     

Transient infrared spectroscopy for detection of toxigenic fungi in corn: potential for on-line evaluation

An urgent need for rapid sensors to detect contamination of food grains by toxigenic fungi such as Aspergillus flavus prompted research and development of Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) as a highly sensitive probe for fungi growing on the surfaces of individual corn kernels. However, the photoacoustic technique has limited potential for screening bulk corn because currently available photoacoustic detectors can accommodate only a single intact kernel at a time. Transient infrared spectroscopy (TIRS), on the other hand, is a promising new technique that can acquire analytically useful infrared spectra from a moving mass of solid materials. Therefore, the potential of TIRS for on-line, noncontact detection of A. flavus contamination in a moving bed of corn kernels was explored. Early test results based on visual inspection of TIRS spectral differences predict an 85% or 95% success rate in distinguishing healthy corn from grain infected with A. flavus. Four unique infrared spectral features which identified infected corn in FTIR-PAS were also found to be diagnostic in TIRS. Although the technology is still in its infancy, the preliminary results indicate that TIRS is a potentially effective screening method for bulk quantities of corn grain.     

Fate of fumonisin B(1) in corn kernel steeping water containing SO(2)

Six 100 ppm fumonisin B(1) (FB(1)) solutions were prepared by dissolving pure standard in six different solvents containing SO(2). Two of the solvents contained 0.2 or 0.4% SO(2) in distilled water. The other four solvents were obtained by steeping corn kernels at 60 degrees C in a 0.2% SO(2) aqueous solution for 6, 12, 24, or 48 h. After the addition of FB(1), all solutions were maintained at 60 degrees C for 7 days. Fumonisin B(1) content in each solution was determined in triplicate by HPLC. Steeping corn kernels in 0.2% solution at 60 degrees C for 6 h seems to be the most effective treatment to decrease the amount of FB(1).