我们要感谢由Worrell水技术有限责任公司提供的财政支持。商标或专利产品的提及仅是为了进行识别，并不意味着由农业部美国农业部保证或产品的保证。

1.在封闭腔室的顶部空间气体二氧化氯的测定<ol><li>获得的材料:二氧化氯袋（0.5克在选择用于其释放速率的聚合物膜CLO 2浆料（9.5％AI）的（6厘米2）的总表面面积;确切的组分是专有的），玻璃室（19.14 L），并具有可切换气体入口和出口的盖子。 </li><li>附加二氧化氯袋使用双面胶带盖子。 </li><li>通过密封用凡士林盖关闭所述腔室。 </li><li>连接CLO 2气体检测到所述腔室的入口和出口。 注意:这是一个气体循环系统，并在测量时没有发生气体损失。 </li><li>打开的入口和出口气体流量和测量所述室中的二氧化氯浓度为孵育后0，1，2，3，4，24，26，28，和48小时。 </li><li>监测温度和相对湿度（RH）在与回火腔室ATURE和RH数据记录仪。 </li></ol> 2.水果制品和存储<ol><li>从本地零售商获得15千克的新鲜葡萄番茄（ 番茄变种cerasiforme）。确保水果是健康的，没有视觉上的缺陷。 </li><li> 接种的准备 <ol><li>使用从柑橘类水果表面27 大肠杆菌 （野生型）和链格孢的菌株接种。 </li><li>在35℃下对大肠杆菌琼脂（ECA）培养大肠杆菌中27 1天，然后再培养于1天一个新板的生物体。通过与BAC-环采样ECA板，条纹上莱文细菌曙红亚甲基蓝（EMB）琼脂，并在35℃温育24小时，确认生物体;该转反射，金属绿培养是阳性的大肠杆菌 。 </li><li>在马铃薯葡萄糖琼脂培养链格孢 （PDA），在25℃下直至孢子出现。 </li><li>刮除从琼脂板中的大肠杆菌细胞在50mL的无菌蒸馏水直到估计浓度达到9日志CFU / mL的使用具有麦克法兰等价浊度标准的比较。加入1950毫升的含有0.1％无菌水吐温20，使2 L的总的最终接种物。 </li><li>验证通过在埃克·阿加平板稀释电镀的细胞浓度。从培养基中刮去链格孢孢子，并将其暂停到2L含有0.1％Tween-20的无菌蒸馏水中。 注:最终大肠杆菌人口为7.5日志CFU / g，并且所述链格孢人口为5.5日志CFU / g以下。 </li></ol></li><li> 放置7千克西红柿的入10升的不锈钢制锅，其是完全由耐高压加热的袋覆盖。放置在一个安全罩袋和锅。使用从顶部施加，同时轻轻搅拌该扳机喷雾器应用接种物溶液（2L）到水果水果用戴着手套的手。 <ol><li> 5分钟后，将西红柿上灭菌片材的单个层，并允许他们在空气中干燥2小时。把约200g每个水果的成twent四个1磅（1.14〜L）穿孔的蛤壳。 </li></ol></li><li>小心地折污染箔和它们放入钢锅。取下手套，把它们放进锅里。包裹袋高压灭菌和高压釜中于121℃下所有污染的用品25分钟。 </li><li>附着二氧化氯袋至12个蛤壳盖。使用其他12个蛤壳作为对照。称量每个整个翻盖。保存在20°C的水果14天。 </li><li>采取样品第3天，7，10，和14样品3个蛤壳，代表3次重复，每人每天的治疗。 </li></ol>在蛤壳二氧化氯浓度3.监测<ol><li>插入二氧化氯的气体检测器的入口和出口管导入蛤壳的中心，无线TH两个端部之间的2厘米的距离，并采取天3，7，10，和14中的二氧化氯的测量。 </li></ol> 4.确定微生物种群和果实品质属性的<ol><li> 从各搅拌5种水果（约60g）复制在100rpm在无菌取样袋99毫升无菌磷酸钾缓冲液的沿轨道振荡器上1个小时（0.01M，pH 7.2）中。 <ol><li>的缓冲液洗涤板连续稀释液（1-，10-，和100-倍），每次50微升，在ECA（用于大肠杆菌 ），使用螺旋接种仪和PDA（对于链格孢 ）。 </li><li>孵育在35℃下24个小时的ECA板和PDA平板在25℃下3天。读取使用的光学板阅读器中微生物菌落计数。清理所有设备在使用后接触到受污染的水果。 </li></ol></li><li>测量使用制造商的协议的果实硬度测试仪果实硬度。校准测试仪前每次使用。测量水果20为每个复制和表达的结果作为压力力，牛顿（N），由1毫米到压缩果需要（换算成N·米- 1）。 </li><li>称取与水果整个蛤壳式的开始处和在储存过程中和计算相比于初始重量的重量损失。 </li></ol> 5.统计分析<ol><li>复制所有实验一式三份。分析使用方差分析（ANOVA）分析数据。确定由Duncan多范围检验的均值分离;的意义是在p &lt;0.05所定义。 </li></ol>

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M., Ragaert, P., Jeyachchandran, V., Debevere, J., Devlieghere, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shelf-life+of+minimally+processed+lettuce+and+cabbage+treated+with+gaseous+chlorine+dioxide+and+cysteine.">Shelf-life of minimally processed lettuce and cabbage treated with gaseous chlorine dioxide and cysteine.</a> Int J Food Microbiol. 121 (1), 74-83 (2008).</li><li>Mahovic, M. J., Tenney, J. D., Bartz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+chlorine+dioxide+gas+for+control+of+bacterial+soft+rot+in+tomatoes.">Applications of chlorine dioxide gas for control of bacterial soft rot in tomatoes.</a> Plant Dis. 91 (10), 1316-1320 (2007).</li><li>Tan, H. K., Wheeler, W. B., Wei, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+of+chlorine+dioxide+with+amino-acids+and+peptides+-+kinetics+and+mutagenicity+studies.">Reaction of chlorine dioxide with amino-acids and peptides - kinetics and mutagenicity studies.</a> Mutat Res. 188 (4), 259-266 (1987).</li></ol>

The authors declare that they have no competing financial interests.

二氧化氯是防止食品腐烂的理想杀菌剂。然而，在高浓度和非运输的，需要昂贵的发电机或低效的两部分粉末混合不稳定。本研究二氧化氯的稳定，随时可以使用形式的应用，以减少食品腐败和食源性疾病的发生率。相较于目前使用的其他二氧化氯应用技术，这里使用的商业二氧化氯具有成本效益，具有保质期长，且不需要大型发电机或预混。然而，由于二氧化氯的强氧化性，二...

丰富的新鲜水果和蔬菜的饮食可能有助于减少许多疾病，包括冠状动脉心脏疾病和特定类型的癌症1的风险。不过，也有一些食源性致病微生物，如大肠杆菌 ， 沙门氏菌和李斯特菌 ，新鲜水果和蔬菜，可能会导致消费者谁食用被污染的农产品2中患病甚至死亡的消费有关。例如， 大肠杆菌（E.coli）O157:H7爆发已经与葡萄，番茄，草莓和3,4相关联，并且甲型肝炎爆发已与新鲜蓝莓5相关联。此外，微生物污染可导致通过采后腐烂6实质的产品损失。 交链孢是一种重要的植物病原真菌ŧ<html>帽是已知会导致叶斑和其他疾病中的植物7的超过380宿主物种。它已被证明是一种黑斑病 8，茎溃疡病，西红柿9的叶枯病的原因。因此，安全有效的采收后处理净化是需要两个控制食源性致病菌并防止采后腐烂在新鲜的农产品。 低收入和无渣技术是替代消毒剂的新趋势。各种采后杀菌剂已被用于减少腐败菌，防止食源性疾病。臭氧，强大的抗微生物剂，已显示出保留草莓和蓝莓10，11的质量和新鲜度。然而，臭氧可以引起水果表面组织的氧化，可导致变色和风味质量的恶化S = "外部参照"&gt; 12。氯已被用于消毒新鲜农产品，如蓝莓和苹果13。虽然有效，氯可以与含氮化合物或氨反应，导致致癌的副产物14，用于新鲜水果15中的消毒时尤其如此。 二氧化氯（二氧化氯 ），洗手液的替代物，正在为水果和蔬菜16的采后处理批准中国和美国。二氧化氯是一种水溶性氧化剂与氧化能力比的游离氯17的大2.5倍。二氧化氯是在低浓度和具有短接触时间18非常有效。二氧化氯具有用于消毒浓度低毒性和最小的腐蚀性，并且它被认为是最有效的杀菌之一并在各种设置19，20，21使用杀菌剂。 众多的研究结果已经表明，二氧化氯可以控制食源性致病菌和采后腐烂16。例如，二氧化氯的气体已被用于灭活单增李斯特菌 ， 沙门氏菌和大肠杆菌 O157:H7和防止蓝莓，草莓变质22,23。二氧化氯的气体减少了微生物污染的风险，同时保持新鲜水果的属性，并且它是在控制草莓24的采后腐烂有效。然而，在高浓度和不可传输不稳定的，需要在历史上的网站或低效的两部分粉末混合昂贵发电机。 然而，一个新的CLO2产物与现成的，控制释放制剂（ 即，它不要求一个发生器或成分的预混合）已被证明是在控制的预备试验25食品腐败生物和病原体非常有效。它是一种安全，经济高效，无腐蚀性，易于运输和控释二氧化氯的形式，对环境无不良影响。先前的实验已经证明，该缓释CLO 2粉末包裹在过滤材料，并放置在吸塑包装显著减少的新鲜蓝莓和草莓的衰退，减少浆果失水，和采后存储器25，26期间维持果实硬度。最近，一种控释二氧化氯包通过在半透性聚合物膜密封二氧化氯的浆液形式开发的。这项工作的目标是:于:1）监测在两个封闭的容器中二氧化氯的气体释放性和在穿孔的蛤壳，2）调查控制释放二氧化氯袋封闭在上食源性致病菌的容器和葡萄西红柿的衰减的效果，以及3）评价对葡萄西红柿的质量存储控释二氧化氯的影响。

<table><tbody><tr><td>Curoxin&lt;sup&gt;&amp;#174;&lt;/sup&gt; chlorine dioxide pouch</td><td>Worrell Water Technologies</td><td></td><td>Slurry, a.i. 9.5% in sealed semi-permeable polymer film</td></tr><tr><td>Grape tomato</td><td>Santa Sweets, Inc</td><td></td><td>Santa Sweets Authentic&amp;nbsp;</td></tr><tr><td>ClO&lt;sub&gt;2&lt;/sub&gt; gas detector</td><td>Analytical Technology, Inc., Collegeville, PA</td><td></td><td>PortaSens II&amp;nbsp;</td></tr><tr><td>Perforated clamshell</td><td>Packaging Plus LLC, Yakima, WA</td><td></td><td>OSU #1, 1 lb</td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt;&amp;nbsp;</td><td></td><td></td><td>Wild Type (WT) from fruit surface</td></tr><tr><td>Alternaria alternata</td><td></td><td></td><td>from fruit surface</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; agar&amp;nbsp;</td><td>EC Broth, Oxoid, UK</td><td></td><td>EC Broth with 1.5% agar</td></tr><tr><td>Potato dextrose agar&amp;nbsp;</td><td>BD Difco, Sparks, MD</td><td></td><td></td></tr><tr><td>Levine eosin methylene blue agar</td><td>BD Difco, Sparks, MD</td><td></td><td></td></tr><tr><td>Trigger spray bottle&amp;nbsp;</td><td>Impact Products, LLC., Toledo, OH</td><td></td><td></td></tr><tr><td>Sterilized sampling bag&amp;nbsp;</td><td>Fisherbrand, Fisher Scientific, Pittsburgh, PA</td><td></td><td></td></tr><tr><td>Orbit shaker&amp;nbsp;</td><td>New Brunswick Scientific, New Brunswick, NJ</td><td></td><td>Innova 2100</td></tr><tr><td>IUL Instruments Neutec Eddy jet spiral plater inoculation plating system</td><td>Neutec Group Inc., Farmingdale, NY</td><td></td><td></td></tr><tr><td>EZ micro optical plate reader&amp;nbsp;</td><td>Synoptics, Ltd., Cambridge, UK</td><td></td><td>ProtoCOL</td></tr><tr><td>Fruit firmness tester&amp;nbsp;</td><td>Bioworks Inc, Wamego, KS</td><td></td><td>FirmTech 2&amp;nbsp;</td></tr><tr><td>Tinytag temperature and RH data logger</td><td>Gemini Data Loggers, West Sussex, UK</td><td></td><td></td></tr><tr><td>McFarland equivalence turbidity standard</td><td>Fisherbrand, Fisher Scientific, Pittsburgh, PA</td><td></td><td></td></tr></tbody></table>

controlled-release of chlorine dioxide in a perforated packaging system to extend the storage life and improve the safety of grape tomatoes

的控释二氧化氯（CLO 2）袋是由密封二氧化氯的浆液形式成半透聚合物膜显影;袋的释放特性在有或无果的容器进行了监测。将袋固定到含有葡萄西红柿穿孔蛤壳的内部，并在微生物群体，坚固性的效果，并在在20℃下14天的储存期进行评价的重量损失。在3天后，将二氧化氯浓度在蛤壳达到为3.5ppm和保持不变，直到第10天。此后，通过3.08日志CFU降低至2ppm白天14.二氧化氯袋显示出强大的抗微生物活性，从而减少大肠杆菌群体/克和链格孢种群由2.85日志CFU /克后存储14天。的二氧化氯处理也降低软化和体重减轻和延长了的西红柿的整体的货架寿命。我们的研究结果表明，二氧化氯的治疗是用于延长保质期和储存过程中改善的西红柿的微生物安全性而不损害它们的质量是有用的。

二氧化氯的释放显示出在最初的几个小时的线性图案。浓度在第一4小时增加约2.38 PPM /小时。温育24个小时后的释放速度减慢，并且二氧化氯浓度达到25.4 ppm的。然而，浓度趋向于孵育的24个小时（ 图1）后保持稳定。 顶部空间二氧化氯浓度与葡萄西红柿蛤壳为3天和第10天之间约4ppm，它储存?...

Watch this Scientific Journal Video about Controlled-release of Chlorine Dioxide in a Perforated Packaging System to Extend the Storage Life and Improve the Safety of Grape Tomatoes at JoVE.com

Controlled-release of Chlorine Dioxide in a Perforated Packaging System to Extend the Storage Life and Improve the Safety of Grape Tomatoes

Here, we describe a protocol for the application of a novel, slow-release ClO2 product that reduces spoilage and extends the shelf life of fresh fruit. The slow-release ClO2 product was added to standard commercial grape tomato packaging and tested against Escherichia coli and Alternaria alternata.

在穿孔的包装系统二氧化氯的控释，延长贮藏寿命，提高葡萄番茄的安全

A controlled-release chlorine dioxide (ClO2) pouch was developed by sealing a slurry form of ClO2 into semipermeable polymer film; the release properties ...

controlled-release-chlorine-dioxide-perforated-packaging-system-to

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

10.9K 次观看.  USDA, ARS, USHRL. Here, we describe a protocol for the application of a novel, slow-release ClO2 product that reduces spoilage and extends the shelf life of fresh fruit. The slow-release ClO2 product was added to standard commercial grape tomato packaging and tested against Escherichia coli and Alternaria alternata.

视频: Controlled-release of Chlorine Dioxide in a Perforated Packaging System to Extend the Storage Life and Improve the Safety of Grape Tomatoes

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

利用乙烯释放化合物，2-氯乙基膦酸，作为一种工具来研究细菌乙烯反应

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

科研

生物化学

The Portable Chemical Sterilizer (PCS), D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary Service Support Detachments, Combat Support Hospitals, and Area Medical Laboratories to sterilize surgical instruments and to sterilize pathological specimens prior to disposal in operating rooms, emergency treatment areas, and intensive care units. The following ensemble of novel, &#8216;clean and green&#8217; chlorine dioxide technologies are versatile and flexible to adapt to meet a number of critical military needs for decontamination6,15. Specifically, the Portable Chemical Sterilizer (PCS) was invented to meet urgent battlefield needs and close critical capability gaps for energy-independence, lightweight portability, rapid mobility, and rugged durability in high intensity forward deployments3. As a revolutionary technological breakthrough in surgical sterilization technology, the PCS is a Modern Field Autoclave that relies on on-site, point-of-use, at-will generation of chlorine dioxide instead of steam. Two (2) PCS units sterilize 4 surgical trays in 1 hr, which is the equivalent throughput of one large steam autoclave (nicknamed &#8220;Bertha&#8221; in deployments because of its cumbersome size, bulky dimensions, and weight). However, the PCS operates using 100% less electricity (0 vs. 9 kW) and 98% less water (10 vs. 640 oz.), significantly reduces weight by 95% (20 vs. 450 lbs, a 4-man lift) and cube by 96% (2.1 vs. 60.2 ft3), and virtually eliminates the difficult challenges in forward deployments of repairs and maintaining reliable operation, lifting and transporting, and electrical power required for steam autoclaves.

The Portable Chemical Sterilizer PCS, D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

The Portable Chemical Sterilizer (PCS) is a revolutionary, energy-independent, almost waterless sterilization technology for Army medical units. The PCS generates chlorine dioxide from dry reagents mixed with water on-site, at-will, and at point-of-use (PoU) in a plastic suitcase. The Disinfectant-sprayer for Foods and ENvironmentally-friendly Sanitation (D-FENS) and the Disinfectant for ENvironmentally-friendly Decontamination, All-purpose (D-FEND ALL) produce aqueous chlorine dioxide in a collapsible spray bottle and other potential embodiments. These versatile decontamination technologies kill microbes in myriad diverse Dual-use applications for military and civilian consumers.

便携式化学消毒（PCS），D-沼泽，和D-FEND ALL：新型二氧化氯去污技术用于军事

There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary ...

生物工程

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

一氧化氮气体防治害虫实验室熏蒸程序

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

环境科学

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

培养和维护<em&gt;难辨梭状芽孢杆菌</em&gt;在厌氧环境

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

免疫与感染

An Efficient Clearing Protocol for the Study of Seed Development in Tomato (Solanum lycopersicum L.)

Tomato (Solanum lycopersicum L.) is one of the major cash crops worldwide. The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. Visualization of finer embryonic structure within a tomato seed is often hampered by seed coat mucilage, multi-cell-layered integument, and a thick-walled endosperm, which needs to be resolved by laborious embedding-sectioning. A simpler alternative is to employ tissue clearing techniques that turn the seed almost transparent using chemical agents. Although conventional clearing procedures allow deep insight into smaller seeds with a thinner seed coat, clearing tomato seeds continues to be technically challenging, especially in the late developmental stages.
Presented here is a rapid and labor-saving clearing protocol to observe tomato seed development from 3 to 23 days after flowering when embryonic morphology is nearly complete. This method combines chloral hydrate-based clearing solution widely used in Arabidopsis with other modifications, including the omission of Formalin-Aceto-Alcohol (FAA) fixation, the addition of sodium hypochlorite treatment of seeds, removal of the softened seed coat mucilage, and washing and vacuum treatment. This method can be applied for efficient clearing of tomato seeds at different developmental stages and is useful in full monitoring of the developmental process of mutant seeds with good spatial resolution. This clearing protocol may also be applied to deep imaging of other commercially important species in the Solanaceae.

An Efficient Clearing Protocol for the Study of Seed Development in Tomato Solanum lycopersicum L.

The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. This protocol is useful for clearing tomato seeds at different developmental stages to observe the finer embryonic structure.

一种用于研究番茄种子发育的有效清除方案（Solanum lycopersicum L.）

Tomato (Solanum lycopersicum L.) is one of the major cash crops worldwide. The tomato seed is an important model for studying genetics and developmental ...

发育生物学

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

高通量识别对伪单鱼的电阻syringae pv。番茄中的番茄使用苗子洪水测定

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.

Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.

隔离原住民土壤微生物潜力打破生物降解塑料地膜应用于农业

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

生物学

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

拟南芥种子高通量灭菌的标准化方法

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

使用直接输注装置在植物中递送分子

Direct Infusion Device for Molecule Delivery in Plants

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed.
To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant&#39;s trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed.
Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for&#160;testing and facilitate research and screening purposes.

作者聚焦：用于植物分子递送的直接输注装置  

This manuscript describes a novel direct plant infusion device for screening the effectiveness of molecules against the bacteria (Candidatus Liberibacter asiaticus) or its insect vector (Diaphorina citri, Kuwayama) that, in combination, are associated with Huanglongbing citrus disease.

用于植物分子输送的直接输液装置

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but ...

在穿孔的包装系统二氧化氯的控释，延长贮藏寿命，提高葡萄番茄的安全

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一种用于研究番茄种子发育的有效清除方案（Solanum lycopersicum L.）

高通量识别对伪单鱼的电阻syringae pv。番茄中的番茄使用苗子洪水测定

隔离原住民土壤微生物潜力打破生物降解塑料地膜应用于农业

拟南芥种子高通量灭菌的标准化方法

用于植物分子输送的直接输液装置

用于植物分子输送的直接输液装置